Cole, L., Anderson, M., Antin, P. B., & Limesand, S. W. (2009). One process for pancreatic beta-cell coalescence into islets involves an epithelial-mesenchymal transition. The Journal of endocrinology, 203(1), 19-31.
BIO5 Collaborators
Parker B Antin, Sean W Limesand
Islet replacement is a promising therapy for treating diabetes mellitus, but the supply of donor tissue for transplantation is limited. To overcome this limitation, endocrine tissue can be expanded, but this requires an understanding of normal developmental processes that regulate islet formation. In this study, we compare pancreas development in sheep and human, and provide evidence that an epithelial-mesenchymal transition (EMT) is involved in beta-cell differentiation and islet formation. Transcription factors know to regulate pancreas formation, pancreatic duodenal homeobox factor 1, neurogenin 3, NKX2-2, and NKX6-1, which were expressed in the appropriate spatial and temporal pattern to coordinate pancreatic bud outgrowth and direct endocrine cell specification in sheep. Immunofluorescence staining of the developing pancreas was used to co-localize insulin and epithelial proteins (cytokeratin, E-cadherin, and beta-catenin) or insulin and a mesenchymal protein (vimentin). In sheep, individual beta-cells become insulin-positive in the progenitor epithelium, then lose epithelial characteristics, and migrate out of the epithelial layer to form islets. As beta-cells exit the epithelial progenitor cell layer, they acquire mesenchymal characteristics, shown by their acquisition of vimentin. In situ hybridization expression analysis of the SNAIL family members of transcriptional repressors (SNAIL1, -2, and -3; listed as SNAI1, -2, -3 in the HUGO Database) showed that each of the SNAIL genes was expressed in the ductal epithelium during development, and SNAIL-1 and -2 were co-expressed with insulin. Our findings provide strong evidence that the movement of beta-cells from the pancreatic ductal epithelium involves an EMT.
Brown, L. D., Davis, M., Wai, S., Wesolowski, S. R., Hay Jr., W. W., Limesand, S. W., & Rozance, P. J. (2016). Chronically Increased Amino Acids Improve Insulin Secretion, Pancreatic Vascularity, and Islet Size in Growth-Restricted Fetal Sheep. ENDOCRINOLOGY, 157(10), 3788-3799.
Yates, D. T., Cadaret, C. N., Beede, K. A., Riley, H. E., Macko, A. R., Anderson, M. J., Camacho, L. E., & Limesand, S. W. (2016). Intrauterine growth-restricted sheep fetuses exhibit smaller hindlimb muscle fibers and lower proportions of insulin-sensitive Type I fibers near term. American journal of physiology. Regulatory, integrative and comparative physiology, 310(11), R1020-9.
Intrauterine growth restriction (IUGR) reduces muscle mass and insulin sensitivity in offspring. Insulin sensitivity varies among muscle fiber types, with Type I fibers being most sensitive. Differences in fiber-type ratios are associated with insulin resistance in adults, and thus we hypothesized that near-term IUGR sheep fetuses exhibit reduced size and proportions of Type I fibers. Placental insufficiency-induced IUGR fetuses were ∼54% smaller (P
Frost, M., Zehri, A., Limesand, S., Hay, J. W., & Rozance, P. (2012). Differential effects of chronic pulsatile vs. chronic constant maternal hyperglycemia on fetal pancreatic beta-cells. Journal of Pregnancy.
Article ID 812094, 2012
Limesand, S. W., & Anthony, R. V. (2001). Novel activator protein-2α splice-variants function as transactivators of the ovine placental lactogen gene. European Journal of Biochemistry, 268(8), 2390-2401.
PMID: 11298758;Abstract:
Activator protein-2 (AP-2) has been implicated as a transactivator of the human and ovine placental lactogen (oPL) genes. Transcriptional enhancement through an AP-2 cis-acting element has been described for other genes expressed in the placenta, but the AP-2 isoform enhancing expression is species dependent. Transactivation of the oPL minimal promoter (-124 bp to +16 bp) by AP-2 was confirmed by mutational analysis in transiently transfected human choriocarcinoma cells (BeWo). AP-2α was localized in ovine chorionic epithelial cells by immunohistochemistry and a 3-kb transcript was identified by Northern hybridization. Four nearly full-length AP-2 cDNAs were isolated from an ovine placenta cDNA library. Nucleotide sequencing these cDNAs revealed that the AP-2 mRNA expressed in the ovine placenta shares identity with human AP-2α, but variations in the predicted N-terminus were observed, and three unique AP-2α splice-variants were identified. Expression of AP-2α variants in HepG2 cells, devoid of endogenous AP-2, indicates that enhancement through the AP-2 element in the oPL gene minimal promoter was variant dependent. RNA transcripts for all of the ovine AP-2α splice-variants were confirmed in ovine placenta by RT-PCR, and homologs for two variants were found in human placenta. However, only one AP-2α transcript, which shares identity to Xenopus AP-2α, was expressed in BeWo cells. Immunoblot analysis confirmed AP-2α variants in ovine chorionic binucleate cell nuclear extracts, one of which migrates similar to the AP-2α variant identified in BeWo cell nuclear extracts. These data indicate the presence of new mammalian AP-2α splice-variants that augment transactivation of the oPL gene in ovine chorionic binucleate cells.
