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. Author manuscript; available in PMC: 2021 Mar 9.
Published in final edited form as: Curr Top Dev Biol. 2019 Oct 22;136:195–218. doi: 10.1016/bs.ctdb.2019.08.004

Gastrulation in the sea urchin

David R McClay a,*, Jacob Warner b, Megan Martik c, Esther Miranda a, Leslie Slota a
PMCID: PMC7941261  NIHMSID: NIHMS1677273  PMID: 31959288

Abstract

Gastrulation is arguably the most important evolutionary innovation in the animal kingdom. This process provides the basic embryonic architecture, an inner layer separated from an outer layer, from which all animal forms arise. An extraordinarily simple and elegant process of gastrulation is observed in the sea urchin embryo. The cells participating in sea urchin gastrulation are specified early during cleavage. One outcome of that specification is the expression of transcription factors that control each of the many subsequent morphogenetic changes. The first of these movements is an epithelial-mesenchymal transition (EMT) of skeletogenic mesenchyme cells, then EMT of pigment cell progenitors. Shortly thereafter, invagination of the archenteron occurs. At the end of archenteron extension, a second wave of EMT occurs to release immune cells into the blastocoel and primordial germ cells that will home to the coelomic pouches. The archenteron then remodels to establish the three parts of the gut, and at the anterior end, the gut fuses with the stomodaeum to form the through-gut. As part of the anterior remodeling, mesodermal coelomic pouches bud off the lateral sides of the archenteron tip. Multiple cell biological processes conduct each of these movements and in some cases the upstream transcription factors controlling this process have been identified. Remarkably, each event seamlessly occurs at the right time to orchestrate formation of the primitive body plan. This review covers progress toward understanding many of the molecular mechanisms underlying this sequence of morphogenetic events.

1. A sequential overview of sea urchin gastrulation

As a model system for studying gastrulation the sea urchin embryo is valuable for several properties. Those properties include the ability to perform live whole-embryo imaging with many fluorescent probes to reveal molecular dynamics of the process. The system is tractable for experiments that enable assembly of gene regulatory networks, and these provide insight into the control of morphogenetic events. Classically, the system is known for its availability for cell transplantation experiments which open avenues for testing hypotheses about how each of the many events work. These approaches have led to a growing depth of understanding of the steps that lead up to and through gastrulation. As such, sea urchin gastrulation is an excellent model for the study of morphogenesis in the animal kingdom.

To understand sea urchin gastrulation, several species have been utilized including Strongylocentrotus purpuratus and Lytechinus variegatus in the United States, Paracentrotus lividus in Europe, and Hemicentrotus pulcherrimus in Japan. Each of these species has its own advantages for studying morphogenetic events of gastrulation, and for the most part the sequence of events is highly conserved. In the description that follows, the timing refers to hours post fertilization (hpf) in L. variegatus embryos grown at 23°C.

In preparation for gastrulation, a number of important steps occur beginning with oogenesis. Egg-localized maternal factors establish the anterior-posterior axis. After fertilization the dorsal-ventral (aboral-oral) axis is established by Nodal signaling early in cleavage (Duboc, Rottinger, Besnardeau, & Lepage, 2004) and the left-right axis is established later during gastrulation, again by Nodal signaling (Duboc, Rottinger, Lapraz, Besnardeau, & Lepage, 2005). During cleavage and throughout the blastula stage, molecular specification establishes each of the three germ layers by about hatched blastula stage (7.5hpf) and specification continues after that time to program several subtypes of cells within those germ layers. By the mesenchyme blastula stage (9hpf) specification has diversified cells into about 15 different fates, and each lineage has been well documented (Cameron, Hough-Evans, Britten, & Davidson, 1987; Logan & McClay, 1997). Prior to initiation of gastrulation, the embryo is a simple spherical monolayer. A large number of experiments over the last two decades have established models of gene regulatory networks that approximate the molecular sequence in specification of each of the cell types in the embryo as it approaches gastrulation (Davidson & Erwin, 2006; Davidson et al., 2002a; Li, Cui, Peter, & Davidson, 2014; Peter & Davidson, 2017; Su et al., 2009).

At the hatched blastula stage (7.5hpf), the specified cell types present in the embryo can be broadly classified into anterior (animal) cells that will give rise to the ectoderm and neurons and the posterior (vegetal) cells which will give rise to the endoderm and mesoderm (Fig. 1). As the first sign of impending gastrulation the posterior cells change shape and become more columnar in an apical-basal orientation. At about 9hpf the mesenchyme blastula stage is initiated with an epithelial-mesenchyme transition (EMT) and ingression of the mesodermal skeletogenic cells into the blastocoel (Fig. 1A). The 32 ingressed skeletogenic cells become motile, change shape with the cell body moving basally and through the basal lamina. The cells de-adhere from the adherens junction (Saunders & McClay, 2014), and immediately endocytose cadherin from the membrane and coincidentally exocytose proteins needed for mesenchymal migration (Fig. 2). After entering the blastocoel, the skeletogenic cells divide once mitotically, then migrate on the matrix substrate to encircle the base of the invaginating archenteron. The skeletogenic cells then fuse to form a syncytium and begin synthesizing the calcium carbonate skeleton at about 12hpf beginning at two lateral sites in the embryo (Fig. 1B) (McIntyre, Lyons, Martik, & McClay, 2014). The biomineralization sequence is initiated in response to VEGF and FGF signaling from two sites in the ectoderm (Duloquin, Lhomond, & Gache, 2007; Rottinger et al., 2008).

Fig. 1.

Fig. 1

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Major steps in gastrulation. At the mesenchyme blastula stage (A and A′), the skeletogenic cells (red in (A), green in (A′)) undergo an epithelial-mesenchymal transition (EMT) to begin the gastrulation movements. Several hours later as shown in (B and B′), invagination of the archenteron occurs at the posterior end of the embryo (P), beginning with the inbending of the non-skeletal mesoderm (orange) to form the blastopore (B) which remains at the posterior end, and followed by invagination of the endoderm (yellow). As invagination occurs the skeletogenic cells move to surround the base of the archenteron, fuse to form a syncytium, and begin synthesizing the CaCO3 skeleton (green arrows in (B)). Following completion of invagination (C and C′) the anterior end of the archenteron bends toward the ventral side (V) to contact and then fuse with the stomodaeum. That anterior opening becomes the mouth (M), the archenteron differentiates into a three-part gut, and the shape of the embryo changes to conform to the shape of the growing skeletal rods (red in (C), blue in (C′)). This stage is known as the pluteus larva. In the diagrams blue=ectoderm, yellow=endoderm, red and orange=mesoderm, (A) anterior (approximately the animal pole), (P) posterior (approximately the vegetal pole), (V) ventral (also known as oral), (D) dorsal (also known as aboral), (B) blastopore (which later becomes the site of the anus), (M) mouth (derived from the invagination of the stomodaeum). In (A′) the ectoderm is stained with an antibody to β-catenin (red) and an antibody that recognizes skeletogenic cells (green). The pluteus larva in (C′) is stained with a membrane marker (green), a marker specific for the gut (red), and a marker specific for skeletogenic cells (blue).

