Cell diversity and plasticity during atrioventricular heart valve EMTs.

Epithelial-to-mesenchymal transitions (EMTs) of both endocardium and epicardium guide atrioventricular heart valve formation, but the cellular complexity and small scale of this tissue have restricted analyses. To circumvent these issues, we analyzed over 50,000 murine single-cell transcriptomes from embryonic day (E)7.75 hearts to E12.5 atrioventricular canals. We delineate mesenchymal and endocardial bifurcation during endocardial EMT, identify a distinct, transdifferentiating epicardial population during epicardial EMT, and reveal the activation of epithelial-mesenchymal plasticity during both processes. In Sox9-deficient valves, we observe increased epithelial-mesenchymal plasticity, indicating a role for SOX9 in promoting endothelial and mesenchymal cell fate decisions. Lastly, we deconvolve cell interactions guiding the initiation and progression of cardiac valve EMTs. Overall, these data reveal mechanisms of emergence of mesenchyme from endocardium or epicardium at single-cell resolution and will serve as an atlas of EMT initiation and progression with broad implications in regenerative medicine and cancer biology.

Breaking constraint of mammalian axial formulae.

The vertebral column of individual mammalian species often exhibits remarkable robustness in the number and identity of vertebral elements that form (known as axial formulae). The genetic mechanism(s) underlying this constraint however remain ill-defined. Here, we reveal the interplay of three regulatory pathways (Gdf11, miR-196 and Retinoic acid) is essential in constraining total vertebral number and regional axial identity in the mouse, from cervical through to tail vertebrae. All three pathways have differing control over Hox cluster expression, with heterochronic and quantitative changes found to parallel changes in axial identity. However, our work reveals an additional role for Hox genes in supporting axial elongation within the tail region, providing important support for an emerging view that mammalian Hox function is not limited to imparting positional identity as the mammalian body plan is laid down. More broadly, this work provides a molecular framework to interrogate mechanisms of evolutionary change and congenital anomalies of the vertebral column.

A Hox Code Defines Spinocerebellar Neuron Subtype Regionalization.

Coordinated movement requires the integration of many sensory inputs including proprioception, the sense of relative body position and force associated with movement. Proprioceptive information is relayed to the cerebellum via spinocerebellar neurons, located in the spinal cord within a number of major neuronal columns or as various scattered populations. Despite the importance of proprioception to fluid movement, a molecular understanding of spinocerebellar relay interneurons is only beginning to be explored, with limited knowledge of molecular heterogeneity within and between columns. Using fluorescent reporter mice, neuronal tracing, and in situ hybridization, we identify widespread expression of Hox cluster genes within spinocerebellar neurons. We reveal a "Hox code" based on axial level and individual spinocerebellar column, which, at cervico-thoracic levels, is essential for subtype regionalization. Specifically, we show that Hoxc9 function is required in most, but not all, cells of the thoracic spinocerebellar column, Clarke's column, revealing heterogeneity reliant on Hox signatures.

SOX9 modulates the expression of key transcription factors required for heart valve development.

Heart valve formation initiates when endothelial cells of the heart transform into mesenchyme and populate the cardiac cushions. The transcription factor SOX9 is highly expressed in the cardiac cushion mesenchyme, and is essential for heart valve development. Loss of Sox9 in mouse cardiac cushion mesenchyme alters cell proliferation, embryonic survival, and valve formation. Despite this important role, little is known about how SOX9 regulates heart valve formation or its transcriptional targets. Therefore, we mapped putative SOX9 binding sites by ChIP-Seq in E12.5 heart valves, a stage at which the valve mesenchyme is actively proliferating and initiating differentiation. Embryonic heart valves have been shown to express a high number of genes that are associated with chondrogenesis, including several extracellular matrix proteins and transcription factors that regulate chondrogenesis. Therefore, we compared regions of putative SOX9 DNA binding between E12.5 heart valves and E12.5 limb buds. We identified context-dependent and context-independent SOX9-interacting regions throughout the genome. Analysis of context-independent SOX9 binding suggests an extensive role for SOX9 across tissues in regulating proliferation-associated genes including key components of the AP-1 complex. Integrative analysis of tissue-specific SOX9-interacting regions and gene expression profiles on Sox9-deficient heart valves demonstrated that SOX9 controls the expression of several transcription factors with previously identified roles in heart valve development, including Twist1, Sox4, Mecom and Pitx2. Together, our data identify SOX9-coordinated transcriptional hierarchies that control cell proliferation and differentiation during valve formation.

Huntingtin interacting proteins 14 and 14-like are required for chorioallantoic fusion during early placental development.

