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Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1+ haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1+ haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.

The transcriptional repressor Blimp1 controls cell fate decisions in the developing embryo and adult tissues. Here we describe Blimp1 expression and functional requirements within maternal uterine tissues during pregnancy. Expression is robustly up-regulated at early post-implantation stages in the primary decidual zone (PDZ) surrounding the embryo. Conditional inactivation results in defective formation of the PDZ barrier and abnormal trophectoderm invasion. RNA-Seq analysis demonstrates down-regulated expression of genes involved in cell adhesion and markers of decidualisation. In contrast, genes controlling immune responses including IFNγ are up-regulated. ChIP-Seq experiments identify candidate targets unique to the decidua as well as those shared across diverse cell types including a highly conserved peak at the Csf-1 gene promoter. Interestingly Blimp1 inactivation results in up-regulated Csf1 expression and macrophage recruitment into maternal decidual tissues. These results identify Blimp1 as a critical regulator of tissue remodelling and maternal tolerance during early stages of pregnancy., The transcriptional repressor Blimp1/PRDM1 regulates cell fate decisions in the developing embryo and adult tissues. Here the authors show that conditional inactivation within maternal uterine tissues results in a defective primary decidual zone barrier, increased expression of inflammatory cytokines IFN gamma and Csf1, and early embryonic lethality during pregnancy.

The gene regulatory networks that coordinate hematopoietic commitment in the developing murine yolk-sac (YS) remain ill-defined. Here we report that the T-box transcription factor Eomesodermin (Eomes) is transiently expressed in mesodermal progenitors that generate virtually all YS hematopoietic and endothelial cells. Using an embryonic stem cell (ESC) differentiation system, we find that Eomes activity is essential for the production of primitive erythrocytes and definitive hematopoietic progenitors but dispensable for the development of endothelial cells. Bulk RNA-seq and single-cell-RNA-seq experiments demonstrate that in the absence of Eomes function Flk-1+ hematovascular mesoderm is specified but upon further differentiation it is diverted towards an endothelial rather than hematopoietic fate. Utilizing ESC reporter lines, we show that Eomes is expressed prior to both SCL and Runx1 during mesoderm patterning. Interestingly, Eomes activity is dispensable for the expression of SCL but is essential for the normal development of Runx1+ hemogenic endothelium (HE). ATAC-Seq experiments reveal that Eomes governs the accessibility of numerous hematopoietic enhancers that SCL normally utilizes to specify the hematopoietic fate. Finally, ChIP-seq experiments suggest that Eomes coordinates the development of hemogenic competent mesoderm in the context of Activin/Nodal and Tead-Yap signalling at mesodermal stages of development. Collectively, these experiments demonstrate that Eomes sits at the top of the transcriptional hierarchy, functioning upstream of Runx1 expression and SCL functional activity, to promote hemogenic competence of the YS mesodermal lineage. These results suggest that hemogenic competence is endowed earlier during murine embryogenesis than previously appreciated and have widespread implications for the generation of HE from pluripotent stem cell sources.

The essential roles played by Nodal and Bmp signalling during early mouse development have been extensively documented. Here we used conditional deletion strategies to investigate functional contributions made by Nodal, Bmp and Smad downstream effectors during primordial germ cell (PGC) development. We demonstrate that Nodal and its target gene Eomes provide early instructions during formation of the PGC lineage. We discovered that Smad2 inactivation in the visceral endoderm results in increased numbers of PGCs due to an expansion of the PGC niche. Smad1 is required for specification, whereas in contrast Smad4 controls the maintenance and migration of PGCs. Importantly we found that beside Blimp1, down-regulated phosphoSmad159 levels also distinguishes PGCs from their somatic neighbours so that emerging PGCs become refractory to Bmp signalling that otherwise promotes mesodermal development in the posterior epiblast. Thus balanced Nodal/Bmp signalling cues regulate germ cell versus somatic cell fate decisions in the early posterior epiblast.

