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| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online December 7, 2008
doi: 10.1242/10.1242/dev.023564
Review |
Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.
e-mail: ettensohn{at}andrew.cmu.edu
SUMMARY
Significant new insights have emerged from the analysis of a gene regulatory network (GRN) that underlies the development of the endoskeleton of the sea urchin embryo. Comparative studies have revealed ways in which this GRN has been modified (and conserved) during echinoderm evolution, and point to mechanisms associated with the evolution of a new cell lineage. The skeletogenic GRN has also recently been used to study the long-standing problem of developmental plasticity. Other recent findings have linked this transcriptional GRN to morphoregulatory proteins that control skeletal anatomy. These new studies highlight powerful new ways in which GRNs can be used to dissect development and the evolution of morphogenesis.
Introduction
To understand how development is encoded in the genome, biologists are
turning increasingly to system-level approaches. The concept of
transcriptional gene regulatory networks (GRNs) is proving to be a powerful
one in this context. GRNs are ensembles of genes that encode transcription
factors (TFs) and the genes that these proteins regulate. A central component
of GRN analysis is the dissection of the cis-regulatory control systems of
genes. Cis-regulatory systems consist of non-coding DNA sequences that control
when and where genes are transcribed. They are often viewed as modular,
information-processing systems (Davidson,
2006
). GRN analysis attempts to identify not only the functional
interactions among genes, but also the relevant cis-regulatory DNA sequences,
the proteins that bind to these sequences, and the logic by which
cis-regulatory systems control gene transcription.
The GRNs that operate during embryonic development (developmental GRNs) are
highly dynamic. New interactions between genes are continually established as
old interactions are modified or discarded. Inputs from cell signaling
pathways, and intrinsic properties of regulatory networks themselves,
contribute to the dynamic nature of GRNs (see
Davidson, 2006). The genomic
regulatory states of embryonic cells, which are a reflection of the
concentrations and activities of hundreds of TFs and the global patterns of
gene activity they evoke, are thus ever changing.
This review considers recent studies that have applied the GRN concept in
new and informative ways to examine developmental plasticity (that is, the
ability of embryonic cells to switch developmental pathways), morphogenesis,
and the evolution of developmental programs. It focuses on a GRN that controls
skeletogenesis in sea urchins, a group of animals belonging to the phylum
Echinodermata that has proven to be particularly useful for the analysis of
GRNs in early development. GRNs that underlie cell specification are presently
understood in greater detail in the sea urchin than in any other metazoan
embryo, although work is ongoing in several other experimental models
(Koide et al., 2005;
Stathopoulos and Levine, 2005;
Ge et al., 2006;
Satou et al., 2008). For
general reviews of GRNs in early sea urchin development, and of the methods
used to construct and represent GRNs, see Oliveri and Davidson
(Oliveri and Davidson, 2004)
and Ben-Tabou de-Leon and Davidson
(Ben-Tabou de-Leon and Davidson,
2007).
The skeletogenic GRN in sea urchins
In the sea urchin (as in most metazoan embryos), maternal polarity entrains
early patterning (Brandhorst and Klein,
2002; Angerer and Angerer,
2003). Zygotic transcription begins very soon after the egg is
fertilized and reaches a maximal rate during early cleavage. By the 16-cell
stage, different programs of gene expression are already deployed in the
different tiers of blastomeres that are organized along the animal-vegetal
(AV) axis. The late blastula is a mosaic of distinct territories, each of
which is delineated by the domains of expression of many representative genes
(Fig. 1). In most cases, the
different territories of the blastula are not strictly associated with early
cell lineage compartments and their boundaries are not rigidly fixed. GRNs are
currently being developed for many of the early embryonic territories shown in
Fig. 1, although their level of
completeness varies.
The PMC GRN in euechinoid sea urchins: new components, new connections
At present, the best understood GRN in the sea urchin, and probably the
best understood GRN in any embryo, is the network that underlies the
specification and differentiation of skeletogenic cells in euechinoids.
