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In the companion Perspective 'Past and future of human developmental biology' (Hopwood, 2024), historian Nick Hopwood proposes that the field of human developmental biology has gone through periods of attention and neglect. Development invited researchers from the field to respond to this idea. In this article, published to coincide with the 10th anniversary of Development's 'From Stem Cells to Human Development' meeting, researchers from eight countries comment on how they believe their local legal, political, regulatory, societal and technological frameworks are influencing the field's trajectory., Competing Interests: Competing interests A.T.C. is past-president and board member of the International Society for Stem Cell Research. J.H.H. was granted (through the Yeda–Weizmann Institute of Science) patents relevant to the findings and technologies discussed here (naive and naive-like pluripotency and mouse and human bona fide synthetic whole embryo models). J.H.H. is a co-founder and chief scientific advisor of Renewal Bio, which has licensed technologies mentioned above., (© 2024. Published by The Company of Biologists Ltd.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023. Springer Nature Limited.)

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., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

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., Competing Interests: Competing interests The authors declare no competing interests., (© 2023. Published by The Company of Biologists Ltd.)

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., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

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., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Allocation of cells to an endodermal fate in the gastrulating embryo is driven by Nodal signaling and consequent activation of TGFβ pathway. In vitro methodologies striving to recapitulate the process of endoderm differentiation, however, use TGFβ 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 TGFβ 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 TGFβ/SMAD activity, enhanced WNT signaling activity and promoted the generation of DE precursors. Nodal treatment resulted in lower TGFβ/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., (© 2022 Wiley Periodicals LLC.)

The specification of anterior head tissue in the late gastrulation mouse embryo relies on signaling cues from the visceral endoderm and anterior mesendoderm (AME). Genetic loss-of-function studies have pinpointed a critical requirement of LIM homeobox 1 (LHX1) transcription factor in these tissues for the formation of the embryonic head. Transcriptome analysis of embryos with gain-of-function LHX1 activity identified the forkhead box gene, Foxd4, as one downstream target of LHX1 in late-gastrulation E7.75 embryos. Our analysis of single-cell RNA-seq data show Foxd4 is co-expressed with Lhx1 and Foxa2 in the anterior midline tissue of E7.75 mouse embryos, and in the anterior neuroectoderm (ANE) at E8.25 alongside head organizer genes Otx2 and Hesx1 . To study the role of Foxd4 during early development we used CRISPR-Cas9 gene editing in mouse embryonic stem cells (mESCs) to generate bi-allelic frameshift mutations in the coding sequence of Foxd4 . In an in vitro model of the anterior neural tissues derived from Foxd4 -loss of function (LOF) mESCs and extraembryonic endoderm cells, expression of head organizer genes as well as Zic1 and Zic2 was reduced, pointing to a need for FOXD4 in regulating early neuroectoderm development. Mid-gestation mouse chimeras harbouring Foxd4 -LOF mESCs displayed craniofacial malformations and neural tube closure defects. Furthermore, our in vitro data showed a loss of FOXD4 impacts the expression of cranial neural crest markers Twist1 and Sox9 . Our findings have demonstrated that FOXD4 is essential in the AME and later in the ANE for rostral neural tube closure and neural crest specification during head development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 McMahon, Sibbritt, Aryamanesh, Masamsetti and Tam.)

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., Competing Interests: Declaration of interests J.R. and P.P.L.T. are members of the Advisory Board of Developmental Cell., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2021. The Author(s).)

