The role of the basal lamina in mouth formation in the embryo of the starfish Pisaster ochraceus

Details of mouth formation in normal and exogastrulated Pisaster ochraceus larvae have been studied by light microscopy and transmission and scanning electron microscopy. As the archenteron begins to bend, the cells in the presumptive mouth region dissociate and migrate into the blastocoele where they become mesenchyme cells. This leaves a defect in the “blind” endodermal tube, which is covered by a basal lamina. Subsequently this exposed basal lamina bulges to form a blister which appears to extend across the blastocoele to make contact with spikelike projections from the future stomodeal region of the ectoderm. Mesenchyme cell processes are associated with both the basal lamina blister and the ectoderm in this region and may provide both motive power and guidance for contact. Shortly after contact is made the blister of basal lamina from the endoderm fuses with the basal lamina of the ectodermal cells and the ectoderm begins to invaginate. At this time the lateral walls of the presumptive oesophagus are largely formed of naked basal lamina with some loosely associated cells on the endodermal side. Eventually the lateral walls of the proximal part of the oesophagus become cellular, giving rise to an epithelium. A cell plug located between the stomodeum and oesophagus persists for some time before finally breaking down to complete the larval digestive tract. Experiments with exogastrulae suggest that many of these developmental patterns are determined before gastrulation.     

Local cell interactions and the control of gastrulation in the sea urchin embryo

The sea urchin embryo is a good model system for studying the role of mechanical and cell-cell interactions during epithelial invagination, cell rearrangement and mesenchymal patterning in the gastrula. The mechanisms underlying the initial invagination of the archenteron have been surprisingly elusive; several possible mechanisms are discussed. In contrast to its initial invagination, the cellular basis for the elongation of the archenteron is better understood: both autonomous epithelial cell rearrangement and further rearrangement driven by secondary mesenchyme cells appear to be involved. Experiments indicate that patterning of freely migrating primary mesenchyme cells and secondary mesenchyme cells residing in the tip of the archenteron relies to a large extent on information resident in the ectoderm. Interactions between cells in the early embryo and later cell-cell interactions are both required for the establishment of ectodermal pattern information. Surprisingly, in the case of the oral ectoderm the fixation of pattern information does not occur until immediately prior to gastrulation.     

Cellular basis of gastrulation in the sand dollar Scaphechinus mirabilis

The processes of gastrulation in the sand dollar Scaphechinus mirabilis are quite different from those in regular echinoids. In this study, we explored the cellular basis of gastrulation in this species with several methods. Cell-tracing experiments revealed that the prospective endodermal cells were convoluted throughout the invagination processes. Histological observation showed that the ectodermal layer remained thickened, and the vegetal cells retained an elongated shape until the last step of invagination. Further, most of the vegetal ectodermal cells were skewed or distorted. Wedge-shaped cells were common in the vegetal ectoderm, especially at the subequatorial region. In these embryos, unlike the embryos of regular echinoids, secondary mesenchyme cells did not seem to exert the force to pull up the archenteron toward the inner surface of the apical plate. In fact, the archenteron cells were not stretched along the axis of elongation and were in close contact with each other. Here we found that gastrulation was completely blocked when the embryos were attached to a glass dish coated with poly-L-lysine, in which the movement of the ectodermal layer was inhibited. These results suggest that a force generated by the thickened ectoderm, rather than rearrangement of the archenteron cells, may play a key role in the archenteron elongation in S. mirabilis embryos.     

The origin of pigment cells in embryos of the sea urchin Strongylocentrotus purpuratus

A monoclonal antibody (SP1/20.3.1) that recognizes a cell surface epitope expressed by pigment cells in the pluteus larva of Strongylocentrotus purpuratus has been produced. Using indirect immunofluorescence, the epitope is first detected in nonpigmented cells of the vegetal plate after primary mesenchyme ingression. Between the beginning of gastrulation, and when the archenteron is one-third the distance across the blastocoel, SP1/20.3.1-positive cells are free within the blastocoel, at the tip of the archenteron, and dispersed within the blastoderm. Cells at the tip of the archenteron, and mesenchyme near the tip in later stages of gastrulation (secondary mesenchyme), do not express the SP1/20.3.1 antigen. By the completion of gastrulation all SP1/20.3.1-positive cells are dispersed throughout the epidermis. It has been concluded that in S. purpuratus pigment cell precursors are released from the vegetal plate during the initial phase of gastrulation. The cells migrate first to the vegetal ectoderm, and subsequently disperse throughout the ectoderm and develop pigment granules.     