Euechinoids are the largest subclass of modern sea urchins, and this subclass
includes all of those species commonly used for developmental research,
including the purple sea urchin Strongylocentrotus purpuratus, for
which a high-quality genome assembly is available
(Sea Urchin Genome Consortium,
2006). Sea urchins and other echinoderms produce an elaborate
endoskeleton (an internal skeleton) composed of calcite, a form of calcium
carbonate (for more on skeletogenesis in different echinoderms see
Box 1). The morphogenesis of
the embryonic skeleton of euechinoid sea urchins has been a subject of study
for many decades; in part because the skeleton is a highly ordered, beautiful
structure, and because it can easily be visualized in living embryos
(Wilt and Ettensohn,
2007).
The embryonic skeleton of euechinoids is produced by a specialized population of skeletogenic cells called primary mesenchyme cells, or PMCs (Fig. 2). PMCs are derived from four small cells known as micromeres, which are located at the vegetal pole of the 16-cell stage embryo. Among the echinoderms, only sea urchins produce micromeres, which are therefore considered to be a relatively recent invention (see discussion below and Box 1). Each micromere divides unequally, producing a large daughter cell (large micromere) that later gives rise to PMCs, and a small daughter cell (small micromere), which adopts a different fate. PMCs undergo a sequence of striking morphogenetic behaviors that includes epithelial-mesenchymal transition, directional migration, and cell-cell fusion. During gastrulation, they secrete a bilaterally symmetrical pair of skeletal primordia on the oral (ventral) aspect of the blastocoel wall, in close association with specialized ectodermal territories (Fig. 2A). The skeletal rudiments elongate and branch in a characteristic manner and, by the time the early larva begins to feed, a complex, branched network of skeletal rods supports its angular body (Fig. 2B,C).
|
).
β-Catenin is an essential early activator of the micromere-PMC GRN and is
likely to act exclusively and directly via activation of pmar1
(paired-class micromere anti-repressor 1), which encodes a
transcriptional repressor of the homeodomain family
(Kitamura et al., 2002;
Oliveri et al., 2002;
Oliveri et al., 2003;
Nishimura et al., 2004;
Yamazaki et al., 2005).
Although β-catenin protein is present throughout the vegetal region of
the embryo during early cleavage, it activates pmar1 only in the
micromeres, by mechanisms that are not understood. Because Pmar1 functions as
a transcriptional repressor, it was postulated that activation of the
micromere-PMC GRN might be mediated by a double-repression mechanism
(Oliveri et al., 2002). In
support of this view, a recent screen for Pmar1-repressed genes identified
hesC (hairy/Enhancer-of-split C), which encodes a
transcriptional repressor
(Revilla-i-Domingo et al.,
2007). HesC mRNA is ubiquitous in the early embryo, but
is downregulated in vegetal cells, including in the presumptive PMCs.
Morpholino (MO)-mediated knockdown of HesC leads to excessive numbers of
mesenchymal cells and to the upregulation of several early genes in the
network. These findings indicate that a double-repression `gate' mediated by
pmar1 and hesC activates the skeletogenic GRN. The fact that
this GRN can be deployed throughout much of the embryo (for example, as a
consequence of pmar1 overexpression or hesC knockdown)
indicates that any transcriptional activators required to initiate the network
must be widely distributed.
