Neurogenin3 is differentially required for endocrine cell fate specification in the intestinal and gastric epithelium

Abstract

Endocrine cells of the pancreas and the gastrointestinal tract derive from multipotent endodermal stem cells. We have shown previously that the basic helix- loop-helix (bHLH) transcription factor neurogenin3 (ngn3) is required for the specification of the endocrine lineage in uncommitted progenitors in the developing pancreas. We investigate herein the expression and the function of ngn3 in the control of endocrine cell development in the intestinal and gastric epithelium. Our results indicate that as in the pancreas, gastrointestinal endocrine cells derive from ngn3-expressing progenitors. Mice homozygous for a null mutation in ngn3 fail to generate any intestinal endocrine cells, and endocrine progenitor cells are lacking. The other main intestinal epithelial cell types differentiate properly. In contrast, in the glandular stomach, the differentiation of the gastrin- (G cells) and somatostatin (D cells)-secreting cells is impaired whereas serotonin- (enterochromaffin EC cells), histamine- (enterochromaffin-like ECL cells) and ghrelin (X/A cells)-expressing cells are still present. Thus, ngn3 is strictly required for endocrine cell fate specification in multipotent intestinal progenitor cells, whereas gastric endocrine development is both ngn3 dependent and independent.

Fig. 1. ngn3 is expressed in the embryonic and the adult crypt intestinal epithelium. (A–E) Cryosections through the intestines were hybridized with a ngn3 riboprobe (dark blue) and double stained (E) with anti-BrdU antibodies (brown). (F) Immunohistochemical staining for ngn3 protein. (A) ngn3 transcripts are found in scattered cells from the embryonic stage E12.5 onwards in the developing intestine. (B–D) The number of ngn3⁺ cells increases at E14.5–E15.5, and ngn3-expressing cells are distributed from the duodenum (C) to the colon (arrowheads in D). (E and F) In the adult small intestine, ngn3 transcripts (arrowheads in E) and protein (arrows in F) are found exclusively in the proliferative compartment of the crypts mainly in BrdU⁺ cells (E) and never in the villi (F). i,  intestine; p,  pancreas.

Fig. 2. Intestinal activity of the ngn3 promoter. The β-galactosidase protein marks ngn3 progenitor cells and their deriving enteroendocrine cells. A transgenic mouse model where a nuclear LacZ is driven by ngn3 regulatory sequences was generated and the stability of the β-galactosidase protein was used to trace the progeny of ngn3-expressing cells. (A–D) Whole-mount X-Gal-stained E15.5 digestive tract (A) and adult duodenum (B) were sectioned (C and D, respectively); the blue nuclear staining indicates β-galactosidase catalysis of the X-Gal substrate. At E15.5, X-Gal-stained cells are found all along the proximo-distal axis of the gut (A) in the intestinal epithelium (C). (E) Immunofluorescent co-staining for β-galactosidase (red) and ngn3 (green) shows partial overlapping expression (yellow and orange cells, arrows in E). In the adult intestine, β-Gal-labeled cells are found in the crypts (D, red immunofluoresence in F), in dividing PCNA⁺ cells (arrow in F) and also in the villi (D) where they co-stain with the pan-endocrine marker chromogranin A (dark brown cytoplasmic peroxidase staining, arrows in G) in differentiated enteroendocrine cells. The black line in (D) divides the crypt from the villus region. vi, villus; cr, crypt; d, duodenum.

Fig. 3. ngn3 is required for the differentiation of all the endocrine cell lineages in the intestinal epithelium. The comparison of wild-type (A–E) and ngn3 knock-out (A′–E′) intestines at birth (P₁) (A–D and A′–D′) and postnatal stages (E and E′) shows that milk often stagnates in the intestine of the ngn3-deficient newborns (compare A with A′) and that cells expressing the pan-endocrine marker chromogranin A (B) and the endocrine hormones, illustrated for gastrin/CCK (arrowheads in C) and serotonin (arrowheads in D), are observed in wild-type small intestines but are completely missing in ngn3 mutant intestinal epithelium at birth (compare B–D with B′–D′). Enteroendocrine cells are also missing at later stages of development reached by grafting embryonic intestines under the skin of nude mice and recovered after 4 weeks (E and E′). st, stomach; i, intestine.

