Vertebrate organogenesis is a highly complex process involving sequential cascades of transcription factor activation or repression. Interestingly a single developmental control gene can occasionally be essential for the morphogenesis and differentiation of tissues and organs arising from vastly disparate embryological lineages.

The data described in this article refers to Chatterjee et al. (2015) "In vivo genome-wide analysis of multiple tissues identifies gene regulatory networks, novel functions and downstream regulatory genes for Bapx1 and its co-regulation with Sox9 in the mammalian vertebral column" (GEO GSE35649) [1]. Transcriptional profiling combined with genome wide binding data is a powerful tool to elucidate the molecular mechanism behind vertebrate organogenesis. It also helps to uncover multiple roles of a single gene in different organs. In the above mentioned report we reveal the function of the homeobox gene Bapx1 during the embryogenesis of five distinct organs (vertebral column, spleen, gut, forelimb and hindlimb) at a relevant developmental stage (E12.5), microarray analysis of isolated wildtype and mutant cells in is compared in conjunction with ChIP-Seq analysis. We also analyzed the development of the vertebral column by comparing microarray and ChIP-Seq data for Bapx1 with similarly generated data sets for Sox9 to generate a gene regulatory network controlling various facets of the organogenesis.

Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidence that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders.

The receptor tyrosine kinase RET plays a critical role in the fate specification of enteric neural crest-derived cells (ENCDCs) during enteric nervous system (ENS) development. RET loss of function (LoF) is associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. Although the major phenotypic consequences and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, cell type- and state-specific effects are unknown. We performed single-cell RNA sequencing on an enriched population of ENCDCs from the developing GI tract of Ret null heterozygous and homozygous mice at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: 1) Ret-expressing ENCDCs are a heterogeneous population comprising ENS progenitors as well as glial- and neuronal-committed cells; 2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; 3) expression patterns of HSCR-associated and Ret gene regulatory network genes are impacted by Ret LoF; and 4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes during development of the ENS, including the specification of inhibitory neuron subtypes, cell cycle dynamics of ENS progenitors, and the developmental timing of neuronal and glial commitment.

Identifying DNA sequences (enhancers) that direct the precise spatial and temporal expression of developmental control genes remains a significant challenge in the annotation of vertebrate genomes. Locating these sequences, which in many cases lie at a great distance from the transcription start site, has been a major obstacle in deciphering gene regulation. Coupling of comparative genomics with functional validation to locate such regulatory elements has been a successful method in locating many such regulatory elements. But most of these studies looked either at a single gene only or the whole genome without focusing on any particular process. The pressing need is to integrate the tools of comparative genomics with knowledge of developmental biology to validate enhancers for developmental transcription factors in greater detail

Sensorineural hearing loss is one of the most common disabilities in humans. It is estimated that about 278 million people worldwide have slight to extreme hearing loss in both ears, which results in an economic loss for the country and personal loss for the individual. It is thus critical to have a deeper understanding of the causes for hearing loss to better manage and treat the affected individuals. The mouse serves as an excellent model to study and recapitulate some of these phenotypes, identify new genes which cause deafness, and to study their roles in vivo and in detail. Mutant mice have been instrumental in elucidating the function and mechanisms of the inner ear. The development and morphogenesis of the inner ear from an ectodermal layer into distinct auditory and vestibular components depends on well-coordinated gene expression and well-orchestrated signaling cascades within the otic vesicle and interactions with surrounding layers of tissues. Any disruption in these pathways can lead to hearing impairment. This review takes a look at some of the genes and their corresponding mice mutants that have shed light on the mechanism governing hearing impairment (HI) in humans.

QT interval variation is assumed to arise from variation in repolarization as evidenced from rare Na- and K-channel mutations in Mendelian QT prolongation syndromes. However, in the general population, common noncoding variants at a chromosome 1q locus are the most common genetic regulators of QT interval variation. In this study, we use multiple human genetic, molecular genetic, and cellular assays to identify a functional variant underlying trait association: a noncoding polymorphism (rs7539120) that maps within an enhancer of NOS1AP and affects cardiac function by increasing NOS1AP transcript expression. We further localized NOS1AP to cardiomyocyte intercalated discs (IDs) and demonstrate that overexpression of NOS1AP in cardiomyocytes leads to altered cellular electrophysiology. We advance the hypothesis that NOS1AP affects cardiac electrical conductance and coupling and thereby regulates the QT interval through propagation defects. As further evidence of an important role for propagation variation affecting QT interval in humans, we show that common polymorphisms mapping near a specific set of 170 genes encoding ID proteins are significantly enriched for association with the QT interval, as compared to genome-wide markers. These results suggest that focused studies of proteins within the cardiomyocyte ID are likely to provide insights into QT prolongation and its associated disorders.

Previous research in autism and other neurodevelopmental disorders (NDDs) has indicated an important contribution of protein-coding (coding) de novo variants (DNVs) within specific genes. The role of de novo noncoding variation has been observable as a general increase in genetic burden but has yet to be resolved to individual functional elements. In this study, we assessed whole-genome sequencing data in 2671 families with autism (discovery cohort of 516 families, replication cohort of 2155 families). We focused on DNVs in enhancers with characterized in vivo activity in the brain and identified an excess of DNVs in an enhancer named hs737.

