Download Insights From a Sea Lamprey Into the Evolution of Neural Crest

Reference: Biol. Bull. 214: 303–314. (June 2008)
© 2008 Marine Biological Laboratory
Insights From a Sea Lamprey Into the Evolution of
Neural Crest Gene Regulatory Network
TATJANA SAUKA-SPENGLER AND MARIANNE BRONNER-FRASER
Division of Biology 139-74, California Institute of Technology, Pasadena, California 91125
Abstract. The neural crest is a vertebrate innovation that
forms at the embryonic neural plate border, transforms from
epithelial to mesenchymal, migrates extensively throughout
the embryo along well-defined pathways, and differentiates
into a plethora of derivatives that include elements of peripheral nervous system, craniofacial skeleton, melanocytes,
etc. The complex process of neural crest formation is guided
by multiple regulatory modules that define neural crest gene
regulatory network (NC GRN), which allows the neural
crest to progressively acquire all of its defining characteristics. The molecular study of neural crest formation in
lamprey, a basal extant vertebrate, consisting in identification and functional tests of molecular elements at each
regulatory level of this network, has helped address the
question of the timing of emergence of NC GRN and define
its basal state. The results have revealed striking conservation in deployment of upstream factors and regulatory modules, suggesting that proximal portions of the network arose
early in vertebrate evolution and have been tightly conserved for more than 500 million years. In contrast, certain
differences were observed in deployment of some neural crest
specifier and downstream effector genes expected to confer
species-specific migratory and differentiation properties.
nervous system and craniofacial skeleton, including the powerful jaws. The appearance of neural crest cells has been
intimately linked to the evolution of predation in vertebrates
and invention of the “new head” (Gans and Northcutt, 1983;
Glenn Northcutt, 2005; Northcutt, 2005). Thus, elucidating
definitive neural crest cells and the regulatory mechanisms
guiding their formation from an evolutionary perspective is
critical to understanding vertebrate origins.
In the embryo, neural crest cells are initially specified
much earlier than originally thought (Basch et al., 2006).
This specification occurs at the end of gastrulation, when the
region that will form the future neural crest encompasses the
border strip of the embryonic ectoderm and is situated at the
interface between the neural and adjacent non-neural territories. During early steps of neurulation, the competence of
the neural crest-forming region (termed “neural plate
border”) to respond to neural crest-inducing signals is
established by a complex set of molecular signals emanating from surrounding tissues (neural plate, underlying
mesoderm, and prospective epidermis). As neurulation
progresses, neural crest progenitors come to lie within the
dorsal aspect of the newly formed neural tube. Though still
not morphologically distinct at this stage, presumptive neural crest progenitors display the combined expression of a
characteristic suite of transcription factors, termed “neural
crest markers”; notable among these are Slug, Sox9, and
FoxD3 (Nieto et al., 1994; LaBonne and Bronner-Fraser,
2000; Sasai et al., 2001; Spokony et al., 2002; Aybar et al.,
2003; Honore et al., 2003; Lee et al., 2004; Yan et al., 2005;
Sakai et al., 2006). Neural crest cells first become morphologically identifiable as they undergo an epithelial to mesenchymal transition (EMT), lose connections to other neuroepithelial cells, delaminate from the neural tube, and
commence extensive migration throughout the embryo
along well-defined pathways. They initiate migration at
different times depending upon the axial levels from which
Properties of the Vertebrate Neural Crest
The neural crest is a migratory multipotent cell population
first identified in the developing chick embryo by Wilhelm His
in 1868 (His, 1868; Hörstadius, 1950). Considered a vertebrate
invention, it gives rise to a plethora of derivatives, contributing
to many defining features of vertebrates such as the peripheral
Received 7 November 2007; accepted 11 March 2008.
To whom correspondence should be addressed: [email protected];
[email protected]
Abbreviations: EMT, epithelial to mesenchymal transition; MO, morpholino oligonucleotide; NC GRN, neural crest gene regulatory network.
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T. SAUKA-SPENGLER AND M. BRONNER-FRASER
they arise, in a process that proceeds in a temporal sequence
from rostral to caudal. Although the cells generally emerge
after neural tube closure, emigration in some species is
initiated from still-open neural folds prior to completion of
neurulation, as is the case in anuran lissamphibians and
mammals (for review, see Baker and Bronner-Fraser, 1997).
Finally they settle in diverse and sometimes distant destinations in the periphery, where they differentiate into a wide
variety of derivatives. These include sensory, sympathetic,
and enteric neurons and glia, constituting the majority of the
peripheral nervous system (PNS); melanocytes (pigmented
cells); as well as cartilaginous, bony and connective tissue
elements of the craniofacial skeleton; smooth muscle tissue
and mesenchyme contributing to the cardiac outflow tract;
endocrine chromaffin cells; and so on (for review, see Hall,
1988; Baker and Bronner-Fraser, 1997; Le Douarin, 1999).
This wide array of diverse derivatives, ranging from neuronal and non-neuronal cell types to mineralized matrices
like bone and dentine, highlights the extensive multipotency
of neural crest progenitors, giving them one of the characteristics of a stem-cell–like population.
