Microfolds in the suprablastoporal zone of the frog gastrula and their relationship to the geometry and mechanics of gastrulation

Spatial distribution and orientation of microfolds arising during invagination of the outer layer of suprablastoporal zone into the blastopore dorsal lip and changes of the lip shape were studied in Rana ridibunda embryos using statistical analysis of a normal individual variability. Active invagination of the cells into the lip correlated with deviation of the orientation of microfolds from the normal in the points of their intersection with the zone of dorsal lip inflection and their orientation is normalized upon transition of the cells across the inflection zone. Frequency distribution of the angle of microfold deviation from the normal is close to the exponential and, therefore, the angle of deviation is an analog of the potential energy of cells-components of the microfold: the bigger the deviation angle, the higher the potential energy. The minimum potential energy is observed at the normal orientation of microfolds, i.e., when it coincides with the radius of the dorsal lip curvature at the point of intersection with the microfold. The following mechanism of dorsal lip formation has been proposed: equatorial contraction of cells upon their invagination into the dorsal lip causes deviation of cell flux orientation from the normal orientation and the normal orientation is restored through an increase in the local curvature of dorsal lip. When the orientation of cell fluxes is normalized, invagination of cells in the dorsal lip ceases. The wave of normalization overtakes the wave of cell invagination into the dorsal lip at the lip angle length 120 degrees. At this moment, the archenteron roof is mechanically detached from the superficial cells of the suprablastoporal zone and lateral blastopore lips and this determines separation of the presumptive notochord.     

Early Shell Formation During Molluscan Embryogenesis, with New Studies on the Surf Clam, Spisula solidissima

Shell formation in molluscs begins early in embryogenesis duringsome stage of archenteron formation. Ultrastructural informationon early formation of external shells is available from onlya few bivalves and gastropods. Secretion of the very first shellmaterial by shell field epithelial cells is preceded by an invaginationof the dorsal ectoderm in the region of the shell field. A centuryago, this invagination was termed the "shell gland." As a secretoryfunction for this invagination has not yet been demonstratedand as the term "shell gland" has taken on various meaningsin the literature, the invagination will be referred to as theshell field invagination. The opening into the shell field invaginationseems to be circular in gastropods and elongate in bivalves.Accordingly initial organic shell material seems to form a ringin gastropods and a saddle in bivalves. As in adult molluscs,shells of pre-metamorphic molluscs are composed of both organicand inorganiccomponents. Ultrastructural data from bivalvesand gastropods indicate that the initial organic shell materialis secreted just outside the shell field invagination (acrossthe pore). Initial inorganic shell materials have not been localizednor their pathway traced into or through any pre-metamorphicmolluscs. New SEM and TEM data show that the invagination inthe bivalve Spisula solidissima is composed of a wide outerregion and very narrow inner region with the first shell materialforming at the junction between the two. This is unlike ultrastructuraldata available for other species. Many sections give the falseimpressions that: 1) the shell field invagination is closedto the outside and, 2) that the first organic shell materiallines the innermost region of the invagination. It is not clearwhether the cells of the outer invagination in this speciesare shell field cells. It is suggested that they are not.     

Fate maps of ventral and dorsal pancreatic progenitor cells in early somite stage mouse embryos

The origins of liver progenitor cells have been extensively studied, but evidence on the origin of pancreatic precursor cells is currently limited. Pancreatic and duodenal homeobox gene 1 (Pdx1) is one of the earliest known markers for the pancreas. A transgenic mouse line expressing green fluorescent protein (GFP) under the control of the Pdx1 promoter showed that Pdx1/GFP expression was first observed in the mid-region of the anterior intestinal portal (AIP) lip at embryonic day (E) 8.5 at the 5-6 somite stage (ss). The liver progenitors were confirmed to originate from separate domains at the lateral endoderm and the inner part of the medial AIP as previously reported (Tremblay and Zaret, 2005), which turned out to lie caudally to the Pdx1/GFP-expressing domain. To confirm if the early Pdx1/GFP-positive cells give rise to the pancreatic bud, we labeled the cells on the lip of the AIP using the carbocyanine dye CM-DiI and traced their fates in 1-4 ss, 5-6 ss and 7-9 ss E8.5 embryos using an ex utero whole embryo culture method. At 1 ss, the ventral pancreas progenitors were observed in the lateral endoderm, not yet being segregated from the liver or gut progenitors. Cells that contributed solely to the ventral pancreas first appeared at the AIP lip from 5 ss. At 5-6 ss, cells from the medial of the AIP lip contributed to the ventral pancreas. The pancreas fate region become narrower as development progresses. At 7-9 ss, the cells contributing to the ventral pancreas resided in a narrow region of the AIP lip. From 5 ss, the right flanking region contributes to the posterior gut, and the left flanking region contributes to the anterior gut. Dorsal pancreatic progenitors originate from the dorsal endoderm at the 3-6 somite level at 7-9 ss, though they have not yet diverged from the dorsal gut progenitors at this stage.     

