Axin1 and Axin2 regulate the WNT-signaling landscape to promote distinct mesoderm programs.

How distinct mesodermal lineages - extraembryonic, lateral, intermediate, paraxial and axial - are specified from pluripotent epiblast during gastrulation is a longstanding open question. By investigating AXIN, a negative regulator of the WNT/ß-catenin pathway, we have uncovered new roles for WNT signaling in the determination of mesodermal fates. We undertook complementary approaches to dissect the role of WNT signaling that augmented a detailed analysis of Axin1;Axin2 mutant mouse embryos, including single-cell and single-embryo transcriptomics, with in vitro pluripotent Epiblast-Like Cell differentiation assays. This strategy allowed us to reveal two layers of regulation. First, WNT initiates differentiation of primitive streak cells into mesoderm progenitors, and thereafter, WNT amplifies and cooperates with BMP/pSMAD1/5/9 or NODAL/pSMAD2/3 to propel differentiating mesoderm progenitors into either posterior streak derivatives or anterior streak derivatives, respectively. We propose that Axin1 and Axin2 prevent aberrant differentiation of pluripotent epiblast cells into mesoderm by spatially and temporally regulating WNT signaling levels.

Single-cell analysis of bidirectional reprogramming between early embryonic states reveals mechanisms of differential lineage plasticities.

Two distinct fates, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common progenitor cells, the inner cell mass (ICM), in mammalian embryos. To study how these sister identities are forged, we leveraged embryonic (ES) and eXtraembryonic ENdoderm (XEN) stem cells - in vitro counterparts of the EPI and PrE. Bidirectional reprogramming between ES and XEN coupled with single-cell RNA and ATAC-seq analyses uncovered distinct rates, efficiencies and trajectories of state conversions, identifying drivers and roadblocks of reciprocal conversions. While GATA4-mediated ES-to-iXEN conversion was rapid and nearly deterministic, OCT4, KLF4 and SOX2-induced XEN-to-iPS reprogramming progressed with diminished efficiency and kinetics. The dominant PrE transcriptional program, safeguarded by Gata4, and globally elevated chromatin accessibility of EPI underscored the differential plasticities of the two states. Mapping in vitro trajectories to embryos revealed reprogramming in either direction tracked along, and toggled between, EPI and PrE in vivo states without transitioning through the ICM.

Single-Cell Transcriptomics Reveals Early Emergence of Liver Parenchymal and Non-parenchymal Cell Lineages.

The cellular complexity and scale of the early liver have constrained analyses examining its emergence during organogenesis. To circumvent these issues, we analyzed 45,334 single-cell transcriptomes from embryonic day (E)7.5, when endoderm progenitors are specified, to E10.5 liver, when liver parenchymal and non-parenchymal cell lineages emerge. Our data detail divergence of vascular and sinusoidal endothelia, including a distinct transcriptional profile for sinusoidal endothelial specification by E8.75. We characterize two distinct mesothelial cell types as well as early hepatic stellate cells and reveal distinct spatiotemporal distributions for these populations. We capture transcriptional profiles for hepatoblast specification and migration, including the emergence of a hepatomesenchymal cell type and evidence for hepatoblast collective cell migration. Further, we identify cell-cell interactions during the organization of the primitive sinusoid. This study provides a comprehensive atlas of liver lineage establishment from the endoderm and mesoderm through to the organization of the primitive sinusoid at single-cell resolution.

Guts and gastrulation: Emergence and convergence of endoderm in the mouse embryo.

Gastrulation is a central process in mammalian development in which a spatiotemporally coordinated series of events driven by cross-talk between adjacent embryonic and extra-embryonic tissues results in stereotypical morphogenetic cell behaviors, massive cell proliferation and the acquisition of distinct cell identities. Gastrulation provides the blueprint of the body plan of the embryo, as well as generating extra-embryonic cell types of the embryo to make a connection with its mother. Gastrulation involves the specification of mesoderm and definitive endoderm from pluripotent epiblast, concomitant with a highly ordered elongation of tissue along the anterior-posterior (AP) axis. Interestingly, cells with an endoderm identity arise twice during mouse development. Cells with a primitive endoderm identity are specified in the preimplantation blastocyst, and which at gastrulation intercalate with the emergent definitive endoderm to form a mosaic tissue, referred to as the gut endoderm. The gut endoderm gives rise to the gut tube, which will subsequently become patterned along its AP axis into domains possessing unique visceral organ identities, such as thyroid, lung, liver and pancreas. In this way, proper endoderm development is essential for vital organismal functions, including the absorption of nutrients, gas exchange, detoxification and glucose homeostasis.

