Phase Of Gastrulation Research Articles

Cripto-1 as a Key Factor in Tumor Progression, Epithelial to Mesenchymal Transition and Cancer Stem Cells.

Cripto-1 is an essential protein for human development that plays a key role in the early phase of gastrulation in the differentiation of an embryo as well as assists with wound healing processes. Importantly, Cripto-1 induces epithelial to mesenchymal transition to turn fixed epithelial cells into a more mobile mesenchymal phenotype through the downregulation of epithelial adhesion molecules such as E-cadherin, occludins, and claudins, and the upregulation of mesenchymal, mobile proteins, such as N-cadherin, Snail, and Slug. Consequently, Cripto-1’s role in inducing EMT to promote cell motility is beneficial in embryogenesis, but detrimental in the formation, progression and metastasis of malignant tumors. Indeed, Cripto-1 is found to be upregulated in most cancers, such as breast, lung, gastrointestinal, hepatic, renal, cervical, ovarian, prostate, and skin cancers. Through its role in EMT, Cripto-1 can remodel cancer cells to enable them to travel through the extracellular matrix as well as blood and lymphatic vessels to metastasize to different organs. Additionally, Cripto-1 promotes the survival of cancer stem cells, which can lead to relapse in cancer patients.

Caudal duplication syndrome: a literature review and reappraisal of its pathoembryogenesis.

The term caudal duplication syndrome (CDS) was first introduced for complex anomalies of the distal caudal end of the trunk. The pathoembryogenesis of CDS is yet unknown, although a few theories have been proposed. We reviewed the previously proposed pathoembryogenetic theories and suggested a new perspective through the common clinical characteristics shown in CDS cases reported in the literature. We conducted a systematic literature search of the online database PubMed from October 1993 to October 2020, using the search term "caudal duplication syndrome", according to the first mention of this entity. A total of 17 articles with 23 patients were reviewed. The most common manifestations were the duplication of the distal colon, genitourinary organs, and lower spine. Specifically, the duplicated bladders or uteri contacted their counterpart through a septum, and the duplicated bowels ran parallel. More caudal structures, such as the urethra or anus, were formed separately. The duplication seems to be a result of division by an intervening septum or structure in each part. In addition, duplication was not limited to the structures formed from the caudal cell mass (CCM), such as the distal spine and spinal cord, but also included hindgut structures. Moreover, anomalies involving caudal mesenchymal defects were also present. Considering clinical manifestations that are related to all three germ layers and seemingly the overseptation of these germ layers in CDS patients, with supporting data from animal experiments, events such as late-stage errors involving Hensen's node/the primitive streak and the duplication of the CCM with the hyperplasia of the abnormally located central caudal mesenchyme are probable pathoembryogenetic mechanisms for CDS. The "leakage" of the normal growth power of the caudal mesenchyme into the intervening midline space between the two CCMs and consequent weak lateral and caudal pushes of the caudal mesenchyme may explain the association of caudal agenesis or its related anomalies with CDS. We propose a theory that by a molecular interaction, an insult causes late gastrulation phase problems, resulting in ectopic primitive streak formation, and therefore, a duplication of the CCM is induced. Subsequently, the overactivity of abnormally positioned midline mesenchyme between the two CCMs may divide the hindgut derivatives by a central septum. Underactive lateral and caudal pushes of the caudal mesenchyme may lead to an association of features shown in caudal agenesis.

Early Exposure to THC Alters M-Cell Development in Zebrafish Embryos.

