Trans-omic profiling uncovers molecular controls of early human cerebral organoid formation.

Defining the molecular networks orchestrating human brain formation is crucial for understanding neurodevelopment and neurological disorders. Challenges in acquiring early brain tissue have incentivized the use of three-dimensional human pluripotent stem cell (hPSC)-derived neural organoids to recapitulate neurodevelopment. To elucidate the molecular programs that drive this highly dynamic process, here, we generate a comprehensive trans-omic map of the phosphoproteome, proteome, and transcriptome of the exit of pluripotency and neural differentiation toward human cerebral organoids (hCOs). These data reveal key phospho-signaling events and their convergence on transcriptional factors to regulate hCO formation. Comparative analysis with developing human and mouse embryos demonstrates the fidelity of our hCOs in modeling embryonic brain development. Finally, we demonstrate that biochemical modulation of AKT signaling can control hCO differentiation. Together, our data provide a comprehensive resource to study molecular controls in human embryonic brain development and provide a guide for the future development of hCO differentiation protocols.

Wnt dose escalation during the exit from pluripotency identifies tranilast as a regulator of cardiac mesoderm.

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation.

Phenotypic Analysis of Early Neurogenesis in a Mouse Chimeric Embryo and Stem Cell-Based Neuruloid Model.

Analyzing the impact of genetic mutations on early neurogenesis of mammalian embryos in conventional mouse mutant models is laborious and time-consuming. To overcome these constraints and to fast-track the phenotypic analysis, we developed a protocol that harnesses the amenability of engineering genetic modifications in embryonic stem cells from which mid-gestation mouse chimeras and in vitro neuruloids are generated. These stem cell-based chimera and neuruloid experimental models allow phenotyping at early developmental time points of neurogenesis.

Rat epiblast-derived stem cells recapitulate the attributes of pre-gastrulation epiblast.

Iwatsuki and colleagues have generated self-renewing pluripotent stem cells from the pre-gastrulation epiblast of the rat embryo and from other cellular sources: rat embryonic stem cells (rESCs) and epiblast-like cells derived from the rESCs. These rat epiblast-derived stem cells (rEpiSCs) display germ-line competence that is characteristic of mouse formative stem cells and early signature of specification of germ layer lineages typical of primed state mouse epiblast stem cells.

Time space and single-cell resolved tissue lineage trajectories and laterality of body plan at gastrulation.

Understanding of the molecular drivers of lineage diversification and tissue patterning during primary germ layer development requires in-depth knowledge of the dynamic molecular trajectories of cell lineages across a series of developmental stages of gastrulation. Through computational modeling, we constructed at single-cell resolution, a spatio-temporal transcriptome of cell populations in the germ-layers of gastrula-stage mouse embryos. This molecular atlas enables the inference of molecular network activity underpinning the specification and differentiation of the germ-layer tissue lineages. Heterogeneity analysis of cellular composition at defined positions in the epiblast revealed progressive diversification of cell types. The single-cell transcriptome revealed an enhanced BMP signaling activity in the right-side mesoderm of late-gastrulation embryo. Perturbation of asymmetric BMP signaling activity at late gastrulation led to randomization of left-right molecular asymmetry in the lateral mesoderm of early-somite-stage embryo. These findings indicate the asymmetric BMP activity during gastrulation may be critical for the symmetry breaking process.

Technical challenges of studying early human development.

Recent years have seen exciting progress across human embryo research, including new methods for culturing embryos, transcriptional profiling of embryogenesis and gastrulation, mapping lineage trajectories, and experimenting on stem cell-based embryo models. These advances are beginning to define the dynamical principles of development across stages, tissues and organs, enabling a better understanding of human development before birth in health and disease, and potentially leading to improved treatments for infertility and developmental disorders. However, there are still significant roadblocks en route to this goal. Here, we highlight technical challenges to studying early human development and propose ways and means to overcome some of these constraints.

Programming of Multicellular Patterning with Mechano-Chemically Microstructured Cell Niches.

Multicellular patterning of stem-cell-derived tissue models is commonly achieved via self-organizing activities triggered by exogenous morphogenetic stimuli. However, such tissue models are prone to stochastic behavior, limiting the reproducibility of cellular composition and forming non-physiological architectures. To enhance multicellular patterning in stem cell-derived tissues, a method for creating complex tissue microenvironments endowed with programmable multimodal mechano-chemical cues, including conjugated peptides, proteins, morphogens, and Young's moduli defined over a range of stiffnesses is developed. The ability of these cues to spatially guide tissue patterning processes, including mechanosensing and the biochemically driven differentiation of selected cell types, is demonstrated. By rationally designing niches, the authors engineered a bone-fat assembly from stromal mesenchyme cells and regionalized germ layer tissues from pluripotent stem cells. Through defined niche-material interactions, mechano-chemically microstructured niches enable the spatial programming of tissue patterning processes. Mechano-chemically microstructured cell niches thereby offer an entry point for enhancing the organization and composition of engineered tissues, potentiating structures that better recapitulate their native counterparts.