The use of single-cell RNA-sequencing (scRNA-seq) allows observation of different cells at multi-tiered complexity in the same microenvironment. To get insights into cell identity using scRNA-seq data, we present Cepo, which generates cell-type-specific gene statistics of differentially stable genes from scRNA-seq data to define cell identity. When applied to multiple datasets, Cepo outperforms current methods in assigning cell identity and enhances several cell identification applications such as cell-type characterisation, spatial mapping of single cells and lineage inference of single cells., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Rett Syndrome (RTT) is an X linked neurodevelopmental disorder caused by mutations in the methyl-CpG-binding protein 2 ( MECP2 ) gene, resulting in severe cognitive and physical disabilities. Despite an apparent normal prenatal and postnatal development period, symptoms usually present around 6 to 18 months of age. Little is known about the consequences of MeCP2 deficiency at a molecular and cellular level before the onset of symptoms in neural cells, and subtle changes at this highly sensitive developmental stage may begin earlier than symptomatic manifestation. Recent transcriptomic studies of patient induced pluripotent stem cells (iPSC)-differentiated neurons and brain organoids harbouring pathogenic mutations in MECP2, have unravelled new insights into the cellular and molecular changes caused by these mutations. Here we interrogated transcriptomic modifications in RTT patients using publicly available RNA-sequencing datasets of patient iPSCs harbouring pathogenic mutations and healthy control iPSCs by Weighted Gene Correlation Network Analysis (WGCNA). Preservation analysis identified core gene pathways involved in translation, ribosomal function, and ubiquitination perturbed in some MECP2 mutant iPSC lines. Furthermore, differential gene expression of the parental fibroblasts and iPSC-derived neurons revealed alterations in genes in the ubiquitination pathway and neurotransmission in fibroblasts and differentiated neurons respectively. These findings might suggest that global translational dysregulation and proteasome ubiquitin function in Rett syndrome begins in progenitor cells prior to lineage commitment and differentiation into neural cells.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder, mostly caused by mutations in MECP2 . The disorder mainly affects girls and it is associated with severe cognitive and physical disabilities. Modeling RTT in neural and glial cell cultures and brain organoids derived from patient- or mutation-specific human induced pluripotent stem cells (iPSCs) has advanced our understanding of the pathogenesis of RTT, such as disease-causing mechanisms, disease progression, and cellular and molecular pathology enabling the identification of actionable therapeutic targets. Brain organoid models that recapitulate much of the tissue architecture and the complexity of cell types in the developing brain, offer further unprecedented opportunity for elucidating human neural development, without resorting to conventional animal models and the limited resource of human neural tissues. This review focuses on the new knowledge of RTT that has been gleaned from the iPSC-based models as well as limitations of the models and strategies to refine organoid technology in the quest for clinically relevant disease models for RTT and the broader spectrum of neurodevelopmental disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Haase, Coorey, Riley, Cantrill, Tam and Gold.)

Stem cell-based embryo models open an unprecedented avenue for modeling embryogenesis, cell lineage differentiation, tissue morphogenesis, and organogenesis in mammalian development. Experimentation on these embryo models can lead to a better understanding of the mechanisms of development and offers opportunities for functional genomic studies of disease-causing mechanisms, identification of therapeutic targets, and preclinical modeling of advanced therapeutics for precision medicine. An immediate challenge is to create embryo models of high fidelity to embryogenesis and organogenesis in vivo, to ensure that the knowledge gleaned is biologically meaningful and clinically relevant., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Determining genes that orchestrate cell differentiation in development and disease remains a fundamental goal of cell biology. This study establishes a genome-wide metric based on the gene-repressive trimethylation of histone H3 at lysine 27 (H3K27me3) across hundreds of diverse cell types to identify genetic regulators of cell differentiation. We introduce a computational method, TRIAGE, which uses discordance between gene-repressive tendency and expression to identify genetic drivers of cell identity. We apply TRIAGE to millions of genome-wide single-cell transcriptomes, diverse omics platforms, and eukaryotic cells and tissue types. Using a wide range of data, we validate the performance of TRIAGE in identifying cell-type-specific regulatory factors across diverse species including human, mouse, boar, bird, fish, and tunicate. Using CRISPR gene editing, we use TRIAGE to experimentally validate RNF220 as a regulator of Ciona cardiopharyngeal development and SIX3 as required for differentiation of endoderm in human pluripotent stem cells. A record of this paper's transparent peer review process is included in the Supplemental Information., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The extensive array of basic helix-loop-helix (bHLH) transcription factors and their combinations as dimers underpin the diversity of molecular function required for cell type specification during embryogenesis. The bHLH factor TWIST1 plays pleiotropic roles during development. However, which combinations of TWIST1 dimers are involved and what impact each dimer imposes on the gene regulation network controlled by TWIST1 remain elusive. In this work, proteomic profiling of human TWIST1-expressing cell lines and transcriptome analysis of mouse cranial mesenchyme have revealed that TWIST1 homodimers and heterodimers with TCF3, TCF4, and TCF12 E-proteins are the predominant dimer combinations. Disease-causing mutations in TWIST1 can impact dimer formation or shift the balance of different types of TWIST1 dimers in the cell, which may underpin the defective differentiation of the craniofacial mesenchyme. Functional analyses of the loss and gain of TWIST1-E-protein dimer activity have revealed previously unappreciated roles in guiding lineage differentiation of embryonic stem cells: TWIST1-E-protein heterodimers activate the differentiation of mesoderm and neural crest cells, which is accompanied by the epithelial-to-mesenchymal transition. At the same time, TWIST1 homodimers maintain the stem cells in a progenitor state and block entry to the endoderm lineage., (Copyright © 2020 American Society for Microbiology.)