Defining the Gene Regulatory Network Underlying
Neural Crest Formation
The formation of neural crest is a multi-step process,
extending over long periods of embryogenesis and spanning
different developmental stages. Whereas the first steps are
undertaken at the end of gastrulation, final differentiation of
some of the derivatives and formation of sensory structures
is accomplished during the terminal steps of organogenesis.
These complex events are orchestrated, both temporally and
spatially, by well-defined regulatory modules. As a result of
the successive sequence of regulatory events, the neural
crest progressively acquires all of its defining characteristics—(1) embryonic origin at the neural plate border; (2)
capacity to undergo an EMT to detach from the neural tube,
change shape, and acquire new sets of cell surface adhesion
molecules and signaling receptors that allow appropriate
responses to guidance cues in the environment; (3) migration to diverse destinations; and (4) differentiation to form
characteristic derivatives. Thus, subdivision of the processes that generate a multipotent bona fide neural crest cell
parallels the network partition into temporarily distinct regulatory steps (Fig. 1A). At each level, these steps involve a
different and sometimes reiterated molecular subset.
The inductive interactions that define a broad territory
containing cells competent to form neural crest first involve
extracellular signaling molecules (e.g., Wnt, BMP, FGF,
Delta) emanating from surrounding tissues (e.g., underlying
mesoderm and prospective epidermis) (Roelink and Nusse,
1991; Hollyday et al., 1995; Saint-Jeannet et al., 1997;
Sasai and De Robertis, 1997; LaBonne and Bronner-Fraser,
Patterning Signals
Patterning Signals
BMP Fgf
Wnt
BMP
Delta/Notch
Msx
Dlx
Delta/Notch
Neural Plate Border Specifiers
Neural Plate Border Specifiers
Pax3/7
Wnt
Pax3/7
Zic
Msx
Dlx
Zic
Early Neural Crest Specifiers
Neural Crest Specifiers
Id
c-Myc
AP-2
Id
c-Myc
AP-2
Snail
Sox8/9/10
Snail FoxD3 Twist
Ets-1
Late Neural Crest Specifiers
FoxD3
SoxE
Twist
Ets-1
Neural Crest Effector Genes
Col2a1
Npn
Cad type I&II
A
Robo
Ngn
Neural Crest Effector Genes
c-Ret
Col2a1
B
Npn
Cad type I&II
Robo
Ngn
c-Ret
Figure 1. Diagram of neural crest gene regulatory network (NC GRN) in gnathostomes (A) and lamprey
(B). The NC GRN is subdivided in spatio-temporally defined regulatory modules, and each step parallels a
different phase in formation of multipotent migratory neural crest cells, as they progressively acquire their
defining characteristics.
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NEURAL CREST GRN IN LAMPREY
1998; Nguyen et al., 1998; Mayor and Aybar, 2001; Endo et
al., 2002; Garcia-Castro et al., 2002; Monsoro-Burq et al.,
2003; Glavic et al., 2004a, b). The action of this initial
regulatory module establishes the neural plate border by
upregulating within it a set of transcription factors including
but probably not limited to Msx1/2, Pax3/7, and Zic family
members (Bang et al., 1999; Luo et al., 2001; Aruga et al.,
2002; Tribulo et al., 2003; Kaji and Artinger, 2004; Monsoro-Burq et al., 2005; Sato et al., 2005; Basch et al., 2006).
Their collective expression in a large subset of the border
cell population confers to these cells a molecular apparatus
that enables them to respond to further bona fide neuralcrest-specifying signals. These genes, termed “neural plate
border specifiers,” form a regulatory module within the
neural crest gene regulatory network (NC GRN) and function in combination with signaling molecules to in turn
upregulate a new subcircuit of transcription factors, termed
“neural crest specifier genes.” These guide the specification
of bona fide neural crest cells. Neural crest specifiers (which
include but are most likely not limited to Snail/Slug, AP-2,
FoxD3, Twist, Id, cMyc, and Sox9/10 transcription factors)
regulate a complex suite of developmental events leading to
acquisition of neural crest fate (Dottori et al., 2001; Sasai et
al., 2001; Spokony et al., 2002; Aoki et al., 2003; Aybar et
al., 2003; Bellmeyer et al., 2003; Honore et al., 2003;
Knight et al., 2003; Barrallo-Gimeno et al., 2004; Lee et al.,
2004; O’Brien et al., 2004; Cheung et al., 2005; Knight et
al., 2005; Light et al., 2005; Taylor and Labonne, 2005; Yan
et al., 2005; Montero-Balaguer et al., 2006). Thus, these
represent a third regulatory module in NC GRN. These
transcription factors act together to regulate and maintain
each other’s expression, in an interlinked and hierarchical
manner the exact nature of which has yet to be defined.
Their ultimate role is to render the neural crest migratory
and multipotent, and they do so by upregulating specific
downstream effector genes. They directly regulate such
downstream targets as small GTPases and cadherins, mediating the onset of the neural crest cell migration by mediating changes in cell shape and adhesion properties. For
example, the zinc finger transcription factor Slug has been
shown to be involved in the neural crest EMT by directly
regulating mediators of cell motility and adhesion (Batlle et
al., 2000; Cano et al., 2000; Nieto, 2002; Taneyhill et al.,
2007). Direct regulatory relationships between this set of
transcription factors and a number of signaling receptors
expressed on migrating neural crest remain to be elucidated.
