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During embryonic development, the vertebrate embryonic epiblast is divided into two parts including neural and superficial ectoderm. The neural plate border (NPB) is a narrow transitional area which locates between these parts and contains multipotent progenitor cells. Despite its small size, the cellular heterogeneity in this region produces specific differentiated cells. Signaling pathways, transcription factors, and the expression/repression of certain genes are directly involved in these differentiation processes. Different factors such as the Wnt signaling cascade, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) signaling, and Notch, which are involved in various stages of the growth, proliferation, and differentiation of embryonic cells, are also involved in the determination and differentiation of neural plate border stem cells. Therefore, it is essential to consider the interactions and temporospatial coordination related to cells, tissues, and adjacent structures. This review examines our present knowledge of the formation of the neural plate border and emphasizes the requirement for interaction between different signaling pathways, including the BMP and Wnt cascades, the expression of its special target genes and their regulations, and the precise tissue crosstalk which defines the neural crest fate in the ectoderm at the early human embryonic stages.

The lack of a widely accessible method for expressing genes of interest in wild-type embryos is a fundamental obstacle to understanding genetic regulation during embryonic development. In particular, only a few methods are available for introducing gene expression vectors into cells prior to neural tube closure, which is a period of drastic development for many tissues. In this study, we present a simple technique for injecting vectors into the amniotic cavity and allowing them to reach the ectodermal cells and the epithelia of endodermal organs of mouse embryos at E8.0 via in utero injection, using only a widely used optical fiber with an illuminator. Using this technique, retroviruses can be introduced to facilitate the labeling of cells in various tissues, including the brain, spinal cord, epidermis, and digestive and respiratory organs. We also demonstrated in utero electroporation of plasmid DNA into E7.0 and E8.0 embryos. Taking advantage of this method, we reveal the association between Ldb1 and the activity of the Neurog2 transcription factor in the mouse neocortex. This technique can aid in analyzing the roles of genes of interest during endo- and ectodermal development prior to neural tube closure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The ectoderm is the outermost of the three germ layers of the early embryo that arise during gastrulation. Once the germ layers are established, the complex interplay of cellular proliferation, differentiation, and migration results in organogenesis. The ectoderm is the progenitor of both the surface ectoderm and the neural ectoderm. Notably, the surface ectoderm develops into the epidermis and its associated appendages, nails, external exocrine glands, olfactory epithelium, and the anterior pituitary. Specification, development, and homeostasis of these organs demand a tightly orchestrated gene expression program that is often dictated by epigenetic regulation. In this review, we discuss the recent discoveries that have highlighted the importance of chromatin regulatory mechanisms mediated by transcription factors, histone and DNA modifications that aid in the development of surface ectodermal organs and maintain their homeostasis post-development., (Copyright © 2024 Elsevier Inc. All rights reserved.)

As tissues grow and change shape during animal development, they physically pull and push on each other, and these mechanical interactions can be important for morphogenesis. During Drosophila gastrulation, mesoderm invagination temporally overlaps with the convergence and extension of the ectodermal germband; the latter is caused primarily by Myosin II-driven polarised cell intercalation. Here, we investigate the impact of mesoderm invagination on ectoderm extension, examining possible mechanical and mechanotransductive effects on Myosin II recruitment and polarised cell intercalation. We find that the germband ectoderm is deformed by the mesoderm pulling in the orthogonal direction to germband extension (GBE), showing mechanical coupling between these tissues. However, we do not find a significant change in Myosin II planar polarisation in response to mesoderm invagination, nor in the rate of junction shrinkage leading to neighbour exchange events. We conclude that the main cellular mechanism of axis extension, polarised cell intercalation, is robust to the mesoderm invagination pull. We find, however, that mesoderm invagination slows down the rate of anterior-posterior cell elongation that contributes to axis extension, counteracting the tension from the endoderm invagination, which pulls along the direction of GBE., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The transgenic (TG) zebrafish allows researchers to bio-image specific biological phenomena in cells and tissues in vivo . We established TG lines to monitor changes in the ovaries of live fish. The original TG line with ovarian fluorescence was occasionally established. Although the cDNA integrated into the line was constructed for the expression of enhanced green fluorescent protein (EGFP) driven by the medaka β- actin promoter, the expression of EGFP is restricted to the oocytes and gills in adult fish. Furthermore, we found that germinal vesicles (GVs) in oocytes of the established line can be observed by relatively strong fluorescence around the GV. In this study, we tried to capture the dynamic processes of germinal vesicle breakdown (GVBD) during meiotic cell division using the GV fluorescent oocytes. As a result, GV migration and GVBD could be monitored in real time. We also succeeded in observing actin filaments involved in the migration of GV to the animal pole. This strain can be used for education in the process of oocyte meiotic cell division.

Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos 1-8 . Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells 9-17 . Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis., (© 2023. The Author(s).)

The vertebrate inner ear arises from a pool of progenitors with the potential to contribute to all the sense organs and cranial ganglia in the head. Here, we explore the molecular mechanisms that control ear specification from these precursors. Using a multiomics approach combined with loss-of-function experiments, we identify a core transcriptional circuit that imparts ear identity, along with a genome-wide characterization of noncoding elements that integrate this information. This analysis places the transcription factor Sox8 at the top of the ear determination network. Introducing Sox8 into the cranial ectoderm not only converts non-ear cells into ear progenitors but also activates the cellular programs for ear morphogenesis and neurogenesis. Thus, Sox8 has the unique ability to remodel transcriptional networks in the cranial ectoderm toward ear identity.