The endoderm: a divergent cell lineage with many commonalities.

The endoderm is a progenitor tissue that, in humans, gives rise to the majority of internal organs. Over the past few decades, genetic studies have identified many of the upstream signals specifying endoderm identity in different model systems, revealing them to be divergent from invertebrates to vertebrates. However, more recent studies of the cell behaviours driving endodermal morphogenesis have revealed a surprising number of shared features, including cells undergoing epithelial-to-mesenchymal transitions (EMTs), collective cell migration, and mesenchymal-to-epithelial transitions (METs). In this Review, we highlight how cross-organismal studies of endoderm morphogenesis provide a useful perspective that can move our understanding of this fascinating tissue forward.

The emergent landscape of the mouse gut endoderm at single-cell resolution.

Here we delineate the ontogeny of the mammalian endoderm by generating 112,217 single-cell transcriptomes, which represent all endoderm populations within the mouse embryo until midgestation. We use graph-based approaches to model differentiating cells, which provides a spatio-temporal characterization of developmental trajectories and defines the transcriptional architecture that accompanies the emergence of the first (primitive or extra-embryonic) endodermal population and its sister pluripotent (embryonic) epiblast lineage. We uncover a relationship between descendants of these two lineages, in which epiblast cells differentiate into endoderm at two distinct time points-before and during gastrulation. Trajectories of endoderm cells were mapped as they acquired embryonic versus extra-embryonic fates and as they spatially converged within the nascent gut endoderm, which revealed these cells to be globally similar but retain aspects of their lineage history. We observed the regionalized identity of cells along the anterior-posterior axis of the emergent gut tube, which reflects their embryonic or extra-embryonic origin, and the coordinated patterning of these cells into organ-specific territories.

Ex Utero Culture and Imaging of Mouse Embryos.

Mouse genetic approaches when combined with live imaging tools are revolutionizing our current understanding of mammalian developmental biology. The availability and improvement of a wide variety of genetically encoded fluorescent proteins have provided indispensable tools to visualize cells and subcellular features in living organisms. It is now possible to generate genetically modified mouse lines expressing several spectrally distinct fluorescent proteins in a tissue-specific or -inducible manner. Such reporter-expressing lines make it possible to image dynamic cellular behaviors in the context of living embryos undergoing normal or aberrant development. As with all viviparous mammals, mouse embryos develop within the uterus, and so live imaging experiments require culture conditions that closely mimic the in vivo environment. Over the past decades, significant advances have been made in developing conditions for culturing both pre- and postimplantation-stage mouse embryos. In this chapter, we discuss routine methods for ex utero culture of preimplantation- and postimplantation-stage mouse embryos. In particular, we describe protocols for collecting mouse embryos of various stages, setting up culture conditions for their ex utero culture and imaging, and using laser scanning confocal microscopy to visualize live processes in mouse embryos expressing fluorescent reporters.

Lights, Camera, Action! Visualizing the Cellular Choreography of Mouse Gastrulation.

When it comes to live imaging, the mouse has always played catch-up with models like the zebrafish or fruit fly. Recent work reports a technical tour de force toward the in toto visualization of mouse early post-implantation embryo development at an unprecedented spatio-temporal resolution.

A Gata4 nuclear GFP transcriptional reporter to study endoderm and cardiac development in the mouse.

