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PLENARY TALK ABSTRACTS
Evening Opening Session: Gene Networks and Functional Genomics
Hemichordate neurulation and the origin of the chordate body plan
Hiroshi Wada
University of Tsukuba, Tsukuba, Japan
The origin of the body plan of our own phylum, Chordata, is one of the most fascinating questions in
evolutionary biology. Yet, after more than a century debate, the evolutionary origins of the neural
tube and notochord, the defining features of chordates, remain unclear. The collar cord of
hemichordates have been proposed as predecessor of the neural tube, but this has not been
supported by molecular evidence. Here, we examined the development of the hemichordate collar
cord and found the shared gene expression patterns between hemichordate and chordate
neurulations. Moreover, we found that the nearby endoderm of the collar cord secreted Hedgehog
molecules and the collar cord cells could receive the signal. Our data suggest that the anterior
endoderm functions as an organizer to pattern the overlying collar cord, similar to the relationship
between the notochord and neural tube in chordates. We propose that the gene regulatory
networks underling the development of the hemichordate anterior endoderm and collar cord were
co-opted by chordates for development of the notochord and the neural tube.
Development and evolution of the cardiogenic mesoderm in chordates
Lionel Christiaen
New York University, New York, NY
Craniofacial and cardiac muscles play fundamental roles in animal physiology and have diversified
and adapted substantially in higher vertebrates. Recent studies have uncovered a common clonal
origin of the heart and subsets of head muscles in amniotes [1, 2]. We recently showed that the
cardiogenic mesoderm of the ascidian Ciona intestinalis gives birth to both the heart and atrial
siphon muscles (ASM), which express Islet and Tbx1, two markers of the second heart field and
branchiomeric muscle precursors in amniotes [3]. We proposed that the existence of a population of
cardiopharyngeal mesoderm precursors is an innovation of the monophyletic group comprising
tunicates and vertebrates [4]. We found that, in Ciona larvae, the ASM-specific transcription factor
COE (Collier/Olf/Ebf) is both necessary and sufficient to inhibit heart fate specification and promote
ASM development within the cardiogenic lineage. Using Fluorescence Activated Cell Sorting
(FACS) and whole genome transcription profiling by microarray analysis, we investigated the
transcriptional changes that underlie heart vs ASM fate specification. Our results suggest that the
common heart and ASM progenitors (i.e. the trunk ventral cells) activate an heart-like regulatory
program, while COE triggers a skeletal muscle program at the expense of the pre-existing cardiac
profiles. Finally, we present evidence that key cardiac regulators inhibit COE expression in the heart
precursors, thus uncovering mutually exclusive regulatory inputs that contribute to a dual heart vs
ASM fate specification within the cardiogenic lineage. 1. Lescroart, F., et al., Development, 2010.
137(19): p. 3269-79. 2. Nathan, E., et al., Development, 2008. 135(4): p. 647-57. 3. Stolfi, A., et al.,
Science, 2010. 329(5991): p. 565-8. 4. Tolkin, T. and L. Christiaen, Curr Top Dev Biol, 2012. 100: p. 10742.
1 Plenary Session I Genomic Logic and Cell Specification
Integration of canonical and non-canonical Wnt signaling pathways patterns the
neuroectoderm along the anterior-posterior axis of sea urchin embryos
Ryan Range, Robert Angerer and Lynne Angerer
National Institute of Dental and Craniofacial Research, National Institutes of Health
Bethesda, MD
Patterning the neuroectoderm along the anterior-posterior (AP) axis is a critical event in the early
development of deuterostome embryos. However, the mechanisms that regulate the specification
and patterning of the neuroectoderm are incompletely understood. Remarkably, the anterior
neuroectoderm (ANE) of the deuterostome sea urchin embryo expresses many of the same
transcription factors and secreted modulators of Wnt signaling as the early vertebrate ANE
(forebrain/eye field). Moreover, as is the case in vertebrate embryos, confining the ANE to the
anterior end of the embryo requires a Wnt signaling-dependent mechanism. Here we use
morpholino- or dominant negative-mediated interference to knock down the activities of the different
Wnt signaling pathway branches. Our results show that the early sea urchin embryo integrates
information from the Wnt/β-catenin, Wnt/Fzl5/8-JNK, and Fzl1/2/7-PKC pathways to provide precise
spatiotemporal control of neuroectoderm patterning along its anterior-posterior axis. Together,
through the Wnt1 and Wnt8 ligands, they orchestrate a progressive posterior-to-anterior wave of respecification that restricts the initial, ubiquitous, maternally specified, ANE regulatory state to the
most anterior blastomeres. In these anterior cells, the Wnt receptor antagonist, Dkk1, protects the
ANE fate through a negative feedback mechanism. Because these different Wnt pathways
converge on the same cell fate specification process, our data suggest they may function as
integrated components of an interactive Wnt signaling network. Our findings also provide strong
support for the idea that the sea urchin anterior neuroectoderm regulatory state and the patterning
mechanisms that position and define its borders represent an ancient regulatory mechanism that
was present in the common echinoderm/vertebrate ancestor.
A regulatory fate map for the developing gut
Isabelle S. Peter
California Institute of Technology, Pasadena CA
Cells which form the sea urchin embryonic gut are progressively specified during development
according to their position in the mature organ. Morphologically, the gut consists of three
compartments, the fore-, mid- and hindgut, which are separated by two contractile sphincters. The
cells which compose these compartments derive from two cell lineages which just prior to
gastrulation express different regulatory states. Over the past few years we have solved the
regulatory processes which determine the early specification of endodermal cells. The sufficiency of
these mechanisms was recently demonstrated using a Boolean computational model of the early
endomesoderm GRN. Based on these insights, we are currently identifying the regulatory
apparatus which determines spatial complexity during later gut development. Our results indicate
not only the different regulatory domains which subdivide the cells of the mature gut, but also the
temporal and spatial order in which these different cell fates become established and distinguished
during gut development.
2 Conservation and Divergence of a Gene Regulatory Network that Controls Gut Patterning in
Deuterostomes
M. Ina Arnone and Rosella Annunziata
Stazione Zoologica Anton Dohrn, Napoli, Italy
How to make a functional gut from a primitive archenteron? How the mechanisms involved in gut
patterning evolved? To answer these questions, we used a system approach aimed to elucidate the
GRN involved in patterning and regionalization of the endoderm in the sea urchin embryo, to
assess the degree of conservation of this network amongst the deuterostome lineage, and to gain
insight into the origin and evolution of the Parahox genes leading to the chordate lineage. We in fact
demonstrated that two of the three sea urchin ParaHox genes – discovered through the S.
purpuratus genome project–, SpLox and SpCdx, are expressed in a spatial and temporally collinear
fashion within the developing digestive tube and play a key role in partitioning of the mid- and hindgut. The two ParaHox genes are involved in mutual regulation: the posterior SpCdx gene is not
expressed in the absence of the anterior SpLox gene and the expression domain of the anterior
SpLox gene is not restricted posteriorly when the posterior SpCdx gene is silenced. Moreover,
several signaling events are involved in patterning of the larval gut, as, e.g., SpWnt10 mediates
SpCdx repressive functions in the most posterior hindgut, while retinoic acid signaling appears to
control gut identity along the anterior/posterior axis. A comparison with vertebrates showed a
striking conservation of topology of gene expression and signaling events between sea urchin and
mouse, thus suggesting the existence of an ancient “kernel” of genes involved in gut patterning
processes among deuterostomes. However, when we extended the analysis of this GRN to the
starfish P. miniata, despite the high conservation of topology of gene expression, we surprisingly
found considerable divergence at the level of gene interactions.
Properties of gene regulatory network governing the specification of sea urchin oral
ectoderm.
Enhu Li and Eric Davidson
California Institute of Technology, Pasadena CA
The Nodal signaling pathway is known from earlier work to be an essential mediator of oral
ectoderm specification in the sea urchin embryo, and indirectly, of aboral ectoderm specification as
well. Following expression of the Nodal ligand in the future oral ectoderm during cleavage, a
sequence of regulatory gene activations occurs within this territory which depends directly or
indirectly on nodal gene expression. Here we describe additional regulatory genes that contribute to
the oral ectoderm regulatory state during specification in Strongylocentrotus purpuratus, and show
how their spatial expression changes dynamically during development. By means of system wide
perturbation analyses we have significantly improved current knowledge of the epistatic relations
amongst the regulatory genes of the oral ectoderm. From these studies there emerge diverse
circuitries relating downstream regulatory genes directly and indirectly to Nodal signaling. A key
intermediary regulator, the role of which had not previously been discerned, is the not gene. In
addition to activating several genes earlier described as targets of Nodal signaling, the not gene
product acts to repress other oral ectoderm genes, contributing crucially to the bilateral spatial
organization of the embryonic oral ectoderm. In addition, a de-repressing mechanism is employed
to control oral ectodermal gene expression. Two repressors, Sp-Ets4 and Sp-Sip1, were found to
repress expression of oral ectodermal genes gsc and foxg, and the stomodeal gene bra. Clearance
of sip1 and ets4 thus are required to provide temporal and spatial restriction on the expression of
some oral ectodermal genes.
3 Primordial germ cell specification in the sea urchin
Mamiko Yajima and Gary Wessel
Brown University, Providence RI
The process of germ line determination uses many conserved genes yet is highly variable in
mechanism. Often closely related animals utilize one or another of the two basic mechanisms:
Flies, worms, fish, and frogs use mechanisms based on inheritance of maternal determinates that
direct those inherited cells to become germ cells, whereas mice and other tetrapods use inductive
mechanisms for germ cell fate. Echinoderms are positioned at the base of Deuterostomia and are
critical to understanding these evolutionary transitions, yet the detailed mechanism of germ line
specification is not known in any member of the phyla. Here we demonstrate that small micromeres
(SMics), formed at the 5th cell division of the sea urchin embryo, illustrate many typical features of
inherited primordial germ cell (PGC) specification; SMics autonomously expressed germ line genes
in isolated culture, which included selective Vasa protein accumulation and transcriptional activation
of nanos, their descendants were passively displaced toward the animal pole by secondary
mesenchyme cells (SMCs) and the elongating archenteron during gastrulation, Cadherin (G-form)
has an important role in their specification and clustering phenotype, and a left/right integration into
the future adult anlagen appears to be controlled by a late developmental mechanism. These
results suggest that sea urchin SMics share many more typical characters of PGCs than previously
thought, and implies a more widely conserved system of germ line specification among metazoans.
We hypothesize that the sea urchin embryo has recently acquired changes to adopt a more
autonomous manner of PGC specification. This evolutionarily distinct feature of sea urchin embryos
among echinoderms will be a useful tool to reveal mechanisms of germ line evolution and the
multipotent cell specification program that might be widely conserved throughout the animal
kingdom.
New Linkages in the Mesodermal GRN
Andrew Ransick, Stefan Materna and Eric H. Davidson
California Institute of Technology, Pasadena CA
Euechinoids have evolved mechanisms for initial specification of skeletogenic and chromogenic
mesodermal lineages during the middle cleavage stages. The emergent GRN linkages that
account for specification of these derived mesodermal cell types are relatively well studied. We
refocus now on resolving the Meso-GRN linkages relating to subdivision (or polarization) of the
primary non-skeletogenic mesodermal territory along the oral-aboral axis, an important
consequence of which is specification of an oral sector that later yields the pleisiomorphic
mesodermal lineages for muscle cells, blastocoelar mesenchyme and coeloms. With the recent
insight that an important role is played here by the homeodomain factor Sp-not, a Nodal target gene
orthologous to the notochord gene of vertebrates, our focus is entrained on how the core regulatory
factors of mesodermal founder cells respond to Sp-not, and how the interactions amongst these
regulatory factors drive specification processes within the mesodermal territory.
4 Plenary Session II Genomics, Transcriptomics, and BioInformatics
Sequencing more echinoderm genomes.
R. Andrew Cameron, Q. Tu, P. Kudtarkar, Q. Yuan, U-J. Kim and E. H. Davidson
California Institute of Technology, Pasadena CA
While the genome of the purple sea urchin, Strongylocentrotus purpuratus, continues to be the
reference for studies in cell and developmental biology, sequence information is accumulating for 6
other echinoderms. With the recent addition of a sea cucumber and a brittle star to the panel of
sequencing projects at Human Genome Sequencing Center, Baylor College of Medicine (HGSC) all
classes of eleutherozoan echinoderms are now represented. This diverse collection of data sets will
invite new looks at questions of comparative genomics ranging from the evolution of gene families
to the form of the ancestral echinoderm genome. Furthermore, comparative sequence analyses
previously possible with only isolated sequence regions such as BAC inserts can now be applied to
whole genomes. A first look at some details of inter-specific genome comparisons will be presented.
The status of the various sequencing projects will be discussed along with a description of the
datasets currently available. The progress on the construction of the new web site, Echinobase, to
accommodate these data will be reported.
MicroRNAs regulate cell specification and cell movement in the purple sea urchin
Jia L. Song, Nadezda Stepicheva, and Archana Siddam
University of Delaware, Newark, DE, United States
Development of complex multicellular organisms requires careful regulation at the transcriptional as
well as post-transcriptional levels. Post-transcriptional gene regulation is in part mediated by a class
of 21-25 nucleotides long non-coding RNAs known as microRNAs (miRNAs). While we have made
great progress in understanding the gene regulatory network of the early developmental events in
the purple sea urchin, Strongylocentrotus purpuratus, not much is known about the posttranscriptional regulation mediated by miRNAs. miRNAs are essential for sea urchin gastrulation, a
developmental stage where regulation of cell movement, cell proliferation, and cell specification
need to be carefully controlled to ensure a successful embryo (Song et al. 2012). The current study
tests the hypothesis that early developmental pathways and cellular movements are in part
regulated by a few key miRNAs. Our laboratory examines the functions of four important miRNAs
previously identified to rescue developmental defects induced by the global depletion of miRNAs in
the early embryo. We use loss-of-function and gain-of-function reagents against specific miRNAs to
examine their functions, identify potential miRNA gene targets using candidate gene approach and
high throughput proteomic technology, and validate miRNA targets with reporter constructs and
mutagenesis. We discovered that miR-31 affect cell specification of skeletogenic mesenchyme cells
and identified Alx1 as a direct target. We also identified functional miRNA binding sites within the
3’untranslated regions of cadherin and β-catenin, proteins that are critical for cell movement and
cell specification. These results integrate the regulatory roles of miRNAs to existing gene regulatory
networks to provide a more comprehensive understanding of early developmental processes.
5 A screen for skeletal patterning genes in sea urchin embryos
Cynthia A. Bradham1, Micheal Piacentino1, Finnegan Hewitt1, Arlene Reyna1, Hajerah
Hameeduddin1, Christy J. Li1, Jia J. Yu1, Vijeta J. Patel1, James Chaves1, Patrick J Ferrell1, Evan
Bardot1, David J. Lee1, Sarah J. Cho1, Amanda B. Core1, Matthew J Tse1, Ekaterina J. Olenik1,
Jasmin J. Coulomb-Huntington1, and Albert J. Poustka2
1
Boston University, Boston, MA; 2Max-Planck Institute, Berlin, Germany
Skeletal patterning in sea urchin embryos is regulated by cues within the ectoderm that direct the
position of the skeleton-secreting PMCs. We performed a screen to identify those cues based on
the hypothesis that patterning genes are absent from the ectoderm in embryos treated with either
nickel chloride or SB203580 (SB). Both treatments perturb the skeletal pattern, provoke abnormally
long filopodia from the PMCs, and function within the ectoderm. However, the two agents have
opposite effects on Nodal, the ventral-inducing signal. This implied that patterning genes could be
identified as a set of genes downregulated by both nickel and SB compared to controls, which we
predicted to be a minority set distinct from DV specification genes. We tested this hypothesis by
cDNA sequencing and differential analysis of control, nickel-, and SB-treated embryos at late
gastrula stage. We found that only 1.5% of scaffolds were downregulated by both perturbants. We
focused on the genes encoding cell surface and secreted proteins, which were enriched 2-fold
compared to a random set and represented the majority of the candidates. Using loss of function
(LOF) analysis, we tested 10 candidates and found that each is required for normal PMC
positioning and skeletal patterning, while none impacted the DV axis, as assessed by ciliary band
labeling. These results indicate that the patterning defects we observe are not derivative to DV
defects. We scored large numbers of morphant embryos for PMC positioning and skeletal
patterning defects; the results show that the defects arise from PMC positioning gaps, indicating
that the genes function to direct PMC localization rather than interfering with biomineralization;
indeed, skeletons are produced in LOF morphants in all cases, and element losses often correlate
with PMC positioning gaps. Thus, we have identified a novel cohort of genes that direct PMC
positioning to regulate skeletal patterning.
Direct downregulation of deadenylase activity in small micromeres as a mechanism for
specifying the germ line
S. Zachary Swartz1, A.M. Reich, P.M. Milos2, A. Hamdoun3 and Gary Wessel1
1
Brown University, Providence, RI; 2Helicos BioSciences Corporation, Cambridge, MA;
3
Scripps Institution of Oceanography, La Jolla, CA
The germ line contains stem cells responsible for transmitting all heritable information between
generations. A widely conserved strategy for segregating primordial germ cells (PGCs) in
development involves localized determinants, often called a “germ plasm,” which direct the cells
that inherit it toward germ line fate. Our investigations have not revealed a localized germ plasm in
the early embryo of the sea urchin S. purpuratus . However, several mRNAs commonly found in
germ plasm are broadly distributed in the early embryo, followed by later restriction to the small
micromeres (SMMs), the likely PGCs. To better understand the initial segregation of PGCs in the
sea urchin, we performed deep sequencing and differential expression analysis of FACS-isolated
SMMs. We identified a set of genes whose mRNAs are broadly-distributed in eggs and early
embryos, but later restricted to the SMMs. Further, synthetic injected RNAs predicted to be
nonspecifically-regulated by the embryo also become restricted to the PGCs. This suggests a
general post-transcriptional mechanism that retains mRNAs in the PGCs, and degrades them in
somatic cells. We found that the CCR4-NOT deadenylase complex member Cnot6, its core poly-A
nuclease, is depleted in the SMMs but present in all other cells of the embryo. This observation
suggests a mechanism for the stabilization of germ line mRNAs in the PGCs but turnover
6 elsewhere. Knockdown of Cnot6 results in broad accumulation of transcripts normally restricted to
the PGCs. Finally, the 3’UTR of Cnot6 contains putative Nanos response elements (NREs), which
are required for its exclusion from the SMMs and may be targeted by Nanos, which is selectively
expressed in the SMMs. We thus provide the first evidence that primordial germ cells have directly
repressed deadenylase activity. We propose that this characteristic protects them from
differentiation by retentaining mRNAs of the early totipotent embryo.
Time, clock and neurosensory cells
Paola Oliveri
University College London, London, UK.
Almost all living organisms show circadian rhythmicity as a result of the evolutionary pressure
exerted by the earth’s rotation. The circadian clock is an endogenous time-keeping mechanism that
regulates daily physiological and behavioural processes. The molecular clockwork is encoded in the
genome: it is composed by a transcriptional-translational oscillator (TTO) based on a negative
feedback loop and is entrained to environmental cues such as light. At cellular level, metazoan
relay on either localized or dispersed sensory cells to entrain either central or dispersed clocks. To
better understand metazoan evolution of circadian clocks, we are studying molecular and cellular
components of sea urchin circadian clock in embryos, larvae and adults. Genome analysis of sea
urchin clock genes shows a highly complex “clock” tool-kit with both protostome and deuterostome
components. Our gene expression data show no evidence of robust circadian oscillation in
embryos. On the contrary, fully differentiated larvae show circadian oscillation of gene expression at
different light regimes. In situ hybridization, identified a subset of larval sensory neurons as a
potential central clock cells. These cells arise from the apical domain of the sea urchin embryo and
are characterized by a unique molecular signature. This study sheds light on evolution of the
circadian clock regulatory network and evolution of neurosensory cells.
Plenary Session III Gametogenesis, Fertilization and Early Development
Polar Expansion and Cytokinesis.
Heather F.M. Gudejko and David R. Burgess
Boston College, Chestnut Hill, MA
We revisit the question of polar expansion during cytokinesis. Our earlier studies showed dramatic
movements of membrane rafts containing signaling molecules toward the furrow. We have further
studied membrane dynamics during division. We wished to reconcile our earlier studies on
movement to the furrow of lipid raft containing membrane and the lack of mobility of lipid rafts.
Using fluorescent cholera toxin subunit B as a marker for the plasma membrane rafts, we document
expansion of the plasma membrane at the polar regions of the cell during division, likely due to new
membrane being inserted into the plasma membrane at the poles. New membrane addition at the
poles is dependent on anaphase onset and astral MTs, which are known to reach the poles prior to
the equatorial cortex. This new membrane is compositionally unique from the original zygote
membrane and is added significantly earlier during cytokinesis than the new membrane that is
added after mitotic exit at the cleavage furrow. Our findings suggest that polar expansion via
addition of new membrane forces the original cell membrane containing lipid rafts to flow into the
furrow during cytokinesis. It is likely that the inward inflection of the furrow is due to the joint action
of the contractile ring and flow of the plasma membrane into the equatorial region.
7 Protein-protein interactions during male pronuclear envelope formation revealed by FRETFLIM
Richard D. Byrne1, Christopher Applebee1, Dominic L. Poccia2, and Banafshe Larijani1
1
Cancer Research UK, London, UK; 2Amherst College, Amherst, MA
In the sea urchin, once a sperm has entered the egg it undergoes a number of transformations
before male pronuclear fusion with the female pronucleus. Initially, the sperm mitochondrion and
flagellum are lost. Formation of the male pronucleus (MPN) is preceded by vesiculation of the
sperm nuclear envelope (NE). During vesiculation, sperm chromatin decondenses and a new NE is
assembled mostly from egg endoplasmic reticulum (ER) membranes, which envelop the chromatin.
In addition, a second membrane population found in the egg cortex, MV1, is indispensible for NE
formation. MV1 is enriched in PLCγ, its putative upstream activator src family kinase 1 (SFK1) and
its substrate PtdIns(4,5)P2. The hydrolysis of PtdIns(4,5)P2 by PLCγ yields large quantities of the
fusogenic lipid diacylglycerol, leading to the fusion of juxtaposed nuclear envelope precursor
membranes.
Here, using time-resolved FRET we examine for the first time endogenous protein-protein
interactions taking place on the surface of MV1 during the formation of the MPN in vivo and in vitro.
The temporal association and activation of PLCγ by SFK1 during NE assembly is demonstrated, as
is the resulting SFK-dependent phosphorylation of PLCγ on Y783. These FRET studies are
corroborated by complementary biochemical experiments utilising pharmacological inhibitors and
antibodies that together demonstrate a pivotal role for SFK1 activity in MPN formation. We
demonstrate further enhancements of time-resolved FRET with which we have probed the potential
interaction between Ran GTPase (also enriched on MV1) and PLCγ during NE formation. We
believe time-resolved FRET is a generic approach to monitor endogenous protein-protein
interactions during embryogenesis that is of benefit to the sea urchin community as a whole.