Development of primordial germ cells (PGCs) is required for reproduction. During PGC development in mammals, major epigenetic remodeling occurs, which is hypothesized to establish an epigenetic landscape for sex-specific germ cell differentiation and gametogenesis. In order to address the role of embryonic ectoderm development (EED) and histone 3 lysine 27 trimethylation (H3K27me3) in this process, we created an EED conditional knockout mouse and show that EED is essential for regulating the timing of sex-specific PGC differentiation in both ovaries and testes, as well as X chromosome dosage decompensation in testes. Integrating chromatin and whole genome bisulfite sequencing of epiblast and PGCs, we identified a poised repressive signature of H3K27me3/DNA methylation that we propose is established in the epiblast where EED and DNMT1 interact. Thus, EED joins DNMT1 in regulating the timing of sex-specific PGC differentiation during the critical window when the gonadal niche cells specialize into an ovary or testis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neurulation is the process in early vertebrate embryonic development during which the neural plate folds to form the neural tube. Spinal neural tube folding in the posterior neuropore changes over time, first showing a median hinge point, then both the median hinge point and dorsolateral hinge points, followed by dorsolateral hinge points only. The biomechanical mechanism of hinge point formation in the mammalian neural tube is poorly understood. Here we employ a mechanical finite element model to study neural tube formation. The computational model mimics the mammalian neural tube using microscopy data from mouse and human embryos. While intrinsic curvature at the neural plate midline has been hypothesized to drive neural tube folding, intrinsic curvature was not sufficient for tube closure in our simulations. We achieved neural tube closure with an alternative model combining mesoderm expansion, nonneural ectoderm expansion, and neural plate adhesion to the notochord. Dorsolateral hinge points emerged in simulations with low mesoderm expansion and zippering. We propose that zippering provides the biomechanical force for dorsolateral hinge point formation in settings where the neural plate lateral sides extend above the mesoderm. Together, these results provide a perspective on the biomechanical and molecular mechanism of mammalian spinal neurulation.

Brachyury is a T-box family transcription factor and plays pivotal roles in morphogenesis. In sea urchin embryos, Brachyury is expressed in the invaginating endoderm, and in the oral ectoderm of the invaginating mouth opening. The oral ectoderm is hypothesized to serve as a signaling center for oral (ventral)-aboral (dorsal) axis formation and to function as a ventral organizer. Our previous results of a single-cell RNA-seq (scRNA-seq) atlas of early Strongylocentrotus purpuratus embryos categorized the constituent cells into 22 clusters, in which the endoderm consists of three clusters and the oral ectoderm four clusters (Foster et al., 2020). Here we examined which clusters of cells expressed Brachyury in relation to the morphogenesis and the identity of the ventral organizer. Our results showed that cells of all three endoderm clusters expressed Brachyury in blastulae. Based on expression profiles of genes involved in the gene regulatory networks (GRNs) of sea urchin embryos, the three clusters are distinguishable, two likely derived from the Veg2 tier and one from the Veg1 tier. On the other hand, of the four oral-ectoderm clusters, cells of two clusters expressed Brachyury at the gastrula stage and genes that are responsible for the ventral organizer at the late blastula stage, but the other two clusters did not. At a single-cell level, most cells of the two oral-ectoderm clusters expressed organizer-related genes, nearly a half of which coincidently expressed Brachyury. This suggests that the ventral organizer contains Brachyury-positive cells which invaginate to form the stomodeum. This scRNA-seq study therefore highlights significant roles of Brachyury-expressing cells in body-plan formation of early sea urchin embryos, though cellular and molecular mechanisms for how Brachyury functions in these processes remain to be elucidated in future studies., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Understanding human organ formation is a scientific challenge with far-reaching medical implications 1,2 . Three-dimensional stem-cell cultures have provided insights into human cell differentiation 3,4 . However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction 5,6 , neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior-posterior axis depends on neural ectoderm geometry in addition to molecular gradients 7 . Our approach provides a new route to the study of human organ morphogenesis in health and disease., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

PRC2-mediated epigenetic function involves the interaction with long non-coding RNAs (lncRNAs). Although the identity of some of these RNAs has been elucidated in the context of developmental programs, their counterparts in postmitotic adult tissue homeostasis remain uncharacterized. To this aim, we used terminally differentiated postmitotic skeletal muscle cells in which oxidative stress induces the dynamic activation of PRC2-Ezh1 through Embryonic Ectoderm Develpment (EED) shuttling to the nucleus. We identify lncRNA Malat-1 as a necessary partner for PRC2-Ezh1-dependent response to oxidative stress. We show that in this pathway, PRC2-EZH1 dynamic assembly, and in turn stress induced skeletal muscle targeted genes repression, depends specifically on Malat-1. Our study reports about PRC2-RNA interactions in the physiological context of adaptive oxidative stress response and identifies the first lncRNA involved in PRC2-Ezh1 function., (© 2021. The Author(s).)

Branchio-oto-renal syndrome (BOR) is a disorder characterized by hearing loss, and craniofacial and/or renal defects. Variants in the transcription factor Six1 and its co-factor Eya1, both of which are required for otic development, are linked to BOR. We previously identified Sobp as a potential Six1 co-factor, and SOBP variants in mouse and humans cause otic phenotypes; therefore, we asked whether Sobp interacts with Six1 and thereby may contribute to BOR. Co-immunoprecipitation and immunofluorescence experiments demonstrate that Sobp binds to and colocalizes with Six1 in the cell nucleus. Luciferase assays show that Sobp interferes with the transcriptional activation of Six1+Eya1 target genes. Experiments in Xenopus embryos that either knock down or increase expression of Sobp show that it is required for formation of ectodermal domains at neural plate stages. In addition, altering Sobp levels disrupts otic vesicle development and causes craniofacial cartilage defects. Expression of Xenopus Sobp containing the human variant disrupts the pre-placodal ectoderm similar to full-length Sobp, but other changes are distinct. These results indicate that Sobp modifies Six1 function and is required for vertebrate craniofacial development, and identify Sobp as a potential candidate gene for BOR., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

During vertebrate development, the formation of the central nervous system (CNS) is initiated by neural induction and patterning of the embryonic ectoderm. We previously reported that Cdc2-like kinase 2 (Clk2) promotes neural development in Xenopus embryos by regulating morphogen signaling. However, the functions of other Clk family members and their roles in early embryonic development remain unknown. Here, we show that in addition to Clk2, Clk1 and Clk3 play a role in the formation of neural tissue in Xenopus. clk1 and clk3 are co-expressed in the developing neural tissue during early Xenopus embryogenesis. We found that overexpression of clk1 and clk3 increases the expression of neural marker genes in ectodermal explants. Furthermore, knockdown experiments showed that clk3 is required for the formation of neural tissues. These results suggest that Xenopus Clk3 plays an essential role in promoting neural development during early embryogenesis., Competing Interests: Declaration of competing interest To the best of our knowledge, the named authors have no conflict of interest, financial or otherwise., (Copyright © 2021 Elsevier Inc. All rights reserved.)