Ultrastructural Peculiarities of the Embryogenesis of the Brittle Star Amphipholis kochii (Lutken, 1972)

This paper addresses morphogenetic processes and cell differentiation during embryogenesis of the brittle star Amphipholis kochii at the ultrastructural level. The radial cleavage is not strictly determined. Embryos are covered with a thick hyaline envelope and contain numerous yolk granules and small lipid drops. Blastulae feature a thick blastoderm with extensive intercellular cavities, which are retained in the crest epithelium of late gastrulae. Embryonic cells have single cilia with long cross-striated rootlets associated with the Golgi apparatus. Depolarized cells of the primary mesenchyme with a well-developed rough endoplasmic reticulum differentiate into sclerenchyme syncytium. Gastrulation occurs by invagination. Secondary mesenchymal cells emigrate from the archenteron tip to differentiate into amebocytes, which contain a well-developed Golgi apparatus and numerous mitochondria. The endoderm is formed of cubic cells with numerous yolk granules and rare microvilli. Flattened cells of the dorsal and ventral ectoderm contain a small amount of yolk. Yolk utilization during embryogenesis occurs by intracellular lysosomal digestion with selective exocytosis of toposomes.Original Russian Text Copyright © 2005 by Biologiya Morya, Gliznutsa, Dautov.     

Spatio-temporal expression of pamlin during early embryogenesis in sea urchin and importance of N-linked glycosylation for the glycoprotein function

Expression of pamlin, a heterotrimeric primary mesenchyme cell (PMC) adhesion glycoprotein, and its role during early embryogenesis were examined using immunochemistry and microinjection of pamlin to tunicamycin-treated embryos of the sea urchin, Hemicentrotus pulcherrimus. Pamlin faintly detected in egg cortex before fertilization was strongly expressed in the hyaline layer after fertilization. The embryonic apical surface retained pamlin throughout early embryogenesis, whereas pamlin on the basal surface showed a dynamic change of spatio-temporal distribution from morula to gastrula stage. Pamlin distributed on the entire basal surface of the ectoderm before onset of invagination gradually disappeared from the presumptive archenteron during gastrulation, and then was restricted to the apical tuft region and the PMC sessile sites in early gastrulae. Tunicamycin, an inhibitor of N-glycosydically linked carbohydrate formation, inhibited PMC migration and gastrulation. Tunicamycin also inhibited the assembly of mannose moieties of 180 and 52 kDa subunits of pamlin. Pamlin microinjection to the tunicamycin-treated embryos rescued them from this morphogenetic disturbance. PMCs did not bind to pamlin isolated from the tunicamycin-treated embryos. The present study indicated that pamlin plays an essential role in PMC migration, its termination and gastrulation, and the presence of N-glycosydically linked carbohydrate moieties that contain mannose are necessary to preserve the biological function of pamlin.     

Pattern of Brachyury gene expression in starfish embryos resembles that of hemichordate embryos but not of sea urchin embryos.

Echinoderms, hemichordates and chordates are deuterostomes and share a number of developmental features. The Brachyury gene is responsible for formation of the notochord, the most defining feature of chordates, and thus may be a key to understanding the origin and evolution of the chordates. Previous studies have shown that the ascidian Brachyury (As-T and Ci-Bra) is expressed in the notochord and that a sea urchin Brachyury (HpTa) is expressed in the secondary mesenchyme founder cells. A recent study by [Tagawa et al. (1998)], however, revealed that a hemichordate Brachyury (PfBra) is expressed in a novel pattern in an archenteron invagination region and a stomodaeum invagination region in the gastrula. The present study demonstrated that the expression pattern of Brachyury (ApBra) of starfish embryos resembles that of PfBra in hemichordate embryos but not of HpTa in sea urchin embryos. Namely, ApBra is expressed in an archenteron invagination region and a stomodaeum invagination region.     

