Download Where do referees go

Research Interests - David R. Shook
Integrated Analysis of Morphogenic Machines
In much the same sense that conserved developmental genes, like the HOX genes, pattern
early embryos, there are a conserved set of morphogenic processes or “machines” that shape
their basic body plan. These morphogenic machines (coordinated cell behaviors within a
tissue that cause a shape change or reorganization of the embryo) are of fundamental
importance to understanding development and the basis for variation in development across
species. However, there is currently only a very poor understanding of the cellular
mechanisms driving these machines, and of their contextual dependence on embryonic
architecture (the physical organization and biomechanical properties of the embryo (reviewed
in Keller et al., 2003)). I am therefore investigating the morphogenic machines driving
morphogenesis at the tissue, cell and molecular levels, and characterizing variations in these
machines among species. My focus is on the machines driving morphogenesis during
gastrulation and neurulation in amphibians. My long-term goal is to develop a better
understanding of general morphogenic design principles that can be broadly applied across
the chordates and other metazoans to explain the evolutionary basis for changes in
morphogenesis.
Morphogenesis depends on several embryonic properties, including molecular patterning,
cellular processes and force-generating mechanisms, and embryonic architecture. To gain
insights into the relationships between these properties I use a comparative approach to find
variations in the type or details of the morphogenic machine used for a particular task. I then
study the properties associated with these variant machines in detail, and compare them to
those for already well characterized machines, such as those in the frog Xenopus laevis, to
understand the basis of the variations. This is a powerful approach, similar to a genetic one, in
that I can discern components of morphogenesis and ask about their functional relationship.
Because I look at several levels of organization, my results integrate genetic, epigenetic and
evolutionary components. My comparative approach is complementary to a genetic approach,
in that I can look at variations in the expression and function of candidate genes for
associations with variations in morphogenic machines, and as I do so, provide mechanistic
explanations for the effects of genetic disruptions. My approach also allows me to investigate
the basis of variations in cell behaviors that may be cryptic, or otherwise difficult to study in
traditional model systems, and will provide a better understanding of the range of cell
behaviors that cells are capable of. Understanding how variations in molecular and cellular
pathways and embryonic architecture can produce these different cells behaviors will provide
a better understanding of how cells can transition between these different behaviors, both
evolutionarily and pathologically.
The remarkable conservation of patterning genes that specify the form of developing
metazoan embryos is at odds with our observation that different species of amphibians show
substantial variation in the morphogenic machines they use to produce a tadpole (Keller and
Shook, 2004). At some point down stream of the patterning genes there must be a divergence
in the regulatory pathways of different species such that they direct cellular processes
differently. Our understanding of morphogenic machines and morphogenic diseases depends
on inferences from model systems; thus learning how homologous pathways diverge has
strong relevance for the accuracy of those inferences. Studying the cell biological basis for
1
variation in diverse morphogenic machines thus has strong biomedical relevance, e.g. in terms
of understanding cell behaviors like metastasis. Finding treatments for most diseases, which
have complex causes involving the interaction of components over many levels of
organization, will require a better understanding of such interactions, and the possible
outcomes of variations in them.
Gastrulation in amphibians is a great arena in which to study cell behaviors. Amphibians
are one of the most varied groups in terms of reproductive strategies, and thus of embryonic
architecture. This in turn is strongly correlated with variations in gastrulation mechanisms.
My research focuses on variations in force producing mechanisms driving gastrulation. The
large size of amphibian cells allows me to easily visualize both the localization of components
within cells, as well as the behaviors and movements of the cells themselves. Visualization of
cell movements and morphogenic machines is further facilitated because amphibian embryos
are uniquely amenable to microsurgery and explants survive well in culture, making it easy
for me to image otherwise hidden regions of the embryo (see movies 13_1 to 13_14 at
http://www.gastrulation.org). I can also compare the operation of these machines in isolation
by explanting tissues, or in novel contexts, by transplanting them. I can compare machines in
the context of different embryonic architectures or different complements of other machines by
looking at similar machines operating in different species. Looking at a morphogenic
movement in different contexts allows me to discern individual machines and understand
their dependence on context, to understand which machines actually produce specific
morphogenic movements. The system I have developed to look at cell behaviors and force
production by subduction allows me to take advantage of all these features.
