Polarised cell intercalation during Drosophila axis extension is robust to an orthogonal pull by the invaginating mesoderm.

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.

Small Molecules Promote the Rapid Generation of Dental Epithelial Cells from Human-Induced Pluripotent Stem Cells.

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.

Unlocking trophectoderm mysteries: In vivo and in vitro perspectives on human and mouse trophoblast fate induction.

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.

Chromatin landscape instructs precise transcription factor regulome during embryonic lineage specification.

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.

Hypoblast from human pluripotent stem cells regulates epiblast development.

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 embryos1-8. 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 cells9-17. 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.

An ex vivo system to study cellular dynamics underlying mouse peri-implantation development.

Upon implantation, mammalian embryos undergo major morphogenesis and key developmental processes such as body axis specification and gastrulation. However, limited accessibility obscures the study of these crucial processes. Here, we develop an ex vivo Matrigel-collagen-based culture to recapitulate mouse development from E4.5 to E6.0. Our system not only recapitulates embryonic growth, axis initiation, and overall 3D architecture in 49% of the cases, but its compatibility with light-sheet microscopy also enables the study of cellular dynamics through automatic cell segmentation. We find that, upon implantation, release of the increasing tension in the polar trophectoderm is necessary for its constriction and invagination. The resulting extra-embryonic ectoderm plays a key role in growth, morphogenesis, and patterning of the neighboring epiblast, which subsequently gives rise to all embryonic tissues. This 3D ex vivo system thus offers unprecedented access to peri-implantation development for in toto monitoring, measurement, and spatiotemporally controlled perturbation, revealing a mechano-chemical interplay between extra-embryonic and embryonic tissues.

YAP1 regulates the self-organized fate patterning of hESC-derived gastruloids.

The gastrulation process relies on complex interactions between developmental signaling pathways that are not completely understood. Here, we interrogated the contribution of the Hippo signaling effector YAP1 to the formation of the three germ layers by analyzing human embryonic stem cell (hESC)-derived 2D-micropatterned gastruloids. YAP1 knockout gastruloids display a reduced ectoderm layer and enlarged mesoderm and endoderm layers compared with wild type. Furthermore, our epigenome and transcriptome analysis revealed that YAP1 attenuates Nodal signaling by directly repressing the chromatin accessibility and transcription of key genes in the Nodal pathway, including the NODAL and FOXH1 genes. Hence, in the absence of YAP1, hyperactive Nodal signaling retains SMAD2/3 in the nuclei, impeding ectoderm differentiation of hESCs. Thus, our work revealed that YAP1 is a master regulator of Nodal signaling, essential for instructing germ layer fate patterning in human gastruloids.

Multi-layered regulation of neuroectoderm differentiation by retinoic acid in a primitive streak-like context.

The formation of the primitive streak (PS) and the subsequent induction of neuroectoderm are hallmarks of gastrulation. Combining an in vitro reconstitution of this process based on mouse embryonic stem cells (mESCs) with a collection of knockouts in reporter mESC lines, we identified retinoic acid (RA) as a critical mediator of early neural induction triggered by TGFß or Wnt signaling inhibition. Single-cell RNA sequencing analysis captured the temporal unfolding of cell type diversification, up to the emergence of somite and neural fates. In the absence of the RA-synthesizing enzyme Aldh1a2, a sensitive RA reporter revealed a hitherto unidentified residual RA signaling that specified neural fate. Genetic evidence showed that the RA-degrading enzyme Cyp26a1 protected PS-like cells from neural induction, even in the absence of TGFß and Wnt antagonists. Overall, we characterized a multi-layered control of RA levels that regulates early neural differentiation in an in vitro PS-like system.

A single-cell RNA-seq analysis of Brachyury-expressing cell clusters suggests a morphogenesis-associated signal center of oral ectoderm in sea urchin embryos.

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.

ECM-integrin signalling instructs cellular position sensing to pattern the early mouse embryo.

