TATA-binding associated factors have distinct roles during early mammalian development.

Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome.

Gastrulation-stage gene expression in Nipbl+/- mouse embryos foreshadows the development of syndromic birth defects.

In animal models, Nipbl deficiency phenocopies gene expression changes and birth defects seen in Cornelia de Lange syndrome, the most common cause of which is Nipbl haploinsufficiency. Previous studies in Nipbl+/- mice suggested that heart development is abnormal as soon as cardiogenic tissue is formed. To investigate this, we performed single-cell RNA sequencing on wild-type and Nipbl+/- mouse embryos at gastrulation and early cardiac crescent stages. Nipbl+/- embryos had fewer mesoderm cells than wild-type and altered proportions of mesodermal cell subpopulations. These findings were associated with underexpression of genes implicated in driving specific mesodermal lineages. In addition, Nanog was found to be overexpressed in all germ layers, and many gene expression changes observed in Nipbl+/- embryos could be attributed to Nanog overexpression. These findings establish a link between Nipbl deficiency, Nanog overexpression, and gene expression dysregulation/lineage misallocation, which ultimately manifest as birth defects in Nipbl+/- animals and Cornelia de Lange syndrome.

Graded FGF activity patterns distinct cell types within the apical sensory organ of the sea anemone Nematostella vectensis.

Bilaterian animals have evolved complex sensory organs comprised of distinct cell types that function coordinately to sense the environment. Each sensory unit has a defined architecture built from component cell types, including sensory cells, non-sensory support cells, and dedicated sensory neurons. Whether this characteristic cellular composition is present in the sensory organs of non-bilaterian animals is unknown. Here, we interrogate the cell type composition and gene regulatory networks controlling development of the larval apical sensory organ in the sea anemone Nematostella vectensis. Using single cell RNA sequencing and imaging approaches, we reveal two unique cell types in the Nematostella apical sensory organ, GABAergic sensory cells and a putative non-sensory support cell population. Further, we identify the paired-like (PRD) homeodomain gene prd146 as a specific sensory cell marker and show that Prd146+ sensory cells become post-mitotic after gastrulation. Genetic loss of function approaches show that Prd146 is essential for apical sensory organ development. Using a candidate gene knockdown approach, we place prd146 downstream of FGF signaling in the apical sensory organ gene regulatory network. Further, we demonstrate that an aboral FGF activity gradient coordinately regulates the specification of both sensory and support cells. Collectively, these experiments define the genetic basis for apical sensory organ development in a non-bilaterian animal and reveal an unanticipated degree of complexity in a prototypic sensory structure.

TBXT dose sensitivity and the decoupling of nascent mesoderm specification from EMT progression in 2D human gastruloids.

In the nascent mesoderm, TBXT expression must be precisely regulated to ensure that cells exit the primitive streak and pattern the anterior-posterior axis, but how varying dosage informs morphogenesis is not well understood. In this study, we define the transcriptional consequences of TBXT dosage reduction during early human gastrulation using human induced pluripotent stem cell models of gastrulation and mesoderm differentiation. Multi-omic single-nucleus RNA and single-nucleus ATAC sequencing of 2D gastruloids comprising wild-type, TBXT heterozygous or TBXT null human induced pluripotent stem cells reveal that varying TBXT dosage does not compromise the ability of a cell to differentiate into nascent mesoderm, but instead directly influences the temporal progression of the epithelial-to-mesenchymal transition with wild type transitioning first, followed by TBXT heterozygous and then TBXT null. By differentiating cells into nascent mesoderm in a monolayer format, we further illustrate that TBXT dosage directly impacts the persistence of junctional proteins and cell-cell adhesions. These results demonstrate that epithelial-to-mesenchymal transition progression can be decoupled from the acquisition of mesodermal identity in the early gastrula and shed light on the mechanisms underlying human embryogenesis.

A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

Embryonic spatiotemporal expression pattern of Folded gastrulation suggests roles in multiple morphogenetic events and regulation by AbdA.

