Cleavage and gastrulation in the shrimp Penaeus (Litopenaeus) vannamei (Malacostraca, Decapoda, Dendrobranchiata)

While most malacostracan crustaceans develop through superficial cleavage, in the Amphipoda, Euphausiacea, and Dendrobranchiata (Decapoda) cleavage is complete. Euphausiaceans and dendrobranchiate shrimp share a similar early cleavage pattern, early cleavage arrest and ingression of mesendoderm progenitor cells, a ring of crown cells (prospective naupliar mesoderm) around the blastopore, and hatching as a nauplius larva. Yet recent phylogenies do not support a close relationship between Euphausiacea and Decapoda. In addition, some variation is reported in the timing of mesendoderm cell arrest and number of crown cells for a number of dendrobranchiates. To determine the representative pattern of development in the Dendrobranchiata, embryos of the Pacific white shrimp Penaeus (Litopenaeus) vannamei were stained with Sytox Green to label chromosomes and nuclei and examined with confocal microscopy. The early cleavage pattern, mesendoblast arrest and subsequent ingression at the 32-cell stage, presence of 8 initial crown cells, and fates of the mesendoblasts are the same for P. vannamei (family Peneaeidae) and Sicyonia ingentis (family Sicyoniidae). The lineage of the primordial endoderm cells differs from that reported for P. kerathurus. These characters were discussed in the context of the evolution of development in the Dendrobranchiata and in comparison to the Euphausiacea.     

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.     

Cleavage and gastrulation in the shrimp Sicyonia ingentis: invagination is accompanied by oriented cell division.

Embryos of the penaeoidean shrimp Sicyonia ingentis were examined at intervals during cleavage and gastrulation using antibodies to beta-tubulin and DNA and laser scanning confocal microscopy. Cleavage occurred in a regular pattern within four domains corresponding to the 4-cell-stage blastomeres and resulted in two interlocking bands of cells, each with similar spindle orientations, around a central blastocoel. Right-left asymmetry was evident at the 32-cell-stage, and mirror-image embryos occurred in a 50:50 ratio. Gastrulation was initiated by invagination into the blastocoel at the 62-cell-stage of two mesendoderm cells, which arrested at the 32-cell-stage. Further invagination and expansion of the archenteron during gastrulation was accompanied by rapid and oriented cell division. The archenteron was composed of presumptive naupliar mesoderm and the blastopore was located at the site of the future anus of the nauplius larva. In order to trace cell lineages and determine axial relationships, single 2- and 4-cell-stage blastomeres were microinjected with rhodamine-dextran. The results showed that the mesendoderm cells which initiated gastrulation were derived from the vegetal 2-cell-stage blastomere, which could be distinguished by its slightly larger size and the location of the polar bodies. The mesendoderm cells descended from a single vegetal blastomere of the 4-cell-stage. This investigation provides the first evidence for oriented cell division during gastrulation in a simple invertebrate system. Oriented cell division has previously been discounted as a potential morphogenetic force, and may be a common mechanism of invagination in embryos that begin gastrulation with a relatively small number of cells.     

Conserved patterns of cell movements during vertebrate gastrulation

Vertebrate embryogenesis entails an exquisitely coordinated combination of cell proliferation, fate specification and movement. After induction of the germ layers, the blastula is transformed by gastrulation movements into a multilayered embryo with head, trunk and tail rudiments. Gastrulation is heralded by formation of a blastopore, an opening in the blastula. The axial side of the blastopore is marked by the organizer, a signaling center that patterns the germ layers and regulates gastrulation movements. During internalization, endoderm and mesoderm cells move via the blastopore beneath the ectoderm. Epiboly movements expand and thin the nascent germ layers. Convergence movements narrow the germ layers from lateral to medial while extension movements elongate them from head to tail. Despite different morphology, parallels emerge with respect to the cellular and genetic mechanisms of gastrulation in different vertebrate groups. Patterns of gastrulation cell movements relative to the blastopore and the organizer are similar from fish to mammals, and conserved molecular pathways mediate gastrulation movements.     

