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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.)

The first lineage differentiation in mammals gives rise to the inner cell mass and the trophectoderm (TE). In mice, TEAD4 is a master regulator of TE commitment, as it regulates the expression of other TE-specific genes and its ablation prevents blastocyst formation, but its role in other mammals remains unclear. Herein, we have observed that TEAD4 ablation in two phylogenetically distant species (bovine and rabbit) does not impede TE differentiation, blastocyst formation and the expression of TE markers, such as GATA3 and CDX2, although a reduced number of cells in the inner cell mass was observed in bovine TEAD4 knockout (KO) blastocysts. Transcriptional analysis in bovine blastocysts revealed no major transcriptional effect of the ablation, although the expression of hypoblast and Hippo signalling-related genes tended to be decreased in KO embryos. Experiments were conducted in the bovine model to determine whether TEAD4 was required for post-hatching development. TEAD4 KO spherical conceptuses showed normal development of the embryonic disc and TE, but hypoblast migration rate was reduced. At later stages of development (tubular conceptuses), no differences were observed between KO and wild-type conceptuses., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Human induced pluripotent stem cells (iPSCs) have great potential in research, but pluripotency testing faces challenges due to non-standardized methods and ambiguous markers. Here, we use long-read nanopore transcriptome sequencing to discover 172 genes linked to cell states not covered by current guidelines. We validate 12 genes by qPCR as unique markers for specific cell fates: pluripotency (CNMD, NANOG, SPP1), endoderm (CER1, EOMES, GATA6), mesoderm (APLNR, HAND1, HOXB7), and ectoderm (HES5, PAMR1, PAX6). Using these genes, we develop a machine learning-based scoring system, "hiPSCore", trained on 15 iPSC lines and validated on 10 more. hiPSCore accurately classifies pluripotent and differentiated cells and predicts their potential to become specialized 2D cells and 3D organoids. Our re-evaluation of cell fate marker genes identifies key targets for future studies on cell fate assessment. hiPSCore improves iPSC testing by reducing time, subjectivity, and resource use, thus enhancing iPSC quality for scientific and medical applications., (© 2024. The Author(s).)

The developing placenta, which in mice originates through the extraembryonic ectoderm (ExE), is essential for mammalian embryonic development. Yet unbiased characterization of the differentiation dynamics of the ExE and its interactions with the embryo proper remains incomplete. Here we develop a temporal single-cell model of mouse gastrulation that maps continuous and parallel differentiation in embryonic and extraembryonic lineages. This is matched with a three-way perturbation approach to target signalling from the embryo proper, the ExE alone, or both. We show that ExE specification involves early spatial and transcriptional bifurcation of uncommitted ectoplacental cone cells and chorion progenitors. Early BMP4 signalling from chorion progenitors is required for proper differentiation of uncommitted ectoplacental cone cells and later for their specification towards trophoblast giant cells. We also find biphasic regulation by BMP4 in the embryo. The early ExE-originating BMP4 signal is necessary for proper mesoendoderm bifurcation and for allantois and primordial germ cell specification. However, commencing at embryonic day 7.5, embryo-derived BMP4 restricts the primordial germ cell pool size by favouring differentiation of their extraembryonic mesoderm precursors towards an allantois fate. ExE and embryonic tissues are therefore entangled in time, space and signalling axes, highlighting the importance of their integrated understanding and modelling in vivo and in vitro., (© 2024. The Author(s).)

To implant in the uterus, mammalian embryos form blastocysts comprising trophectoderm (TE) surrounding an inner cell mass (ICM), confined to the polar region by the expanding blastocoel. The mode of implantation varies between species. Murine embryos maintain a single layered TE until they implant in the characteristic thick deciduum, whereas human blastocysts attach via polar TE directly to the uterine wall. Using immunofluorescence (IF) of rapidly isolated ICMs, blockade of RNA and protein synthesis in whole embryos, or 3D visualization of immunostained embryos, we provide evidence of multi-layering in human polar TE before implantation. This may be required for rapid uterine invasion to secure the developing human embryo and initiate formation of the placenta. Using sequential fluorescent labeling, we demonstrate that the majority of inner TE in human blastocysts arises from existing outer cells, with no evidence of conversion from the ICM in the context of the intact embryo., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The first cell differentiation event that occurs in the embryo determines the inner cell mass (ICM) and the trophectoderm (TE). In the mouse, glucose (GLC) is essential for this process, while oxygen tension (O 2 ) also interferes with TE formation. The roles of GLC and O 2 in this event in bovine embryos are not completely elucidated. We hypothesized that the absence of glucose and a higher O 2 tension negatively impact ICM and TE cell allocation in the bovine embryo. The objective of this study was to evaluate the effect of GLC within different O 2 levels on the formation of the TE. In vitro-produced embryos were cultured in serum-free KSOM medium and randomly submitted to treatments on the day of IVC, according to a 2x2 factorial model, in which GLC (present [+GLC] or absent [-GLC]) and O 2 (low [5%O 2 ] or high [20%O 2 ]) were the independent variables. Cleavage and blastocyst rates were obtained at D4 and D8, respectively. Embryos at D8 were subjected to autofluorescence analysis to quantitate NADH and FAD + or fixed for GATA3 and YAP1 immunostaining using a laser scanning confocal microscope. Total, TE, and ICM cell counts were obtained. Embryos were also harvested for gene expression quantification of GATA3, YAP1, SOX2, CDX2, TFAP2C and OCT4. Results indicate that there was an effect of O 2 (p = 0.018) on cleavage rates, although no differences were observed in blastocyst rates. NADH was higher in -GLC compared to + GLC (p = 0.014) and no differences in FAD+ were observed. Total cell count data were not different between variables. There was an increase in the ICM cell count in the +GLC 5%O 2 condition compared to the other three conditions. No effects of GLC, O 2 , or their interactions were observed on TE cell count or the TE/total cell ratio. CDX2 (p = 0.007) and TFAP2C (p = 0.038) were increased in -GLC 20%O 2 compared to + GLC 20%O 2 . SOX2 was decreased in +GLC 20%O 2 compared to + GLC 5%O 2 (p = 0.027) or compared to -GLC 20%O 2 (p = 0.005). GATA3, YAP1, and OCT4 genes did not present differences among conditions. In conclusion, both GLC and high oxygen tension did not impair TE formation and TE cell number, although a +GLC-low oxygen environment led to a higher number of ICM cells. Interestingly, the expression of TE-related gene CDX2 was increased in the absence of glucose within higher O 2 tension. Our results implicate that according to the oxygen tension used in IVC, glucose can exert different effects on blastocyst cell allocation or gene expression., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background Aims: Substrate elasticity may direct cell-fate decisions of stem cells. However, it is largely unclear how matrix stiffness affects the differentiation of induced pluripotent stem cells (iPSCs) and whether this is also reflected by epigenetic modifications., Methods: We cultured iPSCs on tissue culture plastic (TCP) and polydimethylsiloxane (PDMS) with different Young's modulus (0.2 kPa, 16 kPa or 64 kPa) to investigate the sequel on growth and differentiation toward endoderm, mesoderm and ectoderm., Results: Immunofluorescence and gene expression of canonical differentiation markers were hardly affected by the substrates. Notably, when we analyzed DNA methylation profiles of undifferentiated iPSCs or after three-lineage differentiation, we did not see any significant differences on the three different PDMS elasticities. Only when we compared DNA methylation profiles on PDMS-substrates versus TCP we did observe epigenetic differences, particularly on mesodermal differentiation., Conclusions: Stiffness of PDMS substrates did not affect directed differentiation of iPSCs, whereas the moderate epigenetic differences on TCP might also be attributed to other chemical parameters., Competing Interests: Declaration of competing interest WW is a founder of Cygenia GmbH, which provides services for various epigenetic signatures (www.cygenia.com). The medical faculty of RWTH-Aachen has claimed patent application for GermLayerTracker and MEM, KZ and WW are co-applicants for the patent., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