In Drosophila, the signaling pathway activated by the ligand Folded gastrulation (Fog) is among the few known G protein-coupled receptor (GPCR) pathways to regulate cell shape change with a well-characterized role in gastrulation. However, an understanding of the spectrum of morphogenetic events regulated by Fog signaling is still lacking. Here, we present an analysis of the expression pattern and regulation of fog using a genome-engineered Fog::sfGFP line. We show that Fog expression is widespread and in tissues previously not associated with the signaling pathway including germ cells, trachea, and amnioserosa. In the central nervous system (CNS), we find that the ligand is expressed in multiple types of glia indicating a prominent role in the development of these cells. Consistent with this, we have identified 3 intronic enhancers whose expression in the CNS overlaps with Fog::sfGFP. Further, we show that enhancer-1, (fogintenh-1) located proximal to the coding exon is responsive to AbdA. Supporting this, we find that fog expression is downregulated in abdA mutants. Together, our study highlights the broad scope of Fog-GPCR signaling during embryogenesis and identifies Hox gene AbdA as a novel regulator of fog expression.

Anp32e protects against accumulation of H2A.Z at Sox motif containing promoters during zebrafish gastrulation.

Epigenetic regulation of chromatin states is crucial for proper gene expression programs and progression during development, but precise mechanisms by which epigenetic factors influence differentiation remain poorly understood. Here we find that the histone variant H2A.Z accumulates at Sox motif-containing promoters during zebrafish gastrulation while neighboring genes become transcriptionally active. These changes coincide with reduced expression of anp32e, the H2A.Z histone removal chaperone, suggesting that loss of Anp32e may lead to increases in H2A.Z binding during differentiation. Remarkably, genetic removal of Anp32e in embryos leads to H2A.Z accumulation prior to gastrulation and developmental genes become precociously active. Accordingly, H2A.Z accumulation occurs most extensively at Sox motif-associated genes, including many which are normally activated following gastrulation. Altogether, our results provide compelling evidence for a mechanism in which Anp32e preferentially restricts H2A.Z accumulation at Sox motifs to regulate the initial phases of developmental differentiation in zebrafish.

Single-cell transcriptomics illuminates regulatory steps driving anterior-posterior patterning of Drosophila embryonic mesoderm.

Single-cell technologies promise to uncover how transcriptional programs orchestrate complex processes during embryogenesis. Here, we apply a combination of single-cell technology and genetic analysis to investigate the dynamic transcriptional changes associated with Drosophila embryo morphogenesis at gastrulation. Our dataset encompassing the blastoderm-to-gastrula transition provides a comprehensive single-cell map of gene expression across cell lineages validated by genetic analysis. Subclustering and trajectory analyses revealed a surprising stepwise progression in patterning to transition zygotic gene expression and specify germ layers as well as uncovered an early role for ecdysone signaling in epithelial-to-mesenchymal transition in the mesoderm. We also show multipotent progenitors arise prior to gastrulation by analyzing the transcription trajectory of caudal mesoderm cells, including a derivative that ultimately incorporates into visceral muscles of the midgut and hindgut. This study provides a rich resource of gastrulation and elucidates spatially regulated temporal transitions of transcription states during the process.

Real-time monitoring of an endogenous Fgf8a gradient attests to its role as a morphogen during zebrafish gastrulation.

Morphogen gradients impart positional information to cells in a homogenous tissue field. Fgf8a, a highly conserved growth factor, has been proposed to act as a morphogen during zebrafish gastrulation. However, technical limitations have so far prevented direct visualization of the endogenous Fgf8a gradient and confirmation of its morphogenic activity. Here, we monitor Fgf8a propagation in the developing neural plate using a CRISPR/Cas9-mediated EGFP knock-in at the endogenous fgf8a locus. By combining sensitive imaging with single-molecule fluorescence correlation spectroscopy, we demonstrate that Fgf8a, which is produced at the embryonic margin, propagates by diffusion through the extracellular space and forms a graded distribution towards the animal pole. Overlaying the Fgf8a gradient curve with expression profiles of its downstream targets determines the precise input-output relationship of Fgf8a-mediated patterning. Manipulation of the extracellular Fgf8a levels alters the signaling outcome, thus establishing Fgf8a as a bona fide morphogen during zebrafish gastrulation. Furthermore, by hindering Fgf8a diffusion, we demonstrate that extracellular diffusion of the protein from the source is crucial for it to achieve its morphogenic potential.