Archenteron elongation in the sea urchin embryo is a microtubule-independent process

Earlier studies using colchicine (L. G. Tilney and J. R. Gibbins, 1969, J. Cell Sci. 5, 195-210) had suggested that intact microtubules (MTs) are necessary for archenteron elongation during the second phase of sea urchin gastrulation (secondary invagination), presumably by allowing secondary mesenchyme cells (SMCs) to extend their long filopodial processes. In light of subsequently discovered effects of colchicine on other cellular processes, the role of MTs in archenteron elongation in the sea urchin, Lytechinus pictus, has been reexamined. Immunofluorescent staining of ectodermal fragments and isolated archenterons reveals a characteristic pattern of MTs in the ectoderm and endoderm during gastrulation. Ectodermal cells exhibit arrays of MTs radiating away from the region of the basal body/ciliary rootlet and extending along the periphery of the cell, whereas endodermal cells exhibit a similar array of peripheral MTs emanating from the region of the apical ciliary rootlet facing the lumen of the archenteron. MTs are found primarily at the bases of the filopodia of normal SMCs. beta-Lumicolchicine (0.1 mM), an analog of colchicine which does not bind tubulin, inhibits secondary invagination, indicating that the effects previously ascribed to the disruption of MTs are probably due to the effects of colchicine on other cellular processes. The MT inhibitor nocodazole (5-10 micrograms/ml) added prior to secondary invagination does not prevent gastrulation or spontaneous exogastrulation, even though indirect immunofluorescence indicates that cytoplasmic MTs are completely disrupted in drug-treated embryos. Transverse tissue sections indicate that a comparable amount of cell rearrangement occurs in nocodazole-treated and control embryos. Significantly, SMCs in nocodazole-treated embryos often detach prematurely from the tip of the gut rudiment and extend abnormally large broad lamellipodial protrusions but are also capable of extending long slender filopodia comparable in length to those of control embryos. These results indicate that cytoplasmic MTs are not essential for either filopodial extension by SMCs or for the active epithelial cell rearrangement which accompanies elongation during sea urchin gastrulation.     

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.     

Jun N‐terminal kinase activity is required for invagination but not differentiation of the sea urchin archenteron

Although sea urchin gastrulation is well described at the cellular level, our understanding of the molecular changes that trigger the coordinated cell movements involved is not complete. Jun N‐terminal kinase (JNK) is a component of the planar cell polarity pathway and is required for cell movements during embryonic development in several animal species. To study the role of JNK in sea urchin gastrulation, embryos were treated with JNK inhibitor SP600125 just prior to gastrulation. The inhibitor had a limited and specific effect, blocking invagination of the archenteron. Embryos treated with 2 μM SP600125 formed normal vegetal plates, but did not undergo invagination to form an archenteron. Other types of cell movements, specifically ingression of the skeletogenic mesenchyme, were not affected, although the development and pattern of the skeleton was abnormal in treated embryos. Pigment cells, derived from nonskeletogenic mesenchyme, were also present in SP600125‐treated embryos. Despite the lack of a visible archenteron in treated embryos, cells at the original vegetal plate expressed several molecular markers for endoderm differentiation. These results demonstrate that JNK activity is required for invagination of the archenteron but not its differentiation, indicating that in this case, morphogenesis and differentiation are under separate regulation. genesis 53:762–769, 2015. © 2015 Wiley Periodicals, Inc.     