In other recent work, Oliveri and co-workers
(Oliveri et al., 2008)
expanded the micromere-PMC GRN by carrying out MO-mediated knockdowns of many
of the TFs that are expressed selectively in this lineage, including several
that had not previously been analyzed. Quantitative polymerase chain reaction
(QPCR) was used to assess the effects of such gene knockdowns on the
expression of many of the regulatory (TF-encoding) genes in the network and on
the expression of a smaller sampling of biomineralization-related genes. This
work has highlighted the progressive, temporal elaboration of the GRN and the
many feedback interactions that are associated with it, some of which are
shown in Fig. 5. A small set of
key, early regulatory genes, including alx1 (aristaless-like
homeobox 1), ets1 (E26 transformation specific 1) and
tbr (T-brain), is activated in the large micromere progeny,
through the pmar1/hesC double-repression system. These early
TFs, in turn, activate a set of later regulatory genes, including erg
(ets-related gene), hex (hematopoietically expressed
homeobox), tgif (TG-interacting factor), and several
others. Many of the regulatory genes in the network engage in mutual, positive
interactions that probably stabilize the system (an example shown in
Fig. 5 is the positive
regulatory interaction between tgif and hex). The TFs
encoded by these genes activate terminal differentiation genes
(biomineralization genes), often via `feed-forward' interactions in which, for
example, TF-A provides a positive input into gene B, and TF-A and the
TF encoded by gene B both provide positive inputs into gene
C. Several instances of such feed-forward interactions are
illustrated in Fig. 5 [for
example, the positive input from ets1 (TF-A) to alx1 (gene
B) is accompanied by positive inputs from both transcription factors
to dri (gene C)].
| Box 1. Comparative aspects of echinoderm skeletogenesis
The calcified endoskeleton is a distinctive feature of the echinoderm
phylum and appeared during the early Cambrian period, at least 520 million
years ago (Bottjer et al.,
2006
Sea urchin species typically used for developmental and genomic studies are
members of subclass Euechinoidea. Relatively little attention has been paid to
cidaroid urchins (subclass Perischoechinoidea), a group that includes only a
handful of extant species. Cidaroid urchins are of interest because they are
the basal group within the class, and all extant sea urchin species are
believed to have radiated from a cidaroid-like ancestral stock that survived
the Permian-Triassic extinction (Smith et
al., 2006
|
The terminal genes in the GRN, which encode proteins that mediate
biomineralization, were recently surveyed in a genome-wide analysis
(Livingston et al., 2006).
Based on this and earlier work (reviewed by
Wilt and Ettensohn, 2007),
approximately 30 biomineralization proteins have now been identified
(Table 1). Fifteen of these
proteins are spicule matrix proteins, a family of secreted proteins that are
localized within the biomineral. Spicule matrix proteins have a characteristic
structure that consists of a single C-lectin domain and often (but not always)
a region of short repeats that are rich in proline, glycine, glutamine and/or
asparagine residues (Illies et al.,
2002). A small family of acidic, serine-rich, PMC-specific
transmembrane proteins has also been identified, and the founding member (P16)
has been shown to play an important role in skeletal rod elongation
(Cheers and Ettensohn, 2005).
Other biomineralization-related proteins include several collagens and a
PMC-specific carbonic anhydrase
(Livingston et al., 2006). The
identification of the complete repertoire of biomineralization-related genes
has provided an unparalleled picture of the terminal output of this GRN. In
addition, the identification of these genes has revealed features of their
evolution and of the evolution of biomineralization mechanisms more generally
(see Box 2).
|
|
|
;
Revilla-I-Domingo et al.,
2004; Minokawa et al.,
2005; Amore and Davidson,
2006; Ochiai et al.,
2008), but most genes in the network have not been analyzed in
this regard. Exhaustive cis-regulatory analysis of every gene in the network
may not be feasible or informative, but additional studies of this kind are
certainly warranted. Evolutionary insights from the skeletogenic GRN
The construction of detailed, developmental GRNs is important because these networks allow one to move beyond whether an individual gene, gene expression pattern or anatomical structure is shared between two organisms, and to consider the extent to which large blocks of genomic regulatory circuitry have been conserved. As a corollary, it is now possible to dissect in detail the changes in genomic regulatory mechanisms that have occurred during evolution, by carefully comparing the architecture of related GRNs in different species. The establishment of links between GRNs and the anatomy of organisms is also very important, because morphological variation within populations (a substrate for natural selection) may ultimately be interpretable in terms of the properties of GRNs.
Evolutionary changes in skeletogenic GRN architecture
Comparative studies of skeletogenesis in various echinoderms support the
view that two heterochronic shifts in the deployment of the skeletogenic GRN
(that is, changes in the developmental timing of GRN deployment) occurred
following the emergence of the sea urchin lineage. The first imported an
ancestral, adult program of skeletogenesis into the late embryo. The second
shifted this program even earlier in embryogenesis, and was associated with
the invention of micromeres and an early-ingressing, skeletogenic mesenchyme
(Fig. 6).
