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First published online 3 January 2007
doi: 10.1242/dev.02763

Development 134, 515-523 (2007)
Published by The Company of Biologists 2007
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Nkx2.2-repressor activity is sufficient to specify {alpha}-cells and a small number of ß-cells in the pancreatic islet

Michelle J. Doyle1, Zoe L. Loomis2 and Lori Sussel2,*

1 Program in Molecular Biology, University of Colorado at Denver Health Sciences Center, Aurora, CO 80045, USA.
2 Department of Biochemistry and Molecular Genetics, University of Colorado at Denver Health Sciences Center, Aurora, CO 80045, USA.


Figure 1
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  		Fig. 1. Nkx2.2 derivatives are active in pancreatic cell lines. (A) Schematic of Nkx2.2-dominant-derivative transgenes. (B) The Nkx2.2-dependent luciferase reporter (pFoxLuc1-7xNk2prl) was co-transfected with pcDNA3 alone (light grey), pcDNA3-2.2hdVP16 (dark grey) or pcDNA3-2.2hdEnR (white) expression plasmids in ßTC3- or PANC cells, and is activated with Nkx2.2hdVP16. Error bars represent the standard error (s.e.m.) of three independent experiments. 2.2HD, Nkx2.2 homeodomain; myc, myc epitope tag; VP16, VP16 activation domain from herpes simplex virus; EnR, Engrailed repressor domain.

 

Figure 2
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  		Fig. 2. Nkx2.2-derivative transgenes are expressed in patterns similar to endogenous Nkx2.2 when expressed under the control of the Pdx1 promoter. (A) Nkx2.2hdVP16 mRNA is expressed throughout the pancreatic epithelium at E10.5 (L7319), E12.5 (L7318) and E15.5 (L7318), and is restricted to endocrine cells at E18.5 (L7319). (B) Nkx2.2hdEnR mRNA is detected in the pancreatic epithelium at E10.5, E12.5 and E15.5, and strong expression is detected in the endocrine tissue at E18.5 (all L7414). Both transgenes are expressed in a manner similar to Nkx2.2 throughout development; however, a small amount of transgene expression is detectable in acinar cells. (C) Real-time PCR quantification of Nkx2.2-repressor and Nkx2.2-activator transgene expression compared to endogenous Nkx2.2 expression. Percentages indicate percent transgene expression relative to endogenous Nkx2.2. Nkx2.2hdVP16: line 7319 (n=5), line 7318 (n=4); Nkx2.2hdEnR: line 7414 (n=4), line 7660 (n=3).

 

Figure 3
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  		Fig. 3. The Nkx2.2-repressor transgene partially rescues the Nkx2.2-null pancreas phenotype. (A-J) Immunofluorescence staining for insulin (red), glucagon (blue) and amylase (green) (A,C,E,G,I), or ghrelin (red; B,D,F,H,J) on E18.5 pancreata for wild-type (A,B), Nkx2.2-null (C,D), Nkx2.2-/-;Nkx2.2hdEnR (Line 7414; E,F), Nkx2.2-/-;Nkx2.2hdVP16 (Line 7318; G,H) and Nkx2.2-/-; Nkx2.2hdCon (Line 5635; I,J) embryos (20x magnification) demonstrates that the Nkx2.2-repressor transgene can restore glucagon- and insulin-positive cells. (K-M) Quantitative PCR for glucagon (K), insulin (L) and ghrelin (M) on E16.5 pancreatic RNA isolated from wild-type (black bars), Nkx2.2-/- (dark grey bars) and Nkx2.2-/-; Nkx2.2hdEnR (light grey bars) embryos. (N) Summary of cell counting data demonstrates that insulin-positive cell numbers are restored to approximately 20% of wild-type levels and glucagon-positive cell numbers are restored to wild-type levels (E18.5, n=3).

 

Figure 4
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  		Fig. 4. Rescued glucagon-producing cells have normal {alpha}-cell characteristics. (A-D) Immunofluorescence staining for glucagon (red) and ghrelin (green) at E12.5 and E18.5 on wild-type (A,C) and Nkx2.2-/-;Nkx2.2hdEnR (B,D) pancreata. There are a small number of glucagon-ghrelin co-staining cells (yellow) in wild-type and rescued pancreata (20x magnification). (E,F) Glucagon-positive cells (green) co-express the {alpha}-cell marker Pax6 (red) in wild-type (E) and rescued (F) islets at E18.5. (40x magnification.)

 

Figure 5
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  		Fig. 5. Rescued insulin-positive cells are present by E13.5 and do not abnormally co-express the hormone ghrelin. (A,B) Insulin-positive (red, arrows) cells are found at E13.5 in Nkx2.2-/-;Nkx2.2hdEnR embryos (n=4 embryos each). (C,D) Insulin (red) is not co-expressed with ghrelin (green) at E18.5 in either wild-type (C) or Nkx2.2-/-;2.2hdEnR (D) embryos. (20x magnification.)

 

Figure 6
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  		Fig. 6. Immature ß-cells are restored in Nkx2.2-null embryos carrying the Nkx2.2-repressor transgene. Confocal images of immunofluorescence staining on E18.5 wild-type or Nkx2.2-/-; Nkx2.2hdEnR islets. (A-D) Co-expression of insulin (red) with Nkx6.1 (green; A,B) or Pdx1 (green; C,D). (E-H) MafA and Glut2 (green) are co-expressed with insulin-positive (red) cells in wild-type (E,G) islets, but neither is co-expressed with rescued insulin-positive cells (F,H). Inset in F shows a rare insulin-positive cell co-expressing MafA. (I,J) Glucagon (red) and Glut2 (green) immunostaining. No Glut2-positive cells are found in Nkx2.2-/-; 2.2hdEnR islets (H,J; 40x magnification).

 

Figure 7
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  		Fig. 7. Nkx2.2 interacts with Grg3 in the embryonic pancreas. (A-D) RNA in situ hybridization for Grg3 at E12.5 (A) and E16.5 (C,E,F), and for Nkx2.2 at E12.5 (B) and E16.5 (D). Adjacent sections demonstrate similar expression patterns throughout pancreas development. In C and D, outlines represent selected overlapping expression areas. (E,F) Combined RNA in situ hybridization and immunohistochemistry on E16.5 pancreata demonstrate overlapping expressions between Grg3 (blue) and glucagon (brown; arrows in E), and distinct expression domains between Grg3 (blue) and amylase (brown; F). (G) Co-immunoprecipitation for Nkx2.2 and Grg3: PANC cell lysates were incubated with Grg3 antibody and were western blotted with antibodies against Nkx2.2. Lanes 1 and 4: vector-only control; lanes 2 and 5: Nkx2.2 transfected with Grg3; lanes 3 and 6: Nkx2.2{Delta}TN transfected with Grg3. Lanes 1-3 are input samples and lanes 4-6 are Grg3 pull-down samples. Nkx2.2 is co-immunoprecipitated with Grg3 (lane 5).

 

Figure 8
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  		Fig. 8. A model of Nkx2.2 activity in the developing pancreas. Nkx2.2 functions as a repressor to specify and differentiate the glucagon-producing {alpha}-cell population. Nkx2.2 also functions as a repressor to specify the early insulin-positive population and/or the immature Pdx1low, Nkx6.1+, MafA-, Glut2- insulin-producing ß-cells, but this activity is not sufficient to produce a fully differentiated mature Pdx1high, Nkx6.1+, MafA+, Glut2+ ß-cell. We propose that Nkx2.2-activator function will be required for this later differentiation step.

 




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