Eomes restricts Brachyury functions at the onset of mouse gastrulation.

Mammalian specification of mesoderm and definitive endoderm (DE) is instructed by the two related Tbx transcription factors (TFs) Eomesodermin (Eomes) and Brachyury sharing partially redundant functions. Gross differences in mutant embryonic phenotypes suggest specific functions of each TF. To date, the molecular details of separated lineage-specific gene regulation by Eomes and Brachyury remain poorly understood. Here, we combine mouse embryonic and stem-cell-based analyses to delineate the non-overlapping, lineage-specific transcriptional activities. On a genome-wide scale, binding of both TFs overlaps at promoters of target genes but shows specificity for distal enhancer regions that is conferred by differences in Tbx DNA-binding motifs. The unique binding to enhancer sites instructs the specification of anterior mesoderm (AM) and DE by Eomes and caudal mesoderm by Brachyury. Remarkably, EOMES antagonizes BRACHYURY gene regulatory functions in coexpressing cells during early gastrulation to ensure the proper sequence of early AM and DE lineage specification followed by posterior mesoderm derivatives.

The ever-growing world of gastruloids: autogenous models of mammalian embryogenesis.

During early development, extrinsic cues prompt a collection of pluripotent cells to begin the extensive process of cellular differentiation that gives rise to all tissues in the mammalian embryo, a process known as gastrulation. Advances in stem cell biology have resulted in the generation of stem cell-based in vitro models of mammalian gastrulation called gastruloids. Gastruloids and subsequent gastruloid-based models are tractable, scalable and more accessible than mammalian embryos. As such, they have opened an unprecedented avenue for modelling in vitro self-organisation, patterning and fate specification. This review focuses on discussing the recent advances of this rapidly moving research area, clarifying what structures they model and the underlying signal hierarchy. We highlight the exciting potential of these models and where the field might be heading.

Single-cell transcriptome atlas of Drosophila gastrula 2.0.

During development, positional information directs cells to specific fates, leading them to differentiate with their own transcriptomes and express specific behaviors and functions. However, the mechanisms underlying these processes in a genome-wide view remain ambiguous, partly because the single-cell transcriptomic data of early developing embryos containing accurate spatial and lineage information are still lacking. Here, we report a single-cell transcriptome atlas of Drosophila gastrulae, divided into 77 transcriptomically distinct clusters. We find that the expression profiles of plasma-membrane-related genes, but not those of transcription-factor genes, represent each germ layer, supporting the nonequivalent contribution of each transcription-factor mRNA level to effector gene expression profiles at the transcriptome level. We also reconstruct the spatial expression patterns of all genes at the single-cell stripe level as the smallest unit. This atlas is an important resource for the genome-wide understanding of the mechanisms by which genes cooperatively orchestrate Drosophila gastrulation.

An atlas of rabbit development as a model for single-cell comparative genomics.

Traditionally, the mouse has been the favoured vertebrate model for biomedical research, due to its experimental and genetic tractability. However, non-rodent embryological studies highlight that many aspects of early mouse development, such as its egg-cylinder gastrulation and method of implantation, diverge from other mammals, thus complicating inferences about human development. Like the human embryo, rabbits develop as a flat-bilaminar disc. Here we constructed a morphological and molecular atlas of rabbit development. We report transcriptional and chromatin accessibility profiles for over 180,000 single cells and high-resolution histology sections from embryos spanning gastrulation, implantation, amniogenesis and early organogenesis. Using a neighbourhood comparison pipeline, we compare the transcriptional landscape of rabbit and mouse at the scale of the entire organism. We characterize the gene regulatory programmes underlying trophoblast differentiation and identify signalling interactions involving the yolk sac mesothelium during haematopoiesis. We demonstrate how the combination of both rabbit and mouse atlases can be leveraged to extract new biological insights from sparse macaque and human data. The datasets and computational pipelines reported here set a framework for a broader cross-species approach to decipher early mammalian development, and are readily adaptable to deploy single-cell comparative genomics more broadly across biomedical research.