The major genetic risk factors for Hirschsprung disease (HSCR) are three common polymorphisms within cis-regulatory elements (CREs) of the receptor tyrosine kinase gene RET, which reduce its expression during enteric nervous system (ENS) development. These risk variants attenuate binding of the transcription factors RARB, GATA2, and SOX10 to their cognate CREs, reduce RET gene expression, and dysregulate other ENS and HSCR genes in the RET-EDNRB gene regulatory network (GRN). Here, we use siRNA, ChIP, and CRISPR-Cas9 deletion analyses in the SK-N-SH cell line to ask how many additional HSCR-associated risk variants reside in RET CREs that affect its gene expression. We identify 22 HSCR-associated variants in candidate RET CREs, of which seven have differential allele-specific in vitro enhancer activity, and four of these seven affect RET gene expression; of these, two enhancers are bound by the transcription factor PAX3. We also show that deleting multiple variant-containing enhancers leads to synergistic effects on RET gene expression. These, coupled with our prior results, show that common sequence variants in at least 10 RET enhancers affect HSCR risk, seven with experimental evidence of affecting RET gene expression, extending the known RET-EDNRB GRN to reveal an extensive regulatory code modulating disease risk at a single gene.

This work pertains to GEO submission GSE36672, in vivo and in vitro genome wide binding (ChIP-Seq) of Bapx1/Nkx3.2 and Sox9 proteins. We have previously shown that data from a genome wide binding assay combined with transcriptional profiling is an insightful means to divulge the mechanisms directing cell type specification and the generation of tissues and subsequent organs [1]. Our earlier work identified the role of the DNA-binding homeodomain containing protein Bapx1/Nkx3.2 in midgestation murine embryos. Microarray analysis of EGFP-tagged cells (both wildtype and null) was integrated using ChIP-Seq analysis of Bapx1/Nkx3.2 and Sox9 DNA-binding proteins in living tissue.

Disruptions in gene regulatory networks (GRNs), driven by multiple deleterious variants, potentially underlie complex traits and diseases. Hirschsprung disease (HSCR), a multifactorial disorder of enteric nervous system (ENS) development, is associated with at least 24 genes and seven chromosomal loci, with RET and EDNRB as its major genes. We previously demonstrated that RET transcription in the ENS is controlled by an extensive GRN involving the transcription factors (TFs) RARB, GATA2 and SOX10 and other HSCR genes. We now demonstrate, using human and mouse cellular and animal models, that EDNRB is transcriptionally regulated in the ENS by GATA2, SOX10 and NKX2.5 TFs. Significantly, RET and EDNRB expression is regulated by their shared use of GATA2 and SOX10, and in turn, these TFs are controlled by EDNRB and RET in a dose-dependent manner. This study expands the ENS development GRN to include both RET and EDNRB, uncovers the mechanistic basis for RET-EDNRB epistasis and emphasizes how functionally different genes associated with a complex disorder can be united through a common GRN.

Hirschsprung disease (HSCR) is associated with deficiency of the receptor tyrosine kinase RET, resulting in loss of cells of the enteric nervous system (ENS) during fetal gut development. The major contribution to HSCR risk is from common sequence variants in RET enhancers with additional risk from rare coding variants in many genes. Here, we demonstrate that these RET enhancer variants specifically alter the human fetal gut development program through significant decreases in gene expression of RET, members of the RET-EDNRB gene regulatory network (GRN), other HSCR genes, with an altered transcriptome of 2,382 differentially expressed genes across diverse neuronal and mesenchymal functions. A parsimonious hypothesis for these results is that beyond RET's direct effect on its GRN, it also has a major role in enteric neural crest-derived cell (ENCDC) precursor proliferation, its deficiency reducing ENCDCs with relative expansion of non-ENCDC cells. Thus, genes reducing RET proliferative activity can potentially cause HSCR. One such class is the 23 RET-dependent transcription factors enriched in early gut development. We show that their knockdown in human neuroblastoma SK-N-SH cells reduces RET and/or EDNRB gene expression, expanding the RET-EDNRB GRN. The human embryos we studied had major remodeling of the gut transcriptome but were unlikely to have had HSCR: thus, genetic or epigenetic changes in addition to those in RET are required for aganglionosis.

Embryogenesis is an intricate process involving multiple genes and pathways. Some of the key transcription factors controlling specific cell types are the Sox trio, namely, Sox5, Sox6, and Sox9, which play crucial roles in organogenesis working in a concerted manner. Much however still needs to be learned about their combinatorial roles during this process. A developmental genomics and systems biology approach offers to complement the reductionist methodology of current developmental biology and provide a more comprehensive and integrated view of the interrelationships of complex regulatory networks that occur during organogenesis. By combining cell type-specific transcriptome analysis and in vivo ChIP-Seq of the Sox trio using mouse embryos, we provide evidence for the direct control of Sox5 and Sox6 by the transcriptional trio in the murine model and by Morpholino knockdown in zebrafish and demonstrate the novel role of Tgfb2, Fbxl18, and Tle3 in formation of Sox5, Sox6, and Sox9 dependent tissues. Concurrently, a complete embryonic gene regulatory network has been generated, identifying a wide repertoire of genes involved and controlled by the Sox trio in the intricate process of normal embryogenesis.