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.)

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.

During the formation of the neural tube, the primordium of the vertebrate central nervous system, the actomyosin activity of cells in different regions drives neural plate bending. However, how the stiffness of the neural plate and surrounding tissues is regulated and mechanically influences neural plate bending has not been elucidated. Here, we used atomic force microscopy to reveal the relationship between the stiffness of the neural plate and the mesoderm during Xenopus neural tube formation. Measurements with intact embryos revealed that the stiffness of the neural plate was consistently higher compared with the non-neural ectoderm and that it increased in an actomyosin activity-dependent manner during neural plate bending. Interestingly, measurements of isolated tissue explants also revealed that the relationship between the stiffness of the apical and basal sides of the neural plate was reversed during bending and that the stiffness of the mesoderm was lower than that of the basal side of the neural plate. The experimental elevation of mesoderm stiffness delayed neural plate bending, suggesting that low mesoderm stiffness mechanically supports neural tube closure. This study provides an example of mechanical interactions between tissues during large-scale morphogenetic movements., (© 2024 The Author(s). Development, Growth & Differentiation published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Developmental Biologists.)

Embryos, originating from fertilized eggs, undergo continuous cell division and differentiation, accompanied by dramatic changes in transcription, translation, and metabolism. Chromatin regulators, including transcription factors (TFs), play indispensable roles in regulating these processes. Recently, the trophoblast regulator TFAP2C was identified as crucial in initiating early cell fate decisions. However, Tfap2c transcripts persist in both the inner cell mass and trophectoderm of blastocysts, prompting inquiry into Tfap2c's function in post-lineage establishment. In this study, we delineate the dynamics of TFAP2C during the mouse peri-implantation stage and elucidate its collaboration with the key lineage regulators CDX2 and NANOG. Importantly, we propose that de novo formation of H3K9me3 in the extraembryonic ectoderm during implantation antagonizes TFAP2C binding to crucial developmental genes, thereby maintaining its lineage identity. Together, these results highlight the plasticity of the chromatin environment in designating the genomic binding of highly adaptable lineage-specific TFs and regulating embryonic cell fates., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by 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.)

In recent years, the pursuit of inducing the trophoblast stem cell (TSC) state has gained prominence as a compelling research objective, illuminating the establishment of the trophoblast lineage and unlocking insights into early embryogenesis. In this review, we examine how advancements in diverse technologies, including in vivo time course transcriptomics, cellular reprogramming to TSC state, chemical induction of totipotent stem-cell-like state, and stem-cell-based embryo-like structures, have enriched our insights into the intricate molecular mechanisms and signaling pathways that define the mouse and human trophectoderm/TSC states. We delve into disparities between mouse and human trophectoderm/TSC fate establishment, with a special emphasis on the intriguing role of pluripotency in this context. Additionally, we re-evaluate recent findings concerning the potential of totipotent-stem-like cells and embryo-like structures to fully manifest the trophectoderm/trophoblast lineage's capabilities. Lastly, we briefly discuss the potential applications of induced TSCs in pregnancy-related disease modeling., Competing Interests: Declaration of interests Prof. Buganim holds a patent regarding the production of human TSCs with GOKM factors., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Human-induced pluripotent stem cells (hiPSCs) offer a promising source for generating dental epithelial (DE) cells. Whereas the existing differentiation protocols were time-consuming and relied heavily on growth factors, herein, we developed a three-step protocol to convert hiPSCs into DE cells in 8 days. In the first phase, hiPSCs were differentiated into non-neural ectoderm using SU5402 (an FGF signaling inhibitor). The second phase involved differentiating non-neural ectoderm into pan-placodal ectoderm and simultaneously inducing the formation of oral ectoderm (OE) using LDN193189 (a BMP signaling inhibitor) and purmorphamine (a SHH signaling activator). In the final phase, OE cells were differentiated into DE through the application of Purmorphamine, XAV939 (a WNT signaling inhibitor), and BMP4. qRT-PCR and immunostaining were performed to examine the expression of lineage-specific markers. ARS staining was performed to evaluate the formation of the mineralization nodule. The expression of PITX2, SP6, and AMBN, the emergence of mineralization nodules, and the enhanced expression of AMBN and AMELX in spheroid culture implied the generation of DE cells. This study delineates the developmental signaling pathways and uses small molecules to streamline the induction of hiPSCs into DE cells. Our findings present a simplified and quicker method for generating DE cells, contributing valuable insights for dental regeneration and dental disease research.