Expression patterns of twist and snail in Tribolium (Coleoptera) suggest a homologous formation of mesoderm in long and short germ band insects

The mesodermal region in Drosophila is determined by a maternally derived morphogenetic gradient system which specifies the different cell fates along the dorsoventral axis, including the prospective mesodermal cells at the ventral side of the embryo. There are at least two zygotic target genes, twist and snail, which are required for mesoderm formation in Drosophila. To analyze whether a similar mode of mesoderm specification might also apply to short germ band insect embryos, we have cloned twist and snail- related gene fragments from the flour beetle Tri-bolium and have analyzed their expression pattern. Both genes are expressed in a ventral stripe at early blastoderm stage, which is restricted to the region of the developing germ rudiment. The cells expressing the two genes are those that invaginate during gastrulation, indicating that the early stages of mesoderm specification are indeed very similar between the two species. Interestingly, both genes are also expressed during germband extension in a subregion of the growth zone of the embryo which forms the mesodermal cells. This suggests that the expression of the two genes is required for mesoderm formation both at early blastoderm stage and during germband elongation until the end of the segmental growth process. © 1994 Wiley-Liss, Inc.     

Characterization of <Emphasis Type="Italic">twist</Emphasis> and <Emphasis Type="Italic">snail</Emphasis> gene expression during mesoderm and nervous system development in the polychaete annelid <Emphasis Type="Italic">Capitella</Emphasis> sp. I

To investigate the evolutionary history of mesoderm in the bilaterian lineage, we are studying mesoderm development in the polychaete annelid, Capitella sp. I, a representative lophotrochozoan. In this study, we focus on the Twist and Snail families as candidate mesodermal patterning genes and report the isolation and in situ expression patterns of two twist homologs (CapI-twt1 and CapI-twt2) and two snail homologs (CapI-sna1 and CapI-sna2) in Capitella sp. I. CapI-twt1 is expressed in a subset of mesoderm derivatives during larval development, while CapI-twt2 shows more general mesoderm expression at the same stages. Neither twist gene is detected before the completion of gastrulation. The two snail genes have very distinct expression patterns. At cleavage and early gastrula stages, CapI-sna1 is broadly expressed in precursors of all three germ layers and becomes restricted to cells around the closing blastopore during late gastrulation; CapI-sna2 expression is not detected at these stages. After gastrulation, both snail genes are expressed in the developing central nervous system (CNS) at stages when neural precursor cells are internalized, and CapI-sna1 is also expressed laterally within the segmental mesoderm. Based on the expression patterns in this study, we suggest a putative function for Capitella sp. I twist genes in mesoderm differentiation and for snail genes in regulating CNS development and general cell migration during gastrulation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Embryologische Untersuchungen an der holoblastischen Ontogenese vonPenaeus trisulcatus Leach (Crustacea,Decapoda)