Against this evolutionary backdrop, recent studies have explored changes
that have taken place in the architecture and deployment of the skeletogenic
GRN during the last 
500 million years. It has been known for some time
that several biomineralization proteins are used in both the adult and the
embryo of the sea urchin, pointing to similarities in these two programs of
skeletogenesis (Wilt and Ettensohn,
2007). There are minor differences in the utilization of these
genes in the embryo and adult, the functional significance of which is
unclear. For example, the sm30 (spicule matrix protein, 30
kd) gene family consists of six clustered paralogous genes, some of which
are expressed selectively in either the adult or the embryonic skeletal tissue
of sea urchins (Livingston et al.,
2006).
|
|
) have recently examined the expression of several upstream
transcriptional regulators of the micromere-PMC GRN during early phases of
adult skeletogenesis. In sea urchins and other echinoderms that exhibit
indirect development (that is, that form feeding larvae that bear little
resemblance to the corresponding adult forms) the adult body arises from a
larval structure known as the echinus rudiment. During metamorphosis, most of
the larval tissues die and the juvenile sea urchin emerges from the remnants
of the larval body. Skeletogenesis begins within specific regions
(skeletogenic centers) of the juvenile sea urchin while it is still growing
within the feeding larva. Gao and co-workers used whole-mount in situ
hybridization (WMISH) to show that several TFs used during embryonic
skeletogenesis, including ets1, alx1, erg, hex, tgif, jun
(ju-nana) and dri (dead-ringer), are also expressed
selectively within the skeletogenic centers of the juvenile sea urchin. Other
TFs of the embryonic GRN, however, including tbr, tel
(transforming-Ets-leukemia), foxB (forkhead box B),
foxO and foxN2/3, are absent from the skeletogenic centers
of the juvenile, or are expressed at levels too low to be detected by WMISH.
These findings suggest that the heterochronic shift in the deployment of the
GRN into the embryo involved the importation of many regulatory components
that were originally used for skeletogenesis in the adult. In addition, new
regulatory connections appear to have been added, based on the finding that
some TFs appear to be used only during the embryonic phase of skeletogenesis.
One significant difference reported by Gao et al. was the absence of
pmar1 expression in juvenile skeletogenic centers. A potential caveat
is that pmar1 is expressed very transiently during PMC specification,
and a brief period of expression in juvenile skeletogenic cells would probably
have been difficult to detect. Nevertheless, the data suggest that the
upstream regulation of the embryonic network, i.e. the double-repression
system based on pmar1 and hesC, was probably an evolutionary
add-on. Other findings, discussed below, bolster the view that the invention
of this system required the forging of a new link between two pre-existing
programs: an ancestral (adult) biomineralization GRN and an even more ancient
maternal patterning system based on the polarized degradation of
β-catenin (Ettensohn et al.,
2007).
Looking even deeper in evolutionary time, comparisons between the sea
urchin GRN and the skeletogenic GRNs of other classes of echinoderms are now
being drawn. Starfish are a distant relative of sea urchins within the
Echinodermata. Gao et al. (Gao and
Davidson, 2008) examined the expression of several TFs in the
juvenile skeletogenic centers of starfish by WMISH and found evidence of the
expression of ets1, alx1, hex and dri, which are therefore
likely to be ancient regulatory components, but not of foxB or
tbr. The lack of tbr expression in the juvenile skeletogenic
centers of both sea urchins and starfish strongly suggests that the
recruitment of this gene into the large micromere-PMC GRN occurred relatively
recently. Indeed, Hinman et al. (Hinman et
al., 2007) have found that in starfish, tbr is expressed
throughout the endomesoderm in a pattern very different from that observed in
sea urchins. tbr orthologs are broadly expressed throughout the
endomesoderm in several vertebrates and in other invertebrate deuterostomes,
suggesting that this is the ancestral pattern. In starfish, tbr
regulates several genes involved in specification of the endomesoderm,
including delta, otx (orthodenticle homeobox),
gatae (GATA-binding transcription factor E), foxa
and bra (brachyury)
(Hinman and Davidson, 2007;
Hinman et al., 2007). Hinman
and co-workers (Hinman et al.,
2007) have shown that, in the case of otx, this
regulatory interaction is direct, and propose that in sea urchins the link
between these two genes has been replaced by other changes in GRN
architecture, thereby allowing tbr to change its developmental
role.