Feathers originate as protofeathers before birds, in pterosaurs and basal dinosaurs. What characterizes a feather is not only its outgrowth, but its barb cells differentiation and a set of beta-corneous proteins. Reticula appear concomitantly with feathers, as small bumps on plantar skin, made only of keratins. Avian scales, with their own set of beta-corneous proteins, appear more recently than feathers on the shank, and only in some species. In the chick embryo, when feather placodes form, all the non-feather areas of the integument are already specified. Among them, midventral apterium, cornea, reticula, and scale morphogenesis appear to be driven by negative regulatory mechanisms, which modulate the inherited capacity of the avian ectoderm to form feathers. Successive dermal/epidermal interactions, initiated by the Wnt/β-catenin pathway, and involving principally Eda/Edar, BMP, FGF20 and Shh signaling, are responsible for the formation not only of feather, but also of scale placodes and reticula, with notable differences in the level of Shh, and probably FGF20 expressions. This sequence is a dynamic and labile process, the turning point being the FGF20 expression by the placode. This epidermal signal endows its associated dermis with the memory to aggregate and to stimulate the morphogenesis that follows, involving even a re-initiation of the placode., Competing Interests: Declaration of competing interest The author declares no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Cell fate determination in mammalian development is complex and precisely controlled and accumulating evidence indicates that epigenetic mechanisms are crucially involved. N 4 -acetylcytidine (ac 4 C) is a recently identified modification of messenger RNA (mRNA); however, its functions are still elusive in mammalian. Here, we show that N-acetyltransferase 10 (NAT10)-mediated ac 4 C modification promotes ectoderm differentiation of human embryonic stem cells (hESCs) by acetylating nuclear receptor subfamily 2 group F member 1 (NR2F1) mRNA to enhance translation efficiency (TE). Acetylated RNA immunoprecipitation sequencing (acRIP-seq) revealed that levels of ac 4 C modification were higher in ectodermal neuroepithelial progenitor (NEP) cells than in hESCs or mesoendoderm cells. In addition, integrated analysis of acRIP-seq and ribosome profiling sequencing revealed that NAT10 catalysed ac 4 C modification to improve TE in NEP cells. RIP-qRT-PCR analysis identified an interaction between NAT10 and NR2F1 mRNA in NEP cells and NR2F1 accelerated the nucleus-to-cytoplasm translocation of yes-associated protein 1, which contributed to ectodermal differentiation of hESCs. Collectively, these findings point out the novel regulatory role of ac 4 C modification in the early ectodermal differentiation of hESCs and will provide a new strategy for the treatment of neuroectodermal defects diseases., (© 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Mechanisms specifying amniotic ectoderm and surface ectoderm are unresolved in humans due to their close similarities in expression patterns and signal requirements. This lack of knowledge hinders the development of protocols to accurately model human embryogenesis. Here, we developed a human pluripotent stem cell model to investigate the divergence between amniotic and surface ectoderms. In the established culture system, cells differentiated into functional amnioblast-like cells. Single-cell RNA sequencing analyses of amnioblast differentiation revealed an intermediate cell state with enhanced surface ectoderm gene expression. Furthermore, when the differentiation started at the confluent condition, cells retained the expression profile of surface ectoderm. Collectively, we propose that human amniotic ectoderm and surface ectoderm are specified along a common nonneural ectoderm trajectory based on cell density. Our culture system also generated extraembryonic mesoderm-like cells from the primed pluripotent state. Together, this study provides an integrative understanding of the human nonneural ectoderm development and a model for embryonic and extraembryonic human development around gastrulation.

The neural tube, the embryonic precursor to the brain and spinal cord, begins as a flat sheet of epithelial cells, divided into non-neural and neural ectoderm. Proper neural tube closure requires that the edges of the neural ectoderm, the neural folds, to elevate upwards and fuse along the dorsal midline of the embryo. We have previously shown that members of the claudin protein family are required for the early phases of chick neural tube closure. Claudins are transmembrane proteins, localized in apical tight junctions within epithelial cells where they are essential for regulation of paracellular permeability, strongly involved in apical-basal polarity, cell-cell adhesion, and bridging the tight junction to cytoplasmic proteins. Here we explored the role of Claudin-3 (Cldn3), which is specifically expressed in the non-neural ectoderm. We discovered that depletion of Cldn3 causes folic acid-insensitive primarily spinal neural tube defects due to a failure in neural fold fusion. Apical cell surface morphology of Cldn3-depleted non-neural ectodermal cells exhibited increased membrane blebbing and smaller apical surfaces. Although apical-basal polarity was retained, we observed altered Par3 and Pals1 protein localization patterns within the apical domain of the non-neural ectodermal cells in Cldn3-depleted embryos. Furthermore, F-actin signal was reduced at apical junctions. Our data presents a model of spina bifida, and the role that Cldn3 is playing in regulating essential apical cell processes in the non-neural ectoderm required for neural fold fusion., Competing Interests: Declaration of competing interest The authors have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ectodermal appendages in mammals, such as teeth, mammary glands, sweat glands and hair follicles, are generated during embryogenesis through a series of mesenchymal-epithelial interactions. Canonical Wnt signaling and its inhibitors are implicated in the early steps of ectodermal appendage development and patterning. To study the activation dynamics of the Wnt target and inhibitor Dickkopf4 (Dkk4) in ectodermal appendages, we used CRSIPR/Cas9 to generate a Dkk4-Cre knock-in mouse (Mus musculus) line, where the Cre recombinase cDNA replaces the expression of endogenous Dkk4. Using Cre reporters, the Dkk4-Cre activity was evident at the prospective sites of ectodermal appendages, overlapping with the Dkk4 mRNA expression. Unexpectedly, a predominantly mesenchymal cell population in the embryo posterior also showed Dkk4-Cre activity. Lineage-tracing suggested that these cells are likely derived from a few Dkk4-Cre-expressing cells in the epiblast at early gastrulation. Finally, our analyses of Dkk4-Cre-expressing cells in developing hair follicle epithelial placodes revealed intra- and inter-placodal cellular heterogeneity, supporting emerging data on the positional and transcriptional cellular variability in placodes. Collectively, we propose the new Dkk4-Cre knock-in mouse line as a suitable model to study Wnt and DKK4 inhibitor dynamics in early mouse development and ectodermal appendage morphogenesis., (© 2023 The Authors. genesis published by Wiley Periodicals LLC.)

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).)

Objective: To investigate the potential value of exosomes derived from rat ectoderm mesenchymal stem cells (EMSCs-exo) for repairing secondary spinal cord injury., Methods: EMSCs-exo were obtained using ultracentrifugation from EMSCs isolated from rat nasal mucosa, identified by transmission electron microscope, nanoparticle tracking analysis (NTA), and Western blotting, and quantified using the BCA method. Neonatal rat microglia purified by differential attachment were induced with 100 μg/L lipopolysaccharide (LPS) and treated with 37.5 or 75 mg/L EMSCs-exo. PC12 cells were exposed to 400 μmol/L H 2 O 2 and treated with EMSCs-exo at 37.5 or 75 mg/L. The protein and mRNA expressions of Arg1 and iNOS in the treated cells were determined with Western blotting and qRT- PCR, and the concentrations of IL- 6, IL-10, and IGF-1 in the supernatants were measured with ELISA. The viability and apoptosis of PC12 cells were detected using CCK-8 assay and flow cytometry., Results: The isolated rat EMSCs showed high expressions of nestin, CD44, CD105, and vimentin. The obtained EMSCs-exo had a typical cup-shaped structure under transmission electron microscope with an average particle size of 142 nm and positivity for CD63, CD81, and TSG101 but not vimentin. In LPS-treated microglia, EMSCs-exo treatment at 75 mg/L significantly increased Arg1 protein level and lowered iNOS protein expression ( P < 0.05). EMSCs-exo treatment at 75 mg/L, as compared with the lower concentration at 37.5 mg/L, more strongly increased Arg1 mRNA expression and IGF-1 and IL-10 production and decreased iNOS mRNA expression and IL-6 production in LPS-induced microglia, and more effectively promoted cell survival and decreased apoptosis rate of H 2 O 2 -induced PC12 cells ( P < 0.05)., Conclusion: EMSCs-exo at 75 mg/L can effectively reduce the proportion of M1 microglia and alleviate neuronal apoptosis under oxidative stress to promote neuronal survival, suggesting its potential in controlling secondary spinal cord injury.