A major challenge in cell and developmental biology is the automated identification and quantitation of cells in complex multilayered tissues. We developed CytoCensus: an easily deployed implementation of supervised machine learning that extends convenient 2D ‘point-and-click’ user training to 3D detection of cells in challenging datasets with ill-defined cell boundaries. In tests on such datasets, CytoCensus outperforms other freely available image analysis software in accuracy and speed of cell detection. We used CytoCensus to count stem cells and their progeny, and to quantify individual cell divisions from time-lapse movies of explanted Drosophila larval brains, comparing wild-type and mutant phenotypes. We further illustrate the general utility and future potential of CytoCensus by analysing the 3D organisation of multiple cell classes in Zebrafish retinal organoids and cell distributions in mouse embryos. CytoCensus opens the possibility of straightforward and robust automated analysis of developmental phenotypes in complex tissues., eLife digest There are around 200 billion cells in the human brain that are generated by a small pool of rapidly dividing stem cells. For the brain to develop correctly, these stem cells must produce an appropriate number of each type of cell in the right place, at the right time. However, it remains unclear how individual stem cells in the brain know when and where to divide. To answer this question, Hailstone et al. studied the larvae of fruit flies, which use similar genes and mechanisms as humans to control brain development. This involved devising a new method for extracting the brains of developing fruit flies and keeping the intact tissue alive for up to 24 hours while continuously imaging individual cells in three dimensions. Manually tracking the division of each cell across multiple frames of a time-lapse is extremely time consuming. To tackle this problem, Hailstone et al. created a tool called CytoCensus, which uses machine learning to automatically identify stem cells from three-dimensional images and track their rate of division over time. Using the CytoCensus tool, Hailstone et al. identified a gene that controls the diverse rates at whichstem cells divide in the brain. Earlier this year some of the same researchers also published a study showing that this gene regulates a well-known cancer-related protein using an unconventional mechanism. CytoCensus was also able to detect cells in other developing tissues, including the embryos of mice. In the future, this tool could aid research into diseases that affect complex tissues, such as neurodegenerative disorders and cancer.

Reciprocal inductive interactions between the embryonic and extraembryonic tissues establish the anterior–posterior (AP) axis of the early mouse embryo. The anterior visceral endoderm (AVE) signaling center emerges at the distal tip of the embryo at embryonic day 5.5 and translocates to the prospective anterior side of the embryo. The process of AVE induction and migration are poorly understood. Here we demonstrate that the T-box gene Eomesodermin (Eomes) plays an essential role in AVE recruitment, in part by directly activating the homeobox transcription factor Lhx1. Thus, Eomes function in the visceral endoderm (VE) initiates an instructive transcriptional program controlling AP identity.

The T-box transcription factor (TF) Eomes is a key regulator of cell fate decisions during early mouse development. The cis-acting regulatory elements that direct expression in the anterior visceral endoderm (AVE), primitive streak (PS) and definitive endoderm (DE) have yet to be defined. Here, we identified three gene-proximal enhancer-like sequences (PSE_a, PSE_b and VPE) that faithfully activate tissue-specific expression in transgenic embryos. However, targeted deletion experiments demonstrate that PSE_a and PSE_b are dispensable, and only VPE is required for optimal Eomes expression in vivo. Embryos lacking this enhancer display variably penetrant defects in anterior-posterior axis orientation and DE formation. Chromosome conformation capture experiments reveal VPE-promoter interactions in embryonic stem cells (ESCs), prior to gene activation. The locus resides in a large (500 kb) pre-formed compartment in ESCs and activation during DE differentiation occurs in the absence of 3D structural changes. ATAC-seq analysis reveals that VPE, PSE_a and four additional putative enhancers display increased chromatin accessibility in DE that is associated with Smad2/3 binding coincident with transcriptional activation. By contrast, activation of the Eomes target genes Foxa2 and Lhx1 is associated with higher order chromatin reorganisation. Thus, diverse regulatory mechanisms govern activation of lineage specifying TFs during early development., Summary: Expression of the mouse T-box factor Eomes is controlled by a key gene-proximal enhancer-like element, with changes in chromatin accessibility influencing its activity in definitive endoderm.

Instructive programmes guiding cell-fate decisions in the developing mouse embryo are controlled by a few so-termed master regulators. Genetic studies demonstrate that the T-box transcription factor Eomesodermin (Eomes) is essential for epithelial-to-mesenchymal transition, mesoderm migration and specification of definitive endoderm during gastrulation. Here we report that Eomes expression within the primitive streak marks the earliest cardiac mesoderm and promotes formation of cardiovascular progenitors by directly activating the bHLH (basic-helix-loop-helix) transcription factor gene Mesp1 upstream of the core cardiac transcriptional machinery. In marked contrast to Eomes/Nodal signalling interactions that cooperatively regulate anterior-posterior axis patterning and allocation of the definitive endoderm cell lineage, formation of cardiac progenitors requires only low levels of Nodal activity accomplished through a Foxh1/Smad4-independent mechanism. Collectively, our experiments demonstrate that Eomes governs discrete context-dependent transcriptional programmes that sequentially specify cardiac and definitive endoderm progenitors during gastrulation.