Protein Interaction and Phosphorylation Dynamics at Fertilization
Kathy Foltz
UC Santa Barbara, Santa Barbara, CA
The activation of development at fertilization is a complex, exquisitely choreographed collection of
events in response to productive interaction with a single sperm. The sea urchin egg is a key model
system for studying this phenomenon, and many foundational discoveries about fertilization were
first described in this echinoderm. Egg activation clearly depends on the sperm-induced rise in
cytosolic Ca2+ levels. However, little is understood about the precise regulatory mechanism of
Ca2+ release and how this mediates the coordinated events subsequent to insemination. While
individual proteins have been identified and characterized as important, a comprehensive overview
of the signaling and other networks is lacking. Because the events of early egg activation are rapid
and independent of transcription and translation, we have applied proteomic methodologies to the
maternal proteins that mediate the fertilization response through post-translational modifications
and subcellular relocalization. We have designed and carried out large-scale biochemical screens
to identify candidate proteins involved in egg activation. The first screen uses affinity capture
assays to select specific, fertilization-dependent interactions with Src Family Kinases (SFKs), key
players in mediating the initial, sperm-induced Ca2+ release. The second screen identifies proteins
that undergo a quantitative change in phosphorylation state in response to fertilization. Finally, the
third screen is based on high throughput, unbiased protein-protein interactions that occur over time
post-fertilization. Candidates are then collectively evaluated to identify those proteins that emerge
8 as strong hits from all three screens. Currently, these candidates are being characterized in an
effort to rigorously probe their roles in egg activation and the egg-to-embryo transition.
Translational control following fertilization
Julia Morales, Vlad Costache, Sandrine Boulben, Erwan Corre, Gildas Le Corguillé, Virginie
Glippa, Robert Bellé, Odile Mulner-Lorillon, and Patrick Cormier
CNRS/UPMC, Roscoff, France
Half of the variation in proteins concentration in a cell can be attributed to differences in translation
rates as determined by global quantification of gene expression. Therefore, protein synthesis
represents an import step in gene expression regulation. Early embryogenesis is highly dependent
on translational regulatory cascades, and relies on the use of maternally stored mRNA in the
cytoplasm rather than transcription of zygotic genes. In sea urchin, protein synthesis is low in
unfertilized eggs and is stimulated rapidly following fertilization. Protein synthesis is necessary for
the onset of first cell divisions. Sea urchin embryos offer therefore an elegant model to elucidate the
network of actors involved in translational control during early development. We have shown that
fertilization triggers an activation of the translational machinery acting upon several translation
factors, at both levels of initiation (eIF4E, eIF4G, eIF2alpha, 4E-BP) and elongation (eEF2) steps.
Modulation of the translational machinery leads to the translation of maternally stored mRNA. We
have undertaken the analysis of translated mRNAs, which are recruited into polysomes, following
fertilization. All together, our results have given new insights for the understanding of translational
control of gene expression during early development.
Structure, function and regulation of multidrug transporters in sea urchins
Amro Hamdoun, Tufan Gokirmak, Lauren Shipp, Joseph Campanale, Kristen Whalen, and Gary
Moy
Scripps Institute of Oceanography, La Jolla, CA
Multidrug (MDR) efflux transporters are conserved membrane proteins that use ATP to move
molecules out of cells, against their concentration gradients. Unlike other enzymes and membrane
channels, MDR transporters are not specific for single substrates, but rather bind and transport a
diverse array of moderately hydrophobic molecules. While polyspecific transport is advantageous
for broad-spectrum xenobiotic efflux, it could come at a cost in development, where inductive
processes depend on precise gradients of morphogens that are themselves potential substrates of
these transporters. To understand these competing functions, we are investigating how MDR
transporters are controlled in development. In sea urchin embryogenesis, MDR transporter genes
are used extensively with 20 ABCB, ABCC and ABCG genes expressed through the early prism
stage. Transporters predicted to efflux signaling molecules have spatially restricted expression, as
is the case for ABCC5a in veg 2 cells. In contrast, xenobiotic transporters, such as ABCB1a, are
expressed more ubiquitously on apical surfaces of ectodermal cells. Efflux activity can be increased
dramatically by insertion of intracellular stores of transporters in the plasma membrane and their
precise spatial positioning on tips of microvilli. Conversely, activity can decrease in specific subsets
of cells, as seen during the endocytic down-regulation of MDR activity in small micromeres. While
the function of key MDR transporters, such as ABCB1a, appears to be conserved, mutations in
drug binding domains subtly modify substrate selectivity. Collectively, these expression patterns,
ontogenetic switches and structure-function relationships shed light on the diverse range of cellular
and evolutionary processes by which plasma membrane function is finely tuned in development.
9 Spindle positioning mechanisms in ascidian embryos
Remi Dumollard, Janet Chenevert, Francois Prodon, Celine Hebras, Karen Lee, Christian Sardet
and Alex McDougall
Centre National de la Recherche (CNRS), Villefranche-sur-Mer, France.
During asymmetric cell division (ACD) cortical cues regulate the orientation of the cell division plane
creating molecularly-distinct daughter cells. In ascidian embryos a cortical macroscopic structure
(the Centrosome-Attracting Body or CAB: see Hibino et al., 1998) containing polarity proteins
PAR3, PAR6 and aPKC (Patalano et al., 2006) causes three successive unequal cleavages in germ
cell precursors from the 8-64 cell stage (Negishi et al., 2007). This leads to the segregation of 40
maternal mRNAs (PEMs) that are tethered to the CAB into the prospective germ cells.
By combining fast 4D fluorescence imaging at the 16-cell stage we found that the crucial process
regulating unequal cleavage in the ascidian is active during mitosis rather than interphase as
previously thought. For example, the mitotic spindle initially forms far from the CAB before one pole
of the mitotic spindle (which we call the CAB spindle pole) moves towards the CAB at
prometaphase causing the whole spindle to position asymmetrically in the blastomere. The CAB
spindle pole then remains tethered near the cortical CAB throughout cytokinesis thereby ensuring
that the CAB and all the maternal PEM mRNAs are inherited by only one daughter cell (the smaller
of the two sister cells). Knockdown of PEM1 completely abolished spindle movement towards the
CAB. Interestingly, PEM1 has recently been shown to bind Plk1 (Negishi et al., 2011). We also
find that Plk1and other cell cycle-regulated kinases such as Aurora are enriched in the CAB. More
recently we have also been studying the orientation and timing of all cell divisions during ascidian
embryogenesis up to the gastrula stage. Some of our more recent unpublished findings will be
shown.
Activation of ERK Kinase at fertilization in sea urchin eggs
Micheal J. Whitaker, Mark, Levasseur, Marwa Eliwa, Marwa and Ali Etfat
Newcastle University, Newcastle upon Tyne, United Kingdom
Fertilization in sea urchin eggs leads to cell cycle resumption from a G1 arrest. The fertilizing sperm
triggers a large and transient increase in intracellular calcium and a marked increase in cytoplasmic
pH through activation of the phosphoinositide signalling pathway. These changes result in the
initiation of DNA synthesis. Using fluorescent reporters and real time imaging, we have shown that
the onset of DNA synthesis is the result of activation of an ERK signalling pathway that leads to
activation of Cdk2/cyclin E. We are interested in how ERK activity is controlled by the Ca and pH
ionic signals at fertilization. The calcium signal triggers the reorganisation of microtubules that
underlies pronuclear migration. We find that the pH signal plays a role in activating the ERK
pathway at fertilization. We shall discuss how the two ionic signals combine to define a determinate
model of egg activation at fertilization and provide a historical perspective on theories of sea urchin
egg activation.
10 Plenary Session IV Environmental Responses
Evolution of food-induced developmental plasticity in echinoid plutei
Diane K. Adams1, Noemi M. Nowakowski2, Aditya J. Sethi1, and Lynne M. Angerer1
1
National Institute of Dental and Craniofacial Research, National Institutes of Health
Bethesda, MD; 2American University, Washington, DC, United States
While it has been well established that environmentally-induced variability in phenotype is important
for adaptation and evolutionary processes, it is less clear how developmental responses
themselves arose and evolved. Recent work in Strongylocentrotus purpuratus showed that foodinduced dopamine signaling alters development to inhibit arm elongation during the pre-feeding
larval stage. Elucidating part of the mechanism allowed us to probe Echinoidea to determine when,
and eventually how, the ability to alter development in response to food evolved. Within the regular
urchins, multiple species showed a dopamine-mediated developmental response to food,
supporting an ancestral origin within this group. However, while Lytechinus variegatus carolinus
demonstrated the response to food, L. variegatus variegatus failed to respond to the test species
of food. The underlying dopamine-signaling mechanism, however, remains intact, suggesting a
recet loss of the food response in the subspecies. Outside of the regular urchins, larvae of multiple
sand dollars and a basal pencil urchin lacked both the developmental response to food as well as a
detectable dopamine-signaling pathway. Thus, the developmental response to food appears to be
an innovation of the regular urchins. In order to address how this innovation occurred, we are now
identifying signaling and transcription factor targets of the dopamine signal and developing
techniques for late stage-specific perturbations using photoactivatable morpholinos. With more the
mechanism and new tools in hand, echinoids will provide a rich system to answer how
developmental responses to the environment can evolve from innovation to loss.
The feeding larva as a simple model to characterize immune response in the sea urchin
Jonathan P. Rast, Katherine Buckley, Eric Ho, Catherin Schrankel and Guizhi Wang
Sunnybrook Research Institute, Toronto, ON
The sea urchin genome sequence reveals a complex and novel immune system. Nonetheless
important regulators of development and function are shared between vertebrate and sea urchin
immunocytes, suggesting that at some level divergent protective strategies are integrated within
conserved regulatory mechanisms. To understand the nature of this homology, the regulatory
interconnections within these systems must be compared. The sea urchin embryo and larva provide
a morphologically simple experimental model in which to characterize immune gene regulatory
networks. The feeding larva has at least five morphologically distinct types of immune reactive cells
that each undergo characteristic patterns of cell migration and interactions in response to a gutassociated bacterial infection. A distinctive program of immune gene expression accompanies this
response. Early after bacterial exposure, IL-17 genes are expressed transiently in the gut
epithelium. Hours later immunocytes move to the gut and express a suite of regulatory and effector
genes while more peripheral immunocytes express other effectors. We are developing an immune
gene regulatory model to provide a causal framework for organism-level control of gut immune
reactions. This approach will be used to determine which aspects of the immune system are
conserved among deuterostomes and how these conserved regulatory programs interface with
divergent recognition and effector mechanisms. We have also identified divergent and inducible
immune mediators from infection time course RNA-seq data that are not present in unchallenged
transcriptome datasets. These finding can be integrated with the developmental gene networks
that establish this immunocyte system in the embryo. Gene expression profiles suggest that
11 immunity in the sea urchin larva represents a minimal version of what is present in the more
complex adult. Further regulatory mechanisms are likely intercalated into the system as complexity
increases with animal size and in the context of evolution.
Cellular mechanisms of negligible aging in sea urchins
Colin Du, Arielle Anderson, Mae Lortie, Rachel Parsons and Andrea Bodnar
Bermuda Institute of Ocean Sciences, St. George’s, Bermuda
The life history of sea urchins is fundamentally different from humans and traditional model
organisms used to study the process of aging. Sea urchins grow indeterminately, reproduce
throughout their lifespan and some species have been shown to exhibit negligible senescence. In
addition to these properties, different species of sea urchins are reported to have very different
natural lifespans ranging nearly two orders of magnitude. Thus sea urchins provide a unique model
to investigate cellular mechanisms underlying lifespan determination and negligible senescence. To
gain insight into the biological changes that accompany aging in these animals, we are investigating
genomic profiles in tissues from young and old urchins of species with different life spans; shortlived Lytechinus variegatus, long-lived Strongylocentrotus franciscanus and Strongylocentrotus
purpuratus which has an intermediate lifespan. Using microarrays and qRT-PCR, age-related
changes in gene expression were examined in muscle, esophagus and nerve tissue of S.
purpuratus and muscle tissue of S. franciscanus. The results indicate age-related changes in gene
expression involving many key cellular functions such as the ubiquitin-proteasome pathway, DNA
metabolism, Notch signaling and adaptation to hypoxia. To determine if changes in gene
expression result in the predicted cellular outcomes current studies focus on characterizing protein
homeostasis, tissue regeneration and cellular oxidative stress. Indicators of oxidative damage to
DNA, protein and lipid (8-hydroxydeoxyguanosine, protein carbonyls, 4-hydroxynonenal and
lipofuscin) are not consistently increased in sea urchin tissues with age, however the tissues of
longer-lived species have less oxidative damage than that of the shorter-lived species. Further
studies will determine the mechanisms that prevent the accumulation of oxidative damage with age
and whether overall levels of cellular oxidative damage play a role in lifespan determination. Given
the close phylogenetic relationship between sea urchins and humans, understanding these
mechanisms may reveal approaches to lessen accumulation of age-related damage in human
tissues.
Plenary Session V Patterning
Encoding Anatomy: Linking Developmental Gene Regulatory Networks to Morphogenesis
Charles A. Ettensohn
Carnegie Mellon University, Pittsburgh, PA
A central challenge of developmental and evolutionary biology is to understand how anatomy is
encoded in the genome. Insights in this area will require a detailed understanding of specific
morphogenetic processes at the molecular, cellular, and tissue levels and the elucidation of the
genomic regulatory circuitry that drives the expression of genes that control morphogenesis. I will
discuss the development of the endoskeleton of sea urchins as a model for developing an
integrated view of the genomic regulatory control of morphogenesis. The dynamic cellular behaviors
that underlie skeletogenesis are well understood, and a complex transcriptional gene regulatory
network that underlies the specification of embryonic skeletogenic cells (primary mesenchyme cells,
or PMCs) is currently being elucidated. Many genes that play a direct role in skeletal
12 morphogenesis have been identified and transcriptional inputs into these genes have been defined.
This work establishes a unique picture of the genomic regulatory control of a major morphogenetic
process. Furthermore, because echinoderms exhibit diverse programs of skeletal development,
these findings offer new insights concerning the relationship between gene network evolution and
morphological evolution.
Complex regulation of Wnt signaling controls the size of the anterior neuroectoderm in sea
urchin embryos
Ryan C Range and Lynne M Angerer
National Institute of Dental and Craniofacial Research, National Institutes of Health
Bethesda, MD
Recent studies from our lab have shown that sea urchin embryos use the integrated activities of at
least three Wnt signaling pathways to restrict the initially ubiquitous anterior neuroectoderm (ANE)
regulatory state towards the anterior end of the embryo. This process starts before 32-cell stage in
the posterior half and continues in most of the anterior half until early mesenchyme blastula stage
when it is blocked by the Wnt antagonist, Dkk1, which is expressed throughout the ANE. Control of
the last phase of restriction that establishes the final size of the ANE the secreted Wnt regulators,
sFrp1/5 and Dkk3, which are co-expressed in a small region within the ANE. Expression of each of
these regulatory genes requires the cardinal ANE factors, Six3 and FoxQ2, and is rigidly maintained
via auto-repressive and cross-repressive mechanisms involving interactions with Wnt signaling
components. These results raise the possibility that sFRP1/5 and Dkk3 work together to regulate
ANE border position by controlling Wnt signaling and that loss of either one might have similar
effects on the size of the ANE domain. This is the case because when either sFrp1/5 or Dkk3, or
their upstream regulator, FoxQ2, is knocked down, both inner and outer regions of the ANE are
significantly enlarged, indicating that the sFRP-Dkk3 system is required for the final phase of ANE
restriction. We propose that a precisely controlled balance between the sFRP1/5-Dkk3 system that
potentiates ANE restriction and Dkk1 that attenuates it determines the size of the ANE in the sea
urchin blastula.
Mechanisms regulating establishment of left-right asymmetry in the sea urchin embryo
Nathalie Bessodes, Emmanuel Haillot, Véronique Duboc, Eric Rottinger, François Lahaye and
Thierry Lepage.
CNRS Observatoire Océanologique Villefranche sur Mer France.
Left-right asymmetry is an essential feature of development in most bilaterian animals. How leftright asymmetries arise from embryos that are initially bilaterally symmetrical and how the left-right
axis aligns consistently with the antero-posterior and dorsal-ventral axes are questions that have
recently become the subject of intensive research. Specification of the left-right axis can be
conceptually divided into four distinct steps. The first step involves a directional symmetry-breaking
event and in many species is regulated by a left-right organizer, (the node in the mouse, Küpffer
vesicle in zebrafish, and archenteron roof in Xenopus). The second step in left-right axis
determination involves establishment of asymmetric gene expression on the left and/or right side of
the embryo in response to the flow of laterality information from the organizer. During the third step,
left-right information is transferred from the organizer to more distant tissues.
Sea urchin development offers a striking example of left-right asymmetry. Like most echinoderms,
sea urchins develop indirectly and their larvae undergo a metamorphosis during which most larval
tissues are replaced by adult tissues generated from an imaginal disk called the adult rudiment, that
13 forms exclusively on the left side of an otherwise bilaterally symmetric larva. We showed previously
that nodal expression on the right side of the ectoderm of the larva regulates left-right asymmetry by
repressing formation of the rudiment but the mechanisms that restrict nodal expression to the right
side remained unknown.
We attempted to dissect the molecular cascade that directs nodal expression to the right side of the
sea urchin larva and identified some of the pathways responsible for initiation, restriction and
propagation of left-right asymmetry in the sea urchin embryo. We will present the results of this
study.
Eph and Ephrin signaling functions in the specification and patterning of pigment cells of
the sea urchin embryo
Robert D. Burke, Valerie J. Taylor, Valerie J. and Oliver Krupke
University of Victoria, Victoria, BC, Canada
Early in embryonic development, sea urchin embryos elaborate a population of pigmented
mesoderm cells that distribute themselves throughout aboral ectoderm where they function as the
innate immune system of the larva. Prior to migration from the vegetal plate, pigment cell
precursors express the Eph receptor, Sp-Eph. The ligand, Sp-Efn, is expressed throughout the
aboral ectoderm and presumptive ciliary band. There appears to be a gradient of ligand with the
highest levels of Sp-Efn posteriorly and at arm tips. Interfering with expression of either Sp-Eph or
Sp-Efn with morpholino anti-sense oligonucleotides alters pigment cell distribution and morphology.
Treating embryos with an Eph kinase inhibitor (NVP BHG 712) has a similar affect on pigment cell
morphology and distribution. Ectopic expression of Sp-Efn throughout the embryo causes pigment
cells to migrate onto the apical surface of the ectoderm and enter the oral ectoderm. We conclude
that pigment cells are guided to regions with high levels of Sp-Efn by forward signaling through SpEph. Knocking down expression of Sp-Eph, or treatment with NVP, reduces the number of pigment
cells. Pigment cell number is reduced only if the embryos are treated with the inhibitor during the
first 12 hours of development, indicating that kinase activity is necessary for pigment cell
specification. Sp-Eph is expressed during cleavage by small micromeres, suggesting that Sp-Eph
signaling initiates a non-autonomous effect on pigment cell specification.
Large and small micromere morphogenesis in sea urchin patterning
David R. McClay, David R. Garfield, Megan Martik, Lindsay Saunders, Dede Lyons, and Dan
McIntyre
Duke University, Durham, NC, United States
The small micromeres arise at the vegetal pole from an unequal 5th cleavage, and most of their
progeny eventually move to the left coelomic pouch. We show that the small micromeres reach the
coelomic pouches via a directed homing mechanism. Small micromeres home to the coelomic
pouches from any ectopic location in the early embryo. In a multi-generation genetic experiment we
provide strong evidence indicating that progeny of the small micromeres become the primordial
germ cells of the embryo. The large micromeres that result from the asymmetric 5th cleavage
become the skeletogenic cells. In a series of experiments, we show that components of the
micromere GRN control several discrete events leading up to skeletogenesis. These include five
distinct sub-circuits that together control the ingression of the PMCs, including the invasion of the
PMCs through the basal lamina. The PMCs then fuse, and a different subcircuit of the GRN
controls that fusion. Finally, the PMCs build the skeleton. That function is known to require
patterning input from the ectoderm. We show how signaling inputs establish the border ectoderm
14 specification that is necessary for release of the short-range spatial signals used by PMCs in
constructing the skeleton. The border ectoderm intersects with the ciliary band and at that site
signals are released enabling PMCs to initiate skeletogenesis. From that origin, patterning of the
skeleton unfolds using the border ectoderm and the ciliary band as signaling sources. The initial
morphogenetic and patterning sequences of the ectoderm and micromere lineages will be
described.
Plenary Session VI Evolution
Insights into the Development and Evolution of Multicellularity from The Genome of the
Ctenophore, Mnemiopsis leidyi
J.F. Ryan1, K. Pang2, C.E. Schnitzler 3, W.E. Browne4, B.J. Koch 3, A-D Nguyen3, R.T. Moreland3,
J.C. Mullikin3, T.G. Wolfsberg3, A.D. Baxevanis 3 M.Q Martindale2
1
NHGRI/NIH and Sars Intl. Centre for Marine Mol. Biol.; 2Kewalo Marine Lab, Univ. Hawaii; 3Natl.
Human Genome Res. Inst., NIH and 4Univ. Miami, Florida
Ctenophores are one of the most distant relatives of bilateral animals (bilaterians) and as such, are
critical for reconstructing ancient metazoan ancestors. However, the exact phylogenetic position of
these elaborate animals has been in dispute for over a century. We used next-generation
sequencing technologies to sequence, assemble, and annotate the ~150-megabase genome of the
ctenophore Mnemiopsis leidyi collected in the waters of Woods Hole. While many components of
key protein families and regulatory pathways are present in Mnemiopsis, there are notable
absences; for example, there are no Hox genes of discernible microRNAs in Mnemiopsis, and
elements of the microprocessor complex are altogether missing. We conducted a range of
phylogenomic analyses using a large set of EST and full-genome data. Our analyses provides no
support for a sister relationship between ctenophores and cnidarians (i.e., Coelenterata); instead, it
confirms the sister relationship between cnidarians and bilaterians. The most supported position for
Ctenophora is sister to the rest of animals, but there is also evidence for sponges branching at the
base or even a basal branch consisting of a sponge/ctenophore clade. All three of these branching
positions are in concordance with gene family and signaling pathway studies as well as our
genomic inventories, which show that many genes present in cnidarians and bilaterians (e.g., Hox
genes) are absent in both sponges and ctenophores. Indeed, many genes involved in the formation
of mesoderm in bilaterians are absent from the Mnemiopsis genome. The result is surprising given
that Mnemiopsis and other ctenophores possess definitive mesodermal musculature. Our data
indicate one of three intriguing possibilities, either: (1) Mnemiopsis has lost fundamental
components of its myogenic network yet still maintains this pathway, (2) the mesoderm of
ctenophores evolved independently of other animals, (3) or many genes that were hitherto thought
to be essential for the evolution of the mesoderm evolved after ctenophores diverged from the rest
of animals. Our work enhances the genomic resolution at the base of the animal tree and shows
that several signaling and developmental pathways expanded after ctenophores and sponges
diverged from the rest of animals. We also used blastomere isolation and transcriptomics to gain
insight into the segregation of developmental potential during early embryogenesis of these
animals.
