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, 28 (10), 3208-18

Requirement of the Tissue-Restricted Homeodomain Transcription Factor Nkx6.3 in Differentiation of Gastrin-Producing G Cells in the Stomach Antrum

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Requirement of the Tissue-Restricted Homeodomain Transcription Factor Nkx6.3 in Differentiation of Gastrin-Producing G Cells in the Stomach Antrum

Michael Y Choi et al. Mol Cell Biol.

Abstract

Many homeodomain transcription factors function in organogenesis and cell differentiation. The Nkx family illustrates these functions especially well, and the Nkx6 subfamily controls differentiation in the central nervous system and pancreas. Nkx6.3, a recent addition to this subfamily, overlaps Nkx6.1 and Nkx6.2 in expression in the hindbrain and stomach. Nkx6.3 transcripts localize in the epithelium of the most distal stomach region, the antrum and pylorus; expression in the adult intestine is lower and confined to the proximal duodenum. Nkx6.3(-)(/)(-) mice develop and grow normally, with a grossly intact stomach and duodenum. These mice show markedly reduced gastrin mRNA, many fewer gastrin-producing (G) cells in the stomach antrum, hypogastrinemia, and increased stomach luminal pH, with a corresponding increase in somatostatin mRNA levels and antral somatostatin-producing (D) cells. They express normal levels of other transcription factors required for gastric endocrine cell differentiation, Pdx1, Pax6, and Ngn3; conversely, Ngn3(-)(/)(-) mice, which also show reduced gastrin levels, express Nkx6.3 normally. These studies implicate Nkx6.3 as a selective regulator of G- and D-cell lineages, which are believed to derive from a common progenitor, and suggest that it operates in parallel with Ngn3.