The invention of a new cell lineage
The evolutionary invention of the pmar1/hesC double-repression
system paralleled the invention of micromeres. In euechinoid sea urchins, the
mitotic spindle of each of the four vegetal blastomeres of the
eight-cell-stage embryo interacts with the vegetal cortex and becomes
positioned near to the vegetal pole, which results in an unequal cell
division. A similar process occurs at the fifth cleavage division, when each
micromere divides unequally to produce large and small daughter cells. These
unequal cell divisions appear to be necessary for PMC specification, as
chemical treatments that equalize the divisions also block PMC formation
(Langelan and Whiteley, 1985).
These observations suggest that: (1) the molecular events that activate the
PMC GRN in the large micromeres are functionally linked to unequal
(asymmetric) cell division, by a molecular mechanism that is presently
unknown; and (2) the cell biological mechanisms that produce unequal divisions
evolved in parallel with the appearance of the new regulatory linkages in the
GRN. Cidaroid sea urchins may represent a transitional state in this respect,
as they form variable numbers of micromeres during cleavage and lack an early
skeletogenic mesenchyme (Box
1). One very unusual feature of the pmar1 gene may be
related to its relatively recent recruitment into the PMC GRN; in all three
species of euechinoid urchins that have been examined, the pmar1
locus consists of several (at least ten) nearly identical, tandem copies of
the gene (Nishimura et al.,
2004; Ettensohn et al.,
2007; Sea Urchin Genome
Sequencing Consortium, 2006). This suggests that recent
duplications of the pmar1 gene might have been associated with its
shift in developmental function.
| Box 2. The evolution of biomineralization proteins
Biomineralization is regulated by secreted proteins that control the growth
and physical properties of the material
(Baeuerlein, 2007
|
The skeletogenic GRN and developmental plasticity
GRNs are a powerful tool with which to address the long-standing problem of
developmental plasticity. Early experimental embryological manipulations of
sea urchin embryos were the first to lead to an appreciation of regulative
development (Driesch, 1892;
Hörstadius, 1939). The
plasticity of sea urchin development seems at odds, however, with clear
evidence that: (1) the fates of blastomeres are biased at early stages; (2)
embryonic patterning is entrained by molecular asymmetries within the
unfertilized egg; and (3) distinct domains of differential gene expression
arise very early in development. Despite early patterning processes, cell
specification in early sea urchin embryos remains strikingly labile. This
feature of early embryogenesis is not unique to echinoderms. Recent studies of
early mammalian development also suggest that early developmental biases and
regulative properties may co-exist
(Zernicka-Goetz, 2006). The
inescapable conclusion from work with the sea urchin is that early
developmental GRNs are conditionally deployed and subject to extensive
modifications by extrinsic signals. One hypothesis is that GRNs become less
labile after feedback interactions are established among regulatory genes in
the network, a state that may render networks relatively refractory to
reprogramming.
|
Some populations of cells retain the capacity to activate the micromere-PMC
GRN even after the onset of gastrulation. Microsurgical removal of PMCs at the
early-gastrula stage triggers a conversion of non-skeletogenic mesoderm (NSM)
cells to the PMC fate. Transfating is followed by the synthesis of a complete,
well-patterned skeleton, albeit in a delayed fashion (reviewed by
Ettensohn, 1992). Recent
analysis has shown that NSM transfating is associated with the activation of
many (probably all) of the downstream biomineralization genes in the
micromere-PMC GRN (Ettensohn et al.,
2007). Significantly, several of the regulatory genes of the
skeletogenic GRN are normally expressed both by PMCs and NSM cells, including
ets1, erg, tel, hex, snail, foxN2/3 and foxO, which suggests
that there are many similarities in the genomic regulatory states of these two
cell types (Fig. 8A). One
critical, early transcriptional regulator that is normally absent from NSM
cells, however, is the homeodomain protein Alx1
(Ettensohn et al., 2003).