15 The feeding trochophore of the polychaete Hydroides elegans and its
evolutionary/developmental relevance
Arenas-Mena, Cesar
CSI-CUNY, New York, NY
The study of the indirectly developing polychaete Hydroides elegans provides insight into regulatory
circuits of development and their evolutionary interdependence. We have characterized the
expression of brachury, FoxA1, FoxA2, Otx, GataE, GataC, Blimp, T-brain, Tbx2/3, Brain, Sall,
Snail and several differentiation genes. The expression of several transcription factors is invariably
initiated in specific blastomeres, but it is oftentimes very dynamic and does not follow a lineagebound pattern. The expression during gastrulation, neural development, and endoderm
specification consolidates various synexpression groups; for example, the coexpression of FoxA
and brachyury in the blastopore and secondarily formed anus of protostomes and mouth of
deuterostomes suggests shared regulatory circuits for the making of oral and anal openings in
protostomes and deuterostomes. The deployment of some transcription factors correlates with
distinct developmental modes among bilaterians; for example, brachyury anticipates
morphogenesis in endodermal and ectodermal precursors, but it is not expressed in endodermal
precursors of spiralians that lack a feeding trochophore and do not form a functional gut during
embryogenesis. The dorsal side of the Hydroides embryo expresses the transcription factor Tbx2/3
in all three germ layers and the similar expression found in sea urchins suggests shared
dorsa/aboral identity specification in deuterostomes and protostomes. Feeding-dependent posterior
growth elongation driven by multipotent precursors generates the segmented/reproductive side of
the animal and involves sall and probably Hox cluster gene expression. The expression of the
histone variant H2A.Z is invariably associated with transcriptionally multipotent cells and declines in
differentiating cells of Hydroides and purpuratus. H2A.Z is also activated in differentiated larval
regions that apparently reengage in development during the larva to adult transformation. The
histone variant H2A.Z is associated with regulatory DNA and promotes a loose chromatin
conformation that is accessible to transcription factors. We have initiated the cis-regulatory analysis
of H2A.Z in sea urchins and polychaetes.
Nematostella, Crepidula and Mnemiopsis: Oh My!
Joel Smith, Antje Fischer, Sarah Tulin, and Molly Phillips, Molly
Marine Biological Laboratory, Woods Hole, MA
The bilaterian body plan, based on orthogonal body axes and three germ layers, provides the
foundation for tremendous morphological diversity. We are interested in understanding the
evolutionary origins of this spectacularly successful body plan and have chosen three organisms
from diverse phyla as test subjects. Our goal is to determine the gene regulatory network underlying
pattern formation along embryonic axes and the specification of germ layers in each of these model
systems. We performed RNA-seq, using NIST-established internal standards to compute absolute
quantification, in high-resolution time series and perturbation experiments to define gene models in
each species and to identify core regulatory genes for continuing detailed studies, including
ultimately cis-regulatory analyses.
16 Molgulid Ascidians have a Radical Heterochronic Shift in their Metamorphic Gene Network
Billie J. Swalla1, Max Maliska1, Elijah Lowe1, and Titus C. Brown2
1
University of Washington, Seattle, WA; 2Michigan State University, East Lansing, MI
Transcriptome and genome data offer an exciting new approach to examine the origin and evolution
of the chordate body plan. Chordate body plan evolution can be studied with two tunicate species
with radically different larval body plans—the tailed ascidian Molgula oculata and the tailless M.
occulta. Tailed M. oculata embryos have 40 notochord cells that are converged and extended, tail
muscle cells flanking the notochord, and the otolith, a gravity sensory organ located in the head.
The tailless M. occulta does not form a tail in their larval stage, and have only 20 notochord cells
that do not converge and extend during larval development. We show by transcriptome analyses
that the ascidian metamorphosis program begins earlier in molgulid ascidians. This radical
heterochronic shift has been documented in another tailless ascidian, Molgula tectiformis , and is
now reported for both the tailed, Molgula oculata and tailless Molgula occulta . Further functional
data is necessary to determine if this pronounced heterochrony is the necessary preadaptation for
tailless tadpole to evolve in molgulid ascidians. However, we forecast that these studies will
facilitate the elucidation of the metamorphic signal in ascidian tadpole larvae, which is still currently
unknown.
Gene regulatory network changes associated with the evolution of a novel cell populations
in echinoderms
Veronica Hinman, Department of Biology, Carnegie Mellon, Pittsburgh, PA
A central problem in the evolution of development field is to understand how the great diversity of
animal life can be built using the same genetic “toolkit”. Much of this diversity must be driven by
changes in the way that orthologous genes are used during development; in other words through
rewiring of gene regulatory networks (GRNs). We are using the specification of mesoderm at the
vegetal pole of different groups of echinoderms (sea urchins and sea stars) as a model system to
understand how GRNs are rewired, and the developmental consequence of this rewiring. This
system is especially powerful because the specification of this vegetal pole territory is
extraordinarily well known in sea urchins, and yet these organisms have cell types that are novel to
this group, in particular the micromeres which are needed to not only make the larval skeletal but
are an important early signaling center. I will describe the GRN for specification of mesoderm in sea
star embryos with an emphasis on explaining how GRNs are reorganized to allow for changes in
specification, cell type and hence novelty. Our analysis shows that, despite the different cell fates
seen in the mesoderm of sea urchins and sea stars, most of the regulatory genes that drive the
development of these territories are conserved across echinoderms and very similar fate lockdown
and exclusion subcircuits are activated. Specific differences in the context of cell signaling
meanwhile, particularly, Delta:Notch can explain much of the difference in territory refinement.
Origin and evolution of the endoderm gene regulatory network: Insights from regulation of
Disheveled along the animal-vegetal axis
Athula H. Wikramanayake
The University of Miami, Coral Gables, FL
The animal-vegetal (AV) axis is a maternally established polarity present in most animal ova that
strongly influences early pattern formation in embryos. In several bilaterian taxa and in cnidarians
localized activation of Wnt/beta-catenin signaling along the AV axis specifies endoderm indicating
that this pathway played a key role in the evolution of an ancient endoderm Gene Regulatory
17 Network (GRN) over 600 mya. Strikingly however, Wnt/beta-catenin signaling-dependent
endoderm specification occurs at the vegetal pole in bilaterians and at the animal pole in cnidarians.
These and other observations have led us to propose that during metazoan evolution a shift in the
site of activation of localized Wnt/beta-catenin signaling led to the redeployment of the endoderm
GRN from the animal pole of the last common ancestor of cnidarians and bilaterians to the vegetal
pole of the urbilaterian. It is known that during early embryogenesis in sea urchins and in
Nematostella, Wnt/beta-catenin signaling is induced by the localized “activation” of Disheveled, a
central regulator of the Wnt pathway. Hence, elucidating how Dvl is locally activated in bilaterian
and non-bilaterian embryos is crucial to reconstruct the evolution of the endoderm GRN. In sea
urchins, Dvl accumulates in puncta in a vegetal cortical domain (VCD) of unfertilized eggs and this
structure strongly correlates with vegetally restricted activation of Wnt/beta-catenin signaling.
Moreover, VCD associated Dvl undergoes differential post-translational modification indicating that
molecules co-localized with Dvl in VCD puncta may function to regulate Dvl activity in the VCD, and
subsequently in vegetal blastomeres during activation of Wnt/beta-catenin signaling. To identify
candidate molecules that regulate Dvl activity in sea urchins we used Dvl Co-Immunoprecipitation in
combination with mass spectrometry using lysates from eggs, isolated egg cortices and 16-cell
stage micromeres. Functional analysis of these proteins and their roles in regulating Dvl activity in
eggs and embryos will be discussed.
18 CONCURRENT SESSION ABSTRACTS
Concurrent Session I GRN: Cis and Trans Regulation
Evolution of the gene regulatory network orchestrating myogenesis: insights from the sea
urchin embryo.
Carmen Andrikou and Maria Ina Arnone
Stazione Zoologica Anton Dohrn di Napoli, Naples, Italy
The process of specifying muscle cells is highly regulated and, although the gene repertoire of the
involved regulators is remarkably similar among different animals, the driving force of myogenesis
lies in the regulatory network that interconnect these genes (GRN) and not in the genes
themselves. To better understand the evolution of the GRN involved in the myogenic process, a
basal deuterostome phylum, such as Echinodermata, needs to be studied. Our aim is to dissect the
GRN responsible for the specification and differentiation of the esophageal muscle cell type in the
sea urchin embryo. We have so far cloned and characterized the spatial and temporal expression of
genes known to have a myogenic role in other phyla, such as MyoD, Mef2, Six1/2, FoxF, MyoR,
Twist etc. Moreover, lineage analysis using dynamic expression profiles of putative regulatory and
terminal differentiation genes, such as Myosin Heavy Chain, revealed the molecular fingerprint of
the myoblast precursors highlighting the importance of the Fox family factors including FoxY ,
whose role was known to be restricted to the small micromere lineage. Perturbation analysis
experiments also confirmed the key role of some of these genes, such as FoxC and FoxF , and
placed them at the top of the myogenic GRN hierarchy. Another thing emerged from this study is
that only one MyoD paralog (MyoD2) is a myogenic regulatory factor while the previously
characterized SUM1 was coopted to serve the skeletogenic lineage. Also, we demonstrated that
the timing of emergence of muscle progenitors is controlled by an FGFR mediated signaling
pathway, occurring in the early stages of development. In conclusion, using both lineage and
perturbation analyses, we were able to identify the major players of the GRN governing myogenesis
in the sea urchin embryo, which allowed us to unravel interesting evolutionary implications.
Cis-regulatory analysis of the cyclin D gene in Strongylocentrotus purpuratus
Christopher M. McCarty and James A. Coffman
Mount Desert Island Biological Laboratory, Salisbury Cove, ME; and The University of Maine,
Orono, ME
Genes of the cyclin D family regulate the cell cycle, growth and differentiation, and are frequently
mis-regulated in cancers. Our aim is to identify and characterize all potential cis-regulatory module
(CRM)-bearing sequences of SpcycD. Hypothesized CRM-bearing regions consisting of
sequences that are either conserved in cyclin D genes in other organisms or which contain known
binding sites for transcription factors have been incorporated into GFP reporter constructs. Using
conventional methods of cis-regulatory analysis, we found that two regions upstream of the SpcycD
promoter have distinct activity patterns. One of these, a 3.8 kb sequence located 4.5 kb upstream of
the 5’ untranslated region (UTR), becomes active by early blastula stage; the other, a 2.1 kb
sequence directly adjacent to the 5’ UTR, becomes active between late blastula and early gastrula
stage. Currently, the entire SpcycD gene is being subjected to a comprehensive cis-regulatory
analysis using a new high throughput approach developed by Nam et al. (Proc. Natl. Acad. Sci,
USA, 107: 3930-3935, 2010). This protocol confirmed the activity of the two previously-identified
regions, and identified several additional ones. Using this technique, we are testing 12 regions
19 possessing conserved sequences and/or binding sites for clusters of transcription factors, along
with 11 additional regions comprising the remaining sequence not contained in these 12. Once we
have identified which regions are active, we will compare the temporal activity pattern of these
regions to that of both endogenous SpcycD, and that of a co-injected SpcycD BAC. This will allow
us to test the hypothesis that the active regions we have identified are sufficient to regulate the
temporal expression of SpcycD during embryonic development in S. purpuratus, and allow us to
elucidate the role of each region in regulating this pattern.
Runx is a Linchpin for Genomic Control of Cell Cycle Development
James A. Coffman
Mount Desert Island Biological Laboratory, Salisbury Cove, ME
After the initial cleavage stage of embryogenesis the cell cycle becomes non-autonomous,
dependent on intercellular signaling and controlled by the genomically-encoded developmental
program. The regulatory linkages that mediate this transition are still largely unknown. In published
work we have shown that cell division becomes dependent at blastula stage on the Runx
transcription factor Runt-1, which is required for the expression of known mitogenic genes such as
cycD, PKC, and several wnts. More recently a microarray analysis showed that an akt (PKB) gene
is significantly underexpressed in blastula stage runt-1 morphants. Interrogation of the genome
revealed the existence of two akt genes, Sp-akt-1 and Sp-akt-2. While both genes are expressed
maternally, akt-2 at several-fold higher levels than akt-1, transcripts of both undergo marked Runt1-dependent blastula stage accumulation. Inhibition of Akt activity, either via knockdown of akt-2 or
by over-expression of a dominant negative (kinase-dead) mutant of akt-1, arrests cell division at
blastula stage. Development of both akt-2 and runt-1 morphants is partially rescued by
overexpression of akt-1 mRNA. Together these findings indicate that akt is essential for the
continuance of cell cycling beyond cleavage stage, and is part of a Runx-dependent battery of
mitogenic signaling genes that function antagonistically to GSK-3.
MicroRNA-31 is important for the proper specification of the sea urchin
Nadezda Stepicheva and Jia L. Song
University of Delaware, Newark, DE, United States
The microRNAs (miRNAs) are small endogenous RNAs that regulate the translation and stability of
target mRNAs. We previously showed that knockdown of key enzymes of miRNA biogenesis
pathway, such as Drosha and Dicer, resulted in a range of developmental defects in the purple sea
urchin embryos. These developmental defects could be rescued by the exogenous addition of four
most abundant miRNAs, including evolutionary conserved microRNA-31 (miR-31). We hypothesize
that miR-31 regulates essential developmental pathways that are important for proper cell
specification. miR-31 knockdown resulted in dose-dependent developmental defects in the early
embryos, including an overall delay in development, a reduction in embryonic size, formation of cell
clumps, and an increased number of cells in the blastocoelar space of the gastrula embryo. These
defects could be partly rescued by the exogenous addition of the Dicer substrate double-stranded
miRNA-31. To investigate the molecular mechanism of the observed phenotype, we are currently
using different cell specific molecular markers to identify and characterize the different cell types
present in the blastocoelar space of the miR-31 knockdown embryos. We observed decreased
numbers of SM50-positive skeletogenic mesenchyme cells and bioinformatically identified Pmar
and Alx1 3’untranslated regions (3’UTR) to contain potential miR-31 binding sites. Pmar is a pivotal
transcription repressor that indirectly mediates activation of Alx1, a transcription factor that
regulates the skeletogenic fate specification in the sea urchin embryo. Reporter constructs
20 containing Pmar and Alx1 3’ UTRs were generated to test their regulation by miR-31. Preliminary
data suggest that the 3’UTR of Alx1 contains functional miR-31 binding sites. This study contributes
to the understanding of miR-31 function during early embryogenesis and integrates the regulatory
roles of miRNAs into the endomesodermal gene regulatory network.
Constructing regulatory state domains using gene expression patterns in the developing
gut.
Jonathan Valencia, Cathy Dong, Isabelle Peter, and Eric Davidson
California Institute of Technology, Pasadena, CA
The GRN specifying early endoderm formation in the sea urchin embryo is well understood but less
is known about the GRNs governing the regionalization of the larval gut. Identifying all regulatory
genes involved in gut development at the systems level is the first and most crucial step towards
solving the underlying specification GRNs. The following criteria were used to identify candidate
transcription factor genes: 1) genes previously known to be expressed either in endoderm
precursors or in the developing archenteron, 2) regulatory genes orthologous to genes involved in
endoderm specification or gut development in other species, and 3) regulatory genes expressed in
the relevant time window during development, between 24hpf (the time just prior to the onset of
gastrulation) and 72hpf (the time at which the three compartments of the gut within the pluteus larva
have formed) as determined by RNA-sequencing data. We have currently assayed approximately
60 genes from these categories using whole-mount in-situ hybridization (WMISH) experiments and
have confirmed that 80% of these are expressed in the developing gut. About 30 of these genes
have been analyzed by detailed time-course WMISH experiments at six-hour intervals between 2472hpf, to detect the dynamic changes in spatial gene expression during gut development.
Interestingly, our results show that several sea urchin regulatory genes orthologous to genes
expressed in developing vertebrate endodermal organs are expressed also in the developing gut of
the sea urchin embryo. For example, hnf1-al and hnf4, whose orthologs are expressed in the
vertebrate liver, are expressed on the oral side of the midgut and also share a similar dynamic
pattern of spatial expression throughout gut development, suggesting a conserved functional and/or
regulatory connection between these genes.
Concurrent Session II Gametes, Fertilization & Cleavage
Nanos is extra cool!
Nathalie Oulhen1, Mamiko Yajima1, Jia Song2, Naoaki Sakamoto3, Takashi Yamamoto3, and Gary
Wessel1
1
Brown University, Providence, RI; 2University of Delaware, Newark, DE; 3Hiroshima University,
Higashi-Hiroshima, Japan
Nanos is a translational regulator required for the survival and maintenance of primordial germ cells
during embryogenesis. It is usually expressed uniquely in germ cells, and is often “toxic” if active
elsewhere in the embryo. Three nanos homologs are present in the genome of the sea urchin
Strongylocentrotus purpuratus (Sp), each nanos mRNA accumulates specifically in the small
micromeres (SMM), the lineage that contributes to the germline. Using a reporter gene construct to
identify nanos transcriptional regulation, we consistently found transcription outside of the small
micromeres. Instead, we found that the 3’UTR of nanos2 is sufficient for protein accumulation
selectively in the SMM lineage: microinjection into a Sp fertilized egg of an RNA that contains the
GFP open reading frame followed by the Sp nanos2 3’UTR leads to selective protein enrichment in
21 the small micromeres at the blastula stage. The same result was seen with nanos2 from the sea
urchin Hemicentrotus pulcherrimus (Hp). In both species, the 5’UTR alone was not sufficient for the
SMM localization, but it always increased the SMM protein enrichment when present with the
3’UTR. We defined an element conserved between Hp and Sp nanos2 3’UTR that is necessary and
sufficient for protein enrichment in the SMM, and refer to it as GNARLE (Global Nanos Associated
RNA Lability Element). We found that the nanos2 3’UTR is essential for the selective RNA retention
in the SMM, and that GNARLE is required but not sufficient for this process. Surprisingly, injection
of an RNA containing a fused GFP - Sp Nanos2 ORF followed by a Sp nanos2 3’UTR deleted in the
GNARLE also leads to a selective protein enrichment in the small micromeres. These results show
that a combination of selective RNA retention, translational control and protein stability
mechanisms, instills selective nanos expression in the presumptive germ line lineage.
Characterization of sea urchin Mos in Go arrested eggs.
Michelle M. Roux-Osovitz1, Derek Pan2; Erin Baker1, and Kathy R. Foltz2
1
St. Petersburg College, Clearwater, FL; 2UC Santa Barbara, Santa Barbara, CA
Sea urchin eggs have the unusual characteristic of completing meiosis prior to fertilization and
arresting in a haploid Go state. The maintenance of this arrest and regulation of reentry into Sphase is not well understood. In vertebrate oocytes, meiotic arrest is controlled by cytostatic factor
(CSF), of which the protein Mos is the only characterized component. Mos is a highly conserved
MAP kinase kinase kinase which maintains arrest through stabilization of cell cycle regulators.
Given the unique Go arrest of urchin eggs, we asked whether mature eggs express Mos and if this
expression is responsible for mitotic arrest prior to fertilization. The Strongylocentrotus purpuratus
genome contains a Mos-like sequence, and based on this, the full length SpMos cDNA was cloned
and sequenced. The kinase domain shares strong identity with all known Mos proteins. However,
SpMos lacks the canonical Mos phosphorylation site that regulates degradation and the protein has
an extended N-terminus. Recombinant SpMos protein was tested for the ability to arrest cell cycle
progression in dividing Xenopus blastomeres, the gold-standard assay for canonical Mos function.
Both full-length and an N-terminal truncation of SpMos, but not control protein, halted cell division in
a dose-dependent manner. Q-rtPCR revealed that SpMos mRNA is present in unfertilized sea
urchin eggs, decreases after fertilization, and then increases at the blastula-gastrula transition.
Preliminary data indicate that the SpMos protein is present in unfertilized eggs as well, and that it
decreases soon after fertilization, consistent with a role in the regulation of cell cycle arrest.
Experiments are underway to test the ability of SpMos to arrest dividing sea urchin blastomeres.
These data collectively support the emerging view that the Mos protein plays a key role in not only
meiotic cell cycle regulation, but also in controlling transitions from Go arrest to S phase.
NAADP-dependent Ca2+ signaling and the role of ARCs and TPCs
Isabela Ramos and Gary Wessel
Brown University, Providence, RI, United States
A number of messengers able to activate Ca 2+ channels were described for the first time in
echinoderm eggs, such as IP3, cADPR and NAADP, and this system continues to be a vital tool for
investigating such events. Recent studies have identified that NAADP triggers Ca2+ release from
acidic vesicles, through a new class of calcium channels, the two-pore channels (TPCs) and that,
remarkably, both cADPR and NAADP are synthesized by the same family of enzymes, the ADPribosyl cyclases (ARCs). The aim of this project is to gain insights into the mechanisms of action of
TPCs and ARCs using the sea star P. miniata as a model. One ARC and three TPC isoforms were
found in ovarian transcriptomes of the sea star P. miniata. PmTPCs share 37-43% similarity with
22 their correspondents from sea urchin and human, whereas PmARC shares higher similarity with the
sea urchin β-ARC (43%) and human CD38 (human homologous of β-ARC). PmTPC1 and PmTPC3
were detected in high amounts in young, immature and mature oocytes/eggs and co-localize to
PmARC in the plasma membrane, cortical granules and endocytic/ recycling vesicles in the
peripheral cytoplasm, suggesting an involvement with intracellular trafficking and vesicle formation
during oogenesis. In contrast, PmTPC2 is not found significantly in oocytes, instead, its highest
expression is found in migrating mesenchymal cells at mid-gastrula, also co-localizing with PmARC.
To further address the functional roles of the different PmTPC isoforms and PmARC, we are
currently working on the ablation of PmTPCs and PmARC translation by injecting young/ immature
oocytes with inhibitory morpholinos. By studying the abnormalities caused by these knock-down
perturbations, we expect to gain insights into the link between NAADP induced Ca 2+ signals and
the regulation of oogenesis and early embryogenesis.
