Ran Wang

Spatial Transcriptome for the Molecular Annotation of Lineage Fates and Cell Identity in Mid-gastrula Mouse Embryo

Gastrulation of the mouse embryo entails progressive restriction of lineage potency and the organization of the lineage progenitors into a body plan. Here we performed a high-resolution RNA sequencing analysis on single mid-gastrulation mouse embryos to collate a spatial transcriptome that correlated with the regionalization of cell fates in the embryo. 3D rendition of the quantitative data enabled the visualization of the spatial pattern of all expressing genes in the epiblast in a digital whole-mount in situ format. The dataset also identified genes that (1) are co-expressed in a specific cell population, (2) display similar global pattern of expression, (3) have lineage markers, (4) mark domains of transcriptional and signaling activity associated with cell fates, and (5) can be used as zip codes for mapping the position of single cells isolated from the mid-gastrula stage embryo and the embryo-derived stem cells to the equivalent epiblast cells for delineating their prospective cell fates.

Dual Roles of Histone H3 Lysine 9 Acetylation in Human Embryonic Stem Cell Pluripotency and Neural Differentiation

Background: H3K9Ac is an epigenetic mark representing transcriptionally active chromatin. Results: Inhibition of HDAC activity promotes pluripotency maintenance at the initiation of hESC differentiation and inhibits neural differentiation during neural commitment stage. Conclusion: H3K9Ac plays dual roles in hESC pluripotency maintenance and neural differentiation through regulating different targets. Significance: This work provides insight into the epigenetic mechanisms underlying neural differentiation of hESCs associated with histone acetylation patterns. 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.

AF9 promotes hESC neural differentiation through recruiting TET2 to neurodevelopmental gene loci for methylcytosine hydroxylation

AF9 mutations have been implicated in human neurodevelopmental diseases and murine Af9 mediates histone methylation during cortical neuron generation. However, AF9 function and related mechanisms in human neurodevelopment remain unknown. Here we show that AF9 is necessary and sufficient for human embryonic stem cell (hESC) neural differentiation and neurodevelopmental gene activation. The 5-methylcytosine (5mC) dioxygenase TET2, which was identified in an AF9-associated protein complex, physically interacted with AF9. Both AF9 and TET2 co-localized in 5-hydroxymethylcytosine (5hmC)-positive hESC-derived neurons and were required for appropriate hESC neural differentiation. Upon binding to AAC-containing motifs, AF9 recruited TET2 to occupy the common neurodevelopmental gene loci to direct 5mC-to-5hmC conversion, which was followed by sequential activation of neural target genes and hESC neural commitment. These findings define an AF9–TET2 regulatory complex for modulating human neural development and reveal a novel mechanism by which the AF9 recognition specificity and TET2 hydroxylation activity cooperate to control neurodevelopmental gene activation.

Ectodermal progenitors derived from epiblast stem cells by inhibition of Nodal signaling

The ectoderm has the capability to generate epidermis and neuroectoderm and plays imperative roles during the early embryonic development. Our recent study uncovered a region with ectodermal progenitor potential in mouse embryo at embryonic day 7.0 and revealed that Nodal inhibition is essential for its formation. Here, we demonstrate that through brief inhibition of Nodal signaling in vitro, mouse embryonic stem cell (ESC)-derived epiblast stem cells (ESD-EpiSCs) could be committed to transient ectodermal progenitor populations, which possess the ability to give rise to neural or epidermal ectoderm in the absence or presence of BMP4, respectively. Mechanistic studies reveal that BMP4 recruits distinct transcriptional targets in ESD-EpiSCs and ectoderm-like cells. Furthermore, FGF-Erk signaling may also be alleviated during the generation of ectoderm-like cells. Thus, our data suggest that instructive interactions among several extracellular signals participate in the commitment of ectoderm from ESD-EpiSCs, which shed new light on the understanding of the formation of ectoderm during the gastrulation in early mouse embryo development.