casanova plays an early and essential role in endoderm formation in zebrafish.

The cellular and molecular mechanisms that regulate endoderm development in vertebrates have only recently begun to be explored. Here we show that the zebrafish locus casanova plays an early and essential role in this process. casanova mutants lack a gut tube and do not express any molecular markers of endoderm differentiation. The early endodermal expression of genes such as axial, gata5, and fkd2 does not initiate in casanova mutants, indicating that the endoderm is defective from the onset of gastrulation. Mosaic analysis demonstrates that casanova functions cell autonomously within the endodermal progenitors. We also report the isolation of a zebrafish homologue of Mixer, a gene important for early endoderm formation in Xenopus. casanova does not encode zebrafish Mixer, and mixer expression is normal in casanova mutants, indicating that casanova acts downstream of, or parallel to, mixer to promote endoderm formation. We further find that the forerunner cells, a specialized group of noninvoluting dorsal mesendodermal cells, do not form in casanova mutants. Studies of casanova mutants do not support an important role for the forerunner cells in either dorsal axis or tail development, as has been previously proposed. In addition, although different populations of mesodermal precursors are generated normally in casanova mutants, morphogenetic defects in the heart, vasculature, blood, and kidney are apparent, suggesting a possible role for the endoderm in morphogenesis of these organs.     

A revised fate map for amphioxus and the evolution of axial patterning in chordates

The chordates include vertebrates plus two groups of invertebrates(the cephalochordates and tunicates). Previous embryonic fatemaps of the cephalochordate amphioxus (Branchiostoma) were influencedby preconceptions that early development in amphioxus and ascidiantunicates should be fundamentally the same and that the earlyamphioxus embryo, like that of amphibians, should have ventralmesoderm. Although detailed cell lineage tracing in amphioxushas not been done because of limited availability of the embryosand because cleavage is radial and holoblastic with the blastomeresnearly equal in size and not tightly adherent until the mid-blastulastage, a compilation of data from gene expression and function,blastomere isolation and dye labeling allows a more realisticfate map to be drawn. The revised fate map is substantiallydifferent from that of ascidians. It shows (1) that the anteriorpole of the amphioxus embryo is offset dorsally from the animalpole only by about 20°, (2) that the ectoderm/mesendodermboundary (the future rim of the blastopore) is at the equatorof the blastula, which approximately coincides with the 3rdcleavage plane, and (3) that there is no ventral mesoderm duringthe gastrula stage. Involution or ingression of cells over theblastopore lip is negligible, and the blastopore, which is posterior,closes centripetally as if by a purse string. During the gastrulastage, the animal pole shifts ventrally, coming to lie about20° ventral to the anterior tip of the late gastrula/earlyneurula. Comparisons of the embryos of amphioxus and vertebratesindicate that in spite of large differences in the mechanicsof cleavage and gastrulation, anterior/posterior and dorsal/ventralpatterning occur by homologous genetic mechanisms. Therefore,the small, nonyolky embryo of amphioxus is probably a reasonableapproximation of the basal chordate embryo before the evolutionof determinate cleavage in the tunicates and the evolution largeamounts of yolk in basal vertebrates.     