Studies using early-stage avian embryos have substantially impacted developmental biology, through the availability of simple culture methods and easiness in tissue manipulation. However, the regulations underlying brain and head development, a central issue of developmental biology, have not been investigated systematically. Yoshihi et al. (2022a) devised a technique to randomly label the epiblast cells with a green fluorescent protein before their development into the brain tissue. This technique was combined with grafting a node or node-derived anterior mesendoderm labeled with a cherry-colored fluorescent protein. Then cellular events were live-recorded over 18 hours during the brain and head development. The live imaging-based analyses identified previously undescribed mechanisms central to brain development: all anterior epiblast cells have a potential to develop into the brain tissues and their gathering onto a proximal anterior mesendoderm forms a brain primordium whereas the remaining cells develop into the covering head ectoderm. The analyses also ruled out the direct participation of the node's activity in the brain development. Yoshihi et al. (2022a) also demonstrate how the enigmatic data from classical models can be reinterpreted in the new model.This chapter was adapted from Yoshihi K, Iida H, Teramoto M, Ishii Y, Kato K, Kondoh H. (2022b). Epiblast cells gather onto the anterior mesendoderm and initiate brain development without the direct involvement of the node in avian embryos: Insights from broad-field live imaging. Front Cell Dev Biol. 10:1019845. doi: 10.3389/fcell.2022.1019845., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

In vertebrate development, ectoderm is specified into neural plate (NP), neural plate border (NPB), and epidermis. Although such patterning is thought to be achieved by molecular concentration gradients, it has been revealed, mainly by in vitro analysis, that mechanical force can regulate cell specification. During in vivo patterning, cells deform and migrate, and this applies force to surrounding tissues, shaping the embryo. However, the role of mechanical force for cell specification in vivo is largely unknown. In this study, with an aspiration assay and atomic force microscopy, we have demonstrated that tension on ectodermal cells decreases laterally from the midline in Xenopus early neurula. Ectopically applied force laterally expanded the neural crest (NC) region, a derivative of the NPB, whereas force relaxation suppressed it. Furthermore, force application activated both the FGF and Wnt pathways, which are required for NC formation during neuroectodermal patterning. Taken together, mechanical force is necessary for NC formation in order to regulate signaling pathways. Furthermore, molecular signals specify the NP and generate force on neighboring tissue, the NPB, with its closure. This force activates signals, possibly determining the appropriate width of a narrow tissue, the NC.

Inhibition of the Embryonic Ectoderm Development (EED) subunit in Polycomb Repressive Complex 2 (PRC2) can inhibit tumor growth. In this paper, we selected six experimentally designed EED competitive Inhibitors of the triazolopyrimidine derivatives class. We investigated the difference in the binding mode of the natural substrate to the Inhibitors and the effects of differences in the parent nuclei, heads, and tails of the Inhibitors on the inhibitory capacity. The results showed that the binding free energy of this class of Inhibitors was close to or lower compared to the natural substrate, providing an energetic basis for competitive inhibition. For the Inhibitors, the presence of a strong negatively charged group at the 6-position of the parent nucleus or the 8'-position of the head would make the hydrogen atom on the head imino group prone to flip, resulting in the vertical movement of the parent nucleus, which significantly decreased the inhibitory ability. When the 6-position of the parent nucleus was a nonpolar group, the parent nucleus would move horizontally, slightly decreasing the inhibitory ability. When the 8'-position of the head was methylene, it formed an intramolecular hydrophobic interaction with the benzene ring on the tail, resulting in a significant increase in inhibition ability.

Background: FOXI3 is a forkhead family transcription factor that is expressed in the progenitors of craniofacial placodes, epidermal placodes, and the ectoderm and endoderm of the pharyngeal arch region. Loss of Foxi3 in mice and pathogenic Foxi3 variants in dogs and humans cause a variety of craniofacial defects including absence of the inner ear, severe truncations of the jaw, loss or reduction in external and middle ear structures, and defects in teeth and hair., Results: To allow for the identification, isolation, and lineage tracing of Foxi3-expressing cells in developing mice, we targeted the Foxi3 locus to create Foxi3 GFP and Foxi3 CreER mice. We show that Foxi3 GFP mice faithfully recapitulate the expression pattern of Foxi3 mRNA at all ages examined, and Foxi3 CreER mice can trace the derivatives of pharyngeal arch ectoderm and endoderm, the pharyngeal pouches and clefts that separate each arch, and the derivatives of hair and tooth placodes., Conclusions: Foxi3 GFP and Foxi3 CreER mice are new tools that will be of use in identifying and manipulating pharyngeal arch ectoderm and endoderm and hair and tooth placodes., (© 2023 American Association for Anatomy.)

