Papizan JB, Singer RA, Tschen SI, Dhawan S, Friel JM, Hipkens SB, Magnuson MA, Bhushan A, Sussel L. Nkx2.2 repressor complex regulates islet β-cell specification and prevents β-to-α-cell reprogramming. (2011) Genes Dev 25: 2291-305
Show Abstract · Added January 8, 2012Regulation of cell differentiation programs requires complex interactions between transcriptional and epigenetic networks. Elucidating the principal molecular events responsible for the establishment and maintenance of cell fate identities will provide important insights into how cell lineages are specified and maintained and will improve our ability to recapitulate cell differentiation events in vitro. In this study, we demonstrate that Nkx2.2 is part of a large repression complex in pancreatic β cells that includes DNMT3a, Grg3, and HDAC1. Mutation of the endogenous Nkx2.2 tinman (TN) domain in mice abolishes the interaction between Nkx2.2 and Grg3 and disrupts β-cell specification. Furthermore, we demonstrate that Nkx2.2 preferentially recruits Grg3 and HDAC1 to the methylated Aristaless homeobox gene (Arx) promoter in β cells. The Nkx2.2 TN mutation results in ectopic expression of Arx in β cells, causing β-to-α-cell transdifferentiation. A corresponding β-cell-specific deletion of DNMT3a is also sufficient to cause Arx-dependent β-to-α-cell reprogramming. Notably, subsequent removal of Arx in the β cells of Nkx2.2(TNmut/TNmut) mutant mice reverts the β-to-α-cell conversion, indicating that the repressor activities of Nkx2.2 on the methylated Arx promoter in β cells are the primary regulatory events required for maintaining β-cell identity. | Publication | 22056672 (PMID)
PMC3219233 (PMCID)
10.1101/gad.173039.111 (DOI) |
Potter LA, Choi E, Hipkens SB, Wright CV, Magnuson MA. A recombinase-mediated cassette exchange-derived cyan fluorescent protein reporter allele for Pdx1. (2011) Genesis :
Show Abstract · Added January 8, 2012Fluorescent protein (FP) reporter alleles are useful both for identifying and purifying specific cell populations in the mouse. Here, we report the generation of mouse embryonic stem cells that contain a pancreatic and duodenal homeobox 1 (Pdx1) loxed cassette acceptor (Pdx1(LCA) ) allele and the use of recombinase-mediated cassette exchange to derive mice that contain a Pdx1(CFP) (Cerulean) reporter allele. Mice with this allele exhibited cyan fluorescence within the previously well-characterized Pdx1 expression domain in posterior foregut endoderm. Immunolabeling showed that endogenous Pdx1 was coexpressed with CFP at all time points examined. Furthermore, fluorescence-activated cell sorting was used to isolate CFP-positive cells from E11.5 and E18.5 embryonic tissues using both 405 and 445 nm lasers, although the latter resulted in a nearly 50-fold increase in emission intensity. The Pdx1(CFP) allele will enable the isolation of specific foregut endoderm and pancreatic cell populations, both alone and in combination with other FP reporter alleles. genesis 00:1-9, 2012. © 2011 Wiley Periodicals, Inc. | Publication | 21913313 (PMID)
10.1002/dvg.20804 (DOI) |
Porat S, Weinberg-Corem N, Tornovsky-Babaey S, Schyr-Ben-Haroush R, Hija A, Stolovich-Rain M, Dadon D, Granot Z, Ben-Hur V, White P, Girard CA, Karni R, Kaestner KH, Ashcroft FM, Magnuson MA, Saada A, Grimsby J, Glaser B, Dor Y. Control of pancreatic β cell regeneration by glucose metabolism. (2011) Cell Metab 13: 440-9
Show Abstract · Added January 8, 2012Recent studies revealed a surprising regenerative capacity of insulin-producing β cells in mice, suggesting that regenerative therapy for human diabetes could in principle be achieved. Physiologic β cell regeneration under stressed conditions relies on accelerated proliferation of surviving β cells, but the factors that trigger and control this response remain unclear. Using islet transplantation experiments, we show that β cell mass is controlled systemically rather than by local factors such as tissue damage. Chronic changes in β cell glucose metabolism, rather than blood glucose levels per se, are the main positive regulator of basal and compensatory β cell proliferation in vivo. Intracellularly, genetic and pharmacologic manipulations reveal that glucose induces β cell replication via metabolism by glucokinase, the first step of glycolysis, followed by closure of K(ATP) channels and membrane depolarization. Our data provide a molecular mechanism for homeostatic control of β cell mass by metabolic demand. | Publication | 21459328 (PMID)
10.1016/j.cmet.2011.02.012 (DOI) |
Chen SX, Osipovich AB, Ustione A, Potter LA, Hipkens S, Gangula R, Yuan W, Piston DW, Magnuson MA. Quantification of factors influencing fluorescent protein expression using RMCE to generate an allelic series in the ROSA26 locus in mice. (2011) Dis Model Mech :