Gastrulation in the Sea Urchin,Strongylocentrotus purpuratus,Is Disrupted by the Small Laminin Peptides YIGSR and IKVAV

Laminin is present on the apical and basolateral sides of epithelial cells of very early sea urchin blastulae. We investigated whether small laminin-peptides, known to have cell binding activities, alter the development of sea urchin embryos. The peptide YIGSR-NH₂ (850 μM) and the peptide PA22-2 (5 μM), which contains the peptide sequence IKVAV (Tashiro et al., J. Biol. Chem. 264, 16174, 1989), typically blocked archenteron formation when added to the sea water soon after fertilization. At lower doses, the YIGSR peptide allowed invagination of the archenteron but blocked archenteron extension and differentiation and evagination of the feeding arms. The effect of YIGSR and PA22-2 peptides declined when added to progressively older stages until no effect was seen when added at the mesenchyme blastula stage (24 hours after fertilization). Control peptides GRGDS, YIGSE, and SHA22, a dodeca-peptide with a scrambled IKVAV sequence, had no effect on development. The YIGSK peptide containing a conserved amino acid modification had only a small effect on gastrulation. The results suggest that YIGSR and IKVAV peptides specifically disrupt cell/extracellular matrix interactions required for normal development of the archenteron and feeding arms. Our recent finding that YTGIR is at the cell binding site of the B1 chain of S. purpuratus laminin supports this conclusion. Evidently, laminin or other laminin-like molecules are among the many extracellular matrix components needed for the invagination and extension of the archenteron during the gastrulation movements of these embryos.     

Skeletogenic potential of induced secondary mesenchyme cells derived from the presumptive ectoderm in echinoid embryos

 During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC. Received: 14 November 1996 / Accepted: 30 December 1996     

TEMPORAL RELATIONS BETWEEN EXTENSION OF ARCHENTERON ROOF AND REALIZATION OF NEURAL INDUCTION DURING GASTRULATION OF NEWT EMBRYO

This investigation was performed in order to analyze the basic relationships between the archenteron roof and the overlying ectoderm in primary induction in the Cynopus (Triturus) pyrrhogaster embryo.
The part of the archenteron roof that is active in inducing capacity extends linearly after invagination at the speed of 0.15 mm per hr at 23°C until stage 13b. The period of contact at each position of the presumptive neuro-ectoderm with the active archenteron roof could be estimated by the formula described in the Discussion.
Pieces of the presumptive neuro-ectoderm were isolated from gastrulae at three developmental stages and cultured separately in Holtfreter solution after being divided caudo-cranially into 4 parts. The result showed that some of them were able to differentiate into neural tissues even in the mid-gastrula stage and that the presumptive neuro-ectoderm acquired the capacity to differentiate into neural tissue along a caudocranial axis from the part adjacent to the blastopore during gastrulation.
It could be estimated that 3 hr of contact with the active archenteron roof is sufficient for the presumptive neuro-ectoderm to differentiate into neural tissue.
The present study also showed that the neuralizing capacity of the whole prospective neuro-ectodermal area has already been determined before the end of stage 13, i.e., within less than 14 hr after first contact of the ectoderm with the active archenteron roof at 23°C.     

Behavior of pigment cells closely correlates the manner of gastrulation in sea urchin embryos

To know whether behavior of pigment cells correlates the process of gastrulation or not, gastrulating embryos of several species of regular echinoids (Anthocidaris crassispina, Mespilia globulus and Toxopneustes pileolus) and irregular echinoids (Clypeaster japonicus and Astriclypeus manni) were examined. In M. globulus and A. crassispina, the archenteron elongated stepwise like in well-known sea urchins. In the embryos of both species, fluorescent pigment cells left the archenteron tip and migrated into the blastocoel during gastrulation. In T. pileolus, C. japonicus and A. manni, on the other hand, the archenteron elongated at a constant rate throughout gastrulation. In these species, no pigment cell was observed at the archenteron tip during invagination processes; pigment cells began to migrate in the ectoderm from the vegetal pole side toward the apical plate without entering the blastocoel. These results clearly indicate that the behavior of pigment cells closely correlated the manner of gastrulation. Further, it was examined whether the archenteron cells are rearranged during invagination, by comparing the number of cells observed on cross sections of the archenteron at the early and late gastrula stages. The rearrangement was not conspicuous in A. crassispina and M. globulus, in which archenteron elongated stepwise. In contrast, the archenteron cells were remarkably rearranged in C. japonicus, alothough the archenteron elongated continuously. Thus, neither the behavior of pigment cells nor the manner of gastrulation matches the current taxonomic classification of echinoids.     