Primary Invagination of the Vegetal Plate During Sea Urchin Gastrulation

The initial phase of echinoid gastrulation, primary invagination,involves an inpocketing of a monolayered epithelium. To gaininformation about the nature of the mechanical forces that areresponsible for primary invagination, several experimental approacheshave been taken, using the transparent embryos of the sea urchin,Lytechinus pictus, as the principal material. Vegetal platesisolated microsurgically well before the onset of gastrulationwill invaginate normally, demonstrating that the forces responsiblefor primary invagination are generated by the cells in the vegetal to of the embryo. As shown by serial reconstructions of L.pictus embryos, relatively few cells (about 100) take part inprimary invagination. Both the number of cells and the totalvolume of tissue in the wall of the archenteron increase withtime. Even so, it can be shown that very little movement ofcells over the lip of the blastopore takes place during primaryinvagination, and this process is best viewed as a simple inpocketingof the vegetal epithelium. The cells in the wall of the archenteronhave a distinctive shape; they are elongated along their apico-basalaxes and frequently have enlarged, rounded, basal ends. However,they do not undergo any dramatic changes in shape during primaryinvagination. In particular, there is only a slight decreasein the height of the cells (length along the apico-basal axis),a result that is inconsistent with the hypothesis that invaginationis due to cell rounding (Gustafson and Wolpert, 1967). Examinationof L. pictus and Strongylocentrotus purpuratus gastrulae bytransmission electron microscopy reveals that cells in the wallof the archenteron continue to be joined by typical junctionalcomplexes during primary invagination. In addition, the morphologyof the junctional complex at the gastrula stage is more elaboratethan previously described. Sparse bands of micronlaments areassociated with the plasma membrane at the level of the junctionalcomplexes in both endodermal and ectodermal cells. These andother relevant data on early echinoid gastrulation are discussedin relation to several possible mechanisms of epithelial morphogenesis.     

Gastrulation in Calcareous Sponges: In Search of Haeckel's Gastraea

Haeckel's studies of development in calcareous sponges (1872)led him to develop the "Gastraea Theory," which proposes thatthe ancestral mode of germ layer formation, or gastrulation,was by invagination to produce a functional gut. His observationsthat gastrulation in the Calcarea occurs by invagination ofa ciliated larva upon settlement and metamorphosis were supportedby remarkable photomicrographs of the stage by Hammer in 1908.Although no later work found the same stage, these conceptsare repeated in texts today. We have re-examined embryogenesisand metamorphosis in Sycon sp. cf. S. raphanus in order to understandwhen gastrulation occurs. Almost all larvae settle on theirciliated anterior pole and metamorphose into a bilayered juvenilewhose interior cells rapidly differentiate into choanocytesand other cells of the young sponge. After a four-year searchwe have found the transitory stage shown by Hammer in whichthe anterior cells invaginate into the posterior half of thelarva. The hole closes and it is not until some days later thatthe sponge forms an osculum at its apical pole. To understandwhether invagination comprises gastrulation and if the holecan be considered to be a blastopore we have carried out a reviewof the literature dealing with this brief moment in calcaroneansponge development. Despite the intrigue of this type of metamorphosis,we conclude that gastrulation occurs earlier, during formationof the two cellular regions of the larva, and that metamorphosisinvolves the reorganization of these already differentiatedregions. Considering the pivotal position occupied by the Calcareaas the possible sister-group to all other Metazoa, these resultscall for a reassessment of germ layer formation and of the relationshipsof the primary germ layers among basal metazoan phyla.     

In vitro organogenesis of pancreas in Xenopus laevis dorsal lips treated with retinoic acid

Dorsal lips of Xenopus laevis may differentiate into pancreas after treatment with retinoic acid in vitro. The dorsal lip region is fated to be dorsal mesoderm and anterior endoderm. Dorsal lip cells isolated from stage 10 early gastrula differentiate into tissues such as notochord, muscle and pharynx. However, in the present study, dorsal lips treated with 10(-4) M retinoic acid for 3 h differentiated into pancreas-like structures accompanied by notochord and thick endodermal epithelium. Sections of the explants showed that some cells gathered and formed an acinus-like structure as observed under microscopes. In addition to the morphological changes, expressions of the pancreas-specific molecular markers, XIHbox8 and insulin, were induced in retinoic acid-treated dorsal lip explants. Therefore, it is suggested that retinoic acid may induce the dorsal lip cells to differentiate into a functional pancreas. However, continuous treatment with retinoic acid did not induce pancreas differentiation at any concentration. Dorsal lips treated with retinoic acid within 5 h after isolation differentiated into pancreas-like cells, while those treated after 15 h or more did not. The present study provided a suitable test system for analyzing pancreas differentiation in early vertebrate development.     

