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.

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.

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.

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.

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.

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.

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.

Ethical issues in human organoid and gastruloid research.

Research involving human organoids and gastruloids involves ethical issues associated with their derivation as well as their current and future uses. These include unique issues related to the extent of maturation that can be achieved in vitro or through chimeric research, as well as fundamental ethical considerations such as those concerning the provenance of human biomaterials and the use of gene-editing technologies. Many of these issues are not specifically addressed by existing ethics oversight mechanisms, but these mechanisms might be easily extended to help ensure that human organoid and related research moves forward in an ethically appropriate manner.

CoRest1 regulates neurogenesis in a stage-dependent manner.

BACKGROUND: Developmental processes, including neuronal differentiation, require precise regulation of transcription. The RE-1 silencing transcription factor (Rest), is often called a "master neuronal regulator" due to its large number of neural-specific targets. Rest recruits CoRest (Rcor) and Sin3 corepressor complexes to gene regulatory sequences. CoRest not only associates with Rest, but with other transcription regulators. In this study, we generated zebrafish rcor1 mutants using transcription activator-like effector nucleases (TALENS), to study its requisite role in repression of Rest target genes as well as Rest-independent Rcor1 developmental functions. RESULTS: While rcor1 mutants have a slight decrease in fitness, most survived and produced viable offspring. We examined expression levels of RE1-containing genes in maternal zygotic rcor1 (MZrcor1) mutants and found that Rcor1 is generally not required for the repression of Rest target genes at early stages. However, MZrcor1 mutants undergo more rapid neurogenesis compared to controls. We found that at gastrula stages, Rcor1 acts as a repressor of her gene family, but at later stages, her6 decreased in the MZrcor1 mutant. CONCLUSIONS: Based on these findings, the central role of CoRest1 in neurogenesis is likely due to a Rest-independent role rather than as a Rest corepressor.

Roles for Xenopus aquaporin-3b (aqp3.L) during gastrulation: Fibrillar fibronectin and tissue boundary establishment in the dorsal margin.

Aquaporins and aquaglyceroporins are a large family of membrane channel proteins that allow rapid movement of water and small, uncharged solutes into and out of cells along concentration gradients. Recently, aquaporins have been gaining recognition for more complex biological roles than the regulation of cellular osmotic homeostasis. We have identified a specific expression pattern for Xenopus aqp3b (also called aqp3.L) during gastrulation, where it is localized to the sensorial (deep) layer of the blastocoel roof and dorsal margin. Interference with aqp3b expression resulted in loss of fibrillar fibronectin matrix in Brachet's cleft at the dorsal marginal zone, but not on the free surface of the blastocoel. Detailed observation showed that the absence of fibronectin matrix correlated with compromised border integrities between involuted mesendoderm and noninvoluted ectoderm in the marginal zone. Knockdown of aqp3b also led to delayed closure of the blastopore, suggesting defects in gastrulation movements. Radial intercalation was not affected in aqp3b morphants, while the data presented are consistent with impeded convergent extension movements of the dorsal mesoderm in response to loss of aqp3b. Our emerging model suggests that aqp3b is part of a mechanism that promotes proper interaction between cells and the extracellular matrix, thereby playing a critical role in gastrulation.

A dynamic intracellular distribution of Vangl2 accompanies cell polarization during zebrafish gastrulation.

During vertebrate gastrulation, convergence and extension movements elongate embryonic tissues anteroposteriorly and narrow them mediolaterally. Planar cell polarity (PCP) signaling is essential for mediolateral cell elongation underlying these movements, but how this polarity arises is poorly understood. We analyzed the elongation, orientation and migration behaviors of lateral mesodermal cells undergoing convergence and extension movements in wild-type zebrafish embryos and mutants for the Wnt/PCP core component Vangl2 (Trilobite). We demonstrate that Vangl2 function is required at the time when cells transition to a highly elongated and mediolaterally aligned body. vangl2 mutant cells fail to undergo this transition and to migrate along a straight path with high net speed towards the dorsal midline. Instead, vangl2 mutant cells exhibit an anterior/animal pole bias in cell body alignment and movement direction, suggesting that PCP signaling promotes effective dorsal migration in part by suppressing anterior/animalward cell polarity and movement. Endogenous Vangl2 protein accumulates at the plasma membrane of mesenchymal converging cells at the time its function is required for mediolaterally polarized cell behavior. Heterochronic cell transplantations demonstrated that Vangl2 cell membrane accumulation is stage dependent and regulated by both intrinsic factors and an extracellular signal, which is distinct from PCP signaling or other gastrulation regulators, including BMP and Nodals. Moreover, mosaic expression of fusion proteins revealed enrichment of Vangl2 at the anterior cell edges of highly mediolaterally elongated cells. These results demonstrate that the dynamic Vangl2 intracellular distribution is coordinated with and necessary for the changes in convergence and extension cell behaviors during gastrulation.