PLCgamma, G-protein of the Galpha-q type and cADPr pathway are associated to trigger the
fertilization Ca2+ signal in the sea urchin
Brigitte CIAPA
CNRS, Paris, France
In all species, fertilization triggers in the egg a rapid and transient increase of intracellular free
calcium (Cai), but how this signal is generated following sperm and egg interaction has not been
clearly characterised yet. In sea urchin, a signalling pathway involving tyrosine kinase and
PLCgamma has been proposed to be at the origin of the fertilization Cai signal. We found that
injection of src homology-2 (SH2) domains of the sea urchin PLCgamma inhibits in a competitive
manner the endogenous PLCgamma, alters both the amplitude and duration of the fertilization Cai
wave, but does not abrogate it. Our results suggest that PLCgamma acts in conjunction with a
cADPr pathway and G-proteins of the Galpha-q type to trigger the fertilization Cai wave, but
reinforce a crucial role of PLCgamma during mitosis and cytokinesis.
Comparative analysis of echinoderm ovary transcriptomes
Adrian Reich and Gary Wessel
Brown University, Providence, RI, United States
Echinodermata is a diverse phylum that spans 500 million years of evolution but the vast majority of
known genetic information within the phylum is from a single species of sea urchin, S. purpuratus.
In the current study we have sequenced and assembled de novo transcriptomes of ovaries of
twenty different species, representing all five families of echinoderms. The goals of this dataset are
to identify orthologous genes, resolve the phylogenetic relationships of extant echinoderms, identify
rapidly evolving genes, and to provide a resource for the community. A variety of analysis pipelines
were implemented for each transcriptome dataset including protein prediction, gene ontology
annotation, and BLAST comparison to the NCBI non-redundant protein database. For each
transcriptome, we select transcript variants from all assembled contigs by first choosing the contig
with the highest fold coverage amongst isotigs and second by removing any contigs that did not
have a close hit to NCBI nr (e-value > 0.0001). Comparative analyses of these transcripts and
genes from S. purpuratus has revealed significant transitions of gene sequences within the
echinoderm phylum.
23 Concurrent Session III Patterning Mechanisms
Maintenance of Axial Patterning in the Sea Urchin Embryo: A Role of Wnt1 Signaling
Zheng Wei, Ryan Range, Ryan; Lynne Angerer, Lynne and Robert Angerer
NIDCR/NIH, Bethesda, MD, United States
The initial patterning along the anterior-posterior (AP) and dorsal-ventral (DV) axes of sea urchin
embryos depends on Wnt and Nodal signaling, respectively, and occurs before gastrulation. The
secondary DV axial patterning relies on a Wnt-dependent process that removes a suppressor of
nodal expression from non-anterior ectoderm. Here we report that unexpectedly, later, when
gastrulation begins, Wnt signaling continues to affect Nodal signaling, not by supporting it, but
rather by preventing nodal expression in the ventral-posterior region of the embryo. This region
normally gives rise to the posterior-transverse ciliary band, the supra-anal ectoderm and ventral
endoderm. When Wnt1 is knocked down, expression of nodal and its target genes, gsc and bra,
extends ectopically on the ventral side toward the blastopore. As a consequence, initial fates of
cells in this region are changed to oral ectoderm, as shown by lineage tracing. Strikingly, the ciliary
band, which forms adjacent to the nodal expression domain, is shifted significantly in the dorsal
direction, toward and sometimes beyond the position of the blastopore. This results in the
blastopore and stomodeal regions being positioned in the same ventral plane instead of
approximately at a 90 degree angle. Before gastrulation, wnt1 expression is radial in posterior
blastomeres. But when gastrulation begins, it is lost from the dorsal side by a Nodal-dependent
process and maintained only on the ventral side, where it suppresses nodal. Thus, Wnt- and Nodaldependent processes mutually antagonize each other to maintain the body plan established at
earlier stages by these same pathways.
Frizzled function in Wnt/Planar Cell Polarity and Wnt/beta-catenin signaling during
gastrulation in the cnidarian Nematostella vectensis
Naveen M. Wijesena and Athula H. Wikramanayake
University of Miami, Coral Gables, FL, United States
The evolution of gastrulation was arguably a key innovation that enabled metazoan diversification.
The developmental mechanisms that mediated induction of cell fate specification and archenteron
formation during gastrulation are unknown, but one crucial step was likely the co-option of a
molecular anisotropy present in early metazoan ova. An increased understanding of mechanisms
specifying developmental polarity in eggs and embryos in early diverging metazoan taxa may
provide insights into the origins of these mechanisms. We previously showed that Wnt/ß-catenin
signaling is required for endoderm specification in Nematostella, and that blocking this pathway had
no effect on primary archenteron invagination. Conversely, we have shown that initial archenteron
invagination in Nematostella is mediated by NvStrabismus signaling in the Wnt/PCP pathway
without effect on Wnt/ß-catenin signaling. This experimental uncoupling of endoderm specification
from initial archenteron invagination in Nematostella provides evidence for the independent
evolution of these two processes. However, it is not clear how these two pathways are spatially
coordinated during development. Here we present experimental evidence for coordination of the
two Wnt pathways during gastrulation by NvFlamingo (NvFmi). NvFmi is localized to the animal
pole in eggs and early embryos, and becomes restricted to the apical side of invaginating cells at
the blastopore. Morpholino-mediated NvFmi-knockdown blocked both ß-catenin signaling
dependent endoderm cell fate specification and archenteron invagination. Overexpression of the
carboxy-terminal intracellular domain of NvFmi had no effect on primary archenteron invagination
but these embryos were unable to form an organized endodermal epithelium suggesting that this
24 construct was affecting ß-catenin dependent cell fate specification. Based on this and other
evidence we propose that NvFmi functions as a scaffold to coordinate the two Wnt pathways during
endoderm cell fate specification and primary archenteron invagination in Nematostella. The
implications of these findings for the evolution of gastrulation will be discussed. Supported by the
NSF.
Genomic control of neural development: Mapping regulatory states of the sea urchin apical
organ
Avi Lerner and Oliveri, Paola
University College London, United Kingdom
The sea urchin apical organ constitutes a fundamental part of the larval nervous system and forms
a neuro-sensory structure capable of sensing environmental cues and coordinating swimming
behaviour. How the genome controls the development of this anterior neuronal structure is unclear.
To shed light on the specification and patterning of the apical organ, we analysed the dynamic of its
regulatory states. We overlaid spatio-temporal expression data of regulatory and downstream
genes onto cellular resolution digital maps of the apical organ at different developmental stages.
Our results show the spatial organisation of the apical domain is far more complex than previously
thought. Early patterning confers oral-aboral polarity and refines the apical organ into multiple
concentric sub–domains with unique molecular signatures. Post-gastrulation, a number of genes
begin to be expressed in individual cells along the aboral edge of the apical organ. Multiple
regulatory states begin to emerge in a highly dynamic fashion in these individual cells and together
with the appearance of the serotonergic nervous system illustrate an additional layer of spatial
complexity that exists in the apical organ. Furthermore, a potential circadian photoreceptor (dcry)
has been identified in a subset of these cells, lending support for a sensory function of this
structure. dcry shows partial co-expression with both mox and the photoreceptor differentiation
transcript factor z167 and in the pluteus larvae becomes restricted to a specific subset of
serotonergic neurons, proving the existence of at least two different types of serotonergic neurons.
Opposing Nodal and BMP Signals Regulate Left-Right Asymmetry in the Sea Urchin Larva
Yi-Hsien Su and Yi-Jyun Lu
Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan
Nodal and BMP signals are important for establishing left-right (LR) asymmetry in vertebrates. In
sea urchins, Nodal signaling prevents the formation of the rudiment on the right side. However, the
opposing pathway to Nodal signaling during LR axis establishment is not clear. Here, we revealed
that BMP signaling is activated in the left coelomic pouch, specifically in the veg2 lineage, but not in
the small micromeres. By perturbing BMP activities, we demonstrated that BMP signaling is
required for activating the expression of the left-sided genes and the formation of the left-sided
structures. On the other hand, Nodal signals on the right side inhibit BMP signaling, and control LR
asymmetric separation and apoptosis of the small micromeres. Our findings show that BMP
signaling is the positive signal for left-sided development in sea urchins, suggesting that the
opposing roles of Nodal and BMP signals in establishing LR asymmetry are conserved in
deuterostomes.
25 A computational model for BMP 2/4 movement in sea urchin embryos
Daphne Schatzberg, Heather Hardway, Tasso J. Kaper, and Cynthia A. Bradham
Boston University, Boston, MA, United States
Spatial patterns of bone morphogenetic proteins (BMPs) are critical for correct axis formation in
developing organisms. The formation of such patterns relies on regulation of BMP signaling by
extracellular proteins, in particular by Sog/Chordin and Tsg, which bind BMP and prevent it from
signaling, as well as Tolloid, a protease that cleaves Sog/Chordin and releases BMP to bind its
receptor. In Drosophila, BMP is expressed broadly in the dorsal territory, but signals only in a
narrow region at the dorsal midline. This restriction of BMP signaling is a consequence of Sog- and
Tsg-mediated facilitated diffusion. In the sea urchin, BMP 2/4 is expressed in the ventral ectoderm,
but signals broadly in the dorsal ectoderm. Here we explore the seemingly paradoxical behavior of
BMP movement in the two organisms using computational approaches. Using a reaction-diffusionbased mathematical model, we demonstrate that despite the opposed production and signaling
domains of BMP 2/4 in sea urchins, dorsal BMP localization can occur but requires relatively fast
formation of the BMP-Chd-Tsg ternary complex and relatively slow BMP receptor binding compared
to intermediate complex formation. Within the range of realistic parameter sets considered, a small
percent (5.17%) result in high levels of dorsal receptor-bound BMP for the sea urchin model. Only a
fraction of parameter sets, 0.04%, replicate the correct proportions of BMP signaling along the DV
axis in both sea urchins and flies. When considering parameters that are successful for both
species, the computational model displays optimal performance when the levels of Tolloid are
substantially lower in sea urchins than in flies.
Notch2 and BMP5-8 are each required for normal skeletal patterning as well as left-right
asymmetric Nodal expression in Lytechinus variegatus embryos
Michael Piacentino, Vijeta Patel, Finnegan Hewitt, Jia Yu, James Chaves, and Cynthia Bradham
Boston University, Boston, MA, United States
Skeletal patterning in sea urchin embryos involves communication between the pattern-dictating
ectoderm and the skeletogenic primary mesenchyme cells (PMCs); however, the molecular
mechanisms underlying this process remain unknown. To identify the extracellular molecules
involved, we performed an Illumina sequencing-based screen that has identified several skeletal
patterning candidate genes. Among these candidates are Notch2 and BMP5-8. Notch and BMP are
highly conserved signaling molecules involved in developmental patterning and left-right asymmetry
in vertebrates. Loss of function analyses using splice-blocking morpholino-substituted antisense
oligonucleotide (MO) microinjections confirm that each signal is required for proper skeletal
patterning. Morphometric analysis of LOF morphants shows that Notch2 is required for right side
skeletal development, while BMP5-8 is required for the left side. This suggested that both signals
interact with Nodal, which specifies the left-right (LR) axis throughout deuterostomes and is
expressed on the right side in sea urchin embryos. To assay Nodal expression in our patterning
morphants, we employed a Nodal::GFP BAC wherein GFP expression is regulated by the Nodal
cis-regulatory apparatus. Coinjection of this BAC with gene-specific MOs demonstrates that Notch2
is required for right side Nodal expression, while BMP5-8 is required to prevent randomization of
Nodal expression, consistent with findings in vertebrates. Together these data show that Notch2
and BMP5-8 are required to regulate right-side Nodal expression. Finally, we will report
experiments that test whether late Nodal signaling contributes to skeletal patterning.
26 Concurrent Session IV Imaging and Image Processing
A quantitative framework to define normal embryonic development and its range of variation
Paul Villoutreix1, Louise Duloquin1, Barbara Rizzi1, Emmanuel Faure2, Thierry Savy2, and Nadine
Peyriéras1
1
CNRS, Gif sur yvette, France; 2CNRS Paris, France
Performing quantitative approaches in developmental biology requires appropriate data and the
design of specific methodologies and tools. Live organisms’ microscopy imaging and automatic
image reconstructions open the way to spatiotemporal measurements of cell behaviors. This
quantitative data gives the opportunity to revisit some ‘supposed to be known’ mechanisms of early
development and to investigate interindividual variability. We compared six digital reconstructions
of Paracentrotus lividus embryos, obtained through the algorithmic processing of 3D+time image
data sets of fluorescently labeled embryos, observed from the 32-cell stage (4hpf) to hatching
(around 500 cells, 10 hpf). We established the complete lineage, throughout this developmental
period, of small and large micromeres, macromeres veg1 and veg2, and mesomeres. In addition to
the identification of the cell nuclei position, we performed the algorithmic segmentation of cell
membranes. From this large amount of data, we extracted a list of biologically meaningful
parameters, relevant to describe individual cells and their relationship to their neighborhood. We
measured a different variability between cell populations within the embryo as well as a growing
variability in the parameters values indicating a divergence in the individual cell behaviors as they
differentiate into more specific cell types. Because of the observed spatiotemporal variability, we
concluded that the sea urchin cell lineage cannot be described as ‘an invariant cell lineage’.
Consequently the cell-to-cell comparison between individuals is not possible. Our methodology
based on the lineage of single cells, allowed us to compare groups of cells of the same clonal origin
along their cell cycles. Similar values for specific parameters indicated some coherence within cell
populations, revealing asymmetries. From this quantitative study, we generated several maps, and
their integration provides a description of normal embryonic development with its range of
parameters values, accounting for intrinsic variability. This methodology is a way toward a
multiscale study of variation.
A Systems-Level Analysis of Ingression, an Epithelial to Mesenchymal Transition
Lindsay R. Saunders, and David R. McClay
Duke University, Durham, NC
The sea urchin Gene Regulatory Network (GRN) is a powerful tool in the quest for understanding
how a single fertilized egg develops into the complex body plan of an embryo and can be a tool for
understanding cell rearrangements at gastrulation. The first morphological movement to shape the
urchin embryo is Primary Mesenchyme Cell (PMC) ingression, an epithelial to mesenchymal
transition (EMT) where an epithelial cell detaches from neighboring cells and becomes
mesenchymal and free moving. Much is known about the cell biology of an EMT and discrete cell
biological events can be scored as intermediate EMT phenotypes. These include apical
constriction, de-adhesion, basement membrane invasion, acquisition of directed motility, and loss of
apical-basal polarity. To examine control of each event, we transplanted fluorescently labeled PMC
precursors containing knockdowns to each of 12 transcription factors in the PMC GRN into host
embryos and measured EMT failure for those five ingression components. In a computationally
unsupervised series of quantitative analyses, intermediate EMT phenotypes were scored over a two
hour time-course for hundreds of transplants. From quantified phenotypic data we built five unique
subcircuits within the PMC GRN that demonstrate distinct transcriptional control over the five cell
27 biological events of EMT. In most cases, perturbation of any one transcription factor resulted in a
unique phenotype that shared characteristics with its upstream regulators and downstream targets.
For example, Erg is a known regulator of both Hex and FoxN2/3 and all three shared a motility
phenotype. Additionally, Hex and Erg both regulated apical constriction but only Hex knockdown
prevented invasion, while FoxN2/3 was the lone regulator of cell polarity. Thus a new role for the
PMC GRN has been developed that connects nodes directly to five discrete EMT events and
provides an explanation for morphogenetic control within the context of that PMC GRN.
ABC-transporter activity regulates the left/right patterning and the migratory behavior of sea
urchin small micromeres
Joseph P. Campanale, Jose A. Espinoza, Tufan Gokirmak and Amro Hamdoun
Scripps Institution of Oceanography, La Jolla, CA, United States
Embryonic cells use ATP-binding cassette (ABC) transporters to establish morphogenetic gradients
and secrete chemoattractants. At their formation, small micromeres (SMMs) undergo a 60-65%
reduction in the activity of ABC-transporters. We hypothesized that this loss of ABC-transporter
activity in SMMs could be required for detection of chemoattractants necessary for their migration to
coelomic pouches (CPs). Consistent with this hypothesis, we found that while 92% of control SMMs
segregated in either a 3/5 or a 4/4 (left/right CP) pattern, only 40% and 57% of the SMMs in ABCB
or ABCC inhibitor treated embryos segregated normally. In 2% of (ABCB) and 16% (ABCC)
inhibited embryos, all eight SMMs were on one CP. To investigate the role of ABC-transporters in
segregation we time-lapsed SMMs expressing GFP-tagged pleckstrin homology domain, which
marks phosphatidylinositol-(4,5)-bisphosphate (PIP2). At the onset of gastrulation control SMMs
have strong membrane localization of PIP2, and they transition from epithelial morphology with
microvilli to mesenchymal morphology with pseudopodia. This involves SMMs jockeying to the tip of
the archenteron. SMMs actively position themselves from left to right across the archenteron,
moving as much as 35 µm from one forming CP to the other. Control SMMs move at an average
speed of 0.21 µm/min and extend both short (<10 min) and long (>30 min) lived PIP2 rich
filopodia. Preliminary time-lapse videos of embryos grown in ABCB inhibitors indicate that these
SMMs do not transition to become mesenchyme, and do not move as rapidly, moving at only 0.1
µm/min. These results suggest that crawling, perhaps in response to a transporter-secreted signal,
orients SMMs along the left/right axis. In the absence of ABC-transporter activity segregation could
be passive, with SMMs carried at random to forming CPs after gastrulation.
Gastrulation in high-resolution: New insights into an important process of development.
Megan L. Martik and David R. McClay
Duke University, Durham, NC, United States
Gastrulation is a complex orchestration of movements by cells that are specified early in
development. Until now, it was thought that lateral rearrangement of endoderm cells by convergent
extension was the main contributor to archenteron elongation. Our project characterizes, at high
resolution, the repertoire of cellular movements contributing to the length of gut. We have
performed cell transplantation to live image and analyze a subset of labeled Veg endoderm cells
and have found that the Veg2 cells remain contiguous throughout extension, so that, if convergent
extension is present, it is not a major contributor to elongation. We have also found, unexpectedly,
that endoderm cells proliferate as they move to elongate the archenteron.
Our descriptive studies of the cellular processes during gastrulation have allowed us to begin
investigating their molecular control. The endomesoderm gene regulatory network (GRN) describes
28 the cell fate specification of the future larval gut; however, the GRN does not describe specific cell
biological events driving morphogenesis. We plan to dissect the transcriptional circuitry of the GRN
responsible for the cell biological events, such as changes in polarity and adhesion, of gastrulation.
Our ability to connect the endomesoderm GRN to the morphogenetic events of gastrulation will
provide a framework for characterizing this remarkable sequence of cell movements in the simplest
of deuterostome models at an unprecedented scale.
Activation of multidrug efflux transport by microvillar tip localization
Kristen Whalen and Amro Hamdoun
Scripps Institution of Oceanography, La Jolla, United States
Fertilization is characterized by structural and functional changes of the cell surface. In sea urchins,
these changes include polymerization of cortical actin and a coincident, switch-like increase in the
activity of the multidrug efflux transporter ABCB1a. However, it is unclear how cortical
reorganization leads to changes in membrane transport physiology. We used 3D super-resolution
fluorescence microscopy to resolve the fine-scale movements of the transporter along polymerizing
actin filaments and show that efflux activity is established after ABCB1a translocates to microvillar
tips. After fertilization, microvillar length increases from 370 nm in the egg to a maximum of 3.71 µm
(10-fold increase) at 45 min post fertilization (pf). Correspondingly, we see the microvillar tip
accumulation of actin-associated ABCB1a go from 15% at 15 min pf to 49% by 60 min pf over the
period when the switch in efflux activity occurs. Inhibition of actin polymerization or the loss of actin
bundle formation prevents tip-localization, resulting in both patching of ABCB1a at the cell surface
and decreased efflux activity. Alternatively, enhanced polymerization results in the accumulation of
ABCB1a in the microvillar membrane. These results indicate that a dynamic actin cytoskeleton is
required for precise positioning of ABCB1a at the microvillus tip, since the loss of actin treadmilling
leads to the build-up of transporter on the microvillus. Finally, interference with Rab11 GTPase, a
regulator of apical recycling, inhibits complete activation of efflux activity in embryos.
Immunolocalization studies concluded Rab11 colocalizes with ABCB1a in the egg, however, Rab11
does not show microvillar tip accumulation, indicating the role of this regulator may facilitate
insertion of the transporter into the membrane rather its movement along microvilli. In sum, these
results provide a critical first step toward understanding the role of microvilli in regulating the traffic
and positioning of membrane transporters critical for cellular protection.
Concurrent Session V Neurogenesis/Ciliary Band
Ciliary band formation provides a unique model of Eph-Ephrin controlled apical constriction
during embryonic development.
Oliver A. Krupke and Robert D. Burke
University of Victoria, Victoria, BC, Canada
Early in embryonic development, sea urchin embryos form a specialized region of ectoderm, the
ciliary band. This continuous 4 to 5 cell-wide strip forms a raised ridge encircling the oral ectoderm
and functions as the principal swimming and feeding organ of the larva. Cells of this region are
elongated with a narrow apical end bearing a single cilium and occur as a simple sheet of epithelial
cells overlying a basement membrane. During its differentiation a dramatic and coordinated apical
constriction occurs in all ciliary band cells. Inhibiting actomyosin contractility prevents this
constriction and blocks ciliary band formation. Apical constriction of these cells is also dependent
on Ephrin forward signaling through Eph and knocking down expression of either Eph or Efn or
29 inhibiting Eph kinase function results in a significant reduction in apical constriction. This shape
change appears to depend on phosphorylation of focal adhesion kinase that requires Ephrin
signaling through Eph. Concomitant with this cell shape change is an outward buckling of the entire
cell sheet to form a ridge that becomes the ciliary band. This contrasts starkly to widely studied
models where apical constriction results in an inward buckling of a cell sheet. We hypothesize that
apical constriction is not the principle force underlying shape change in the cell sheet that forms the
ciliary band. We further hypothesize that this change in cell shape is controlled in part by forward
signaling by Ephrin through Eph. The simplicity of this system provides an excellent model to study
the contribution of apical constriction to cell shape change.
TGF-ß signals regulate the dorsoventral patterning of neurogenic ectoderm of the sea urchin
embryo.