These receptors, such as Robo, Neuropilin or Eph receptors,
mediate various chemoattractant and chemorepellant cues
present in the environment, setting the neural crest on a
precisely defined migratory path.
Other neural crest specifiers such as Sox9 and Sox10
persist in the postmigratory neural crest, where they regulate
processes of terminal differentiation. For example, Sox10
directly regulates expression of dopachrome tautomerase
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(Dct/Trp2) during melanocyte differentiation (Potterf et al.,
2001; Ludwig et al., 2004) and expression of several myelin
genes like protein zero (P(0)) gene, myelin basic protein
(MBP), or proteolipid protein (PLP) during glial differentiation (Peirano et al., 2000; Stolt et al., 2002). Similarly,
Sox9 has been shown to directly bind to the Col2a1 promoter and in this way control development of neural crestderived cartilage (Lefebvre et al., 1997; Tsuda et al., 2003).
Thus, the neural crest cell population is defined by a
combination of its embryonic origin, change from epithelial
to mesenchymal morphology, acquisition of a migratory
state, multipotency, and molecular markers. In addition,
most neural crest cells have extensive proliferative ability
and well-regulated population size control. Each of the
regulatory modules constituting the NC GRN (Fig.1A) confers one or more of these characteristics to the bona fide
neural crest cell. In other words, the temporal and spatial
developmental sequence of neural crest formation is precisely paralleled by a sequence of regulatory modules
within NC GRN. Signaling and neural plate border modules
reflect establishment of the competence to form neural crest
at the border. Subsequently, different subsets of neural crest
specifier modules define neural crest cell fate through regulation of downstream effectors that control EMT (Slug,
FoxD3, etc.), proliferation and population size (c-Myc, Id
etc.), and migration and terminal differentiation (Sox9,
Sox10), etc.
Neural Crest and Non-Vertebrate Chordates
Traditionally, the neural crest has been considered an
evolutionary innovation of vertebrates, since nonvertebrate
chordates lack bona fide neural crest and are filter-feeders.
A recent study in the colonial ascidian Ecteinascidia identified a population of migratory pigment cells that expresses
some neural crest markers, suggesting that the neural crest
might not have evolved de novo, but from a population of
neural tube cells that possesses a subset of their molecular
and migratory properties (Jeffery et al., 2004). These cells
may be an evolutionary intermediate to the bona fide vertebrate neural crest, possibly utilizing the same gene batteries as partial elements of the NC GRN to regulate migration
and pigment cell differentiation while lacking factors that
maintain multipotency or guide overt proliferation. Consequently, the evolution of neural crest was likely driven by
changes at the gene-regulatory level. These may include
addition of novel interactions, deployment or co-option of
ancestral gene batteries used elsewhere in the embryo, or
invention of new genetic cascades. Any one or some combination of these events could render novel developmental
potential to the regulatory network. However, the genetic
changes that enabled the emergence of the neural crest cell
remain elusive. Current data suggest either that protochordates and vertebrates diverged before the NC GRN evolved,
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T. SAUKA-SPENGLER AND M. BRONNER-FRASER
or in the less parsimonious scenario, that a NC GRN was
lost in the former.
In contrast to urochordates, cephalochordates have retained many features of the basal chordate body plan and are
likely to approximate the ancestor of both urochordates and
vertebrates. At the end of gastrulation in amphioxus, signaling molecules like BMP, Notch, and Wnt are expressed in
a pattern closely resembling that of vertebrates. Furthermore, homologs of neural plate border specifiers Msx, Zic,
and Pax3/7 are present within the neural plate border territory in late gastrula/early neurula, suggesting that the initial
steps of the border patterning and specification are already
present in cephalochordates (Holland et al., 1999; Sharman
et al., 1999; Gostling and Shimeld, 2003). In contrast, no
neural crest specifiers, with the exception of Snail, are
deployed at the neural plate border (Langeland et al., 1998;
Yu et al., 2002; ; Meulemans and Bronner-Fraser, 2002,
2004; Meulemans et al., 2003).
According to recent molecular phylogenies, urochordates
are the sister group to the vertebrates (Delsuc et al., 2006).
In Ciona intestinalis, the only urochordate for which comprehensive functional and gene expression data are available, homologs of neural plate border specifiers Pax3/7, Zic,
and Msx are expressed in the epidermal cells at the lateral
edges of the forming neural plate. Studies in other ascidian
species show homologous border specifier expression patterns (Ma et al., 1996; Wada and Saiga, 2002). Similar to
what happens in amphioxus, lateral neural plate cells express the Ciona homolog of Snail, whereas AP-2 and Id
homologs (EMC) are expressed only in the dorsal epidermis. SoxE is not expressed at early embryonic stages,
whereas FoxD shows limited expression in the nervous
system in the posterior neural plate cells and anterior neural
cord (Wada et al., 1997; Wada and Saiga, 2002; Imai et al.,
2002, 2004; Satou et al., 2003).
