neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas

Abstract

In the mammalian pancreas, the endocrine cell types of the islets of Langerhans, including the alpha-, beta-, delta-, and pancreatic polypeptide cells as well as the exocrine cells, derive from foregut endodermal progenitors. Recent genetic studies have identified a network of transcription factors, including Pdx1, Isl1, Pax4, Pax6, NeuroD, Nkx2.2, and Hlxb9, regulating the development of islet cells at different stages, but the molecular mechanisms controlling the specification of pancreatic endocrine precursors remain unknown. neurogenin3 (ngn3) is a member of a family of basic helix-loop-helix transcription factors that is involved in the determination of neural precursor cells in the neuroectoderm. ngn3 is expressed in discrete regions of the nervous system and in scattered cells in the embryonic pancreas. We show herein that ngn3-positive cells coexpress neither insulin nor glucagon, suggesting that ngn3 marks early precursors of pancreatic endocrine cells. Mice lacking ngn3 function fail to generate any pancreatic endocrine cells and die postnatally from diabetes. Expression of Isl1, Pax4, Pax6, and NeuroD is lost, and endocrine precursors are lacking in the mutant pancreatic epithelium. Thus, ngn3 is required for the specification of a common precursor for the four pancreatic endocrine cell types.

Figure 1

Expression of ngn3 during pancreas development. (a) Expression in the pancreatic bud (arrowhead) shown by whole-mount RNA in situ hybridization at E9.5. (b–f) Cryosections through the pancreas hybridized with an ngn3 riboprobe (dark blue) and counterstained with Safranin (b–d) or double-stained with anti-glucagon and anti-insulin antibodies (brown; e and f). ngn3 transcripts are found in cells either scattered or clustered in the pancreatic epithelium at E10.5 (b and e). A large number of ngn3 + cells are found associated with pancreatic ducts at E15.5 (c). (d) A high magnification of a transverse section through a duct at E17.5 containing an ngn3-expressing cell (arrowhead). ngn3+ cells are distinct from insulin- and glucagon-producing cells, suggesting that ngn3 is transiently expressed in endocrine precursor cells (e and f). However, we cannot completely rule out that a few cells strongly stained for ngn3 also produce glucagon or insulin. No ngn3 transcripts are detected in the exocrine tissue.

Figure 2

Targeted disruption of the ngn3 gene. (a) Structure of wild-type and targeted ngn3 locus. The ngn3 coding sequence (filled box) was deleted from amino acid 60 to the stop codon at amino acid 214, including the bHLH domain, and was replaced by a PGK-neo selection cassette (NEO). Restriction enzymes: H, HindIII; K, KpnI; N, NotI; X, XhoI; Xb, XbaI. (b) Genotype analysis by Southern blotting of two ES cell lines, one wild-type (lane 1) and one with a targeted ngn3 allele (lane 2), hybridized with a 0.4-kb 3′ external genomic fragment. (c) Genotyping by PCR of a litter from a heterozygous intercross. The two sets of primers used to identify the wild-type (WT) and mutant alleles (generating a 0.2- and a 0.7-kb PCR product, respectively) are indicated by arrows in a. (d) This image of two 3-day-old littermates, one wild-type (upper) and one ngn3-deficient (lower), shows the reduced size of the mutant animal. (e) Pancreas (p), spleen (s), and duodenum (d) of postnatal day 2 wild-type and ngn3 mutant mice. The mutant pancreas does not have any obvious morphological abnormality.

Figure 3

ngn3 is required for the differentiation of the four pancreatic endocrine cell lineages. Islets of Langerhans (arrows in a) and cells expressing insulin (b and f), glucagon (c, g, arrowheads in h), somatostatin (d), PP (e), and Isl1 (arrowheads in i) are observed in wild-type pancreas (a–h) but are completely missing in ngn3 mutant pancreas at birth (compare a–e with a′–e′) and also missing at early stages of differentiation: E15.5 (f, f′, g, and g′), E10.5 (i and i′), and E9.5 (h and h′). (a and a′) Hematoxylin-eosin (HE) staining.

Figure 4

Lack of expression of several essential regulators of pancreatic development in ngn3 mutant mice. In wild-type pancreas, Pdx1 is initially expressed in endodermal progenitors in the pancreatic bud (a), whereas Pax4 (b), Pax6 (c), and NeuroD (d) are expressed in differentiating endocrine cells, and p48 (e) is expressed in the differentiating exocrine tissue. In ngn3-deficient mice, Pdx1 (a′) remains expressed in pancreatic progenitors, and p48 remains expressed in exocrine cells (e′), whereas expression of Pax4 (b′), Pax6 (c′), and NeuroD (d′) is completely missing, suggesting that ngn3 is essential for the specification or differentiation of endocrine precursors. An asterisk (*) marks the background in the lumen of the pancreatic bud also observed in control experiments. Expression of NeuroD in enteroendocrine cells of the gut (arrowheads in d) is also lost in ngn3 mutant mice (d′). (a–c and a′–c′) Pancreas at E10.5. (d–e and d′–e′) Pancreas at E15.5.

Figure 5

The pancreas of ngn3 mutants lacks endocrine precursors. An RNA probe recognizing both wild-type (a) and mutant (a′) ngn3 transcripts in the neural tube labels endocrine precursors in wild-type pancreas at E10.5 (b) but no cells in ngn3 mutant pancreas at the same stage (b′). Prox1 (29) transcripts mark the pancreatic epithelium in adjacent sections of the same embryos (c and c′). The absence of ngn3+ cells in mutant pancreas is unlikely to be due to their death, because no increase in apoptosis was detected in E10.5 mutant (d′) versus wild-type (d) pancreas. (d and d′) Gut cells undergoing apoptosis in the same sections shown in e and e′ are used as control for the TUNEL reaction. sc, spinal cord.

Figure 6

Pancreatic exocrine cells are affected by the loss of ngn3. Ultrastructural analysis of exocrine pancreas at postnatal day 1 shows an accumulation of secretory granules (arrowheads) in acinar cells in ngn3 mutant animals (b) and not in wild-type littermates (a).