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First published online 30 May 2007
doi: 10.1242/dev.002691

Development 134, 2491-2500 (2007)
Published by The Company of Biologists 2007
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The transcription factors Nkx6.1 and Nkx6.2 possess equivalent activities in promoting beta-cell fate specification in Pdx1+ pancreatic progenitor cells

Shelley B. Nelson, Ashleigh E. Schaffer and Maike Sander*

Department of Developmental and Cell Biology, University of California at Irvine, 4203 McGaugh Hall, Irvine, CA 92697-2300, USA.


Figure 1
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  		Fig. 1. Nkx6.1 is expressed in similar domains to Ngn3 and Pdx1 during pancreas development. (A-I) Immunofluorescence for the indicated proteins in the developing mouse pancreas at E10.5 (dorsal bud; A,D,G), E12.5 (B,H), E14.5 (C,E,I) and E18.5 (F). (A) Ngn3 (red) is co-expressed with Pdx1 (green) at E10.5 (arrows). (B) At E12.5, a subset of Ngn3+ cells does not express Pdx1 (arrowhead), but some Ngn3+ cells maintain Pdx1 expression (arrow). (C) At E14.5, the majority of Ngn3+ cells express low levels of Pdx1. However, some Ngn3+ cells are Pdx1-negative (arrowhead) and some express high levels of Pdx1 (arrow). (D-F) Nkx6.1 (red) is co-expressed with Pdx1 (green) at E10.5 (D), E14.5 (E) and E18.5 (F). Notably, Nkx6.1 is absent from Pdx1+ acini at E14.5 (E, arrow). (G-I) Ngn3 (green) is co-expressed with Nkx6.1 (red) at E10.5 (G, arrows), E12.5 (H) and E14.5 (I). Scale bar: 50 µm.

 

Figure 2
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  		Fig. 2. Characterization of Ngn3-Nkx6.1 and Pdx1-Nkx6.1 transgenic mice. (A) The Ngn3-Nkx6.1 transgenic construct comprises the Ngn3 promoter fused to Nkx6.1 cDNA followed by an IRES-eGFP-polyA cassette. (B) In Ngn3-Nkx6.1 embryos at E14.5, 60% of the transgene-expressing;GFP+ cells express Ngn3 (arrows). (C,D) Whereas 26% of Ngn3+ cells are co-positive for Nkx6.1 in wild-type embryos at E16.5 (C, arrows), this percentage is increased to 61% in Ngn3-Nkx6.1 embryos (D, arrows). (E) In the Pdx1-Nkx6.1 transgenic construct, the Pdx1 promoter drives the expression of Nkx6.1 cDNA and an IRES-lacZ-polyA cassette. (F-I) lacZ expression in Pdx1-Nkx6.1 mice is detected in the ventral (vb) and dorsal (db) pancreatic anlagen at E10.5 (F), in the entire pancreatic epithelium at E14.5 (G), and in a subset of pancreatic cells at E18.5 (H) and postnatal day (P)6 (I). (J-L) ß-galactosidase (ß-gal) and Pdx1 are co-expressed in pancreata from Pdx1-Nkx6.1 mice at E14.5 (J, ß-gal in green; K, Pdx1 in red; L, merged). However, some Pdx1+ cells are negative for the ß-gal reporter (L, arrows). db/vb, dorsal/ventral pancreatic anlagen. Scale bars: 50 µm in B for B-D and in J for J-L.

 

Figure 3
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  		Fig. 3. Characterization of Ngn3-Nkx6.1 and Pdx1-Nkx6.1 transgene expression in the Nkx6.1 mutant background at E14.5. (A-D) Immunofluorescence detection of both Nkx6.1 (red; absent in Nkx6.1-null mice, B) and Ngn3 (green) shows strong co-expression in wild-type (A) and Nkx6.1-/-;Ngn3-Nkx6.1 (C) pancreata, but weak or no co-expression in Nkx6.1-/-;Pdx1-Nkx6.1 (D) pancreata. A subset of Ngn3+ cells is not targeted by the Ngn3-Nkx6.1 transgene (C). (E,F,H) Nkx6.1 (red; absent in Nkx6.1-null mice, F) is co-expressed with Pdx1 (green) in pancreata from wild-type (E) and Nkx6.1-/-;Pdx1-Nkx6.1 (H) embryos. Notably, Pdx1+ acinar cells do not express Nkx6.1 protein in Nkx6.1-/-;Pdx1-Nkx6.1 embryos (H, arrows). (G) Nkx6.1-/-;Ngn3-Nkx6.1 embryos display a subset of Nkx6.1+ cells (red) with no expression of Pdx1 (green), and another subset with weak Pdx1 expression. (I,J,L) Nkx6.1 (red; absent in Nkx6.1-null mice, J) is co-expressed with insulin (Ins, green) in pancreata from wild-type (I) and Nkx6.1-/-;Pdx1-Nkx6.1 (L, arrows) embryos. (K) Occasional co-localization of Nkx6.1 (red) and insulin (green) is also observed in Nkx6.1-/-;Ngn3-Nkx6.1 pancreata (arrow). Scale bar: 50 µm.

