Nkx2.2 regulates cell fate choice in the enteroendocrine cell lineages of the intestine

Abstract

Nkx2.2 is a homeodomain-containing transcription factor essential for pancreatic islet cell specification. In this study we investigate the role of Nkx2.2 within the small intestine. We have determined that Nkx2.2 is expressed at the onset of intestinal epithelial cell differentiation in specific intestinal cell populations, including a subset of enteroendocrine cells. Similar to its role in the pancreatic islet, Nkx2.2 regulates cell fate choices within the intestinal enteroendocrine population; in the Nkx2.2 null mice, several hormone-producing enteroendocrine cell populations are absent or reduced and the ghrelin-producing cell population is upregulated. The remaining intestinal cell populations, including the paneth cells, goblet cells, and enterocytes appear to be unaffected by the loss of Nkx2.2. Furthermore, similar to the pancreatic islet, Nkx2.2 appears to function upstream of Pax6 in regulating intestinal cell fates; Pax6 mRNA and protein expression is decreased in the Nkx2.2 null mice. These studies identify a novel role for Nkx2.2 in intestinal endocrine cell development and reveal the regulatory similarities between cell type specification in the pancreatic islet and in the enteroendocrine population of the intestine.

Figure 1. Nkx2.2 is expressed in the developing intestine

RNA in situ analyses of Nkx2.2 expression at different embryonic stages. a) e12.5; b and c) e13.5; and c) e15.5. The boxed area is shown in higher magnification in d. d) higher magnification of box shown in c. dp=dorsal pancreas, vp=ventral pancreas, s=stomach, i=intestine.

Figure 2. Nkx2.2 is expressed in the glucagon-producing enteroendocrine cells

RNA in situ analysis of Nkx2.2 (purple) combined with immunoperoxidase staining of glucagon-expressing cells (brown) at e18.5. a) Co-staining of Nkx2.2 and glucagon in enteroendocrine cells of the small intestine. The boxed areas are shown in higher magnification in b-d. b and c) Nkx2.2-positive, glucagon-positive enteroendocrine cells (arrowheads). d) Nkx2.2-positive, glucagon-negative intestinal cell (arrows). e) Co-staining of Nkx2.2 and glucagon in the pancreatic islet. The boxed area is shown in higher magnification in f.

Figure 3. In the Nkx2.2 null mice, many intestinal hormone-producing enteroendocrine populations are reduced and ghrelin-producing cells are significantly upregulated

a-h) Immunofluorescence analysis of e18.5 wild type (a, c, e, g and i) and Nkx2.2 null (b, d, f, h, j) mice. a,b) Synaptophysin (green) costained with DAPI (blue). Inset contains a higher magnification of a synaptophysin-positive cell. c,d) Serotonin (green) with a higher magnification inset of a serotonin-positive cell. e,f) CCK (green) with a higher magnification inset of a CCK-positive cell. ; g, h) GIP (green) with a higher magnification inset of a GIP-positive cell. i,j) Ghrelin expression (green) with higher magnifcation insets of ghrelin-positive cells (green) costained with synaptophysin (red). k, l) RNA in situ analysis of ghrelin expression at e18.5.

Figure 4. Quantification of hormone expression in e18.5 Nkx2.2 null small intestine

Real time PCR analysis of relative hormone expression in the Nkx2.2 wild type and mutant small intestine. Note the change of scale for the comparative ghrelin expression. See Table 3 for numbers tested. Fold change normalized to wild type; * p < 0.01; ** p < 0.001.

Figure 5. Nkx2.2 does not regulate the formation of non-enteroendocrine cell populations in the small intestine

Analysis of intestinal cell populations in e18.5 embryos. a,b) H&E staining of wild type and mutant tissue. c,d) RNA in situ of villin-expressing intestinal epithelial cells. e,f) Alcian blue staining of intestinal goblet cells. g,h) RNA in situ analysis of IFABP-expressing enterocytes. i,j) RNA in situ analysis of cryptdin1-producing paneth cells.

Figure 6. Intestinal transcription factor expression in Nkx2.2 wild type and mutant small intestine

RNA in situ analysis of transcription factor expression in wild type (a,c,e,g, i and k) and Nkx2.2 null (b,d,f,h,j, and l) embryos. a and b) tissue derived from e18.5 embryos; c-l) tissue derived from e15.5 embryos. m and n) Immunofluorescent staining of Pax6 protein in wild type and Nkx2.2 null e18.5 embryos. Boxed area is shown in higher magnification in o and p. Arrows point to Pax6-immunoreactive cells.

Figure 6. Intestinal transcription factor expression in Nkx2.2 wild type and mutant small intestine

RNA in situ analysis of transcription factor expression in wild type (a,c,e,g, i and k) and Nkx2.2 null (b,d,f,h,j, and l) embryos. a and b) tissue derived from e18.5 embryos; c-l) tissue derived from e15.5 embryos. m and n) Immunofluorescent staining of Pax6 protein in wild type and Nkx2.2 null e18.5 embryos. Boxed area is shown in higher magnification in o and p. Arrows point to Pax6-immunoreactive cells.