During embryonic development, cells differentiate into a variety of distinct cell types and subtypes with diverse transcriptional profiles. To date, transcriptomic signatures of different cell lineages that arise during development have been only partially characterized. Here we used single-cell RNA-seq to perform transcriptomic analysis of over 20,000 cells disaggregated from the trunk region of zebrafish embryos at the 30 hpf stage. Transcriptional signatures of 27 different cell types and subtypes were identified and annotated during this analysis. This dataset will be a useful resource for many researchers in the fields of developmental and cellular biology and facilitate the understanding of molecular mechanisms that regulate cell lineage choices during development., Competing Interests: The authors have declared that no competing interests exist.

The spatiotemporal coordination of multiple morphogens is essential for embryonic patterning yet poorly understood. During neural crest (NC) formation, dynamic bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and WNT signals cooperate by acting on mesoderm and ectoderm. Here, we show that Fhl3, a scaffold LIM domain protein, modulates BMP gradient interpretation during NC induction. During gastrulation, low BMP signaling neuralizes the neural border (NB) ectoderm, while Fhl3 enhances Smad1 intracellular response in underlying paraxial mesoderm, triggering the high WNT8 signals needed to pattern the NB. During neurulation, fhl3 activation in NC ectoderm promotes simultaneous high BMP and BMP-dependent WNT activity required for specification. Mechanistically, Fhl3 interacts with Smad1 and promotes Smad1 binding to wnt8 promoter in a BMP-dependent manner. Consequently, differential Fhl3 expression in adjacent cells ensures a finely tuned coordination of BMP and WNT signaling at several stages of NC development, starting by positioning the NC-inducing mesoderm center under competent NB ectoderm., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Problems of cell biology and the molecular controls underpinning them have been studied in the remarkably versatile Xenopus systems for many years. This versatility is showcased in several accompanying protocols, which are introduced here. One protocol demonstrates how the Xenopus embryonic ectoderm can be used to study the effects of mechanical cell deformation; another illustrates how the developing eye can be used as a platform for determining cell-cycle length. Two protocols show how extracts from Xenopus embryos can be exploited to characterize the behavior of specific intracellular proteins-specifically, to determine protein phosphorylation status and the ability to bind to chromatin. Finally, because specific antibodies to Xenopus proteins are pivotal reagents for cell biology and biochemistry applications, four protocols describing how to generate, purify, and assay the specificity of antibodies raised against Xenopus proteins are included in hopes of stimulating the expansion of these critical resources across the Xenopus community., (© 2021 Cold Spring Harbor Laboratory Press.)

Specification of Sox2 + proneurosensory progenitors within otic ectoderm is a prerequisite for the production of sensory cells and neurons for hearing. However, the underlying molecular mechanisms driving this lineage specification remain unknown. Here, we show that the Brg1-based SWI/SNF chromatin-remodeling complex interacts with the neurosensory-specific transcriptional regulators Eya1/Six1 to induce Sox2 expression and promote proneurosensory-lineage specification. Ablation of the ATPase-subunit Brg1 or both Eya1/Six1 results in loss of Sox2 expression and lack of neurosensory identity, leading to abnormal apoptosis within the otic ectoderm. Brg1 binds to two of three distal 3' Sox2 enhancers occupied by Six1, and Brg1-binding to these regions depends on Eya1-Six1 activity. We demonstrate that the activity of these Sox2 enhancers in otic neurosensory cells specifically depends on binding to Six1. Furthermore, genome-wide and transcriptome profiling indicate that Brg1 may suppress apoptotic factor Map3k5 to inhibit apoptosis. Together, our findings reveal an essential role for Brg1, its downstream pathways, and their interactions with Six1/Eya1 in promoting proneurosensory fate induction in the otic ectoderm and subsequent neuronal lineage commitment and survival of otic cells., Competing Interests: The authors declare no competing interest.

The generation of the components that make up the embryonic body axis, such as the spinal cord and vertebral column, takes place in an anterior-to-posterior (head-to-tail) direction. This process is driven by the coordinated production of various cell types from a pool of posteriorly-located axial progenitors. Here, we review the key features of this process and the biology of axial progenitors, including neuromesodermal progenitors, the common precursors of the spinal cord and trunk musculature. We discuss recent developments in the in vitro production of axial progenitors and their potential implications in disease modelling and regenerative medicine., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

We previously identified the protein Lbh as necessary for cranial neural crest (CNC) cell migration in Xenopus through the use of morpholinos. However, Lbh is a maternally deposited protein and morpholinos achieve knockdowns through prevention of translation. In order to investigate the role of Lbh in earlier embryonic events, we employed the new technique "Trim-Away" to degrade this maternally deposited protein. Trim-Away utilizes the E3 ubiquitin ligase trim21 to degrade proteins targeted with an antibody and was developed in mammalian systems. Our results show that Xenopus is amenable to the Trim-Away technique. We also show that early knockdown of Lbh in Xenopus results in defects in gastrulation that present with a decrease in fibronectin matrix assembly, an increased in mesodermal cell migration and decrease in endodermal cell cohesion. We further show that the technique is also effective on a second abundant maternal protein PACSIN2. We discuss potential advantages and limit of the technique in Xenopus embryos as well as the mechanism of gastrulation inhibition., (Copyright © 2020 Elsevier Inc. All rights reserved.)

At implantation, the mouse embryo undergoes a critical transformation which requires the precise spatiotemporal control of signalling pathways necessary for morphogenesis and developmental progression. The role played by such signalling pathways during this transition are largely unexplored, due to the inaccessibility of the embryo during the implantation when it becomes engulfed by uterine tissues. Genetic studies demonstrate that mutant embryos for BMPs die around gastrulation. Here we have aimed to dissect the role of BMPs during pre-to post-implantation transition by using a protocol permitting the development of the embryo beyond implantation stages in vitro and using stem cells to mimic post-implantation tissue organisation. By assessing both the canonical and non-canonical mechanisms of BMP, we show that the loss of canonical BMP activity compromises the extra-embryonic ectoderm development. Our analyses demonstrate that BMP signalling maintains stem cell populations within both embryonic/extra-embryonic tissues during pre-to post-implantation development. These results may provide insight into the role played by BMP signalling in controlling early embryogenesis., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Published by Elsevier Inc.)