What if stem cells turn into embryos in a dish?

Recent studies show that pluripotent stem cells can undergo self-organized development in vitro into structures that mimic the body plan of the post-implantation embryo. Modeling human embryogenesis in a dish opens up new possibilities for the study of early development and developmental disorders, but it may also raise substantial ethical concerns.

Glypican4 modulates lateral line collective cell migration non cell-autonomously.

Collective cell migration is an essential process during embryonic development and diseases such as cancer, and still much remains to be learned about how cell intrinsic and environmental cues are coordinated to guide cells to their targets. The migration-dependent development of the zebrafish sensory lateral line proves to be an excellent model to study how proteoglycans control collective cell migration in a vertebrate. Proteoglycans are extracellular matrix glycoproteins essential for the control of several signaling pathways including Wnt/ß-catenin, Fgf, BMP and Hh. In the lateral line primordium the modified sugar chains on proteoglycans are important regulators of cell polarity, ligand distribution and Fgf signaling. At least five proteoglycans show distinct expression patterns in the primordium; however, their individual functions have not been studied. Here, we describe the function of glypican4 during zebrafish lateral line development. glypican4 is expressed in neuromasts, interneuromast cells and muscle cells underlying the lateral line. knypekfr6/glypican4 mutants show severe primordium migration defects and the primordium often U-turns and migrates back toward the head. Our analysis shows that Glypican4 regulates the feedback loop between Wnt/ß-catenin/Fgf signaling in the primordium redundantly with other Heparan Sulfate Proteoglycans. In addition, the primordium migration defect is caused non-cell autonomously by the loss of cxcl12a-expressing muscle precursors along the myoseptum via downregulation of Hh. Our results show that glypican4 has distinct functions in primordium cells and cells in the environment and that both of these functions are essential for collective cell migration.

New insights into the maternal to zygotic transition.

The initial phases of embryonic development occur in the absence of de novo transcription and are instead controlled by maternally inherited mRNAs and proteins. During this initial period, cell cycles are synchronous and lack gap phases. Following this period of transcriptional silence, zygotic transcription begins, the maternal influence on development starts to decrease, and dramatic changes to the cell cycle take place. Here, we discuss recent work that is shedding light on the maternal to zygotic transition and the interrelated but distinct mechanisms regulating the onset of zygotic transcription and changes to the cell cycle during early embryonic development.

Mechanics of blastopore closure during amphibian gastrulation.

Blastopore closure in the amphibian embryo involves large scale tissue reorganization driven by physical forces. These forces are tuned to generate sustained blastopore closure throughout the course of gastrulation. We describe the mechanics of blastopore closure at multiple scales and in different regions around the blastopore by characterizing large scale tissue deformations, cell level shape change and subcellular F-actin organization and by measuring tissue force production and structural stiffness of the blastopore during gastrulation. We find that the embryo generates a ramping magnitude of force until it reaches a peak force on the order of 0.5µN. During this time course, the embryo also stiffens 1.5 fold. Strain rate mapping of the dorsal, ventral and lateral epithelial cells proximal to the blastopore reveals changing patterns of strain rate throughout closure. Cells dorsal to the blastopore, which are fated to become neural plate ectoderm, are polarized and have straight boundaries. In contrast, cells lateral and ventral to the blastopore are less polarized and have tortuous cell boundaries. The F-actin network is organized differently in each region with the highest percentage of alignment occurring in the lateral region. Interestingly F-actin was consistently oriented toward the blastopore lip in dorsal and lateral cells, but oriented parallel to the lip in ventral regions. Cell shape and F-actin alignment analyses reveal different local mechanical environments in regions around the blastopore, which was reflected by the strain rate maps.

Sebox regulates mesoderm formation in early amphibian embryos.

BACKGROUND: Mix/Bix genes are important regulators of mesendoderm formation during vertebrate embryogenesis. Sebox, an additional member of this gene family, has been implicated in endoderm formation during early embryogenesis in zebrafish. However, it remains unclear whether Sebox plays a unique role in early Xenopus embryos. RESULTS: In this study, we provide evidence that Sebox is uniquely required for the formation of mesoderm during early Xenopus embryogenesis. Sebox is dynamically expressed in the involuted mesoderm during gastrulation. It is activated by Nodal/Activin signaling and modulated by zygotic Wnt/ß-catenin signaling. Overexpression of Sebox perturbs movements during convergent extension and inhibits the expression of mesodermal, but not endodermal, genes induced by Nodal/Activin signaling. Depletion of Sebox using a specific morpholino increases the expression of noncanonical wnt5a, wnt5b, and wnt11b. Depletion of Sebox also up-regulates the expression of pcdh8.2, a paraxial mesoderm-specific protocadherin, in a Wnt11B-dependent manner. Sebox morphants display reduced development of the head and notochord. CONCLUSIONS: Our findings illustrate that Sebox, a unique member of the Mix/Bix gene family, functions downstream of Nodal/Activin signaling and is required for the proper expression of noncanonical Wnt ligands and the normal development of mesoderm in Xenopus.

