Pamela Hoodless

Hepatocytes differentiate from definitive endoderm in response to external signaling cues. However, it is unclear how the hepatic lineage is specified and how transcription factors shape the chromatin to allow gene expression. In this study, we used a human stem cell model of liver development, differentiating cells stepwise into endodermal and hepatic progenitors combined with whole-genome epigenomic mapping. To assess the chromatin dynamics during differentiation, we mapped the histone modifications H3K4me1, H3K4me3, H3K27ac, H3K27me3 and H3K36me3 using ChIP-seq at four time points. We used this data to identify 655,208 enhancers and 20,517 promoters and define their activation state across the differentiation. Enhancers were found to exhibit dynamic regulation, with many primed at the definitive endoderm. Tracing the fate of the primed enhancers, we identified hepatic-specific enhancers, which gained active histone modifications, in contrast to non-hepatic enhancers, that subsequently lost H3K4me1 after hepatic specification. Promoters were more stable than enhancers, but also showing dynamic patterns in their regulation. To identify regulating transcription factors, we performed ChIP-seq for two identified predicted regulators, TBX3 and HNF4A, before and after hepatic specification. TBX3, and to lesser extend HNF4A, bound to the hepatic enhancers and formed an interdependent network with known pioneer factors. Mechanistically, TBX3 bound both hepatic enhancers and promoters prior to H3K4 methylation, likely recruiting methyltransferase complexes through interactions with RbBP5. However, TBX3 binding was transient and H3K4 methylation was observed after eviction of TBX3. Interestingly, a subset of promoters that specifically gained H3K4me3 in hepatoblasts and showed immediate upregulation of gene expression was specifically bound by TBX3. This was in contrast to the majority of identified promoters that gained active histone modifications early on but required the activation of associated enhancers to increase gene expression. This suggests that TBX3 plays a role in identifying and priming hepatic enhancers and promoters during specification. These data revealed a very dynamic developmental chromatin landscape, that allowed us to identify TBX3 as a potential driver of hepatic cell identity. Our comprehensive developmental histone modification dataset will serve as a resource for researchers studying the possible reactivation of developmental processes in cancer or regeneration.

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The cellular complexity and small scale of the mouse embryonic liver and heart valves have constrained analyses probing cell type diversity and differentiation during their development. To address this, we have analyzed 29 single-cell RNA-seq libraries from embryonic day (E)7.5 to E12.5 and have validated the spatiotemporal distribution of identified cell lineages by histology. To assess liver development, we have analyzed 45,334 cells and have detailed the developmental trajectories taken during the early emergence of liver parenchymal and non-parenchymal cell lineages. These analyses describe the development of hepatoblasts, liver endothelium, and mesenchyme from endoderm progenitor specification at E7.5 to liver bud formation at E10.5. Our data detail the divergence of vascular and sinusoidal endothelia, the specification of hepatoblasts, and the emergence of distinct mesothelial cell types. We further identify a novel, hybrid hepatomesenchymal cell type that we hypothesize plays a role in liver bud formation. To delineate atrioventricular (AV) valve development, we have analyzed 48,822 cells from publicly available and newly generated single-cell datasets. These data provide insight into the epithelial-to-mesenchymal transformations from endocardium (EndMT) and epicardium (EpiMT) that contribute to AV mesenchyme during development. During EndMT, we detail the bifurcation of valve mesenchymal and endocardial lineages, which our data suggest involves the sporadic activation of epithelial-mesenchymal plasticity. During EpiMT, scRNA-seq data and histology identify a unique activated epicardial population that emerges at the onset of this process. We further use our transcriptomic data to deconvolve signals that may influence the initiation and progression of EndMT and EpiMT.Lastly, we have assessed the role of AV mesenchymal master regulator SOX9 during EndMT by comparing Sox9 endothelial-specific mutant and wildtype AV canals. Our data show developmental arrest during EndMT without Sox9, including the failure of endocardial transdifferentiation to mesenchyme and the accumulation of endocardium exhibiting epithelial-mesenchymal plasticity.These data reveal surprising diversity during the emergence of liver and AV cell lineages. As a resource, they will serve as comprehensive single-cell atlases of liver and AV lineage establishment for researchers studying the development of these tissues or the reactivation of developmental processes in disease and regenerative contexts.

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Cardiac malformations affect approximately 1% of human newborns and a large number of these are due to defects in the heart valves and septum. It has been suggested that cardiac valve diseases, which make up about one third of all cardiovascular defects, arise from underlying developmental malformations that occur during embryogenesis. Interestingly, the development of the heart valves (cardiac cushions) and tissues that form cartilage templates (such as the limb) share a number of key TFs, such as TWIST1, SOX9, and NFATC1 suggesting that they have similar transcriptional programs. It has been proposed that regulatory networks involved in cartilage formation, are also active during valve development and disease. The transcription factor SOX9 has an essential role in heart valve and cartilage formation and its loss leads to major congenital abnormalities in the embryo. Regardless of this critical role, little is known about how SOX9 regulates heart valve development or its transcriptional targets. Therefore, to identify transcriptional targets of SOX9 and elucidate the role of SOX9 in the developing valves, we have used ChIP-Seq on the E12.5 atrioventricular canal (heart valves) and limb buds. Comparisons of SOX9DNA-binding regions among tissues revealed both context-dependent and context–independent SOX9 interacting regions. Context-independent SOX9 binding suggests that SOX9 may play a role in regulating proliferation-associated genes across many tissues. Generation of two endothelial specific Sox9 mutants uncovers two potential roles for SOX9 in heart valve formation: first in the initial formation of valve mesenchyme and later in the survival and differentiation of valve mesenchyme. Analysis of tissue-specific SOX9-DNA binding regions with gene expression profiles from Sox9 mutant heart valves indicates that SOX9 directly regulates a collection of transcription factors known to be important for heart development. Taken together, this study identified that SOX9 controls transcriptional hierarchies involved in proliferation across tissues and heart valve differentiation. SOX9 transcriptional targets identified in this data could be used as predictive factors of heart valve disease, or as targets for new therapeutic strategies for disease and congenital defects.

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No abstract available.

Malformations of the cardiovascular system are the most common type of birth defect in humans, affecting predominantly the formation of valves and septa. While many studies have addressed the role of specific genes during valve and septa formation, a global understanding is still largely incomplete. To address this deficit we have undertaken a genome-wide transcriptional profiling of the developing heart in the mouse. We generated and analyzed 19 Serial Analysis of Gene Expression (SAGE) libraries representing different regions of the mouse heart at multiple stages of embryonic development.We speculated that genes important for heart valve development would be differentially expressed in the valve forming regions, and have dynamic temporal expression patterns. We used our dataset to identify a novel list of valve enriched genes. Using k-means cluster analysis we also uncovered 14 distinct temporal gene expression patterns in the developing valves. Unique temporal expression patterns were found to be enriched for specific signalling pathway members and functional categories such as signal transduction, transcription factor activity, proliferation and apoptosis. The most highly expressed transcription factor within the developing valves was found to be Twist1. Analysis of gene expression changes in the Twist1 null developing valves revealed a novel phenotype consistent with a role of TWIST1 in controlling differentiation of mesenchymal cells following their transformation from endothelium in the mouse. Our data suggests that TWIST1 directly activates valve specific and cell motility gene expression in the atrio-ventricular canal, while suppressing expression of valve maturation markers. This work provides the first comprehensive temporal and spatial gene expression dataset for heart development during formation of the heart valves. It is a valuable resource for the elucidation of the molecular mechanisms underlying heart development.

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No abstract available.