An aggregation of human embryonic and trophoblast stem cells reveals the role of trophectoderm on epiblast differentiation.

The interactions between extra-embryonic tissues and embryonic tissues are crucial to ensure proper early embryo development. However, the understanding of the crosstalk between the embryonic tissues and extra-embryonic tissues is lacking, mainly due to ethical restrictions, difficulties in obtaining natural human embryos, and lack of appropriate in vitro models. Here by aggregating human embryonic stem cells (hESCs) with human trophoblast stem cells (hTSCs), we revealed the hESCs robustly self-organized into a unique asymmetric structure which the primitive streak (PS) like cells exclusively distributed at the distal end to the TS-compartment, and morphologically flattened cells, presumed to be the extra-embryonic mesoderm cells (EXMC) like cells, were induced at the proximal end to hTSCs. Our study revealed two potential roles of extra-embryonic trophectoderm in regulating the proper PS formation during gastrulation and EXMCs induction from the human epiblast.

Progesterone Receptor Modulates Extraembryonic Mesoderm and Cardiac Progenitor Specification during Mouse Gastrulation.

Progesterone treatment is commonly employed to promote and support pregnancy. While maternal tissues are the main progesterone targets in humans and mice, its receptor (PGR) is expressed in the murine embryo, questioning its function during embryonic development. Progesterone has been previously associated with murine blastocyst development. Whether it contributes to lineage specification is largely unknown. Gastrulation initiates lineage specification and generation of the progenitors contributing to all organs. Cells passing through the primitive streak (PS) will give rise to the mesoderm and endoderm. Cells emerging posteriorly will form the extraembryonic mesodermal tissues supporting embryonic growth. Cells arising anteriorly will contribute to the embryonic heart in two sets of distinct progenitors, first (FHF) and second heart field (SHF). We found that PGR is expressed in a posterior-anterior gradient in the PS of gastrulating embryos. We established in vitro differentiation systems inducing posterior (extraembryonic) and anterior (cardiac) mesoderm to unravel PGR function. We discovered that PGR specifically modulates extraembryonic and cardiac mesoderm. Overexpression experiments revealed that PGR safeguards cardiac differentiation, blocking premature SHF progenitor specification and sustaining the FHF progenitor pool. This role of PGR in heart development indicates that progesterone administration should be closely monitored in potential early-pregnancy patients undergoing infertility treatment.

The extra-embryonic area opaca plays a role in positioning the primitive streak of the early chick embryo.

Classical studies have established that the marginal zone, a ring of extra-embryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the chick embryo, is important in setting embryonic polarity by positioning the primitive streak, the site of gastrulation. The more external extra-embryonic region (area opaca) was thought to have only nutritive and support functions. Using experimental embryology approaches, this study reveals three separable functions for this outer region. First, juxtaposition of the area opaca directly onto the area pellucida induces a new marginal zone from the latter; this induced domain is entirely posterior in character. Second, ablation and grafting experiments using an isolated anterior half of the blastoderm and pieces of area opaca suggest that the area opaca can influence the polarity of the adjacent marginal zone. Finally, we show that the loss of the ability of such isolated anterior half-embryos to regulate (re-establish polarity spontaneously) at the early primitive streak stage can be rescued by replacing the area opaca by one from a younger stage. These results uncover new roles of chick extra-embryonic tissues in early development.

Cell-cell contact landscapes in Xenopus gastrula tissues.

Molecular and structural facets of cell-cell adhesion have been extensively studied in monolayered epithelia. Here, we perform a comprehensive analysis of cell-cell contacts in a series of multilayered tissues in the Xenopus gastrula model. We show that intercellular contact distances range from 10 to 1,000 nm. The contact width frequencies define tissue-specific contact spectra, and knockdown of adhesion factors modifies these spectra. This allows us to reconstruct the emergence of contact types from complex interactions of the factors. We find that the membrane proteoglycan Syndecan-4 plays a dominant role in all contacts, including narrow C-cadherin-mediated junctions. Glypican-4, hyaluronic acid, paraxial protocadherin, and fibronectin also control contact widths, and unexpectedly, C-cadherin functions in wide contacts. Using lanthanum staining, we identified three morphologically distinct forms of glycocalyx in contacts of the Xenopus gastrula, which are linked to the adhesion factors examined and mediate cell-cell attachment. Our study delineates a systematic approach to examine the varied contributions of adhesion factors individually or in combinations to nondiscrete and seemingly amorphous intercellular contacts.