Abnormal Paraventricular Nucleus of Hypothalamus and Growth Retardation Associated with Loss of Nuclear Receptor Gene COUP-TFII

The paraventricular nucleus of hypothalamus plays important roles in the regulation of energy balance and fetal growth. However, the molecular mechanisms underlying its formation and function have not been clearly elucidated. Various mutations in the human COUP-TFII gene, which encodes a nuclear receptor, result in growth retardation, congenital diaphragmatic hernia and congenital heart defects. Here, we show that COUP-TFII gene is expressed in the developing hypothalamus in mouse. The ventral forebrain-specific RXCre/+; COUP-TFIIF/F mutant mice display growth retardation. The development of the paraventricular nucleus of hypothalamus is compromised in the COUP-TFII mutant mainly because of increased apoptosis and mis-migration of the Brn2+ neurons. Moreover, hypoplastic anterior pituitary with blood cell clusters and shrunken posterior pituitary lacking AVP/OT neuron innervations are observed in the mutant, indicating the failure of formation of the hypothalamic-pituitary axis. Mechanistic studies show that the expression of Bdnf and Nrp1 genes is reduced in the mutant embryo, and that Bdnf is a direct downstream target of the COUP-TFII protein. Thus, our findings provide a novel functional validation that COUP-TFII gene promotes the expression of Bdnf and Nrp1 genes to ensure the appropriate morphogenesis of the hypothalamic-pituitary axis, especially the paraventricular nucleus of hypothalamus, and to prevent growth retardation.

Silencing of developmental genes by H3K27me3 and DNA methylation reflects the discrepant plasticity of embryonic and extraembryonic lineages

Dear Editor, One of the most important topics in mammalian embryogenesis is the generation of multiple cell lineages. Briefly, one single-cell totipotent zygote develops into the inner cell mass (ICM) and trophectoderm (TE) at the blastocyst stage. Afterwards, the ICM further generates the epiblast cells and finally forms multiple somatic cell lineages, while the TE develops into extraembryonic ectoderm (ExE) cells and eventually forms the placental tissue. The highly ordered programming of mammalian embryo development is spatial-temporally regulated by epigenetic mechanisms. Previous work has revealed that the ICM and TE remain largely epigenetically indistinguishable, 2 even though there exist significant transcriptional distinctions. Recently we revealed the spatial-specific transcriptome of key development-related genes (DRGs) in mouse E7.0 gastrula, and we also noticed that these DRG-silenced gastrula regions possess distinct developmental potencies. However, whether there are any distinctions of epigenetic mechanisms underlying the region-specific distribution of DRGs in post implantation embryos and how these epigenetic distinctions contribute to the regionalized developmental potential in mouse embryos remain unclear. Here, we focused on two major epigenetic silencing mechanisms, H3K27me3 and DNA methylation (DNAme), in mouse post implantation embryos (Fig. 1a and Supplementary information, Figure S1A and B), which have been well characterized in preimplantation embryos. 2 The embryos were dissected into ExE and epiblast (Epi) at three consecutive stages, encompassing the last homogeneous epiblast stage (E6.5), the initial regionalized stage (E7.0) and the three germ-layer formation stage (E7.5) (Fig. 1a and Supplementary information, Figure S1B). According to the regionalized developmental propensity, the E7.0 and E7.5 epiblasts were further dissected into anterior (A), posterior (P), and anterior mesoderm (AM, for E7.5 only) parts for high-fidelity epigenetic profiling, which can be indicated by region-specific marker expression (Supplementary information, Figure S1C and D). Given the limited cell number of early post implantation embryos, modified chromatin immunoprecipitation adapted for 10 cells was utilized to profile the modification pattern of H3K27me3 and reduced representation bisulfite sequencing was used to profile DNA methylome. Two biological replicates with high reproducibility (Supplementary information, Figure S1E, F and Table S1) were generated for each sample. We observed weak dynamics of DNAme at post implantation stage from E6.5 to E7.5 stage (Fig. 1b). Noticeably, the ExE cells possessed lower DNAme level than the intraembryonic cells. Meanwhile, pervasive remodeling of H3K27me3 was identified during the development of the post implantation embryo (Fig. 1b and Supplementary information, Figure S2A). Clustering analysis revealed that significant distinctions of H3K27me3 and DNAme exist between pre and post implantation embryos (Supplementary information, Figure S2B and C). Interestingly, genomic distributions of H3K27me3 and DNAme exhibited negative correlation at multiple genomic regions, such as CpG islands (CGIs) and intracisternal A-particle (IAP) regions (Fig. 1b). Strikingly, most de novo H3K27me3 of post implantation embryos occurred at the regulatory genomic regions, such as CGIs. Moreover, weak dynamics of H3K27me3 can be identified at the repetitive regions, such as endogenous retrovirus K regions (ERVKs). Meanwhile, the increment of DNAme was observed at both the regulatory regions and repetitive elements. The different dynamics of H3K27me3 and DNAme suggested distinct roles of these two epigenetic modifications in mouse embryonic development. Differentially methylated region (DMR) analysis revealed that more unique methylation regions (4,862, change in methylation > 0.15, q-value < 0.05) can be identified in embryonic cells than in ExE cells (1,194; Supplementary information, Figure S2D). Surprisingly, most unique methylation regions in ExE were enriched for embryonic DRGs, such as neuron differentiation-related genes. In contrast, in the embryonic cells, specific lineage-related developmental genes were devoid of DNAme, and regions with higher DNAme were mostly surrounded by the genes associated with proteolysis and metabolic processes (Supplementary information, Figure S2E). Moreover, few DMRs were detected between intraembryonic cells (such as P and A; Supplementary information, Figure S2D). Given the generation of multiple lineages in the embryonic cells, alternative epigenetic silencing mechanisms must be employed to direct the diversification of embryonic lineages. It has been reported that H3K27me3 at high CpG content promoters (HCP) in the embryonic stem cells are vital for the regulation of developmental genes. As to the mouse embryos, we found that the preference for different types of promoters was newly established in the post implantation embryos, and the preference of H3K27me3 for HCPs was much higher in embryonic cells of the post implantation embryos compared to the ExE (Fig. 1d; Supplementary information, Figure S2F and G). Given that the HCP-related genes are generally associated with ubiquitously expressed house-keeping genes and key developmental genes and DRGs are uniquely methylated in the ExE cells, the higher preference of H3K27me3 for HCPs in embryonic cells may be the alternative epigenetic mechanism to direct the generation of multiple embryonic lineages. By mapping H3K27me3 distribution and DNAme profiles around all 13,117 HCPs, we found that 13.4% (1,760/13,117) of HCPs exhibited differential distribution of H3K27me3 between ExE and embryonic cells (Fig. 1e and Supplementary information, Table S2). Further analysis revealed that among these 1,760 HCPs,

