Project description:Esophageal atresia (EA) with or without tracheoesophageal fistula (TEF) are tracheoesophageal (TE) birth defects which affect 1 in 3000 live births in the United States. EA occurs when the esophagus fails to form a continuous tube from the oral cavity to the stomach and TEF occurs when there is an abnormal connection between the trachea and esophagus. The etiology of EA/TEF arises when the common foregut tube fails to properly separate into the esophagus and respiratory tract during early embryogenesis. This process is essential for proper organ function and involves complex molecular and morphological processes. As part of a multi-institutional project aiming to identify genes causing tracheoesophageal defects, we identified two EA/TEF patients who presented with different mutations in SMAD6, a BMP signaling inhibitor. We used a CRISPR-based approach to knock down smad6 in frog embryos and observed separation defects of the esophagus and trachea, supporting the conclusion that SMAD6 mutations in humans may cause EA/TEF. To investigate the role of SMAD6 during human development, we used CRISPR to generate human pluripotent stem cell lines with loss-of-function alleles in SMAD6. The human foregut is comprised of an inner layer of endoderm-derived epithelium surrounded by mesenchyme, and it is not known if SMAD6 functions in either or both layers. To elucidate this, we investigated the role of SMAD6 in either foregut epithelium or mesenchyme, or both in combination. Our analysis of human SMAD6-/- tracheoesophageal epithelium and mesenchyme reveals an increase in BMP/TGFβ signaling, consistent with its role as a repressor of these pathways. We also observed patterning defects in both tissue types; SMAD6 mutant endoderm shows increased expression of distal gut tube markers and SMAD6 mutant mesenchyme shows increased expression of cardiac markers. Furthermore, we observed cell differentiation changes, where SMAD6-/- mesenchyme had decreased ability to form CD31-positive vasculature, as well as changes in genes involved in processes such as cell migration and extracellular matrix deposition, which are critical for mesenchyme morphogenesis and differentiation during the process of TE separation. Our results suggest SMAD6 mutations impact the identity and function of human engineered TE tissue. By identifying pathways significant for TE development during early embryogenesis, our results contribute to understanding how patient mutations cause congenital malformations.

Project description:Background: CrohnM-bM-^@M-^Ys disease is presently an incurable inflammatory bowel disease. Fistulae are extensions of the intestinal tract that may form connections with other organs. These are a common complication of CrohnM-bM-^@M-^Ys disease affecting up to 50% of patients with the most common including perianal and rectovaginal fistulae. The dysregulated growth observed in fistulae shares several major characteristics seen in the development of tumours. These similarities include epithelial-to-mesenchymal transition (EMT), invasive cell growth, increased extracellular matrix production and remodelling, up-regulated local expression of growth factors such as IGF-1 and the down-regulation of apoptosis pathways. Although several susceptibility loci have been described within CrohnM-bM-^@M-^Ys disease there is no individual gene or mutation that identifies susceptibility or the subsequent formation of fistulae within all people. Copy-number variation (CNV) is one mechanism that may explain much of this genetic complexity. CNV may be caused by a variety of mechanisms such as cycles of chromosomal breakage/fusion/bridging that are typical within chronically inflamed tissue. Interestingly CNV shows locus-specific mutation rates between individuals higher than that of SNPs and has been associated with complex Mendelian traits including disease susceptibility. In this current study we performed array comparative genomic hybridisation (aCGH) analysis of active fistulae resected from patients as part of a previous surgical intervention. As the control for comparison we employed tissue taken from the same patient within the same surgery at an uninvolved site of the gastrointestinal tract. This matched control was employed to better investigate CNV specific to the fistula tissue of each individual avoiding complications associated with the large number of CNV present within the healthy population. Major question addressed by the work: What are the differences in genetic copy-number within localised intestinal fistula tissue of individuals with CrohnM-bM-^@M-^Ys disease compared to healthy intestinal tissue from the same individual? Samples were selected from patients presenting with CrohnM-bM-^@M-^Ys Disease fistulae at Eastern Health Department of Gastroenterology and Hepatology (Arnold Street, Box Hill, Victoria, Australia). The 8 samples used in this study were formalin-fixed paraffin-embedded (FFPE) specimens following a surgical intervention to remove fistula tissue. To focus on changes within the individual patient we used tissue taken from the same surgery at an uninvolved region of the intestine as the matched control for CNV analysis.

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Tracheoesophageal fistula characterisation

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