Cannabis is one of the most commonly used illicit recreational drugs that is often taken for medicinal purposes. The psychoactive ingredient in cannabis is Δ9-Tetrahydrocannabinol (Δ9-THC, hereafter referred to as THC), which is an agonist at the endocannabinoid receptors CB1R and CB2R. Here, we exposed zebrafish embryos to THC during the gastrulation phase to determine the long-term effects during development. We specifically focused on reticulospinal neurons known as the Mauthner cells (M-cell) that are involved in escape response movements. The M- cells are born during gastrulation, thus allowing us to examine neuronal morphology of neurons born during the time of exposure. After the exposure, embryos were allowed to develop normally and were examined at two days post-fertilization for M-cell morphology and escape responses. THC treated embryos exhibited subtle alterations in M-cell axon diameter and small changes in escape response dynamics to touch. Because escape responses were altered, we also examined muscle fiber development. The fluorescent labelling of red and white muscle fibers showed that while muscles were largely intact, the fibers were slightly disorganized with subtle but significant changes in the pattern of expression of nicotinic acetylcholine receptors. However, there were no overt changes in the expression of nicotinic receptor subunit mRNA ascertained by qPCR. Embryos were allowed to further develop until 5 dpf, when they were examined for overall levels of movement. Animals exposed to THC during gastrulation exhibited reduced activity compared with vehicle controls. Together, these findings indicate that zebrafish exposed to THC during the gastrula phase exhibit small changes in neuronal and muscle morphology that may impact behavior and locomotion.

Comparative analysis of human and mouse development: From zygote to pre-gastrulation.

Development of the mammalian embryo begins with formation of the totipotent zygote during fertilization. This initial cell is able to give rise to every embryonic tissue of the developing organism as well as all extra-embryonic lineages, such as the placenta and the yolk sac, which are essential for the initial patterning and support growth of the fetus until birth. As the embryo transits from pre- to post-implantation, major structural and transcriptional changes occur within the embryonic lineage to set up the basis for the subsequent phase of gastrulation. Fine-tuned coordination of cell division, morphogenesis and differentiation is essential to ultimately promote assembly of the future fetus. Here, we review the current knowledge of mammalian development of both mouse and human focusing on morphogenetic processes leading to the onset of gastrulation, when the embryonic anterior-posterior axis becomes established and the three germ layers start to be specified.

Setting up for gastrulation: D. melanogaster.

Drosophila melanogaster embryos develop initially as a syncytium of totipotent nuclei and subsequently, once cellularized, undergo morphogenetic movements associated with gastrulation to generate the three somatic germ layers of the embryo: mesoderm, ectoderm, and endoderm. In this chapter, we focus on the first phase of gastrulation in Drosophila involving patterning of early embryos when cells differentiate their gene expression programs. This patterning process requires coordination of multiple developmental processes including genome reprogramming at the maternal-to-zygotic transition, combinatorial action of transcription factors to support distinct gene expression, and dynamic feedback between this genetic patterning by transcription factors and changes in cell morphology. We discuss the gene regulatory programs acting during patterning to specify the three germ layers, which involve the regulation of spatiotemporal gene expression coupled to physical tissue morphogenesis.

Active Tension Network model suggests an exotic mechanical state realized in epithelial tissues.

Mechanical interactions play a crucial role in epithelial morphogenesis, yet understanding the complex mechanisms through which stress and deformation affect cell behavior remains an open problem. Here we formulate and analyze the Active Tension Network (ATN) model, which assumes that the mechanical balance of cells within a tissue is dominated by cortical tension and introduces tension-dependent active remodeling of the cortex. We find that ATNs exhibit unusual mechanical properties. Specifically, an ATN behaves as a fluid at short times, but at long times supports external tension like a solid. Furthermore, an ATN has an extensively degenerate equilibrium mechanical state associated with a discrete conformal - “isogonal” - deformation of cells. The ATN model predicts a constraint on equilibrium cell geometries, which we demonstrate to approximately hold in certain epithelial tissues. We further show that isogonal modes are observed in the fruit y embryo, accounting for the striking variability of apical areas of ventral cells and helping understand the early phase of gastrulation. Living matter realizes new and exotic mechanical states, the study of which helps to understand biological phenomena.