Suppressing Nodal Signaling Activity Predisposes Ectodermal Differentiation of Epiblast Stem Cells

Summary The molecular mechanism underpinning the specification of the ectoderm, a transient germ-layer tissue, during mouse gastrulation was examined here in a stem cell-based model. We captured a self-renewing cell population with enhanced ectoderm potency from mouse epiblast stem cells (EpiSCs) by suppressing Nodal signaling activity. The transcriptome of the Nodal-inhibited EpiSCs resembles that of the anterior epiblast of embryonic day (E)7.0 and E7.5 mouse embryo, which is accompanied by chromatin modifications that reflect the priming of ectoderm lineage-related genes for expression. Nodal-inhibited EpiSCs show enhanced ectoderm differentiation in vitro and contribute to the neuroectoderm and the surface ectoderm in postimplantation chimeras but lose the propensity for mesendoderm differentiation in vitro and in chimeras. Our findings show that specification of the ectoderm progenitors is enhanced by the repression of Nodal signaling activity, and the ectoderm-like stem cells provide an experimental model to investigate the molecular characters of the epiblast-derived ectoderm.

Sequential formation and resolution of multiple rosettes drive embryo remodelling after implantation

The morphogenetic remodelling of embryo architecture after implantation culminates in pro-amniotic cavity formation. Despite its key importance, how this transformation occurs remains unknown. Here, we apply high-resolution imaging of embryos developing in vivo and in vitro, spatial RNA sequencing and 3D trophoblast stem cell models to determine the sequence and mechanisms of these remodelling events. We show that cavitation of the embryonic tissue is followed by folding of extra-embryonic tissue to mediate the formation of a second extra-embryonic cavity. Concomitantly, at the boundary between embryonic and extra-embryonic tissues, a hybrid 3D rosette forms. Resolution of this rosette enables the embryonic cavity to invade the extra-embryonic tissue. Subsequently, β1-integrin signalling mediates the formation of multiple extra-embryonic 3D rosettes. Podocalyxin exocytosis leads to their polarized resolution, permitting the extension of embryonic and extra-embryonic cavities and their fusion into a unified pro-amniotic cavity. These morphogenetic transformations of embryogenesis reveal a previously unappreciated mechanism for lumen expansion and fusion.Using time-lapse microscopy and transcriptome analysis of the post-implantation mouse embryo, Christodoulou et al. show that cavity fusion occurs through the formation and polarized resolution of multiple, multicellular three-dimensional rosettes.

Publisher Correction: Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures

In the version of this Technical Report originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Report.

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.