Recently, gene interactions were tested in the ascidian
Ciona intestinalis using morpholino-mediated gene knockdown (Imai et al., 2006). Of these, only the activation of
Snail by Zic is reminiscent of the vertebrate NC GRN.
Conversely, other interactions related to later events in
nervous system formation that occur after neural crest induction in vertebrates appear to be conserved between urochordates and vertebrates. For example, inactivation of
Ciona Msxb leads to upregulation of SoxB2, whereas inactivation of FoxD-a/b in dorsal nerve cord precursors results
in depletion of ZicL expression. The latter interaction could
correspond to the regulation of the later onset of genes like
Zic1 and Pax3/7 by neural crest specifiers such as FoxD3,
c-Myc, and Id, as well as neural marker Sox2, within the
roofplate, as suggested by our experiments in lamprey
(Sauka-Spengler et al., 2007). Thus, this interaction could
be a part of an ancient cascade, present in urochordates and
conserved in vertebrates, perhaps involved in specification
of different neuronal subtypes. In contrast, neural crest
specific interactions are absent in Ciona; although homologous genes are present, their function is often in the opposite
direction to that of their vertebrate orthologs. For instance,
Ciona FoxD-a/b acts as a negative regulator of AP2 and Id
(EMC), whereas the zebrafish ortholog, FoxD3, is a positive
regulator of AP2 expression in the neural crest (Imai et al.,
2006; Stewart et al., 2006). Similarly, depletion of AP2 in
Ciona leads to the upregulation of Id (EMC), contrary to
what would be expected for the interaction between these
two specifiers during neural crest induction in vertebrates.
These observations suggest that recruitment of supplementary transcription factor(s) into the regulatory cascade must
have occurred during the transition from protochordates to
vertebrates.
Lamprey as a Model for Studying Neural Crest
Evolution
Since the hypothetical NC GRN does not appear to be in
place in nonvertebrate chordates, critical unresolved questions concerning the evolution of the neural crest remain:
What did the archetypical vertebrate neural crest gene network look like? When did it first appear in the vertebrate
lineage? Is there divergence between jawless and jawed
vertebrates or a shared genetic architecture of the NC GRN
in their last common ancestor?
The degree to which the vertebrate NC GRN is conserved
between vertebrate groups may inform on the basal state of
the neural crest gene regulatory interactions. In fact, functional evidence for different aspects of this putative network
often conflicts between gnathostome models. For example,
knocking down the neural crest specifier genes AP2, FoxD3,
or Sox10, in Xenopus disrupts neural crest induction,
whereas in zebrafish, knock-down of orthologous molecules
impinges on differentiation but does not affect early induction events. Resolving differences between model systems
requires an understanding of the primitive neural crest network and how it may have been altered in different vertebrate lineages.
A successful approach to gaining insight into the level of
conservation, general architecture, and evolutionary history
of a prototypic vertebrate network is to interrogate such a
network in a basal vertebrate. To this end, we opted to test
the evolutionary history of the NC GRN and, thus, vertebrate origins, by investigating the presence of this hypothetical network in the basal-most extant vertebrate, the lamprey. Lampreys are jawless vertebrates (agnathans) and the
most primitive representatives of the cyclostome branch
from which it is feasible to obtain embryos routinely. Recent fossil finds suggest that modern lampreys are “living
fossils,” that strikingly reflect the primitive vertebrate condition and therefore occupy an important position, closely
mimicking characters of the common ancestor of jawless
and jawed vertebrates (Gess et al., 2006; Janvier, 2006). As
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NEURAL CREST GRN IN LAMPREY
a basal vertebrate, lamprey possesses a bona fide neural
crest and many neural crest-derived structures found in
gnathostomes, though they lack several important neural
crest derivatives, including sympathetic chain ganglia
(Johnels, 1956; Butler and Hodos, 1996). DiI-labeling experiments show that lamprey neural crest cells migrate
along pathways similar to those seen in higher vertebrates,
with the exception of the migratory pattern within the hindbrain region (Horigome et al., 1999; McCauley and Bronner-Fraser, 2003). At least one neural crest specifier gene,
SoxE1, has an analogous function in formation of neuralcrest-derived cartilage of the pharyngeal arches (McCauley
and Bronner-Fraser, 2006). However, molecular profiling of
the lamprey neural crest was until recently limited to only a
few scarce examples describing late-migrating neural crest
cells and their derivatives. The data suggested that several
genes were expressed in patterns analogous to those in
gnathostomes (Newth, 1950; Myojin et al., 2001; Neidert et
al., 2001; Meulemans and Bronner-Fraser, 2002; Meulemans et al., 2003; McCauley and Bronner-Fraser, 2003,
2006). However, little or no molecular information about
early steps in neural crest specification in the lamprey was
available, leaving unanswered the intriguing question of
whether the molecular mechanisms of neural crest specifi-
307
cation and their underlying regulatory sequence are similar
or different between jawed and jawless vertebrates.