Integration of Computational Analysis and Spatial Transcriptomics in Single-cell Studies.

Recent advances of single-cell transcriptomics technologies and allied computational methodologies have revolutionized molecular cell biology. Meanwhile, pioneering explorations in spatial transcriptomics have opened up avenues to address fundamental biological questions in health and diseases. Here, we review the technical attributes of single-cell RNA sequencing and spatial transcriptomics, and the core concepts of computational data analysis. We further highlight the challenges in the application of data integration methodologies and the interpretation of the biological context of the findings.

Mammalian gastrulation: signalling activity and transcriptional regulation of cell lineage differentiation and germ layer formation.

The interplay of signalling input and downstream transcriptional activity is the key molecular attribute driving the differentiation of germ layer tissue and the specification of cell lineages within each germ layer during gastrulation. This review delves into the current understanding of signalling and transcriptional control of lineage development in the germ layers of mouse embryo and non-human primate embryos during gastrulation and highlights the inter-species conservation and divergence of the cellular and molecular mechanisms of germ layer development in the human embryo.

Spatial molecular anatomy of germ layers in the gastrulating cynomolgus monkey embryo.

During mammalian embryogenesis, spatial regulation of gene expression and cell signaling are functionally coupled with lineage specification, patterning of tissue progenitors, and germ layer morphogenesis. While the mouse model has been instrumental for understanding mammalian development, comparatively little is known about human and non-human primate gastrulation due to the restriction of both technical and ethical issues. Here, we present a spatial and temporal survey of the molecular dynamics of cell types populating the non-human primate embryos during gastrulation. We reconstructed three-dimensional digital models from serial sections of cynomolgus monkey (Macaca fascicularis) gastrulating embryos at 1-day temporal resolution from E17 to E21. Spatial transcriptomics identifies gene expression profiles unique to the germ layers. Cross-species comparison reveals a developmental coordinate of germ layer segregation between mouse and primates, and species-specific transcription programs during gastrulation. These findings offer insights into evolutionarily conserved and divergent processes during mammalian gastrulation.

Mouse organogenesis atlas at single-cell resolution.

The generation of spatial transcriptomes of whole embryo has been limited in scale and resolution due to various technological restrictions. In this issue of Cell, Chen et al. introduce a DNA nanoball-based sample-capture technology for spatial transcriptome analysis to generate a molecular atlas of mouse organogenesis at single-cell resolution.

Identification and Visualization of Protein Expression in Whole Mouse Embryos by Immunofluorescence.

Mouse embryo studies are pivotal for the understanding of early development. Analysis of the spatial and temporal changes of protein expression during development of a mouse embryo allows us to identify the genetic basis of errors of development in animal disease models. Immunofluorescence is a powerful technique to study the localization and variation in expression pattern of specific proteins in cells, tissues, and organs. Detecting the antigens with their specific antibodies labeled with fluorescent probes allows visualization of proteins at the cellular level. Here, we provide the optimized protocol of immunostaining whole mouse embryos at embryonic stages E7.5 to E11.5.

Exploring Chromatin Accessibility in Mouse Epiblast Stem Cells with ATAC-Seq.

The assay for transposase-accessible chromatin using sequencing (ATAC-seq) is used to identify open chromatin regions in cells. This can be used to identify putative regulatory regions, determine dynamics and mechanisms of transcription factors when coupled with ChIP-seq and predict interactions between proteins and chromatin. Compared to previous methods, MNase-seq and DNase-seq, ATAC-seq requires only 50,000 cells, orders of magnitude fewer cells. In addition, the ATAC-seq protocol takes one day to progress from cells to sequencing ready libraries.

Grafting of Epiblast Stem Cell into the Epiblast and Whole-Embryo Imaging to Unveil Lineage Competence.

Here we describe a method to engraft epiblast stem cells (EpiSC) into the epiblast of gastrulation-stage mouse embryo to test the lineage propensity acquired by the EpiSCs during in vitro culture under different signaling conditions. After dissection and grafting, the recipient embryos can be grown in whole-embryo culture for up to 48 h and the contribution of the EpiSC-derived cells to tissues in the recipient embryo is assessed by light sheet 3D microscopy.

Differential impact of TGFß/SMAD signaling activity elicited by Activin A and Nodal on endoderm differentiation of epiblast stem cells.