Developmental genes are often regulated by multiple elements with overlapping activity. Yet, in most cases, the relative function of those elements and their contribution to endogenous gene expression remain poorly characterized. An example of this phenomenon is that distinct sets of enhancers have been proposed to direct Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary. Using in vivo CRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the apical ectodermal ridge, the control of Fgf8 expression appears distributed between different enhancers. In contrast, we find that in the midbrain-hindbrain boundary, one of the three active enhancers is essential while the other two are dispensable. We further dissect the essential midbrain-hindbrain boundary enhancer to reveal that it is also composed by a mixture of essential and dispensable modules. Cross-species transgenic analysis of this enhancer suggests that its composition may have changed in the vertebrate lineage.

Wnt/β-catenin signaling controls many biological processes for the generation and sustainability of proper tissue size, organization and function during development and homeostasis. Consequently, mutations in the Wnt pathway components and modulators cause diseases, including genetic disorders and cancers. Targeted treatment of pathway-associated diseases entails detailed understanding of the regulatory mechanisms that fine-tune Wnt signaling. Here, we identify the neurotrophin receptor-associated death domain (Nradd), a homolog of p75 neurotrophin receptor (p75 NTR ), as a negative regulator of Wnt/β-catenin signaling in zebrafish embryos and in mammalian cells. Nradd significantly suppresses Wnt8-mediated patterning of the mesoderm and neuroectoderm during zebrafish gastrulation. Nradd is localized at the plasma membrane, physically interacts with the Wnt receptor complex and enhances apoptosis in cooperation with Wnt/β-catenin signaling. Our functional analyses indicate that the N-glycosylated N-terminus and the death domain-containing C-terminus regions are necessary for both the inhibition of Wnt signaling and apoptosis. Finally, Nradd can induce apoptosis in mammalian cells. Thus, Nradd regulates cell death as a modifier of Wnt/β-catenin signaling during development.

Collective migration of cohesive tissues is a fundamental process in morphogenesis and is particularly well illustrated during gastrulation by the rapid and massive internalization of the mesoderm, which contrasts with the much more modest movements of the ectoderm. In the Xenopus embryo, the differences in morphogenetic capabilities of ectoderm and mesoderm can be connected to the intrinsic motility of individual cells, very low for ectoderm, high for mesoderm. Surprisingly, we find that these seemingly deep differences can be accounted for simply by differences in Rho-kinases (Rock)-dependent actomyosin contractility. We show that Rock inhibition is sufficient to rapidly unleash motility in the ectoderm and confer it with mesoderm-like properties. In the mesoderm, this motility is dependent on two negative regulators of RhoA, the small GTPase Rnd1 and the RhoGAP Shirin/Dlc2/ArhGAP37. Both are absolutely essential for gastrulation. At the cellular and tissue level, the two regulators show overlapping yet distinct functions. They both contribute to decrease cortical tension and confer motility, but Shirin tends to increase tissue fluidity and stimulate dispersion, while Rnd1 tends to favor more compact collective migration. Thus, each is able to contribute to a specific property of the migratory behavior of the mesoderm. We propose that the "ectoderm to mesoderm transition" is a prototypic case of collective migration driven by a down-regulation of cellular tension, without the need for the complex changes traditionally associated with the epithelial-to-mesenchymal transition., Competing Interests: The authors have declared that no competing interests exist.

The olfactory epithelia arise from morphologically identifiable structures called olfactory placodes. Sensory placodes are generally described as being induced from the ectoderm suggesting that their development is separate from the coordinated cell movements generating the central nervous system. Previously, we have shown that the olfactory placodes arise from a large field of cells bordering the telencephalic precursors in the neural plate, and that cell movements, not cell division, underlie olfactory placode morphogenesis. Subsequently by image analysis, cells were tracked as they moved in the continuous sheet of neurectoderm giving rise to the peripheral (olfactory organs) and central (olfactory bulbs) nervous system (Torres-Paz and Whitlock, 2014). These studies lead to a model whereby the olfactory epithelia develop from within the border of the neural late and are a neural tube derivative, similar to the retina of the eye (Torres-Paz and Whitlock, 2014; Whitlock, 2008). Here we show that randomly generated clones of cells extend across the morphologically differentiated olfactory placodes/olfactory bulbs, and test the hypothesis that these structures are patterned by a different level of distal-less (dlx) gene expression subdividing the anterior neurectoderm into OP precursors (high Dlx expression) and OB precursors (lower Dlx expression). Manipulation of DLX protein and RNA levels resulted in morphological changes in the size of the olfactory epithelia and olfactory bulb. Thus, the olfactory epithelia and bulbs arise from a common neurectodermal region and develop in concert through coordinated morphological movements.

Background: Hedgehog signaling has various regulatory functions in tissue morphogenesis and differentiation. To investigate its involvement in anterior pituitary precursor development and the lens precursor potential for anterior pituitary precursors, we investigated Talpid mutant Japanese quail embryos, in which hedgehog signaling is defective., Results: Talpid mutants develop multiple pituitary precursor-like pouches of variable sizes from the oral ectoderm (OE). The ectopic pituitary pouches initially express the pituitary-associated transcription factor (TF) LHX3 similarly to Rathke's pouch, the genuine pituitary precursor. The pouches coexpress the TFs SOX2 and PAX6, a signature of lens developmental potential. Most Talpid mutant pituitary pouches downregulate LHX3 expression and activate the lens-essential TF PROX1, leading to the development of small lens tissue expressing α-, β-, and δ-crystallins. In contrast, mutant Rathke's pouches express a lower level of LHX3, which is primarily localized in the cytoplasm, and activate the lens developmental pathway., Conclusions: Hedgehog signaling in normal embryos regulates the development of Rathke's pouch in two steps. First, by confining Rathke's pouch development in a low hedgehog signaling region of the OE. Second, by sustaining LHX3 activity to promote anterior pituitary development, while inhibiting ectopic lens development., (© 2020 Wiley Periodicals LLC.)

