Parker B Antin
Publications
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.
In situ hybridization (ISH) in embryos allows the visualization of specific RNAs as a readout of gene expression during normal development or after experimental manipulations. ISH using short DNA probes containing locked nucleic acid nucleotides (LNAs) holds the additional advantage of allowing the detection of specific RNA splice variants or of closely related family members that differ in only short regions, creating new diagnostic and detection opportunities. Here we describe methods for using short (14-24 nt) DNA probes containing LNA nucleotides to detect moderately to highly expressed RNAs in whole chick embryos during the first 5 days of embryonic development. The protocol is easily adaptable for use with embryos of other vertebrate species.
PMID: 9417129;Abstract:
To investigate the regulation of calpastatin gene expression, we isolated bovine heart calpastatin cDNAs and 5'-regions of the calpastatin gene. Analysis of 5'-cDNA sequence identified a new translation initiation site that is in frame and 204 nucleotides upstream of the previously designated start site. Conceptual translation from this upstream AUG produces a protein containing 68 additional N-terminal amino acids. This 'XL' region contains three potential PKA phosphorylation sites but shares no homology with other regions of calpastatin or with any known protein. Immunoblot studies demonstrated that heart and liver contain a calpastatin protein of 145 kDa on SDS-polyacrylamide gel electrophoresis that comigrates with full- length bacterially expressed calpastatin and calpastatin produced by coupled in vitro transcription-translation from the upstream AUG. An antibody raised against the XL region recognized the 145-kDa band, demonstrating that the upstream AUG is utilized and that the 145-kDa band represents full-length calpastatin in vivo. Transient transfection assays demonstrated that sequence within 272 nucleotides upstream of transcription initiation of the calpastatin gene is sufficient to direct moderate level transcription. Promoter sequences further upstream act to inhibit or stimulate transcriptional activity. Exposure of transfected cells to dibutyryl cAMP resulted in a 7-20-fold increase in promoter activity for constructs containing at least 272 nucleotides of upstream promoter sequence. Deletion analysis indicates that at least one cAMP-responsive element resides within 102 nucleotides of transcription initiation.