Shunsuke Yaguchi1, Junko Yaguchi1, Noriyo Takeda2, and Kazuo Inaba1
1
University of Tsukuba, Shizuoka, Japan; 2Tohoku University, Aomori, Japan
In normal sea urchin embryos, the serotonergic neurons are differentiated only at dorsal edge of the
anterior neurogenic ectoderm although almost all of the cell fate at this region will be specified as
neurons in pre-signaling condition. We previously suggested that Nodal, which is diffused from
ventral side, is involved in patterning the neurogenic ectoderm, but the detailed mechanism is
unclear. Here we show that Nodal signaling controls the expression of genes encoding transcription
factors, FoxQ2 and Homeobrain (Hbn), resulting in patterning the neurogenic ectoderm. FoxQ2 and
Hbn are required for the specification of neurogenic ectoderm and serotonergic neurons,
respectively. In normal embryos, foxQ2 and hbn are expressed at the same broad region in animal
half of unhatched blastulae. Then, foxQ2 expression is restricted to the animal pole, but hbn
expression is progressively shifted towards dorsal side and disappeared from ventral side by
mesenchyme blastula. In Nodal morphants, the size of foxQ2-positive region becomes smaller than
that of normal embryos, and hbn-expressing cells surround that area. In contrast, in Lefty
morphants, in which Nodal signaling is enhanced, the restriction of foxQ2 expression from hatching
blastula does not occur and thus foxQ2 region remains bigger than that of normal embryos. hbn
expression is invariant at unhatched blastula of the morphants but it disappeared after
mesenchyme blastula. These data together with Nodal diffusion pattern in normal embryos indicate
that Nodal signaling functions in maintaining foxQ2 expression and suppressing hbn expression at
the ventral side of neurogenic ectoderm. Because BMP2/4 is also required for hbn expression at
dorsal side after mesenchyme blastula, TGF-ß family signals, which are essential for dorsoventral
axis formation, are required for the precise patterning of neurogenic ectoderm.
Formation of larval GABAergic nervous system in sea urchin
Katow, Hideki
Tohoku University
Larval swimming activity of sea urchin is regulated by serotonergic and dopaminergic neural
systems. However, none of above regulatory system inhibits the swimming activity perfectly,
suggesting the presence of a third system. Among classical neurotransmitters in sea urchin,
GABAergic system has not been examined. Here, we report formation of the system and its role in
larval swimming regulation. GABA-expressing cells were first seen in blastocoelar cells from prism
stage and accumulate at the circumoral ciliary band by 2-arm pluteus stage. Inhibition of GABA
synthesis by 3-mercaptopropinic acid (3-MPA) resulted in conspicuous decreasing of larval
swimming activity. The inhibition was more severe in older larvae than younger ones. The older
larvae developed ciliary band-associated exo-larval network of glutamic acid decarboxylase (GAD)-
30 expressing cells. Inhibition of GABAA receptor (GABAAR) activity was also resulted in decreased
larval swimming activity, suggesting the presence of GABA-GABAAR signal transmission pathway,
which was supported by close localization of GABAAR-associated protein (GABARAP) to GABAGABAAR. GAD gene activity and the protein expression were detected, at least, from morula stage
and on the entire ectoderm from swimming blastula stage and localized to the blastocoelar cells
and on the apical side of the ciliary band by 2-arm pluteus stage. During exo-GAD cell network
formation, GAD cells express Unc-5 and egress to the apical surface of larva from 2-arm pluteus
stage. The egression was followed by oriented migration on Netrin matrix that was formed on the
apical side of hyaline layer at the place where the exo-GAD cell network is formed. The migration
was inhibited by a Netrin peptide that is known to mediate Netrin/Unc-5 interaction during
serotonergic axon extension in sea urchin larvae.
Embryonic localization of Coup-TF splice variants and their possible role in the oral
ectoderm
Constantin N. Flytzanis, Ioannis Tsironis, Maria Aivalioti, and Christina Kalogirou, Christina
University of Patras, Patras, Greece
COUP-TFs (Chicken Ovalbumin Upstream Promoter-Transcription Factors) are orphan nuclear
receptors, members of the steroid-thyroid-retinoic acid receptor superfamily. A characteristic
COUP-TF splice site, downstream of the DNA binding domain (DBD), within the carboxyl terminal
extension of the DBD, is conserved within metazoans. In the Mediterranean sea urchin
Paracentrotus lividus, a small 63bp exon embedded within an approximate 12kbp intron is
alternatively spliced at this site, generating two transcripts and consequently two protein products.
This in frame insertion of the 21aa exon, alters the in vitro DNA binding properties of the large
protein variant rendering it incapable of binding to its response element. Western blot analysis
shows that both protein variants are found in embryonic and adult tissues of the sea urchin
P.lividus. In situ hybridizations reveal that both transcripts are co-expressed in the cells of the
putative and the definitive oral ectoderm during embryonic development. Egg injections of in vitro
synthesized RNAs, show the presence of both protein variants in the nuclei of the early embryo
blastomeres and the characteristic Coup-TF association with the condensed chromatin during
mitosis. In addition, Coup-TF morpholino injected embryos fail to express early ciliary band
markers.
Cilia-mediated differential labelling of the ectodermal epithelium and ciliated band cells by
DiI, DiO, and DiD
Bruce P. Brandhorst
University of British Columbia, Vancouver, BC
We have observed that fluorescent dyes such as FM1-43 and TO-PRO-3 (which are able to
penetrate TRP channels) rapidly accumulate in cells of the blastocoelar cell network (BCN) of S.
purpuratus prisms and plutei (but the membrane dyes appear more slowly in endocytic vesicles of
ectodermal cells). Dyes having larger diameters are excluded from the BCN, suggesting uptake via
channel pores. To assess a possible role of cilia in dye uptake, we incubated embryos in the
lipophilic carbocyanine dyes DiI, DiO, DiD dissolved in SW. Some aboral ectodermal cells and
ciliated band cells became labelled (including their cilia). Some cells were labelled with only one
dye, some with two or three dyes, and many remained unlabelled (after an hour); the fraction of
labelled cell was not dependent on labelling incubation time. Labelling was eliminated after
deciliation induced by brief hypertonic shock. These observations suggest a capacity of
neighbouring cells to distinguish between these similar dyes present at similar concentrations,
31 implying differences in the properties of their cilia. Dye labelling of processes extending from
ectodermal cells to the BCN and of some BCN cells (especially those near tips of arms) was
observed, suggesting possible cilia-mediated transfer of the dyes to the BCN. Deciliation did not
prevent rapid uptake of FM1-43 into the BCN, indicating it is not mediated by ectodermal cilia.
Immunostaining for acetylated tubulin detected scattered cilia within the blastocoel but their
association with the BCN is uncertain. Incubation of living embryos with Tubulin-tracker labelled
cilia on the surface of embryos but not inside the blastocoel or lumen of the gut. Treatment of
embryos with FM1-43, DiI, DiO, and DiD (as well as various channel inhibitors) disrupts swimming,
often reversibly. Supported by a grant from NSERC.
Hedgehog signaling is dependent on motile cilia in the sea urchin embryo.
Jacob Warner and David R. McClay
University Program in Genetics and Genomics, Duke University, Durham, NC
A recent frontier in Hedgehog (Hh) signaling research is the requirement of the primary cilium and
intra-flageller transport (IFT) proteins for signal transduction. Studies in vertebrates have shown that
proper trafficking of Hh pathway components within the primary cilium is essential for Hh signaling.
This machinery is not necessary, however, for Hh signal transduction in protostomes such as
Drosophila. As a basal deuterostome, the sea urchin occupies a unique phylogenetic position and
can provide crucial insight into the evolution of hedgehog signaling. Here we provide evidence that,
as in vertebrates, Hh signaling in the sea urchin relies on the presence of cilia. It has been
previously shown by Robert Morris and John Scholey that Kinesin II is essential for cilia assembly.
Indeed, knockdown of Kinesin II using an antibody phenocopies Hh morphants and leads to
downregulation of the conserved Hh target Patched. These findings lead to a model of sea urchin
Hh signaling that closely resembles the pathway as it functions in vertebrates. Furthermore,
analysis of the cilia on Hh receiving cells reveal that they exhibit a 9+2 microtubule morphology
consistent with motile cilia and implies that motile cilia are also capable of carrying out Hh signal
transduction. This work indicates that the requirement of cilia for Hh signal transduction is not
specific to vertebrates or primary cilia, but extends to other phyla and ciliary morphologies.
Concurrent Session VI Emerging Topics
Bicistronic and Antisense Expression Mediated by Pantropic Retroviruses in Sea Urchin
Embryos
Pinet, Kaylinnette J., Amanda B. Core, Evan Conaway, Arlene Reyna, Arlene and Cynthia
Bradham
Boston University, Boston, MA
Sea urchins are an important developmental model organism that offers advantages for systems
level studies, including transparency, morphological simplicity, rapid development, and the
availability of synchronously developing embryos in enormous numbers. We have previously
engineered self-inactivating (SIN) pantropic retroviruses (PRV) for sea urchin embryos in an effort
to produce a new tool for systems-level approaches in this developmental model organism. We
showed that SIN PRVs infect sea urchin eggs with high efficiency and genomically integrate gene
expression cassettes that include the LvOtx cis-regulatory module 3 (Otx CRM3). In the current
study, we extend our previous efforts by generating SIN PRVs that drive expression of LvDelta
antisense (AS) sequences and by incorporating an internal ribosomal entry sequence (IRES) from
cricket paralysis virus (CrPV) to promote bicistronic gene expression in infected embryos. We
32 optimized both AS and bicistronic expression driven by LvOtx CRM3. The addition of these
technologies expands the possibilities for retrovirally-mediated transduction by providing an
approach for perturbing gene function, which will be important for generating large scale samples of
perturbed embryos, and by providing the opportunity to express bicistronic messages, which will be
valuable for functional studies.
Conservation and Fine Tuning of Polyspecific Efflux Activity in Sea Urchin MDR
Transporters
Tufan Gokirmak1, Joseph P. Campanale1, Lauren E. Shipp1, Adam M. Reitzel2, Gary M. Moy1,
Houchao Tao1, and Amro Hamdoun1
1
Scripps Institution of Oceanography, UCSD, La Jolla, CA, United States; 2Woods Hole
Oceanographic Institution, Woods Hole, MA, United States
Sea urchin embryos have dynamic multidrug resistance (MDR)-type efflux activities during
development, but the corresponding transporters remained to be characterized. In this study, we
identified four transporters that appear structurally and functionally similar to human MDRtransporters. Three efflux transporters, Sp-ABCB1a, Sp-ABCB4a and Sp-ABCG2a, localized to the
apical membranes of blastulae and effluxed canonical substrates of their human MDR homologs.
For instance, expression of Sp-ABCB1a and Sp-ABCB4a reduced intracellular calcein accumulation
by 87% and 82% compared to controls. Similarly, expression of Sp-ABCG2 reduced mitoxatrone
accumulation by 30%. The fourth protein, Sp-ABCC1 localized to the basolateral membranes and
thus did not participate in apical efflux. Although our characterization showed that sea urchin MDRtransporters are polyspecific efflux pumps with similar topologies, localizations, and efflux activities
to mammalian homologs, phylogenetic analyses indicate that they lack direct orthology. To
understand how this polyspecific efflux activity is conserved despite the lack of orthology, we
compared the drug binding domains of sea urchin and mouse ABCB1a transporters the latter of
which was structurally characterized with stereoisomeric cyclic peptide inhibitors (QZ59-RRR and
QZ59-SSS). Our functional and protein sequence comparisons showed that stereoselective
inhibition of Sp-ABCB1a by QZ59 enantiomers is reversed relative to the mouse homolog and that
there are four non-conservative substitutions in helices 6 and 12 of sea urchin and mouse drug
binding domains, which interact directly with QZ59s. Next, we investigated the role of these
substitutions in maintenance or diversification of efflux activity by murinizing these residues. We
found that these substitutions are involved in determining stereoselectivity of QZ59s, but that they
do not affect the calcein-am efflux function. Thus, while the broad substrate recognition of MDRtransporters appears to be conserved through evolution, the primary structure of the drug binding
domains may be fine-tuned for the unique landscape of endogenous and environmental substrates
in different organisms.
Sea urchin twins for investigating biological variability
Dimitri Fabrèges, Louise Duloquin, Paul Villoutreix, Thierry Savy, and Nadine Peyriéras
CNRS, Gif-sur-Yvette, France
Every biologist who has worked with living organisms has noticed that variability is a rule although
ill-defined and ill-quantified. We investigated the phenotypic variability of developing sea urchins
Paracentrotus lividus from the 3D+time imaging of fluorescently labeled embryos. The
quantification of cell displacements and cell shape changes using the automated processing of
image data was applied to populations of siblings or twins (2-cell stage dissociation) to identify
different components of the phenotypic variabiality. The possibility to compare populations of
siblings and populations of sibling twins leading to normal and fertile specimens is a very valuable
33 model for a quantitative approach of variability. Developing twins with chromatin (H2B-mCherry)
and membrane (eGFP-Ras) stains were observed by Selective Plane Illumination Microscopy
(SPIM/DSLM) during early stages. Twins morphology was strikingly different from control embryos
and displayed a highly variable cellular organization. At the 16-cell stage, the twins were cupshaped with a variable number of mesomeres/macromeres/micromeres and presented abnormal
cell-cell contacts (e.g. mesomeres/micromeres). By the 60-cell stage the twin embryos became flat
with variable geometry but converged to a normal blastula shape before hatching. Thereafter,
apart from their smaller size, twins did not show macroscopic differences compared to control
embryos. The phenotypic variability defined as the measurable difference between two individuals
is expected to correlate with 1) transmitted differences (genetic polymorphism, epigenetic variability,
random protein distributions ...) so that normal siblings differ from the 1-cell stage; 2) acquired
differences through biological processes stochasticity; and 3) environmental variability which could
induce different responses at different levels (cellular, sub cellular …). We tried to minimize the
latter and derived different parameters from the automated cell lineage and cell shape
reconstructions. We then compared twin embryos to measure acquired variability and expect to
deduce the transmitted variability by comparing twin pairs.
An ancient function for IL-17 in gut-associated antibacterial immune response
Eric C.H. Ho, Kate Buckley, Guizhi Wang, and Jonathan Rast
University of Toronto/Sunnybrook Research Institute, Toronto, ON, Canada
In order to maintain a homeostatic gut microflora, a robust gut immune system that balances
immune defense and tolerance is required. Our aim is to characterize the immune regulatory
system that coordinates interactions between the gut epithelium and the mesodermal immunocytes
using the purple sea urchin larva as a model. We approach this problem with three interrelated
strategies: (1) Characterization of larval immunocytes based on both cellular behaviour (e.g.,
phagocytic competence and surveillance activity) and gene expression; (2) Analysis of a model gutassociated immune response using the marine bacterium Vibrio diazotrophicus and (3) Regulatory
analysis of Interleukin-17 (IL-17), a multigene family of pro-inflammatory cytokines. We have
identified and characterized five larval immunocyte morphotypes using time-lapse microscopy and
gene expression profiling. These cell types can be characterized by their phagocytic and motile
behaviours; or via physical attributes such as granularity. Nine IL-17 sub-groups made up of 32
genes are encoded in the purple sea urchin. Based on expression data from our larval infection
model, we have identified a sub-group (SpIL-17-I) that exhibits early expression and is then quickly
attenuated in the gut epithelium. A second divergent IL-17 sub-group (SpIL17-IV) encoded by a
single gene is expressed in the gut epithelium later in the course of infection response. We have
initiated cis-regulatory analysis of the IL-17-I sub-group. BAC-based GFP transgenes and smaller
subclones recapitulate endogenous IL-17 expression in response to bacteria. We are tracing
regulatory inputs that control activity downstream of bacterial recognition as well as those
responsible for attenuation of IL-17 signal. Although IL-17 expression in mammals is predominately
studied in the context of Th17 T cells, epithelial expression as is seen in the sea urchin has recently
been shown to play a critical role in gut immune response in mouse.
Enterocoely, metamerism and deuterostome homology
Valerie Morris
University of Sydney, Sydney, Australia
Hyman (1955) describes deuterostomes as enterocoelous coelomates. During enterocoely in the
abbreviated development of the sea urchin Holopneustes purpurescens (Morris 2012), a self-
34 renewing population of ectodermal cells involutes at the blastopore and transitions to a population
of coelomic cells, probably undergoing asymmetric cell divisions. The coelomic population is
regionalized in an antero-posterior direction, becoming the source of a growth zone for the
metameric series in each of the echinoid rays. The metamerism provides a link with the chordate
axis and tentacle-bearing hemichordates. The challenge will be understand how the related genes
of a possibly common growth zone produce the markedly different morphological outcomes in
deuterostomes.
References:
Hyman LH (1955) The invertebrates: Echinodermata IV. New York, McGraw-Hill.
Morris VB (2012) Early development of coelomic structures in an echinoderm larva and a similarity with
coelomic structures in a chordate embryo. Dev. Genes Evol. (in press).
An Examination of the Nuclear Transport Proteins of the Sea Urchin Genome in the Context
of Deuterostome Evolution
Christine Byrum
College of Charleston, Charleston, SC
Nuclear transport is critical to the survival of any eukaryote. To traverse the nuclear membrane
barrier, cargo pass through cylindrical aqueous channels called nuclear pore complexes (NPC).
Although small molecules (<40 kDa) can freely diffuse through the NPC, translocation of larger
macromolecules (e.g. RNA and proteins) requires active transport and is mediated by a group of
proteins called the karyopherins. By forming low affinity, but highly specific bonds with a group of
proteins present in the NPC (the phenylalanine-glycine nucleoporins), the karyopherins are able to
shuttle macromolecules to and from the nucleus. Prior to public release of the sea urchin genome,
members of the Sea Urchin Genome Consortium made a concerted effort to annotate all of the
predicted genes. Key gene families were completed, however the karyopherins remained poorly
characterized. In reviewing relevant sea urchin studies, we have encountered only one paper (Song
and Wessel, 2007) that has examined expression of a sea urchin karyopherin (Sp-Exportin5). Our
work identifies karyopherin sequences present in the S. purpuratus genome and provides the most
comprehensive analysis of the evolution of karyopherins in the Deuterostomata to date. Within the
karyopherin alpha (KPNA) family, we have found that expansion of this gene family likely occurred
in the vertebrate line. Because the karyopherins selectively transport specific cargo types,
differential expression of these molecules (e.g. transcription factors) could play an important role in
the regulation of developmental processes. Simplicity of the sea urchin model would make it a
particularly attractive subject for these sorts of investigations.
Concurrent Session VII Evolution II
Embryonic development of the slate pencil urchin Eucidaris tribuloides: Re-booting research
on cidaroids
Eric Erkenbrack and Eric H. Davidson
Caltech, Pasadena, CA, United States
The Echinoidea are comprised of two sub-classes, cidaroids and euechinoids. These two clades
last shared a common ancestor over 270 million years ago. Morphologically, cidaroid embryos
exhibit pigment cells and larval skeletons, but lack hallmark euechinoid traits, such as invariable
numbers of small and large micromeres and a precociously ingressing skeletogenic mesenchyme
(SM). With the arrival of large RNA-seq and genomic sequence datasets, we are now in an
excellent position to begin studying echinoids at many different levels. During the last several years,
35 we have focused on the embryonic development of the slate pencil urchin Eucidaris tribuloides and
here present WMISH, qPCR and MASO perturbation data. We show that pregastrular spatial
regulatory states of endodermal territories are similar to those found in euechinoids. But, as
expected, we found many differences in SM specification in Eucidaris, including no traces of the
initial skeletogenic GRN activation mechanism that euechinoids use, nor early expression of alx1.
Surprisingly, though, we also found that specification of non-skeletogenic mesoderm (NSM) of the
vegetal plate is just as different: in pre-gastrular Eucidaris embryos there are no molecular signs of
the polarization of oral-aboral mesoderm regulatory states characteristic of euechinoids. These
preliminary data indicate that while the endodermal GRN is at least in part shared across these
subclasses, there exist major differences in pregastrular mesoderm specification, indicating
significant differences in GRN structure. These aspects of the euechinoid GRN may be derived
features that evolved in the period immediately following divergence from the common
archaeocidarid ancestor.
Gene expression during regeneration of a crinoid, Oxycomanthus japonicus
Mariko Kondo, Shusaku Ueda, Rikai Sawafuji, Toko Tsurugaya, Akhito Omori, and Koji Akasaka
MMBS, The Univ. of Tokyo, Miura, Kanagawa, Japan
Crinoids (sea lilies and feather stars) are potentially important model organisms for research,
although sea urchins and sea stars have been studied as main representative echinoderms. They
are considered the group of animals that have branched the earliest from the other echinoderms
and are indispensable to understand the evolution of echinoderms as well as deuterostomes.
With the exception of sea urchins, echinoderms in general exhibit a strong capacity for
regeneration. Feather stars also are reported to regenerate most of its body parts. In natural
conditions, it appears that they self-amputate (autotomize) their arms, and arm regeneration is
observed very frequently. To study the course of regeneration, autotomy is induced by pinching the
arms in the laboratory. There have been morphological or histological studies on crinoid
regeneration, but only few with any molecular data.
We have isolated from the feather star, Oxycomanthus japonicus, several genes that are generally
considered markers for stem cells, including the vasa gene, those involved in retinoic acid signaling,
and Hox genes. We are currently analyzing the temporal and spatial expression of these genes to
test their involvement in regeneration. Our results show the presence of undifferentiated cells in the
arm, and a possible indication of a proximal-distal axis in the pinnules.
Evolution of spicule matrix proteins
Brian Livingston
California State University Long Beach, Long Beach, CA
The Strongylocentrotus purpuratus skeletal elements have been shown to contain a large family of
spicule matrix proteins characterized as secreted proteins with a C-type lectin domain and an acidic
repetitive low complexity domain. Homologous proteins in other euechinoids have different numbers
of repeats and the amino acid sequence differs between species, although the repeats retain the
same general characteristics. These differences reflect the large amount of variation in proteins
involved in biomineralization across more distantly related animal taxa. This variation and the
apparent independent origin of matrix proteins in different taxa suggests that the overall
characteristics of the proteins rather than the primary sequence seems to be what is selected for. In
order to study the evolution of these proteins in echinoderms, we looked for spicule matrix proteins
in the brittle star Ophiocoma wendtii, which forms an embryonic skeleton similar to that seen in
urchins, in addition to the adult skeleton. We generated a transcriptome database of sequences
36 expressed at the gastrula stage of development when a skeleton begins to form. We queried this
with S. purpuratus sequences encoding spicule matrix proteins, and found some sequences
encoding C-type lectin domains, but no acidic repetitive domains similar to that seen in urchins. We
then isolated the occluded proteins from the adult skeleton. After separating the occluded matrix
proteins from Ophiocoma wendtii by SDS-PAGE, bands were excised and subjected to proteomic
analysis. De novo amino acid sequences obtained were used to query our transcriptome database.
We have identified a number of proteins present in the O. wendtii skeletal proteome as well as
partial sequences encoding known functional domains. We have compared this proteome to that of
the S. purpuratus skeletal proteome. We are also currently examining adult transcriptome and
genomic databases to further identify and characterize the brittle star skeletal proteome.