The amino acid L-proline exhibits growth factor-like properties during development - from improving blastocyst development to driving neurogenesis in vitro. Addition of 400 μM L-proline to self-renewal medium drives naïve mouse embryonic stem cells (ESCs) to early primitive ectoderm-like (EPL) cells - a transcriptionally distinct primed or partially primed pluripotent state. EPL cells retain expression of pluripotency genes, upregulate primitive ectoderm markers, undergo a morphological change and have increased cell number. These changes are facilitated by a complex signalling network hinging on the Mapk, Fgfr, Pi3k and mTor pathways. Here, we use a factorial experimental design coupled with statistical modelling to understand which signalling pathways are involved in the transition between ESCs and EPL cells, and how they underpin changes in morphology, cell number, apoptosis, proliferation and gene expression. This approach reveals pathways which work antagonistically or synergistically. Most properties were affected by more than one inhibitor, and each inhibitor blocked specific aspects of the naïve-to-primed transition. These mechanisms underpin progression of stem cells across the in vitro pluripotency continuum and serve as a model for pre-, peri- and post-implantation embryogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The Foxi3 transcription factor, expressed in the neural plate border at the end of gastrulation, is necessary for the formation of posterior placodes and is thus important for ectodermal patterning. We have created two knock-in mouse lines expressing GFP or a tamoxifen-inducible Cre recombinase to show that Foxi3 is one of the earliest genes to label the border between the neural tube and epidermis, and that Foxi3-expressing neural plate border progenitors contribute primarily to cranial placodes and epidermis from the onset of expression, but not to the neural crest or neural tube lineages. By simultaneously knocking out Foxi3 in neural plate border cells and following their fates, we show that neural plate border cells lacking Foxi3 contribute to all four lineages of the ectoderm - placodes, epidermis, crest and neural tube. We contrast Foxi3 with another neural plate border transcription factor, Zic5, the progenitors of which initially contribute broadly to all germ layers until gastrulation and gradually become restricted to the neural crest lineage and dorsal neural tube cells. Our study demonstrates that Foxi3 uniquely acts early at the neural plate border to restrict progenitors to a placodal and epidermal fate., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The vertebrate 'neural plate border' is a transient territory located at the edge of the neural plate containing precursors for all ectodermal derivatives: the neural plate, neural crest, placodes and epidermis. Elegant functional experiments in a range of vertebrate models have provided an in-depth understanding of gene regulatory interactions within the ectoderm. However, these experiments conducted at tissue level raise seemingly contradictory models for fate allocation of individual cells. Here, we carry out single cell RNA sequencing of chick ectoderm from primitive streak to neurulation stage, to explore cell state diversity and heterogeneity. We characterise the dynamics of gene modules, allowing us to model the order of molecular events which take place as ectodermal fates segregate. Furthermore, we find that genes previously classified as neural plate border 'specifiers' typically exhibit dynamic expression patterns and are enriched in either neural, neural crest or placodal fates, revealing that the neural plate border should be seen as a heterogeneous ectodermal territory and not a discrete transitional transcriptional state. Analysis of neural, neural crest and placodal markers reveals that individual NPB cells co-express competing transcriptional programmes suggesting that their ultimate identify is not yet fixed. This population of 'border located undecided progenitors' (BLUPs) gradually diminishes as cell fate decisions take place. Considering our findings, we propose a probabilistic model for cell fate choice at the neural plate border. Our data suggest that the probability of a progenitor's daughters to contribute to a given ectodermal derivative is related to the balance of competing transcriptional programmes, which in turn are regulated by the spatiotemporal position of a progenitor., Competing Interests: AT, AB, EH, CC, NL, JB, AS No competing interests declared, (© 2023, Thiery et al.)

The nervous system has various types of cells derived from three neuroectodermal regions: neural plate (NP), neural crest (NC), and preplacodal ectoderm (PPE). Differentiation of these regions is regulated by various morphogens. However, regulatory mechanisms of morphogen distribution in neural patterning are still debated. In general, an extracellular component, heparan sulfate (HS), is essential to regulate morphogen gradients by modulating morphogen binding. The present study focused on an HS modification enzyme, heparan sulfate 6-O-sulfotransferase 1 (Hs6st1), which is highly expressed during the neurula stage in Xenopus. Our present in situ hybridization analysis revealed that Hs6st1 is expressed in the lateral sensorial layer of neuroectoderm. Overexpression of Hs6st1 expands Sox3 (NP marker gene) expression, and slightly dampens FoxD3 (NC marker) expression. Hs6st1 knockout using the CRISPR/Cas9 system also expands the neural plate region, followed by retinal malformation. These results imply that 6-O sulfation, mediated by Hs6st1, selectively regulates morphogen distribution required for neuroectodermal patterning. Among morphogens required for patterning, Fgf8a accumulates on Hs6st1-expressing cells, whereas a secreted BMP antagonist, Noggin, diffuses away from those cells. Thus, cell-autonomous 6-O sulfation of HS at the sensorial layer of neuroectoderm also affects neuroectodermal patterning in neighboring regions, including neural plate and neural crest, not only through accumulation, but also through dispersal of specific morphogens., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dynamic chromatin accessibility regulates stem cell fate determination and tissue homeostasis via controlling gene expression. As a histone-modifying enzyme that predominantly mediates methylation of lysine 27 in histone H3 (H3K27me1/2/3), Polycomb repressive complex 2 (PRC2) plays the canonical role in targeting developmental regulators during stem cell differentiation and transformation. Embryonic ectoderm development (EED), the core scaffold subunit of PRC2 and as an H3K27me3-recognizing protein, has been broadly implicated with PRC2 stabilization and allosterically stimulated PRC2. Accumulating evidences from experimental data indicate that EED-associating epigenetic modifications are indispensable for stem cell maintenance and differentiation into specific cell lineages. In this review, we discuss the most updated advances to summarize the structural architecture of EED and its contributions and underlying mechanisms to mediating lineage differentiation of different stem cells during epigenetic modification to expand our understanding of PRC2., (© 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Of all the cell types arising from the neural crest, ectomesenchyme is likely the most unusual. In contrast to the neuroglial cells generated by neural crest throughout the embryo, consistent with its ectodermal origin, cranial neural crest-derived cells (CNCCs) generate many connective tissue and skeletal cell types in common with mesoderm. Whether this ectoderm-derived mesenchyme (ectomesenchyme) potential reflects a distinct developmental origin from other CNCC lineages, and/or epigenetic reprogramming of the ectoderm, remains debated. Whereas decades of lineage tracing studies have defined the potential of CNCC ectomesenchyme, these are being revisited by modern genetic techniques. Recent work is also shedding light on the extent to which intrinsic and extrinsic cues determine ectomesenchyme potential, and whether maintenance or reacquisition of CNCC multipotency influences craniofacial repair., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

During development of the vertebrate sensory system, many important components like the sense organs and cranial sensory ganglia arise within the head and neck. Two progenitor populations, the neural crest, and cranial ectodermal placodes, contribute to these developing vertebrate peripheral sensory structures. The interactions and contributions of these cell populations to the development of the lens, olfactory, otic, pituitary gland, and cranial ganglia are vital for appropriate peripheral nervous system development. Here, we review the origins of both neural crest and placode cells at the neural plate border of the early vertebrate embryo and investigate the molecular and environmental signals that influence specification of different sensory regions. Finally, we discuss the underlying molecular pathways contributing to the complex vertebrate sensory system from an evolutionary perspective, from basal vertebrates to amniotes., Competing Interests: Conflict of interest No conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Pharyngeal arch artery (PAA) progenitors undergo proliferative expansion and angioblast differentiation to build vessels connecting the heart with the dorsal aortae. However, it remains unclear whether and how these two processes are orchestrated. Here we demonstrate that Tmem88 is required to fine-tune PAA progenitor proliferation and differentiation. Loss of zebrafish tmem88a/b leads to an excessive expansion and a failure of differentiation of PAA progenitors. Moreover, tmem88a/b deficiency enhances cyclin D1 expression in PAA progenitors via aberrant Wnt signal activation. Mechanistically, cyclin D1-CDK4/6 promotes progenitor proliferation through accelerating the G1/S transition while suppressing angioblast differentiation by phosphorylating Nkx2.5/Smad3. Ectodermal Wnt2bb signaling is confined by Tmem88 in PAA progenitors to ensure a balance between proliferation and differentiation. Therefore, the proliferation and angioblast differentiation of PAA progenitors manifest an inverse relationship and are delicately regulated by cell cycle machinery downstream of the Tmem88-Wnt pathway., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