The developmental potential of cells, termed pluripotency, is highly dynamic and progresses through a continuum of naive, formative and primed states. Pluripotency progression of mouse embryonic stem cells (ESCs) from naive to formative and primed state is governed by transcription factors (TFs) and their target genes. Genomic techniques have uncovered a multitude of TF binding sites in ESCs, yet a major challenge lies in identifying target genes from functional binding sites and reconstructing dynamic transcriptional networks underlying pluripotency progression. Here, we integrated time-resolved 'trans-omic' datasets together with TF binding profiles and chromatin conformation data to identify target genes of a panel of TFs. Our analyses revealed that naive TF target genes are more likely to be TFs themselves than those of formative TFs, suggesting denser hierarchies among naive TFs. We also discovered that formative TF target genes are marked by permissive epigenomic signatures in the naive state, indicating that they are poised for expression prior to the initiation of pluripotency transition to the formative state. Finally, our reconstructed transcriptional networks pinpointed the precise timing from naive to formative pluripotency progression and enabled the spatiotemporal mapping of differentiating ESCs to their in vivo counterparts in developing embryos., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Assays for transposase-accessible chromatin sequencing (ATAC-seq) provides an innovative approach to study chromatin status in multiple cell types. Moreover, it is also possible to efficiently extract differentially accessible chromatin (DACs) regions by using state-of-the-art algorithms (e.g. DESeq2) to predict gene activity in specific samples. Furthermore, it has recently been shown that small dips in sequencing peaks can be attributed to the binding of transcription factors. These dips, also known as footprints, can be used to identify trans-regulating interactions leading to gene expression. Current protocols used to identify footprints (e.g. pyDNAse and HINT-ATAC) have shown limitations resulting in the discovery of many false positive footprints. We generated a novel approach to identify genuine footprints within any given ATAC-seq dataset. Herein, we developed a new pipeline embedding DACs together with bona fide footprints resulting in the generation of a Pre dictive gene regulatory Net work (PreNet) simply from ATAC-seq data. We further demonstrated that PreNet can be used to unveil meaningful molecular regulatory pathways in a given cell type.

