Maternal mRNA deadenylation and allocation via Rbm14 condensates facilitate vertebrate blastula development.

Early embryonic development depends on proper utilization and clearance of maternal transcriptomes. How these processes are spatiotemporally regulated remains unclear. Here we show that nuclear RNA-binding protein Rbm14 and maternal mRNAs co-phase separate into cytoplasmic condensates to facilitate vertebrate blastula-to-gastrula development. In zebrafish, Rbm14 condensates were highly abundant in blastomeres and markedly reduced after prominent activation of zygotic transcription. They concentrated at spindle poles by associating with centrosomal γ-tubulin puncta and displayed mainly asymmetric divisions with a global symmetry across embryonic midline in 8- and 16-cell embryos. Their formation was dose-dependently stimulated by m6 A, but repressed by m5 C modification of the maternal mRNA. Furthermore, deadenylase Parn co-phase separated with these condensates, and this was required for deadenylation of the mRNAs in early blastomeres. Depletion of Rbm14 impaired embryonic cell differentiations and full activations of the zygotic genome in both zebrafish and mouse and resulted in developmental arrest at the blastula stage. Our results suggest that cytoplasmic Rbm14 condensate formation regulates early embryogenesis by facilitating deadenylation, protection, and mitotic allocation of m6 A-modified maternal mRNAs, and by releasing the poly(A)-less transcripts upon regulated disassembly to allow their re-polyadenylation and translation or clearance.

SWI/SNF complexes are required for retinal pigmented epithelium differentiation and for the inhibition of cell proliferation and neural differentiation programs.

During embryonic development, tissue-specific transcription factors and chromatin remodelers function together to ensure gradual, coordinated differentiation of multiple lineages. Here, we define this regulatory interplay in the developing retinal pigmented epithelium (RPE), a neuroectodermal lineage essential for the development, function and maintenance of the adjacent retina. We present a high-resolution spatial transcriptomic atlas of the developing mouse RPE and the adjacent ocular mesenchyme obtained by geographical position sequencing (Geo-seq) of a single developmental stage of the eye that encompasses young and more mature ocular progenitors. These transcriptomic data, available online, reveal the key transcription factors and their gene regulatory networks during RPE and ocular mesenchyme differentiation. Moreover, conditional inactivation followed by Geo-seq revealed that this differentiation program is dependent on the activity of SWI/SNF complexes, shown here to control the expression and activity of RPE transcription factors and, at the same time, inhibit neural progenitor and cell proliferation genes. The findings reveal the roles of the SWI/SNF complexes in controlling the intersection between RPE and neural cell fates and the coupling of cell-cycle exit and differentiation.

Gain-of-function mutations in Trim71 linked to congenital hydrocephalus.

The genetic basis of congenital hydrocephalus is only partially understood. A new study in PLOS Biology reports a potential gain-of-function pathological mechanism of congenital hydrocephalus in mouse embryonic stem cells that involves Wnt-ß-catenin signaling pathway regulation.

Author Correction: Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Embryonic vascular establishment requires protein C receptor-expressing endothelial progenitors.

Vascular establishment is one of the early events in embryogenesis. It is believed that vessel-initiating endothelial progenitors cluster to form the first primitive vessel. Understanding the molecular identity of these progenitors is crucial in order to elucidate lineage hierarchy. In this study, we identify protein C receptor (Procr) as an endothelial progenitor marker and investigate the role of Procr+ progenitors during embryonic vascular development. Using a ProcrmGFP-2A-lacZ reporter, we reveal a much earlier Procr expression (embryonic day 7.5) than previously acknowledged (embryonic day 13.5). Genetic fate-mapping experiments using ProcrCre and ProcrCreER demonstrate that Procr+ cells give rise to blood vessels throughout the entire embryo proper. Single-cell RNA-sequencing analyses place Procr+ cells at the start of endothelial commitment and maturation. Furthermore, targeted ablation of Procr+ cells results in failure of vessel formation and early embryonic lethality. Notably, genetic fate mapping and scRNA-seq pseudotime analysis support the view that Procr+ progenitors can give rise to hemogenic endothelium. In this study, we establish a Procr expression timeline and identify Procr+ vessel-initiating progenitors, and demonstrate their indispensable role in establishment of the vasculature during embryo development.

Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

During post-implantation development of the mouse embryo, descendants of the inner cell mass in the early epiblast transit from the naive to primed pluripotent state1. Concurrently, germ layers are formed and cell lineages are specified, leading to the establishment of the blueprint for embryogenesis. Fate-mapping and lineage-analysis studies have revealed that cells in different regions of the germ layers acquire location-specific cell fates during gastrulation2-5. The regionalization of cell fates preceding the formation of the basic body plan-the mechanisms of which are instrumental for understanding embryonic programming and stem-cell-based translational study-is conserved in vertebrate embryos6-8. However, a genome-wide molecular annotation of lineage segregation and tissue architecture of the post-implantation embryo has yet to be undertaken. Here we report a spatially resolved transcriptome of cell populations at defined positions in the germ layers during development from pre- to late-gastrulation stages. This spatiotemporal transcriptome provides high-resolution digitized in situ gene-expression profiles, reveals the molecular genealogy of tissue lineages and defines the continuum of pluripotency states in time and space. The transcriptome further identifies the networks of molecular determinants that drive lineage specification and tissue patterning, supports a role of Hippo-Yap signalling in germ-layer development and reveals the contribution of visceral endoderm to the endoderm in the early mouse embryo.

Using Single-Cell and Spatial Transcriptomes to Understand Stem Cell Lineage Specification During Early Embryo Development.

Embryonic development and stem cell differentiation provide a paradigm to understand the molecular regulation of coordinated cell fate determination and the architecture of tissue patterning. Emerging technologies such as single-cell RNA sequencing and spatial transcriptomics are opening new avenues to dissect cell organization, the divergence of morphological and molecular properties, and lineage allocation. Rapid advances in experimental and computational tools have enabled researchers to make many discoveries and revisit old hypotheses. In this review, we describe the use of single-cell RNA sequencing in studies of molecular trajectories and gene regulation networks for stem cell lineages, while highlighting the integratedexperimental and computational analysis of single-cell and spatial transcriptomes in the molecular annotation of tissue lineages and development during postimplantation gastrulation.

Dynamics of Wnt activity on the acquisition of ectoderm potency in epiblast stem cells.

During embryogenesis, the stringent regulation of Wnt activity is crucial for the morphogenesis of the head and brain. The loss of function of the Wnt inhibitor Dkk1 results in elevated Wnt activity, loss of ectoderm lineage attributes from the anterior epiblast, and the posteriorisation of anterior germ layer tissue towards the mesendoderm. The modulation of Wnt signalling may therefore be crucial for the allocation of epiblast cells to ectoderm progenitors during gastrulation. To test this hypothesis, we examined the lineage characteristics of epiblast stem cells (EpiSCs) that were derived and maintained under different signalling conditions. We showed that suppression of Wnt activity enhanced the ectoderm propensity of the EpiSCs. Neuroectoderm differentiation of these EpiSCs was further empowered by the robust re-activation of Wnt activity. Therefore, during gastrulation, the tuning of the signalling activities that mediate mesendoderm differentiation is instrumental for the acquisition of ectoderm potency in the epiblast.

VGLL4 plays a critical role in heart valve development and homeostasis.

Heart valve disease is a major clinical problem worldwide. Cardiac valve development and homeostasis need to be precisely controlled. Hippo signaling is essential for organ development and tissue homeostasis, while its role in valve formation and morphology maintenance remains unknown. VGLL4 is a transcription cofactor in vertebrates and we found it was mainly expressed in valve interstitial cells at the post-EMT stage and was maintained till the adult stage. Tissue specific knockout of VGLL4 in different cell lineages revealed that only loss of VGLL4 in endothelial cell lineage led to valve malformation with expanded expression of YAP targets. We further semi-knockout YAP in VGLL4 ablated hearts, and found hyper proliferation of arterial valve interstitial cells was significantly constrained. These findings suggest that VGLL4 is important for valve development and manipulation of Hippo components would be a potential therapy for preventing the progression of congenital valve disease.