Current Work: I am currently investigating the cellular mechanism, biomechanics and
patterning of a force producing morphogenic machine, “subduction”, that drives gastrulation
in the salamander Ambystoma mexicanum. Subduction involves apical constriction, ingression
and epithelial to mesenchymal transition (EMT) just inside the blastopore, adjacent to the
vegetal endoderm (Shook et al., 2002). Subduction resembles primitive streak ingression in
amniotes (Shook and Keller, 2003) and is very different from the mediolateral cell intercalation
behavior (MIB) used by the frog, Xenopus laevis (Keller et al., 2000). In collaboration with Lance
Davidson, I have developed a force measuring assay and have shown that subduction is a
force producing machine. I have also shown that it is dependent on a myosin II based
contraction mechanism, probably in the apical cortex of apically constricting cells, and that
actin is preferentially localized to the apical cortex of cells entering the region where
subduction behaviors begin. In collaboration with Bill Bement, I am using a probe for
activated RhoA to visualize the activation pattern of the force producing mechanism in live
explants undergoing subduction.
Subduction occurs in the same spatial and temporal progression during gastrulation as
MIB in Xenopus, to produce similar biomechanical forces to achieve the same morphogenic
end, namely involution and blastopore closure. This suggests that I have an example of a
homologous upstream patterning pathway, which somehow diverges to produce different cell
behaviors. To understand both the initial patterning and the divergence of the downstream
regulatory pathway, I am investigating the specification of superficial presumptive
mesodermal cells to learn how and when they are directed to ingress and undergo EMT. I
have demonstrated that the apical constriction and EMT associated with subduction occur
progressively, beginning at one edge of the presumptive mesoderm, and appears to be pre2
patterned, as it occurs in explants with the normal time course even when portions of the
tissue that would go through subduction earlier are removed (Shook et al., 2002). Whereas
Ambystoma ingresses most of its large field of superficial presumptive mesoderm during
gastrulation, X. laevis ingresses its few superficial presumptive mesodermal cells during
neurulation (Shook et al., 2004). This delay suggests a change in the timing of mesodermal
specification and differentiation, e.g. to ingress and go through EMT in these cells. To
determine which genes direct these behaviors, I am testing candidates likely to be involved,
based on work in Xenopus and other systems. In Xenopus, these cells express the mesodermal
marker Xbra, rather than the endodermal marker Xsox17, expressed by the adjacent epithelial
cells, prior to ingressing (my unpublished results). I am working to determine when
mesodermal marker expression begins in the ingressing cells, and to determine the expression
of the A. mexicanum homologs in subducting cells of that species. In X. laevis, the EMT
regulator Xsnail appears to be expressed in the ingressing cells (Essex et al., 1993). I am
therefore also looking at the expression of Snail of in cells fated to subduct in A. mexicanum, in
collaboration with Dr. Marianne Bronner-Fraser. Differences in expression of these and other
candidate genes that are correlated with the timing of ingression in the two species will
suggest involvement in its regulation, which can be tested using antimorphic genetic reagents
(e.g. dominant negative constructs and antisense technologies).
Comparing the morphogenic machines that close the blastopore in different species has
brought to light a previously poorly understood morphogenic machine and provided a robust
model system in which to study it. X. laevis is thought to use a robust anisotropic (dorsally
focused) axial convergence and extension machine to close its blastopore during gastrulation,
but the use of dorsally focused convergence and extension is delayed until after gastrulation in
the marsupial frog, Gastrotheca riobambae (del Pino, 1996), and is used only weakly during
gastrulation in other anurans, such as the dart frog Epipedobates tricolor and the direct
developing frog Eleutherodactylus coqui (Shook & Elinson, unpublished results). E. tricolor, E.
coqui and G. riobambae instead use a robust “isotropic convergence” machine that converges
equally around the blastopore, leading to isotropic blastopore closure without substantial axial
extension (Shook, unpublished results), a behavior that is only cryptically expressed in X.
laevis gastrulae (Keller and Danilchik, 1988). Isotropic convergence may be an ancestral
mechanism for amphibians, and perhaps chordates generally. I am therefore collaborating
with Dr. Eugenia del Pino to continue characterizing morphogenesis in G. riobambae and E.
tricolor, and with Dr. Rick Elinson in E. coqui, to develop a model system to study the cell
behavioral basis of the isotropic convergence machine, and with Dr. Chris Lowe to look at
isotropic convergence in hemichordate gastrulation. I will also look more closely at X. laevis to
understand how it works there.
Planned Research: I will continue the work begun in the Keller lab, and expand to other
morphogenic machines and amphibian species as the size of my lab expands. My specific aims
reflect the three aspects of my work: determining the cellular mechanisms underlying the cell
behaviors driving morphogenic machines and the biomechanical effects they produce;
understanding the specification and patterning of cell behaviors; and exploration of further
phylogenetic variations in the morphogenic machines used to drive morphogenic movements.