Huntington disease (HD) is an adult-onset neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms that is caused by a CAG expansion in the HTT gene. Palmitoylation is the addition of saturated fatty acids to proteins by DHHC palmitoylacyl transferases. HTT is palmitoylated by huntingtin interacting proteins 14 and 14-like (HIP14 and HIP14L or ZDHHC17 and 13 respectively). Mutant HTT is less palmitoylated and this reduction of palmitoylation accelerates its aggregation and increases cellular toxicity. Mouse models deficient in either Hip14 (Hip14(-/-)) or Hip14l (Hip14l(-/-)) develop HD-like phenotypes. The biological function of HTT palmitoylation and the role that loss of HTT palmitoylation plays in the pathogenesis of HD are unknown. To address these questions mice deficient for both genes were created. Loss of Hip14 and Hip14l leads to early embryonic lethality at day embryonic day 10-11 due to failed chorioallantoic fusion. The chorion is thickened and disorganized and the allantois does not fuse correctly with the chorion and forms a balloon-like shape compared to Hip14l(-/-); Hip14(+/+) littermate control embryos. Interestingly, the Hip14(-/-) ; Hip14(-/-) embryos share many features with the Htt(-/-) embryos, including folding of the yolk sac, a bulb shaped allantois, and a thickened and disorganized chorion. This may be due to a decrease in HTT palmitoylation. In Hip14(-/-); Hip14l(-/-) mouse embryonic fibroblasts show a 25% decrease in HTT palmitoylation compared to wild type cells. This is the first description of a double PAT deficient mouse model where loss of a PAT or multiple PATs results in embryonic lethality in mammals. These results reinforce the physiological importance of palmitoylation during embryogenesis.

Hippo signaling influences HNF4A and FOXA2 enhancer switching during hepatocyte differentiation.

Cell fate acquisition is heavily influenced by direct interactions between master regulators and tissue-specific enhancers. However, it remains unclear how lineage-specifying transcription factors, which are often expressed in both progenitor and mature cell populations, influence cell differentiation. Using in vivo mouse liver development as a model, we identified thousands of enhancers that are bound by the master regulators HNF4A and FOXA2 in a differentiation-dependent manner, subject to chromatin remodeling, and associated with differentially expressed target genes. Enhancers exclusively occupied in the embryo were found to be responsive to developmentally regulated TEAD2 and coactivator YAP1. Our data suggest that Hippo signaling may affect hepatocyte differentiation by influencing HNF4A and FOXA2 interactions with temporal enhancers. In summary, transcription factor-enhancer interactions are not only tissue specific but also differentiation dependent, which is an important consideration for researchers studying cancer biology or mammalian development and/or using transformed cell lines.

A Notch-dependent transcriptional hierarchy promotes mesenchymal transdifferentiation in the cardiac cushion.

BACKGROUND: Valvuloseptal defects are the most common congenital heart defects. Notch signaling-induced endothelial-to-mesenchymal transition (EMT) in the atrioventricular canal (AVC) cushions at murine embryonic day (E)9.5 is a required step during early valve development. Insights to the transcriptional network that is activated in endocardial cells (EC) during EMT and how these pathways direct valve maturation are lacking. RESULTS: We show that at E11.5, AVC-EC retain the ability to undergo Notch-dependent EMT when explanted on collagen. EC-Notch inhibition at E10.5 blocks expression of known mesenchymal genes in E11.5 AVC-EC. To understand the genetic network and AVC development downstream of Notch signaling beyond E9.5, we constructed Tag-Seq libraries corresponding to different cell types of the E11.5 AVC and atrium in wild-type mice and in EC-Notch inhibited mice. We identified 1,400 potential Notch targets in the AVC-EC, of which 124 are transcription factors (TF). From the 124 TFs, we constructed a transcriptional hierarchy and identify 10 upstream TFs within the network. CONCLUSIONS: We validated 4 of the upstream TFs as Notch targets that are enriched in AVC-EC. Functionally, we show these 4 TFs regulate EMT in AVC explant assays. These novel signaling pathways downstream of Notch are potentially relevant to valve development.

Co-ordinating Notch, BMP, and TGF-ß signaling during heart valve development.