Vertebrate Hox genes are critical for the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here, we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that the HoxC gene cluster was co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of the HoxC cluster led to mice lacking nails (anonychia), a condition stronger than the previously reported loss of function of Hoxc13 , which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two mammalian-specific ectodermal enhancers located upstream of the HoxC gene cluster, which together regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with the mammalian taxon to provide the level of HOXC proteins necessary for the full development of hair and nail., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The Six1 transcription factor plays a major role in craniofacial development. Mutations in SIX1 and its co-factor, EYA1, are causative for about 50% of Branchio-otic/Branchio-oto-renal syndrome (BOR) patients, who are characterized by variable craniofacial, otic and renal malformations. We previously screened for other proteins that might interact with Six1 to identify additional genes that may play a role in BOR, and herein characterize the developmental role of one of them, Microspherule protein 1 (Mcrs1). We found that in cultured cells, Mcrs1 bound to Six1 and in both cultured cells and embryonic ectoderm reduced Six1-Eya1 transcriptional activation. Knock-down of Mcrs1 in embryos caused an expansion of the domains of neural plate genes and two genes expressed in both the neural plate and neural crest (zic1, zic2). In contrast, two other genes expressed in pre-migratory neural crest (foxd3, sox9) were primarily reduced. Cranial placode genes showed a mixture of expanded and diminished expression domains. At larval stages, loss of Mcrs1 resulted in a significant reduction of otic vesicle gene expression concomitant with a smaller otic vesicle volume. Experimentally increasing Mcrs1 above endogenous levels favored the expansion of neural border and neural crest gene domains over cranial placode genes; it also reduced otic vesicle gene expression but not otic vesicle volume. Co-expression of Mcrs1 and Six1 as well as double knock-down and rescue experiments establish a functional interaction between Mcrs1 and Six1 in the embryo, and demonstrate that this interaction has an important role in the development of craniofacial tissues including the otic vesicle., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Neuroectoderm formation is the first step in development of a proper nervous system for vertebrates. The developmental decision to form a non-neural ectoderm versus a neural one involves the regulation of BMP signaling, first reported many decades ago. However, the precise regulatory mechanism by which this is accomplished has not been fully elucidated, particularly for transcriptional regulation of certain key transcription factors. BMP4 inhibition is a required step in eliciting neuroectoderm from ectoderm and Foxd4l1.1 is one of the earliest neural genes highly expressed in the neuroectoderm and conserved across vertebrates, including humans. In this work, we focused on how Foxd4l1.1 downregulates the neural repressive pathway. Foxd4l1.1 inhibited BMP4/Smad1 signaling and triggered neuroectoderm formation in animal cap explants of Xenopus embryos. Foxd4l1.1 directly bound within the promoter of endogenous neural repressor ventx1.1 and inhibited ventx1.1 transcription. Foxd4l1.1 also physically interacted with Xbra in the nucleus and inhibited Xbra-induced ventx1.1 transcription. In addition, Foxd4l1.1 also reduced nuclear localization of Smad1 to inhibit Smad1-mediated ventx1.1 transcription. Foxd4l1.1 reduced the direct binding of Xbra and Smad1 on ventx1.1 promoter regions to block Xbra/Smad1-induced synergistic activation of ventx1.1 transcription. Collectively, Foxd4l1.1 negatively regulates transcription of a neural repressor ventx1.1 by multiple mechanisms in its exclusively occupied territory of neuroectoderm, and thus leading to primary neurogenesis. In conjunction with the results of our previous findings that ventx1.1 directly represses foxd4l1.1, the reciprocal repression of ventx1.1 and foxd4l1.1 is significant in at least in part specifying the mechanism for the non-neural versus neural ectoderm fate determination in Xenopus embryos.

Understanding limb development not only gives insights into the outgrowth and differentiation of the limb, but also has clinical relevance. Limb development begins with two paired limb buds (forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER). As a transitional embryonic structure, the AER undergoes four stages and contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cell activities by secretory signalling and the activation of gene expression. Within the scope of proximodistal patterning, it is understood that while fibroblast growth factors (FGFs) function sequentially over time as primary components of the AER signalling process, there is still no consensus on models that would explain proximodistal patterning itself. In anteroposterior patterning, the AER has a dual-direction regulation by which it promotes the sonic hedgehog (Shh) gene expression in the zone of polarizing activity (ZPA) for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates Engrailed-1 (En1) expression, and thus represses Wnt family member 7a (Wnt7a) expression in the ventral ectoderm by the expression of Fgfs, Sp6/8, and bone morphogenetic protein (Bmp) genes. The AER also plays a vital role in shaping the individual digits, since levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. In summary, the knowledge of crosstalk within AER among the three main axes is essential to understand limb growth and pattern formation, as the development of its areas proceeds simultaneously.

Background: The frontonasal ectodermal zone (FEZ) is a signaling center that regulates patterned development of the upper jaw, and Sonic hedgehog (SHH) mediates FEZ activity. Induction of SHH expression in the FEZ results from SHH-dependent signals from the brain and neural crest cells. Given the role of miRNAs in modulating gene expression, we investigated the extent to which miRNAs regulate SHH expression and FEZ signaling., Results: In the FEZ, the miR-199 family appears to be regulated by SHH-dependent signals from the brain; expression of this family increased from HH18 to HH22, and upon activation of SHH signaling in the brain. However, the miR-199 family is more broadly expressed in the mesenchyme of the frontonasal process and adjacent neuroepithelium. Downregulating the miR-199 genes expanded SHH expression in the FEZ, resulting in wider faces, while upregulating miR-199 genes resulted in decreased SHH expression and narrow faces. Hypoxia inducible factor 1 alpha (HIF1A) and mitogen-activated protein kinase kinase kinase 4 (MAP3K4) appear to be potential targets of miR-199b. Reduction of MAP3K4 altered beak development but increased apoptosis, while reducing HIF1A reduced expression of SHH in the FEZ and produced malformations independent of apoptosis., Conclusions: Our results demonstrate that this miRNA family appears to participate in regulating SHH expression in the FEZ; however, specific molecular mechanisms remain unknown., (© 2020 Wiley Periodicals LLC.)