EDA is a tumor necrosis factor (TNF) family member, which functions together with its cognate receptor EDAR during ectodermal organ development. Mutations of EDA have long been known to cause X-linked hypohidrotic dysplasia in humans characterized by primary defects in teeth, hair and sweat glands. However, the structural information of EDA interaction with EDAR is lacking and the pathogenic mechanism of EDA variants is poorly understood. Here, we report the crystal structure of EDA C-terminal TNF homology domain bound to the N-terminal cysteine-rich domains of EDAR. Together with biochemical, cellular and mouse genetic studies, we show that different EDA mutations lead to varying degrees of ectodermal developmental defects in mice, which is consistent with the clinical observations on human patients. Our work extends the understanding of the EDA signaling mechanism, and provides important insights into the molecular pathogenesis of disease-causing EDA variants., (© 2023. The Author(s).)

Sterile alpha and HEAT/Armadillo motif-containing protein (SARM) is conserved in evolution and negatively regulates TRIF-dependent Toll signaling in mammals. The SARM protein from Litopenaeus vannamei and its Drosophila orthologue Ectoderm-expressed (Ect4) are also involved in immune defense against pathogen infection. However, the functional mechanism of the protective effect remains unclear. In this study, we show that Ect4 is essential for the viral load in flies after a Drosophila C virus (DCV) infection. Viral load is increased in Ect4 mutants resulting in higher mortality rates than wild-type. Overexpression of Ect4 leads to a suppression of virus replication and thus improves the survival rate of the animals. Ect4 is required for the viral induction of STAT-responsive genes, TotA and TotM . Furthermore, Ect4 interacts with Stat92E, affecting the tyrosine phosphorylation and nuclear translocation of Stat92E in S2 cells. Altogether, our study identifies the adaptor protein Ect4 of the Toll pathway contributes to resistance to viral infection and regulates JAK/STAT signaling pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Huang, Wang, Xu, Gong, Hu, Liu, Su, Anjum, Deng, Yang, Liu, Jiao and Chen.)

Background: Despite their high repair capability, bone defects still present a major challenge in orthopedic tissue engineering. Osteoblast differentiation is central to the treatment of bone defects. METHODS AND RESULTS: We used nasal mucosal-derived ectoderm mesenchymal stem cells (EMSCs) to promote osteogenic differentiation by co-culturing MC3T3-E1 cells. Our results showed that MC3T3-E1/EMSCs co-culture upregulated bone-related proteins and transglutaminase 2 (TG2) and increased alkaline phosphatase (ALP) activity and bone nodule formation relative to controls. Furthermore, our results showed that EMSC-derived sonic hedgehog (Shh) accounted for the enhanced MC3T3-E1 differentiation because inhibiting Shh signaling substantially reduced osteogenic differentiation., Conclusion: Altogether, these results suggest that EMSCs differentiated into osteoblast cells and supported MC3T3-E1 differentiation. Thus, EMSCs may be a promising cell source for treating bone-related diseases., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The inner ear is derived from the otic placode, one of the numerous cranial sensory placodes that emerges from the pre-placodal ectoderm (PPE) along its anterior-posterior axis. However, the molecular dynamics underlying how the PPE is regionalized are poorly resolved. We used stem cell-derived organoids to investigate the effects of Wnt signaling on early PPE differentiation and found that modulating Wnt signaling significantly increased inner ear organoid induction efficiency and reproducibility. Alongside single-cell RNA sequencing, our data reveal that the canonical Wnt signaling pathway leads to PPE regionalization and, more specifically, medium Wnt levels during the early stage induce (1) expansion of the caudal neural plate border (NPB), which serves as a precursor for the posterior PPE, and (2) a caudal microenvironment that is required for otic specification. Our data further demonstrate Wnt-mediated induction of rostral and caudal cells in organoids and more broadly suggest that Wnt signaling is critical for anterior-posterior patterning in the PPE., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Limb buds develop as bilateral outgrowths of the lateral plate mesoderm and are patterned along three axes. Current models of proximal to distal patterning of early amniote limb buds suggest that two signals, a distal organizing signal from the apical epithelial ridge (AER, Fgfs) and an opposing proximal (retinoic acid [RA]) act early on pattern this axis., Results: Transcriptional analysis of stage 51 Xenopus laevis hindlimb buds sectioned along the proximal-distal axis showed that the distal region is distinct from the rest of the limb. Expression of capn8.3, a novel calpain, was located in cells immediately flanking the AER. The Wnt antagonist Dkk1 was AER-specific in Xenopus limbs. Two transcription factors, sall1 and zic5, were expressed in distal mesenchyme. Zic5 has no described association with limb development. We also describe expression of two proximal genes, gata5 and tnn, not previously associated with limb development. Differentially expressed genes were associated with Fgf, Wnt, and RA signaling as well as differential cell adhesion and proliferation., Conclusions: We identify new candidate genes for early proximodistal limb patterning. Our analysis of RA-regulated genes supports a role for transient RA gradients in early limb bud in proximal-to-distal patterning in this anamniote model organism., (© 2022 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Placodes are embryonic structures originating from the rostral ectoderm that give rise to highly diverse organs and tissues, comprising the anterior pituitary gland, paired sense organs and cranial sensory ganglia. Their development, including the underlying gene regulatory networks and signalling pathways, have been for the most part characterised in animal models. In this Review, we describe how placode development can be recapitulated by the differentiation of human pluripotent stem cells towards placode progenitors and their derivatives, highlighting the value of this highly scalable platform as an optimal in vitro tool to study the development of human placodes, and identify human-specific mechanisms in their development, function and pathology., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