Epigenetic Control of Cell Potency and Fate Determination during Mammalian Gastrulation.

Pluripotent embryonic stem cells have a unique and characteristic epigenetic profile, which is critical for differentiation to all embryonic germ lineages. When stem cells exit the pluripotent state and commit to lineage-specific identities during the process of gastrulation in early embryogenesis, extensive epigenetic remodelling mediates both the switch in cellular programme and the loss of potential to adopt alternative lineage programmes. However, it remains to be understood how the stem cell epigenetic profile encodes pluripotency, or how dynamic epigenetic regulation helps to direct cell fate specification. Recent advances in stem cell culture techniques, cellular reprogramming, and single-cell technologies that can quantitatively profile epigenetic marks have led to significant insights into these questions, which are important for understanding both embryonic development and cell fate engineering. This review provides an overview of key concepts and highlights exciting new advances in the field.

The single-cell and spatial transcriptional landscape of human gastrulation and early brain development.

The emergence of the three germ layers and the lineage-specific precursor cells orchestrating organogenesis represent fundamental milestones during early embryonic development. We analyzed the transcriptional profiles of over 400,000 cells from 14 human samples collected from post-conceptional weeks (PCW) 3 to 12 to delineate the dynamic molecular and cellular landscape of early gastrulation and nervous system development. We described the diversification of cell types, the spatial patterning of neural tube cells, and the signaling pathways likely involved in transforming epiblast cells into neuroepithelial cells and then into radial glia. We resolved 24 clusters of radial glial cells along the neural tube and outlined differentiation trajectories for the main classes of neurons. Lastly, we identified conserved and distinctive features across species by comparing early embryonic single-cell transcriptomic profiles between humans and mice. This comprehensive atlas sheds light on the molecular mechanisms underlying gastrulation and early human brain development.

Regulation of anterior neurectoderm specification and differentiation by BMP signaling in ascidians.

The most anterior structure of the ascidian larva is made of three palps with sensory and adhesive functions essential for metamorphosis. They derive from the anterior neural border and their formation is regulated by FGF and Wnt. Given that they also share gene expression profiles with vertebrate anterior neural tissue and cranial placodes, their study should shed light on the emergence of the unique vertebrate telencephalon. We show that BMP signaling regulates two phases of palp formation in Ciona intestinalis. During gastrulation, the anterior neural border is specified in a domain of inactive BMP signaling, and activating BMP prevented its formation. During neurulation, BMP defines ventral palp identity and indirectly specifies the inter-papilla territory separating the ventral and dorsal palps. Finally, we show that BMP has similar functions in the ascidian Phallusia mammillata, for which we identified novel palp markers. Collectively, we provide a better molecular description of palp formation in ascidians that will be instrumental for comparative studies.

Base editing-mediated one-step inactivation of the Dnmt gene family reveals critical roles of DNA methylation during mouse gastrulation.

During embryo development, DNA methylation is established by DNMT3A/3B and subsequently maintained by DNMT1. While much research has been done in this field, the functional significance of DNA methylation in embryogenesis remains unknown. Here, we establish a system of simultaneous inactivation of multiple endogenous genes in zygotes through screening for base editors that can efficiently introduce a stop codon. Embryos with mutations in Dnmts and/or Tets can be generated in one step with IMGZ. Dnmt-null embryos display gastrulation failure at E7.5. Interestingly, although DNA methylation is absent, gastrulation-related pathways are down-regulated in Dnmt-null embryos. Moreover, DNMT1, DNMT3A, and DNMT3B are critical for gastrulation, and their functions are independent of TET proteins. Hypermethylation can be sustained by either DNMT1 or DNMT3A/3B at some promoters, which are related to the suppression of miRNAs. The introduction of a single mutant allele of six miRNAs and paternal IG-DMR partially restores primitive streak elongation in Dnmt-null embryos. Thus, our results unveil an epigenetic correlation between promoter methylation and suppression of miRNA expression for gastrulation and demonstrate that IMGZ can accelerate deciphering the functions of multiple genes in vivo.