Mammalian blastocysts are composed of two distinct cell lineages, namely the inner cell mass (ICM) and trophectoderm (TE). TE cells that give rise to the embryonic placenta are marked by an exclusive expression of the key determinant transcription factor, CDX2. Although Hippo signaling pathway is known to be responsible for this TE-specific expression of CDX2, the upstream regulator of this pathway in mammalian embryos is still controversial. In the present study, the involvement of the small molecular G protein, RHOA, in TE cell-fate decision in cattle was investigated. Inhibition of RHOA by the specific inhibitor, C3 transferase (C3), severely impaired the blastocyst formation. Further, C3 treatment significantly decreased the number of blastomeres with nuclearized YAP1, the prominent effector of Hippo pathway. An artificial isolation of ICM cells from blastocysts followed by the continuing culture to regenerate TE cells was conducted and showed that TE re-emergence from the isolated ICM is governed by Hippo pathway and suppressed by C3 treatment like that observed in developing embryos. Finally, the long-term exposure to C3 suggests the presence of alternative regulators of CDX2 expression other than RHOA signaling because there were still CDX2-positive cells after C3 treatment. These results demonstrated that RHOA signaling plays a significant role in TE cell-fate decision by regulating Hippo pathway in cattle., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Embryonic development is particularly vulnerable to stress and DNA damage, as mutations can accumulate through cell proliferation in a wide number of cells and organs. However, the biological effects of chronic exposure to ionising radiation (IR) at low and moderate dose rates (< 6 mGy/h) remain largely controversial, raising concerns for environmental protection. The present study focuses on the molecular effects of IR (0.005 to 50 mGy/h) on zebrafish embryos at the gastrula stage (6 hpf), at both the transcriptomics and epigenetics levels. Our results show that exposure to IR modifies the expression of genes involved in mitochondrial activity from 0.5 to 50 mGy/h. In addition, important developmental pathways, namely, the Notch, retinoic acid, BMP and Wnt signalling pathways, were altered at 5 and 50 mGy/h. Transcriptional changes of genes involved in the morphogenesis of the ectoderm and mesoderm were detected at all dose rates, but were prominent from 0.5 to 50 mGy/h. At the epigenetic level, exposure to IR induced a hypomethylation of DNA in the promoter of genes that colocalised with both H3K27me3 and H3Kme4 histone marks and correlated with changes in transcriptional activity. Finally, pathway enrichment analysis demonstrated that the DNA methylation changes occurred in the promoter of important developmental genes, including morphogenesis of the ectoderm and mesoderm. Together, these results show that the transcriptional program regulating morphogenesis in gastrulating embryos was modified at dose rates greater than or equal to 0.5 mGy/h, which might predict potential neurogenesis and somitogenesis defects observed at similar dose rates later in development.

The mechanisms underlying mammalian neural tube closure remain poorly understood. We report a unique cellular process involving multicellular rosette formation, convergent cellular protrusions, and F-actin cable network of the non-neural surface ectodermal cells encircling the closure site of the posterior neuropore, which are demonstrated by scanning electron microscopy and genetic fate mapping analyses during mouse spinal neurulation. These unique cellular structures are severely disrupted in the surface ectodermal transcription factor Grhl3 mutants that exhibit fully penetrant spina bifida. We propose a novel model of mammalian neural tube closure driven by surface ectodermal dynamics, which is computationally visualized., (Copyright © 2020 Elsevier Inc. All rights reserved.)

R-spondins are a family of secreted proteins that play important roles in embryonic development and cancer. R-spondins have been shown to modulate the Wnt pathway; however, their involvement in other developmental signaling processes have remained largely unstudied. Here, we describe a novel function of Rspo2 in FGF pathway regulation in vivo Overexpressed Rspo2 inhibited elongation of Xenopus ectoderm explants and Erk1 activation in response to FGF. By contrast, the constitutively active form of Mek1 stimulated Erk1 even in the presence of Rspo2, suggesting that Rspo2 functions upstream of Mek1. The observed inhibition of FGF signaling was accompanied by the downregulation of the FGF target genes tbxt/brachyury and cdx4 , which mediate anterioposterior axis specification. Importantly, these target genes were upregulated in Rspo2-depleted explants. The FGF inhibitory activity was mapped to the thrombospondin type 1 region, contrasting the known function of the Furin-like domains in Wnt signaling. Further domain analysis revealed an unexpected intramolecular interaction that might control Rspo2 signaling output. We conclude that, in addition to its role in Wnt signaling, Rspo2 acts as an FGF antagonist during mesoderm formation and patterning., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Epithelial bending is a fundamental process that shapes organs during development. Previously known mechanisms involve cells locally changing shape from columnar to wedge-shaped. Here we report a different mechanism that occurs without cell wedging. In mammalian salivary glands and teeth, we show that initial invagination occurs through coordinated vertical cell movement: cells towards the periphery of the placode move vertically upwards while their more central neighbours move downwards. Movement is achieved by active cell-on-cell migration: outer cells migrate with apical, centripetally polarised leading edge protrusions but remain attached to the basal lamina, depressing more central neighbours to "telescope" the epithelium downwards into underlying mesenchyme. Inhibiting protrusion formation by Arp2/3 protein blocks invagination. FGF and Hedgehog morphogen signals are required, with FGF providing a directional cue. These findings show that epithelial bending can be achieved by a morphogenetic mechanism of coordinated cell rearrangement quite distinct from previously recognised invagination processes.