Summary Embryonic development of holoblastic Penaeidae to a free-swimming nauplius stage is completed within 12h overnight. Difficulty of obtaining material accounts for the lack of a previous investigation ofPenaeus.Development is not influenced by yolk and appears to be very primitive and clarifies some fundamental principles of arthropod development.A comprehensive morphogenetic-ontological comparison of patterns of development in arthropods becomes possible, including phylogenetic considerations.Cleavage is total without early blastomere-inequality and there are no signs of spiral cleavage.The 16-cell-stage consists of equal blastomeres. During the cleavage to this stage polarity of the embryo can be recognized for the first time by the retarded division of a blastomere which marks the vegetal pole. This is due to a graded retardation of cleavage along the animal-vegetal axis.The result of the 5th cleavage is a 30-cell-blastula—two voluminous cells with retarded division mark the vegetal pole.During the 6th cleavage the vegetal pole looses its spherical shape and gets more and more flattened, both the voluminous undivided cells (Mesendoderm) invaginate.The blastoporal aperture, situated in the sagittal axis is surrounded by 8 division-delayed cells, which get displaced as prospective nauplius-mesoderm into the gastrulation cavity as gastrulation advances. They form the inner wall of the invagination gastrula.The occurence of a classic invagination-gastrula can be regarded as an ancestral attribute in the sense of a recapitulation of a phylogenetic stage.Mesendoderm (the first two blastomeres which invaginate at the vegetal pole) differentiates by two divisions at the bottom of the invagination-cavity into: five yolk endoderm cells, a primordial endoderm cell, a primordial mesoderm cell and the primordial germ cell.The position of these elements is preserved in the succeeding development-stages of the embryo.The division delayed primordial cells are displaced in the direction of the blastopore in consequence of the increase of yolk-endoderm cells constituting a pyramide-like projection into the gastrocoel-cavity. The blastopore survives during early development and allows a good orientation of the embryo to be made.The outlines of the naupliar appendages are folded out in a lateral position. Nauplius mesoderm follows the outgrowths as prospective appendage musculature. At the animal pole neuro-ectoderm (presumptive naupliar nervous system) is displaced into the interior in a second invagination in the sagittal axis, followed by immediate closure of the cleft.The undivided primordial cells of the mesendoderm pyramid are displaced with the enlargement of the yolk endoderm without changing their position in the immediate vicinity of the blastopore.Situated in the outgrowing postmandibular rudiment the primordial cells start their division activity which can be observed very clearly.The primordial endoderm cell constitutes the beginning of the midgutrudiment epithelium. The primordial mesoderm cell divides into the initials of the teloblastic mesoderm. The characteristically situated primordial germ cell divides maintaining the same position in the embryo.In the ventral ectoderm of the postmandibular rudiment the formation of the ectodermal growth zone can be observed. It corresponds to the ectoteloblastic region of the meroblastic arthropods and is arranged symmetrically around the mediocentral ectoderm cell.Before hatching, the nauplius-body is bent ventrally in the surrounding egg membrane with the naupliar appendages projecting body dorsally.After hatching, the embryo extends and the position of appendages changes into ventrocaudal position.In the area of the still visible remainder of the blastopore, at the end of the postmandibular rudiment, the anus develops in advanced stages.The mouth is formed by an ectodermal groove just behind the labrum.Contrary to what might be expected theoretically, the ontogenetic development ofPenaeus does not follow the principles of protostomial development, and if this principle is applied rigidly, the natural relationships between invertebrate phyla would be destroyed. The relationships between the blastopore and anus is therefore not a suitable criterion.This phenomenon of holoblastic development can be brought in full accordance with the well-known facts of meroblastic development in arthropods.

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Embryologische Untersuchungen an der holoblastischen Ontogenese vonPenaeus trisulcatus Leach (Crustacea, Decapoda)