A gene regulatory network anchored by LIM homeobox 1 for embryonic head development.

Development of the embryonic head is driven by the activity of gene regulatory networks of transcription factors. LHX1 is a homeobox transcription factor that plays an essential role in the formation of the embryonic head. The loss of LHX1 function results in anterior truncation of the embryo caused by the disruption of morphogenetic movement of tissue precursors and the dysregulation of WNT signaling activity. Profiling the gene expression pattern in the Lhx1 mutant embryo revealed that tissues in anterior germ layers acquire posterior tissue characteristics, suggesting LHX1 activity is required for the allocation and patterning of head precursor tissues. Here, we used LHX1 as an entry point to delineate its transcriptional targets and interactors and construct a LHX1-anchored gene regulatory network. Using a gain-of-function approach, we identified genes that immediately respond to Lhx1 activation. Meta-analysis of the datasets of LHX1-responsive genes and genes expressed in the anterior tissues of mouse embryos at head-fold stage, in conjunction with published Xenopus embryonic LHX1 (Xlim1) ChIP-seq data, has pinpointed the putative transcriptional targets of LHX1 and an array of genetic determinants functioning together in the formation of the mouse embryonic head.

Transcriptome analysis reveals determinant stages controlling human embryonic stem cell commitment to neuronal cells.

Proper neural commitment is essential for ensuring the appropriate development of the human brain and for preventing neurodevelopmental diseases such as autism spectrum disorders, schizophrenia, and intellectual disorders. However, the molecular mechanisms underlying the neural commitment in humans remain elusive. Here, we report the establishment of a neural differentiation system based on human embryonic stem cells (hESCs) and on comprehensive RNA sequencing analysis of transcriptome dynamics during early hESC differentiation. Using weighted gene co-expression network analysis, we reveal that the hESC neurodevelopmental trajectory has five stages: pluripotency (day 0); differentiation initiation (days 2, 4, and 6); neural commitment (days 8-10); neural progenitor cell proliferation (days 12, 14, and 16); and neuronal differentiation (days 18, 20, and 22). These stages were characterized by unique module genes, which may recapitulate the early human cortical development. Moreover, a comparison of our RNA-sequencing data with several other transcriptome profiling datasets from mice and humans indicated that Module 3 associated with the day 8-10 stage is a critical window of fate switch from the pluripotency to the neural lineage. Interestingly, at this stage, no key extrinsic signals were activated. In contrast, using CRISPR/Cas9-mediated gene knockouts, we also found that intrinsic hub transcription factors, including the schizophrenia-associated SIX3 gene and septo-optic dysplasia-related HESX1 gene, are required to program hESC neural determination. Our results improve the understanding of the mechanism of neural commitment in the human brain and may help elucidate the etiology of human mental disorders and advance therapies for managing these conditions.

Mediator Med23 deficiency enhances neural differentiation of murine embryonic stem cells through modulating BMP signaling.

Unraveling the mechanisms underlying early neural differentiation of embryonic stem cells (ESCs) is crucial to developing cell-based therapies of neurodegenerative diseases. Neural fate acquisition is proposed to be controlled by a 'default' mechanism, for which the molecular regulation is not well understood. In this study, we investigated the functional roles of Mediator Med23 in pluripotency and lineage commitment of murine ESCs. Unexpectedly, we found that, despite the largely unchanged pluripotency and self-renewal of ESCs, Med23 depletion rendered the cells prone to neural differentiation in different differentiation assays. Knockdown of two other Mediator subunits, Med1 and Med15, did not alter the neural differentiation of ESCs. Med15 knockdown selectively inhibited endoderm differentiation, suggesting the specificity of cell fate control by distinctive Mediator subunits. Gene profiling revealed that Med23 depletion attenuated BMP signaling in ESCs. Mechanistically, MED23 modulated Bmp4 expression by controlling the activity of ETS1, which is involved in Bmp4 promoter-enhancer communication. Interestingly, med23 knockdown in zebrafish embryos also enhanced neural development at early embryogenesis, which could be reversed by co-injection of bmp4 mRNA. Taken together, our study reveals an intrinsic, restrictive role of MED23 in early neural development, thus providing new molecular insights for neural fate determination.