alx1 expression is activated early in the transfating response
(Fig. 8B-D) and this activation
is essential for a complex suite of downstream, PMC-specific behaviors. In the
micromere-PMC lineage, alx1 is regulated by the maternal
β-catenin-based patterning system through pmar1, which is likely
to be a direct target of β-catenin. During transfating, however,
alx1 is activated by novel, pmar1-independent inputs
(Ettensohn et al., 2007).
Consistent with the pivotal role of alx1 in re-programming NSM cells,
ectopic expression of this protein is sufficient to cause NSM cells to express
a PMC fate, and to trigger morphogenetic behaviors that are characteristic of
PMCs, as well as the activation of downstream biomineralization genes
(Fig. 8E). NSM transfating
therefore exemplifies a situation in which many components of a particular GRN
are already deployed as part of an initial regulatory state. A key subcircuit
is missing, however: one that provides essential inputs into cell-specific
morphoregulatory genes and the cell behaviors they control.
|
). The
invention of an early-ingressing skeletogenic mesenchyme may have been
accompanied by the creation of new cellular interactions that suppressed the
skeletogenic potential of the ancestral population, by inactivating a key,
Alx1-mediated subcircuit of the skeletogenic GRN. According to this view, the
plasticity of NSM cells is a manifestation of a primordial developmental
function (and a primordial genomic regulatory state), which has been overlaid
by more recent modifications. A prediction of this model is that regulatory
inputs into the alx1 gene in cidaroids will prove to be similar to
those in transfating, skeletogenic NSM cells of euechinoids, and will be
similarly independent of the pmar1/hesC double-repression
system that operates in micromeres.
NSM cells are not the only cells in the gastrula-stage embryo that can
express a skeletogenic fate, and in some cases it appears that the change in
genomic regulatory state is more extensive than is observed during NSM
transfating. The surgical removal of both PMCs and NSM cells during
gastrulation leads to the transfating of endoderm cells to a PMC fate
(McClay and Logan, 1996). The
regulatory state of endoderm cells at the gastrula stage is clearly very
different from that of NSM cells, and it will be instructive to investigate
the changes in GRN architecture that accompany this particular
re-specification process. Even more extensive re-wiring of GRNs is likely to
underlie skeletogenesis during the remarkable process of larval cloning; i.e.,
the budding of complete, new individuals from small regions of advanced larvae
(Eaves and Palmer, 2003;
Vaughn and Strathmann,
2008).
Competition between GRNs
The specification of embryonic cells can be viewed as a competition between
GRNs. The most dominant networks may be those with the most robust
positive-feedback interactions or the greatest ability to repress other
genetic programs (Niwa, 2007).
At the heart of genomic reprogramming is the ability of one transcriptional
GRN to dominate another. In the sea urchin, there are several examples of such
dominance at work. The best understood of these are associated with the
repression of the GRN that governs the specification of pigment cells, a major
sub-population of cells within the NSM. The NSM arises at the blastula stage
from a torus-shaped region located between the prospective PMCs and the
prospective endoderm. The initial specification of pigment cells within this
territory is dependent upon a local Delta signal presented by adjacent
prospective PMCs (Sherwood and McClay,
1999; Sweet et al.,
2002). This signal acts through the ubiquitous Notch receptor and
impinges directly on the key regulatory gene gcm (glial cells
missing) via Su(H) (Suppressor of Hairless) target sites in the
cis-regulatory apparatus of this gene
(Ransick and Davidson, 2006).
gcm, in turn, provides essential inputs into several terminal
differentiation genes that regulate pigment biosynthesis
(Calestani et al., 2003).