Fig. 2.

Fig. 2

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Sequence of skeletogenic EMT. Shown from top to bottom are single images from a time lapse movie of an EMT (images of the region shown in the diagrammed insert in (A)). A single micromere at the 16-cell stage, previously injected with cadherin-GFP RNA, expresses cadherin GFP in adherens junctions (green arrow). From top to bottom cells 1, 2, and 3 were tracked. Cell 1 is the first to pull its cytoplasmic tail away from the adherens junctions and when that happens an intracellular vesicle of cadherin GFP appears (arrowhead in frame (B′)). Shortly thereafter an intracellular vesicle appears in cell 2 (right arrowhead in frame C′). Cell 3 is last to leave the adherens junction and internalize the fluorescent cadherin (arrowhead in frame (D′)). Finally, the adherens junction is no longer present when the skeletogenic cells from the transplant complete the EMT (frame (E, E′)).

Pigment cells, the second mesodermal cell type to undergo an EMT, enter the blastocoel between 12 and 13hpf. Upon arrival in the blastocoel the pigment cells migrate to the dorsal side where they re-enter the ectoderm via a mesoderm to epithelial transition. Shortly after the pigment cells initiate their EMT, invagination of the archenteron begins. Non-skeletal mesoderm cells (NSM) at the center of the vegetal plate change shape as the first evidence of that invagination. Some of these NSM cells extend their cell bodies inward in a maneuver that caused these cells to be called “bottle cells” based on their shape (Kimberly & Hardin, 1998). The bottle cells initiate an inward bending of the archenteron and the surrounding NSM cells adopt a keystone shape which establishes the inbending dome. Mechanical properties involving the extracellular matrix are thought to participate in this bending process (Davidson, Koehl, Keller, & Oster, 1995; Davidson, Oster, Keller, & Koehl, 1999). The archenteron then advances, first with the invagination of additional NSM cells (cells that will later form the coelomic pouches, two immune cell types, primordial germ cells and muscle cell progenitors). Endoderm invagination follows and begins at about 13hpf. These cells push the mesoderm cells upward through the blastocoel (Fig. 1B and B′).

Several types of cell movements are necessary to conduct the invagination. The arrival of endoderm cells from lateral positions on the vegetal plate results, at least in part, from the force derived from the spreading cell sheet of the vegetal plate. This spreading force is not globally provided as demonstrated by Ettensohn (1985b), who observed that isolated vegetal half embryos continued to invaginate, demonstrating that the mechanical force had to be local. Presumably, that force is generated in part by the resolution of a previously thickened vegetal plate which undergoes cell shape changes, and these cause a localized spreading, creating the force allowing cells to move through the blastopore and into the lengthening archenteron. Additional forces for lengthening the archenteron are generated by repeated cycles of filopodial extension and contraction by NSM cells, and by convergent-extension movements of the endoderm (Fig. 3). Filopodial extension occurs cyclically; NSM cell filopodia make contact with the basal lamina on the sides of the blastocoel, briefly exert a pulling force on the archenteron, then the filopodia are released and retracted. This extension, pull, and retraction cycle continues throughout archenteron extension. As the endoderm cells invaginate, the diameter of the gut narrows from about 32 cells surrounding the blastopore to about eight cells per diameter along the archenteron (Ettensohn, 1985a). That narrowing is the result of convergent-extension (Martik & McClay, 2017). From time lapse movies, the convergent-extension operates as if each cluster of cells around the base of the blastopore converges about twice (Fig. 3A, A′, B, B′) (Martik & McClay, 2017). Though convergent-extension accounts for much of the lengthening of the archenteron, that morphogenetic movement occurs somewhat differently than the process seen in frogs and fish (contrast Fig. 3C with D). Further aiding archenteron elongation, as invagination proceeds, the endoderm cells divide on average one time each and the orientation of the divisions are biased along the long axis of the archenteron. The divisions therefore add to the lengthening of the archenteron though quantitative analyzes established that the oriented divisions contribute only about 10% of the total length of the archenteron (Martik & McClay, 2017).

Fig. 3.

Fig. 3

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Invagination of the archenteron. Shown are an early frame from a time lapse movie (A, A′) and a later frame of the same movie (B, B′). In the experiment, a single cell at the 32-cell stage was replaced by a cell expressing Histone RFP and membrane GFP. Progeny of that cell are seen to be entering the archenteron at the beginning of gastrulation. Later in gastrulation convergent-extension aligns the cells of that cluster into a single file. The diagram in (C) and (D) illustrate the two extreme forms of convergent extension. In (C), modeled after sea urchins, cells converge from a patch of cells as those cells move in the direction of extension and establish single files. In (D), modeled after zebrafish and frog, cells approach from 90 degrees and randomly merge to extend the tissue. From Martik, M.L., & McClay, D.R. (2017). New insights from a high-resolution look at gastrulation in the sea urchin, Lytechinus variegatus. Mechanisms of Development 148, 3–10.

The final 1/3 of archenteron extension relies heavily on filopodial contraction. In a series of experiments filopodia were severed with a laser each time they were extended. As a consequence, the archenteron did not proceed to more than 2/3 its final length (Hardin & McClay, 1990). Normally, the filopodia cause the archenteron to stretch, so the cells along the gut become anisotropic to account for much of the final third of the archenteron extension (Fig. 4D, inset). The filopodia continue to pull the gut toward the anterior end of the embryo until a “target” is reached at the anterior end. Experiments showed that if the target site is moved out of reach by distorting the ectoderm into a sausage shape, the filopodia continue to pull the archenteron for hours longer than the usual endpoint (Hardin & McClay, 1990). If the sausage shape of the embryo is relaxed at any time to allow the tip of the archenteron to contact the target, the filopodia attach and terminate their cycle of extension, attachment, contraction, and release (Hardin & McClay, 1990). Following the termination of archenteron extension, the anisotropic shape of the cells along the archenteron is resolved to be more isotropic due at least in part to the late addition of Veg1 hindgut cells at the posterior end (Fig. 4A–F and F inset) (Logan & McClay, 1997; Miller & McClay, 1997). Archenteron extension lasts for a total of about 5h from 12 to 17hpf. The target is reached at about 15hpf and during the next 2h the hindgut cells move into the archenteron.

Fig. 4.

Fig. 4

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Veg1 cells enter the archenteron late in gastrulation. A single Veg1 blastomere was labeled with diI at the 60-cell stage. In the sequence shown, the progeny of that blastomere eventually enter the archenteron late in gastrulation, after the Veg2 endoderm and mesoderm produce much of the length of the archenteron. Upon entering the archenteron, the Veg1 progeny align in a file (as in Fig. 3B), and their entry allows the stretched anisotropic Veg2 cells to return to isotropy. The inserts in (D) and (F) show embryos stained with β-catenin at the same stages shown in (D) and (F), respectively. In the (D) inset many of the cells are stretched and anisotropic (arrow) while later, as Veg1 cells enter the archenteron, most cells return to isotropy (arrow). The green lines in the insets mark length vs width of single cells. From Logan, C. Y., & McClay, D. R. (1997). The allocation of early blastomeres to the ectoderm and endoderm is variable in the sea urchin embryo. Development, 124, 2213–2223; Miller, J. R., & McClay, D. R. (1997). Characterization of the role of cadherin in regulating cell adhesion during sea urchin development. Developmental Biology, 192, 323–339.