Phenotypic defects in newborn Gammarus fossarum (Amphipoda) following embryonic exposure to fenoxycarb

During morphogenesis numerous morphogenetic factors ensure the production of a target phenotype. By disrupting these processes, a toxic exposure during this period could cause an increase of phenotypic defects. In the present study, embryos of the freshwater amphipod Gammarus fossarum were exposed throughout the embryogenesis to increasing concentrations of fenoxycarb (0, 0.5µgL−1, 5µgL−1 and 50µgL−1), a growth regulator insecticide analog of the insect juvenile hormone. In addition, to identify morphogenesis’ sensitive period, embryos were exposed during either early or late embryonic development to 5µgL−1 of fenoxycarb. In newborn individuals from exposed embryos, three phenotypes were investigated: i) eye pigmentation, ii) length of the antenna and gnathopod of both left and right sides and iii) midgut tissue state. Developmental homeostasis was assessed by measuring fluctuating asymmetry and inter-individual variance of both the antenna and gnathopod. Exposure to 5µgL−1 and 50µgL−1 fenoxycarb throughout the embryonic development induced a delayed hatching and altered appendages size. Moreover, exposure to 5µgL−1 throughout the embryogenesis and during the gastrulation phase impaired eye pigmentation, while exposure to 50µgL−1 resulted in increased tissue damages of the midgut. No significant increase of fluctuating asymmetry was observed in exposed individuals, neither for the antenna nor for the gnathopod. These results demonstrate that fenoxycarb can alter embryonic development of G. fossarum without disrupting developmental homeostasis.

Wnt, Frizzled, and sFRP gene expression patterns during gastrulation in the starfish Patiria (Asterina) pectinifera

By the initial phase of gastrulation, Wnt pathway regulation mediates endomesoderm specification and establishes the animal-vegetal axis, thereby leading to proper gastrulation in starfish. To provide insight into the ancestral mechanism regulating deuterostome gastrulation, we identified the gene expression patterns of Wnt, Frizzled (Fz), and secreted frizzled-related protein (sFRP) family genes, which play a role in the initial stage of the Wnt pathway, in starfish Patiria (Asterina) pectinifera embryos using whole mount in situ hybridization. We identified ten Wnt, four Fz, and two sFRP paralogues. From the hatching blastula to the late gastrula stage, the majority of the Wnt genes and both Fz5/8 and sFRP1/5 were expressed in the posterior and anterior half of the embryo, respectively. Wnt8, Fz1, and Fz4 showed restricted expression in the lateral ectoderm. On the other hand, several genes were expressed de novo in the restricted domain of the archenteron at the late gastrula stage. These results suggest that the canonical and/or non-canonical Wnt pathway might implicate endomesoderm specification, anterior-posterior axis establishment, anterior-posterior patterning, and archenteron morphogenesis in the developmental context of starfish embryos. From comparison with the expression patterns observed in Patria miniata, we consider that the Wnt pathway is conserved among starfishes.

Collective Epithelial and Mesenchymal Cell Migration During Gastrulation

Gastrulation, the process that puts the three major germlayers, the ectoderm, mesoderm and endoderm in their correct topological position in the developing embryo, is characterised by extensive highly organised collective cell migration of epithelial and mesenchymal cells. We discuss current knowledge and insights in the mechanisms controlling these cell behaviours during gastrulation in the chick embryo. We discuss several ideas that have been proposed to explain the observed large scale vortex movements of epithelial cells in the epiblast during formation of the primitive streak. We review current insights in the control and execution of the epithelial to mesenchymal transition (EMT) underlying the formation of the hypoblast and the ingression of the mesendoderm cells through the streak. We discuss the mechanisms by which the mesendoderm cells move, the nature and dynamics of the signals that guide these movements, as well as the interplay between signalling and movement that result in tissue patterning and morphogenesis. We argue that instructive cell-cell signaling and directed chemotactic movement responses to these signals are instrumental in the execution of all phases of gastrulation.

The influence of scaffold elasticity on germ layer specification of human embryonic stem cells

Mechanical forces are critical to embryogenesis, specifically, in the lineage-specification gastrulation phase, whereupon the embryo is transformed from a simple spherical ball of cells to a multi-layered organism, containing properly organized endoderm, mesoderm, and ectoderm germ layers. Several reports have proposed that such directed and coordinated movements of large cell collectives are driven by cellular responses to cell deformations and cell-generated forces. To better understand these environmental-induced cell changes, we have modeled the germ layer formation process by culturing human embryonic stem cells (hESCs) on three dimensional (3D) scaffolds with stiffness engineered to model that found in specific germ layers. We show that differentiation to each germ layer was promoted by a different stiffness threshold of the scaffolds, reminiscent of the forces exerted during the gastrulation process. The overall results suggest that three dimensional (3D) scaffolds can recapitulate the mechanical stimuli required for directing hESC differentiation and that these stimuli can play a significant role in determining hESC fate.

