Primate-specific ZNF808 is essential for pancreatic development in humans [RNA-seq]
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ABSTRACT: Functional investigation of the role primate-specific gene ZNF808 over pancreas differentiation. Loss of function of ZNF808 is a cause of pancreatic agenesis, which is a failure of pancreas development.
Project description:Functional investigation of the role primate-specific gene ZNF808 over pancreas differentiation. Loss of function of ZNF808 is a cause of pancreatic agenesis, which is a failure of pancreas development.
Project description:Identifying genes linked to extreme phenotypes in humans has the potential to highlight biological processes not shared with all other mammals. Here, we report the identification of homozygous loss-of-function variants in the primate-specific gene ZNF808 as a cause of pancreatic agenesis. ZNF808 is a member of the KRAB zinc finger protein family, a large and rapidly evolving group of epigenetic silencers which target transposable elements. We show that loss of ZNF808 in vitro results in aberrant activation of regulatory potential contained in the primate-specific transposable elements it represses during early pancreas development. This leads to inappropriate specification of cell fate with induction of genes associated with liver identity. Our results highlight the essential role of ZNF808 in pancreatic development in humans and the contribution of primate-specific regions of the human genome to congenital developmental disease.
Project description:Skin biopsies were obtained from a patient with Mitchell-Riley syndrome caused by a homozygous frame-shift mutation (c.1129C>T) in the RFX6 gene that leads to a premature stop codon (p.Arg377X). The patient suffered severe pancreatic agenesis, in common with other Mitchell-Riley syndrome patients. Fibroblasts from the biopsy were reprogrammed to generate a human induced pluripotent stem cell (hiPSC) line (MRS2-6). To assess the effects of the mutant RFX6 allele on pancreas formation and identify direct targets of the transcription factor RFX6, MRS2-6 and H9 control human embryonic stem cells (hESC) were differentiated into pancreatic progenitors. Samples were harvested for RNA isolation and whole transcriptome analysis at days 4 (definitive endoderm), 7 and 8 (gut tube), and 12 (pancreatic progenitors).
Project description:To interrogate the alternative fates of pancreas and liver in the earliest stages of human organogenesis, we developed laser capture, RNA amplification, and computational analysis of deep sequencing. Pancreas-enriched gene expression was less conserved between human and mouse than for liver. The dorsal pancreatic bud was enriched for components of Notch, Wnt, BMP, and FGF signaling, almost all genes known to cause pancreatic agenesis or hypoplasia, and over 30 unexplored transcription factors. SOX9 and RORA were imputed as key regulators in pancreas compared with EP300, HNF4A, and FOXA family members in liver. Analyses implied that current in vitro human stem cell differentiation follows a dorsal rather than a ventral pancreatic program and pointed to additional factors for hepatic differentiation. In summary, we provide the transcriptional codes regulating the start of human liver and pancreas development to facilitate stem cell research and clinical interpretation without inter-species extrapolation.
Project description:During embryogenesis, the pancreas develops from separate dorsal and ventral buds, which fuse to form the mature pancreas. Little is known about the functional differences between these two buds or the relative contribution of cells derived from each portion to the pancreas after fusion. To follow the fate of dorsal or ventral bud derived cells in the pancreas after fusion, we produced chimeric Elas-GFP transgenic/wild type embryos in which either dorsal or ventral pancreatic bud cells expressed GFP. We found that ventral pancreatic cells migrate extensively into the dorsal pancreas after fusion, whereas the converse does not occur. Moreover, we found that annular pancreatic tissue is composed exclusively of ventral pancreas derived cells. To identify ventral pancreas specific genes that may play a role in pancreatic bud fusion, we isolated individual dorsal and ventral pancreatic buds, prior to fusion, from stage 38/39 Xenopus laevis tadpoles and compared their gene expression profiles. Morpholino-mediated knockdown of one of these ventral specific genes, transmembrane 4 superfamily member 3 (tm4sf3), inhibited dorsal-ventral pancreatic bud fusion as well as acinar cell differentiation. Conversely, overexpression of tm4sf3 promoted the development of annular pancreas. Our results are the first to define molecular and behavioral differences between the dorsal and ventral pancreas, and suggest an unexpected role for the ventral pancreas in pancreatic bud fusion. Experiment Overall Design: We analyzed two samples of dorsal and two samples of ventral pancreatic buds.