Germ Layer Interactions in Pattern Formation of Amphibian Mesoderm during Primary Embryonic Induction

Mesodermal differentiation of dorsal marginal zone (DMZ) before and after invagination was analyzed by a series of combination experiments with different kinds of ectoderm.
Lower DMZ of early gastrula didn't show any axial-mesoderm (notochord and somitic mesoderm) but lateral mesoderm (mesenchyme, mesothelium, or blood cells) in combinant with non-competent ventral ectoderm, while combinant with competent ectoderm was found to have well-differentiated axial-mesoderm with deutero-spinocaudal neurals. The axial-mesoderms have origin in the ectoderm. Uninvaginated DMZ of middle gastrula also showed difference in mesodermal differentiation between competent and non-competent ectoderms; axial-mesoderm differentiation was much better in competent than in non-competent. The axial-mesoderm originated from the uninvaginated DMZ. Archenteron roof of late gastrula showed regional difference in mesodermal differentiation in both combinants with competent and non-competent. The present study further demonstrated that there was regionality in promoting effect of induced neurectoderm on axial-mesoderm differentiation of invaginated archenteron roof.
The present experiments suggest that the cranio-caudal and dorso-ventral axis formations of amphibian mesoderm are finally determined by sequential and reciprocal interactions between the mesodermal anlage and the overlying ectoderm. It should be also shown that lower DMZ acts to trigger a series of the sequential interactions during primary embryonic induction.     

The structure and activities of echinonectin: a developmentally regulated cell adhesion glycoprotein with galactose-specific lectin activity.

The extracellular matrix of the sea urchin embryo contains a 230 kD homodimeric glycoprotein known as echinonectin (EN). EN contains a cell attachment domain as well as a galactose-specific lectin activity. Cell attachment to EN is differentially regulated in the three primary germ layers, endoderm, ectoderm and mesoderm. Prior to gastrulation all embryonic cells adhere equally to EN-coated substrates, but during gastrulation primary mesenchyme cells lose affinity for EN, ectoderm cells increase their binding to the molecule, and cells of the endoderm maintain a similar or slightly lowered level of binding. The mechanisms governing these adhesive changes and the specific functions they serve in development are not currently understood. They are timed to coincide with distinct morphogenetic events such as primary mesenchyme cell ingression and archenteron formation, suggesting that regulated adhesion to EN plays at least a permissive role in early morphogenesis.     

Mesendoderm cells induce oriented cell division and invagination in the marine shrimp Sicyonia ingentis

The mesendoderm (ME) cells are the two most vegetal blastomeres in the early developing embryo of the marine shrimp Sicyonia ingentis. These two cells enter mitotic arrest for three cycles after the 5th cell cycle (32-cell stage) and ingress into the blastocoel at the 6th cycle (62-cell stage). Circumjacent to the ingressing ME cells are nine presumptive naupliar mesoderm (PNM) cells that exhibit a predictable pattern of spindle orientation into the blastopore, followed by invagination. We examined the role of ME cells and PNM cells in gastrulation using blastomere recombinations and confocal microscopy. Removal of ME progenitors prevented gastrulation. Removal of any other blastomeres, including PNM progenitors, did not interfere with normal invagination. Altered spindle orientations occurred in blastomeres that had direct contact with one of the ME cells; one spindle pole localized to the cytoplasmic region closest to ME cell contact. In recombined embryos, this resulted in an extension of the region of ME-embryo contact. Our results show that ME cells direct the spindle orientations of their adjacent cells and are consistent with a mechanism of oriented cell division being a responsible force for archenteron elongation.