Evolutionary co-option of Delta gene at the cis-regulatory level
Feng Gao and Eric H. Davidson
California Institute of Technology, Pasadena, CA, United States
The major mechanism of evolutionary change in GRN structure is co-option of regulatory and
signaling genes to expression in new spatial/temporal domains of the developing organism. This
means change of cis-regulatory modules at the sequence level, so that they respond to different
regulatory states; or alternately, changes in the cis-regulatory modules of genes encoding the
spatial allocation of regulatory states. An excellent example is the use of Delta-Notch signaling to
promote mesoderm specification in sea urchins, but to promote endoderm specification in sea stars
(the sea urchin mode is the derived co-option). Sea stars and sea urchins shared a last common
ancestor about 500 million years ago. To determine what happened in the lineage leading to sea
urchins, we are carrying out a cis-regulatory study of sea star delta, for comparison to sea urchin
delta, including cross-specific transfer of expression constructs. Current results show that though it
is expressed quite differently in sea stars, a cis-regulatory module of sea star delta produces
expression in sea urchin skeletogenic lineages, though no such lineage exists in sea stars.
Evolution of pluteus larvae and interaction between epidermis and mesenchyme
Yoshiaki Morino, Hiroyuki Koga, and Hiroshi Wada
University of Tsukuba, Tsukuba, Japan
For the pluteus evolution, acquisition of larval skeleton is one of the essential steps. Recent studies
suggested that adult skeletogenic program was co-opted for larval skeletogenesis. We showed the
heterochronic activation of VEGF signaling is the key event for the acquisition of larval skeleton.
However, the acquisition of larval skeleton is not sufficient for the evolution of pluteus. For example,
sea cucumber have skeleton in larval stage, but they are not pluteus larvae. Not only larval
skeleton, but also elongation of spicule and formation of pluteus arm are also needed for acquisition
of pluteus larvae shape. Formation of pluteus arm is established by the interaction between the
epidermis and mesenchyme. Several genes, fgf/fgf-r2 pea3, pax2/5/8, sprouty, otp, tetraspanin are
expressed in epidermis or mesenchyme located in the tip of arm in sea urchin. It is suggested that
these genes are involved in such interaction and formation of pluteus arm.
In this study, we examined expression pattern of fgfA, fgf-r1, fgf-r2, pax2/5/8, pea3, sprouty,
tetraspanin, and otp in starfish Asterina pectinifera, brittle star Ophiothrix exigua and sea cucumber
Holothuria leucospilota. Based on expression patterns of these genes, we will discuss evolution of
pluteus larvae.
37 Evolution of innate immune receptors in the deuterostomes
Katherine M. Buckley and Jonathan P. Rast
University of Toronto, Sunnybrook Research Institute, Toronto, ON, Canada
The purple sea urchin genome sequence has revolutionized our understanding of animal immunity.
Several other genomes from important phyla have since been sequenced that further contribute to
our knowledge, including three additional sea urchin species, five sequences from the
Xenacoelomorpha, the lamprey and several protostome sequences. In the purple sea urchin,
immune recognition is mediated by homologs of the pattern recognition receptors that form the core
of vertebrate innate immunity. However, in contrast to vertebrates, in which these receptors are
found in small gene families (10-20 genes), the sea urchin genome encodes highly expanded and
diversified multigene families for several types of innate immune receptors. TLRs are the most
tractable of these families to study due to their simple intron-exon architecture and stereotypic
protein structure. Our analyses show that the complex sea urchin TLR family is largely derived
from independent expansions in the echinoderm lineages, although a small TLR family with
structural similarity to Drosophila Toll can be traced to an ancient eumetazoan ancestor. Patterns
of selection indicate that the sea urchin TLRs are rapidly evolving and that the ligand-binding
ectodomains are subject to strong positive selection in spatially clustered regions that differ from
those of vertebrates. Representatives of most of the S. purpuratus TLR subfamilies are found in all
three sea urchin genomes, although the Lytechinus variegatus TLR family is considerably smaller.
A role for these receptors in immune defense is suggested by their similarity to TLRs in vertebrates,
sequence diversity, and expression in immunologically active tissues. These analyses identify
quickly evolving TLR subfamilies that are likely to have novel immune recognition functions and
other, more stable, subfamilies that may function more similarly to those of vertebrates. These
bioinformatics comparisons among sea urchin species will help target experimental approaches to
understand function of this novel immune system.
Concurrent Session VIII Membranes, Cytoskeleton and Polarity
Actin dynamics in the sea urchin egg
Andrea Ellis and Charles B. Shuster
New Mexico State University, Las Cruces, NM
The actin cytoskeleton of the early sea urchin embryo is organized into a thin, isotropic cortex with
many interspersed microvilli. And while much is known regarding the upstream and proximal
regulators of actin polymerization during cell migration, it is unclear how these regulators contribute
to actin organization early in development or how these regulators are themselves modulated by
the cell cycle. In an effort to understand actin organization and function during the embryonic cell
cycle, we employed GFP-Lifeact to follow actin filament dynamics during the first embryonic cell
division. In addition to the contractile ring, we were able to identify four distinct populations of actin
filaments in sea urchin eggs, all of which were subject to changes in CDK1 activity. Upon entry into
mitosis, microvilli were found to be short and dense leading up to mitotic exit, at which time they
elongated at a rate of approximately 340 nm/minute. A thin submembranous population of actin
filaments could also be detected that underwent a transient thinning upon anaphase onset, only to
recover shortly before cytokinesis. A bright ring of nuclear actin was observed that collapsed
approximately 2-3 minutes prior to nuclear envelope breakdown. Lastly, GFP-Lifeact revealed an
explosive elaboration of cytoplasmic actin occurring just prior to the metaphase-anaphase
transition. The polymerization of cytoplasmic actin appeared to initiate at the cell surface, moving in
wave towards the cell center. However, kymograph analysis suggested that these filaments were
38 not being transported inward, but were instead stationary, disassembling during cytokinesis. Our
current efforts are focused on identifying the proximal regulators of these different actin populations,
but injection of C3 transferase ablated all actin-based structures, implicating RhoA-dependent actin
modulators as likely candidates.
Programmed cell fusion of PMCs during skeletal development in the sea urchin Lytechinus
variegatus
Deirdre C. Lyons and David R. McClay
Department of Biology, Duke University, Durham NC
Skeletogenesis in sea urchin embryos occurs after the development of a multinucleate syncytium
that will surround the larval skeleton. Syncytium formation requires a cell-cell fusion event within the
primary mesenchyme cell (PMC) population. The PMC lineage undergoes multiple specificationstate transitions, some of which are described by the PMC gene regulatory network (GRN). The
necessary fusogenic kernel remains to be defined. Previously, the transcription factors FoxN2/3,
Alx1 and Twist were shown to be necessary for PMC fusion. We developed a transplant-based
fusion assay using control and morpholino-carrying micromeres to study the events of PMC fusion
in vivo. Using this assay we tested 13 genes expressed in PMCs for a role in fusion and found that
only a handful are necessary for this process. Not all of these nodes are directly connected to one
another according to the GRN, suggesting that fusion is controlled by convergent regulatory
pathways. These fusion morphants provide an entry point for studying the cell biological
mechanisms controlling fusion in the embryo.
Effects of Early Disruption in Polarity on Later Development in the Sea Urchin Embryo
Kathleen Moorhouse, Heather F.M. Gudejko, Lea M. Alford, and David R. Burgess
Boston College, Chestnut Hill, MA, United States
Establishment and maintenance of cell polarity has become an increasingly interesting biological
question in a diversity of cell types and has been found to play a role in variety of biological
functions. Previously, it was thought that the sea urchin embryo remained relatively unpolarized
until the first asymmetric division at the 16cell stage of development. However, there is mounting
evidence to suggest that polarity is established much earlier. We analyzed roles of the cell polarity
regulators, the PAR complex proteins, and how their disruption in early development affects later
developmental milestones such as blastula and gastrula formation. We found that PAR6 along with
aPKC and CDC42 localize to the apical cortex (free surface) as early as the 2-cell stage of
development and this localization is retained through the blastula stage. Uniquely, in early cleavage
stage embryos PAR1 and PAR6 also appear to colocalize in the apical cortex. PAR6, aPKC, and
CDC42 are anchored in the cortex by myosin as disruption of myosin light chain kinase activity with
ML-7 and ML-9 resulted in cytoplasmic pooling. Additionally, pulse treatments with ML-7 and ML-9
prevented the embryos from reaching the gastrula stage, while the Rho-kinase inhibitor, H1152, did
not have this effect. This same pulse with ML-7 disrupted PAR6 localization at the fertilized egg and
maintained this disruption through the first cleavage division. Interestingly, aPKC inhibition early in
development prevented blastula formation, but not effect micromere formation. These observations
suggest that disruptions of the polarity complex in the early embryo can have a significant impact on
the ability of the embryo to reach later critical stages in development.
39 Mechanisms of cilia growth and differentiation
Robert L. Morris, Leah M. Williams, Else M. Doherty, Nidal A. Sholi, and Ali M. McCarthy
Wheaton College, Norton, MA, United States
In most sea urchin and sand dollar embryos, the onset of ciliogenesis occurs on all blastomeres
simultaneously just prior to embryo hatching and is regulated thereafter in a tissue-specific manner
to generate cilia with different lengths and behaviors. To understand the process of ciliary
formation and differentiation during development, we have begun to characterize the morphological,
behavioral, functional, and molecular differences between cilia subtypes that arise on echinoid
embryos. At hatching, every blastomere possesses the same type of long motile cilium to propel
the embryo. Such cilia grow in a stepwise manner sometimes to full length where they linger and
sometimes in repeating cycles of incomplete growth and retraction. Shortly thereafter, while lateral
regions continue to produce long motile cilia, other regions modulate the ciliogenic program is in
three ways. First, primary mesenchyme cells retract their motile cilia as the cells ingress and
suppress ciliogenesis at least until spiculogenesis onset. Next, as invagination begins, cells of the
vegetal plate retract their motile cilia and replace them with short cilia. Such short cilia come to line
the archenteron as invagination proceeds. Finally, cells of the animal plate grow their cilia to
approximately double-length and these apical tuft cilia lose motility. Light microscopic and genomic
approaches suggest that switching of ciliary subtypes appears to involve retraction of the ciliary
axoneme entirely into the cytoplasm, disassembly of the axoneme, and subsequent regrowth of a
new cilium from a cytoplasm containing an altered suite of ciliary proteins. The ability of metal ions
to animalize or vegetalize echinoderm embryos offers the opportunity to enrich for cilia subtypes
that usually exist in small numbers and thereby help us better understand how the ciliogenic
program is modulated in specific regions to generate the spectrum of cilia morphologies, motilities,
and functions found in different tissues.
Investigating the molecular determinants for polarity in the sea urchin egg
Lingyu Wang1, Albert Poustka2, and Athula Wikramanayake1
1
University of Miami, Coral Gables, FL, United States; 2Max-Planck Institut für Molekulare Genetik,
Berlin, Germany
The Dishevelled (Dsh) protein in the Wnt signaling pathway is essential for endomesoderm
specification in sea urchin embryos. Dsh is highly enriched in a vegetal cortical domain (VCD) of the
unfertilized egg, and several lines of evidence indicate that activation of Dsh in the Wnt pathway
during endomesoderm specification requires the VCD. First, while Dsh is required for activation of
beta-catenin signaling in vegetal cells, overexpression of Dsh is not sufficient for ectopic
endomesoderm formation in mesomeres (Weitzel et al). Second, a differentially modified form of
Dsh accumulates in the VCD and this domain is selectively inherited by the vegetal blastomeres
that nuclearize beta-catenin in early embryos (Peng et al, submitted). Moreover, embryological
extirpation of the VCD from eggs blocks activation of beta-catenin signaling in embryos developing
from VCD minus eggs (Croce et al). The mechanisms that tether Dsh to the VCD and activate it in
the Wnt pathway during early development are unknown, but this information is critical for
understanding early pattern formation in sea urchin embryos. To identify candidate molecules with
potential roles in regulating Dsh function in early embryos, we carried out two separate molecular
screens. To identify all RNAs enriched in the egg cortex, we did a large-scale RNA-seq screen. To
identify candidate proteins that regulate Dsh activity, we performed Dsh co-immunoprecipitation
using egg and isolated cortex lysates. These screens have identified several intriguing candidate
proteins, some of which are enriched in the VCD. We will discuss our ongoing studies to
characterize the respective roles of these candidate factors in regulating Dsh localization and
activation in eggs and early embryos.
40 References:
Weitzel, H et al. 2004. Differential stability of beta-catenin along an embryonic axis mediated by
Dishevelled. Development. 131.
Croce, J et al. 2011. Wnt6 activates endoderm in the sea urchin gene regulatory network. Development.
138.
Reconstruction of a Digital Sea Urchin Embryo during Early Development
Barbara Rizzi1, Louise Duloquin1, Thierry Savy2, Paul Villoutreix1, Emmanuel Faure2 and Nadine
Peyriéras1
1
Team "Multiscale Dynamics in Animal Morphogenesis", CNRS-NED, Gif sur Yvette, France; 2ISCPIF, Paris Île-de-France Complex Systems Institute, Paris, France
In vivo imaging systems offer to biologists the opportunity of examining cells for a long period of
time, under conditions allowing a normal development of the embryo. Nevertheless, understanding
the morphodynamics underlying the animal embryogenesis requires a quantitative analysis of cell
behavior that still remains a challenging issue. We present a chain of image processing algorithms
to reconstruct a digital embryo from time lapse Multi Photon Laser Scanning Microscopy images,
encompassing the Paracentrotus lividus embryo development during the period 32-cell stage (4hpf)
- hatching (around 500 cells 10 hpf). Our reconstruction provides cell positions and trajectories over
time as well as cell shapes. The embryo is fluorescently labeled to allow the simultaneous
acquisition of cell nuclei and membranes images. If the huge amount and the complexity of this kind
of data need to be treated automatically, the quality of images often limits the quantity of
informations that can be extracted. Typically 3-D LSM images of cells in living organisms are
characterized by low contrast. Nuclei appear clustered and membranes exhibit poorly defined or
absent boundaries, preventing the cell detection and the shape reconstruction. Therefore our
strategy consists in removing the noise with an edge-preserving filtering method, the geodesic
mean curvature flow. Cell positions are detected by identifying cell centers as local maxima of a
smoothed-simplified version of the nuclei images. Cell membranes segmentation is then achieved
using a modified version of the Subjective Surfaces method, whose peculiarity lies in its ability to
complete missing contours. Results are checked, annotated and validated by visual inspection
through the custom designed interactive visualization platform Mov-IT. Our strategy provides
measures on cell volumes, neighbors, orientations, shape descriptors that can be combined with
the cell lineage tree to allow a quantitative description of the embryogenesis.
Concurrent Session IX Morphogenesis
VEGF Signaling Plays Multiple Roles in the Migration and Differentiation of Primary
Mesenchyme Cells in the Sea Urchin Embryo
Ashrifia Adomako-Ankomah, and Charles A. Ettensohn
Carnegie Mellon University, Pittsburgh, PA, United States
Growth factor signaling pathways provide essential migration and differentiation cues to mesoderm
cells during embryonic development in many metazoans. Recent studies have implicated the VEGF
and FGF pathways in providing guidance and differentiation cues to primary mesenchyme cells
(PMCs) during skeletogenesis in embryos of the sea urchin Paracentrotus lividus (Duloquin et al.,
2007, Rottinger et al., 2008), though their relative contributions to these processes are presently
unknown. In this study, we use two-color, fluorescent in situ hybridization to show that in the sea
urchin Lytechinus variegatus, FGF and VEGF ligands are expressed in distinct domains in the
embryonic ectoderm. As in P. lividus, PMC migration is disrupted in L. variegatus VEGF morphants
41 and these embryos fail to form skeletal elements. Unlike in P. lividus, however, PMC migration is
unaffected in L. variegatus FGF morphants and well-patterned skeletal elements form. We show
that a second-generation VEGFR inhibitor (axitinib) phenocopies VEGF knockdown and can be
used to specifically block VEGF signaling at selected developmental stages. Results show that
VEGF signaling is essential not only for the initial phase of PMC migration (subequatorial ring
formation), but also for the late phase (migration toward the animal pole). However, we find that
VEGF signaling is not required for PMC fusion. Importantly, we also identify a role for VEGF
signaling in skeletogenesis that can be separated from its effects on PMC migration. Inhibition of
VEGF signaling after the completion of PMC migration causes significant defects in skeletogenesis,
and selectively blocks the elongation of specific skeletal rods. Nanostring nCounter analysis of ~100
genes in the PMC gene regulatory network shows a decrease in the expression of many genes with
proven or predicted roles in biomineralization in VEGF morphants. Our work therefore leads to a
better understanding of the role played by growth factors in gastrulation and skeletogenesis.
SLC26a2 is required for ventral sulfated proteoglycans and for ventral skeletal patterning in
sea urchin embryos
Finnegan J. Hewitt, Christy Li, and Cynthia A. Bradham
Boston University, Boston, MA, United States
The sea urchin larval skeleton is secreted by PMCs but the skeletal pattern is dictated by
ectodermal cues that regulate PMC positioning. We performed a differential screen for skeletal
patterning genes in sea urchin embryos, and thereby identified SLC26a2 (SLC), a sulfatetransporting channel. SLC is required for normal skeletal patterning in vertebrates since it promotes
normal sulfation of proteoglycans which in turn promote FGF and Ihh signaling. In sea urchin
embryos, SLC loss of function (LOF) via splice-blocking morpholino-substituted oligo injection
results in embryos with dramatic losses of ventral and animal elements, but does not impact DV
specification. SLC is expressed in the ventral ectoderm, and that expression pattern overlaps with
the pattern of sulfated proteoglycans (sPGs), which are strongly enriched in the ventral territory as
assessed by staining with either Alcian blue or Cuprolinic acid. SLC LOF inhibits ventral sPG
accumulation, ventral PMC localization, and ventral skeletal development, suggesting that SLCdependent sPGs direct PMCs to the ventral ectoderm, promoting skeletal development in this
territory.
Spatial and temporal profiling of ATP-binding cassette (ABC) transporters reveals a role for
ABCC5a in archenteron fusion
Lauren E. Shipp, Gary W. Moy, and Amro Hamdoun
Scripps Institution of Oceanography, La Jolla, CA
ATP-binding cassette (ABC) transporters are necessary for morphogenesis and protection in
embryos. Few studies have identified which ABC transporters regulate development. Using qPCR,
we surveyed expression of ABCB, ABCC, and ABCG transporters during the first 60 h of S.
purpuratus development (egg-early prism stage). Embryos expressed 20 transporters with
predicted functions in cell signaling, lysosomal and mitochondrial homeostasis, potassium channel
regulation, pigmentation, and xenobiotic efflux. The predicted xenobiotic transporter ABCB1a was
strongly expressed in all cells throughout development, and ABCB1a fluorescent protein (FP)
fusions localized to apical cell membranes. In contrast, mRNA of ABCC5a , a possible cGMP
and/or glycosaminoglycan transporter, was undetectable until hatching, after which it was detected
exclusively in the veg2 lineage. ABCC5a FP fusions localized to basolateral membranes.
Consistent with their predicted functions, over-expression of FP-ABCB1a reduced accumulation of
42 xenobiotics, while FP-ABCC5a did not. Collectively, these results suggest that in S. purpuratus ,
ABCB1a is protective while ABCC5a may have a developmental function. To test this hypothesis,
we knocked-down ABCC5a with morpholinos and found that these embryos develop elongated
archenterons that fail to fuse with the stomodeum in the animal-pole ectoderm. At 72 h, when
control embryos are plutei, ABCC5a-morpholino embryos have reduced skeletons, and their
archenterons have elongated to the extent that they protrude outward from the animal pole by
>50 µm, surrounded by a layer of animal-pole ectoderm. Since mammalian ABCC5a transports
cGMP and/or glycosaminoglycans, we are investigating the possibility that Sp -ABCC5a effluxes
cyclic nucleotides, or heteropolysaccharide components of the extracellular matrix that could be
required for archenteron fusion.
Micromere” formation and endomesoderm specification in the primitive echinoid
Prionocidaris baculosa
Atsuko Yamazaki1, Yumi Kidachi1, and Takuya Minokawa2
1
Aomori University, Aomori, Japan; 2Research center for Marine Biology, Tohoku University,
Aomori, Japan
To estimate ancestral endomesoderm specification mechanism in echinoid, the cidaroid (a primitive
group of echinoid) is considered to provide valuable information. In the present study, we chose an
indirect-developing cidaroid Prionocidaris baculosa as an experimental model, and examined the
blastomere composition and expression profiles of endomesoderm specification gene orthologs.
We found that blastomere composition in the 16-cell-stage Prionocidaris embryos was different
from that of the indirect-developing echinoids belonging to Euechinoidea, a derived group of the
echinoids. The sizes of the blastomeres in the 16-cell-stage embryo varied, and no embryos formed
a “micromere quartet,” a group of four equal-sized micromeres. The smallest blastomere was
usually located around the vegetal pole. We also found significant differences in early expression
profile of wnt8 orthologs of the Prionocidaris and euechinoids. Unlike euechinoids, the expression of
Prionocidaris wnt8 ortholog was not detected at the 16-cell stage; it began at the 32-cell stage in
the broad area containing the vegetal pole. However, in later stages, the expression profile of
Prionocidaris wnt8 was similar to that of euechinoid ortholog. Based on the results from expression
analyses and functional analyses for endomesoderm specification gene orthologs, we propose a
hypothesis for the evolution of endomesoderm specification mechanism in echinoids.
The Role of Twisted Gastrulation in Sea Urchin Embryo Development
Patrick S. Ferrell, Amanda B. Core, Ian Murray, Christie Li, Heather Hardaway, Tasso Kaper and
Cynthia Bradham, Cynthia
Boston University, Boston, MA, United States
Bone Morphogenetic Protein (BMP) is required for dorsal specification in the embryos of both
Drosophila and sea urchins; however BMP is expressed in domains that are spatially distinct from
those in which it signals in both organisms. BMP protein translocates across the embryo in a
complex with two other proteins, Chordin (Chd) and Twisted Gastrulation (Tsg); this process is
currently modeled as diffusive. Tsg has not been previously studied in sea urchins. This study,
therefore, characterizes the role of Tsg in the development of the sea urchin embryo. QPCR
analysis indicates that LvTsg is expressed throughout the time period during which BMP2/4 protein
is translocated. Spatial analysis of Tsg expression by whole-mount in situ hybridization shows that
Tsg is broadly expressed in the embryo in the early stages of development, with no spatial
restriction along the dorsal/ventral axis. Knockdown of Tsg by the injection of an antisense
morpholino-substituted oligo (MO) resulted in radialized embryos, indicating that loss of Tsg
43 function results in ventralization of the embryo. Misexpression of Tsg by injection of mRNA at high
doses also resulted in embryos with an expanded ventral region, although in these morphants, the
DV axis remains intact. These results indicate that correct levels of Tsg expression are required for
proper specification of the dorsal/ventral axis, and are consistent with its involvement in the
movement of BMP2/4. Thus, Tsg functions in BMP transport and DV specification appear to be
conserved in sea urchin embryos. Finally, the results herein inform a mathematical model of the
movement of BMP2/4 in sea urchin embryos.