Show Abstract · Added February 23, 2011Fluorescent proteins (FPs) have great utility in identifying specific cell populations and in studying cellular dynamics in the mouse. To quantify the factors that determine both the expression and relative brightness of FPs in mouse embryonic stem cells (mESCs) and in mice, we generated eight different FP-expressing ROSA26 alleles using recombinase-mediated cassette exchange (RMCE). These alleles enabled us to analyze the effects on FP expression of a translational enhancer and different 3'-intronic and/or polyadenylation sequences, as well as the relative brightness of five different FPs, without the confounding position and copy number effects that are typically associated with randomly inserted transgenes. We found that the expression of a given FP can vary threefold or more depending on the genetic features present in the allele. The optimal FP expression cassette contained both a translational enhancer sequence in the 5'-untranslated region (UTR) and an intron-containing rabbit β-globin sequence within the 3'-UTR. The relative expressed brightness of individual FPs varied up to tenfold. Of the five different monomeric FPs tested, Citrine (YFP) was the brightest, followed by Apple, eGFP, Cerulean (CFP) and Cherry. Generation of a line of Cherry-expressing mice showed that there was a 30-fold variation of Cherry expression among different tissues and that there was a punctate expression pattern within cells of all tissues examined. This study should help investigators make better-informed design choices when expressing FPs in mESCs and mice. | Publication | 21324933 (PMID)
10.1242/dmm.006569 (DOI) |
Gu Y, Lindner J, Kumar A, Yuan W, Magnuson MA. Rictor/mTORC2 Is Essential for Maintaining a Balance Between {beta}-Cell Proliferation and Cell Size. (2011) Diabetes :
Show Abstract · Added February 23, 2011OBJECTIVE We examined the role of Rictor/mammalian target of rapamycin complex 2 (mTORC2), a key component of the phosphotidylinositol-3-kinase (PI3K)/mTORC2/AKT signaling pathway, in regulating both β-cell mass and function. RESEARCH DESIGN AND METHODS Mice with β-cell-specific deletions of Rictor or Pten were studied to determine the effects of deleting either or both genes on β-cell mass and glucose homeostasis. RESULTS Rictor null mice exhibited mild hyperglycemia and glucose intolerance caused by a reduction in β-cell mass, β-cell proliferation, pancreatic insulin content, and glucose-stimulated insulin secretion. Islets from these mice exhibited decreased AKT-S473 phosphorylation and increased abundance of FoxO1 and p27 proteins. Conversely, Pten null (βPtenKO) mice exhibited an increase in β-cell mass caused by increased cellular proliferation and size. Although β-cell mass was normal in mice lacking both Rictor and Pten (βDKO), their β-cells were larger than those in the βPtenKO mice. Even though the β-cell proliferation rate in the βDKO mice was lower than in the βPtenKO mice, there was a 12-fold increase the phosphorylation of AKT-T308. CONCLUSIONS PI3K/AKT signaling through mTORC2/pAKT-S473 plays a key role in maintaining normal β-cell mass. The phosphorylation of AKT-S473, by negatively regulating that of AKT-T308, is essential for maintaining a balance between β-cell proliferation and cell size in response to proliferative stimuli. | Publication | 21266327 (PMID)
10.2337/db10-1194 (DOI) |
Huppert SS, Magnuson MA. New complexity in differentiating stem cells toward hepatic and pancreatic fates. (2009) Sci Signal 2: pe50
Show Abstract · Added February 23, 2011The differentiation of hepatic and pancreatic progenitor cells during embryogenesis is determined by inductive factors secreted by neighboring cells. These factors stimulate and repress the expression of key regulatory genes in progenitor cells, thereby establishing unique genetic programs that determine cell fate. The signaling network that controls liver and pancreas development is highly dynamic with respect to both concentration and timing of exposure to several key inductive factors. Not only do large changes occur within short time frames, multiple signaling pathways also converge on the same target genes. Given the intense effort under way to generate certain differentiated cell types from both embryonic and induced pluripotent stem cells, greater understanding of how different inductive signals interact with each other may be essential for the eventual success of such efforts. | Publication | 19671927 (PMID)
10.1126/scisignal.283pe50 (DOI) |
Burlison JS, Long Q, Fujitani Y, Wright CV, Magnuson MA. Pdx-1 and Ptf1a concurrently determine fate specification of pancreatic multipotent progenitor cells. (2008) Dev Biol 316: 74-86
Show Abstract · Added April 8, 2010The pancreas is derived from a pool of multipotent progenitor cells (MPCs) that co-express Pdx-1 and Ptf1a. To more precisely define how the individual and combined loss of Pdx-1 and Ptf1a affects pancreatic MPC specification and differentiation we derived and studied mice bearing a novel Ptf1a(YFP) allele. While the expression of Pdx-1 and Ptf1a in pancreatic MPCs coincides between E9.5 and 12.5 the developmental phenotypes of Pdx-1 null and Pdx-1; Ptf1a double null mice are indistinguishable, and an early pancreatic bud is formed in both cases. This finding indicates that Pdx-1 is required in the foregut endoderm prior to Ptf1a for pancreatic MPC specification. We also found that Ptf1a is neither required for specification of Ngn3-positive endocrine progenitors nor differentiation of mature beta-cells. In the absence of Pdx-1 Ngn3-positive cells were not observed after E9.5. Thus, in contrast to the deletion of Ptf1a, the loss of Pdx-1 precludes the sustained Ngn3-based derivation of endocrine progenitors from pancreatic MPCs. Taken together, these studies indicate that Pdx-1 and Ptf1a have distinct but interdependent functions during pancreatic MPC specification. | Publication | 18294628 (PMID)
PMC2425677 (PMCID)
10.1016/j.ydbio.2008.01.011 (DOI) |
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