After the archenteron reaches its final length the anterior end is the first to be remodeled. Several types of cells leave the archenteron tip via EMT to invade the blastocoel. Among these, eight primordial germ cells go through a typical EMT, migrate a short distance, and enter the coelomic pouches (Campanale et al., 2014; Martik & McClay, 2015). About 16 presumptive immune cells also go through an EMT at the tip of the archenteron and migrate through the blastocoel until they attach themselves to the extracellular matrix surrounding the growing skeleton and to the outside of the gut tube. These cells will then engage in immunosurveillance during the larval stage (Ch Ho et al., 2016). Later, muscle cells delaminate and spread to wrap around the foregut and are patterned into circumesophageal rings by Hedgehog signaling (Walton, Warner, Hertzler, & McClay, 2009).

Meanwhile, coelomic pouches form as outpockets from the mesoderm at the tip of the archenteron and later “pinch off” to form lumens separated from the gut. These structures eventually house the primordial germ cells, the left of which becomes incorporated into the rudiment that will form the adult following metamorphosis. Finally, at about 23hpf the anterior end of the endodermal gut contacts and fuses with the invaginating stomodaeum to establish the through-gut (Gustafson & Wolpert, 1963). Little is currently understood about the mechanisms of this deuterostome fusion event, though it is known that the stomodaeum is specified by oral ectoderm autonomously long before the stomodaeum contacts the foregut (Gross & McClay, 2001; Oliveri, Walton, Davidson, & McClay, 2006). Once the anterior end of the gut is remodeled, the stomodaeum-anterior gut fusion involves loss of the basement membranes separating the two invaginations, and then fusion of the two epithelia. Further morphogenetic events establish sphincters between the foregut and midgut, between the midgut and hindgut, and at the anal opening. Fig. 1C and C′ illustrate a 24hpf pluteus larva showing how the skeleton growth distorts the A-P and D-V axis, and also the appearance of the differentiated gut within the blastocoel.

Much of the above was established by observation, experiments using classic and modern imaging techniques, or classical embryological experiments. Over the past two decades a strong effort by many in the sea urchin community have added insights into some of the molecular mechanisms necessary for the gastrulation process. Below, we review advances toward understanding those molecular mechanisms.

2. Setting the stage for gastrulation: Specification of the vegetal plate

Most of the vegetal half of the embryo (the progeny of the micromeres and the macromeres of the 16-cell stage embryo) will later be internalized during gastrulation movements. Between the 16-cell stage and mesenchyme blastula stage (2.5–9hpf) the vegetal half of the embryo is specified to produce cells that will later differentiate into a number of cell types prior to gastrulation: primordial germ cells (8 cells at the beginning of gastrulation), skeletogenic cells (32 cells), non-skeletal mesoderm cells (~64 cells), and endoderm cell precursors (~160 cells). The non-skeletal mesoderm cells further separate into pigment cell precursors (~20 cells), immune cell precursors (~20 cells), coelomic pouch cell precursors (~18 cells), and pharyngeal muscle precursors (~6 cells). Each of these cell types divides once more during or just after their gastrulation movements. Specification of these several cell types has been examined in some detail. Gene regulatory networks operating during cleavage have been extensively characterized over the past 20 years (Croce & McClay, 2010; Davidson et al., 2002a, 2002b; Duboc et al., 2010; Li et al., 2014; Li, Materna, & Davidson, 2012; Oliveri, Tu, & Davidson, 2008; Peter & Davidson, 2010; Saudemont et al., 2010; Sethi, Angerer, & Angerer, 2009; Sethi, Wikramanayake, Angerer, Range, & Angerer, 2012). Those studies and others established GRN models of the sequence of specification and signaling that programs each of the cell types listed above for their future differentiation, and for their specific role in gastrulation movements (Figs. 5 and 6). For some of the cell types the specification is known in enough detail to know how particular gastrulation movements are programmed. Two examples are illustrated. Without going into detail on all the steps of specification there are several important steps that establish the several cell types in the vegetal plate that will later engage in the gastrulation movements. Fig. 5 illustrates the specification sequence of the skeletogenic cells. The Wnt pathway initiates this lineage by the asymmetrical fourth cleavage, so the skeletogenic cell progenitors are initially programmed toward their eventual fate by activation of a repressor (Pmar1) that, in turn, represses a repressor (HesC) (Logan, Miller, Ferkowicz, & McClay, 1999; Oliveri, Davidson, & McClay, 2003; Revilla-i-Domingo et al., 2007). Repression of HesC expression results in activation of skeletogenic cell specification. The GRN model in Fig. 5 shows the progression of that network activation with time reflected (HPF) from top to bottom at the right. At about 9hpf, two events happen. A number of differentiation genes begin to be activated by network transcription factors, and subsets of those factors direct the epithelial-mesenchymal transition which consists of (1) acquisition of motility, (2) cell shape change, (3) cell polarity change, (4) invasion of the cell body through the basement membrane, and (5) de-adhesion from the adherens junction. Those movements, also illustrated above in Fig. 2, are the morphogenetic output driven by the transcription factors of the specified skeletogenic cells.

Fig. 5.

Fig. 5

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Skeletogenic cell gene regulatory network (GRN). Shown is a model of the GRN experimentally established in Lytechinus variegatus. It is very similar to the skeletogenic cell GRN established for Strongylocentrotus purpuratus (Oliveri et al., 2008). In the Biotapestry diagram shown, specification begins in the micromeres at fourth cleavage (embryo to upper left). Genes are depicted as horizontal lines and their activities are depicted as lines with arrows (activators) or blocks (repressors) on the other genes in the network. The genes are drawn from top to bottom based on time of first expression as seen by in situ hybridization and/or QPCR, with approximate times in hours. Post fertilization times provided on the right side of the diagram.Activities directed downward advance the GRN while activities directed upward provide feedback in the system. At about 9hpf EMT begins and at the same time the skeletogenic cells begin to differentiate. The model shows the collective group of transcription factors that activate expression of a number of known differentiation genes, and separately, those transcription factors that control each of the EMT component activities. Thick lines represent activities that have been confirmed or established experimentally in L. variegatus. Thin lines are drawn where genes are present in L. variegatus, but the connections have been established only for S. purpuratus. Several genes are missing from the Lytechinus model that are in the S. purpuratus GRN model. Those genes are present but not expressed in the L. variegatus skeletogenic cells. Two genes are in L. variegatus model are not present in, or yet found, in S. purpuratus. Ubiq are assumed ubiquitously expressed drivers of genes where no other activator has been discovered. Mat are maternal inputs of early expressed genes. Circles represent formation of complexes or activities along a signal transduction pathway. Slashes represent intercellular signal transmission. Delta and Wnt8 are produced by the early skeletogenic cells. These signals act on the macromeres and their progeny as modeled in Fig. 6. From Saunders, L.R., & McClay, D.R. (2014). Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition. Development, 141, 1503–1513.