 

Figure 4
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  		Fig. 4. Expression of Nkx6.1 in the Pdx1+ domain can fully restore insulin expression in Nkx6.1 mutant embryos. (A-F) Immunofluorescence detection of insulin (Ins, green) and glucagon (Glc, red). (B,E) Nkx6.1-/- embryos have reduced insulin but normal glucagon cell numbers at E14.5 (B) and E18.5 (E) compared to wild type (A,D). (C,F) Expression of the Pdx1-Nkx6.1 transgene in Nkx6.1-/- embryos restores the formation of insulin+ cells at E14.5 (C), and restores normal islet morphology and size at E18.5 (F). (G) Morphometric quantification of the insulin+ area over total pancreatic area at E14.5 reveals an almost complete absence of insulin+ cells in Nkx6.1-/- embryos and a significant rescue of insulin+ cells in a subset of Nkx6.1-/- embryos carrying the Pdx1-Nkx6.1 transgene (n=4). (H) At E18.5, the insulin+ area is reduced by a threefold average in Nkx6.1-/- embryos (n=5) compared to wild-type mice (n=5). The Pdx1-Nkx6.1 transgene restores the insulin+ area in Nkx6.1-/- mutants to wild-type values in two embryos, whereas five embryos showed partial or no rescue (n=7). Scale bar: 50 µm.

 

Figure 5
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  		Fig. 5. Characterization of Pdx1-Nkx6.2 transgenic mice. (A) The Pdx1-Nkx6.2 transgene contains the Pdx1 promoter fused to Nkx6.2 cDNA followed by an IRES-lacZ-polyA cassette. (B) Mirroring the Pdx1+ domain, lacZ expression in Pdx1-Nkx6.2 mice is detected in the ventral (vb) and dorsal (db) pancreas anlagen at E10.5. (C-H) In Pdx1-Nkx6.2 mice, expression of ß-galactosidase (ß-gal, red) and Pdx1 (green) overlaps completely at E14.5 (C-E), whereas a subset of Pdx1+ cells fails to express ß-gal at E18.5 (F-H, arrows in H). db/vb, dorsal/ventral pancreatic anlagen Scale bars: 50 µm.

 

Figure 6
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  		Fig. 6. Expression of Nkx6.2 in the Pdx1+ domain restores insulin expression in Nkx6.1 mutant embryos. (A-F) Immunofluorescence detection of insulin (Ins, green) and glucagon (Glc, red). (B,E) Nkx6.1-/- embryos have reduced insulin but normal glucagon cell numbers at E14.5 (B) and E18.5 (E) compared to wild type (A,D). (C,F) Expression of the Pdx1-Nkx6.2 transgene in Nkx6.1-/- embryos restores the formation of insulin+ cells at E14.5 (C) and restores normal islet morphology and size at E18.5 (F). (G) Morphometric quantification of the insulin+ area over total pancreatic area in E18.5 embryos reveals an average threefold reduction of insulin+ area in Nkx6.1-/- (n=5) compared to wild-type mice (n=5). The Pdx1-Nkx6.2 transgene restores the insulin+ area in Nkx6.1-/- mutants to wild-type values in two embryos, whereas three embryos showed partial or no rescue (n=5). (Compare with Fig. 4.) Scale bar: 50 µm.

 

Figure 7
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  		Fig. 7. Expression of Nkx6.1 or Nkx6.2 in the Pdx1 domain restores Myt1 expression in Nkx6.1 mutant embryos. (A-D) Immunofluorescence detection of Myt1 (red) and insulin (Ins, blue) in pancreata from wild-type (A), Nkx6.1-/- (B), Nkx6.1-/-;Pdx1-Nkx6.1 (C) and Nkx6.1-/-;Pdx1-Nkx6.2 (D) embryos at E14.5. (B-D) Nkx6.1-/- embryos have reduced Myt1 expression (B), which is restored by expression of the Pdx1-Nkx6.1 (C) or Pdx1-Nkx6.2 (D) transgenes. (E) Morphometric quantification of the number of Myt1+ cells over total pancreatic area in E14.5 embryos reveals an average reduction of Myt1+ cells in Nkx6.1-/- to 14% of wild-type numbers (n=3). The Pdx1-Nkx6.1 or Pdx1-Nkx6.2 transgene restores the number of Myt1+ cells in Nkx6.1-/- mutants to 75% and 70% of wild-type values, respectively (n=3). Values in E represent averages ±s.e.m. Scale bar: 50 µm.

 

Figure 8
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  		Fig. 8. Expression of Nkx6.1 or Nkx6.2 in the Pdx1+ domain restores beta-cell development and maturation in Nkx6.1 mutant embryos. Immunofluorescence detection of Glut2 (A-D, red), MafA (E-H, red), Nkx6.1 (I-K, red) or ß-galactosidase (ß-gal; L, red) with insulin (Ins, green) in pancreata from wild-type (A,E,I), Nkx6.1-/- (B,F,J), Nkx6.1-/-;Pdx1-Nkx6.1 (C,G,K) and Nkx6.1-/-;Pdx1-Nkx6.2 (D,H,L) embryos at E18.5. (A,B) Whereas Glut2 expression is confined to insulin+ cells in wild-type embryos (A), insulin+ cells are Glut2-negative in Nkx6.1-/- mutants (B). (B) Notably, Nkx6.1-/- embryos display clusters of Glut2+, but insulin-negative, cells adjacent to the insulin+ domain. (C,D) The Pdx1-Nkx6.1 and Pdx1-Nkx6.2 transgenes restore the formation of insulin/Glut2-co-expressing cells in the Nkx6.1-deficient background. Some insulin+/Glut2-negative as well as Glut2+/insulin-negative cells remain in transgene-expressing embryos. (F) In Nkx6.1-/- embryos, insulin+ cells do not express MafA. (G,H) MafA expression is restored in the majority of, but not in all, insulin+ cells by the Pdx1-Nkx6.1 and Pdx1-Nkx6.2 transgenes (arrows point to MafA-negative/insulin+ cells). (K,L) Sustained expression of the Pdx1-Nkx6.1 and Pdx1-Nkx6.2 transgenes in rescued insulin+ cells is confirmed by the detection of Nkx6.1 (K) or ß-gal (L), respectively. Scale bar: 50 µm.

 

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© The Company of Biologists Ltd 2007