Zusammenfassung Eine embryologische Untersuchung der holoblastischen Penaeiden, die innerhalb 12 h über Nacht ein freies Naupliusstadium entwikkeln, steht bisher infolge der schwierigen Materialbeschaffung aus.Die übersichtliche, vom Dotter unbeeinflu\te Ontogenese ist als au\ergewöhnlich ursprünglich zu werten. An diesem holoblastischen Keim lassen sich die Grundprinzipien erhellen, die für die Arthropoden-Frühontogenese als primÄr gelten müssen. Daher ergeben sich Möglichkeiten für einen umfassenden morphologisch-ontogenetischen Vergleich aller Arthropoden-Ontogenesen und damit für deren phylogenetische Interpretation.Die Furchung verlÄuft zunÄchst total adÄqual jedoch lassen sich bei einer kritischen Diskussion bisher keine signifikanten AnklÄnge an die Spiralfurchung nachweisen.Die 16-Zell-Blastula besteht noch aus gleichgro\en Blastomeren, jedoch lÄ\t die Teilung zum Stadium 16 erstmals die PolaritÄt des Keimes durch Teilungsverzögerung einer Blastomere erkennen. Diese bezeichnet den vegetativen Pol; ein abnehmendes TeilungsgefÄlle in animal-vegetativer Richtung ist die Ursache.Aus der 5. Teilung resultiert eine 30-Zellen-Blastula, wobei zwei voluminöse, teilungsverzögerte Blastomeren den vegetativen Pol bezeichnen. Im Laufe der 6. Teilung flacht sich der vegetative Pol ab; die beiden umfangreichen, ungeteilt gebliebenen Zellen (Mesentoderm) invaginieren.Der in der Sagittalachse liegende Blastoporus-Spalt wird von acht in ihrer Teilung verzögerten Zellen eingefa\t, die im Zuge der fortschreitenden Gastrulation als prospektives Nauplius-Mesoderm ebenfalls nach innen verlagert werden. Sie bilden die laterale Innenwand der Invaginationsgastrula. Das Auftreten einer klassischen Invaginationsgastrula ist als ancestrales Merkmal im Sinne einer Rekapitulation zu deuten.Das Mesentoderm (die beiden Gastrulations-Initialen des vegetativen Pols) differenziert sich in zwei Teilungsschritten am Dach der Invaginationshöhle in fünf Dotterentodermzellen, eine Urentodermzelle, eine Urmesodermzelle und die Urkeimzelle. Die Lage dieser Elemente bleibt in der folgenden Keimesentwicklung erhalten.Die teilungsretardierten Urzellen werden durch Vermehrung der Dotterentodermzellen, welche eine ins Gastrocoel ragende Pyramide aufbauen, auf den Blastoporus zu mitverlagert. Dieser persistiert wÄhrend der gesamten Entwicklung, wodurch eine gute Orientierungsmöglichkeit geboten wird.Die Anlagen der Nauplius-ExtremitÄten falten sich lateral aus.Naupliusmesoderm (Material der Gastrulationshöhle) folgt den Ausbuchtungen als prospektive ExtremitÄten-Muskulatur.Am Apikaipol gelangt durch Spaltbildung unter Invagination und folgendem Spaltschlu\ in der Sagittalachse Neuroektoderm (prospektives naupliales Nervensystem) ins Innere.Die ungeteilten Urzellen der Mesentoderm-Pyramide werden ohne LageverÄnderung mit der Ausdehnung der Dotterentodermzellen in unmittelbare NÄhe zum Blastoporus verlagert. In der auswachsenden postmandibularen Schwanzknospe setzt ihre gut verfolgbare Teilungs-AktivitÄt ein.Aus der Urentodermzelle wird der Beginn des Mitteldarmrohres angelegt.Die Urmesodermzelle teilt sich in die Initialen des Sprossungsmesoderm. Die in charakteristischer Position liegende Urkeimzelle teilt sich unter Beibehaltung der Lage.Im ventralen Ektodermbereich der Schwanzknospe formiert sich in Symmetrie zu einer mediozentral liegenden Zelle das der ektoteloblastischen Region der Meroblastier Ähnliche Sprossungsektoderm.Der Naupliuskörper ist vor dem Schlüpfen in der Eihülle ventral eingekrümmt; die ExtremitÄten überragen ihn nach dorsal.Nach dem Schlüpfen streckt sich der Keim und die ExtremitÄten richten sich nach ventrocaudal um.Im Bereich des sichtbar persistierenden Blastoporus, am Ende der Schwanzknospe, entsteht spÄter der After; die Mundöffnung senkt sich hinter der Oberlippe vom Ektoderm ein.Die Ontogenese des ArthropodenPenaeus richtet sichnicht nach dem von der Theorie geforderten protostomialen Typ. Eine Einteilung der Metazoen in Proto- und Deuterostomier würde natürliche Verwandtschaftsbeziehungen zerstören und ist deshalb ungeeignet.Die VorgÄnge der holoblastischen Ontogenese können mit den bei meroblastischen Crustaceen-Ontogensen gewonnenen Daten weitgehend in Einklang gebracht werden.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft. Meinem Lehrer, Herrn Prof. Dr. Rolf Slewing, möchte ich für wertvolle Anregungen danken. Herr Prof. Dr. José M^a San Feliu ermöglichte mir die Materialbeschaffung am Laboratorio de Investigaciones Pesqueras in Grao de Castellon de la Plana, Spanien. Ihm gilt mein herzlicher Dank     

Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation

Knowledge of the molecular mechanisms regulating cell ingression, epithelial–mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo.     