Neurulation and the Neural Crest Gene Regulatory
Network in Lamprey
To address the question of whether deployment and function of lamprey cognates of gnathostome neural crest genes
are evolutionarily conserved, we performed moderatethroughput transcriptome analysis, identifying more than 50
genes involved in neural crest formation in lamprey. We
have isolated and examined the expression patterns of homologs of patterning molecules from each regulatory module in NC GRN— early signaling pathways, neural-plateborder specifiers, neural crest specifiers, and effector
genes—and then tested functional relationships between a
subset of these genes (Sauka-Spengler et al., 2007).
The expression pattern of lamprey neural crest homologs
was examined from late gastrulation (E4) through early
ammocoete larval stages (Fig. 2). Similar to its development
in fish, the lamprey neural tube is formed as a result of the
secondary neurulation, which involves ectoderm thickening,
followed by cavitation (Damas, 1944; Lowery and Sive,
2004). Lamprey embryos develop very slowly, and the
course of neurulation and early neural crest induction can be
Figure 2. Expression of neural plate border and neural crest specifiers during early neural crest patterning
in lamprey embryos. Dorsal view of E4 and E4.5 lamprey late gastrula/early open neural plate neurula, and
lateral view of E5.5–E7.5 lamprey neurulae; anterior is to the top. DlxB and MsxA share expression in the
ventrolateral ectoderm, but DlxB transcripts are excluded from the neural plate border, whereas MsxA transcripts
are present there. ZicA and Pax3/7 transcripts are present throughout the neural plate and its border, where they
overlap with MsxA expression. At E4.5, initial expression of early neural crest specifiers overlaps with that of
neural-plate-border specifiers within the border territory. AP2 expression spans the non-neural ectoderm plus
neural plate border; Id is found at lower levels in the non-neural ectoderm, and strongly upregulated at the
border; whereas n-Myc is confined to the open neural plate, including neural plate border. AP-2 persists in the
premigratory neural crest at E5.5–E6, where it is co-expressed with late neural crest specifiers FoxD-A and
SoxE1 (white arrowheads) in a pattern complementary to that of the neural marker SoxB1, which labels the neural
tube proper but is excluded from the bulging neural crest (black arrowheads). Id is expressed in the dorsal aspect
of the neural tube/prospective neural crest along the entire anteroposterior axis. Both early (n-Myc) and late
neural crest specifiers (FoxD-A) are expressed by migrating cephalic neural crest in late neurulae (white arrows).
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T. SAUKA-SPENGLER AND M. BRONNER-FRASER
followed in a temporally detailed manner. The neural plate
border is distinct at E4 –E4.5, when the dorsal ectoderm has
thickened along the anteroposterior axis of the embryo and
a shallow neural groove appears in the midline. At E4.5, the
edges of the neural plate (neural folds) are drawn together in
the shape of a rod. By E5, the neural rod is almost entirely
detached from the dorsal epithelial layer, except at the most
posterior level of neuropore. At about E6, the rod cavitates
to form a columnar neural tube, and the head extends. Thus,
the process of early neural crest formation is spread over a
period of more than 2 days and spans the stages of neural
rod thickening (E5), cavitation (E6), and onset of neural
crest migration (E6.5)— encompassing the induction, premigratory, and early migration phases of neural crest formation. Because the neural folds never elevate and form a
morphologically distinct prospective neural crest population
prior to migration, these cells can best be identified by using
a number of genetic markers. With these markers, the cells
are apparent as two independent stripes at the edges of the
condensing neural rod (Sauka-Spengler et al., 2007).
Signals at the interface
Neural crest induction is mediated by a variety of signaling pathways, including Wnt, BMP, Fgf and Notch/Delta,
although no single or combination of signaling molecules
has been shown to function identically across vertebrates.
To examine whether deployment of these genes is conserved in a basal vertebrate, we isolated and examined the
expression pattern of six Wnt homologs: Wnt4 –9. We found
that at least two of them, Wnt6 and Wnt8, are expressed in
a pattern consistent with involvement in the early processes
of neural crest induction. Namely, Wnt6 is expressed in the
neural plate border at the onset of neurulation (E4) and in
the dorsolateral edges of the forming neural tube at E5
(neural crest progenitor region), whereas Wnt8 is absent
from a neural plate border at E4 but at E5 is expressed in the
non-neural ectoderm, directly adjacent to the neural crest
progenitor region.
We also found that two lamprey BMP2/4 homologs,
BMP2/4a and BMP2/4b, exhibit divergent patterns of expression during neurulation, consistent with their proposed
role in neural crest induction in jawed vertebrates. BMP2/4a
is expressed at lower levels throughout the non-neural
ectoderm at E4, with more prominent expression at the
border between the neural and non-neural ectoderm,
highly reminiscent of that reported in Xenopus and zebrafish. In contrast, BMP2/4b is expressed at low levels
in the neural plate itself, including the border territory.
After neurulation, both BMP genes become relegated to
the dorsal neural tube.
The members of the Delta/Notch signaling pathway are
also expressed in the E4 neurula—Delta in a punctate
pattern within the non-neural ectoderm surrounding the
neural plate and Notch ubiquitously present throughout the
embryo, with slightly higher levels in the non-neural ectoderm and the neural plate border.