Color Photographic Index of Fall Chinook Salmon Embryonic Development and Accumulated Thermal Units

BackgroundKnowledge of the relationship between accumulated thermal units and developmental stages of Chinook salmon embryos can be used to determine the approximate date of egg fertilization in natural redds, thus providing insight into oviposition timing of wild salmonids. However, few studies have documented time to different developmental stages of embryonic Chinook salmon and no reference color photographs are available. The objectives of this study were to construct an index relating developmental stages of hatchery-reared fall Chinook salmon embryos to time and temperature (e.g., degree days) and provide high-quality color photographs of each identified developmental stage.Methodology/Principal FindingsFall Chinook salmon eggs were fertilized in a hatchery environment and sampled approximately every 72 h post-fertilization until 50% hatch. Known embryonic developmental features described for sockeye salmon were used to describe development of Chinook salmon embryos. A thermal sums model was used to describe the relationship between embryonic development rate and water temperature. Mean water temperature was 8.0°C (range; 3.9–11.7°C) during the study period. Nineteen stages of embryonic development were identified for fall Chinook salmon; two stages in the cleavage phase, one stage in the gastrulation phase, and sixteen stages in the organogenesis phase. The thermal sums model used in this study provided similar estimates of fall Chinook salmon embryonic development rate in water temperatures varying from 3.9–11.7°C (mean = 8°C) to those from several other studies rearing embryos in constant 8°C water temperature.Conclusions/SignificanceThe developmental index provides a reasonable description of timing to known developmental stages of Chinook salmon embryos and was useful in determining developmental stages of wild fall Chinook salmon embryos excavated from redds in the Columbia River. This index should prove useful to other researchers who wish to approximate fertilization dates of Chinook salmon eggs from natural redds, assuming the thermal history of embryos is known.

Early Development and Cleavage Pattern of the Japanese Purple Mussel,Septifer virgatus

Despite easy access to bivalves, few studies have examined the development of these animals, at least in part because most bivalve eggs are very small. In addition, annotating cells of the early bivalve embryo is difficult because few landmarks are present. We conducted detailed cell annotations of the Japanese purple mussel, Septifer virgatus, during early embryogenesis because of its relatively large eggs (ca. 130 microm in diameter). Septifer virgatus underwent the unique cell division profile reported for four other bivalve species, suggesting that the cleavage pattern itself is important for bivalve morphogenesis. The shell field invagination was led by 2d (X) lineage cells, supporting the hypothesis that lineage cells differentiate into cells excreting the shell matrix. The large egg size enabled us to trace cell movements in the early gastrulation phase, during which the invagination of the archenteron was initiated by 4d (M) lineage cells. These observations will serve as a basis for future analyses of S. virgatus embryogenesis and will contribute to understanding the evolution of the molluscan body plan, which is achieved by modification of early embryogenesis.

Axial differentiation and early gastrulation stages of the pig embryo

Differentiation of the principal body axes in the early vertebrate embryo is based on a specific blueprint of gene expression and a series of transient axial structures such as Hensen's node and the notochord of the late gastrulation phase. Prior to gastrulation, the anterior visceral endoderm (AVE) of the mouse egg-cylinder or the anterior marginal crescent (AMC) of the rabbit embryonic disc marks the anterior pole of the embryo. For phylogenetic and functional reasons both these entities are addressed here as the mammalian anterior pregastrulation differentiation (APD). However, mouse and rabbit show distinct structural differences in APD and the molecular blueprint, making the search of general rules for axial differentiation in mammals difficult. Therefore, the pig was analysed here as a further species with a mammotypical flat embryonic disc. Using light and electron microscopy and in situ hybridisation for three key genes involved in early development ( sox17, nodal and brachyury), two axial structures of early gastrulation in the pig were identified: (1) the anterior hypoblast (AHB) characterised by increased cellular height and density and by sox17 expression, and (2) the early primitive streak characterised by a high pseudostratified epithelium with an almost continuous but unusually thick basement membrane, by localised epithelial–mesenchymal transition, and by brachyury expression in the epiblast. The stepwise appearance of these two axial structures was used to define three stages typical for mammals at the start of gastrulation. Intriguingly, the round shape and gradual posterior displacement of the APD in the pig appear to be species-specific (differing from all other mammals studied in detail to date) but correlate with ensuing specific primitive streak and extraembryonic mesoderm development. APD and, hence, the earliest axial structure presently known in the mammalian embryo may thus be functionally involved in shaping extraembryonic membranes and, possibly, the specific adult body form.