Fig. 6.

Fig. 6

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Endoderm specification leading to gastrulation. Shown is a simplified model of the GRN that was identified through a number of perturbation experiments. It reflects experiments conducted in L. variegatus and is similar to the GRN identified in S. purpuratus (Peter & Davidson, 2011). Known maternal inputs are shown at the top. Endomesoderm specification occurs between fourth and eighth cleavages (the embryo diagrammed at the upper left is at sixth cleavage. The yellow cells are the Veg1 cells and the orange cells are Veg2 cells). A late arrival of β-catenin, after the HesC repressor is activated in macromeres, excludes macromeres from a skeletogenic fate (Revilla-i-Domingo, Oliveri, & Davidson, 2007). Later, macromere progeny remaining in direct contact with the Delta-signaling micromeres for more than 3h (Croce & McClay, 2010), become the non-skeletogenic mesoderm. Macromere progeny that escape the Delta signal (first Veg1 at sixth cleavage, and later upper Veg2 at eighth cleavage) become the endoderm. The lower Veg2 progeny resulting from eighth cleavage become the non-skeletal mesoderm (specification not shown). Specification of endoderm cells leads to activation of a number of differentiation genes, and to proteins necessary for morphogenesis of the three parts of the gut. Genes are represented as horizontal bars with output arrows. Gene activities are depicted as line with arrows representing activating activities, and bars representing repression activity. Thick lines were established experimentally in L. variegatus and largely agree with S. purpuratus GRN models. Thin lines were established experimentally in S. purpuratus but both genes involved are expressed in endoderm in L. variegatus. In the pregastrula endoderm a several Wnts are present so cWnt is used to represent the activity of one or more of these.

The epithelial-mesenchymal transition of the skeletogenic cells begins 3h before all other gastrulation movements allowing analysis of that event to be studied independent of other gastrulation movements. As revealed in the GRN analysis, in the 2h prior to skeletogenic cell EMT (7–9hpf) 10 transcription factors are uniquely activated in the skeletogenic cell GRN, with three others upstream of those (Fig. 5) (Oliveri et al., 2008; Wu & McClay, 2007; Wu, Yang, & McClay, 2008; and unpublished comparisons of the S. purpuratus GRN connections in L. variagatus). Those transcription factors were systematically knocked down and assays were developed to ask if absence of that transcription had an impact on the EMT, and if so, how (Saunders & McClay, 2014). Using this approach, several distinct transcriptional subcircuits illustrated in Fig. 5 were found to separately control (1) onset of motility, (2) cell shape change, (3) polarity change, (4) invasion through the basement membrane, and (5) de-adhesion of the skeletogenic cells from the adherens junction. Together these several cell biological functions constitute the EMT. Thus, of the 13 transcription factors that are upstream of the EMT, at least 10 of them control component parts of the EMT morphogenetic event (Saunders & McClay, 2014). The transcription factors are upstream of the actual cell biological processes that conduct the EMT, but they provide a means for identifying those cell biological components that are driven by the GRN.

At the cell biological level, a number of EMT properties have been identified. The emerging skeletogenic cells invade through a remodeled basement membrane. If stained with an antibody to laminin, the basement membrane loses laminin at the site of invasion at the time the cells move through. That loss is cell autonomous in that if micromeres are moved to ectopic positions the laminin is eliminated from that ectopic site at the time of EMT (Saunders & McClay, 2014). A number of experiments utilized time-lapse movies to assess the progress of the EMT using transplanted micromeres that were fluorescently labeled in unlabeled host embryos so when the micromere progeny (the skeletogenic cells) went through the EMT it was easy to visualize detailed aspects of that morphogenetic movement. The cells first become motile while still part of the blastula monolayer. At about the same time the laminin hole appears in the basement membrane. It should be noted that the entire basement membrane is not eliminated as the “hole” still contains some basement membrane components. The cell then changes shape and much of the cell body breaches the hole and moves to the other side of the basement membrane. At this point the tail of the cell is still attached to the adherens junction distorting the cell shape. Finally, the tail of the cell releases from the adherens junction and the tail withdraws into the now mesenchymal skeletogenic cell. In cells expressing fluorescent Cadherin, strong staining is present in the adherens junction until that release (Miller & McClay, 1997; Saunders & McClay, 2014). As soon as the cell is released the Cadherin is endocytosed and the fluorescence quickly lost, presumably via lysosomal hydrolysis (Fig. 2). When Cadherin-GFP-expressing skeletogenic cells release their cytoplasmic tail from the adherens junction, all the fluorescence is taken up by the ingressed skeletal cell indicating that no Cadherin and no pinched off part of a cell is left behind (Saunders & McClay, 2014).

Once inside the blastocoel the 32 skeletogenic cells divide once, then rearrange themselves to surround the invaginating archenteron at the posterior end of the blastocoel. They then fuse to form into a syncytium, and at two lateral sites of the syncytium nuclei gather into a cluster, prompted by release of VEGF and FGF by the ectoderm just above the clusters. Skeletogenesis is initiated at these two sites (arrows in Fig. 1B). Several reviews and accounts of skeletogenesis document this process in some detail (Ettensohn & Dey, 2017; Guss & Ettensohn, 1997; Hodor & Ettensohn, 1998; Lyons, Martik, Saunders, & McClay, 2014; Malinda & Ettensohn, 1994; McIntyre et al., 2014; Piacentino et al., 2016; Piacentino, Ramachandran, & Bradham, 2015; Rafiq, Cheers, & Ettensohn, 2012; Rafiq, Shashikant, McManus, & Ettensohn, 2014; Sharma & Ettensohn, 2010).

3. The pigment cells invade the blastocoel shortly after the skeletogenic cells

The Delta-Notch pathway with signaling from the micromeres induces the non-skeletogenic mesoderm starting at sixth cleavage a process that will later result in mesoderm being separated from endomesoderm. Endoderm specification continues in endomesoderm cells that lose contact with the Delta-expressing skeletogenic cells between the sixth and eighth cleavage, thereby subdividing the mesoderm from specification of the endoderm. Delta from micromeres (Fig. 5) activates Notch in cells that will become the endomesoderm, and later some of those cells will be further specified as mesoderm (Fig. 6) (Sherwood & McClay, 1997, 1999; Sweet, Gehring, & Ettensohn, 2002). Endomesoderm specification also requires Wnt signaling (Fig. 6).