Cleft lip is one of the most common human birth defects. However, there remain a limited number of mouse models of cleft lip that can be leveraged to characterize the genes and mechanisms that cause this disorder. Crosstalk between epithelial and mesenchymal cells underlies formation of the face and palate, but the basic molecular events mediating this crosstalk remain poorly understood. We previously demonstrated that mice lacking the epithelial-specific splicing factor Esrp1 have fully penetrant bilateral cleft lip and palate. In this study, we further investigated the mechanisms leading to cleft lip as well as cleft palate in both existing and new Esrp1 mutant mouse models. These studies included a detailed transcriptomic analysis of changes in ectoderm and mesenchyme in Esrp1 -/- embryos during face formation. We identified altered expression of genes previously implicated in cleft lip and/or palate, including components of multiple signaling pathways. These findings provide the foundation for detailed investigations using Esrp1 mutant disease models to examine gene regulatory networks and pathways that are essential for normal face and palate development - the disruption of which leads to orofacial clefting in human patients., Competing Interests: Competing interestsY.X. is a scientific cofounder of Panorama Medicine. All other authors declare no competing interests., (© 2020. Published by The Company of Biologists Ltd.)

Epibranchial placodes are the geniculate, petrosal and nodose placodes that generate parts of cranial nerves VII, IX and X, respectively. How the three spatially separated placodes are derived from the common posterior placodal area is poorly understood. Here, we reveal that the broad posterior placode area is first patterned into a Vgll2 + /Irx5 + rostral domain and a Sox2 + /Fgf3 + /Etv5 + caudal domain relative to the first pharyngeal cleft. This initial rostral and caudal patterning is then sequentially repeated along each pharyngeal cleft for each epibranchial placode. The caudal domains give rise to the neuronal and non-neuronal cells in the placode, whereas the rostral domains are previously unrecognized structures, serving as spacers between the final placodes. Notch signalling regulates the balance between the rostral and caudal domains: high levels of Notch signalling expand the caudal domain at the expense of the rostral domain, whereas loss of Notch signalling produces the converse phenotype. Collectively, these data unravel a new patterning principle for the early phases of epibranchial placode development and a role for Notch signalling in orchestrating epibranchial placode segregation and differentiation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

In the last decades, the cephalochordate amphioxus has reached a peculiar place in research laboratories as an excellent animal model to answer Evo/Devo questions. Nevertheless, mainly due to its restricted spawning season and to the small size of its embryos, only a few basic techniques in developmental biology could be used until recently. Fortunately, these last years, and thanks to the development of high-throughput techniques, new technical approaches have been possible, such as comparative transcriptomics and/or genomics. However, classic micromanipulation techniques are still difficult to apply. Here we present simple protocols for the manipulation of amphioxus embryos. First, we present the spawning induction method used with the European amphioxus species Branchiostoma lanceolatum. Second, we explain simple methods to manipulate the developing amphioxus embryo during the first steps of its development (before the hatching stage). These methods open many technical possibilities for future functional studies. Thus, we present here a simple technique to efficiently dechorionate a large number of embryos, we detail a protocol for the dissociation of cells during the first steps of the embryonic development and, finally, we describe micromanipulation approaches for tissue isolation during the gastrula stage.

Sensory placodes and neural crest cells are among the key cell populations that facilitated the emergence and diversification of vertebrates throughout evolution. Together, they generate the sensory nervous system in the head: both form the cranial sensory ganglia, while placodal cells make major contributions to the sense organs-the eye, ear and olfactory epithelium. Both are instrumental for integrating craniofacial organs and have been key to drive the concentration of sensory structures in the vertebrate head allowing the emergence of active and predatory life forms. Whereas the gene regulatory networks that control neural crest cell development have been studied extensively, the signals and downstream transcriptional events that regulate placode formation and diversity are only beginning to be uncovered. Both cell populations are derived from the embryonic ectoderm, which also generates the central nervous system and the epidermis, and recent evidence suggests that their initial specification involves a common molecular mechanism before definitive neural, neural crest and placodal lineages are established. In this review, we will first discuss the transcriptional networks that pattern the embryonic ectoderm and establish these three cell fates with emphasis on sensory placodes. Second, we will focus on how sensory placode precursors diversify using the specification of otic-epibranchial progenitors and their segregation as an example., (© 2020 Elsevier Inc. All rights reserved.)

Estrogen related receptor beta (Esrrb) is an orphan nuclear receptor that is required for self-renewal and pluripotency in mouse embryonic stem (ES) cells. However, in the early post-implantation mouse embryo, Esrrb is specifically expressed in the extraembryonic ectoderm (ExE) and plays a crucial role in trophoblast development. Previous studies showed that Esrrb is also required to maintain trophoblast stem (TS) cells, the in vitro stem cell model of the early trophoblast lineage. In order to identify regulatory targets of Esrrb in vivo, we performed microarray analysis of Esrrb-null versus wild-type post-implantation ExE, and identified 30 genes down-regulated in Esrrb-mutants. Among them is Bmp4, which is produced by the ExE and known to be critical for primordial germ cell (PGC) specification in vivo. We further identified an enhancer region bound by Esrrb at the Bmp4 locus by performing Esrrb ChIP-seq and luciferase reporter assay using TS cells. Finally, we established a knockout mouse line in which the enhancer region was deleted using CRISPR/Cas9 technology. Both Esrrb-null embryos and enhancer knockout embryos expressed lower levels of Bmp4 in the ExE, and had reduced numbers of PGCs. These results suggested that Esrrb functions as an upstream factor of Bmp4 in the ExE, regulating proper PGC development in mice., (Copyright © 2019 Elsevier Inc. All rights reserved.)

During vertebrate embryogenesis, precise regulation of gene expression is crucial for proper cell fate determination. Much of what we know about vertebrate development has been gleaned from experiments performed on embryos of the amphibian Xenopus laevis ; this review will focus primarily on studies of this model organism. An early critical step during vertebrate development is the formation of the three primary germ layers-ectoderm, mesoderm, and endoderm-which emerge during the process of gastrulation. While much attention has been focused on the induction of mesoderm and endoderm, it has become clear that differentiation of the ectoderm involves more than the simple absence of inductive cues; rather, it additionally requires the inhibition of mesendoderm-promoting genes. This review aims to summarize our current understanding of the various inhibitors of inappropriate gene expression in the presumptive ectoderm.