Figures

FIG. 1.
FIG. 1.
Expression of Nkx6.3 in the stomach. (A) Northern analysis of Nkx6.3 expression in adult mouse organs (top), showing the highest levels in the stomach, and in adult stomach and intestinal segments (bottom). The data confirm stomach (Sto) expression and reveal trace levels in the duodenum (Duo) but absence in the jejunum (Je), ileum (Il), cecum (Ce), or colon (Co). 18S or 28S RNA serves as a loading control. Sk., skeletal. (B) Relative expression of Nkx6.3 mRNAs in isolated forestomach (fs), upper (uc) and lower (lc) corpus, antrum-pylorus (a/p), and duodenum (du) samples from adult wild-type mice, as measured by qRT-PCR. All results were normalized to the level of GAPDH mRNA, and each segment is represented relative to an assigned wild-type antrum expression value of 1.0. The diagram depicts the anatomy and results schematically. (C) In situ hybridization on adult wild-type stomach tissue localizes Nkx6.3 transcripts in the epithelial compartment, especially in cells at the bases of antral gland units. Arrowheads mark the continuous line of Nkx6.3 expression along the base of the antral mucosa. (D) Higher resolution of the boxed area in panel C, showing Nkx6.3 expression in epithelial cells at the bottoms of glands. Arrowheads point to groups of Nkx6.3-expressing cells; expression is absent in the submucosa and the upper regions of glands. (E) Magnified image of the boxed area in panel D, showing nests of epithelial cells at the gland base, bordering the submucosa. Arrowheads point to cells with the highest Nkx6.3 signal level. (F) Graphic depiction of an antral gland unit where G and D cells occupy the base.
FIG. 2.
FIG. 2.
Targeted disruption of the mouse Nkx6.3 gene. (A) Gene-targeting strategy to replace exons 2 and 3 in the mouse Nkx6.3 locus with a PGK-Neor cassette. Also shown are the locations of the 5′ and 3′ flanking probes used for Southern analysis and predicted band sizes in EcoRI- or NcoI-digested DNA. Wt, wild type; Vec., vector; KO, knockout. (B) Southern blot assay of ES cell DNA digested with EcoRI and interrogated with the 5′ probe. (C) Southern blot assay of mouse tail DNA digested with NcoI and interrogated with the 3′ probe. (D) PCR analysis of mouse tail DNA to distinguish Nkx6.3 wild-type (+/+), heterozygote (+/−), and mutant (−/−) animals. (E) Northern analysis of RNA from the distal stomachs of adult wild-type and Nkx6.3 knockout mice, demonstrating the absence of Nkx6.3 mRNA in the latter. 18S and 28S RNA species are visible on the blot and confirmed equal loading. (F) qRT-PCR of cDNA from adult wild-type and Nkx6.3-null stomachs, showing that Nkx6.3 mRNA is virtually undetectable with primers from the 5′ and 3′ ends. Expr, expression.
FIG. 3.
FIG. 3.
Normal features of the gastro-duodenal junction, duodenum, and stomach in Nkx6.3-deficient adult (8- to 12-week-old) mice. (A, B) H&E staining at the pyloric junction of wild-type (WT) (A) and knockout (KO) (B) adult mice shows normal tissue architecture and morphology in Nkx603-null mice. py, pylorus; in, intestine, br, Brünner's glands. (C to H) H&E (C, D), Alcian blue (E, F), and PAS (G, H) staining of control (C, E, G) and knockout (D, F, H) mouse duodenums reveals normal organization and goblet cell frequency. (I, J) PAS staining demonstrates normal morphology in wild-type (I) and mutant (J) Brünner's glands. br, Brünner's glands; vi, villus. (K to M) Transmission electron micrographs of Brünner's glands cells (K, several cells facing a small central lumen pointed to by the white arrowhead), enterocytes (L), and the intestinal apical brush border (labeled with black arrowheads and magnified in panel M), revealing normal features of each compared to wild-type samples (not shown). Mucosal glands in the gastric corpus (N, O) or antrum (P to S) of the adult wild-type (N, P, R) or Nkx6.3-null (O, Q, S) stomach, stained with H&E (N to Q) or PAS (R, S) to show the normal glandular organization and dominant epithelial cell lineages. (T to V) Transmission electron micrographs of representative foveolar (T), parietal (U), and chief (V) cells in Nkx6.3-deficient mice, which show normal ultrastructural features compared to wild-type specimens (not shown). The white arrowheads in panel T point to mucin-laden granules at the apex of a foveolar (pit) cell.
FIG. 4.
FIG. 4.
Decreased number of gastric G cells in the absence of Nkx6.3. (A, B) Immunostaining for chromogranins A and B in stomach glands from wild-type (WT) (A) and knockout (KO) (B) mice. Arrowheads point to the single cells shown at a higher resolution in each inset. (C) Transmission electron micrographs of the cytoplasm, including secretory granules, of representative wild-type (top) and Nkx6.3−/− (bottom) gastric endocrine cells. (D to H) Gastrin immunostaining shows that the Nkx6.3-null antrum is virtually devoid of G cells, as confirmed by cell counts averaged from multiple animals (H). Low-magnification views (D, E) illustrate the antrum (an), with the duodenal (du) junction marked by arrowheads. (I to Q) Immunostaining for somatostatin (I to K), serotonin (L to N), and ghrelin (O to Q), with cell counts from identical vertical tissue sections represented in bar graphs. Photographs were taken from antral glands, and all counts were obtained in the antrum, the region with the highest Nkx6.3 expression level. Somatostatin-expressing D cells are increased about threefold in the Nkx6.3−/− antrum, whereas serotonin- and ghrelin-producing cell numbers are normal. Bar graphs represent means ± standard deviations; P values were obtained by unpaired t test. All analyses were conducted with adult mice between 8 and 12 weeks of age.
FIG. 5.
FIG. 5.
gastrin mRNA expression is abrogated in the Nkx6.3-null antrum, and Nkx6.3 and gastrin colocalize in antral epithelial cells. (A to C) Relative expression of gastrin, somatostatin, and ghrelin mRNAs in the isolated lower (L.) corpus, antrum, and duodenum (Duod) from adult (8- to 12-week-old) wild-type (Wt) and Nkx6.3 mutant (knockout [Ko]) mice, as measured by qRT-PCR. All results were normalized to the level of GAPDH mRNA, and each segment is represented relative to an assigned wild-type antral expression value of 1.0; high gastrin levels in the wild-type antrum necessitated the use of three different y-axis scales in panel A. gastrin expression is dramatically reduced in the Nkx6.3-null antrum (A), whereas somatostatin expression is increased 2.5-fold (B) and ghrelin levels are unchanged (C). (D to I) Nkx6.3 in situ hybridization (D, F, H) followed by gastrin immunostaining (E, G, I) on the same sections. Almost all G cells coexpress Nkx6.3; conversely, cells with especially high Nkx6.3 signals coexpress gastrin, as appreciated best in high-resolution photomicrographs (F to I). Matching arrowhead colors designate Nkx6.3 in situ hybridization (F, H) and gastrin immunostaining (G, I) on the same cells. (J) qRT-PCR analysis of gastrin, somatostatin, and ghrelin mRNA levels in 293TD epithelial cells in response to forced Nkx6.3 expression (Expr) by plasmid transfection. The results represent a composite of five data points from two independent experiments. All bars (A to C, J) reflect the mean ± the standard deviation.
FIG. 6.
FIG. 6.
Nkx6.3 and Ngn3 affect G-cell differentiation through independent pathways. (A) Summary table of mutant mouse strains reported to have altered G-cell numbers and their effects on other gastric endocrine cell lineages. Empty boxes represent absence of published data. KO, knockout; Ref. reference. Pdx1, Pax6, and Ngn3 also function in organs other than the stomach. Effects on D-cell density in mice with gastrin or its receptor knocked out are opposite to those in Nkx6.3−/− mice. (B to D) Relative expression levels of Pdx1 (B), Pax6 (C), and Ngn3 (D) mRNAs are unchanged in adult Nkx6.3−/− GI segments compared to those in wild-type (Wt) mice, as measured by qRT-PCR. Results were normalized to the level of GAPDH mRNA, and each segment is represented relative to an assigned wild-type antrum expression value of 1.0. (E) Nkx6.3 mRNA levels are unchanged in the neonatal Ngn3-null antrum compared to that in littermate controls. Bar graphs show the mean ± the standard deviation. L., lower; Duod, duodenum.

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