Three hours after the EMT of skeletogenic cells is initiated the pigment cell progenitors undergo an EMT and enter the blastocoel. The timing of this event is coincident with the earliest visual evidence of invagination of the archenteron (primary invagination), and by the time the primary invagination has established a dome at the vegetal plate, the pigment cells have completed their EMT. Once inside the blastocoel most pigment cells immediately migrate toward the posterior end of the embryo where they undergo a mesenchymal-to-epithelial transition and re-invade the ectoderm. They selectively invade the dorsal ectoderm somehow using Eph and ephrin proteins as a mechanism to provide that selectivity (Krupke, Zysk, Mellott, & Burke, 2016). The EMT of the pigment cells looks similar to the EMT of skeletogenic cells in time-lapse movies; however, most of the transcription factors found to participate in controlling skeletogenic cell EMT are not expressed by pigment cells (D. McClay Unpublished Data). Thus, this EMT must occur with a largely different set of controlling transcription factors.

4. Mechanistic studies of primary invagination

At 12hpf, at the same time the pigment cells undergo their EMT, the remaining non-skeletogenic cells undergo cell shape changes to produce the initial inbending of the archenteron. The site of the inbending varies with species, being at the middle of the vegetal plate in L. variegatus, and offset toward the oral side in S. purpuratus. Veg2 cells that receive the Notch signal continuously for about 3h become the non-skeletogenic mesoderm cells, and of all macromere progeny only those from the lower tier of Veg2 after eighth cleavage satisfy this requirement (Croce & McClay, 2010; Materna, Ransick, Li, & Davidson, 2013; Ransick & Davidson, 2006, 2012). A separate signaling input, this time from Nodal, separates the ventral (oral) from dorsal (aboral) non-skeletogenic mesoderm (Duboc et al., 2010). Much of the dorsal NSM gives rise to the pigment cells. Pigment cells are not major participants in the primary inbending of the archenteron because by that time the pigment cells have largely removed themselves from the vegetal plate by EMT. Several mechanisms have been proposed to cause the initial inbending, one involving bottle cells (Hardin & Keller, 1988), and others involving extraembryonic matrix (Lane, Koehl, Wilt, & Keller, 1993), RhoA (Beane, Gross, & McClay, 2006) or by several other proposed mechanisms involving mechanical properties (Davidson et al., 1995). Details of the initial inbending sequence likely involve processes known in other systems to control cell shape change but there is much to learn about the process that initiates this conserved deuterostome event.

5. Specification of endoderm

Following the initial inbending by the NSM, the archenteron lengthens by addition of endoderm cells from the vegetal plate. Those endoderm cells move toward the blastopore, pass through it, and contribute to the lengthening archenteron. At a molecular level, much has been learned about how the endoderm cells are specified prior to and during invagination. Fig. 6 is a GRN model of endoderm specification. Initially, macromeres and their progeny (yellow and orange cells in the diagram at top left of Fig. 6) begin specification as endomesoderm through a combination of Wnt and Delta-Notch signaling. At sixth cleavage the Veg1 cells (the yellow cells in Fig. 6, and the upper progeny of division of macromeres at sixth cleavage), are removed from the source of Delta signaling and proceed to become endoderm (though some Veg1 progeny also become ectoderm). The lower macromere progeny, the Veg2 cells (orange cells in Fig. 6), continue to receive Delta for two more cleavages and at eighth cleavage the upper Veg2 cells are removed from Delta as a consequence of a meridional cleavage, and because these cells receive fewer than 3h of continuous Delta signaling, these cells become endoderm, while the lower tier of Veg2 cells continue to receive Delta for 3h and as a consequence become the non-skeletogenic mesoderm (Croce & McClay, 2010). Fig. 6 is a general model for events leading to endoderm specification and morphogenesis. For simplification, parts of specification leading to mesoderm specification are excluded.

Endoderm-activating genes of the endomesoderm (the genes illustrated in the early endomesoderm portion of the model), direct the upper Veg2 cells toward an endoderm fate once those cells are removed from the Delta signal. Among the first genes activated in the definitive endoderm is brachyury (bra) which, in invertebrates, is expressed in the endoderm (Fig. 6). In the sea urchin bra is expressed 4h prior to the onset of invagination in a ring that will later become the blastopore (Fig. 7) (Croce, Lhomond, & Gache, 2001; Gross & McClay, 2001). Shortly thereafter, foxA is expressed in the same region (Oliveri et al., 2006). These two transcription factors eventually are expressed in all endoderm cells with foxA expression remaining on, once activated, while bra expression is restricted to the ring of cells surrounding the blastopore throughout gastrulation (Fig. 7). Since all endoderm cells pass through the blastopore this means that presumptive endoderm cells activate bra expression as they approach the blastopore and extinguish bra expression as they enter the archenteron (Gross & McClay, 2001). Recently, it was learned that Wnts also display dynamic expression changes throughout gastrulation (Fig. 7) (also see Robert, Lhomond, Schubert, & Croce, 2014). As with the skeletogenic GRN, endoderm specification leads to two outcomes: activation of genes involved in differentiation and activation of genes expressing proteins involved in morphogenesis. Specification events even later in gastrulation include several evolutionarily conserved pathways with expression of transcription factors that are also expressed in vertebrate guts and pancreas (Cole, Rizzo, Martinez, Fernandez-Serra, & Arnone, 2009; Perillo, Wang, Leach, & Arnone, 2016).

Fig. 7.

Fig. 7

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The blastopore is a dynamic structure both for specification and morphogenesis. (A) Brachyury is present in cells just outside the blastopore from mesenchyme blastula, through gastrulation (B), as seen with an antibody to Brachyury. (C) In a double in situ of an embryo at late mesenchyme blastula, cells just outside the blastopore express brachyury (red), a few cells express bra and foxA (yellow), and still other cells express only foxA (green). The foxA-expressing cells previously had expressed bra, then both bra and foxA, and then bra expression was extinguished. This pattern is followed by all endoderm cells sequentially as they approach and move through the blastopore. (D–F) Expression of Wnts is dynamic at the blastopore. Shown here are wnt1 (green) and wnt8 (red) expression at three stages, before, at the beginning and at the end of gastrulation. Here, as the cells move into the blastopore and archenteron, wnt1 and wnt8 expression moves from cell to cell, counter to the direction to cell movement toward the blastopore. lv, lateral view; vv, vegetal view; MB, mesenchyme blastula; EB, early blastula; HB, hatched blastula; LG, late gastrula. Panels (A) and (B): From Gross, J. M., & McClay, D. R. (2001). The role of Brachyury (T) during gastrulation movements in the sea urchin Lytechinus variegatus. Developmental Biology, 239, 132–147.)