The GATA zinc-finger transcription factor GATA4 is expressed in a variety of tissues during mouse embryonic development and in adult organs. These include the primitive endoderm of the blastocyst, visceral endoderm of the early post-implantation embryo, as well as lateral plate mesoderm, developing heart, liver, lung and gonads. Here, we generate a novel Gata4 targeted allele used to generate both a Gata4H2B-GFP transcriptional reporter and a Gata4FLAG fusion protein to analyse dynamic expression domains. We demonstrate that the Gata4H2B-GFP transcriptional reporter faithfully recapitulates known sites of Gata4 mRNA expression and correlates with endogenous GATA4 protein levels. This reporter labels nuclei of Gata4 expressing cells and is suitable for time-lapse imaging and single cell analyses. As such, this Gata4H2B-GFP allele will be a useful tool for studying Gata4 expression and transcriptional regulation.This article has an associated First Person interview with the first author of the paper.

Loss of Apela Peptide in Mice Causes Low Penetrance Embryonic Lethality and Defects in Early Mesodermal Derivatives.

Apela (also known as Elabela, Ende, and Toddler) is a small signaling peptide that activates the G-protein-coupled receptor Aplnr to stimulate cell migration during zebrafish gastrulation. Here, using CRISPR/Cas9 to generate a null, reporter-expressing allele, we study the role of Apela in the developing mouse embryo. We found that loss of Apela results in low-penetrance cardiovascular defects that manifest after the onset of circulation. Three-dimensional micro-computed tomography revealed a higher penetrance of vascular remodeling defects, from which some mutants recover, and identified extraembryonic anomalies as the earliest morphological distinction in Apela mutant embryos. Transcriptomics at late gastrulation identified aberrant upregulation of erythroid and myeloid markers in mutant embryos prior to the appearance of physical malformations. Double-mutant analyses showed that loss of Apela signaling impacts early Aplnr-expressing mesodermal populations independently of the alternative ligand Apelin, leading to lethal cardiac defects in some Apela null embryos.

Visualizing endoderm cell populations and their dynamics in the mouse embryo with a Hex-tdTomato reporter.

Live imaging is the requisite tool for studying cell behaviors driving embryonic development and tissue formation. Genetically encoded reporters expressed under cell type-specific cis-regulatory elements that drive fluorescent protein expression at sufficient levels for visualization in living specimens have become indispensable for these studies. Increasingly dual-color (red-green) imaging is used for studying the coordinate behaviors of two cell populations of interest, identifying and characterizing subsets within broader cell populations or subcellular features. Many reporters have been generated using green fluorescent protein (GFP) due to its brightness and developmental neutrality. To compliment the large cohort of available GFP reporters that label cellular populations in early mouse embryos, we have generated a red fluorescent protein (RFP)-based transgenic reporter using the red fluorescent tdTomato protein driven by cis-regulatory elements from the mouse Hex locus. The Hex-tdTomato reporter predominantly labels endodermal cells. It is a bright RFP-based reporter of the distal visceral endoderm (DVE)/anterior visceral endoderm (AVE), a migratory population within the early post-implantation embryo. It also labels cells of the definitive endoderm (DE), which emerges at gastrulation. Dual-color visualization of these different early endodermal populations will provide a detailed understanding of the cellular behaviors driving key morphogenetic events involving the endoderm.

The p53 Family Coordinates Wnt and Nodal Inputs in Mesendodermal Differentiation of Embryonic Stem Cells.

In this study, we outline a regulatory network that involves the p53 tumor suppressor family and the Wnt pathway acting together with the TGF-ß pathway in mesendodermal differentiation of mouse and human embryonic stem cells. Knockout of all three members, p53, p63, and p73, shows that the p53 family is essential for mesendoderm specification during exit from pluripotency in embryos and in culture. Wnt3 and its receptor Fzd1 are direct p53 family target genes in this context, and induction of Wnt signaling by p53 is critical for activation of mesendodermal differentiation genes. Globally, Wnt3-activated Tcf3 and nodal-activated Smad2/3 transcription factors depend on each other for co-occupancy of target enhancers associated with key differentiation loci. Our results therefore highlight an unanticipated role for p53 family proteins in a regulatory network that integrates essential Wnt-Tcf and nodal-Smad inputs in a selective and interdependent way to drive mesendodermal differentiation of pluripotent cells.

GFRA2 Identifies Cardiac Progenitors and Mediates Cardiomyocyte Differentiation in a RET-Independent Signaling Pathway.