Allocation of cells to an endodermal fate in the gastrulating embryo is driven by Nodal signaling and consequent activation of TGFß pathway. In vitro methodologies striving to recapitulate the process of endoderm differentiation, however, use TGFß family member Activin in place of Nodal. This is despite Activin not known to have an in vivo role in endoderm differentiation. In this study, five epiblast stem cell lines were subjected to directed differentiation using both Activin A and Nodal to induce endodermal fate. A reporter line harboring endoderm markers FoxA2 and Sox17 was further analyzed for TGFß pathway activation and WNT response. We demonstrated that Activin A-treated cells remain more primitive streak-like when compared to Nodal-treated cells that have a molecular profile suggestive of more advanced differentiation. Activin A elicited a robust TGFß/SMAD activity, enhanced WNT signaling activity and promoted the generation of DE precursors. Nodal treatment resulted in lower TGFß/SMAD activity, and a weaker, sustained WNT response, and ultimately failed to upregulate endoderm markers. This is despite signaling response resembling more closely the activity seen in vivo. These findings emphasize the importance of understanding the downstream activities of Activin A and Nodal signaling in directing in vitro endoderm differentiation of primed-state epiblast stem cells.

Early human embryonic development: Blastocyst formation to gastrulation.

There has been recent renewed interest in studying human early embryonic development. The advent of improved culture conditions to maintain blastocysts in vitro for an extended period and the emerging stem-cell-based models of the blastocyst and peri-implantation embryos have provided new information that is relevant to early human embryogenesis. However, the mechanism of lineage development and embryonic patterning, and the molecular pathways involved in their regulation, are still not well understood. Interest in human embryonic development has been reinvigorated recently given numerous technical advances. In this review, Rossant and Tam discuss new insights into human embryogenesis gathered from successes in culturing human embryos in vitro and stem-cell-based embryo models. Then they outline what questions still need answering.

Elucidation of Gene Expression Patterns in the Craniofacial Tissues of Mouse Embryos by Wholemount In Situ Hybridization.

Analysis of animal models allows a deeper understanding of craniofacial development in health and diseases of humans. Wholemount in situ hybridization (WISH) is an informative technique to visualize gene expression in tissues across the developmental stages of embryos. The principle of WISH is based on the complementary binding (hybridization) of the DNA/RNA probe to the target transcript. The bound probe can then be visualized by an enzymatic color reaction to delineate the expression pattern of transcripts within a tissue. Here we describe an optimized method to perform in situ hybridization in mouse embryos.

Visualization of the Cartilage and Bone Elements in the Craniofacial Structures by Alcian Blue and Alizarin Red Staining.

Craniofacial morphogenesis is underpinned by orchestrated growth and form-shaping activity of skeletal and soft tissues in the head and face. Disruptions during development can lead to dysmorphology of the skull, jaw, and the pharyngeal structures. Developmental disorders can be investigated in animal models to elucidate the molecular and cellular consequences of the morphogenetic defects. A first step in determining the disruption in the development of the head and face is to analyze the phenotypic features of the skeletal tissues. Examination of the anatomy of bones and cartilage over time and space will identify structural defects of head structures and guide follow-up analysis of the molecular and cellular attributes associated with the defects. Here we describe a protocol to simultaneously visualize the cartilage and bone elements by Alcian blue and Alizarin red staining, respectively, of wholemount specimens in mouse models.

A cis-regulatory-directed pipeline for the identification of genes involved in cardiac development and disease.

BACKGROUND: Congenital heart diseases are the major cause of death in newborns, but the genetic etiology of this developmental disorder is not fully known. The conventional approach to identify the disease-causing genes focuses on screening genes that display heart-specific expression during development. However, this approach would have discounted genes that are expressed widely in other tissues but may play critical roles in heart development. RESULTS: We report an efficient pipeline of genome-wide gene discovery based on the identification of a cardiac-specific cis-regulatory element signature that points to candidate genes involved in heart development and congenital heart disease. With this pipeline, we retrieve 76% of the known cardiac developmental genes and predict 35 novel genes that previously had no known connectivity to heart development. Functional validation of these novel cardiac genes by RNAi-mediated knockdown of the conserved orthologs in Drosophila cardiac tissue reveals that disrupting the activity of 71% of these genes leads to adult mortality. Among these genes, RpL14, RpS24, and Rpn8 are associated with heart phenotypes. CONCLUSIONS: Our pipeline has enabled the discovery of novel genes with roles in heart development. This workflow, which relies on screening for non-coding cis-regulatory signatures, is amenable for identifying developmental and disease genes for an organ without constraining to genes that are expressed exclusively in the organ of interest.