Patterning and Nuclear β-Catenin Expression in the Colonic Adenoma-Carcinoma Sequence: Analogies with Embryonic Gastrulation

Patterning is a process by which ordered arrangements of cells and tissue structure are attained. The term derived from developmental biology is also useful for the study of colonic carcinogenesis, in which the patterning of neoplastic tubules is necessary for properties of growth, invasion, and metastasis. Interestingly the nuclear expression and transcriptional activity of beta-catenin, a major oncoprotein in colonic carcinogenesis, is decisive for the first patterning of a tubule in embryogenesis, which creates the primitive gut and is called the gastrulation. Thus, basic patternings of embryogenesis and carcinogenesis might be linked. To test this hypothesis we compared morphological patterns and immunohistochemical beta-catenin stainings in colonic adenomas and adenocarcinomas with the gastrulation steps. Two analogies were found: 1) the patterning of invasion with reconstruction in adenocarcinomas corresponded to the epithelio-mesenchymal transition, ingression, and rearrangement of cells during the first phase of gastrulation; and 2) the patterning of tubular branching in adenomas and adenocarcinomas resembled the endodermal invagination during the second phase. The intratumorous distribution and intensity of nuclear beta-catenin expression was significantly correlated with the two patternings, similar to the findings in gastrulation. The results indicate microenvironmental regulations of nuclear beta-catenin expression and a return of neoplastic cells to embryonic transcriptional susceptibilities during colonic carcinogenesis.

The βL Integrin Subunit Is Necessary for Gastrulation in Sea Urchin Embryos

Integrins are a family of cell adhesion molecules reported to mediate cellular interactions essential for normal embryonic morphogenesis. Here we describe a β integrin subunit that is expressed during early embryogenesis in the sea urchin embryo and appears to be necessary for normal development. The deduced amino acid sequence of βL is similar to vertebrate β integrin subunits, but is most closely related to the sea urchin βG subunit. Northern blots show that βL is expressed at all stages with maximum expression beginning during gastrulation. Immunolocalization andin situRNA hybridization show that in blastulae βL is expressed in the blastoderm and by the ring of bottle cells in the vegetal plate during the initial phase of gastrulation. Presumptive secondary mesenchyme cells express high levels of βL throughout elongation of the archenteron and in the pluteus βL is expressed by blastocoelar cells, skeletal mesenchyme, and pigment cells. Antibodies and Fab fragments against βL block spreading of dissociated embryonic cells on RGD (arginine–glycine–aspartate)-containing substrates. Treating embryos with anti-βL antibodies blocks the initial phase of gastrulation and interferes with the organization of actin filaments. Prior to gastrulation, the antibodies cause thickening of the blastoderm and later in development defects in skeletal patterning result. Probing for antibody in treated embryos indicates that it penetrates the ectoderm to cells within the blastocoel and is actively endocytosed. We propose that βL forms receptors that bind to RGD-containing ligands and anchors actin filaments. These receptors appear to be essential in several aspects of morphogenesis.