A-P gut patterning by retinoic acid predates chordates.
Rossella Annunziata1, Libero Petrone2, Salvatore D'Aniello1, and Maria Ina Arnone1
1
Stazione Zoologica Anton Dohrn, Naples, Italy; 2University College London, London, United
Kingdom
Retinoic acid (RA) is a vitamin-A derived morphogen with a known role in chordate axial patterning
and organ formation. Hundreds of genes encoding for transcription factors, enzymes and
neurotransmitters are regulated by RA, a very famous example being Hox genes. For a long time
RA signaling has been considered a vertebrate and then a chordate specific feature; subsequently,
the finding of RA machinery components in the genome of several non chordate organisms,
strongly suggested that RA signaling role in development originated much earlier in bilaterian
evolution. Despite the importance of the above mentioned discovery, very rare and incomplete
reports are available about the involvement of RA in developmental processes outside vertebrates.
In the sea urchin S. purpuratus genome, key components of the RA pathway have been identified,
among which the two receptors RAR and RXR. Interestingly, all of them show expression in the gut
of the developing embryo, suggesting a very likely role for RA signaling in the gut patterning
process. We treated sea urchin embryos with exogenous RA and discovered alteration in transcript
distribution along the A-P axis of the gut, such as reduction or ectopic expression of key regulators
and posterior shift in the expression domains of midgut specific terminal differentiation genes. With
this work we present the first, and, up to now, only demonstration of a role for RA in the
development of a non chordate organism, providing significant information about the reconstruction
of RA signaling evolution, and a simple tool, the sea urchin embryo, to explore the still poorly
understood RA mechanism of action.
Concurrent Session X Embryos and informatics in education and
outreach
Sea urchin supply corresponding to the purpose for more convenience from a marine
laboratory
Masato Kiyomoto
Ochanomizu University, Tokyo, Japan
The sea urchin is a wonderful material for observing animal development. But, for unfamiliar people,
it might be hard to get, to keep, and to use. Usually expensive aquarium is necessary to keep
sensitive mature urchins. Using recent transportation business, it is possible to send materials on
the appointed day. You can receive it two hours before your experiment. Or you don't have to
prepare the spawning. Unfertilized eggs and sperm are possible to preserved for several days or
weeks. These optional supplies corresponding to the purpose would make this material more useful
in various fields, though it is not still the best way to maintain the quality of gametes, so far, for the
specialists of sea urchin research.
44 We have supported high schools from the two projects supported by CoREF (Consortium for
Renovating Education of the Future), one was an easy and simple fertilization experiment without
taking care of urchins for the beginners and the other was a larval culture in a small bottle by each
student to observe the metamorphosis to juveniles. And we start new projects, one is for the
experimental course in the universities (Supply of Marine Bioresource supported by the Ministry of
Education, Culture, Sports, Science & Technology in Japan) and the other is for all elementary and
secondary educations (as A Gift from the Sea supported by The Nippon Foundation). Both include
gametes, embryos, and larvae to observe several important stages.
Using Sea Urchin Embryogenesis to Teach about Climate Change and Ocean Acidification.
John Henson
Dickinson College, Carlisle, PA
Global climate change is arguably the biggest challenge that humans currently face and former
NAS President and current Science Editor Bruce Alberts has admonished all scientists to
incorporate sustainability into their research and/or teaching. Sea urchin embryogenesis offers a
straightforward means of teaching about climate change and ocean acidification as has been
demonstrated by the Stanford University Virtual Urchin Acid Ocean website. We have developed a
teaching lab exercise in which Lytechinus pictus embryogenesis under control and CO2-based
acidified conditions is monitored over a four day period. Embryos were maintained in air-tight
containers and were monitored using simple brightfield or darkfield optics. More sophisticated
experiments involved immunofluorescent localization of cytoskeletal and neuronal markers followed
by imaging with epifluorescence and confocal laser scanning microscopy. We chose pH 7.6 and
7.8 experimental conditions to compare with pH 8.0 and 8.1 controls in order to emphasize the
impact of acidification on overall morphogenesis in general and skeletogenesis in particular. In this
talk I will also discuss other interactive methods for teaching about climate change to include
climate modeling and data analysis and presentation. Using marine models in the MBL Logan Science Journalism Program
Brad Shuster1 and David Burgess2
1
New Mexico State University, Las Cruces, NM and 2Boston College, Chestnut Hill, MA
Rapid advances in the fields of cell, molecular and developmental biology present challenges to
science journalists attempting to describe these findings to the general public. These challenges are
further compounded by fact that science writers working in either traditional or online media
generally do not have an educational background in science. Now in its twenty-sixth year, the
Logan Science Journalism Program brings science writers to the MBL for a hands-on “boot camp”
focused on either biomedical or environmental science. Since assuming the directorship of the
biomedical program in 2009, we have redirected the emphasis of the course from standard
molecular biology techniques and genetic model systems to a program where marine invertebrate
embryos are featured prominently as models for cell and developmental biology. Science writers
engage in the imaging, biochemical, and bioinformatic approaches used routinely in the lab, while
keeping lectures on biological concepts and techniques to a bare minimum. By keeping the
schedule flexible and providing time for discussions about the culture and politics of science, the
program seeks to give journalists an appreciation for how science is actually carried out, as well as
the value of basic science and non-mammalian models for biomedical research. 45 Genomics for undergrads: achieving simple goals with complex tools
Robert Morris
Wheaton College, Norton, MA
Given the great complexity of genomics tools and tremendous depth of genomic data, wading into
genomic analysis can be daunting for experienced researchers and outright paralytic to
undergraduates. Nevertheless, with clear instructions that target specific learning goals, students
can delve into genomics and realize important lessons about this growing field. To this end, I will
present an integrated set of tools to introduce genomics to undergraduates that includes one book,
two microscopy labs, and a genome browser exercise.
Genomics and Bioinformatics for Undergraduates
Billie J. Swalla1, Andrea B. Kohn2, and Leonid L. Moroz2
1
University of Washington, WA, United States; and University of Florida, FL, United States
Teaching undergraduates about genomics and bioinformatics can be challenging. I’ve developed a
Bioinformatics exercise that can be done in about an hour with undergraduates that I will describe
and share. It is best done with every student using their own computers, either their laptops, or in a
computer lab. Once they do the exercise, then they do a project that consists of a “virtual”
developmental biology experiment. They need to use their knowledge of development from a
model system, and design an experiment in a different animal. They must describe a known gene,
tell what it does, and the gene structure in a model system, then predict where it will be expressed
in the embryos of their species. Students have been very clever with this and I will share some of
the projects that have been done. I have also taught two genomics apprenticeships at FHL where
we taught undergraduates how to look for orthologous genes and annotate the genome of a local
ctenophore, Pleurobrachia bachei. Students had individual projects, but also contributed to the
entire project of annotating the genome. These apprenticeships have been very successful, both in
the research that was done and in inspiring the students to go on to graduate school. I will share
some of the highlights of the ctenophore genome and how we structured the course over the
quarter. Digital specimens for teaching Deuterostomian early embryogenesis
Nadine Peyriéras
CNRS, Gif-sur-Yvette, France
We investigate new tools to achieve the reconstruction of model organisms’ multiscale dynamics in
the context of a complex systems approach of Deuterostomian embryogenesis. The cellular
organization is taken as the necessary integrator of microscopic (molecular & genetic) and
macroscopic (mechanical constraints) processes. The reconstruction of the cell lineage tree
deployed in 3D+time and annotated with cell membrane and nucleus shape leads to digital
specimens that are the basis for further modeling cell dynamics. The interactive visualization of
digital specimens provides invaluable new pedagogical tools. Further enriching the 3D+time cell
lineage tree with gene expression data and interaction networks architecture is then a step for the
integration of cellular, molecular and genetic dynamics. Confronting numerical simulations and
reconstructed digital specimens will be ideal tools to explore the parameters space leading to a
normal or pathological development.
46 POSTER ABSTRACTS
The evolution of ciliary signaling
Monika Abedin and Jeremy Reiter
Department of Biochemistry, University of California, San Francisco
Cilia, evolutionarily ancient structures that project from the cell surface into the extracellular
environment, are critical sensors of external cues. In animals, they detect light, odors, soluble
chemicals and mechanical forces, and defects in cilia cause a range of diseases. Vertebrate cilia
respond to intercellular signaling molecules such as Hedgehog, a regulator of embryonic
development. Despite its important roles in embryogenesis and disease, little is known about the
origins and evolution of ciliary signaling outside of vertebrates. Understanding how cilia evolved
mechanisms to receive and transduce signals will help identify core ciliary functions fundamental to
animal biology. To obtain a more complete picture of the ciliary signaling pathways present in
animals, gain insights into the evolutionary origins of these pathways, and perhaps identify novel
ciliary signaling mechanisms, I will identify and determine the phylogenetic distribution of ciliary
signaling proteins in two non-vertebrate lineages: (1) choanoflagellates, the closest known relative
of animals and (2) sea urchins, a marine invertebrate. To this end, I will isolate cilia from the
choanoflagellate Salpingoeca rosetta and from embryos of the sea urchin Strongylocentrotus
purpuratus. After analyzing the cilia by mass spectrometry, I will identify putative signaling proteins
and determine their phylogenetic distribution using a combination of BLAST and HMM-based
analyses. I expect to find three classes of proteins: ciliary signaling proteins that are conserved
among animals and choanoflagellates, those that are conserved between sea urchins and
vertebrates but not choanoflagellates, and proteins that are unique to each lineage. This work will
broaden our understanding of cilia-dependent signal transduction and potentially identify novel, core
signaling molecules.
Investigating adult skeletogenesis during arm regeneration of the brittle star Amphiura
filiformis
Anna Czarkwiani, Olga Ortega-Martinez, David Dylus and Paola Oliveri
University College London, London, United Kingdom
Background and aims: The brittle star Amphiura filiformis is emerging as a useful model organism
for regeneration studies due to the ease of maintenance, manipulation both in vivo and in vitro,
great regenerative potential and the availability of an RNA sequence database. The aim of this
project is to attract attention to brittle stars as model organisms by optimizing experimental
procedures and studying regeneration and adult skeletogenesis.
Methods: Several methods have been used to evaluate the brittle star as a potential model
organism for regeneration studies including morphological observations, gene cloning, whole-mount
in situ hybridization (WMISH) and quantitative PCR (QPCR) for studies of gene expression.
Moreover, functional molecular analysis techniques such as electroporation on blastema and
chemical inhibition of signalling pathway on explants have been tested.
Results: An overview of the regenerative mechanism of Amphiura has been compiled along with a
detailed characterization of skeleton formation. WMISH protocol was optimized by completing
several control experiments and spatial patterns of differentiation markers and a few regulatory
genes were investigated. Preliminary temporal expression data have been obtained using QPCR
for the undifferentiated and differentiated regenerating arm segments. Explants were used to
investigate the effects of FGF inhibition on the expression of skeletogenic genes.
47 Summary and conclusions: Experimentally validated methods for studying gene expression and
results of the morphological observations will be useful for future researchers wishing to use the
brittle star as a model organism and study the regeneration and adult skeletogenesis.
Evolution of Larval Skeletogenesis: A Comparative Approach.
David Dylus, Paula Oliveri and Anna Czarkwiani
University College London, London, United Kingdom
Gene regulatory networks (GRNs) are maps of interacting transcription factors, describing precisely
the development of an animal at the genome level. Due to the complexity of the GRN architecture,
functional modifications are difficult to predict. In order to understand how modifications can drive
the same developmental process between species, we are undertaking a comparative analysis for
larval skeleton formation in echinoderms. Due to its unique phylogenetic position and being in the
sea urchin the most-complete developmental GRN, we are aiming to resolve whether larval
skeletogenesis is regulated through a similar GRN in the ophiuroid Amphiura filiformis. This brittle
star provides an excellent comparative system since it forms a larval skeleton, the early spicules
derive from primary mesenchyme cells and some transcriptome data is available. To obtain an
initial understanding of the difference in network architecture for the development of the skeleton,
we first, analyzed A. filiformis development and thoroughly compared it with the sea urchin. Based
on a preselected candidate list, we then identified more than 40 orthologous genes of the sea
urchin skeletogenic lineage in the brittle star using reciprocal blast. We studied their temporal
expression profile, using QPCR, and compared them with the sea urchin one. Finally, we cloned
several of these genes using RACE and degenerate PCR and collected spatial data using WMISH.
Our results propose that, although A. filiformis produces an embryonic skeleton, the emerging
regulatory state dynamic is strikingly different and suggest that a strongly diverged network
architecture is controlling the early specification events. Interestingly, along with several other
observed differences, the sea urchin pmar1/hesC double negative gate, for instance, is not
conserved in A. fifliformis. Our data are preliminary to a full GRN study for skeletogenesis in the
brittle star and will contribute to the understanding of network rewiring.
The Role of Wnt/Planar Cell Polarity (PCP) components in Nematostella gastrulation
Erica B. Flores, and Athula Wikramanayake
University of Miami, Coral Gables, FL
Wnt signaling is known to be critical for developmental processes in most multicellular animals,
particularly in early embryo patterning and morphogenesis. One essential event is gastrulation,
which establishes the main body axis and segregates the primary germ layers for future
differentiation. In the starlet anemone, Nematostella vectensis, both Wnt/β-catenin and Wnt/PCP
(planar cell polarity) signaling are involved in coordinating this complex process through endoderm
specification and archenteron invagination, respectively. Both of these pathways utilize Frizzled (Fz)
as a transmembrane receptor and the cytoplasmic protein Dishevelled (Dsh) as a signal transducer.
The lesser studied Wnt/PCP pathway functions to establish cell polarity and coordinate cytoskeletal
changes. One core component of Wnt/PCP signaling is Strabismus (Stbm), a 4-pass
transmembrane protein that, along with Dsh, is known to be involved in convergence and extension
movements during gastrulation and neural tube closure in vertebrates. Both of these
morphogenetic processes involve polarized cell behaviors. Interactions between Dsh and Stbm
have been observed, but their exact role in these events is not clear. Phosphorylation of Dsh and
Stbm are known to occur during signaling, and investigation of this post-translation modification in
48 Nematostella embryos can provide insight into their role during critical developmental events. We
have carried out 2D Western blot analysis of NvDsh and NvStbm to correlate post-translational
modifications occurring in these two proteins at critical developmental stages. Moreover, we have
developed NvDsh and NvStbm Co-Immunoprecipitation protocols to identify Dsh-Stbm interactions
during these critical developmental stages and also to identify novel Dsh and Stbm interacting
proteins. We will present our results from these preliminary studies.
Decision Making to Decision Taking: Understanding the Transition Between Inductive and
Inherited Mechanisms of Germ Line Determination
Tara Fresques, Adrian Reich, and Gary Wessel
Brown University, Wessel Lab, Providence, United States
Embryos set aside primordial germ cells early in their development, and these germ line stem cells
ultimately give rise to gametes in the adult organism. Germ cells can be specified by either
inductive mechanisms, where cell-to-cell interactions induce a set of cells to take on the germ cell
fate, or germ cells can be specified by inherited mechanisms, where germ cell determinants from
the zygote are selectively accumulated by a subset of embryonic cells that then take on the germ
cell fate.
Although the basal mechanism for germ line determination for the metazoan phylogenetic tree is
likely inductive, many clades contain species’ that utilize each type of germ line specification
mechanisms. The echinoderm clade, for example, contains species that specify their germ lines by
each of these distinct mechanisms. By performing in situ and immunolocalization experiments on
these two species we have begun to determine when two key germline determinants, Nanos and
Vasa, begin to accumulate in putative primordial germ cells. From this data we show that S.
purpuratus, a species of sea urchin, accumulates germline determinants in the small micromeres at
the 32-cell stage and seem to specify their germ lines with a more cell autonomous and inheritedlike mechanism. On the other hand, P. miniata, more commonly known as the sea star, doesn’t
show accumulation of key germline determinants until much later in development, approximately 4
days post-fertilization, which is more reminiscent of an induced mechanism of germ line
determination. We propose these closely related species represent a transition between the two
mechanisms of germ line determination and can be used to study the evolutionary changes from
inductive to inherited mechanisms of germ line specification.
Expression of Aminopeptidase N Genes During Sea Urchin Development
Eric P. Ingersoll, and Diana L. Drab
Penn State Abington, Abington, PA, United States
Aminopeptidase N (APN) is an exopeptidase that has been shown to play a role in the development
of nematodes and fruit files. In previous studies, we examined the expression of one APN gene
during sea urchin development. We have searched the sea urchin genome and found a number of
additional APN genes. After careful examination of these sequences, we found that only about
seven sequences represented full-length APN genes with the rest lacking important functional
motifs and probably representing either gene fragments or pseudogenes. In this study, we
determined which of these additional APN genes is expressed during embryonic development. We
used reverse transcription PCR to detect gene-specific mRNAs throughout development. Our
studies have identified five APN genes that are expressed in Strongylocentrotus purpuratus
embryos. Each of these genes shows a different temporal expression pattern during embryonic
development, including one gene that appears to be expressed only at the blastula stage. We were
49 unable to detect expression of these genes in Lytechinus variegatus embryos. In the future, we will
determine the spatial expression patterns and developmental functions of these genes.
Study on the formation of the anteroposterior body axis of the sea cucumber Apostichopus
japonicus
Mani Kikuchi
Department of Biological Sciences, Graduate School of Science, the University of Tokyo, Tokyo,
Japan
Echinoderms show bilateral symmetry at the larval stage. However, in the adult, they exhibit
pentameral symmetry. So far, anterior-posterior (A-P) axis of the echinoderm remains to be a major
issue. As the other major groups of the deuterostomes (Hemichordates and Chordates) are
fundamentally bilateral throughout their development, and some fossil records imply that the basal
echinoderms had triradiate body, it is generally regarded that the pentameral body patterns of the
echinoderms had been evolved from bilateral ancestors. However, the origin and evolution of the
pentameral echinoderm body pattern is still controversial. Among the five extant echinoderm
classes, Holothurians (sea cucumbers) possess apparent anterior-posterior (A-P) axis in the adult
body. It has been suggested that Holothurians evolved secondary anterior-posterior (A-P) axis. We
assumed that Hox cluster genes are involved in the A-P axis patterning in the adult of Holothurians
as well as in the larval development. We isolated Hox genes (Aj-Hox1, Aj-Hox5, Aj-Hox7, Aj-Hox8,
Aj-Hox9/10, Aj-Hox11/13a, Aj-Hox11/13b, Aj-Hox11/13c) from Apostichopus japonicus and
performed whole-mount in situ hybridization at the different developmental stages including
juvenile. We will discuss the spatially sequential and the collinear arrangement of expression
domains in the larva and the juvenile.
Experimental replication of larval expression of alx1 in starfish
Hiroyuki Koga and Hiroshi Wada
University of Tsukuba, Tsukuba, Ibaraki, Japan
Pluteus larva of sea urchin and brittle star is thought to have derived from ancestral auriclaria-type
larva. In this evolution, heterochrony of skeletal element played crucial role. Indeed, Pluteus larvae
have well-developed skeleton, whereas starfish larvae bipinnaria do not have such skeletal
elements. One of the important genetic difference is larval expression of alx1 gene encoding a
transcription factor which play a central role in skeletogenic gene regulatory network. We previously
reported that heterochronic shift of alx1 from adult to embryo have occurred during the evolution.
Here, we demonstrated replication of larval expression of alx1 in starfish larva by inducing mRNA
and assessed its effects on genotype by RNA sequencing, as well as on phenotype. We report
effects of ectopic expression of alx1 in starfish embryogenesis. Some of upregulated genes showed
similarity to skeletogenic genes of sea urchin.
The sea urchin embryo and the translational control of gene expression : A role for Mnk in
the regulation of protein synthesis required for the cell divisions of early development
Olile Mulner-Lorillon, Laure Sultan, Virginie Glippa, Robert Belle and Patrick Cormier.
CNRS/UPMC, Roscoff, France
Global and selective protein synthesis modifications following fertilization of sea urchin eggs occur
independently from transcription. This makes the sea urchin embryo a canonical model to study the
translational network controlling gene expression. A well-established regulator of translational
50 control is the initiation factor, eIF4E (eukaryotic initiation factor 4E), which binds the cap-structure of
mRNA and recruits all the other factors needed to activate initiation. Our data in sea urchin embryo
have shown that eIF4E availability relied not only on eIF4E dissociation from an inhibitory complex
with 4E-BP proteins (eIF4E-binding proteins) as demonstrated so far in various models, but also
from 4E-BP degradation, both events depending on the PI3K/Akt/mTOR-regulated phosphorylation
status of 4E-BP. In mammalian cells, it was also proposed that eIF4E activity could be regulated by
eIF4E phosphorylation by Mnk (MAPK-interacting kinase). However the precise role of Mnk and/or
of eIF4E phosphorylation on translational control remains to be elucidated. We report here data
sustaining a role for eIF4E phosphorylation by Mnk in the control of proper cell division in early
development. Based on immunodetection experiments, we showed that two eIF4E isoforms were
present in sea urchin embryos. Following fertilization, we demonstrated the existence of a
phosphorylated pool of eIF4E. Incubation of the embryos in the presence of CG 57238, a specific
Mnk inhibitor, led to the disappearance of this phospho-eIF4E pool. Furthermore, CG 57238
inhibited the cell division following fertilization. Strikingly, global protein synthesis in the embryos
was poorly impacted by the presence of Mnk inhibitor. We are currently investigating the hypothesis
that the phospho-eIF4E pool could regulate the translation of a selective (or localized) set of
proteins involved in cell division.
The Effects of MLD and SVEP on Triradiate Orientation and PMC Migration in Sea Urchin
Embryos
Ian Murray, Finnegan Hewitt, Joe Perez-Rogers, Michael Piacentino, Hajerah Hameeduddin,
James Chavez, and Cynthia Bradham
Boston University, Boston, MA, United States
Skeletal patterning in sea urchins requires ectodermal instruction of the skeleton-secreting primary
mesenchymal cells (PMCs). At the late gastrula stage, the PMCs migrate to form a ring-and-cords
pattern and begin to secrete the skeleton in the form of two triradiates. A comprehensive screen for
ectodermal genes involved in the direction of PMC migration and positioning identified two adhesion
genes: SVEP, which encodes an adhesion protein involved in epithelial-mesenchymal interactions
in mammalian bone, and MLD, which encodes a protein containing repeating extracellular adhesion
domains. Loss of function (LOF) analyses of PMC positioning indicates both genes are necessary
for ventral localization of PMC in the ring-and-cords pattern, but eventual production of ventral
skeletal elements in these morphants indicates a possible compensatory relationship between
these genes. Analyses of skeletal phenotypes in both morphants indicate that SVEP is required for
normal alignment of the triradiates about the animal-vegetal (AV) axis, while MLD is required for
normal alignment about the left-right (LR) axis. Examination of PMC position and triradiate
orientation in single and combined LOF time course experiments will further characterize the role
these genes play in urchin skeletal patterning.
