Exome sequencing efficacy and phenotypic expansions involving esophageal atresia/tracheoesophageal fistula plus.

Esophageal atresia/tracheoesophageal fistula (EA/TEF) is a life-threatening birth defect that often occurs with other major birth defects (EA/TEF+). Despite advances in genetic testing, a molecular diagnosis can only be made in a minority of EA/TEF+ cases. Here, we analyzed clinical exome sequencing data and data from the DECIPHER database to determine the efficacy of exome sequencing in cases of EA/TEF+ and to identify phenotypic expansions involving EA/TEF. Among 67 individuals with EA/TEF+ referred for clinical exome sequencing, a definitive or probable diagnosis was made in 11 cases for an efficacy rate of 16% (11/67). This efficacy rate is significantly lower than that reported for other major birth defects, suggesting that polygenic, multifactorial, epigenetic, and/or environmental factors may play a particularly important role in EA/TEF pathogenesis. Our cohort included individuals with pathogenic or likely pathogenic variants that affect TCF4 and its downstream target NRXN1, and FANCA, FANCB, and FANCC, which are associated with Fanconi anemia. These cases, previously published case reports, and comparisons to other EA/TEF genes made using a machine learning algorithm, provide evidence in support of a potential pathogenic role for these genes in the development of EA/TEF.

Tracheal separation is driven by NKX2-1-mediated repression of Efnb2 and regulation of endodermal cell sorting.

The mechanisms coupling fate specification of distinct tissues to their physical separation remain to be understood. The trachea and esophagus differentiate from a single tube of definitive endoderm, requiring the transcription factors SOX2 and NKX2-1, but how the dorsoventral site of tissue separation is defined to allocate tracheal and esophageal cell types is unknown. Here, we show that the EPH/EPHRIN signaling gene Efnb2 regulates tracheoesophageal separation by controlling the dorsoventral allocation of tracheal-fated cells. Ventral loss of NKX2-1 results in disruption of separation and expansion of Efnb2 expression in the trachea independent of SOX2. Through chromatin immunoprecipitation and reporter assays, we find that NKX2-1 likely represses Efnb2 directly. Lineage tracing shows that loss of NKX2-1 results in misallocation of ventral foregut cells into the esophagus, while mosaicism for Nkx2-1 generates ectopic NKX2-1/EPHRIN-B2 boundaries that organize ectopic tracheal separation. Together, these data demonstrate that NKX2-1 coordinates tracheal specification with tissue separation through the regulation of EPHRIN-B2 and tracheoesophageal cell sorting.

Delineating the early transcriptional specification of the mammalian trachea and esophagus.

The genome-scale transcriptional programs that specify the mammalian trachea and esophagus are unknown. Though NKX2-1 and SOX2 are hypothesized to be co-repressive master regulators of tracheoesophageal fates, this is untested at a whole transcriptomic scale and their downstream networks remain unidentified. By combining single-cell RNA-sequencing with bulk RNA-sequencing of Nkx2-1 mutants and NKX2-1 ChIP-sequencing in mouse embryos, we delineate the NKX2-1 transcriptional program in tracheoesophageal specification, and discover that the majority of the tracheal and esophageal transcriptome is NKX2-1 independent. To decouple the NKX2-1 transcriptional program from regulation by SOX2, we interrogate the expression of newly-identified tracheal and esophageal markers in Sox2/Nkx2-1 compound mutants. Finally, we discover that NKX2-1 binds directly to Shh and Wnt7b and regulates their expression to control mesenchymal specification to cartilage and smooth muscle, coupling epithelial identity with mesenchymal specification. These findings create a new framework for understanding early tracheoesophageal fate specification at the genome-wide level.

The trachea or windpipe is a tube that connects the throat to the lungs, while the esophagus connects the throat to the stomach. The trachea has cartilage rings that help to ensure clear airflow to the lungs, while the esophagus walls are lined with muscles that help to move food to the stomach. Although there are many differences between them, both the trachea and esophagus form from the same group of cells during development. Proteins called transcription factors help to control the formation of different body parts by switching different groups of genes on and off in different subsets of cells. Existing research has suggested that a transcription factor called NKX2.1 drives trachea formation, while another, called SOX2, is important in esophagus formation. An absence of either of these two proteins is thought to be associated with serious birth defects including loss of the trachea or esophagus, or failure of the two to separate fully. How these two transcription factors interact and drive the development of the trachea and esophagus, however, is currently unclear. Kuwahara et al. used mice to study the role of NKX2.1 and SOX2 in the formation of the trachea and esophagus. The findings identify many new genes that are active in the trachea and esophagus and reveal that NKX2.1 is not a master regulator that controls all of the genes involved in trachea formation. However, NKX2.1 does control several key genes, particularly those involved in forming cartilage in the trachea instead of muscle in the esophagus. The investigation also revealed many genes that are not controlled by NKX2.1 suggesting that other, currently unknown, systems play a major role in trachea formation. More work is required to understand the wider genetic regulators involved in differentiating the trachea from the esophagus. The findings in this study will help researchers to understand birth defects in the trachea and esophagus that result from genetic errors. They also reveal information about gene regulation processes that are relevant to the formation of other body parts and in the context of other diseases. In the long term, they could support regenerative medicine to regrow or replace lost or damaged body parts using lab-grown stem cells.

Adult hippocampal neural stem and progenitor cells regulate the neurogenic niche by secreting VEGF.

The adult hippocampus hosts a population of neural stem and progenitor cells (NSPCs) that proliferates throughout the mammalian life span. To date, the new neurons derived from NSPCs have been the primary measure of their functional relevance. However, recent studies show that undifferentiated cells may shape their environment through secreted growth factors. Whether endogenous adult NSPCs secrete functionally relevant growth factors remains unclear. We show that adult hippocampal NSPCs secrete surprisingly large quantities of the essential growth factor VEGF in vitro and in vivo. This self-derived VEGF is functionally relevant for maintaining the neurogenic niche as inducible, NSPC-specific loss of VEGF results in impaired stem cell maintenance despite the presence of VEGF produced from other niche cell types. These findings reveal adult hippocampal NSPCs as an unanticipated source of an essential growth factor and imply an exciting functional role for adult brain NSPCs as secretory cells.