In the primitive vertebrate gastrula, the boundary between ectoderm and mesoderm is formed by Brachet's cleft. Here we examine Brachet's cleft and its control by Eph/ephrin signaling in Xenopus at the ultrastructural level and by visualizing cortical F-actin. We infer cortical tension ratios at tissue surfaces and their interface in normal gastrulae and after depletion of receptors EphB4 and EphA4 and ligands ephrinB2 and ephrinB3. We find that cortical tension downregulation at cell contacts, a normal process in adhesion, is asymmetrically blocked in the ectoderm by Eph/ephrin signals from the mesoderm. This generates high interfacial tension that can prevent cell mixing across the boundary. Moreover, it determines an asymmetric boundary structure that is suited for the respective roles of ectoderm and mesoderm, as substratum and as migratory layers. The Eph and ephrin isoforms also control different cell-cell contact types in ectoderm and mesoderm. Respective changes of adhesion upon isoform depletion affect adhesion at the boundary to different degrees but usually do not prohibit cleft formation. In an extreme case, a new type of cleft-like boundary is even generated where cortical tension is symmetrically increased on both sides of the boundary., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The development of the amniote limb has been an important model system to study patterning mechanisms and morphogenesis. For proper growth and patterning, it requires the interaction between the distal sub-apical mesenchyme and the apical ectodermal ridge (AER) that involve the separate implementation of coordinated and tissue-specific genetic programs., Results: Here, we produce and analyze the transcriptomes of both distal limb mesenchymal progenitors and the overlying ectodermal cells, following time-coursed dissections that cover from limb bud initiation to fully patterned limbs. The comparison of transcriptomes within each layer as well as between layers over time, allowed the identification of specific transcriptional signatures for each of the developmental stages. Special attention was given to the identification of genes whose transcription dynamics suggest a previously unnoticed role in the context of limb development and also to signaling pathways enriched between layers., Conclusion: We interpret the transcriptomic data in light of the known development pattern and we conclude that a major transcriptional transition occurs in distal limb buds between E9.5 and E10.5, coincident with the switch from an early phase continuation of the signature of trunk progenitors, related to the initial proximo distal specification, to a late intrinsic phase of development., (© 2021 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Pre-placodal ectoderm (PPE), a horseshoe-shaped narrow region formed during early vertebrate development, gives rise to multiple types of sensory organs and ganglia. For PPE induction, a certain level of FGF signal activation is required. However, it is difficult to reproducibly induce the narrow region with variations in gene expression, including FGF, among individuals. An intracellular regulatory factor of FGF signaling, Dusp6, is expressed by FGF signal activation and inactivates a downstream regulator, ERK1/2, in adult tissues; however, its role in early development is not well known. Here, we reveal that Dusp6 is expressed in an FGF-dependent manner in Xenopus PPE. Gain- and loss-of-function experiments showed that Dusp6 is required for expression of a PPE gene, Six1, and patterning of adjacent regions, neural plate, and neural crest. To reveal the importance of Dusp6 in variable FGF production, we performed Dusp6 knockdown with FGF-bead implantation, which resulted in varying Six1 expression patterns. Taken together, these results suggest that Dusp6 is required for PPE formation and that it contributes to the robust patterning of PPE by mediating FGF signaling., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Traumatic brain injury (TBI) leads to cell and tissue impairment, as well as functional deficits. Stem cells promote structural and functional recovery and thus are considered as a promising therapy for various nerve injuries. Here, we aimed to investigate the role of ectoderm-derived frontal bone mesenchymal stem cells (FbMSCs) in promoting cerebral repair and functional recovery in a murine TBI model., Methods: A murine TBI model was established by injuring C57BL/6 N mice with moderate-controlled cortical impact to evaluate the extent of brain damage and behavioral deficits. Ectoderm-derived FbMSCs were isolated from the frontal bone and their characteristics were assessed using multiple differentiation assays, flow cytometry and microarray analysis. Brain repairment and functional recovery were analyzed at different days post-injury with or without FbMSC application. Behavioral tests were performed to assess learning and memory improvements. RNA sequencing analysis, immunofluorescence staining, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to examine inflammation reaction and neural regeneration. In vitro co-culture analysis and quantification of glutamate transportation were carried out to explore the possible mechanism of neurogenesis and functional recovery promoted by FbMSCs., Results: Ectoderm-derived FbMSCs showed fibroblast like morphology and osteogenic differentiation capacity. FbMSCs were CD105, CD29 positive and CD45, CD31 negative. Different from mesoderm-derived MSCs, FbMSCs expressed the ectoderm-specific transcription factor Tfap2β. TBI mice showed impaired learning and memory deficits. Microglia and astrocyte activation, as well as neural damage, were significantly increased post-injury. FbMSC application ameliorated the behavioral deficits of TBI mice and promoted neural regeneration. RNA sequencing analysis showed that signal pathways related to inflammation decreased, whereas those related to neural activation increased. Immunofluorescence staining and qRT-PCR data revealed that microglial activation and astrocyte polarization to the A1 phenotype were suppressed by FbMSC application. In addition, FGF1 secreted from FbMSCs enhanced glutamate transportation by astrocytes and alleviated the cytotoxic effect of excessive glutamate on neurons., Conclusions: Ectoderm-derived FbMSC application significantly alleviated neuroinflammation, brain injury, and excitatory toxicity to neurons, improved cognition and behavioral deficits in TBI mice. Therefore, ectoderm-derived FbMSCs could be ideal therapeutic candidates for TBI which mostly affect cells from the same embryonic origins as FbMSCs., (© 2022. The Author(s).)

Proper ectodermal patterning during human development requires previously identified transcription factors such as GATA3 and p63, as well as positional signalling from regional mesoderm 1-6 . However, the mechanism by which ectoderm and mesoderm factors act to stably pattern gene expression and lineage commitment remains unclear. Here we identify the protein Gibbin, encoded by the Xia-Gibbs AT-hook DNA-binding-motif-containing 1 (AHDC1) disease gene 7-9 , as a key regulator of early epithelial morphogenesis. We find that enhancer- or promoter-bound Gibbin interacts with dozens of sequence-specific zinc-finger transcription factors and methyl-CpG-binding proteins to regulate the expression of mesoderm genes. The loss of Gibbin causes an increase in DNA methylation at GATA3-dependent mesodermal genes, resulting in a loss of signalling between developing dermal and epidermal cell types. Notably, Gibbin-mutant human embryonic stem-cell-derived skin organoids lack dermal maturation, resulting in p63-expressing basal cells that possess defective keratinocyte stratification. In vivo chimeric CRISPR mouse mutants reveal a spectrum of Gibbin-dependent developmental patterning defects affecting craniofacial structure, abdominal wall closure and epidermal stratification that mirror patient phenotypes. Our results indicate that the patterning phenotypes seen in Xia-Gibbs and related syndromes derive from abnormal mesoderm maturation as a result of gene-specific DNA methylation decisions., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Apical constriction, or a reduction in size of the apical domain, underlies many morphogenetic events during development. Actomyosin complexes play an essential role in apical constriction; however, the detailed analysis of molecular mechanisms is still pending. Here, we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells. Lmo7 is primarily localized at apical junctions and promotes the formation of the dense circumferential actomyosin belt. Strikingly, Lmo7 binds non-muscle myosin II (NMII) and recruits it to apical junctions and the apical cortex. This NMII recruitment is essential for Lmo7-mediated apical constriction. Lmo7 knockdown decreases NMIIA localization at apical junctions and delays neural tube closure in Xenopus embryos. Our findings suggest that Lmo7 serves as a scaffold that regulates actomyosin contractility and apical domain size., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Background: Fibroblast growth factors (Fgfs) are required for survival and organ formation during embryogenesis. Fgfs often execute their functions redundantly. Previous analysis of Fgf3 mutants revealed effects on inner ear formation and embryonic survival with incomplete penetrance., Results: Here, we show that presence of a neomycin resistance gene (neo) replacing the Fgf3 coding region leads to reduced survival during embryogenesis and an increased penetrance of inner ear defects. Fgf3 neo/neo mutants showed reduced expression of Fgf4, which is positioned in close proximity to the Fgf3 locus in the mouse genome. Conditional inactivation of Fgf4 during inner ear development on a Fgf3 null background using Fgf3/4 cis mice revealed a redundant requirement between these Fgfs during otic placode induction. In contrast, inactivation of Fgf3 and Fgf4 in the pharyngeal region where both Fgfs are also co-expressed using a Foxg1-Cre driver did not affect development of the pharyngeal arches. However, these mutants showed reduced perinatal survival., Conclusions: These results highlight the importance of Fgf signaling during development. In particular, different members of the Fgf family act redundantly to guarantee inner ear formation and embryonic survival., (© 2021 American Association for Anatomy.)