A surface marker that distinctly identifies cardiac progenitors (CPs) is essential for the robust isolation of these cells, circumventing the necessity of genetic modification. Here, we demonstrate that a Glycosylphosphatidylinositol-anchor containing neurotrophic factor receptor, Glial cell line-derived neurotrophic factor receptor alpha 2 (Gfra2), specifically marks CPs. GFRA2 expression facilitates the isolation of CPs by fluorescence activated cell sorting from differentiating mouse and human pluripotent stem cells. Gfra2 mutants reveal an important role for GFRA2 in cardiomyocyte differentiation and development both in vitro and in vivo. Mechanistically, the cardiac GFRA2 signaling pathway is distinct from the canonical pathway dependent on the RET tyrosine kinase and its established ligands. Collectively, our findings establish a platform for investigating the biology of CPs as a foundation for future development of CP transplantation for treating heart failure.

Notochord morphogenesis in mice: Current understanding & open questions.

The notochord is a structure common to all chordates, and the feature that the phylum Chordata has been named after. It is a rod-like mesodermal structure that runs the anterior-posterior length of the embryo, adjacent to the ventral neural tube. The notochord plays a critical role in embryonic tissue patterning, for example the dorsal-ventral patterning of the neural tube. The cells that will come to form the notochord are specified at gastrulation. Axial mesodermal cells arising at the anterior primitive streak migrate anteriorly as the precursors of the notochord and populate the notochordal plate. Yet, even though a lot of interest has centered on investigating the functional and structural roles of the notochord, we still have a very rudimentary understanding of notochord morphogenesis. The events driving the formation of the notochord are rapid, taking place over the period of approximately a day in mice. In this commentary, we provide an overview of our current understanding of mouse notochord morphogenesis, from the initial specification of axial mesendodermal cells at the primitive streak, the emergence of these cells at the midline on the surface of the embryo, to their submergence and organization of the stereotypically positioned notochord. We will also discuss some key open questions. Developmental Dynamics 245:547-557, 2016. © 2016 Wiley Periodicals, Inc.

Lhx1 functions together with Otx2, Foxa2, and Ldb1 to govern anterior mesendoderm, node, and midline development.

Gene regulatory networks controlling functional activities of spatially and temporally distinct endodermal cell populations in the early mouse embryo remain ill defined. The T-box transcription factor Eomes, acting downstream from Nodal/Smad signals, directly activates the LIM domain homeobox transcription factor Lhx1 in the visceral endoderm. Here we demonstrate Smad4/Eomes-dependent Lhx1 expression in the epiblast marks the entire definitive endoderm lineage, the anterior mesendoderm, and midline progenitors. Conditional inactivation of Lhx1 disrupts anterior definitive endoderm development and impedes node and midline morphogenesis in part due to severe disturbances in visceral endoderm displacement. Transcriptional profiling and ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) experiments identified Lhx1 target genes, including numerous anterior definitive endoderm markers and components of the Wnt signaling pathway. Interestingly, Lhx1-binding sites were enriched at enhancers, including the Nodal-proximal epiblast enhancer element and enhancer regions controlling Otx2 and Foxa2 expression. Moreover, in proteomic experiments, we characterized a complex comprised of Lhx1, Otx2, and Foxa2 as well as the chromatin-looping protein Ldb1. These partnerships cooperatively regulate development of the anterior mesendoderm, node, and midline cell populations responsible for establishment of the left-right body axis and head formation.

A loss-of-function and H2B-Venus transcriptional reporter allele for Gata6 in mice.