Specific Aims:
3
1) Determine the cellular basis of superficial presumptive mesoderm EMT and of the
associated force producing behavior, using subduction in Ambystoma mexicanum as a model
system. I will: A) test the role of likely candidate molecules (e.g. apical junctional
components, actin, myosin, Rho GTPases) in cell behaviors and force production, using
specific pharmacological and antimorphic genetic inhibitors; B) characterize the localization
of these molecules, using live imaging of GFP or fluorophore tagged variants of these
molecules to observe the dynamics of their expression.
2) Investigate the specification and patterning of the superficial cells of the marginal zone in X.
laevis and A. mexicanum to understand: A) how superficial presumptive mesoderm is
specified differently than the adjacent superficial endoderm; B) how superficial mesoderm is
specified to ingress and undergo EMT, and to do so at the right time and place. I will
initially compare the spatial and temporal expression of candidate patterning genes or other
markers of mesodermal and endodermal fate between species with different layouts of
superficial mesoderm, and/or different timing of ingression. I will test the role of those
expressed in the right time and place with antimorphic techniques.
3) Compare species with diverse gastrulation movements, initially using embryological
approaches, to identify novel morphogenic machines. By comparing the effects of machines
in different contexts, or species that use different sets of machines at a specific stage, I can
determine the biomechanical forces and resulting morphogenic distortions produced by each
machine. A valuable by-product of this comparative approach is the discovery of species
with especially robust versions of a particular machine, species that can be used as model
systems for the detailed study of the cell biology and patterning controlling that machine, as
above.
Funding: I have recently submitted my initial RO1 NIH grant application (10/1/04). The
grant is focused on the cell biological and patterning aspects of my work (Specific Aims 1 and
2). The ACS may also be interested in funding my work on ingression and EMT. Once I have
secured NIH funding, I will acquire NSF funding for my more explicitly comparative
evolutionary research (Specific Aim 3).
My first attempt at grant writing, for postdoctoral funding (NRSA) for my work on
superficial presumptive mesoderm, was funded by the NIH with a high score. I have been
successful in convincing review panels that my approach, described above, has relevance from
the biomedical and human health perspective. The importance of my work with Dr. del Pino
was recognized by the editors of Development, who awarded me a travelling fellowship to set
up my initial collaboration with her in Ecuador. I have participated in writing several RO1s in
the Keller lab, all of which have been funded. I am confident that I will be able to obtain
further funding for my unique, innovative and highly integrated line of research, addressing
as it does fundamental questions about the mechanisms and evolution of morphogenesis,
including questions of basic biomedical interest, such the mechanisms of cell motility,
ingression and EMT.
References
del Pino, E. M. (1996). The expression of brachyury (T) during gastrulation in the marsupial
frog Gastrotheca riobambae. Developmental Biology 177, 64-72.
4
Essex, L. J., Mayor, R., and Sargent, M. G. (1993). Expression of Xenopus snail in mesoderm
and prospective neural fold ectoderm. Developmental Dynamics 198, 108-22.
Keller, R., and Danilchik, M. (1988). Regional expression, pattern and timing of convergence
and extension during gastrulation of Xenopus laevis. Development 103, 193-209.
Keller, R., Davidson, L., Edlund, A., Elul, T., Ezin, M., Shook, D., and Skoglund, P. (2000).
Mechanisms of convergence and extension by cell intercalation. Philosophical Transactions of
the Royal Society of London Series B: Biological Sciences 355, 897-922.
Keller, R., Davidson, L. A., and Shook, D. R. (2003). How we are shaped: The biomechanics of
gastrulation. Differentiation 71, 171–205.
Keller, R., and Shook, D. (2004). Gastrulation in Amphibians. In “Gastrulation: From Cells to
Embryo” (C. D. Stern, Ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Shook, D., and Keller, R. (2003). Mechanisms, mechanics and function of epithelialmesenchymal transitions in early development. Mechanisms of Development 120, 1351-1383.
Shook, D. R., Majer, C., and Keller, R. (2002). Urodeles remove mesoderm from the superficial
layer by subduction through a bilateral primitive streak. Developmental Biology 248, 220-239.
Shook, D. R., Majer, C., and Keller, R. (2004). Pattern and morphogenesis of presumptive
superficial mesoderm in two closely related species, Xenopus laevis and Xenopus tropicalis.
Developmental Biology 270, 163-185.
5