Congenital heart defects affect approximately 1-5 % of human newborns each year, and of these cardiac defects 20-30 % are due to heart valve abnormalities. Recent literature indicates that the key factors and pathways that regulate valve development are also implicated in congenital heart defects and valve disease. Currently, there are limited options for treatment of valve disease, and therefore having a better understanding of valve development can contribute critical insight into congenital valve defects and disease. There are three major signaling pathways required for early specification and initiation of endothelial-to-mesenchymal transformation (EMT) in the cardiac cushions: BMP, TGF-ß, and Notch signaling. BMPs secreted from the myocardium set up the environment for the overlying endocardium to become activated; Notch signaling initiates EMT; and both BMP and TGF-ß signaling synergize with Notch to promote the transition of endothelia to mesenchyme and the mesenchymal cell invasiveness. Together, these three essential signaling pathways help form the cardiac cushions and populate them with mesenchyme and, consequently, set off the cascade of events required to develop mature heart valves. Furthermore, integration and cross-talk between these pathways generate highly stratified and delicate valve leaflets and septa of the heart. Here, we discuss BMP, TGF-ß, and Notch signaling pathways during mouse cardiac cushion formation and how they together produce a coordinated EMT response in the developing mouse valves.

Notch initiates the endothelial-to-mesenchymal transition in the atrioventricular canal through autocrine activation of soluble guanylyl cyclase.

The heart is the most common site of congenital defects, and valvuloseptal defects are the most common of the cardiac anomalies seen in the newborn. The process of endothelial-to-mesenchymal transition (EndMT) in the cardiac cushions is a required step during early valve development, and Notch signaling is required for this process. Here we show that Notch activation induces the transcription of both subunits of the soluble guanylyl cyclase (sGC) heterodimer, GUCY1A3 and GUCY1B3, which form the nitric oxide receptor. In parallel, Notch also promotes nitric oxide (NO) production by inducing Activin A, thereby activating a PI3-kinase/Akt pathway to phosphorylate eNOS. We thus show that the activation of sGC by NO through a Notch-dependent autocrine loop is necessary to drive early EndMT in the developing atrioventricular canal (AVC).

MIST1 regulates the pancreatic acinar cell expression of Atp2c2, the gene encoding secretory pathway calcium ATPase 2.

MIST1 is a transcription factor expressed in pancreatic acinar cells and other serous exocrine cells. Mice harboring a targeted deletion of the Mist1 gene (Mist1(-/-)) exhibit alterations in acinar regulated exocytosis and aberrant Ca(2+) signaling that are normally controlled by acinar cell Ca(2+)-ATPases. Previous studies indicated that total sarcoendoplasmic reticulum Ca(2+)-ATPases (SERCA) and plasma membrane Ca(2+)-ATPases (PMCA) remained unaffected in Mist1(-/-) acinar cultures. Therefore, we have assessed the expression of Atp2c2, the gene that encodes the secretory pathway Ca(2+)-ATPase 2 (SPCA2). We revealed a dramatic decrease in pancreatic expression of Atp2a2 mRNA and SPCA2 protein in Mist1(-/-) mice. Surprisingly, this analysis indicated that the acinar-specific Atp2c2 mRNA is a novel transcript, consisting of only the 3' end of the gene and the protein and localizes to the endoplasmic reticulum. Expression of SPCA2 was also lost in Mist1(-/-) secretory cells of the salivary glands and seminal vesicles, suggesting that Atp2c2 transcription is regulated by MIST1. Indeed, inducible MIST1 expression in Mist1(-/-) pancreatic acinar cells restored normal Atp2c2 expression, supporting a role for MIST1 in regulating the Atp2c2 gene. Based on these results, we have identified a new Atp2c2 transcript, the loss of which may be linked to the Mist1(-/-) phenotype.

Identification of a transcription factor, BHLHB8, involved in mouse seminal vesicle epithelium differentiation and function.

The seminal vesicle is a male accessory sex organ that develops from segments of the Wolffian duct adjacent to the urogenital sinus. It produces most of the seminal plasma in both humans and rodents. To date, very few transcription factors have been linked to the development and differentiation of seminal vesicles. In this study, we have examined the role of basic helix-loop-helix (BHLH) B8 transcription factor expressed at high levels in the adult seminal vesicle and during seminal gland differentiation. Immunofluorescent studies indicate that BHLHB8 is expressed within the epithelial layer of the seminal layer of the seminal vesicle following branching morphogenesis but prior to full maturation of cell morphology and function. Analysis of mice that do not express BHLHB8 (Bhlhb8(-/-)) indicates no deficiency in the initial development of the seminal vesicle. However, morphological and ultrastructural analysis indicates disruption of the epithelial cellular architecture. The seminal vesicle epithelial layer of 2-mo-old Bhlhb8(-/-) mice shows extensive cellular degeneration based on the appearance of reduced microvilli, altered granule size, and dilated endoplasmic reticulum and Golgi apparatus. The seminal vesicle epithelial cells also degenerate prematurely, as evidenced by disruption of nuclear architecture and significant accumulations of autophagic bodies. These results identify BHLHB8 as a regulator in establishing and stabilizing the secreting epithelial cells of the seminal vesicle.