Fate and plasticity of the endoderm in the early chick embryo

In vertebrates, the endoderm is established during gastrulation and gradually becomes regionalized into domains destined for different organs. Here, we present precise fate maps of the gastrulation stage chick endoderm, using a method designed to label cells specifically in the lower layer. We show that the first population of endodermal cells to enter the lower layer contributes only to the midgut and hindgut; the next cells to ingress contribute to the dorsal foregut and followed finally by the presumptive ventral foregut endoderm. Grafting experiments show that some migrating endodermal cells, including the presumptive ventral foregut, ingress from Hensen's node, not directly into the lower layer but rather after migrating some distance within the middle layer. Cell transplantation reveals that cells in the middle layer are already committed to mesoderm or endoderm, whereas cells in the primitive streak are plastic. Based on these results, we present a revised fate map of the locations and movements of prospective definitive endoderm cells during gastrulation.     

Formation of germ layers and roles of the dorsal lip of the blastopore in normally developing embryos of the newt Cynops pyrrhogaster

Three-dimensional relationships between tissues during the formation of germ layers were studied in sections of normally developing embryos of the newt, Cynops pyrrhogaster. In gastrulae, the inner postinvolution layer was not in direct contact with the outer preinvolution layer as a result of the presence of an intervening layer of cells. Only after the formation of the yolk plug, a narrow strip of primitive notochord, which consisted of columnar cells, established a close contact with the central part of the overlaying presumptive neural plate. The primitive notochord was also linked to endoderm at its right and left margins, facing the archenteron. Mesodermal cells other than notochord cells were mesenchymal until the neurula stage, when primitive somites appeared on both sides of the notochord. From a comparison of the relative locations of tissues in embryos at different stages of development, it was shown that the notochord elongates by a remodeling of the mass of the primitive notochord, and that, as the anteriorly directed translocation of the neural area and the invagination of endoderm occur, these processes keep pace with the elongation of the notochord. These observations suggest organizing or guiding roles for the notochord in the formation of germ layers. A role for the dorsal lip of the blastopore as the organizer is discussed in relation to the origin of the notochord.     

Mechanical Coupling between Endoderm Invagination and Axis Extension in Drosophila

How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.     

Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice

During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the retinaldehyde dehydrogenase 2 (Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells. Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx 1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to Pdx 1-deficient mice, the early glucagon-expressing cells do not develop in Raldh2 knockout embryos. Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodomain protein Isl 1, required for the formation of the dorsal mesenchyme, is altered in Raldh2--/-- embryos. The homeobox gene Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in Raldh2--/-- dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx 1 and mesenchymal Isl 1 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx 1 function.     

Opposing Nodal/Vg1 and BMP signals mediate axial patterning in embryos of the basal chordate amphioxus

The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution.     

A conserved role for FGF signaling in chordate otic/atrial placode formation

The widely held view that neurogenic placodes are vertebrate novelties has been challenged by morphological and molecular data from tunicates suggesting that placodes predate the vertebrate divergence. Here, we examine requirements for the development of the tunicate atrial siphon primordium, thought to share homology with the vertebrate otic placode. In vertebrates, FGF signaling is required for otic placode induction and for later events following placode invagination, including elaboration and patterning of the inner ear. We show that results from perturbation of the FGF pathway in the ascidian Ciona support a similar role for this pathway: inhibition with MEK or Fgfr inhibitor at tailbud stages in Ciona results in a larva which fails to form atrial placodes; inhibition during metamorphosis disrupts development of the atrial siphon and gill slits, structures which form where invaginated atrial siphon ectoderm apposes pharyngeal endoderm. We show that laser ablation of atrial primordium ectoderm also results in a failure to form gill slits in the underlying endoderm. Our data suggest interactions required for formation of the atrial siphon and highlight the role of atrial ectoderm during gill slit morphogenesis.     

Spemann's organizer--it's origin and derivatives (cellular-tissue and molecular-genetic aspects)