Expression of signaling molecules (BMP, Wnt, and
Delta) during the first steps of neural crest formation in
lamprey seems to be highly similar to the patterns in jawed
vertebrates, suggesting that signaling cues in lamprey are
present at the proper time and place to have functions in
neural crest specification analogous to those in other vertebrates (Sauka-Spengler et al., 2007).
Neural-plate-border specifiers
The collective expression of neural-plate-border specifiers is thought to uniquely define this territory. In addition to
the border, individual transcription factors that are proposed
to play this role in establishing the competence of the neural
plate border to give rise to the crest are often expressed in
either adjacent non-neural or neural ectoderm as well. Such
factors include the homeobox genes Msx1/2, Pax3/7, and
Dlx3/5, as well as Zic zinc finger transcriptional regulators.
Of the two representatives of the Msx gene family we
isolated, only MsxA is expressed in the early embryo. In the
early neurulae (E4), MsxA is found at the neural plate border
and in the presumptive epidermis; it is then downregulated
in the presumptive neural fold territory (E5) and again
expressed in the most dorsal aspect of the neural tube after
its formation (E6 – 6.5).
In the early neurulae (E4), the lamprey ZicA homolog is
expressed throughout the neural plate, overlapping MsxA at
the neural plate border. With time, its expression is restricted to the dorsal aspect of the forming neural rod and
subsequently to the neural tube along the entire anteroposterior axis. The ZicA- and MsxA-positive border domain also
expresses Pax3/7 and abuts Dlx-positive non-neural ectoderm. While MsxA and DlxA/DlxB genes are downregulated
in the E5 neurula, Pax3/7 and ZicA expression persists in the
dorsal edges of the forming neural rod throughout neurulation. Later, all these genes are co-expressed in the dorsal
neural tube, but they are not observed in migrating neural
crest cells. The expression patterns of MsxA, ZicA, Dlx, and
Pax3/7 genes are found within the neural plate border and
adjacent territories in lamprey, implying that their cooperative presence in this territory is highly conserved in the
early neurula across all vertebrates (Sauka-Spengler et al.,
2007).
To interrogate functional relationships between the components of the network, an antisense morpholino oligonucleotides (MO) approach was used to individually block
translation of three neural-plate-border specifiers (MsxA,
Pax3/7, ZicA). The effect of these perturbations was assessed by in situ hybridization with probes against neural
crest specifier genes (markers of morphologically recognizable bona fide neural crest cells, such as SoxE or FoxD
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NEURAL CREST GRN IN LAMPREY
309
Figure 3. FITC-tagged anti-sense morpholino oligonucleotides (MOs), targeted against neural plate border
and neural crest specifier genes are injected into a single blastomere at the 2-cell stage. Embryos with unilateral
distribution of labeled MOs were selected and analyzed for expression of neural crest markers. FoxD-A
expression was absent in lamprey E5.5 and E6 neurulae after MO-mediated protein knockdown of ZicA and
MsxA (black arrows). The uninjected side serves as an internal control.
family members), but also against other border specifiers
expressed in the dorsal neural tube at the time when neural
crest progenitors emerge (Fig. 3). The depletion of the
neural plate border specifiers Pax3/7, MsxA, and ZicA generally alters the expression of other border specifiers (for
instance, often Pax3/7 and ZicA domains in the neural tube
are expanded). Most importantly, the expression of neural
crest specifiers, FoxD-A and SoxE1, markers of the premigratory neural crest, is markedly reduced (Fig. 3). The high
penetrance of this phenotype in the early emerging neural
crest indicates that altering the crest-competence of the
border territory results in a very reduced bona fide neural
crest population. The overall neural crest population appears diminished and, despite their compensation ability,
the remaining neural crest cells are often unable to make up
for the defect. Accordingly, MO-depleted embryos, lacking
neural plate border specifiers, Pax3/7, ZicA, and MsxA,
show defects in the expression of markers of the migrating
and postmigratory neural crest as well as the defects in
neural crest derivatives and related structure, even after
10 –12 days of development. Our finding that inactivation of
border specifiers MsxA, Pax3/7, and ZicA results in depletion of neural crest specifier expression is consistent with
results obtained in similar experiments in other vertebrates,
suggesting that the more proximal modules of the NC GRN
are highly conserved among jawless and jawed vertebrates
(Sauka-Spengler et al., 2007) (Fig.1B).
Neural crest specifiers
Neural plate border specifiers are thought to upregulate
neural crest specifiers like c-Myc, AP2, Id, Snail, FoxD3,
and SoxE family members, often referred to as “neural crest
markers” since they are expressed by bona fide neural crest
progenitors. These transcription factors are thought to confer delaminating ability and migratory guidance as well as
to specify early cell fate identities. In addition to their
expression in premigratory neural crest, some of these genes
are maintained in the late-migrating and post-migratory
crest cells, directing their ultimate differentiation. In this
respect, the neural crest specifier genes differ from border
specifiers: not only do they tend to be retained in the
migrating neural crest population, but they are also generally thought to function after formation of the crest-competent neural plate border. Interestingly, lamprey neural crest
specifier genes exhibit a biphasic mode of expression: a
subset of them are expressed early in the neural plate border,
while others are found only in the bona fide neural crest
progenitors of the E5.5–E6 neurulae and later in the migrating/differentiation crest.
