7 research outputs found
Glis3 protein expression in E17.5 pancreata.
<p>E17.5 Glis3<sup>GFP/GFP</sup> pancreata were double-stained with antibodies against GFP and DBA (A), or Nkx6.1 (B), Pdx1 (C), Ins (D), or Gcg (E). DAPI and merged images are indicated. Dashed circles and dashed lines indicate pancreatic ducts. Schematic view of tubule structure and developing islets at E17.5 of embryonic pancreatic development is shown on the right: A, (pre) acinar cells; D, (pre) ductal cells; α, ß and PP cells are indicated. DOI <a href="http://dx.doi.org/10.6084/m9.figshare.3189175" target="_blank">10.6084/m9.figshare.3189175</a>.</p
The Spatiotemporal Pattern of Glis3 Expression Indicates a Regulatory Function in Bipotent and Endocrine Progenitors during Early Pancreatic Development and in Beta, PP and Ductal Cells
<div><p>The transcription factor Glis-similar 3 (Glis3) has been implicated in the development of neonatal, type 1 and type 2 diabetes. In this study, we examined the spatiotemporal expression of Glis3 protein during embryonic and neonatal pancreas development as well as its function in PP cells. To obtain greater insights into the functions of Glis3 in pancreas development, we examined the spatiotemporal expression of Glis3 protein in a knockin mouse strain expressing a Glis3-EGFP fusion protein. Immunohistochemistry showed that Glis3-EGFP was not detectable during early pancreatic development (E11.5 and E12.5) and at E13.5 and 15.5 was not expressed in Ptf1a<sup>+</sup> cells in the tip domains indicating that Glis3 is not expressed in multipotent pancreatic progenitors. Glis3 was first detectable at E13.5 in the nucleus of bipotent progenitors in the trunk domains, where it co-localized with Sox9, Hnf6, and Pdx1. It remained expressed in preductal and Ngn3<sup>+</sup> endocrine progenitors and at later stages becomes restricted to the nucleus of pancreatic beta and PP cells as well as ductal cells. Glis3-deficiency greatly reduced, whereas exogenous Glis3, induced Ppy expression, as reported for insulin. Collectively, our study demonstrates that Glis3 protein exhibits a temporal and cell type-specific pattern of expression during embryonic and neonatal pancreas development that is consistent with a regulatory role for Glis3 in promoting endocrine progenitor generation, regulating insulin and Ppy expression in beta and PP cells, respectively, and duct morphogenesis.</p></div
Glis3 protein expression at E15.5 of pancreas development.
<p>E15.5 Glis3<sup>GFP/GFP</sup> pancreata were double-stained with antibodies against GFP (A-G) and Pdx1 (A), Nkx6.1 (B), Ngn3 (C), Sox9 (D), Hnf6 (E), or Ptf1a (M). DAPI stained and merged images are indicated. Boxed areas were enlarged and shown in the panels on the right. Arrows indicate Glis3<sup>+</sup>Pdx1<sup>+</sup> (A), Glis3<sup>+</sup>Nkx6.1<sup>+</sup> (B), Glis3<sup>+</sup>Ngn3<sup>+</sup> (C), Glis3<sup>+</sup>Sox9<sup>+</sup> (D) or Glis3<sup>+</sup>Hnf6<sup>+</sup> (E) cells; arrow heads indicate Ptf1a single positive cells (F). Triple immunostaining of E15.5 Glis3<sup>GFP/GFP</sup> pancreata with anti-GFP, anti-Nkx6.1, and Sox9 (G). Arrows indicate triple positive cells (G, inset). Dashed circles indicate tip domains; dashed lines indicate trunk domains. Schematic view of tubule structure at E15.5 of embryonic pancreatic development is indicated on the right. DOI <a href="http://dx.doi.org/10.6084/m9.figshare.3189172" target="_blank">10.6084/m9.figshare.3189172</a>.</p
PP cells and Ppy are novel targets for Glis3 regulation.
<p>(A) Relative expression of several endocrine marker genes was analyzed by QRT-PCR in the pancreas from 4 weeks old WT and Glis3KO2 mice (n ≥ 4). (B, C) Pancreata from 4 weeks old WT and Glis3KO2 mice were examined by DAPI and FITC-DBA (B) or with antibodies against Ppy and insulin (C). (D-E) Exogenous expression of Glis3 induced Ppy mRNA expression in αTC1-9 and HPDE cells. Cells were infected with pLVX-mCherry, pLVX-mCherry-Glis3, -Pdx1, -MafA, or -NeuroD1 lentivirus and expression of Ppy mRNA was analyzed by QRT-PCR. Data present mean ± SEM. * p <0.05. (F) Schematic of the spatiotemporal pattern of expression of Glis3 during pancreas development. DOI <a href="http://dx.doi.org/10.6084/m9.figshare.3189178" target="_blank">10.6084/m9.figshare.3189178</a>.</p
Glis3 protein is selectively expressed in mouse pancreatic islets and ducts.