Evolutionary origin and diversification of pancreatic cell types
Margherita Perillo, and Maria Ina Arnone
Stazione Zoologica Anton Dohrn, Napoli, Italy
We are interested in the evolutionary origin and diversification of pancreatic cell types. The
pancreas is a mixed exocrine and endocrine gland. Only vertebrates have a complete pancreatic
gland, while it has been found that in invertebrates, pancreatic hormones, such as insulin, are
produced mainly in neurons and sometimes in scattered cells of the gut. It is known that in
vertebrates there are two master regulators of pancreatic development: Ptf1 (pancreatic
transcriptional factor 1), which is also involved in exocrine pancreas differentiation, and Pdx1
51 (pancreatic duodenal homeobox gene 1), which in β-cells (endocrine pancreas) plays a role both in
cell differentiation and in glucose-dependent insulin gene expression. To study the origin of
pancreatic cell types, we examined the expression of the S.purpuratus homologues of Ptf1 and
Pdx1 (SpLox and SpPtf1a, respectively) and their putative target genes during embryo and larva
development in the sea urchin, so far the best basal deuterostome available for functional analysis.
To this end, we studied the spatial profile of the only two insulin-like genes found in the sea urchin
genome, SpIgf1p and SpIlgf2 and we found interesting groups of cells in the larval gut which
express SpIgf1p as well as other pancreatic cell markers. Moreover, using a commercial anti-insulin
antibody, we observed a different spatial expression in the larval gut when comparing feeding
versus starving conditions. On the other hand, we are studying the spatial and temporal expression
of SpPtf1a and its putative target genes (such as a carboxypeptidase and a threonine
dehydrogenase) in order to investigate whether these putative cells can represent an ancestral cell
type of the exocrine pancreas.
Study of the Gene Regulatory Network for Pigment Cells Development in the Sea Urchin
Embryo
Antonio Ortiz and Cristina Calestani
Valdosta State University, Valdosta, GA, United States
In order to better understand the processes of cell specification and differentiation, we study the
Gene Regulatory Network (GRN) playing a role in the development of larval pigment cells, which
are part of the larval immune system. In order to reconstruct the pigment cells GRN we are studying
the cis -regulatory elements controlling the expression of the differentiation gene polyketide
synthase ( pks ) in pigment cells. The pks gene encodes a protein essential for the biosynthesis of
the S. purpuratus pigment known as echinochrome. Pks starts to be expressed during the
blastula stage in non-skeletogenic mesodermal cells and by pluteus stage is expressed exclusively
in pigment cells. The characterization of the regulatory elements that control the transcription of
pks , both spatially and temporally, will allow for further understanding of the developmental gene
regulatory network upstream of this terminal differentiation gene. Our previous studies showed that
DNA-binding sites with homology to Glial cell missing, GataE, and Kruppel-like binding sites
function as positive cis -regulators. Comparative genomics was performed by utilizing the software
FamilyRelationsII, MEME, and SANN in order to predict additional cis -regulatory modules and
putative DNA-binding sites. The MEME analysis was done utilizing five co-expressed pigment cell
genes: flavin-monoxidase 1, 2, 3, sulfotransferase , and pks . In addition, these sequences were
compared across three different sea urchin species: Allocentrotus fragilis, Strongylocentrotus
fransiscanus , and Strongylocentrotus purpuratus . We selected for further studies the putative
transcription factor binding sites for TLX1-NFIC, MYC-MAX, NFK-B, NKX3-1, and FOS. The DNAbinding sites mutagenesis results suggested that TLX1-NFIC, MYC-MAX, and NFK-B are functional
binding sites for positive regulators. The function of NKX3-1 and FOS sites will be discussed. Our
study broadens the knowledge of developmental GRNs structure and function particularly during
the differentiation process.
Lipoxygenase is required for skeletal patterning
Vijeta D. Patel, Joe Perez-Rogers, Finnegan Hewitt, Christy Li, Christy, and Cynthia Bradham,
Boston University, Boston, MA, United States
Skeletal patterning and development rely on ectodermal instruction of the skeleton-secreting
primary mesenchyme cells (PMCs). We performed a differential screen based on high throughout
sequencing of control and perturbed embryos, and thereby identified multiple skeletal patterning
52 genes, including lipoxygenase (LOX). LOX is a leukotriene generator which indirectly impacts
multiple signaling cascades. LOX was knocked down using an antisense morpholino-substituted
oligonucleotide (MO), and skeletal patterning defects were systematically evaluated. The resulting
embryos exhibit defects in triradiate orientation, particularly about the DV axis, loss of the ventral
transverse rods, and defects in 2° skeletogenesis on the left side. Systematic analysis of PMC
positions indicates that LOX is required for ventral PMC positioning, consistent with the absence of
ventral transverse rods in LOX morphants. However, the ciliary band was not affected by LOX
knockdown, indicating that the DV axis was normally specified. Together these data demonstrate
that leukotrienes are required for normal skeletal patterning, particularly in the ventral and left
regions.
Molecular characterization of circadian rhythm in sea urchin
Libero Petrone, Avi Lerner, Agnieszka Chomka, Linda Klotz, Mattias Thurner and Paola Oliveri
University College London, London, United Kingdom
The circadian clock is an endogenous time-keeping mechanism that regulates daily physiological
and behavioural processes. The molecular components of circadian clocks have been extensively
characterized in land organisms such as mammals, insects and plants. The molecular clockwork is
encoded in the genome and is composed by a transcriptional-translational oscillator based on a
delayed negative feedback loop. Many of the “clock” genes that encode the core components of the
clock are highly conserved across animal phyla. Although life, and early circadian clocks, evolved
in the sea, which represents a complex environment characterized by the interplay of multiple
periodicities, little is known about clocks in marine organisms.
We are undertaking a study to determine and characterize the molecular components of sea urchin
circadian clock in larvae and adults. Genome analysis of sea urchin clock genes shows a highly
complex “clock” tool-kit with both protostome and deuterostome components. Interestingly, our
comparative analysis shows a conservation of the positive arm of the transcriptional-translational
oscillator while the negative arm has been extensively diversified during animal evolution. Our
gene expression data show that almost all of the “clock” genes are maternally expressed and decay
around blastula stage. Many of them are also expressed later in development and in the larval
stages. Interestingly, the Drosophila-like cryptocrome (encoding a light-sensing protein) and
timeless genes show localized expression in some cells of the neuro-sensory apical organ
suggesting the presence of a centralized clock. However, we have not found evidence of oscillatory
expression in “clock” genes during embryonic development. On the contrary, larvae with fully
differentiated neuronal structures and autonomous feeding once exposed to different light regimes
show circadian oscillations in gene expression. Moreover, we detected expression of “clock” genes
in adult tissues, in particular the tube feet, which are the major sensory organs, and in the radial
nerves.
Expanding the PMC GRN: Genome-Wide Analysis of Ets1 and Alx1 Targets
Kiran Rafiq and Charles A. Ettensohn
Carnegie Mellon University, Pittsburgh, PA, United States
We use a model morphogenetic process, the formation of the sea urchin embryonic skeleton, as an
experimental system to understand how a complex gene regulatory network (GRN) controls the
development of a major anatomical feature. Here, we link a GRN deployed in skeletogenic primary
mesenchyme cells (PMCs) to genes that control skeletal morphogenesis and uncover
transcriptional regulatory inputs into these genes at the early gastrula stage. We focus on Ets1 and
53 Alx1, two well-characterized transcription factors that provide some of the earliest inputs into the
network and are responsible for the differentiation of the PMC cell lineage into skeletogenic cells. In
our earlier work, we restricted our analysis to downstream genes expressed exclusively by PMCs.
One striking finding from our studies was that the targets of ets1 and alx1 were almost identical. To
provide a comprehensive, genome-wide analysis of the targets of ets1 and alx1 and to shed insight
into the parallel connectivity of these two regulatory genes, we performed paired-end, highthroughput cDNA sequencing (RNA-seq). We used Cufflinks, a free open-source software tool for
comparing gene and transcript expression under control and ets1/alx1 knockdown conditions. The
targets of ets1 and alx1 identified previously were found to represent only a small subset of a much
larger number of functional targets. Analysis of regulatory genes controlled by ets1 and alx1
identified several common targets, including alx4, foxb, hnf1, and nk7, which could perhaps explain
how ets1 and alx1 co-regulate such a large set of common downstream genes. Our
transcriptome analysis also allowed us to identify new classes of proteins that were not previously
known to play a role in skeletogenesis. Thus, our works expands the PMC GRN to a great extent
and continues to establish linkages between this transcriptional network and the morphogenesis of
the skeletal system.
Probing TGF-β and Delta Notch signaling in activation of the non-skeletogenic mesoderm
gene regulatory network.
David Rogers and David R. McClay
Duke University, Durham, NC, United States
The GRN for mesoderm formation is incomplete with many of the non-skeletogenic mesoderm
genes expressed without identified activators, especially in the oral nonskeletogenic mesoderm. In
other cases, the network of the oral mesoderm models Hnf6 in the oral mesoderm as upstream of
GataC. However, Hnf6 appears to be expressed everywhere but the mesoderm prior to the
expression of GataC, and therefore most likely does not regulate GataC, at least directly. In the sea
urchin, TGF-β signaling is conserved and plays a role in patterning all three germ layers, specifically
by initially helping to shape the dorsal (aboral)/ventral (oral) axis, followed by the left/right axis. New
insights can be made to connect TGF-β signaling to the current GRN that is responsible for initiating
mesoderm formation, adding important inputs into the GRN that we know are currently missing and
to refine inputs that may be incorrect. Here, perturbations to not only TGF-β signaling, but also to
Delta Notch signaling, specifically the second Delta signal, identify upstream components and
downstream targets of the mesoderm GRN. Mesoderm formation following perturbations on the oral
mesodermal genes Ese, GataC, and Prox and the aboral mesodermal genes Gcm and Gfi offer
new insights into the mesoderm GRN. As Ese is first expressed as early as eight hours post
fertilization and GataC and Prox are expressed around ten to eleven hours, we expect Ese to be
controlled by earlier signals such as the first Delta signal, while GataC and Prox may be controlled
by later inputs. Gfi is only transiently expressed in the aboral mesodermal territory between eleven
and fourteen hours, an expression domain that may be controlled by later signals like the second
Delta signal. TGF-β signaling appears to help pattern these genes into oral and aboral half rings,
but does not directly control their expression.
54 A novel approach to deliver morpholinos into sea urchin eggs that is amenable to research
by undergraduate students in the context of a small liberal arts institution
Laura Romano, Michelle Clark, and Efua Thompson
Denison University, Granville, OH, United States
Although microinjection is successful in delivering morpholinos into sea urchin eggs, it can be quite
difficult and time-consuming for undergraduate students to become competent in this approach.
Moreover, the necessary supplies and equipment can be prohibitively expensive in the context of a
small liberal arts institution. Thus, we decided to examine the effectiveness of Endo-Porter from
Gene Tools, Inc. in the green urchin, Lytechinus variegatus. Endo-Porter is a peptide that binds to
the plasma membrane and triggers endocytosis of the morpholino, which is eventually released into
the cytosol upon acid-induced permeabilization of the endosome by the poly-cationic form of the
reagent. We tested different concentrations of the reagent, as well as the effect of paraaminobenzoic acid on the survival of the sea urchin embryos at 24 hours post-fertilization. Once we
determined the optimal conditions for survival, we tested the ability of the reagent to deliver control
morpholinos tagged with 3’ fluorescein into sea urchin eggs. Once we confirmed uptake of control
morpholinos by diffuse fluorescence within the cytosol (albeit at a low frequency compared to
microinjection), we tested the ability of the reagent to deliver snail morpholinos tagged with 3’
fluorescein into sea urchin eggs and produce the mutant phenotype as described by Yu et al.,
(2007). We ultimately developed a protocol that delivered the snail morpholinos into sea urchin
eggs and produced the expected mutant phenotype (i.e. a delay in the ingression of primary
mesenchyme cells). In the future, we will continue to modify our protocol in an effort to increase the
rate of delivery so that we can eventually explore the function of various genes in our species of
interest, the pencil urchin, Eucidaris tribuloides.
Characterizing molecular markers for immunocyte lineages in the embryo and larva of
Strongylocentrotus purpuratus
Catherine Schrankel, Guizhi Wang, Katherine Buckley, Eric Ho, Koeun Bae, Cynthia Solek and
Jonathan Rast
University of Toronto, Toronto, CA
Immune response in the purple sea urchin larva is mediated by granular pigment cells and subsets
of blastocoelar cells. Blastocoelar immunocytes develop from a patch of cells in the oral nonskeletogenic mesoderm that co-express orthologs of the vertebrate hematopoietic stem cell
transcription factors Gata1/2/3, Scl/Tal-2/Lyl-1, and Erg/Fli-1 at 24-27 hrs. Following the down
regulation of these factors, precursors migrate into the blastocoel and differentiate into several cell
types. We have defined four classes of blastocoelar immunocytes based on morphology and
behaviour: highly motile amoeboid cells, migratory globular cells, highly phagocytic ovoid cells, and
a subset of network-forming, phagocytic filopodial cells. Terminal gene batteries are needed to
better characterize these lineages and to link them with early developmental programs. Detailed
RNA-Seq analysis has identified over 200 immune-related genes that are constitutively expressed
in the feeding larva. Candidate immunocyte markers identified from these datasets are being
validated by qPCR, WMISH localization, and fluorescent protein reporters. Two examples are
Tecp2, an α-2-macroglobulin-like protein, and MacpfA2, a gene encoding a membrane
attack/perforin domain found in perforin and complement proteins. Tecp2 is transcribed in pigment
cells and follows an expression pattern similar to other pigment-specific genes such as PKS.
MacpfA2 expression, in contrast, initiates much later at 48 hrs and increases during pluteus
development. The number of MacpfA2 positive cells increases from ~2 cells at 48 hrs to ~10 cells at
96 hrs. At 10 days, expression is localized to cells in the blastocoel, arm tips and apex.
55 Fluorescent protein reporter transgenes in combination with time lapse microscopy have identified
motile globular cells as the specific subset of blastocoelar cells that express MacpfA2. MacpfA2
and additional markers will be used to frame a gene regulatory network model for connecting NSM
specification to downstream mechanisms of immunocyte EMT, proliferation, and terminal
differentiation.
Sea Urchin Embryogenesis is Negatively Impacted by Seawater Acidified via CO2
Enrichment: A Laboratory Exercise for Teaching about the Impacts of Climate Change–
Associated Ocean Acidification
Matthew R. Weeks, Mesrob Yeterian and John Henson
Dickinson College, Carlisle, PA, United States
Computer models of future anthropogenic carbon dioxide atmospheric enrichment and climate
change predict a significant acidification of the world’s oceans and current measurements indicate
that this is already occurring. Several recent studies have suggested that the lowered pH and
increased temperatures of near-future climate conditions negatively impact fertilization and
embryonic development in sea urchins. In this study we piloted an educational lab exercise focused
on the observation of sea urchin larvae reared in either control or CO2-induced acidified seawater.
Eggs and sperm shed from Lytechinus pictus were combined for fertilization under control
conditions and then immediately washed into control (pH 8.0 or 8.1) or CO2 acidified Instant Ocean
artificial seawater (pH 7.8 or 7.6) and then cultured at a low density in airtight T25 tissue culture
flasks (to prevent re-equilibration with the atmosphere) at a temperature of 18-20 degrees C for up
to 5 days. Embryonic development was monitored within the flasks using brightfield, darkfield,
phase contrast, and/or relief contrast microscopy and aliquots of embryos were fixed at various time
points for staining with fluorescent markers for nuclei, the cytoskeleton, and neurogenesis. The
cleavage, blastula and early gastrula stages of the control and acidified embryos appeared similar.
However, the process of skeletogenesis in later gastrula and pluteus larvae was clearly inhibited in
the acidified embryos with a dramatic shortening of the post oral arms and the overall skeleton. We
consider this simple lab exercise to be a powerful tool for teaching students at many levels about
the potential impact of climate change-associated ocean acidification on the embryonic
development of an important marine invertebrate.
Characterization of SpNeurabin, a putative scaffolding protein highly enriched in the sea
urchin egg cortex
Wei Wu1, Lingyu Wang1, Albert Poustka2 and Athula Wikramanayake1
1
Department of Biology, University of Miami, Coral Gables, FL 33124,2Evolution and Development
Group, Max-Planck Institut für Molekulare Genetik, Germany
Molecules localized in the egg cortex regulate many important processes during development
including fertilization, egg activation, and early pattern formation. The sea urchin egg provides an
excellent system to study the various functions of the egg cortex since large numbers of egg
cortices can be easily isolated and analyzed to identify critical regulators of developmental
processes of interest. In order to identify candidate molecules enriched in the egg cortex that may
regulate early pattern formation, we compared mRNAs extracted from isolated cortices with mRNAs
from whole eggs using Illumina next-generation sequencing. RNA-seq data showed that
SpNeurabin mRNA is the most highly enriched message in the egg cortex. In vertebrates, Neurabin
is only expressed in the brain and neural tissue, and it is highly concentrated in the synapses of
neurons and in the lamellipodia of the growth cone. Studies have shown that Neurabin plays
important roles in modulating excitatory synaptic transmission and dendritic spine morphology. To
the best of our knowledge, Neurabin expression has not been documented in non-neural cells, and
56 the function of this protein in these cells in not known. To begin to characterize the roles of
SpNeurabin in the sea urchin egg we carried out fluorescence in situ hybridization (FISH) and
immunofluorescence assay (IFA) analysis. These analyses showed that SpNeurabin mRNA and
protein are enriched in the sea urchin egg cortex. Western blot and IFA analysis showed that
SpNeurabin is expressed during early development. Functional studies are proposed to investigate
the roles of SpNeurabin in the egg and during early embryo development.
Exploring the evolution of developmental mode using transcriptome profiling
Jennifer A. Wygoda1, Maria Byrne2, David R. McClay1 and Gregory A. Wray1.
1
Duke University, Durham, NC, United States; and 2University of Sydney, Sydney, Australia
The ancestral mode of development in Echinoids is indirect development through a planktotrophic
pluteus larva. Heliocidaris tuberculata (HT) employs this strategy, while its sister species H.
erythrogramma (HE) is a direct developer with an abbreviated larval stage. In order to gain insight
into how the ancestral, indirect development program was altered to produce direct development,
we are comparing transcriptome profiles of HT, HE, and the out-group indirect developer L.
variegatus (LV) across several developmental stages through metamorphosis. In the first phase of
this study, we used Illumina RNA-seq to measure the transcriptome profiles of 6 distinct
developmental stages of HE. Ongoing analyses are underway to characterize expression dynamics
associated with the transition from larval to juvenile development in HE, and future studies will
compare these findings to the developmental expression profiles of the indirect developers HT and
LV.
Dissecting the Roles of the Actin Nucleation Facilitators the Arp2/3 Complex and Formin in
Structuring the Peripheral Actin Cytoskeleton in Sea Urchin Coelomocytes.
Mesrob Yeterian, Matthew R. Weeks, and John Henson
Dickinson College, Carlisle, PA, United States
We have used sea urchin coelomocytes as a model experimental system for studying the structure
and dynamics of the peripheral actin cytoskeleton. These disc-shaped cells exhibit extensive actinbased centripetal flow and our previous work has demonstrated a clear role for the actin nucleation
facilitator the Arp2/3 complex in the generation of the dendritic array of branched, short actin
filaments present at the cell edge. In the present study we used two recently developed small
molecule inhibitors to examine the potential distinct roles of the Arp2/3 complex and another actin
nucleation facilitator, formin – crucial for the formation of elongate and unbranched filaments.
These experiments involved live cell imaging, immunofluorescent labeling for Arp3 and actin and
critical point dry and rotary shadow (CPD/RS) transmission electron microscopy. In cells treated
with the Arp2/3 complex inhibitor CK666 the peripheral dendritic actin meshwork was replaced with
arcs of elongate actin filaments oriented parallel to the cell edge that continued to undergo
centripetal flow. Wash out of this drug reestablished the dendritic meshwork pattern of actin
polymerization. In cells treated with the formin inhibitor SMIFHD2 the actin peripheral network was
disrupted with cessation of centripetal flow at higher concentrations of the drug. In cells under the
influence of the Apr2/3 inhibitor the formation and centripetal movement of the actin arcs was
inhibited by addition of the formin inhibitor. Taken together these results indicate that both the
Arp2/3 complex and formin participate in the generation of the peripheral actin array in
coelomocytes. Our current working hypothesis is that formin is involved in the formation of elongate
filaments that in turn can serve as the scaffold for filament branches nucleated by the Arp2/3
complex.
57 Phylogenetic, synthetic, and functional analysis of Gustavus, a key player in Vasa protein
regulation
Vanesa Zazueta-Novoa, Vanesa Adrian Reich, and Gary M. Wessel.
Brown University, Providence, RI, United States
Vasa is a conserved DEAD-box RNA helicase associated with germ line development in many
animal species. Although vasa is not essential for normal physiology of every cell type, it is
essential for oocyte development, posterior pattering, primordial germ cells specification, and
fecundity in adult organisms. In the developing Drosophila oocyte, Vasa protein accumulates at the
posterior pole despite a uniform distribution of vasa mRNA. A regulatory balance between Vasa
synthesis and its degradation through the ubiquitination pathway appears to contribute to this
phenotype. Two paralogous E3 ubiquitin ligase specific receptors were identified in Drosophila, fsn
and gustavus, involved in this regulatory balance of Vasa ubiquitylation controlling pole plasm
accumulation. Vasa interacts with Gustavus through both its N-terminal region and its DEAD-box
domain. Gustavus contains B30.2/SPRY sequence comprising a single structure domain that
interacts with Vasa and a SOCS-box that interacts with ElonginB/C-Cullin 5 complex. This complex
links target proteins to the ubiquitin-proteasome machinery for degradation. In the sea urchin S.
purpuratus, vasa mRNA accumulation is uniform in blastula but Vasa protein is only enriched in the
small micromeres and appears to be limited to these by Gustavus activity. In the starfish, Pateria
miniata, vasa is uniformly distributed during cleavage and is enriched in the endoderm during
gastrulation but then becomes enriched in the posterior enterocoel. Blast analysis using Gustavus
protein sequences from all echinoderms showed orthologs gustavus sequences that define a
conserved SOCs box and a more diverse SPRY domain, the region that interacts with vasa. The
goal of this study is to test if Gustavus is regulating Vasa distribution in starfish and if the
conservation among species, of both SPRY and SOCS sequences, is controlling the specific
interaction with Vasa and ElonginB/C-Cullin 5 complex.
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