Xgravin-like (Xgl), a novel putative a-kinase anchoring protein (AKAP) expressed during embryonic development in Xenopus

A-kinase anchoring proteins (AKAPs) are a heterogeneous family of scaffolding proteins that regulate the compartmentalization of signaling components, in particular that of the broad specificity kinase PKA. Here we describe the identification of a new member of this gene family, termed Xenopus gravin-like (Xgl), which encodes a highly acidic protein of 268 kDa that shares extensive homology with human Gravin and murine SSeCKS. Xgl is zygotically expressed in a highly dynamic fashion. During gastrulation Xgl is expressed in posterior mesoderm of the dorsal blastopore lip. During neurulation expression is transiently detected in the forebrain, two bilateral neuroectodermal stripes and the notochord. At tailbud stages expression commences in the mandibular neural crest and the roof of the spinal cord from where neural crest cells migrate into the intersomitic region. In addition expression is detected in the heart and the anterior aspect of the chordoneural hinge.     

Gastrulation in Halocordyle disticha (Hydrozoa,Athecata)

Summary

Previous reports of development in Halocordyle disticha have described gastrulation as occurring by gradual differentiation of the inner and outer cells of the stereoblastula. In 1984, however, Martin and Thomas described an indentation on the surface of the embryo at the time of gastrulation. They hypothesized from morphological data that the indentation represented a blastopore. Here we provide results of marking studies which demonstrate that the indentation is in fact a site of cellular ingression. This is the first example known of gastrulation that involves unipolar ingression in a form with a stereoblastula. Possible functions of gastrulation by unipolar ingression are discussed, and the possible phylogenetic significance of the occurrence of such a mode of gastrulation in H. disticha is considered.     

Cleavage and gastrulation of the dendrobranchiate shrimp Penaeus monodon (Crustacea,Malacostraca, Decapoda)

The cleavage pattern of the black tiger shrimp Penaeus monodon was analyzed from the first division until gastrulation. Observations were based on microscopy combined with the use of fluorescent dyes, histological techniques, and computer based three-dimensional reconstructions. Early cleavage is holoblastic and follows a stereotypic pattern, which largely corresponds to what is known from other dendrobranchiate decapods. However, for the first time in this group, we report the presence of an intracellular structure throughout early development. This intracellular body (icb) marks the lineage of one of the two enlarged and division-delayed mesendoderm cells that initiate gastrulation. The identity of the icb as a natural marker and putative determinant of the germ line and its implications on the establishment of the body axes are discussed. The icb as a landmark reveals that the same stereotypic cell division pattern can lead to different fates of individual cells. Hence, the results of this study permit an additional approach to study the relation between cell lineage pattern and the identity of cell lineages.     

Ultrastructure of putative germ granules in the penaeid shrimp Marsupenaeus japonicus

Knowledge about the specification of the germ line in penaeid shrimp would allow development of techniques to control germ cell formation and/or fate to produce reproductively sterile shrimp for genetic copyright purposes. Recent studies have traced the localization of an RNA–enriched intracellular body (ICB) in the putative germ line of four penaeid shrimp species. It is hypothesized that the ICB may serve as a putative germ granule and marker of germ line fate. In this study semi-thin and ultra-thin sections of Marsupenaeus japonicus embryos were prepared, and the dimensions and ultrastructure of the ICB was examined at different stages of embryogenesis. The ICB was an aggregation of electron dense granules, small vesicles and multi-vesicular bodies (MVBs), similar to germ granules from other species. Lamellar membranes and mitochondria were localized at the periphery of the ICB. Using fluorescence microscopy, microtubules were also observed between the centrosome and the ICB. The localization of the ICB in the D lineage and putative germ cell line, the enrichment of RNA in the ICB, and the ultrastructural similarities to other germ granules characterized in this study support the hypothesis that the ICB contains germ granules.     