Maternal control of axial-paraxial mesoderm patterning via direct transcriptional repression in zebrafish.

Axial-paraxial mesoderm patterning is a special dorsal-ventral patterning event of establishing the vertebrate body plan. Though dorsal-ventral patterning has been extensively studied, the initiation of axial-paraxial mesoderm pattering remains largely unrevealed. In zebrafish, spt cell-autonomously regulates paraxial mesoderm specification and flh represses spt expression to promote axial mesoderm fate, but the expression domains of spt and flh initially overlap in the entire marginal zone of the embryo. Defining spt and flh territories is therefore a premise of axial-paraxial mesoderm patterning. In this study, we investigated why and how the initial expression of flh becomes repressed in the ventrolateral marginal cells during blastula stage. Loss- and gain-of-function experiments showed that a maternal transcription factor Vsx1 is essential for restricting flh expression within the dorsal margin and preserving spt expression and paraxial mesoderm specification in the ventrolateral margin of embryo. Chromatin immunoprecipitation and electrophoretic mobility shift assays in combination with core consensus sequence mutation analysis further revealed that Vsx1 can directly repress flh by binding to the proximal promoter at a specific site. Inhibiting maternal vsx1 translation resulted in confusion of axial and paraxial mesoderm markers expression and axial-paraxial mesoderm patterning. These results demonstrated that direct transcriptional repression of the decisive axial mesoderm gene by maternal ventralizing factor is a crucial regulatory mechanism of initiating axial-paraxial mesoderm patterning in vertebrates.

Multistep molecular mechanism for bone morphogenetic protein extracellular transport in the Drosophila embryo.

In the Drosophila embryo, formation of a bone morphogenetic protein (BMP) morphogen gradient requires transport of a heterodimer of the BMPs Decapentaplegic (Dpp) and Screw (Scw) in a protein shuttling complex. Although the core components of the shuttling complex--Short Gastrulation (Sog) and Twisted Gastrulation (Tsg)--have been identified, key aspects of this shuttling system remain mechanistically unresolved. Recently, we discovered that the extracellular matrix protein collagen IV is important for BMP gradient formation. Here, we formulate a molecular mechanism of BMP shuttling that is catalyzed by collagen IV. We show that Dpp is the only BMP ligand in Drosophila that binds collagen IV. A collagen IV binding-deficient Dpp mutant signals at longer range in vivo, indicating that collagen IV functions to immobilize free Dpp in the embryo. We also provide in vivo evidence that collagen IV functions as a scaffold to promote shuttling complex assembly in a multistep process. After binding of Dpp/Scw and Sog to collagen IV, protein interactions are remodeled, generating an intermediate complex in which Dpp/Scw-Sog is poised for release by Tsg through specific disruption of a collagen IV-Sog interaction. Because all components are evolutionarily conserved, we propose that regulation of BMP shuttling and immobilization through extracellular matrix interactions is widely used, both during development and in tissue homeostasis, to achieve a precise extracellular BMP distribution.

Serotonin receptors are selectively expressed in the avian germ cells and early embryos.

The expression of nine serotonin (5-HT) receptor transcripts was studied using reverse transcription polymerase chain reaction (RT-PCR) in germ cells, cleavage and gastrulation stages of Japanese quail, and qPCR for 5-HT3 and 5-HT4 receptors in oocytes and embryos. We show the presence/absence of nine serotonin transcripts known in birds for receptors 5-HT1A, 5-HT1F, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT6 and 5-HT7A in avian germ cells and early embryos. The absence of 5-HT3 and 5-HT5A in primordial germ cells and of 5-HT3 and 5-HT7A in sperm is characteristic. All transcripts appeared in oocytes at all stages (except for 5-HT3 and 5-HT5A transcripts) and all were present in cleaving embryos and at gastrulation, except for 5-HT3, which was permanently observed as late as in stage 4. Interestingly, 5-HT3 and 5-HT5A receptors accumulated in 3-mm and F1 oocytes but were degraded at ovulation and started to be re-transcribed in cleavage stage II embryos and beyond. The selective appearance of 5-HT receptors in germ cells and early embryos supports the hypothesis that serotonin may act as a signalling molecule at early stages of germ line and embryo differentiation via individual receptors present during different stages, when specialized communication systems are not yet developed.