Early neural crest specifiers, such as AP-2, Id, Snail, or
c-Myc/n-Myc, initiate expression in the E4.5 lamprey neurulae, shortly after the onset of neural plate border specifiers, MsxA, ZicA and Pax3/7, and their expression domains
overlap in the neural plate border. AP-2 and Id are observed
in the non-neural ectoderm and the neighboring border,
similar to their patterns in other vertebrates. Later, they are
restricted to the dorsal neural tube and migrating neural
crest cells (Meulemans and Bronner-Fraser, 2002; Meulemans et al., 2003; Sauka-Spengler et al., 2007). In contrast
to AP-2 and Id, early onset of the expression of n-Myc and
Snail is confined to the neural plate and encompasses the
neural plate border. n-Myc is then maintained in the dorsal
aspect of the neural tube and in neural crest, whereas Snail
expression is expanded broadly to many tissues, including
the dorsal neural tube, neural crest, and mesoderm (SaukaSpengler et al., 2007).
Conversely, late neural crest specifiers in lamprey, such
as FoxD-A and SoxE family members, are upregulated in
either pre-migratory (FoxD-A, SoxE1 and SoxE3) or migrating neural crest cells in the head (SoxE2) and trunk (SoxE2
and SoxE3). Subsequently, they are maintained in different
subsets of neural crest derivatives. In E6.5 neurulae, these
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310
T. SAUKA-SPENGLER AND M. BRONNER-FRASER
factors are co-expressed with n-Myc, AP2, Snail, and Id in
streams of cranial neural crest and early-migrating trunk
neural crest cells. Broad co-expression of neural crest specifiers is less apparent in late-migratory and post-migratory
neural crest as their deployment becomes restricted to particular neural crest subpopulations. FoxD-A, for instance,
strongly labels cranial ganglia in the head and dorsal root
ganglia in the trunk, but is downregulated in chondrogenic
cranial neural crest at E12, whereas n-Myc persists in pharyngeal neural crest where it is co-expressed with SoxE
genes and homologs of Col2a1 (McCauley and BronnerFraser, 2006; Zhang et al., 2006; Sauka-Spengler et al.,
2007).
Co-option of Ets1 and Twist as neural crest specifiers in
higher vertebrates?
In contrast to the expression of Twist and Ets1 in jawed
vertebrates, where they are present in the first bona fide
neural crest progenitors, their lamprey orthologs are expressed only in late-migratory and post-migratory cranial
neural crest (Hopwood et al., 1989; Meyer et al., 1997;
Linker et al., 2000; Remy and Baltzinger, 2000; Tahtakran
and Selleck, 2003; Sauka-Spengler et al., 2007). In jawed
vertebrates, these two transcription factors play important
roles in early specification of neural crest progenitors. In
contrast, in lamprey they are confined to subpopulations of
early-differentiating cells within and lining the branchial
arches, and they persist in the mesenchyme and form buccal
cartilage. Thus, it is likely that these genes have been
co-opted to an earlier function in the gnathostome lineage or
have lost their early specification function in lampreys. This
represents an interesting divergence from other members of
the neural crest specifier regulatory module.
In lamprey, as in jawed vertebrates, inactivation of early
(c-Myc/n-Myc, Id family members or AP-2) and late
(FoxD-A and SoxE representatives) neural crest specifiers
generally affects the expression of all neural crest specifiers
(Spokony et al., 2002; Bellmeyer et al., 2003; Honore et al.,
2003; Kee and Bronner-Fraser, 2005; Light et al., 2005;
O’Donnell et al., 2006; Sauka-Spengler et al., 2007). The
few exceptions occur in zebrafish mutants that sometimes
do not show alteration in expression levels of pre-migratory
and early-migrating neural crest markers, but instead
present defects in selective neural crest derivatives. This
discrepancy is most likely due to the tetraploidy of the
zebrafish genome and compensation by redundant paralogs
(Luo et al., 2001; O’Brien et al., 2004; Yan et al., 2005;
Lister et al., 2006). The rapidly accumulating data from gain
and loss of function experiments suggest that, within their
regulatory module, the neural crest specifiers cross-regulate
extensively to maintain one another’s expression. However,
precise hierarchical relationships among them are still difficult to assign. In lamprey, late neural crest specifiers
(SoxE, FoxD-A) do not seem to control expression of neural-plate-border specifiers when co-localized in the dorsal
neural tube at late neurula stages (E6 –E6.5); similarly, the
inactivation of early neural crest specifiers, such as AP-2
and n-Myc, unlike the inactivation of border specifiers,
causes the expansion of Pax3/7 and ZicA within this territory (Sauka-Spengler et al., 2007). Conversely, it is not
clear if early neural crest specifiers, when co-localized with
neural plate border specifiers at E4.5, during the early steps
of establishment of the border crest-competence, regulate
their expression. The possible existence of the regulatory
feedback loops from early neural crest specifiers to neural
plate border specifiers, which most likely induce their expression initially, remains to be investigated. When direct
temporal interactions are more clearly elucidated, the complex connections between the transcription factors specifying the neural plate border and neural crest will likely need
to be re-visited.