Ultraviolet irradiation of eggs and blastomere isolation experiments suggest that gastrulation in the direct developing ascidian, Molgula pacifica, requires localized cytoplasmic determinants in the egg and cell signaling beginning at the two-cell stage

Gastrulation in the maximum direct developing ascidian Molgula pacifica is highly modified compared with commonly studied "model" ascidians in that endoderm cells situated in the vegetal pole region do not undergo typical invagination and due to the absence of a typical blastopore the involution of mesoderm cells is highly modified. At the gastrula stage, embryos are comprised of a central cluster of large yolky cells that are surrounded by a single layer of ectoderm cells in which there is only a slight indication of an inward movement of cells at the vegetal pole. As a consequence, these embryos do not form an archenteron. In the present study, ultraviolet (UV) irradiation of fertilized eggs tested the possibility that cortical cytoplasmic factors are required for gastrulation, and blastomere isolation experiments tested the possibility that cell signaling beginning at the two-cell stage may be required for the development of the gastrula. Irradiation of unoriented fertilized eggs with UV light resulted in late cleavage stage embryos that failed to undergo gastrulation. When blastomeres were isolated from two-cell embryos, they developed into late cleavage stage embryos; however, they did not undergo gastrulation and subsequently develop into juveniles. These results suggest that cytoplasmic factors required for gastrulation are localized in the egg cortex, but in contrast to previously studied indirect developers, these factors are not exclusively localized in the vegetal pole region at the first stage of ooplasmic segregation. Furthermore, the inability of embryos derived from blastomeres isolated at the two-cell stage to undergo gastrulation and develop into juveniles suggests that important cell signaling begins as early as the two-cell stage in M. pacifica. These results are discussed in terms of the evolution of maximum direct development in ascidians.     

An improved method of cell culture system from eye stalk,hepatopancreas, muscle,ovary, and hemocytes of <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO₂. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO₂ supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.     

The Development of the Brachiopod Crania (Neocrania) anomala (O. F. Müller)and its Phylogenetic Significance

The freely spawned eggs of Crania go through radial cleavage, embolic gastrulation, and the posteroventral part of the archenteron forms mesoderm through modified enterocoely. The blastopore closes in the posterior end of the larva. The ciliated, lecithotrophic larva has four pairs of coelomic pouches and three pairs of dorsal setal bundles. At metamorphosis, the larva curls ventrally by contraction of a pair of midventral muscles, which are extensions of the first pair of coelomic sacs; the larva attaches by the epithelium just behind the closed blastopore. The brachial valve is secreted by the middle part of the dorsal epithelium and the pedicle valve is secreted by the attachment epithelium. The second pair of coelomic sacs develop small attachment areas at the edge of the dorsal valve and become the lophophore coelom (mesocoel); the third pair of coelomic sacs become the body coelom (metacoel) with the adductor muscles. The posterior position of the closing blastopore is characteristic of deuterostomes. The ventral curving of the settling larva and the formation of both valves from dorsal epithelial areas indicate that the brachiopods have a very short ventral side as opposed to the phoronids. It is concluded that both groups have originated from a creeping ancestor with a straight gut.     

Cell and tissue requirements for the gene eed during mouse gastrulation and organogenesis

Summary: Mouse embryos homozygous for the allele eed^{l7Rn5‐3354SB} of the Polycomb Group gene embryonic ectoderm development (eed) display a gastrulation defect in which epiblast cells move through the streak and form extraembryonic mesoderm derivatives at the expense of development of the embryo proper. Here we demonstrate that homozygous mutant ES cells have the capacity to differentiate embryonic cell types both in vitro as embryoid bodies and in vivo as chimeric embryos. In chimeric embryos, eed mutant cells can respond to wild‐type signals and participate in normal gastrulation movements. These results indicate a non–cell‐autonomous function for eed. Evidence of mutant cell exclusion from the forebrain and segregation within somites, however, suggests that eed has cell‐autonomous roles in aspects of organogenesis. A requirement for eed in the epiblast during embryonic development is supported by the fact that high‐contribution chimeras could not be rescued by a wild‐type extraembryonic environment. genesis 31:142–146, 2001. © 2001 Wiley‐Liss, Inc.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

Cell lineage studies in the crayfish Cherax destructor (Crustacea,Decapoda) : germ band formation,segmentation, and early neurogenesis