Xenopus chip for single-egg trapping, in vitro fertilization, development, and tadpole escape.

Xenopus laevis is highly suitable as a toxicology animal model owing to its advantages in embryogenesis research. For toxicological studies, a large number of embryos must be handled simultaneously because they very rapidly develop into the target stages within a short period of time. To efficiently handle the embryos, a convenient embryo housing device is essential for fast and reliable assessment and statistical evaluation of malformation caused by toxicants. Here, we suggest 3D fabrication of single-egg trapping devices in which Xenopus eggs are fertilized in vitro, and the embryos are cultured. We used manual pipetting to insert the Xenopus eggs inside the trapping sites of the chip. By introducing a liquid circulating system, we connected a sperm-mixed solution with the chip to induce in vitro fertilization of the eggs. After the eggs were fertilized, we observed embryo development involving the formation of egg cleavage, blastula, gastrula, and tadpole. After the tadpoles grew inside the chip, we saved their lives by enabling their escape from the chip through reverse flow of the culture medium. The Xenopus chip can serve as an incubator to induce fertilization and monitor normal and abnormal development of the Xenopus from egg to tadpole.

Capillarity and active cell movement at mesendoderm translocation in the Xenopus gastrula.

During Xenopus gastrulation, leading edge mesendoderm (LEM) advances animally as a wedge-shaped cell mass over the vegetally moving blastocoel roof (BCR). We show that close contact across the BCR-LEM interface correlates with attenuated net advance of the LEM, which is pulled forward by tip cells while the remaining LEM frequently separates from the BCR. Nevertheless, lamellipodia persist on the detached LEM surface. They attach to adjacent LEM cells and depend on PDGF-A, cell-surface fibronectin and cadherin. We argue that active cell motility on the LEM surface prevents adverse capillary effects in the liquid LEM tissue as it moves by being pulled. It counters tissue surface-tension effects with oriented cell movement and bulges the LEM surface out to keep it close to the curved BCR without attaching to it. Proximity to the BCR is necessary, in turn, for the maintenance and orientation of lamellipodia that permit mass cell movement with minimal substratum contact. Together with a similar process in epithelial invagination, vertical telescoping, the cell movement at the LEM surface defines a novel type of cell rearrangement: vertical shearing.

The recycling endosome protein Rab25 coordinates collective cell movements in the zebrafish surface epithelium.

In emerging epithelial tissues, cells undergo dramatic rearrangements to promote tissue shape changes. Dividing cells remain interconnected via transient cytokinetic bridges. Bridges are cleaved during abscission and currently, the consequences of disrupting abscission in developing epithelia are not well understood. We show that the Rab GTPase Rab25 localizes near cytokinetic midbodies and likely coordinates abscission through endomembrane trafficking in the epithelium of the zebrafish gastrula during epiboly. In maternal-zygotic Rab25a and Rab25b mutant embryos, morphogenic activity tears open persistent apical cytokinetic bridges that failed to undergo timely abscission. Cytokinesis defects result in anisotropic cell morphologies that are associated with a reduction of contractile actomyosin networks. This slows cell rearrangements and alters the viscoelastic responses of the tissue, all of which likely contribute to delayed epiboly. We present a model in which Rab25 trafficking coordinates cytokinetic bridge abscission and cortical actin density, impacting local cell shape changes and tissue-scale forces.

Surface contraction waves or cell proliferation waves in the presumptive neurectoderm during amphibian gastrulation: Mexican axolotl versus African clawed frog.

This essay represents a critical analysis of the literary data on various types of waves occurring in the amphibian embryos during gastrulation. A surface contraction wave travels through the presumptive neurectoderm during Mexican axolotl gastrulation. This wave coincides temporally and spatially with involution of the inducing chordomesoderm and with the prospective neural plate. By contrast, there is no similar surface contraction wave during African clawed frog gastrulation. However, the clawed frog displays the waves of DNA synthesis and mitosis in the presumptive neurectoderm during gastrulation, whereas no such waves were discovered in axolotl gastrulae. These sets of experimental data are in accordance with the contemporary concept of considerable ontogenetic diversity of the class Amphibia.