In contrast to depletion of border specifiers, long-term
deficiencies in neural-crest-derived structures are less apparent after the loss of function of neural crest specifiers.
Less severe global reductions of neural crest derivatives in
lamprey embryos injected with MOs against neural crest
specifiers are likely to reflect more efficient regulation and
regeneration of the pool of neural crest progenitors by
neighboring precursors. In contrast, depletion of neural
plate border specifiers appears to largely remove the precursor pool. In neural crest specifier morphants, the neural
plate border has already been properly defined and the
initial pool of progenitors established. Thus, redundant
mechanisms responsible for maintenance of the neural crest
population may be sufficient to attenuate and repair longterm effects on neural crest derivatives.
Neural crest effector genes in lamprey
Between lamprey and jawed vertebrates, there are interesting differences in deployment of downstream neural crest
effector genes that function to render the neural crest migratory and multipotent. This is not surprising since different paralogs of effector genes are deployed at different
times and places among different gnathostome models,
likely conferring some species-specific properties. Nevertheless, some general similarities can be ascribed. As in
higher vertebrates, lamprey neural crest specifiers seem to
turn on specific downstream effector genes, such as Type I
and II cadherin representatives; signaling receptors expressed on premigratory and migrating neural crest cells,
such as Neuropilin and Robo1; neurogenic markers such as
Neurogenin; and several collagen molecules, including
Col2a1 (Sauka-Spengler et al., 2007).
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NEURAL CREST GRN IN LAMPREY
Evolutionary Changes to Establish Neural Crest
The detailed comparative expression and functional analysis of the putative neural crest gene regulatory network
(NC GRN) in lamprey shows that most regulatory modules
responsible for bona fide neural crest specification, migration, and differentiation are in place in the representative of
a basal vertebrate lineage. This suggests that these elements
were fixed early in vertebrate evolution. Rescue experiments using co-expression of morpholino nucleotides and
heterospecific messenger RNA (Xenopus and chick Zic1,
Msx1, AP-2, Pax3, Id, and Sox9) demonstrate that homologous proteins from jawed vertebrates can functionally
compensate for the loss of their lamprey orthologs. This
result implies that the protein structure of these transcription
factors has been sufficiently conserved during vertebrate
evolution to be interchangeable in the context of neural crest
induction (Sauka-Spengler et al., 2007).
Evolution of the neural crest was therefore most likely
driven by gene-regulatory changes that may have included
recruitment of additional transcription factors into the regulatory sequence, but also by co-option and deployment of
ancestral gene batteries in a new context. Interestingly,
deployment of the more proximal signaling and neuralplate-border regulatory modules of the NC GRN appears to
be present in protochordates. This suggests an ancestral
presence of the proximal portion of the network prior to the
protochordate/vertebrate split. However, the regulatory connections to neural crest specifiers are clearly lacking, perhaps accounting for the absence of bona fide neural crest
cells in this branch. These results suggest that ancient invention of NC GRN likely occurred during the early Cambrian, within the estimated 200 million years of transition
from protochordates to vertebrates (Meulemans and Bronner-Fraser, 2005; Sauka-Spengler and Bronner-Fraser,
2006; Sauka-Spengler et al., 2007).
Present evidence suggests that the gene regulatory mechanisms guiding formation of neural crest are a vertebrate
synapomorphy. They represent a case of a conserved gene
regulatory network functioning during development of the
vertebrate body plan, which contains at least one “kernel”
(Davidson and Erwin, 2006). While the evolutionarily inflexible neural-plate-border regulatory module is found in
all chordates and plays a role in establishing border identities, its presence alone is insufficient to mediate formation
of a bona fide neural crest. The incorporation of the neural
crest specifier module or modules into the network most
likely led to the vertebrate innovation of the neural crest
kernel interconnecting the two parts—the neural plate border and neural crest specifier modules. Their intricate connections still remain to be elucidated, and will inform on the
detailed regulatory changes that gave rise to vertebrate
neural crest as found today. Sequencing of the lamprey
genome is ongoing. The emerging genomic information
represents a valuable tool that will greatly aid in investigating prospective regulatory regions. This will help to further
characterize regulatory relationships within the neural crest
gene regulatory network and assess direct interactions
therein. Other elements of the network represent “plug-ins”
that could provide signaling inputs (Wnts) or guidance cues
(Neuropilin or Robo signaling cues) and “switches,” possibly co-opted from existing developmental programs to provide regulative mechanisms of cell-cycle, proliferation, and
population-size control (c-Myc/Id battery, for example).
Gene differentiation batteries, which operate in the more
distal downstream modules of the NC GRN, could have
been co-opted from ancient programs for cell differentiation
(i.e., regulation of Trp-2 by Sox10 in melanocyte lineage or
Col2a1 by Sox9 in chondrogenic lineage) or could involve
gaining novel interactions. The data hence suggest that,
although a neural crest gene network was largely fixed at the
base of vertebrates, the possible divergences of more distal
modules could be responsible for establishment of distinct
species-specific traits.
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