The epicardium, the outermost tissue layer that envelops the developing heart and provides essential trophic signals for the myocardium, derives from the pro-epicardial organ (PEO). Two of the three members of the Flrt family of transmembrane glycoproteins, Flrt2 and Flrt3, are strongly co-expressed in the PEO. However, beginning at around day 10 of mouse development, following attachment and outgrowth, Flrt3 is selectively downregulated, and only Flrt2 is exclusively expressed in the fully delaminated epicardium. The present gene-targeting experiments demonstrate that mouse embryos lacking Flrt2 expression arrest at mid-gestation owing to cardiac insufficiency. The defects in integrity of the epicardial sheet and disturbed organization of the underlying basement membrane closely resemble those described in Flrt3-deficient embryos that fail to maintain cell-cell contacts in the anterior visceral endoderm (AVE) signalling centre that normally establishes the A-P axis. Using in vitro and in vivo reconstitution assays, we demonstrate that Flrt2 and Flrt3 are functionally interchangeable. When acting alone, either of these proteins is sufficient to rescue functional activities in the AVE and the developing epicardium.

Gene inactivation studies of mammalian histone and DNA-modifying proteins have demonstrated a role for many such proteins in embryonic development. Post-implantation embryonic lethality implies a role for epigenetic factors in differentiation and in development of specific lineages or tissues. However a handful of chromatin-modifying enzymes have been found to be required in pre- or peri-implantation embryos. This is significant as implantation is the time when inner cell mass cells of the blastocyst exit pluripotency and begin to commit to form the various lineages that will eventually form the adult animal. These observations indicate a critical role for chromatin-modifying proteins in the earliest lineage decisions of mammalian development, and/or in the formation of the first embryonic cell types. Recent work has shown that the two major class I histone deacetylase-containing co-repressor complexes, the NuRD and Sin3 complexes, are both required at peri-implantation stages of mouse development, demonstrating the importance of histone deacetylation in cell fate decisions. Over the past 10 years both genetic and biochemical studies have revealed surprisingly divergent roles for these two co-repressors in mammalian cells. In this review we will summarise the evidence that the two major class I histone deacetylase complexes in mammalian cells, the NuRD and Sin3 complexes, play important roles in distinct aspects of embryonic 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.

Summary Transcriptional heterogeneity within embryonic stem cell (ESC) populations has been suggested as a mechanism by which a seemingly homogeneous cell population can initiate differentiation into an array of different cell types. Chromatin remodeling proteins have been shown to control transcriptional variability in yeast and to be important for mammalian ESC lineage commitment. Here we show that the Nucleosome Remodeling and Deacetylation (NuRD) complex, which is required for ESC lineage commitment, modulates both transcriptional heterogeneity and the dynamic range of a set of pluripotency genes in ESCs. In self-renewing conditions, the influence of NuRD at these genes is balanced by the opposing action of self-renewal factors. Upon loss of self-renewal factors, the action of NuRD is sufficient to silence transcription of these pluripotency genes, allowing cells to exit self-renewal. We propose that modulation of transcription levels by NuRD is key to maintaining the differentiation responsiveness of pluripotent cells., Graphical Abstract Highlights ► NuRD directly regulates the transcription of pluripotency genes ► The repressive activity of NuRD is required for ESC differentiation ► NuRD mediates transcriptional heterogeneity in ESC populations ► Transcription levels are determined by balanced activation and silencing

BackgroundSmad4 mutant embryos arrest shortly after implantation and display a characteristic shortened proximodistal axis, a significantly reduced epiblast, as well as a thickened visceral endoderm layer. Conditional rescue experiments demonstrate that bypassing the primary requirement for Smad4 in the extra-embryonic endoderm allows the epiblast to gastrulate. Smad4-independent TGF-β signals are thus sufficient to promote mesoderm formation and patterning. To further analyse essential Smad4 activities contributed by the extra-embryonic tissues, and characterise Smad4 dependent pathways in the early embryo, here we performed transcriptional profiling of Smad4 null embryonic stem (ES) cells and day 4 embryoid bodies (EBs).ResultsTranscripts from wild-type versus Smad4 null ES cells and day 4 EBs were analysed using Illumina arrays. In addition to several known TGF-β/BMP target genes, we identified numerous Smad4-dependent transcripts that are mis-expressed in the mutants. As expected, mesodermal cell markers were dramatically down-regulated. We also observed an increase in non-canonical potency markers (Pramel7,Tbx3,Zscan4), germ cell markers (Aire,Tuba3a,Dnmt3l) as well as early endoderm markers (Dpp4,H19,Dcn). Additionally, expression of the extracellular matrix (ECM) remodelling enzymesMmp14andMmp9was decreased in Smad4 mutant ES and EB populations. These changes, in combination with increased levels oflaminin alpha1, cause excessive basement membrane deposition. Similarly, in the context of the Smad4 null E6.5 embryos we observed an expanded basement membrane (BM) associated with the thickened endoderm layer.ConclusionSmad4 functional loss results in a dramatic shift in gene expression patterns and in the endodermal cell lineage causes an excess deposition of, or an inability to breakdown and remodel, the underlying BM layer. These structural abnormalities probably disrupt reciprocal signalling between the epiblast and overlying visceral endoderm required for gastrulation.