Development entails patterned emergence of diverse cell types within the embryo. In mammals, cells positioned inside the embryo give rise to the inner cell mass (ICM), which eventually forms the embryo itself. Yet, the molecular basis of how these cells recognise their 'inside' position to instruct their fate is unknown. Here, we show that provision of extracellular matrix (ECM) to isolated embryonic cells induces ICM specification and alters the subsequent spatial arrangement between epiblast (EPI) and primitive endoderm (PrE) cells that emerge within the ICM. Notably, this effect is dependent on integrin ß1 activity and involves apical-to-basal conversion of cell polarity. We demonstrate that ECM-integrin activity is sufficient for 'inside' positional signalling and is required for correct EPI/PrE patterning. Thus, our findings highlight the significance of ECM-integrin adhesion in enabling position sensing by cells to achieve tissue patterning.

Cell geometry, signal dampening, and a bimodal transcriptional response underlie the spatial precision of an ERK-mediated embryonic induction.

Precise control of lineage segregation is critical for the development of multicellular organisms, but our quantitative understanding of how variable signaling inputs are integrated to activate lineage-specific gene programs remains limited. Here, we show how precisely two out of eight ectoderm cells adopt neural fates in response to ephrin and FGF signals during ascidian neural induction. In each ectoderm cell, FGF signals activate ERK to a level that mirrors its cell contact surface with FGF-expressing mesendoderm cells. This gradual interpretation of FGF inputs is followed by a bimodal transcriptional response of the immediate early gene, Otx, resulting in its activation specifically in the neural precursors. At low levels of ERK, Otx is repressed by an ETS family transcriptional repressor, ERF2. Ephrin signals are critical for dampening ERK activation levels across ectoderm cells so that only neural precursors exhibit above-threshold levels, evade ERF repression, and "switch on" Otx transcription.

Human neural tube morphogenesis in vitro by geometric constraints.

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. 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 induction5,6, 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 gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease.

Comparison of Differentiation Pattern and WNT/SHH Signaling in Pluripotent Stem Cells Cultured under Different Conditions.

Pluripotent stem cells (PSCs) are characterized by the ability to self-renew as well as undergo multidirectional differentiation. Culture conditions have a pivotal influence on differentiation pattern. In the current study, we compared the fate of mouse PSCs using two culture media: (1) chemically defined, free of animal reagents, and (2) standard one relying on the serum supplementation. Moreover, we assessed the influence of selected regulators (WNTs, SHH) on PSC differentiation. We showed that the differentiation pattern of PSCs cultured in both systems differed significantly: cells cultured in chemically defined medium preferentially underwent ectodermal conversion while their endo- and mesodermal differentiation was limited, contrary to cells cultured in serum-supplemented medium. More efficient ectodermal differentiation of PSCs cultured in chemically defined medium correlated with higher activity of SHH pathway while endodermal and mesodermal conversion of cells cultured in serum-supplemented medium with higher activity of WNT/JNK pathway. However, inhibition of either canonical or noncanonical WNT pathway resulted in the limitation of endo- and mesodermal conversion of PSCs. In addition, blocking WNT secretion led to the inhibition of PSC mesodermal differentiation, confirming the pivotal role of WNT signaling in this process. In contrast, SHH turned out to be an inducer of PSC ectodermal, not mesodermal differentiation.

Single-cell transcriptome analysis of the zebrafish embryonic trunk.

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.

Temporal transcriptomic profiling reveals dynamic changes in gene expression of Xenopus animal cap upon activin treatment.

Activin, a member of the transforming growth factor-ß (TGF-ß) superfamily of proteins, induces various tissues from the amphibian presumptive ectoderm, called animal cap explants (ACs) in vitro. However, it remains unclear how and to what extent the resulting cells recapitulate in vivo development. To comprehensively understand whether the molecular dynamics during activin-induced ACs differentiation reflect the normal development, we performed time-course transcriptome profiling of Xenopus ACs treated with 50 ng/mL of activin A, which predominantly induced dorsal mesoderm. The number of differentially expressed genes (DEGs) in response to activin A increased over time, and totally 9857 upregulated and 6663 downregulated DEGs were detected. 1861 common upregulated DEGs among all Post_activin samples included several Spemann's organizer genes. In addition, the temporal transcriptomes were clearly classified into four distinct groups in correspondence with specific features, reflecting stepwise differentiation into mesoderm derivatives, and a decline in the regulation of nuclear envelop and golgi. From the set of early responsive genes, we also identified the suppressor of cytokine signaling 3 (socs3) as a novel activin A-inducible gene. Our transcriptome data provide a framework to elucidate the transcriptional dynamics of activin-driven AC differentiation, reflecting the molecular characteristics of early normal embryogenesis.

Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre.

Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.

FGF signalling plays similar roles in development and regeneration of the skeleton in the brittle star Amphiura filiformis.

Regeneration as an adult developmental process is in many aspects similar to embryonic development. Although many studies point out similarities and differences, no large-scale, direct and functional comparative analyses between development and regeneration of a specific cell type or structure in one animal exist. Here, we use the brittle star Amphiura filiformis to characterise the role of the FGF signalling pathway during skeletal development in embryos and arm regeneration. In both processes, we find ligands expressed in ectodermal cells that flank underlying skeletal mesenchymal cells, which express the receptors. Perturbation of FGF signalling showed inhibited skeleton formation in both embryogenesis and regeneration, without affecting other key developmental processes. Differential transcriptome analysis finds mostly differentiation genes rather than transcription factors to be downregulated in both contexts. Moreover, comparative gene analysis allowed us to discover brittle star-specific differentiation genes. In conclusion, our results show that the FGF pathway is crucial for skeletogenesis in the brittle star, as in other deuterostomes, and provide evidence for the re-deployment of a developmental gene regulatory module during regeneration.

Machine learning-assisted high-content analysis of pluripotent stem cell-derived embryos in vitro.

Stem cell-based embryo models by cultured pluripotent and extra-embryonic lineage stem cells are novel platforms to model early postimplantation development. We showed that induced pluripotent stem cells (iPSCs) could form ITS (iPSCs and trophectoderm stem cells) and ITX (iPSCs, trophectoderm stem cells, and XEN cells) embryos, resembling the early gastrula embryo developed in vivo. To facilitate the efficient and unbiased analysis of the stem cell-based embryo model, we set up a machine learning workflow to extract multi-dimensional features and perform quantification of ITS embryos using 3D images collected from a high-content screening system. We found that different PSC lines differ in their ability to form embryo-like structures. Through high-content screening of small molecules and cytokines, we identified that BMP4 best promoted the morphogenesis of the ITS embryo. Our study established an innovative strategy to analyze stem cell-based embryo models and uncovered new roles of BMP4 in stem cell-based embryo models.

MUSE Stem Cells Can Be Isolated from Stromal Compartment of Mouse Bone Marrow, Adipose Tissue, and Ear Connective Tissue: A Comparative Study of Their In Vitro Properties.

The cells present in the stromal compartment of many tissues are a heterogeneous population containing stem cells, progenitor cells, fibroblasts, and other stromal cells. A SSEA3(+) cell subpopulation isolated from human stromal compartments showed stem cell properties. These cells, known as multilineage-differentiating stress-enduring (MUSE) cells, are capable of resisting stress and possess an excellent ability to repair DNA damage. We isolated MUSE cells from different mouse stromal compartments, such as those present in bone marrow, subcutaneous white adipose tissue, and ear connective tissue. These cells showed overlapping in vitro biological properties. The mouse MUSE cells were positive for stemness markers such as SOX2, OCT3/4, and NANOG. They also expressed TERT, the catalytic telomerase subunit. The mouse MUSE cells showed spontaneous commitment to differentiation in meso/ecto/endodermal derivatives. The demonstration that multilineage stem cells can be isolated from an animal model, such as the mouse, could offer a valid alternative to the use of other stem cells for disease studies and envisage of cellular therapies.

Single-cell multiomics sequencing reveals the functional regulatory landscape of early embryos.

Extensive epigenetic reprogramming occurs during preimplantation embryo development. However, it remains largely unclear how the drastic epigenetic reprogramming contributes to transcriptional regulatory network during this period. Here, we develop a single-cell multiomics sequencing technology (scNOMeRe-seq) that enables profiling of genome-wide chromatin accessibility, DNA methylation and RNA expression in the same individual cell. We apply this method to depict a single-cell multiomics map of mouse preimplantation development. We find that genome-wide DNA methylation remodeling facilitates the reconstruction of genetic lineages in early embryos. Further, we construct a zygotic genome activation (ZGA)-associated regulatory network and reveal coordination among multiple epigenetic layers, transcription factors and repeat elements that instruct proper ZGA. Cell fates associated cis-regulatory elements are activated stepwise in post-ZGA stages. Trophectoderm (TE)-specific transcription factors play dual roles in promoting the TE program while repressing the inner cell mass (ICM) program during the ICM/TE separation.