Ocular anterior segment dysgenesis (ASD) is a failure of normal development of anterior structures of the eye, leading to lens opacification. The underlying mechanisms relating to ASD are still unclear. Previous studies have implicated transcriptional factor muscle segment homeobox 2 (Msx2) in ASD. In this study, we used Msx2 conditional knockout (CKO) mice as a model and found that Msx2 deficiency in surface ectoderm induced ASD. Loss of Msx2 function specifically affected lens development, while other eye structures were not significantly affected. Multiple lines of evidence show that calcium signaling pathways are involved in this pathogenesis. Our study demonstrates that Msx2 plays an essential role in lens development by activating a yet undetermined calcium signaling pathway.

The mammalian embryo displays sexual dimorphism in the preimplantation period. Moreover, competence of the embryo to develop is dependent on the sire from which the embryo is derived and can be modified by embryokines produced by the endometrium such as colony stimulating factor 2 (CSF2). The preimplantation period is characterized by large changes in epigenetic modifications of DNA and histones. It is possible, therefore, that effects of sex, sire, and embryo regulatory molecules are mediated by changes in epigenetic modifications. Here it was tested whether global levels of two histone modifications in the trophectoderm of the bovine blastocyst were affected by sex, sire, and CSF2. It was found that amounts of immunolabeled H3K27me3 were greater (P = 0.030) for male embryos than female embryos. Additionally, labeling for H3K27me3 and H3K18ac depended upon the bull from which embryos were derived. Although CSF2 reduced the proportion of embryos developing to the blastocyst, there was no effect of CSF2 on labeling for H3K27me3 or H3K18ac. Results indicate that the blastocyst trophoctoderm can be modified epigenetically by embryo sex and paternal inheritance through alterations in histone epigenetic marks., Competing Interests: The authors have declared that no competing interests exist.

A major challenge in vertebrate evolution is to identify the gene regulatory mechanisms that facilitated the origin of neural crest cells and placodes from ancestral precursors in invertebrates. Here, we show in lamprey, a primitively jawless vertebrate, that the transcription factor Snail is expressed simultaneously throughout the neural plate, neural plate border, and pre-placodal ectoderm in the early embryo and is then upregulated in the CNS throughout neurogenesis. Using CRISPR/Cas9-mediated genome editing, we demonstrate that Snail plays functional roles in all of these embryonic domains or their derivatives. We first show that Snail patterns the neural plate border by repressing lateral expansion of Pax3/7 and activating nMyc and ZicA. We also present evidence that Snail is essential for DlxB-mediated establishment of the pre-placodal ectoderm but is not required for SoxB1a expression during formation of the neural plate proper. At later stages, Snail regulates formation of neural crest-derived and placode-derived PNS neurons and controls CNS neural differentiation in part by promoting cell survival. Taken together with established functions of invertebrate Snail genes, we identify a pan-bilaterian mechanism that extends to jawless vertebrates for regulating neurogenesis that is dependent on Snail transcription factors. We propose that ancestral vertebrates deployed an evolutionarily conserved Snail expression domain in the CNS and PNS for neurogenesis and then acquired derived functions in neural crest and placode development by recruitment of regulatory genes downstream of neuroectodermal Snail activity. Our results suggest that Snail regulatory mechanisms in vertebrate novelties such as the neural crest and placodes may have emerged from neurogenic roles that originated early in bilaterian evolution., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways., (© 2019 Japanese Society of Developmental Biologists.)

The transcription factor p63 is a master regulator of ectoderm development. Although previous studies show that p63 triggers epidermal differentiation in vitro, the roles of p63 in developing embryos remain poorly understood. Here, we use zebrafish embryos to analyze in vivo how p63 regulates gene expression during development. We generate tp63-knock-out mutants that recapitulate human phenotypes and show down-regulated epidermal gene expression. Following p63-binding dynamics, we find two distinct functions clearly separated in space and time. During early development, p63 binds enhancers associated to neural genes, limiting Sox3 binding and reducing neural gene expression. Indeed, we show that p63 and Sox3 are co-expressed in the neural plate border. On the other hand, p63 acts as a pioneer factor by binding non-accessible chromatin at epidermal enhancers, promoting their opening and epidermal gene expression in later developmental stages. Therefore, our results suggest that p63 regulates cell fate decisions during vertebrate ectoderm specification.

The mammalian lip and primary palate form when coordinated growth and morphogenesis bring the nasal and maxillary processes into contact, and the epithelia co-mingle, remodel and clear from the fusion site to allow mesenchyme continuity. Although several genes required for fusion have been identified, an integrated molecular and cellular description of the overall process is lacking. Here, we employ single cell RNA sequencing of the developing mouse face to identify ectodermal, mesenchymal and endothelial populations associated with patterning and fusion of the facial prominences. This analysis indicates that key cell populations at the fusion site exist within the periderm, basal epithelial cells and adjacent mesenchyme. We describe the expression profiles that make each population unique, and the signals that potentially integrate their behaviour. Overall, these data provide a comprehensive high-resolution description of the various cell populations participating in fusion of the lip and primary palate, as well as formation of the nasolacrimal groove, and they furnish a powerful resource for those investigating the molecular genetics of facial development and facial clefting that can be mined for crucial mechanistic information concerning this prevalent human birth defect., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Lack or excess expression of the surface ectoderm-expressed transcription factor Grainyhead-like2 (Grhl2), each prevent spinal neural tube closure. Here we investigate the causative mechanisms and find reciprocal dysregulation of epithelial genes, cell junction components and actomyosin properties in Grhl2 null and over-expressing embryos. Grhl2 null surface ectoderm shows a shift from epithelial to neuroepithelial identity (with ectopic expression of N-cadherin and Sox2), actomyosin disorganisation, cell shape changes and diminished resistance to neural fold recoil upon ablation of the closure point. In contrast, excessive abundance of Grhl2 generates a super-epithelial surface ectoderm, in which up-regulation of cell-cell junction proteins is associated with an actomyosin-dependent increase in local mechanical stress. This is compatible with apposition of the neural folds but not with progression of closure, unless myosin activity is inhibited. Overall, our findings suggest that Grhl2 plays a crucial role in regulating biomechanical properties of the surface ectoderm that are essential for spinal neurulation.