Summary The cell division pattern of the germ band of Cherax destructor is described from gastrulation to segmentation, limb bud formation, and early neurogenesis. The naupliar segments are formed almost simultaneously from scattered ectoderm cells arranged in a V-shaped germ disc, anterior to the blastopore. No specific cell division pattern is recognisable. The post-naupliar segments are formed successively from front to rear. Most post-naupliar material is budded by a ring of about 39 to 46 ectoteloblasts, which are differentiated successively and in situ in front of the telson ectoderm. The ectoteloblasts give rise to 15 descendant cell rows by unequal divisions in an anterior direction, following a mediolateral mitotic wave. Scattered blastoderm cells of non-ectoteloblastic origin in front of the ectoteloblast descendants and behind the mandibular region are also arranged in rows. Despite their different origins, teloblastic and non-teloblastic rows cleave twice by mediolateral mitotic waves to form 4 regular descendant rows each. Thereafter, the resulting grid-like pattern is dissolved by stereotyped differential cleavages. Neuroblasts are formed during these differential cleavages and segmentation becomes visible. Each ectoderm row represents a parasegmental unit. Therefore, the segmental boundary lies within the area covered by the descendants of 1 row. Segmental structures (limbs, ganglia) are composed of derivatives of 2 ectoderm rows. The results are compared with the early development of other crustaceans and insects in relation to mechanisms of germ band formation, segmentation, neurogenesis, and evolution.     

Animal phylogeny in the light of the trochaea theory

Ultrastructural similarities unite Choanoflagellata and Metazoa as the Kingdom Animalia. Mctazoa (Porifera + Placozoa + Gastraeozoa) are characterized by the presence of collagen, septate/tight junctions and spermatozoa. Porifera and Placozoa lack basal lamina, nerve cells and synapses, which characterize Gastraeozoa (Cnidaria + Trochaeozoa). Gnidaria have cnidoblasts and lack the multiciliate cells found in almost all Trochaeozoa (Gastroneuralia + Protornaeozoa). Gastroneuralia (Spiralia + Aschelminthes) have an apical brain and a pair of ventral nerves, a blastopore which becomes mouth and anus, a mouth surrounded by a downstream collecting system of compound cilia, and a mesoderm formed from the blastopore lips. Spiralia (Articulata + Parenchymia + Bryozoa) have spiral cleavage and 4d-cell mesoderm, whereas these characters are lacking in Aschelminthes, which all lack primary larvae. Protornaeozoa (Ctenophora + Notoneuralia) have mesoderm from vegetal cells. Ctenophores have colloblasts. Notoneuralia have a dorsal nervous system behind the apical area and form a new mouth surrounded by an upstream collecting system of single cilia on monociliate cells; the blastopore becomes the anus surrounded by a ring of compound cilia.
These features fit the trochaea theory, which proposes that Gastroneuralia and Notoneuralia evolved independently from the trochaea, a blastaea with the blastopore surrounded by a ring of compound cilia, which were both locomotory and particle collecting.     

Gastrulation in the sea anemone Nematostella vectensis occurs by invagination and immigration: an ultrastructural study

The sea anemone Nematostella vectensis has recently been established as a new model system for the understanding of the evolution of developmental processes. In particular, the evolutionary origin of gastrulation and its molecular regulation are the subject of intense investigation. However, while molecular data are rapidly accumulating, no detailed morphological data exist describing the process of gastrulation. Here, we carried out an ultrastructural study of different stages of gastrulation in Nematostella using transmission electron microscope and scanning electron microscopy techniques. We show that presumptive endodermal cells undergo a change in cell shape, reminiscent of the bottle cells known from vertebrates and several invertebrates. Presumptive endodermal cells organize into a field, the pre-endodermal plate, which undergoes invagination. In parallel, the endodermal cells decrease their apical cell contacts but remain loosely attached to each other. Hence, during early gastrulation they display an incomplete epithelial–mesenchymal transition (EMT). At a late stage of gastrulation, the cells eventually detach and fill the interior of the blastocoel as mesenchymal cells. This shows that gastrulation in Nematostella occurs by a combination of invagination and late immigration involving EMT. The comparison with molecular expression studies suggests that cells expressing snailA undergo EMT and become endodermal, whereas forkhead/brachyury expressing cells at the ectodermal margin of the blastopore retain their epithelial integrity throughout gastrulation.