In the large micromere progeny, Alx1 is required not only to activate a key
subcircuit within the skeletogenic GRN, but also to repress gcm and
downstream differentiation genes of the pigment cell GRN. In the absence of
Alx1, the pigment cell GRN is activated in at least some of these cells as a
consequence of the Delta signal, which is produced by large micromere progeny
in an alx1-independent manner
(Ettensohn et al., 2003;
Ettensohn et al., 2007;
Oliveri et al., 2008). The
mechanism by which Alx1 represses gcm is not known, but the dual role
of this TF in activating one circuit while simultaneously repressing another
is instructive. The foxa gene plays a rather analogous role in the
prospective endoderm, where this gene represses gcm and prevents
prospective endoderm cells from deploying the pigment cell GRN
(Oliveri et al., 2006).
foxa also has positive regulatory inputs into endodermal genes, but
at later stages of development. The term `exclusion effect' has been used to
describe the linking of a particular GRN network to transcriptional repressors
that target regulatory genes required for alternative regulatory states
(Oliveri and Davidson, 2007).
Perhaps no issue will be more important for understanding developmental
plasticity than elucidating the specific regulatory connections between GRNs
that cause one network to be repressed when another is deployed.
|
Another current challenge is to link early GRNs with the specific morphogenetic processes that underlie changes in form. Although the immediate biochemical events that drive changes in cell behavior are likely to be regulated primarily by post-translational mechanisms, it is nevertheless appropriate to view the overall morphogenetic state of a cell as an output of the transcriptional GRNs that operated earlier in the history of that cell lineage. A prerequisite for establishing connections between GRNs and morphogenesis is a thorough understanding of the mechanical basis of specific morphogenetic events at the cellular/tissue level, and knowledge of the specific effector proteins (such as adhesion proteins, cytoskeletal proteins, their regulators, and the like) that mediate such events.
The formation of the endoskeletal system of euechinoid sea urchins is
likely to be the first morphogenetic process to be understood in such a way.
The skeleton determines the angular shape of the sea urchin larva and
influences its swimming and orientation (see
Wilt and Ettensohn, 2007).
Skeletogenesis is very well understood at the cellular level, and recent
studies have shed further light on the molecules that regulate the distinctive
morphogenetic behaviors of the PMCs. The first step in PMC morphogenesis, the
ingression of the cells into the blastocoel via an epithelial-mesenchymal
transition, is associated with the downregulation of cadherin at both
transcriptional and post-translational levels. In the green sea urchin
Lytechinus variegatus, both processes appear to be mediated by
snail, which functions downstream of alx1 in the
micromere-PMC GRN (Wu and McClay,
2007).
Considerable evidence has shown that signals from overlying ectoderm cells
play an important role in PMC migration and in the regulation of
biomineralization-related genes. Recently, the molecular mechanisms of this
interaction were clarified when it was shown that the directional migration of
PMCs is dependent on VEGF (vascular endothelial growth factor) and FGF
(fibroblast growth factor) signaling
(Duloquin et al., 2007;
Rottinger et al., 2008). VEGF
and FGF ligands are expressed in localized regions of the ectoderm that serve
as PMC target sites, and the cognate receptor tyrosine kinases
(vegfr10 and fgfr2) are restricted to PMCs. MO knockdowns
and mRNA misexpression experiments that disrupt either FGF or VEGF signaling
result in aberrant PMC migration and skeletal patterning, and indicate that
these pathways play non-redundant roles. Several potential regulatory inputs
into the sea urchin vegfr gene have been identified
(Oliveri et al., 2008).
Another inroad into an integrated, genomic regulatory view of skeletal
morphogenesis has come from the analysis of the biomineralization proteins
described above. At least two of these are required for skeletal rod growth,
the spicule matrix protein SM50
(Peled-Kamar et al., 2002) and
the novel transmembrane protein P16
(Cheers and Ettensohn, 2005).
p16 is positively regulated by alx1, but other potential
inputs have not yet been examined. The cis-regulatory architecture of
sm50 has been analyzed in considerable detail
(Makabe et al., 1995) and
several regulatory inputs into this gene have been identified
(Oliveri et al., 2008),
although the precise nature of the spatial control of this gene remains
incompletely understood.