6. Cells at the tip of the advancing archenteron are necessary for establishing right-left asymmetry

Nodal is responsible for establishing the dorsal-ventral axis in all three germ layers in the embryo (Duboc et al., 2010, 2004). Nodal signaling is also responsible for establishing right-left asymmetry (Duboc et al., 2005). Nodal signaling from the tip of the archenteron, beginning late in gastrulation, activates Nodal signaling on the right side to establish an asymmetry in specification of the left and right coelomic pouches (Fig. 8) (Bessodes et al., 2012; Warner et al., 2016). Misexpression of Hedgehog compromises that left-right asymmetry (Walton et al., 2009; Warner, Lyons, & McClay, 2012; Warner et al., 2016). This raised the question as to which signal was upstream, Nodal or Hedgehog. A careful study showed that Nodal can initiate right-left in the absence of Hedgehog but the asymmetry cannot be maintained without Hedgehog (Warner et al., 2016). Thus, it appears that Nodal is the symmetry-breaking signal, but, perdurance of Nodal for a period of time is required to stabilize the asymmetry and Hedgehog is necessary for maintenance of that signal. In other deuterostomes, Nodal signaling establishes the left side, while in the sea urchin, Nodal operates on the right side to prevent the right coelomic pouch from becoming part of the rudiment (Duboc et al., 2005).

Fig. 8.

Fig. 8

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The coelomic pouches are specified asymmetrically and are the targets for primordial germ cell homing. (A) A single primordial germ cell (PGC), labeled green with FITC, was placed in an embryo stained with Rhodamine-dextran at the 60-cell stage. During gastrulation the PGC successfully homed to the left coelomic pouch where its two progeny are seen at the pluteus larval stage. (B) During gastrulation left-right asymmetry is established as evidenced by a double in situ of soxE (green) and pitx (red) showing that the left and right coelomic pouches express different molecular markers. The left coelomic pouch will contribute to the rudiment of the adult. (C) Experiments used the homing of PGCs as an assay. Perturbation of transcription factors expressed by the coelomic pouches asked which factors were necessary for homing. Then, experiments asked how necessary factors interacted with each other. Results identified a GRN circuit necessary for production of the homing signal. Shown is that circuit which is both a feed forward and a feedback circuit. Panel (A): From Martik, M. L., & McClay, D. R. (2015). Deployment of a retinal determination gene network drives directed cell migration in the sea urchin embryo. Elife, 4, e08827; Panel (B): From Warner, J. F., Miranda, E. L., & McClay, D. R. (2016). Contribution of hedgehog signaling to the establishment of left-right asymmetry in the sea urchin. Developmental Biology, 411, 314–324; Panel (C): From Martik, M. L., & McClay, D. R. (2015). Deployment of a retinal determination gene network drives directed cell migration in the sea urchin embryo. Elife 4, e08827.

7. Homing of the primordial germ cells to the coelomic pouches

At fifth cleavage, the cells at the vegetal pole of the embryo divide asymmetrically to produce four large and four small micromeres. The small micromeres divide once more before gastrulation and are fated to become the primordial germ cells (PGCs) (Yajima & Wessel, 2011). These cells remain attached to the archenteron during invagination. Then, when the archenteron reaches its full length, the PGCs undergo an EMT, migrate in the blastocoel briefly, then enter the coelomic pouches via a MET (Campanale et al., 2014; Martik & McClay, 2015). Experiments with PGCs revealed several properties of these cells. First, the PGCs are actively retained in the archenteron cell sheet until gastrulation is completed. If PGCs are transplanted to their normal position in a second host embryo this remains true. If, however, the PGCs are transplanted to an ectopic position in the host embryo they undergo an EMT precociously suggesting that in their normal position they are actively restrained from undergoing an EMT until gastrulation is completed (Martik & McClay, 2015). Additionally, if the PGCs are transplanted into an ectopic position, they go through the EMT, then demonstrate a remarkably robust ability to find their way to the coelomic pouches indicating a highly efficient homing mechanism (Martik & McClay, 2015) (Fig. 8). To home correctly to the coelomic pouch, a group of five transcription factors expressed by the coelomic pouch cells were found to establish an upstream control circuit that is necessary for the coelomic pouch cells to release the presumed homing signal. Perturbation of those five transcription factors established that the control circuit is both a classic feed forward and a positive feedback device (Martik & McClay, 2015). In Lytechinus, the PGCs preferentially home to the left coelomic pouch, and that pouch later becomes an important component of the embryonic rudiment which becomes the juvenile at metamorphosis. Thus, like PGCs of many species in the animal kingdom, sea urchin PGCs actively home to a specific target. Presumably, the left coelomic pouch, containing the PGCs, later contributes to the development of the adult gonad after metamorphosis.

8. Summary

Gastrulation in the sea urchin is a beautifully orchestrated sequence of morphogenetic movements. After over a century of study the molecular underpinnings of these processes of specification, morphogenesis, and patterning are beginning to be understood at a molecular level. The lessons we have learned so far have applications and implications across many phyla and biological processes from cancer metastasis to cell specification. Recent advances in high throughput omics and advances in quantitative microscopy will further provide the foundation for future mechanistic studies.

Acknowledgments

The authors thank Esther Miranda for her technical support in all aspects of the research. We also thank the members of the McClay lab for their advice and input. Support for this project was provided by: NIH RO1 HD14483 (to D.R.M.), PO1 HD37105 (to D.R.M.), and NSF fellowship support (to L.S.).