The Hippo pathway, a highly conserved signaling cascade that functions as an integrator of molecular signals and biophysical states, ultimately impinges upon the transcription coactivator Yes-associated protein 1 (YAP). Hippo-YAP signaling has been shown to play key roles both at the early embryonic stages of implantation and gastrulation, and later during neurogenesis. To explore YAP's potential role in neurulation, we used self-organizing neuruloids grown from human embryonic stem cells on micropatterned substrates. We identified YAP activation as a key lineage determinant, first between neuronal ectoderm and nonneuronal ectoderm, and later between epidermis and neural crest, indicating that YAP activity can enhance the effect of BMP4 stimulation and therefore affect ectodermal specification at this developmental stage. Because aberrant Hippo-YAP signaling has been implicated in the pathology of Huntington's Disease (HD), we used isogenic mutant neuruloids to explore the relationship between signaling and the disease. We found that HD neuruloids demonstrate ectopic activation of gene targets of YAP and that pharmacological reduction of YAP's transcriptional activity can partially rescue the HD phenotype., Competing Interests: FP, TH, QT, TL, RD, AB, TC, JL No competing interests declared, NK, KG is part to an application for patent protection of derivatives of the Lats inhibitor TRULI used in this study, FE is a shareholder of RUMI Scientific, AH Is part to to an application for patent protection of derivatives of the Lats inhibitor TRULI used in this study, AB is a co-founder and shareholder of RUMI Scientific, (© 2022, Piccolo et al.)

The facial surface ectoderm is essential for normal development of the underlying cranial neural crest cell populations, providing signals that direct appropriate growth, patterning, and morphogenesis. Despite the importance of the ectoderm as a signaling center, the molecular cues and genetic programs implemented within this tissue are understudied. Here, we show that removal of two members of the AP-2 transcription factor family, AP-2α and AP-2ß, within the early embryonic ectoderm of the mouse leads to major alterations in the craniofacial complex. Significantly, there are clefts in both the upper face and mandible, accompanied by fusion of the upper and lower jaws in the hinge region. Comparison of ATAC-seq and RNA-seq analyses between controls and mutants revealed significant changes in chromatin accessibility and gene expression centered on multiple AP-2 binding motifs associated with enhancer elements within these ectodermal lineages. In particular, loss of these AP-2 proteins affects both skin differentiation as well as multiple signaling pathways, most notably the WNT pathway. We also determined that the mutant clefting phenotypes that correlated with reduced WNT signaling could be rescued by Wnt1 ligand overexpression in the ectoderm. Collectively, these findings highlight a conserved ancestral function for AP-2 transcription factors in ectodermal development and signaling, and provide a framework from which to understand the gene regulatory network operating within this tissue that directs vertebrate craniofacial development., Competing Interests: EV, IM, HP, JT, HL, KJ, TW No competing interests declared, (© 2022, Van Otterloo et al.)

Despite previous intensive investigations on epiblast cell migration in avian embryos during primitive streak development before stage (st.) 4, this migration at later stages of brain development has remained uninvestigated. By live imaging of epiblast cells sparsely labeled with green fluorescence protein, we investigated anterior epiblast cell migration to form individual brain portions. Anterior epiblast cells from a broad area migrated collectively towards the head axis during st. 5-7 at a rate of 70-110 µm/h, changing directions from diagonal to parallel and forming the brain portions and abutting head ectoderm. This analysis revised the previously published head portion precursor map in anterior epiblasts at st. 4/5. Grafting outside the brain precursor region of mCherry-expressing nodes producing anterior mesendoderm (AME) or isolated AME tissues elicited new cell migration towards ectopic AME tissues. These locally convergent cells developed into secondary brains with portions that depended on the ectopic AME position in the anterior epiblast. Thus, anterior epiblast cells are bipotent for brain/head ectoderm development with given brain portion specificities. A brain portion potential map is proposed, also accounting for previous observations., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The polycomb repressive complex 2 (PRC2), which comprised of the core subunits: Enhancer of Zeste Homolog 2 (EZH2), Suppressor of Zeste 12 (SUZ12), and Embryonic Ectoderm Development (EED), is an essential epigenetic gene silencer responsible for depositing repressive histone H3 lysine 27 trimethylation (H3K27me3) marks on chromatin. The aberrant activity of PRC2 is closely involved in tumorigenesis and progression, making its inhibition a viable strategy for epigenetic cancer therapy. Although the clinical development of small PRC2 inhibitors has made impressive progress, with one EZH2 inhibitor approved for cancer therapy and several other candidates in clinical trials, current EZH2 inhibitors are limited to treating certain hematological malignancies and have acquired drug resistance. EED is essential for PRC2 stabilization and allosterically stimulating PRC2 activity because it functions as a scaffold protein and an H3K27me3-recognizing protein. Thus, due to its novel mechanism of action, targeting EED provides a promising new strategy for inhibiting PRC2 function and exhibits the potential to overcome the issues encountered by EZH2 inhibitors. This review provides a comprehensive overview of available cancer therapy strategies that target EED, including allosteric inhibitors, protein-protein interaction (PPI) inhibitors, and proteolysis-targeting chimeras (PROTACs)., 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 © 2022 Elsevier Masson SAS. All rights reserved.)