Clearly these studies are only a beginning. Much remains to be learnt at the cell biological level about the mechanisms of FGF- and VEGF-mediated PMC guidance, and a detailed dissection of the cis-regulatory control of the genes that encode the receptors within the PMC GRN has yet to be carried out. With respect to the biomineralization proteins, important questions remain that concern their biochemical properties, their potential functional redundancy, and the coordinated transcriptional regulation of the cognate genes. Other proteins that play key roles in PMC morphogenesis - for example, proteins that mediate cell-cell fusion - have yet to be identified. Despite these gaps in our understanding, the recent findings are tantalizing because they point the way to an elucidation of the genomic regulatory control of a major morphogenetic process in the embryo. They establish a continuous, if slender, conceptual thread that links the earliest polarity of the egg to the activation and progressive elaboration of a zygotic GRN, which, in turn, controls a complex anatomical feature.
Conclusions
The work discussed here points towards a future that will be exciting and extraordinarily informative. Further efforts will be required to expand and refine GRNs that have already been constructed, and to elucidate networks that operate in other embryonic cell types. All of the essential experimental tools are in place for such analyses, although the detailed dissection of the cis-regulatory elements of genes remains a time-consuming bottleneck. As the body of information increases, it might become necessary to create new kinds of pictorial representations of GRNs that allow researchers to visualize and analyze these complex, dynamic networks.
It seems likely that comparative GRN analysis will emerge as a major
enterprise of evolutionary developmental biology. Echinoderms will continue to
be valuable experimental material for such studies, building on GRNs that are
being constructed in the sea urchin. The phylum has an extensive fossil record
and a robust phylogeny. Moreover, the embryos of many species are readily
available (Foltz et al., 2004)
and comparisons can be drawn over a wide range of evolutionary distances.
Starfish have emerged as the vanguard for this kind of work, but other classes
of echinoderms will follow. The development of new genomic resources for
echinoderms other than euechinoid sea urchins will be pivotal in this
research. Fortunately, such information is likely to become available in the
near future with the emergence of new and more affordable DNA-sequencing
platforms.
Long-standing questions concerning the plasticity and regulative properties of embryos can now be reformulated in the new context of developmental GRNs. The rich history of experimental embryology has uncovered many examples of cellular reprogramming that can now be analyzed in terms of GRN re-wiring. In the sea urchin, recent studies of NSM transfating have revealed that an intimate relationship exists between plasticity and the evolutionary progression of a developmental program. It seems reasonable to suggest that other examples of developmental plasticity will be better understood when placed in an evolutionary context.
An overarching goal will be to develop an integrated view of the genomic
control of anatomy. The assembly and patterning of the euechinoid endoskeleton
is likely to be the first morphogenetic process fully understood in genomic
regulatory terms, but others will surely follow. In the sea urchin, the
invagination and elongation of the archenteron may be the second, as new
molecular players emerge (Beane et al.,
2006; Croce et al.,
2006) and as GRNs continue to be developed for various territories
of the endomesoderm (Davidson et al.,
2002). Work with other experimental models is also pointing in
this direction, as efforts are underway to elucidate the genomic control of
notochord morphogenesis in ascidians
(Davidson and Christiaen,
2006; Munro et al.,
2006; Satou et al.,
2008), ventral furrow formation in Drosophila
(Gong et al., 2004;
Stathopolous and Levine, 2005; Sandmann et
al., 2007), and vertebrate neural crest morphogenesis
(Sauka-Spengler and Bronner-Fraser,
2008). An understanding of how the anatomy of the embryo is
hard-wired in the genome, certainly one of the central problems in all of
developmental and evolutionary biology, now seems accessible.
Footnotes
The author is grateful to V. Hinman and to three anonymous reviewers for their valuable suggestions. Research by the author that was discussed in this review was supported by the National Science Foundation.
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