<p>Pancreata from P7 Glis3<sup>GFP/GFP</sup> mice were examined by immunohistochemistry with antibodies against GFP (A-E), Pdx1 (A), Ppy (B), Gcg (C), Sst (D), Ghrl (E) or stained with DBA or DAPI (F). Boxed areas were enlarged and shown in the panels on the right. Arrows indicate Glis3<sup>+</sup>Pdx1<sup>+</sup> (A), Glis3<sup>+</sup>Ppy<sup>+</sup> (B) or Glis3<sup>+</sup>Sst<sup>+</sup> (D) cells; arrow heads indicate either Glis3 single positive cells (A, B) or Glis3<sup>-</sup>Gcg<sup>+</sup> (C), Glis3<sup>-</sup>Sst<sup>+</sup> (D) or Glis3<sup>-</sup>Ghrl<sup>+</sup> (E) cells. Eighty four percent of Glis3<sup>+</sup> cells were Pdx1<sup>+</sup>. More than 90% of the Ppy<sup>+</sup> cells were Glis3<sup>+,</sup> and more than 94% of the DBA<sup>+</sup> cells were Glis3<sup>+</sup>. Bars indicate 50 μm. DOI <a href="http://dx.doi.org/10.6084/m9.figshare.3189163" target="_blank">10.6084/m9.figshare.3189163</a>.</p
Generation of Glis3-EGFP mice.
<p>(A) Schematic view of the mouse WT Glis3 allele and the targeted allele. EGFP was inserted in frame into exon 11 just before the TGA stop codon to generate a Glis3-EGFP fusion protein. To confirm the targeting in ES clones, genomic Southern was performed with the 5’ and 3’ probes as indicated in the map. (B) Comparison of the transcriptional activity of Glis3-EGFP with that of Flag-Glis3. A luciferase reporter driven by the <i>Ins2</i> promoter (Ins2-Luc) was used to examine the transcription activity in HEK293 cells. Data shown are representative of 3 independent experiments. Each experiment was carried out in triplicate. Data present mean ± SD. There was no statistical difference in reporter activation by Flag-Glis3 and Glis3-GFP. (C) The subcellular localization of Glis3-EGFP in COS-1 cells was analyzed by confocal microscopy. DOI <a href="http://dx.doi.org/10.6084/m9.figshare.3189148" target="_blank">10.6084/m9.figshare.3189148</a>.</p
Glis3 protein was not detectable in E12.5 pancreata, but was selectively expressed at E13.5 of pancreas development.
<p>E12.5 pancreata from Glis3<sup>GFP/GFP</sup> embryo were double-stained with antibodies against GFP (A-B) and Pdx1 (A) or Sox9 (B). E13.5 Glis3<sup>GFP/GFP</sup> pancreata were double-stained with antibodies against GFP (C-G) and Pdx1 (C), Ngn3 (D), Sox9 (E), or Ptf1a (F). Boxed areas were enlarged and shown in the panels on the right. DAPI stained and merged images are indicated. Arrows indicate Glis3<sup>+</sup>Pdx1<sup>+</sup> (C), Glis3<sup>+</sup>Ngn3<sup>+</sup> (D) or Glis3<sup>+</sup>Sox9<sup>+</sup> (E) cells; arrow heads indicate Ptf1a single positive cells (F). Triple staining of E13.5 Glis3<sup>GFP/GFP</sup> pancreata with anti-GFP, anti-Nkx6.1, and Sox9 (G). Arrows indicate triple stained cells (G, inset). Dashed circles indicate tip domains; dashed lines indicate trunk domains. Schematic views (shown on the right) of the primary and secondary transition of pancreas development indicating formation of tubular structures of multipotent progenitor cells (MPC) (E12.5) and the compartmentalization and establishment of multipotent progenitors and (pre)acinar cells (A) at the tip domain and the appearance of bipotent progenitors (BP), endocrine progenitors (EP), and preductal cells (D) in the trunk domain (E13.5), respectively. DOI <a href="http://dx.doi.org/10.6084/m9.figshare.3189169" target="_blank">10.6084/m9.figshare.3189169</a>.</p