Late Specification of Veg1Lineages to Endodermal Fate in the Sea Urchin Embryo

Single blastomeres of the sixth-cleavage veg1and veg2tiers ofStrongylocentrotus purpuratusembryos were labeled with DiI lineage tracer, and the disposition of the progeny was followed through the blastula and gastrula stages in order to determine their respective endodermal and ectodermal contributions. In the endoderm of postgastrula embryos, veg1-derived cells constituted nearly all of the prospective hindgut and about half of the prospective midgut, while veg2-derived cells made up the prospective foregut and half the midgut. Oral veg1clones consistently contributed more cells to endoderm than aboral veg1clones. Oral veg1clones extended along the archenteron up to the foregut region, while aboral veg1clones contributed only small numbers of hindgut cells but large patches of ectoderm cells that extended out to the prospective larval vertex. The oral/aboral asymmetry in veg1allocations was also demonstrated using chimeric embryos, the animal halves of which were labeled with a rhodamine-dextran. Lineages expressing the vegetal plate markerEndo16were more precisely determined by combining lineage tracer injection with whole-mountin situhybridization.Endo16expression was found in all cells that are going to participate in gastrulation. Recruitment of new cells to theEndo16domain occurs in advance of the actual invagination of those cells. During the blastula stagesEndo16expression expands radially until all cells in the veg2lineages express this gene. The first phase of gastrulation, including the normal buckling of the vegetal plate and primary invagination of the archenteron, involves only theEndo16-expressing cells of the veg2lineages. As the archenteron begins to elongate, marking the onset of the second phase of gastrulation, there is an asymmetric expansion ofEndo16into the veg1-derived cells that will contribute to the hindgut and midgut in accordance with lineage tracing observations. The results indicate a relatively late specification of veg1-derived cells, resulting in late recruitment to the periphery of the vegetal plate territory as gastrulation proceeds. Differential recruitment of veg1-derived cells on the oral side of the embryo introduces an oral bias to gastrulation by disproportionately increasing the number of cells on the oral side that are competent to participate in gastrulation.

The evolution of Msx gene function: Expression and regulation of a sea urchin Msx class homeobox gene

Msx-class homeobox genes, characterized by a distinct and highly conserved homeodomain, have been identified in a wide variety of metazoans from vertebrates to coelenterates. Although there is evidence that they participate in inductive tissue interactions that underlie vertebrate organogenesis, including those that pattern the neural crest, there is little information about their function in simple deuterostomes. Both to learn more about the ancient function of Msx genes, and to shed light on the evolution of developmental mechanisms within the lineage that gave rise to vertebrates, we have isolated and characterized Msx genes from ascidians and echinoderms. Here we describe the sequence and expression of a sea urchin (Strongylocentrotus purpuratus) Msx gene whose homeodomain is very similar to that of vertebrate Msx2. This gene, designated SpMsx, is first expressed in blastula stage embryos, apparently in a non-localized manner. Subsequently, during the early phases of gastrulation, SpMsx transcripts are expressed intensely in the invaginating archenteron and secondary mesenchyme, and at reduced levels in the ectoderm. In the latter part of gastrulation, SpMsx transcripts are concentrated in the oral ectoderm and gut, and continue to be expressed at those sites through the remainder of embryonic development. That vertebrate Msx genes are regulated by inductive tissue interactions and growth factors suggested to us that the restriction of SpMsx gene expression to the oral ectoderm and derivatives of the vegetal plate might similarly be regulated by the series of signaling events that pattern these embryonic territories. As a first test of this hypothesis, we examined the influence of exogastrulation and cell-dissociation on SpMsx gene expression. In experimentally-induced exogastrulae, SpMsx transcripts were distributed normally in the oral ectoderm, evaginated gut, and secondary mesenchyme. However, when embryos were dissociated into their component cells, SpMsx transcripts failed to accumulate. These data show that the localization of SpMsx transcripts in gastrulae does not depend on interactions between germ layers, yet the activation and maintenance of SpMsx expression does require cell-cell or cell-matrix interactions.