In 1924 H. Spemann and H. Mangold discovered that a piece of the dorsal lip of a blastopore from Triturus cristatus, after transplantation to the ventral side of another embryo, was able to cause the neighbouring tissues to change their fate and participate in the formation of a new embryo. The dorsal lip was termed "the organizer". Since then, for as long as 75 years, attempts have been made to establish the intimate mechanisms of the organizer activity. However, no real advance was achieved in their understanding. Within the last 15 years, genetic and molecular techniques have been vastly improved, to help in tracing the fate of many cell lineages, and in compiling more exactly the fate maps for different parts of the embryo. Using these data, I have attempted to trace the fate of Spemann's organizer after the early gastrula stage. Analysis of data on inductive abilities of the organizer cells, on the use of markers, and on the observation of expression of specific genes allowed to conclude that Spemann's organizer in amphibia and its homologues in other vertebrates too are heterogeneous: they are composed of distinct cell populations able to induce primarity the development of either the head or trunk parts of the embryo. These population, determined to become the head of the trunk organizers still at the blastula stage, may be located either in the single continuous cell layer (as in amphibia and birds) or separated among different tissue germs (as in mammals). When the dorsal-ventral orientation of the embryo is established and the organizer is switched on the very early invaginating cells of the dorsal blastopore lip (in the case of amphibia) move in advance of the entire invaginating mesoderm and by the end of gastrulation occupy the place just in front of the notochord. It is supposed that the early dorsal lip and the prechordal mesoderm (PCM) are one and the same cell population, i.e. during gastrulation Spemann's organizer transfers from the lip of blastopore to the prechordal zone. The PCM seems to play an exclusive role in the formation of a head in vertebrate, because some mutations in genes expressed in the PCM result in the entire head deletion. It is supposed that spreading of differentiating signals from the PCM occurs along the main body axis in both caudal and rostral directions. After the main body plan formation the PCM is replaced by adenohypophysis. This conclusion is drawn not only from the same topology of both these structures, but also from the similarities of a set of specific genetical markers expressed in these, that makes it possible to suppose the existence of deep connections and succession between them. The adenohypophysis seems to arise directly from the PCM, or cells of the ectoderm influenced by the PCM may be subsequently transformed into humoral cells of adenohypophysis. In this interpretation, adenohypophysis and the much earlier established PCM may be considered as derivatives of Spemann's organizer. This inference is supported by the fact that all the three above structures first originate in vertebrates only.     

Early Development of the Hagfish Pituitary Gland: Evidence for the Endodermal Origin of the Adenohypophysis

The adenohypophysis in early head fold stage hagfish embryosis at first a distinct differentiated thickening of the archentericroof where it contacts the developing infundibular portion ofthe brain. This portion of the archenteron eventually becomesthe dorsal epithelium of the nasopharyngeal duct. The laterdevelopment of the adenohypophysis involves formation of multipleacinar outgrowths of the dorsal nasopharyngeal epithelium whichtogether form a layered mass of follicular tissue adjacent tothe neurohypophysis. This mode of origin of the adenohypophysisby endodermal polyinvagination and delamination differs fromall other vertebrates, including the lampreys. The pertinenceof this new information for considerations of monophyletic versusdiphyletic evolutionary origins of the modern cyclostome groupsis pointed out. The unusual development of the hagfish adenohypophysispresents some new and unanticipated puzzles within the generalquestion of pituitary evolution.     

Embryonic development in two species of scleractinian coral embryos: Symbiodinium localization and mode of gastrulation

Reef-building scleractinian corals widely engage in symbiotic relationships with Symbiodinium dinoflagellates (zooxanthellae), which reside inside cells of the gastrodermis. In most cases, sexually produced larvae acquire their symbionts from the environment in the early developmental stages preceding settlement; however, some scleractinian corals maternally "seed" their oocytes with symbionts, and these symbionts are reported to be restricted to the gastrodermis at the time of its formation (gastrulation). A precise mechanism for how Symbiodinium are translocated to endoderm in these seeded species was previously unknown. In order to examine the process of endoderm formation and Symbiodinium localization during gastrulation, we have examined two species of "robust" clade scleractinians: Fungia scutaria (nonseeded) and Pocillopora meandrina (maternally seeded). We determined that both species, independent of whether or not they are seeded, undergo a "nutritive" stage before gastrulation, wherein lipid-rich cells (F. scutaria) or membrane-bound cellular fragments (P. meandrina) are passed to the blastocoel where they are subsequently taken up by the definitive endoderm. This emergent property of anthozoan development has been co-opted to facilitate the movement of Symbiodinium to the blastocoel (future site of endoderm), in the seeded species, where they are later phagocytosed by the newly formed definitive endoderm. Additionally, both species of robust clade scleractinians examined gastrulate by way of invagination, as do the majority of anthozoans. This invagination differs from the prawn chip-type gastrulation seen in the complex clade corals and provides evidence for a possible linkage between gastrulation type and phylogenetic history.