Mouse gastrulation: Attributes of transcription factor regulatory network for epiblast patterning.

Gastrulation is a key milestone in early mouse development when multipotent epiblast cells are allocated to progenitors of diverse tissue lineages that constitute the ensemble of building blocks of the body plan. The analysis of gene function revealed that the activity of transcription factors is likely to be the fundamental driving force underpinning the lineage specification and tissue patterning in the primary germ layers. The developmental-spatial transcriptome of the gastrulating embryo revealed the concerted and interactive activity of the gene regulatory network anchored by development-related transcription factors. The findings of the network structure offer novel insights into the regionalization of tissue fates and enable tracking of the progression of epiblast patterning, leading to the construction of molecularly annotated fate maps of epiblast during gastrulation.

TGF-ß signaling pathway in early mouse development and embryonic stem cells.

TGF-ß superfamily signaling pathways essentially contribute to the broad spectrum of early developmental events including embryonic patterning, cell fate determination and dynamic movements. In this review, we first introduced some key developmental processes that require TGF-ß signaling to show the fundamental importance of these pathways. Then we discuss how their activities are regulated, and new findings about how the TGF-ß superfamily ligands bind to the chromatin to regulate transcription during embryo development.

Dynamic Heterogeneity of Brachyury in Mouse Epiblast Stem Cells Mediates Distinct Response to Extrinsic Bone Morphogenetic Protein (BMP) Signaling.

Mouse pluripotent cells, such as embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), provide excellent in vitro systems to study imperative pre- and postimplantation events of in vivo mammalian development. It is known that mouse ESCs are dynamic heterogeneous populations. However, it remains largely unclear whether and how EpiSCs possess heterogeneity and plasticity similar to that of ESCs. Here, we show that EpiSCs are discriminated by the expression of a specific marker T (Brachyury) into two populations. The T-positive (T(+)) and the T-negative (T(-)) populations can be interconverted within the same culture condition. In addition, the two populations display distinct responses to bone morphogenetic protein (BMP) signaling and different developmental potentials. The T(-) EpiSCs are preferentially differentiated into ectoderm lineages, whereas T(+) EpiSCs have a biased potential for mesendoderm fates. Mechanistic studies reveal that T(+) EpiSCs have an earlier and faster response to BMP4 stimulation than T(-) EpiSCs. Id1 mediates the commitment of T(-) EpiSCs to epidermal lineage during BMP4 treatment. On the other hand, Snail modulates the conversion of T(+) EpiSCs to mesendoderm fates with the presence of BMP4. Furthermore, T expression is essential for epithelial-mesenchymal transition during EpiSCs differentiation. Our findings suggest that the dynamic heterogeneity of the T(+)/T(-) subpopulation primes EpiSCs toward particular cell lineages, providing important insights into the dynamic development of the early mouse embryo.

Epigenetic regulation of early neural fate commitment.

Early neural fate commitment is a key process in neural development and establishment of the central nervous system, and this process is tightly controlled by extrinsic signals, intrinsic factors, and epigenetic regulation. Here, we summarize the main findings regarding the regulatory network of epigenetic mechanisms that play important roles during early neural fate determination and embryonic development, including histone modifications, chromatin remodeling, DNA modifications, and RNA-level regulation. These regulatory mechanisms coordinate to play essential roles in silencing of pluripotency genes and activating key neurodevelopmental genes during cell fate commitment at DNA, histone, chromatin, and RNA levels. Moreover, we discuss the relationship between epigenetic regulation, signaling pathways, and intrinsic factors during early neural fate specification.