The Initial Phase of Gastrulation in Sea Urchins Is Accompanied by the Formation of Bottle Cells

The morphogenetic processes responsible for the initial phase of gastrulation in sea urchins have yet to be satisfactorily defined. Using conventional and confocal microscopy we have analyzed the buckling of the vegetal plate to form the archenteron in embryos ofStrongylocentrotus purpuratus.The cells of the vegetal plate elongate and a ring of 34 to 36 bottle cells forms within the vegetal plate during invagination. Rhodamine phalloidin staining reveals a reorganization of the actin cytoskeleton associated with these changes in cell shape. During buckling, the ring of bottle cells within the vegetal plate fluoresce intensely at their apical surface and in the narrow neck region. Ionophore A23187 induces precocious buckling and the formation of the ring of bottle cells. The calcium channel blocker verapamil and the calmodulin inhibitor trifluoperazine reversibly inhibit buckling and the formation of the ring of bottle cells. Treatment with antibodies to the apical lamina, which interferes with the initial stage of gastrulation, blocks the appearance of the vegetal plate phalloidin staining. Measurements of the dimensions of cells and an analysis of shape changes suggest that the formation of bottle cells reduces the surface area of the vegetal plate by more than 50%. We propose that actin-mediated changes in cell shape within the vegetal plate are responsible for producing forces which cause buckling of the vegetal plate during the initial phase of gastrulation.

C-otx2 is expressed in two different phases of gastrulation and is sensitive to retinoic acid treatment in chick embryo

We cloned the chick homologue of the mouse Otx2 gene, c-otx2, and analyzed its expression pattern during gastrulation. During mouse embryogenesis, Otx2 expression is first detected in the entire epiblast and after the formation of the primitive streak becomes confined to the most anterior region of the embryo corresponding to presumptive fore- and mid-brain. Similarly, two distinst phases of c-otx2 expression were observed in the chick. c-otx2 transcripts were first detected in the unincubated egg and up to stage XIII, in all epiblast, and forming hypoblast and mesoblast cells. During primitive streak progression, c-otx2 expression becomes progressively restricted to anterior regions and is mainly associated with Hensen's node. When the extension of the streak is maximal, transcripts are only found in Hensen's node. A second phase of c-otx2 expression starts during streak regression. c-otx2 transcripts are lost from the node and present in higher abundance in anterior neuroectoderm and mesendoderm, with the exception of forming notochord and floor plate. The first phase of expression bears strong similarity with that of c-gsc, a gene shown to be a marker for cells that have organizer activity in the chick. Therefore, we compared the expression of the two genes by double staining on the same embryo. This analysis demonstrated that c-otx2 is transcribed first and its expression in the hypoblast precedes that of c-gsc. On the other hand, c-gsc is an earlier marker of primitive streak cells. The expression domains of the two genes transiently overlap in Hensen's node and anterior mesendoderm, whereas only c-otx2 is expressed in neuroectodermal areas. The second phase of c-otx2 expression is sensitive to an early treatment with retinoic acid. This treatment abolishes c-otx2 expression in mesendoderm and restricts it to most anterior regions in the forming neural plate. In conclusion, our results suggest that c-otx2 expression is first associated with cells with an anterior mesendoderm fate and subsequently extends to anterior neuroectoderm.

Secondary mesenchyme of the sea urchin embryo: ontogeny of blastocoelar cells.

Secondary mesenchyme in sea urchin embryos is released into the blastocoel after primary mesenchyme, and although these cells have been recognized for some time, we lack knowledge about many fundamental aspects of their origin and fate. Here we documented the ontogeny of one of the principal, and least well-known, types of cells derived from secondary mesenchyme. The blastocoelar cells arise from mesenchyme released from the tip of the archenteron following the initial phase of gastrulation. The cells migrate with their cell bodies suspended in the blastocoel, rather than being apposed to the basal lamina like primary mesenchyme. The cells extend numerous fine filopodia to form a network of cytoplasmic processes around the gut, along the skeletal rods, and within the larval arms. Once the network is formed, the cells maintain their positions, although they actively translocate vesicles and cytoplasm along their filopodia. Cell counts indicate there is an initial recruitment of cells during gastrulation, followed by a more gradual increase in cell number after the larva begins to feed. Lineage studies in which 16-cell-stage macromeres were injected with horseradish peroxidase indicate that almost all of the macromere-derived mesenchyme forms pigment cells and blastocoelar cells. We propose that blastocoelar cells are a distinct subset of secondary mesenchyme that forms fibroblast-like cells in the blastocoel of sea urchin embryos.