39 research outputs found
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
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
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
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
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
Glis3 associates with Itch, Smurf2, and NEDD4.
<p>A-C. HEK293T cells were transfected with FLAG-Glis3 or the FLAG-Glis3-<i>PY</i><sup><i>461</i></sup> mutant and Myc empty vector, Myc-Itch-C832G, Myc-Smurf2-C716G, or Myc-NEDD4-C867G as indicated. Co-immunoprecipitation was performed using a mouse anti-Myc antibody and immunoprecipitated proteins were examined by Western blot analysis using anti-M2 FLAG-HRP or anti-Myc and goat anti-mouse-HRP antibodies. D. HEK293T cells were transfected with FLAG-Glis3-ΔC480 or its respective <i>PY</i><sup><i>461</i></sup> mutant and Myc empty vector, Myc-Itch-C832G, Myc Smurf2-C716G, or Myc-NEDD4-C867G and co-IPs performed as described for A-C. E. HEK293T cells were transfected with FLAG-Glis3-ΔN496 and Myc empty vector or Myc-Itch-C832G and co-IPs performed as described in A-C. F. HEK293T cells were transfected with FLAG empty vector, FLAG-Glis3 or the FLAG-Glis3-<i>PY</i><sup><i>461</i></sup> mutant as indicated. After 48 h co-immunoprecipitation was performed using a mouse anti-M2 FLAG antibody and immunoprecipitated proteins were examined by Western blot analysis using mouse anti-ITCH primary and goat anti-mouse-HRP antibodies.</p
Itch targets Glis3 for degradation through the 26s proteasome.
<p>A. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch or empty vector. Cells were treated with DMSO, 10 μM MG132, 10 mM ammonium chloride, 200 μM chloroquine, or 100 μM leupeptin as indicated for 7 h. Proteins were analysed by Western blotting using anti-M2 FLAG-HRP antibody or anti-Myc and goat anti-mouse-HRP antibodies. GAPDH is shown as a loading control. B. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch, catalytically inactive Myc-Itch C832G, or empty vector. Cells were treated with 10 μM MG132 or DMSO for 6 h as indicated. Proteins were analysed by western blotting using an anti-M2 FLAG-HRP antibody.</p
HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3) Transcriptional Activity
<div><p>The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a <i>PPxY</i> motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the <i>PPxY</i> motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous <i>Ins2</i> expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.</p></div