References

  1. Beane WS, Gross JM, & McClay DR (2006). RhoA regulates initiation of invagination, but not convergent extension, during sea urchin gastrulation. Developmental Biology, 292, 213–225. [DOI] [PubMed] [Google Scholar]
  2. Bessodes N, Haillot E, Duboc V, Rottinger E, Lahaye F, & Lepage T (2012). Reciprocal signaling between the ectoderm and a mesendodermal left-right organizer directs left-right determination in the sea urchin embryo. PLoS Genetics, 8, e1003121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cameron RA, Hough-Evans BR, Britten RJ, & Davidson EH (1987). Lineage and fate of each blastomere of the eight-cell sea urchin embryo. Genes & Development, 1, 75–85. [DOI] [PubMed] [Google Scholar]
  4. Campanale JP, Gokirmak T, Espinoza JA, Oulhen N, Wessel GM, & Hamdoun A (2014). Migration of sea urchin primordial germ cells. Developmental Dynamics, 243, 917–927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ch Ho E, Buckley KM, Schrankel CS, Schuh NW, Hibino T, Solek CM, et al. (2016). Perturbation of gut bacteria induces a coordinated cellular immune response in the purple sea urchin larva. Immunology and Cell Biology, 94, 861–874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cole AG, Rizzo F, Martinez P, Fernandez-Serra M, & Arnone MI (2009). Two ParaHox genes, SpLox and SpCdx, interact to partition the posterior endoderm in the formation of a functional gut. Development, 136, 541–549. [DOI] [PubMed] [Google Scholar]
  7. Croce J, Lhomond G, & Gache C (2001). Expression pattern of Brachyury in the embryo of the sea urchin Paracentrotus lividus. Development Genes and Evolution, 211, 617–619. [DOI] [PubMed] [Google Scholar]
  8. Croce JC, & McClay DR (2010). Dynamics of Delta/Notch signaling on endomesoderm segregation in the sea urchin embryo. Development, 137, 83–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davidson EH, & Erwin DH (2006). Gene regulatory networks and the evolution of animal body plans. Science, 311, 796–800. [DOI] [PubMed] [Google Scholar]
  10. Davidson LA, Koehl MA, Keller R, & Oster GF (1995). How do sea urchins invaginate? Using biomechanics to distinguish between mechanisms of primary invagination. Development, 121, 2005–2018. [DOI] [PubMed] [Google Scholar]
  11. Davidson LA, Oster GF, Keller RE, & Koehl MA (1999). Measurements of mechanical properties of the blastula wall reveal which hypothesized mechanisms of primary invagination are physically plausible in the sea urchin Strongylocentrotus purpuratus. Developmental Biology, 209, 221–238. [DOI] [PubMed] [Google Scholar]
  12. Davidson EH, Rast JP, Oliveri P, Ransick A, Calestani C, Yuh CH, et al. (2002a). A genomic regulatory network for development. Science, 295, 1669–1678. [DOI] [PubMed] [Google Scholar]
  13. Davidson EH, Rast JP, Oliveri P, Ransick A, Calestani C, Yuh CH, et al. (2002b). A provisional regulatory gene network for specification of endomesoderm in the sea urchin embryo. Developmental Biology, 246, 162–190. [DOI] [PubMed] [Google Scholar]
  14. Duboc V, Lapraz F, Saudemont A, Bessodes N, Mekpoh F, Haillot E, et al. (2010). Nodal and BMP2/4 pattern the mesoderm and endoderm during development of the sea urchin embryo. Development, 137, 223–235. [DOI] [PubMed] [Google Scholar]
  15. Duboc V, Rottinger E, Besnardeau L, & Lepage T (2004). Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo. Developmental Cell, 6, 397–410. [DOI] [PubMed] [Google Scholar]
  16. Duboc V, Rottinger E, Lapraz F, Besnardeau L, & Lepage T (2005). Left-right asymmetry in the sea urchin embryo is regulated by nodal signaling on the right side. Developmental Cell, 9, 147–158. [DOI] [PubMed] [Google Scholar]
  17. Duloquin L, Lhomond G, & Gache C (2007). Localized VEGF signaling from ectoderm to mesenchyme cells controls morphogenesis of the sea urchin embryo skeleton. Development, 134, 2293–2302. [DOI] [PubMed] [Google Scholar]
  18. Ettensohn CA (1985a). Gastrulation in the sea urchin embryo is accompanied by the rearrangement of invaginating epithelial cells. Developmental Biology, 112, 383–390. [DOI] [PubMed] [Google Scholar]
  19. Ettensohn CA (1985b). Mechanisms of epithelial invagination. The Quarterly Review of Biology, 60, 289–307. [DOI] [PubMed] [Google Scholar]
  20. Ettensohn CA, & Dey D (2017). Kirrel L, a member of the Ig-domain superfamily of adhesion proteins, is essential for fusion of primary mesenchyme cells in the sea urchin embryo. Developmental Biology, 421, 258–270. [DOI] [PubMed] [Google Scholar]
  21. Gross JM, & McClay DR (2001). The role of Brachyury (T) during gastrulation movements in the sea urchin Lytechinus variegatus. Developmental Biology, 239, 132–147. [DOI] [PubMed] [Google Scholar]
  22. Guss KA, & Ettensohn CA (1997). Skeletal morphogenesis in the sea urchin embryo: Regulation of primary mesenchyme gene expression and skeletal rod growth by ectoderm-derived cues. Development, 124, 1899–1908. [DOI] [PubMed] [Google Scholar]
  23. Gustafson T, & Wolpert L (1963). Studies on the cellular basis of morphogenesis in the sea urchin embryo. Formation of the coelom, the mouth, and the primary pore canal. Experimental Cell Research, 29, 561–582. [Google Scholar]
  24. Hardin J, & Keller R (1988). The behaviour and function of bottle of cells in gastrulation of Xenopus laevis. Development, 103, 211–230. [DOI] [PubMed] [Google Scholar]
  25. Hardin J, & McClay DR (1990). Target recognition by the archenteron during sea urchin gastrulation. Developmental Biology, 142, 86–102. [DOI] [PubMed] [Google Scholar]
  26. Hodor PG, & Ettensohn CA (1998). The dynamics and regulation of mesenchymal cell fusion in the sea urchin embryo. Developmental Biology, 199, 111–124. [DOI] [PubMed] [Google Scholar]
  27. Kimberly EL, & Hardin J (1998). Bottle cells are required for the initiation of primary invagination in the sea urchin embryo. Developmental Biology, 204, 235–250. [DOI] [PubMed] [Google Scholar]
  28. Krupke OA, Zysk I, Mellott DO, & Burke RD (2016). Eph and Ephrin function in dispersal and epithelial insertion of pigmented immunocytes in sea urchin embryos. Elife, 5, e16000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lane MC, Koehl MA, Wilt F, & Keller R (1993). A role for regulated secretion of apical extracellular matrix during epithelial invagination in the sea urchin. Development, 117, 1049–1060. [DOI] [PubMed] [Google Scholar]
  30. Li E, Cui M, Peter IS, & Davidson EH (2014). Encoding regulatory state boundaries in the pregastrular oral ectoderm of the sea urchin embryo. Proceedings of the National Academy of Sciences of the United States of America, 111, E906–E913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Li E, Materna SC, & Davidson EH (2012). Direct and indirect control of oral ectoderm regulatory gene expression by Nodal signaling in the sea urchin embryo. Developmental Biology, 369, 377–385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Logan CY, & McClay DR (1997). The allocation of early blastomeres to the ectoderm and endoderm is variable in the sea urchin embryo. Development, 124, 2213–2223. [DOI] [PubMed] [Google Scholar]
  33. Logan CY, Miller JR, Ferkowicz MJ, & McClay DR (1999). Nuclear beta-catenin is required to specify vegetal cell fates in the sea urchin embryo. Development, 126, 345–357. [DOI] [PubMed] [Google Scholar]
  34. Lyons DC, Martik ML, Saunders LR, & McClay DR (2014). Specification to biomineralization: Following a single cell type as it constructs a skeleton. Integrative and Comparative Biology, 54, 723–733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Malinda KM, & Ettensohn CA (1994). Primary mesenchyme cell migration in the sea urchin embryo: Distribution of directional cues. Developmental Biology, 164, 562–578. [DOI] [PubMed] [Google Scholar]
