Dataset Information

Combined ectopic expression of Pdx1 and Ptf1a/p48 results in the stable conversion of posterior endoderm into endocrine and exocrine pancreatic tissue.

ABSTRACT: Patterning of the embryonic endoderm into distinct sets of precursor cells involves the precisely regulated activities of key transcription regulators. Ectopic, pan-endodermal activation of XPtf1a/p48 during pancreas precursor cell stages of Xenopus embryogenesis results in an expansion of the pancreatic territory, precisely within the borders of XlHbox8 expression. A combination of both activities is sufficient to expand the pancreatic precursor cell population also into more posterior portions of the endoderm. Both treatments result in the formation of a giant pancreas that persists up to late tadpole stages of development and carries both supernumerary endocrine and exocrine cells. A combination of XPtf1a/p48 and XlHbox8 is thus sufficient to convert nonpancreatic endodermal cells into pancreatic precursor cells.

SUBMITTER: Afelik S

PROVIDER: S-EPMC1475757 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Combined ectopic expression of Pdx1 and Ptf1a/p48 results in the stable conversion of posterior endoderm into endocrine and exocrine pancreatic tissue.

Afelik Solomon S Chen Yonglong Y Pieler Tomas T

Genes & development 20060601 11

Patterning of the embryonic endoderm into distinct sets of precursor cells involves the precisely regulated activities of key transcription regulators. Ectopic, pan-endodermal activation of XPtf1a/p48 during pancreas precursor cell stages of Xenopus embryogenesis results in an expansion of the pancreatic territory, precisely within the borders of XlHbox8 expression. A combination of both activities is sufficient to expand the pancreatic precursor cell population also into more posterior portions ...[more]

PMID: 16751182

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Project description:The aim of the study was to define the specific gene expression signature of pancreatic endoderm cells (PECs), by designing a strategy for generating putative PECs (PDX1+/NKX6-1+, and posterior foregut endoderm (PFG) cells (PDX1+/NKX6-1-). Cell line: PDX1-eGFP reporter cell line (PDXeG clone 170-3) generated using the HUES-4 embryonic stem cell line. Human embryonic stem cells (hESCs) were maintained on irradiated mouse embryonic fibroblasts (MEFs). Generation of a hESC-derived PDX1-eGFP reporter cell line: A PDX1-targeting vector was constructed by inserting an eGFP-pSV40-NeoR reporter cassette into the 5â² untranslated region of the PDX1 gene upstream of the PDX1 start codon (ATG), resulting in an GFP-tagged PDX1 allele. The GFP cassette was flanked by a 12.5kb homologous arm on the 5â direction and by a 3,5kb homologous arm on the 3 direction. The final targeting construct was verified by PCR, restriction analysis and sequencing. After approximately 2 weeks emerging clones were picked, expanded and analyzed by PCR. The Neomycin cassette was deleted by using a CRE expression vector (NLS-CRE-IRES-Puro) that was transiently co-transfected with a DsRED or GFP plasmid into selected targeted cloned. 24 hours post electroporation, GFP/DsRED positive single cells were FACS sorted and plated into 96-well plates. Clones were expanded and characterized for Neo excision by PCR. Differentiation protocol: hESCs cultured on MEFs were grown until 90% confluency and differentiated into definitive endoderm (DE) by using a modified version of the DâAmour protocol (D'Amour et al., 2005). The first day of differentiation, hESCs were cultured in the presence of 100ng/ml human Activin A (Peprotech) and 25ng/ml Wnt3a (R&D systems) in RPMI medium (Life Technologies). The four following days 100ng/ml human Activin A was added together with 1x B27-insulin in RPMI medium (Life Technologies). To generate posterior foregut cells, DE cells were treated with 64 ng/ml bFGF for additional 12 days. To generate pancreatic endoderm cells, DE cells were first treated with 2uM retinoic acid (RA, Sigma Aldrich) in DMEM/F12 medium containing 1x B27 +insulin (Life Technologies) for three days and then finally treated with 64 ng/ml FGF2 in combination with 100 ng/ml Noggin for the remaining 9 days.

Dataset Information

Combined ectopic expression of Pdx1 and Ptf1a/p48 results in the stable conversion of posterior endoderm into endocrine and exocrine pancreatic tissue.

Publications

Combined ectopic expression of Pdx1 and Ptf1a/p48 results in the stable conversion of posterior endoderm into endocrine and exocrine pancreatic tissue.

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