Stomach and intestinal stem cells are located in discrete niches called the isthmus and crypt, respectively. Recent studies have demonstrated a surprisingly conserved role for Wnt signaling in gastrointestinal development. Although intestinal stromal cells secrete Wnt ligands to promote stem cell renewal, the source of stomach Wnt ligands is still unclear. Here, by performing single cell analysis, we identify gastrointestinal stromal cell populations with transcriptome signatures that are conserved between the stomach and intestine. In close proximity to epithelial cells, these perictye-like cells highly express telocyte and pericyte markers as well as Wnt ligands, and they are enriched for Hh signaling. By analyzing mice activated for Hh signaling, we show a conserved mechanism of GLI2 activation of Wnt ligands. Moreover, genetic inhibition of Wnt secretion in perictye-like stromal cells or stromal cells more broadly demonstrates their essential roles in gastrointestinal regeneration and development, respectively, highlighting a redundancy in gastrointestinal stem cell niches.

Human embryonic stem cell-derived beta cells offer a promising cell-based therapy for diabetes. However, efficient stem cell to beta cell differentiation has proven difficult, possibly due to the lack of cross-talk with the appropriate mesenchymal niche. To define organ-specific niche signals, we isolated pancreatic and gastrointestinal stromal cells, and analyzed their gene expression during development. Our genetic studies reveal the importance of tightly regulated Hedgehog signaling in the pancreatic mesenchyme: inactivation of mesenchymal signaling leads to annular pancreas, whereas stroma-specific activation of signaling via loss of Hedgehog regulators, Sufu and Spop, impairs pancreatic growth and beta cell genesis. Genetic rescue and transcriptome analyses show that these Sufu and Spop knockout defects occur through Gli2-mediated activation of gastrointestinal stromal signals such as Wnt ligands. Importantly, inhibition of Wnt signaling in organoid and human stem cell cultures significantly promotes insulin-producing cell generation, altogether revealing the requirement for organ-specific regulation of stromal niche signals.

Hirschsprung disease is characterized by the absence of enteric neurons caused by the defects of enteric neural crest cells, leading to intestinal obstruction. Here, using induced pluripotent stem cell-based models of Hirschsprung and single-cell transcriptomic analysis, we identify a gene set of 118 genes commonly dysregulated in all patient enteric neural crest cells, and suggest HDAC1 may be a key regulator of these genes. Furthermore, upregulation of RNA splicing mediators and enhanced alternative splicing events are associated with severe form of Hirschsprung. In particular, the higher inclusion rate of exon 9 in PTBP1 and the perturbed expression of a PTBP1-target, PKM, are significantly enriched in these patient cells, and associated with the defective oxidative phosphorylation and impaired neurogenesis. Hedgehog-induced oxidative phosphorylation significantly enhances the survival and differentiation capacity of patient cells. In sum, we define various factors associated with Hirschsprung pathogenesis and demonstrate the implications of oxidative phosphorylation in enteric neural crest development and HSCR pathogenesis.

Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. Here, we analyzed pediatric and adult pineoblastoma samples (n = 23) using a combination of genome-wide DNA methylation profiling and whole-exome sequencing or whole-genome sequencing. Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower-grade pineal tumors and normal pineal gland. Recurrent variants were found in genes involved in PKA- and NF-κB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expresion of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain.

Sonic hedgehog medulloblastoma encompasses a clinically and molecularly diverse group of cancers of the developing central nervous system. Here, we use unbiased sequencing of the transcriptome across a large cohort of 250 tumors to reveal differences among molecular subtypes of the disease, and demonstrate the previously unappreciated importance of non-coding RNA transcripts. We identify alterations within the cAMP dependent pathway (GNAS, PRKAR1A) which converge on GLI2 activity and show that 18% of tumors have a genetic event that directly targets the abundance and/or stability of MYCN. Furthermore, we discover an extensive network of fusions in focally amplified regions encompassing GLI2, and several loss-of-function fusions in tumor suppressor genes PTCH1, SUFU and NCOR1. Molecular convergence on a subset of genes by nucleotide variants, copy number aberrations, and gene fusions highlight the key roles of specific pathways in the pathogenesis of Sonic hedgehog medulloblastoma and open up opportunities for therapeutic intervention.

Angiogenesis produces primitive vascular networks that need pruning to yield hierarchically organized and functional vessels. Despite the critical importance of vessel pruning to vessel patterning and function, the mechanisms regulating this process are not clear. Here we show that EphrinB2, a well-known player in angiogenesis, is an essential regulator of endothelial cell death and vessel pruning. This regulation depends upon phosphotyrosine-EphrinB2 signalling repressing c-jun N-terminal kinase 3 activity via STAT1. JNK3 activation causes endothelial cell death. In the absence of JNK3, hyaloid vessel physiological pruning is impaired, associated with abnormal persistence of hyaloid vessels, defective retinal vasculature and microphthalmia. This syndrome closely resembles human persistent hyperplastic primary vitreus (PHPV), attributed to failed involution of hyaloid vessels. Our results provide evidence that EphrinB2/STAT1/JNK3 signalling is essential for vessel pruning, and that defects in this pathway may contribute to PHPV.