Notch signaling controls multiple steps of pancreatic differentiation

Abstract

Multiple cell types of the pancreas appear asynchronously during embryogenesis, which requires that pancreatic progenitor cell potential changes over time. Loss-of-function studies have shown that Notch signaling modulates the differentiation of these progenitors, but it remains unclear how and when the Notch pathway acts. We established a modular transgenic system to heritably activate mouse Notch1 in multiple types of progenitors and differentiated cells. We find that misexpression of activated Notch in Pdx1-expressing progenitor cells prevents differentiation of both exocrine and endocrine lineages. Progenitors remain trapped in an undifferentiated state even if Notch activation occurs long after the pancreas has been specified. Furthermore, endocrine differentiation is associated with escape from this activity, because Ngn3-expressing endocrine precursors are susceptible to Notch inhibition, whereas fully differentiated endocrine cells are resistant.

Fig. 1.

Cre-activated misexpression of activated Notch1. (A) Structure of the Rosa^Notch locus. Transcription (gray arrow) is initially blocked by the STOP cassette, which is deleted by Cre recombinase, permitting transcription of NotchIC and internal ribosome entry sequence–nuclear EGFP (nEGFP). (B) Whole-mount in situ hybridizations (purple staining) for Pdx1 and EGFP at E9.5, when transgene activation is first detected in the pancreatic buds (arrowheads) of bigenic Rosa^Notch;Pdx1-Cre embryos. (C) Gross phenotype of monogenic and bigenic pancreata. (a and c) At E17.5, bigenic pancreas is translucent and cystic. (b and d) Hematoxylin/eosin-stained sections of neonatal pancreata reveal tubular epithelia and absence of normal acinar and islet tissue in Rosaw^Notch;Pdx1-Cre bigenics. ac, acini; d, duodenum; dp, dorsal pancreas; is, islet; s, stomach; vp, ventral pancreas.

Fig. 2.

Early activation of Rosa^Notch inhibits endocrine and exocrine development. (A) Insulin and amylase expression in E15.5 monogenics (a and b) and bigenics (c and d) stained by diaminobenzidene (brown) with hematoxylin counterstain. Bigenic Rosa^Notch;Pdx1-Cre pancreata contain tubular epithelial cells devoid of differentiation markers. Arrowhead in d indicates a cluster of acinar cells in the bigenic pancreas. (B) Confocal immunofluorescence indicates that residual differentiated cells (red) in bigenics (c and d) are largely EGFP-negative (green), indicating that they do not express NotchIC. Green signal in monogenic sections (a and b) represents background autofluorescence. (C) Pdx1 (a and d) and EGFP (b and e) expression detected by confocal immunofluorescence in neonatal pancreata. In Rosa^Notch monogenics, Pdx1 is largely restricted to β cells (arrowhead in a indicates an islet), whereas bigenic pancreata maintain broad Pdx1 expression in EGFP⁺ cells (merged signal in c and f; overlap is yellow).

Fig. 3.

Late activation of Rosa^Notch inhibits endocrine and exocrine development. (A) Embryos were treated with sequential doses of TM at E13.5 and E14.5 and examined at E17.5. Expression of insulin and amylase (brown) is inhibited in bigenics (c and d) compared with wild-type littermates (a and b), concomitant with appearance of undifferentiated epithelial tubules. (B) Embryos were administered a single dose of TM at E11.5 and examined at E15.5 by immunofluorescence for insulin and amylase (red; EGFP is shown in green). EGFP expression in bigenics (c and d) is excluded from differentiated cells.

Fig. 4.

Stages of endocrine lineage commitment responsive to NotchIC inhibition. (A) Ngn3 expression in E15.5 monogenic Rosa^Notch/+ (a) and bigenic Rosa^Notch/+;Pdx1-Cre (b) pancreata. Ngn3⁺ cells are reduced in number in the bigenic and are absent from most epithelial tubules. (B) Glucagon expression in E13.5 monogenic Rosa^Notch;Ngn3-Cre and bigenic pancreata, stained in brightfield (brown, a and b) or by immunofluorescence (red, merged with EGFP in green, c and d). Bigenic embryos exhibit dramatic reduction of glucagon-positive cells, and remaining cells are EGFP-negative, indicating that they do not express NotchIC. (C) Widespread expression of NotchIC in adult β cells, by adult activation of Pdx1-CreER (b), does not affect insulin expression (red; EGFP is shown in green) or islet morphology.

Fig. 5.

Persistence of undifferentiated cells in adult Rosa^Notch;Pdx1-Cre bigenics. (A) Wild-type pancreas (a) comprises islet, acinar, and duct tissue, whereas the bigenic adult pancreata contains large regions of tubular epithelium (b) similar to that seen in embryos. (B) As in the embryo, EGFP⁺ epithelial cells are largely negative for amylase (a and b) and glucagon (e and f) expression, whereas insulin-positive cells frequently coexpress EGFP (c and d), suggesting Rosa^Notch activation subsequent toβ cell differentiation (differentiation markers are shown in red, and EGFP is shown in green). ac, acini; du, duct; is, islet.