Germline development in rat revealed by visualization and deletion of Prdm14.

Primordial germ cells (PGCs), the founder cells of the germline, are specified in pre-gastrulating embryos in mammals, and subsequently migrate towards gonads to mature into functional gametes. Here, we investigated PGC development in rats, by genetically modifying Prdm14, a unique marker and an essential PGC transcriptional regulator. We trace PGC development in rats, for the first time, from specification until the sex determination stage in fetal gonads using Prdm14 H2BVenus knock-in rats. We uncover that the crucial role of Prdm14 in PGC specification is conserved between rat and mice, by analyzing Prdm14-deficient rat embryos. Notably, loss of Prdm14 completely abrogates the PGC program, as demonstrated by failure of the maintenance and/or activation of germ cell markers and pluripotency genes. Finally, we profile the transcriptome of the post-implantation epiblast and all PGC stages in rat to reveal enrichment of distinct gene sets at each transition point, thereby providing an accurate transcriptional timeline for rat PGC development. Thus, the novel genetically modified rats and data sets obtained in this study will advance our knowledge on conserved versus species-specific features for germline development in mammals.

The cellular and molecular mechanisms that establish the mechanics of Drosophila gastrulation.

In this review, we cover advances in the field that have contributed to our mechanistic understanding of how tissues internalize during Drosophila melanogaster gastrulation. The changes in tissue shape and architecture that are associated with mesoderm and endoderm invagination in the early Drosophila embryo are accompanied by cell shape changes which are driven by actomyosin contractility. The activation of signal transduction pathways is patterned by embryonic transcription factors, which define distinct geometries of gene expression and the tissue contractile domains. At the subcellular level, outputs from signaling pathways that activate actomyosin contractility are highly polarized and their behavior is fine-tuned by a balance of both positive and negative regulation. Cells are mechanically linked through adherens junctions, allowing forces that are generated by cells to be integrated across the tissue, ensuring coordinated cell behavior during tissue invagination. The transmission of force between cells also enables mechanical feedback whereby force generation influences both cell and cytoskeletal behavior. Finally, after tissue invagination, mesoderm cells undergo an epithelial-to-mesenchymal transition and cell spreading. We highlight studies that have utilized this model system to uncover fundamental principles at molecular-, cell-, and tissue-levels, which have contributed to our understanding of similar tissue morphogenetic processes across different organisms.

Gastrulation in the sea urchin.

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

Tunicate gastrulation.

Tunicates are a diverse group of invertebrate marine chordates that includes the larvaceans, thaliaceans, and ascidians. Because of their unique evolutionary position as the sister group of the vertebrates, tunicates are invaluable as a comparative model and hold the promise of revealing both conserved and derived features of chordate gastrulation. Descriptive studies in a broad range of tunicates have revealed several important unifying traits that make them unique among the chordates, including invariant cell lineages through gastrula stages and an overall morphological simplicity. Gastrulation has only been studied in detail in ascidians such as Ciona and Phallusia, where it involves a simple cup-shaped gastrula driven primarily by endoderm invagination. This appears to differ significantly from vertebrate models, such as Xenopus, in which mesoderm convergent extension and epidermal epiboly are major contributors to involution. These differences may reflect the cellular simplicity of the ascidian embryo.

Convergent extension in the amphibian, Xenopus laevis.

This review is a comprehensive analysis of the cell biology and biomechanics of Convergent Extension in Xenopus.

Mechanisms of zebrafish epiboly: A current view.

