Project description:The developing vertebrate embryo is exquisitely sensitive to retinoic acid (RA) concentration, particularly during anteroposterior patterning. In contrast to Nodal and Wnt signaling, RA was not previously considered to be an instructive signal in mesoderm formation during gastrulation. Here we show that RARγ is indispensable for the expression of early mesoderm markers and is, therefore, an obligatory factor in mesodermal competence and/or maintenance. We identified several novel targets up-regulated by RAR signaling in the early gastrula that are expressed in the circumblastoporal ring and linked to mesodermal development. Despite overlapping expression patterns of the RA synthetic enzyme, Aldh1a2 and the RA- degrading enzyme, Cyp26a1, RARγ1 functions as a transcriptional activator in early mesoderm development, suggesting that RA ligand is available to the embryo earlier than previously appreciated. RARγ1 is required for cellular adhesion, as revealed by spontaneous dissociation and depletion of N-CAM mRNA in animal caps harvested from RARγ1 knockdown embryos. RARγ1 knockdown obliterates somite boundaries, and causes loss of MYOD protein in the presomitic mesoderm, but ectopic, persistent expression of MYOD protein in the trunk. Thus, RARγ1 is required for stabilizing the mesodermal fate, myogenic commitment, somite boundary formation, and terminal, skeletal muscle differentiation.
Project description:In animal models, Nipbl-deficiency phenocopies gene expression changes and birth defects seen in Cornelia de Lange Syndrome (CdLS), the most common cause of which is Nipbl-haploinsufficiency. Previous studies in Nipbl+/- mice identified aberrant gene expression and heart defects as early as cardiac crescent (CC) stage. Here, we performed single-cell RNA-sequencing on wildtype (WT) and Nipbl+/- mouse embryos at CC- and earlier (gastrulation) stages. Nipbl+/- embryos had fewer mesoderm cells than WT and altered proportions of mesodermal cell subpopulations. These findings were associated with an underexpression of genes implicated in driving specific mesodermal lineages. Nipbl+/- embryos also misexpressed developmentally-critical genes, including the transcription factor, Nanog, and genes governing left-right and anterior-posterior patterning. These events of cell misallocation and transcriptional dysregulation foreshadowed defects in tissue composition and patterning that arise later in Nipbl+/- mice, offering insights into early developmental contributions to birth defects in CdLS.
Project description:We generated loss of function mESC clones by CRISPR-Cas9 to test the function of Evx1 during gastrulation. Evx1Δfs Clone 14 mESCs were differentiated to D4 in serum and compared to unedited mESCs
Project description:The recent identification of novel progenitor populations that contribute to the developing heart in a distinct temporal and spatial manner has fundamentally improved our understanding of cardiac development. However, little remains known about cardiac specification events prior to the establishment of the heart tube, or the mechanisms that direct atrial versus ventricular specification. We have identified a novel progenitor population that gives rise specifically to cardiovascular cells of the ventricles but not the atria, and to the epicardium of the differentiated heart. We determined that this cell population is first specified during gastrulation, when it transiently expresses Foxa2, a gene not previously implicated in cardiac development. Using chimeric mosaic analysis we further demonstrate that Foxa2 is cell-autonomously required for the development of ventricular cells. Finally, we reveal the existence of an analogous Foxa2+ cardiac mesoderm population during in vitro differentiation from embryonic stem cells and illustrate that these cells express genes relevant for heart development. Our data thus describe the first progenitor population identified as early as gastrulation that displays ventricular-specific differentiation potential. Together, these findings provide important new insights into the developmental origin of ventricular and atrial myocytes, and will lead to the establishment of new strategies for generating these cell types from pluripotent stem cells.
Project description:Epigenetic regulation of chromatin states is crucial for proper gene expression programs and progression during development, but precise mechanisms by which epigenetic factors influence differentiation remain poorly understood. Here we find that the histone variant H2A.Z accumulates at Sox motif-containing promoters during zebrafish gastrulation while neighboring genes become transcriptionally active. These changes coincide with reduced expression of anp32e, the H2A.Z histone removal chaperone, suggesting that loss of Anp32e may lead to increases in H2A.Z binding during differentiation. Remarkably, genetic removal of Anp32e in embryos leads to H2A.Z accumulation prior to gastrulation and developmental genes become precociously active. Accordingly, H2A.Z accumulation occurs most extensively at Sox motif-associated genes, including many which are normally activated following gastrulation. Altogether, our results provide compelling evidence for a mechanism in which Anp32e preferentially restricts H2A.Z accumulation at Sox motifs to regulate the initial phases of developmental differentiation in zebrafish.
Project description:Animal responses to their environment rely on activation of sensory neurons by external stimuli. In many sensory systems, however, neurons display basal activity prior to the external stimuli. This prior activity is thought to modulate neural functions, yet its impact on animal behavior remains elusive. Here, we reveal a potential role for prior activity in olfactory receptor neurons (ORNs) in shaping larval olfactory behavior. We show that prior activity in larval ORNs is mediated by the olfactory receptor complex (OR complex). Mutations of Orco, an odorant co-receptor required for OR complex function, cause reduced attractive behavior in response to optogenetic activation of ORNs. Calcium imaging reveals that Orco mutant ORNs fully respond to optogenetic stimulation but exhibit altered temporal patterns of neural responses. These findings together suggest a critical role for prior activity in information processing upon ORN activation in Drosophila larvae, which in turn contributes to olfactory behavior control.
Project description:The pluripotent epiblast gives rise to all tissues and organs in an adult body. Its differentiation starts at gastrulation when the epiblast generates mesoderm and endoderm germ layers through a process called epithelial-mesenchymal transition (EMT). Although gastrulation EMT coincides with loss of epiblast pluripotency, pluripotent cells in development and in culture can adopt either mesenchymal or epithelial morphology. The relationship between epiblast pluripotency and epiblast morphology is not well understood. In this work, using chicken epiblast and human induced pluripotent stem cell (hiPSC) models, we show that pluripotent cells undergo an obligatory mesenchymal-epithelial transition (MET) prior to EMT-associated pluripotency loss. Epiblast MET and the subsequent EMT are two distinct processes. The former, a partial MET, is associated with reversible pluripotency exit; whereas the latter, a full EMT, is associated with complete and irreversible pluripotency loss. We provide evidence that integrin-mediated cell-matrix interaction, but not E-cadherin-mediated cell-cell interaction, is a key player in pluripotency exit regulation. We propose that epiblast partial MET is an evolutionarily conserved process among all amniotic vertebrates and its developmental function is to mediate planar symmetry-breaking within an epithelialized epiblast, taking place after epiblast MET but before gastrulation EMT.
Project description:5'Cap-Analysis of Gene Expression (5' CAGE) from mesodermal and whole embryo RNA at three different time intervals during development.