Single cell RNA-sequencing (scRNA-seq) technology has matured to the point that it is possible to generate large single cell atlases of developing mouse embryos. These atlases allow the dissection of developmental cell lineages and molecular changes during embryogenesis. When coupled with single cell technologies for profiling the chromatin landscape, epigenome, proteome and metabolome, and spatial tissue organisation, these scRNA-seq approaches can now collect a large volume of multi-omic data about mouse embryogenesis. In addition, advances in computational techniques have enabled the inference of developmental lineages of differentiating cells, even without explicitly introduced genetic markers. This Spotlight discusses recent advent of single cell experimental and computational methods, and key insights from applying these methods to the study of mouse embryonic development. We highlight challenges in analysing and interpreting these data to complement and expand our knowledge from traditional developmental biology studies in relation to cell identity, diversity and lineage differentiation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Circular RNAs (circRNA) are a unique class of transcripts that can only be identified from sequence alignments spanning discordant junctions, commonly referred to as backsplice junctions (BSJ). Canonical splicing is also linked with circRNA biogenesis either from the parental transcript or internal to the circRNA, and is not fully utilized in circRNA software. Here we present Ularcirc, a software tool that integrates the visualization of both BSJ and forward splicing junctions and provides downstream analysis of selected circRNA candidates. Ularcirc utilizes the output of CIRI, circExplorer, or raw chimeric output of the STAR aligner and assembles BSJ count table to allow multi-sample analysis. We used Ularcirc to identify and characterize circRNA from public and in-house generated data sets and demonstrate how it can be used to (i) discover novel splicing patterns of parental transcripts, (ii) detect internal splicing patterns of circRNA, and (iii) reveal the complexity of BSJ formation. Furthermore, we identify circRNA that have potential open reading frames longer than their linear sequence. Finally, we detected and validated the presence of a novel class of circRNA generated from ApoA4 transcripts whose BSJ derive from multiple non-canonical splicing sites within coding exons. Ularcirc is accessed via https://github.com/VCCRI/Ularcirc., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 state 1 . 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 gastrulation 2-5 . 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 embryos 6-8 . 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.

Development of an embryo is driven by a series of molecular instructions that control the differentiation of tissue precursor cells and shape the tissues into major body parts. LIM homeobox 1 (LHX1) is a transcription factor that plays a major role in the development of the embryonic head of the mouse. Loss of LHX1 function disrupts the morphogenetic movement of head tissue precursors and impacts on the function of molecular factors in modulating the activity of the WNT signaling pathway. LHX1 acts with a transcription factor complex to regulate the transcription of target genes in multiple phases of development and in a range of embryonic tissues of the mouse and Xenopus. Determining the interacting factors and transcriptional targets of LHX1 will be key to unraveling the ensemble of factors involved in head development and building a head gene regulatory network., (© 2019 Japanese Society of Developmental Biologists.)

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Following implantation, the epiblast (EPI) cells transit from the naive to primed pluripotency, accompanied by dynamic changes in X chromosome activity in females. To investigate the molecular attributes of this process, we performed single-cell RNA-seq analysis of 1,724 cells of E5.25, E5.5, E6.25, and E6.5 mouse embryos. We identified three cellular states in the EPI cells that capture the transition along the pluripotency continuum and the acquisition of primitive streak propensity. The transition of three EPI states was driven by inductive signaling activity emanating from the visceral endoderm (VE). In the EPI of female embryos, X chromosome reactivation (XCR) was initiated prior to the completion of imprinted X chromosome inactivation (XCI), and the ensuing random XCI was highly asynchronous. Moreover, imprinted paternal XCI proceeded faster in the VE than the extraembryonic ectoderm. Our study has provided a detailed molecular roadmap of the emergent lineage commitment before gastrulation and characterized X chromosome dynamics during early mouse development., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Efficient and reliable methods for gene editing are critical for the generation of loss-of-gene function stem cells and genetically modified mice. Here, we outline the application of CRISPR-Cas9 technology for gene editing in mouse embryonic stem cells (mESCs) to generate knockout ESC chimeras for the fast-tracked analysis of gene function. Furthermore, we describe the application of gene editing directly to mouse epiblast stem cells (mEpiSCs) for modelling germ layer differentiation in vitro.

Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (© 2018 Japanese Society of Developmental Biologists.)