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  • Collectively we have evaluated the impact of

    2018-10-20

    Collectively, we have evaluated the impact of a MODY5-causing point mutation (S148L) in the HNF1B gene on human pancreas development using a unique human stem cell model. We report molecular phenotypes (up- and down-regulation of pancreatic genes) when HNF1B is increasingly expressed from days 7 to 10, and observed that these phenotypes extend beyond the expression window of HNF1B. These data provide insights into the long-term impact of the MODY5 mutation on the pancreatic transcriptional network and the subsequent development of human pancreatic progenitors into mature functional endocrine cells. This is in agreement with Haumaitre et al. (2006), who indicated that MODY5 is certainly due to defective morphogenesis of the pancreas. Future efforts to further differentiate these hiPSCs into pancreatic β cmv virus will reveal the impact of transcriptional perturbations on β-cell formation and function.
    Experimental Procedures
    Author Contributions
    Acknowledgments The hiPSC lines used were derived with support from the Joslin DRC iPS Core Facility (NIH5 P30 DK036836-27). FACS analysis was supported by the HSCI/DRC Flow Core (NIH P30DK036836). Microarray studies were performed by the Molecular Genetics Core Facility at Children\'s Hospital Boston, supported by NIH-P50-NS40828 and NIH-P30-HD18655. We thank B. Wagner for assistance with microarray analyses; and K. Tomita, E.K. Tan, N.R. Dunn, Y.-H. Zhou, and Y.-I. Chi for providing plasmids. A.K.K.T. was supported by a Juvenile Diabetes Research Foundation (JDRF) Postdoctoral Fellowship and is currently supported by the Institute of Molecular and Cell Biology (IMCB), , NHG-KTPH SIG/14033, the NUHS-CG Metabolic In-Vitro Core Seed Funding, and the JCO Career Development Award (CDA) 15302FG148, A∗STAR. I.A.V. is supported by an HSCI Sternlicht Director\'s Fund Award and a NIH F31DK098931 award. E.D. is supported by an Advanced JDRF Postdoctoral Fellowship. H.R. is supported by grants from Bergen Forskningsstiftelse (BFS), the Western Norway Regional Health Authority and the Novo Nordisk Foundation. R.N.K. is supported by the HSCI, NIH grants RO1 DK 67536, RO1 DK 055523, and R01103215, and a grant from AstraZeneca.
    Introduction Establishment and maintenance of the adult hematopoietic system requires the generation of hematopoietic stem cells (HSCs) from a unique endothelial cell (hemogenic) in the aorta-gonad-mesonephros (AGM) region of the mammalian embryo (Dzierzak and Speck, 2008). HSCs develop in clusters that bud off from hemogenic endothelium (Bertrand et al., 2010; Boisset et al., 2010), a process termed endothelial to hematopoietic transition (EHT). HSCs migrate to and colonize the fetal liver and, subsequently, the bone marrow (Orkin and Zon, 2008). Whereas major efforts have focused on defining regulatory proteins/networks governing EHT, many questions remain unanswered regarding the molecular constituents and mechanisms. Master regulatory transcription factors co-localize at cis elements of target genes in hematopoietic stem and progenitor cells (HSPCs) to establish genetic networks that control hematopoiesis (Beck et al., 2013; Fujiwara et al., 2009; May et al., 2013; Tripic et al., 2008; Wilson et al., 2010; Wozniak et al., 2008; Yu et al., 2009). The combinatorial mechanisms operating in hemogenic endothelium, and the relationship between mechanisms governing EHT and HSC multi-potency, are unclear. A shared component of the mechanisms involves the transcription factor GATA-2, which is required for definitive hematopoiesis (Tsai et al., 1994). GATA-2 functions in hemogenic endothelium to induce EHT and regulates HSC function (de Pater et al., 2013; Gao et al., 2013; Johnson et al., 2012; Ling et al., 2004; Rodrigues et al., 2005). Since GATA-2 induces EHT, it is instructive to consider factors/signals upstream of GATA-2. Deletion of a cis element 9.5 kb downstream of the Gata2 promoter (+9.5) in mice decreased Gata2 cmv virus expression in AGM hemogenic endothelium, deregulated genes encoding positive regulators of hematopoiesis, and abrogated EHT (Gao et al., 2013). Deletion of a cis element 77 kb upstream of the promoter (−77) reduced Gata2 expression in myelo-erythroid progenitors and impaired progenitor function without affecting EHT (Johnson et al., 2015). The defective HSC generator of +9.5−/− embryos depleted HSPCs in the fetal liver and caused lethality at embryonic day 13–14 (E13–14) (Johnson et al., 2012). Since the +9.5 controls Gata2 expression and EHT (Gao et al., 2013; Hsu et al., 2013), and GATA-2 occupies the +9.5 (Fujiwara et al., 2009; Grass et al., 2006), one aspect of the +9.5 mechanism involves GATA-2-mediated positive autoregulation. Factors implicated upstream of GATA-2 include bone morphogenetic protein 4 (Lugus et al., 2007; Maeno et al., 1996), Notch signaling (Guiu et al., 2013; Robert-Moreno et al., 2008), the Ets factor Etv2 (Liu et al., 2015), and the methylcytosine dioxygenases Tet2/Tet3 (Li et al., 2015).