Dual roles of histone H3 lysine 9 acetylation in human embryonic stem cell pluripotency and neural differentiation.

Early neurodevelopment requires cell fate commitment from pluripotent stem cells to restricted neural lineages, which involves the epigenetic regulation of chromatin structure and lineage-specific gene transcription. However, it remains unclear how histone H3 lysine 9 acetylation (H3K9Ac), an epigenetic mark representing transcriptionally active chromatin, is involved in the neural commitment from pluripotent embryonic stem cells (ESCs). In this study, we demonstrate that H3K9Ac gradually declines during the first 4 days of in vitro neural differentiation of human ESCs (hESCs) and then increases during days 4-8. Consistent with this finding, the H3K9Ac enrichment at several pluripotency genes was decreased, and H3K9Ac occupancies at the loci of neurodevelopmental genes increased during hESC neural commitment. Inhibiting H3K9 deacetylation on days 0-4 by histone deacetylase inhibitors (HDACis) promoted hESC pluripotency and suppressed its neural differentiation. Conversely, HDACi-elicited up-regulation of H3K9 acetylation on days 4-8 enhanced neural differentiation and activated multiple neurodevelopmental genes. Mechanistically, HDACis promote pluripotency gene transcription to support hESC self-renewal through suppressing HDAC3 activity. During hESC neural commitment, HDACis relieve the inhibitory activities of HDAC1/5/8 and thereby promote early neurodevelopmental gene expression by interfering with gene-specific histone acetylation patterns. Furthermore, p300 is primarily identified as the major histone acetyltransferase involved in both hESC pluripotency and neural differentiation. Our results indicate that epigenetic modification plays pivotal roles during the early neural specification of hESCs. The histone acetylation, which is regulated by distinct HDAC members at different neurodevelopmental stages, plays dual roles in hESC pluripotency maintenance and neural differentiation.

Inhibition of transforming growth factor ß (TGF-ß) signaling can substitute for Oct4 protein in reprogramming and maintain pluripotency.

Mouse pluripotent stem cells (PSCs), such as ES cells and induced PSCs (iPSCs), are an excellent system to investigate the molecular and cellular mechanisms involved in early embryonic development. The signaling pathways orchestrated by leukemia inhibitor factor/STAT3, Wnt/ß-catenin, and FGF/MEK/ERK play key roles in the generation of pluripotency. However, the function of TGF-ß signaling in this process remains elusive. Here we show that inhibiting TGF-ß signaling with its inhibitor SB431542 can substitute for Oct4 during reprogramming. Moreover, inhibiting TGF-ß signaling can sustain the pluripotency of iPSCs and ES cells through modulating FGF/MEK/ERK signaling. Therefore, this study reveals a novel function of TGF-ß signaling inhibition in the generation and maintenance of PSCs.

Location of transient ectodermal progenitor potential in mouse development.

Ectoderm is one of the three classic germ layers in the early mouse embryo, with the capacity to develop into both the central nervous system and epidermis. Because it is a transient phase of development with few molecular markers, the early ectoderm is the least understood germ layer in mouse embryonic development. In this work, we studied the differentiation potential of isolated ectoderm tissue in response to BMP signaling at various developmental stages (E6.5, E7.0 and E7.5), and identified a transient region in the anterior-proximal side of the embryo at E7.0 that possesses the ability to become neural or epidermal ectoderm in response to the absence or presence of BMP4, respectively. Furthermore, we demonstrated that inhibition of Nodal signaling could direct the pluripotent E6.5 epiblast cells towards ectoderm lineages during differentiation in explants in vitro. Our work not only improves our understanding of ectodermal layer development in early embryos, but also provides a framework for regenerative differentiation towards ectodermal tissues.