BACKGROUND: The GATA-binding factor 6 (Gata6) gene encodes a zinc finger transcription factor that often functions as a key regulator of lineage specification during development. It is the earliest known marker of the primitive endoderm lineage in the mammalian blastocyst. During gastrulation, GATA6 is expressed in early cardiac mesoderm and definitive endoderm progenitors, and is necessary for development of specific mesoderm and endoderm-derived organs including the heart, liver, and pancreas. Furthermore, reactivation or silencing of the Gata6 locus has been associated with certain types of cancer affecting endodermal organs. RESULTS: We have generated a Gata6(H2B-Venus) knock-in reporter mouse allele for the purpose of labeling GATA6-expressing cells with a bright nuclear-localized fluorescent marker that is suitable for live imaging at single-cell resolution. CONCLUSIONS: Expression of the Venus reporter was characterized starting from embryonic stem (ES) cells, through mouse embryos and adult animals. The Venus reporter was not expressed in ES cells, but was activated upon endoderm differentiation. Gata6(H2B-Venus/H2B-Venus) homozygous embryos did not express GATA6 protein and failed to specify the primitive endoderm in the blastocyst. However, null blastocysts continued to express high levels of Venus in the absence of GATA6 protein, suggesting that early Gata6 transcription is independent of GATA6 protein expression. At early post-implantation stages of embryonic development, there was a strong correlation of Venus with endogenous GATA6 protein in endoderm and mesoderm progenitors, then later in the heart, midgut, and hindgut. However, there were discrepancies in reporter versus endogenous protein expression in certain cells, such as the body wall and endocardium. During organogenesis, detection of Venus in specific organs recapitulated known sites of endogenous GATA6 expression, such as in the lung bud epithelium, liver, pancreas, gall bladder, stomach epithelium, and vascular endothelium. In adults, Venus was observed in the lungs, pancreas, liver, gall bladder, ovaries, uterus, bladder, skin, adrenal glands, small intestine and corpus region of the stomach. Overall, Venus fluorescent protein under regulatory control of the Gata6 locus was expressed at levels that were easily visualized directly and could endure live and time-lapse imaging techniques. Venus is co-expressed with endogenous GATA6 throughout development to adulthood, and should provide an invaluable tool for examining the status of the Gata6 locus during development, as well as its silencing or reactivation in cancer or other disease states.

Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call 'gastruloids'.

SOX17 links gut endoderm morphogenesis and germ layer segregation.

Gastrulation leads to three germ layers--ectoderm, mesoderm and endoderm--that are separated by two basement membranes. In the mouse embryo, the emergent gut endoderm results from the widespread intercalation of cells of two distinct origins: pluripotent epiblast-derived definitive endoderm (DE) and extra-embryonic visceral endoderm (VE). Here we image the trajectory of prospective DE cells before intercalating into the VE epithelium. We show that the transcription factor SOX17, which is activated in prospective DE cells before intercalation, is necessary for gut endoderm morphogenesis and the assembly of the basement membrane that separates gut endoderm from mesoderm. Our results mechanistically link gut endoderm morphogenesis and germ layer segregation, two central and conserved features of gastrulation.

A conditional mutant allele for analysis of Mixl1 function in the mouse.

Mixl1 is the only member of the Mix/Bix homeobox gene family identified in mammals. During mouse embryogenesis, Mixl1 is first expressed at embryonic day (E)5.5 in cells of the visceral endoderm (VE). At the time of gastrulation, Mixl1 expression is detected in the vicinity of the primitive streak. Mixl1 is expressed in cells located within the primitive streak, in nascent mesoderm cells exiting the primitive streak, and in posterior VE overlying the primitive streak. Genetic ablation of Mixl1 in mice has revealed its crucial role in mesoderm and endoderm cell specification and tissue morphogenesis during early embryonic development. However, the early lethality of the constitutive Mixl1(-/-) mutant precludes the study of its role at later stages of embryogenesis and in adult mice. To circumvent this limitation, we have generated a conditional Mixl1 allele (Mixl1(cKO) that permits temporal as well as spatial control of gene ablation. Animals homozygous for the Mixl1(cKO) conditional allele were viable and fertile. Mixl1(KO/KO) embryos generated by crossing of Mixl1(cKO/cKO) mice with Sox2-Cre or EIIa-Cre transgenic mice were embryonic lethal at early somite stages. By contrast to wild-type embryos, Mixl1(KO/KO) embryos contained no detectable Mixl1, validating the Mixl1(cKO) as a protein null after Cre-mediated excision. Mixl1(KO/KO) embryos resembled the previously reported Mixl1(-/-) mutant phenotype. Therefore, the Mixl1 cKO allele provides a tool for investigating the temporal and tissue-specific requirements for Mixl1 in the mouse.