Fig. 4. The intestines of ngn3 mutants lack endocrine progenitors. In wild-type intestines, Math1 (ISH) is expressed in common multipotent progenitors for secretory cells located in the crypts [arrowheads in (A)] as well as in differentiated cells (arrows in A), whereas NeuroD (ISH) is expressed only in post-mitotic enteroendocrine cells (B). Math1 and NeuroD expression are maintained [arrowheads and arrows in (A′)] and turned off (B′), respectively, in the absence of ngn3, suggesting that ngn3 acts downstream of Math1 and upstream of NeuroD in the differentiation of the intestinal endocrine lineage. To address the question of the fate of the ngn3 mutant cells in the gastrointestinal tract, the ngn3 promoter::LacZ and ngn3^+/– transgenic mice were crossed to follow the expression of the β-galactosidase transgene in a ngn3 mutant background. β-Gal⁺ cells [red immunofluorescence, arrowheads in (C)] were absent in the ngn3^–/– intestines (C′), indicating the early function of ngn3 in the determination of the endocrine lineage in the developing intestine. (D) RT–PCR analysis of gene expression in P₁ intestine. Expression of Pax6 is lost in ngn3-deficient intestine. TBP was used as an internal standard.

Fig. 5. Non-endocrine lineages (enterocytes, goblet and Paneth cells) develop normally in ngn3-deficient intestinal epithelium. The development of the intestinal mucosa [compare (A) with (A′)] as well as the differentiation of enterocytes [lactase activity in (B) and (B′)], goblet cells (PAS staining, arrowheads in (A) and (A′)] and Paneth cells [PAS staining, arrowheads in (C) and (C′)] pointing to the Paneth cells localized at the bottom of the crypts] are not affected in ngn3 mutants at birth (A and A′, B and B′) and at postnatal stages [4 weeks intestinal grafts (C) and (C′)], suggesting that ngn3 does not control the determination of these intestinal cytotypes either directly or indirectly. A 60% increase in the number of goblet cells has, however, been observed in ngn3^–/– duodenum [compare (A) and (A′)].

Fig. 6. ngn3 promoter activity in the gastric endocrine lineage. (A) ngn3 regulatory sequences target the expression of the LacZ gene (β-galactosidase activity) in the glandular part (g) of the newborn stomach but not in the squamous epithelium (s) of the forestomach. (B–G) Double stainings for β-galactosidase (blue nuclear staining) and chromogranin A [CA in (B)], gastrin [GAS in (C)], somatostatin [SOM in (D)], serotonin [SER in (E)], ghrelin [GHR in (F)] and histidine decarboxylase [HDC in (G)] (brown peroxidase staining), on sections of newborn (B–F) and adult (G) stomachs. β-galactosidase is expressed in progenitors of the gastric endocrine cells as suggested by the high number of single β-galactosidase-expressing cells (blue enzymatic activity) and the persistence of β-galactosidase activity in differentiated endocrine cells (arrows in B–G).

Fig. 7. Gastric endocrine specification is both ngn3 dependent and independent. Cells expressing the endocrine marker chromogranin A (CA) (A), and the hormones gastrin (B), somatostatin (C), serotonin (D) and ghrelin (E) are detected by immunohistochemistry in the wild-type gastric mucosa at birth. In ngn3 mutants, gastrin-secreting G cells (B′) and somatostatin-secreting D cells (C′) are lost. However, endocrine cells expressing chromogranin A (A′), serotonin (D′) and ghrelin (E′) are still observed. Histidine decarboxylase (HDC), a specific marker of ECL cells, is expressed in both grafted control (F) and ngn3-deficient (F′) stomachs at postnatal stages. The arrowhead in (B′) points to a rare G cell detected in ngn3^–/– stomach.

Fig. 8. Proposed model for the role of ngn3 during endocrine cell differentiation in the gastrointestinal epithelium. (A) ngn3 is required for the differentiation of all the intestinal endocrine cell types and controls endocrine cell fate commitment of Math1-positive multipotent progenitors of the secretory lineages. All the endocrine cell types arise from a common ngn3-specified progenitor. ngn3 could compensate for the absence of NeuroD in enteroendocrine cell types, except for CCK- and secretin-producing cells. (B) ngn3 is essential for the differentiation of G and D cells in the stomach. EC, X/A and ECL cell differentiation occurs in mice lacking ngn3 although they probably derive from ngn3 progenitors. Another gene would control the endocrine determination of these three gastric endocrine cell types. The ngn3-dependent endocrine cell types of the gastrointestinal tract are in red. CCK, cholecystokinin; SEC, secretin; GAS, gastrin; SER, serotonin; GIP, gastric inhibitory peptide; SOM, somatostatin; GLP, glucagon-like peptide; PYY, peptide YY.