  36. Martik ML, & McClay DR (2015). Deployment of a retinal determination gene network drives directed cell migration in the sea urchin embryo. Elife, 4, e08827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Martik ML, & McClay DR (2017). New insights from a high-resolution look at gastrulation in the sea urchin, Lytechinus variegatus. Mechanisms of Development, 148, 3–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Materna SC, Ransick A, Li E, & Davidson EH (2013). Diversification of oral and aboral mesodermal regulatory states in pregastrular sea urchin embryos. Developmental Biology, 375, 92–104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. McIntyre DC, Lyons DC, Martik M, & McClay DR (2014). Branching out: Origins of the sea urchin larval skeleton in development and evolution. Genesis, 52, 173–185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Miller JR, & McClay DR (1997). Characterization of the role of cadherin in regulating cell adhesion during sea urchin development. Developmental Biology, 192, 323–339. [DOI] [PubMed] [Google Scholar]
  41. Oliveri P, Davidson EH, & McClay DR (2003). Activation of pmar1 controls specification of micromeres in the sea urchin embryo. Developmental Biology, 258, 32–43. [DOI] [PubMed] [Google Scholar]
  42. Oliveri P, Tu Q, & Davidson EH (2008). Global regulatory logic for specification of an embryonic cell lineage. Proceedings of the National Academy of Sciences of the United States of America, 105, 5955–5962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Oliveri P, Walton KD, Davidson EH, & McClay DR (2006). Repression of mesodermal fate by foxa, a key endoderm regulator of the sea urchin embryo. Development, 133, 4173–4181. [DOI] [PubMed] [Google Scholar]
  44. Perillo M, Wang YJ, Leach SD, & Arnone MI (2016). A pancreatic exocrine-like cell regulatory circuit operating in the upper stomach of the sea urchin Strongylocentrotus purpuratus larva. BMC Evolutionary Biology, 16, 117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Peter IS, & Davidson EH (2010). The endoderm gene regulatory network in sea urchin embryos up to mid-blastula stage. Developmental Biology, 340, 188–199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Peter IS, & Davidson EH (2011). A gene regulatory network controlling the embryonic specification of endoderm. Nature, 474, 635–639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Peter IS, & Davidson EH (2017). Assessing regulatory information in developmental gene regulatory networks. Proceedings of the National Academy of Sciences of the United States of America, 114, 5862–5869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Piacentino ML, Chung O, Ramachandran J, Zuch DT, Yu J, Conaway EA, et al. (2016). Zygotic LvBMP5–8 is required for skeletal patterning and for left-right but not dorsal-ventral specification in the sea urchin embryo. Developmental Biology, 412, 44–56. [DOI] [PubMed] [Google Scholar]
  49. Piacentino ML, Ramachandran J, & Bradham CA (2015). Late Alk4/5/7 signaling is required for anterior skeletal patterning in sea urchin embryos. Development, 142, 943–952. [DOI] [PubMed] [Google Scholar]
  50. Rafiq K, Cheers MS, & Ettensohn CA (2012). The genomic regulatory control of skeletal morphogenesis in the sea urchin. Development, 139, 579–590. [DOI] [PubMed] [Google Scholar]
  51. Rafiq K, Shashikant T, McManus CJ, & Ettensohn CA (2014). Genome-wide analysis of the skeletogenic gene regulatory network of sea urchins. Development, 141, 950–961. [DOI] [PubMed] [Google Scholar]
  52. Ransick A, & Davidson EH (2006). Cis-regulatory processing of Notch signaling input to the sea urchin glial cells missing gene during mesoderm specification. Developmental Biology, 297, 587–602. [DOI] [PubMed] [Google Scholar]
  53. Ransick A, & Davidson EH (2012). Cis-regulatory logic driving glial cells missing: Self-sustaining circuitry in later embryogenesis. Developmental Biology, 364, 259–267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Revilla-i-Domingo R, Oliveri P, & Davidson EH (2007). A missing link in the sea urchin embryo gene regulatory network: hesC and the double-negative specification of micromeres. Proceedings of the National Academy of Sciences of the United States of America, 104, 12383–12388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Robert N, Lhomond G, Schubert M, & Croce JC (2014). A comprehensive survey of wnt and frizzled expression in the sea urchin Paracentrotus lividus. Genesis, 52, 235–250. [DOI] [PubMed] [Google Scholar]
  56. Rottinger E, Saudemont A, Duboc V, Besnardeau L, McClay D, & Lepage T (2008). FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis [corrected] and regulate gastrulation during sea urchin development. Development, 135, 353–365. [DOI] [PubMed] [Google Scholar]
  57. Saudemont A, Haillot E, Mekpoh F, Bessodes N, Quirin M, Lapraz F, et al. (2010). Ancestral regulatory circuits governing ectoderm patterning downstream of Nodal and BMP2/4 revealed by gene regulatory network analysis in an echinoderm. PLoS Genetics, 6, e1001259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Saunders LR, & McClay DR (2014). Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition. Development, 141, 1503–1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Sethi AJ, Angerer RC, & Angerer LM (2009). Gene regulatory network interactions in sea urchin endomesoderm induction. PLoS Biology, 7, e1000029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Sethi AJ, Wikramanayake RM, Angerer RC, Range RC, & Angerer LM (2012). Sequential signaling crosstalk regulates endomesoderm segregation in sea urchin embryos. Science, 335, 590–593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Sharma T, & Ettensohn CA (2010). Activation of the skeletogenic gene regulatory network in the early sea urchin embryo. Development, 137, 1149–1157. [DOI] [PubMed] [Google Scholar]
  62. Sherwood DR, & McClay DR (1997). Identification and localization of a sea urchin Notch homologue: Insights into vegetal plate regionalization and Notch receptor regulation. Development, 124, 3363–3374. [DOI] [PubMed] [Google Scholar]
  63. Sherwood DR, & McClay DR (1999). LvNotch signaling mediates secondary mesenchyme specification in the sea urchin embryo. Development, 126, 1703–1713. [DOI] [PubMed] [Google Scholar]
  64. Su YH, Li E, Geiss GK, Longabaugh WJ, Kramer A, & Davidson EH (2009). A perturbation model of the gene regulatory network for oral and aboral ectoderm specification in the sea urchin embryo. Developmental Biology, 329, 410–421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Sweet HC, Gehring M, & Ettensohn CA (2002). LvDelta is a mesoderm-inducing signal in the sea urchin embryo and can endow blastomeres with organizer-like properties. Development, 129, 1945–1955. [DOI] [PubMed] [Google Scholar]
  66. Walton KD, Warner J, Hertzler PH, & McClay DR (2009). Hedgehog signaling patterns mesoderm in the sea urchin. Developmental Biology, 331, 26–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Warner JF, Lyons DC, & McClay DR (2012). Left-right asymmetry in the sea urchin embryo: BMP and the asymmetrical origins of the adult. PLoS Biology, 10, e1001404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Warner JF, Miranda EL, & McClay DR (2016). Contribution of hedgehog signaling to the establishment of left-right asymmetry in the sea urchin. Developmental Biology, 411, 314–324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Wu SY, & McClay DR (2007). The snail repressor is required for PMC ingression in the sea urchin embryo. Development, 134, 1061–1070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Wu SY, Yang YP, & McClay DR (2008). Twist is an essential regulator of the skeletogenic gene regulatory network in the sea urchin embryo. Developmental Biology, 319, 406–415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Yajima M, & Wessel GM (2011). Small micromeres contribute to the germline in the sea urchin. Development, 138, 237–243. [DOI] [PMC free article] [PubMed] [Google Scholar]

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