Epiboly is a conserved gastrulation movement describing the thinning and spreading of a sheet or multi-layer of cells. The zebrafish embryo has emerged as a vital model system to address the cellular and molecular mechanisms that drive epiboly. In the zebrafish embryo, the blastoderm, consisting of a simple squamous epithelium (the enveloping layer) and an underlying mass of deep cells, as well as a yolk nuclear syncytium (the yolk syncytial layer) undergo epiboly to internalize the yolk cell during gastrulation. The major events during zebrafish epiboly are: expansion of the enveloping layer and the internal yolk syncytial layer, reduction and removal of the yolk membrane ahead of the advancing blastoderm margin and deep cell rearrangements between the enveloping layer and yolk syncytial layer to thin the blastoderm. Here, work addressing the cellular and molecular mechanisms as well as the sources of the mechanical forces that underlie these events is reviewed. The contribution of recent findings to the current model of epiboly as well as open questions and future prospects are also discussed.

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.

Setting up for gastrulation in zebrafish.

Soon after fertilization the zebrafish embryo generates the pool of cells that will give rise to the germline and the three somatic germ layers of the embryo (ectoderm, mesoderm and endoderm). As the basic body plan of the vertebrate embryo emerges, evolutionarily conserved developmental signaling pathways, including Bmp, Nodal, Wnt, and Fgf, direct the nearly totipotent cells of the early embryo to adopt gene expression profiles and patterns of cell behavior specific to their eventual fates. Several decades of molecular genetics research in zebrafish has yielded significant insight into the maternal and zygotic contributions and mechanisms that pattern this vertebrate embryo. This new understanding is the product of advances in genetic manipulations and imaging technologies that have allowed the field to probe the cellular, molecular and biophysical aspects underlying early patterning. The current state of the field indicates that patterning is governed by the integration of key signaling pathways and physical interactions between cells, rather than a patterning system in which distinct pathways are deployed to specify a particular cell fate. This chapter focuses on recent advances in our understanding of the genetic and molecular control of the events that impart cell identity and initiate the patterning of tissues that are prerequisites for or concurrent with movements of gastrulation.

Zebrafish gastrulation: Putting fate in motion.

Gastrulation entails specification and formation of three embryonic germ layers-ectoderm, mesoderm and endoderm-thereby establishing the basis for the future body plan. In zebrafish embryos, germ layer specification occurs during blastula and early gastrula stages (Ho & Kimmel, 1993), a period when the main morphogenetic movements underlying gastrulation are initiated. Hence, the signals driving progenitor cell fate specification, such as Nodal ligands from the TGF-ß family, also play key roles in regulating germ layer progenitor cell segregation (Carmany-Rampey & Schier, 2001; David & Rosa, 2001; Feldman et al., 2000; Gritsman et al., 1999; Keller et al., 2008). In this review, we summarize and discuss the main signaling pathways involved in germ layer progenitor cell fate specification and segregation, specifically focusing on recent advances in understanding the interplay between mesoderm and endoderm specification and the internalization movements at the onset of zebrafish gastrulation.

Movements of chick gastrulation.

In birds as in all amniotes, the site of gastrulation is a midline structure, the primitive streak. This appears as cells in the one cell-thick epiblast undergo epithelial-to-mesenchymal transition to ingress and form definitive mesoderm and endoderm. Global movements involving tens of thousands of cells in the embryonic epiblast precede gastrulation. They position the primitive streak precursors from a marginal position (equivalent to the situation in anamniotes) along the future antero-posterior axis (typical for amniotes). These epithelial movements continue in modified form during gastrulation, when they are accompanied by collective movements of different class in the forming mesoderm and endoderm. Here I discuss the nature of these collective cell movements shaping the embryo, their interplay with signaling events controlling fate specification and significance in an evolutionary perspective.

Global shape of Toll activation is determined by wntD enhancer properties.

Buffering variability in morphogen distribution is essential for reproducible patterning. A theoretically proposed class of mechanisms, termed "distal pinning," achieves robustness by combining local sensing of morphogen levels with global modulation of gradient spread. Here, we demonstrate a critical role for morphogen sensing by a gene enhancer, which ultimately determines the final global distribution of the morphogen and enables reproducible patterning. Specifically, we show that, while the pattern of Toll activation in the early Drosophila embryo is robust to gene dosage of its locally produced regulator, WntD, it is sensitive to a single-nucleotide change in the wntD enhancer. Thus, enhancer properties of locally produced WntD directly impinge on the global morphogen profile.