Time-aligned hourglass gastrulation models in rabbit and mouse.

The hourglass model describes the convergence of species within the same phylum to a similar body plan during development; however, the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0 and 8.5 and compared the species using a framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell-state compositions at E7.5, supported by the quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages and divergence of primordial germ cell programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a basis for studying the evolution of gastrulation dynamics across mammals.

SRGP-1/srGAP and AFD-1/afadin stabilize HMP-1/⍺-catenin at rosettes to seal internalization sites following gastrulation in C. elegans.

A hallmark of gastrulation is the establishment of germ layers by internalization of cells initially on the exterior. In C. elegans the end of gastrulation is marked by the closure of the ventral cleft, a structure formed as cells internalize during gastrulation, and the subsequent rearrangement of adjacent neuroblasts that remain on the surface. We found that a nonsense allele of srgp-1/srGAP leads to 10-15% cleft closure failure. Deletion of the SRGP-1/srGAP C-terminal domain led to a comparable rate of cleft closure failure, whereas deletion of the N-terminal F-BAR region resulted in milder defects. Loss of the SRGP-1/srGAP C-terminus or F-BAR domain results in defects in rosette formation and defective clustering of HMP-1/⍺-catenin in surface cells during cleft closure. A mutant form of HMP-1/⍺-catenin with an open M domain can suppress cleft closure defects in srgp-1 mutant backgrounds, suggesting that this mutation acts as a gain-of-function allele. Since SRGP-1 binding to HMP-1/⍺-catenin is not favored in this case, we sought another HMP-1 interactor that might be recruited when HMP-1/⍺-catenin is constitutively open. A good candidate is AFD-1/afadin, which genetically interacts with cadherin-based adhesion later during embryonic elongation. AFD-1/afadin is prominently expressed at the vertex of neuroblast rosettes in wildtype, and depletion of AFD-1/afadin increases cleft closure defects in srgp-1/srGAP and hmp-1R551/554A/⍺-catenin backgrounds. We propose that SRGP-1/srGAP promotes nascent junction formation in rosettes; as junctions mature and sustain higher levels of tension, the M domain of HMP-1/⍺-catenin opens, allowing maturing junctions to transition from recruitment of SRGP-1/srGAP to AFD-1/afadin. Our work identifies new roles for ⍺-catenin interactors during a process crucial to metazoan development.

Defined Microenvironments Trigger In Vitro Gastrulation in Human Pluripotent Stem Cells.

Gastrulation is a stage in embryo development where three germ layers arise to dictate the human body plan. In vitro models of gastrulation have been demonstrated by treating pluripotent stem cells with soluble morphogens to trigger differentiation. However, in vivo gastrulation is a multistage process coordinated through feedback between soluble gradients and biophysical forces, with the multipotent epiblast transforming to the primitive streak followed by germ layer segregation. Here, the authors show how constraining pluripotent stem cells to hydrogel islands triggers morphogenesis that mirrors the stages preceding in vivo gastrulation, without the need for exogenous supplements. Within hours of initial seeding, cells display a contractile phenotype at the boundary, which leads to enhanced proliferation, yes-associated protein (YAP) translocation, epithelial to mesenchymal transition, and emergence of SRY-box transcription factor 17 (SOX17)+ T/BRACHYURY+ cells. Molecular profiling and pathway analysis reveals a role for mechanotransduction-coupled wingless-type (WNT) signaling in orchestrating differentiation, which bears similarities to processes observed in whole organism models of development. After two days, the colonies form multilayered aggregates, which can be removed for further growth and differentiation. This approach demonstrates how materials alone can initiate gastrulation, thereby providing in vitro models of development and a tool to support organoid bioengineering efforts.