Direct regulation of intestinal fate by Notch

Abstract

The signals that maintain the proper balance between adult intestinal cell types are poorly understood. Loss-of-function studies have implicated the Notch pathway in the regulation of intestinal fate during development. However, it is unknown whether Notch has a role in maintaining the balance of different cell types in the adult intestine and whether it acts reversibly. To determine whether Notch has a direct effect on intestinal development and adult intestinal cell turnover, we have used a gain-of-function approach to activate Notch. Ectopic Notch signaling in adult intestinal progenitor cells leads to a bias against secretory fates, whereas ectopic Notch activation in the embryonic foregut results in reversible defects in villus morphogenesis and loss of the proliferative progenitor compartment. We conclude that Notch regulates adult intestinal development by controlling the balance between secretory and absorptive cell types. In the embryo, Notch activation perturbs morphogenesis, possibly through effects on stem or progenitor cells.

Fig. 1.

Misexpression of NotchIC in intestinal progenitor cells. (A) Transgenic Fabpl-Cre mice were bred to Z/AP reporter and Rosa-NotchIC strains. Trigenic mice expressing Cre activate NotchIC in a subset of crypt progenitor cells and their progeny through the excision of a “floxed” transcriptional stop sequence (STOP). Transcription of the HPAP transgene is activated in these clones (from the Z/AP reporter allele) by the same mechanism, permitting NotchIC-expressing clones to be recognized by the concomitant expression of HPAP. (B and C) The intestine of an Fabpl-Cre;Z/AP adult mouse stained for HPAP (hematoxylin counterstain). Cre activity in intestinal progenitor cells results in HPAP activity in an arc of cells in villi that have been cut in cross section (B) or in a portion of two adjacent villi derived from the same crypt cut in saggital section (C). (D–G) Reduced goblet cell differentiation (D and E) and endocrine cell differentiation (F and G) in villi exposed to NotchIC. Costaining for HPAP and alcian blue shows a paucity of goblet cells (arrowheads) in HPAP-positive regions of Fabpl-Cre;Rosa-NotchIC;Z/AP trigenic intestines (E) compared with Fabpl-Cre;Z/AP controls (D). Costaining for HPAP and chromogranin A/B shows a paucity of endocrine cells (arrowheads) in HPAP-positive regions of Fabpl-Cre;Rosa-NotchIC;Z/AP trigenic intestines (G) compared FABPCre;Z/AP controls (F). (H) Notch signals block secretory fates in adult intestine. Fabpl-Cre;Rosa-NotchIC;Z/AP and Fabpl-Cre;Z/AP mice were double stained for HPAP and alcian blue (Upper) or chromogranin A/B(Lower). Cells in HPAP⁺ and HPAP^– villus segments were scored separately according to genotype by two independent observers. Each circle indicates data from a single mouse, presented as a percentage of total cells counted. The mean percentage is indicated with a red line. Additional details are listed in the Table 1.

Fig. 2.

Conditional misexpression of NotchIC in the embryonic intestine. (A) Drawing of the embryonic foregut and midgut structures analyzed in this study. The intestinal expression domain of Pdx1 is indicated by blue dots, with the dorsal and ventral pancreas shaded solid blue. (B and C) Whole mount photographs of E18.5 wild type (B) and Pdx1-tTA;tetO-NotchIC bigenic embryos (C), showing loss of pancreatic tissue and defective morphogenesis. The wild-type pylorus (arrow) is obliterated in the bigenic (arrowhead). The small intestine distal to the cyst (C, bracket) appears grossly normal. (D and E) Hematoxylin/eosin (H&E) staining of the peri-pyloric structures in E18.5 wild type (D) and bigenic embryos (E). At this stage, Pdx1 is widely expressed in the pancreas and duodenum (D Inset). In the bigenic embryo, the distal stomach, pancreas, and proximal duodenum have been replaced by a thin-walled cystic epithelium (E). (F) In situ hybridization of duodenum from E12.5 wild-type (Upper) and Pdx1-tTA;tetO-NotchIC (Lower) embryos with a NotchIC antisense probe. Endogenous Notch1 transcripts are found predominantly in the mesenchyme of wild-type intestine (arrowheads), whereas transcripts reflecting transgene expression are also found in the epithelium of Pdx1-tTA;tetO-NotchIC duodenum. (G and H) Pdx1 and Hes1 immunostaining (E12.5). Adjacent sections from a wild-type (Left) or Pdx1-tTA;tetO-NotchIC bigenic embryo (Right) were stained for Pdx1 (G) or Hes1 (H). Pdx1 staining is observed in the branching epithelium of the wild-type pancreas and throughout the duodenal epithelium of both wild-type and bigenic duodenum (G). Hes1 expression in wild-type embryos is observed in the pancreatic epithelium and the duodenal mesenchyme (arrow), but only rarely in the duodenal epithelium. By contrast, Hes1 is expressed throughout the duodenal epithelium of bigenic embryos (arrowheads), indicating Notch pathway activation (H). C, cyst; DP, dorsal pancreas; VP, ventral pancreas; St, stomach; Du, duodenum; Sp, spleen.

Fig. 3.

Constitutive Notch signaling perturbs intestinal morphogenesis; wild-type (Left) and bigenic Pdx1-tTA;tetO-NotchIC (Right) embryos are compared. (A) H&E staining of E18.5 duodenum (in a region distal to the cyst). Compared with wild type, villi from bigenic embryos are blunted and malformed. (B) Disruption of the basal intestinal proliferative layer (E17.5). Pregnant mice were injected with BrdUrd 2 h before killing. Bigenic embryos exhibit a reduced number of BrdUrd⁺ cells compared with wild-type controls. (C) Normal conductin/axin2 expression assessed by in situ hybridization (E18.5). Despite exhibiting villus malformation, conductin/axin2 is expressed in the duodenal crypts of bigenic embryos (arrowheads) with a distribution and level that were comparable with that of wild-type controls. (D and E) TUNEL assays were performed on E15.5 (D) and E18.5 (E) littermates. (D) At E15.5, rare TUNEL⁺ cells (arrowheads) are observed in both wild-type and bigenic embryos; frequent TUNEL⁺ cells in the adjacent liver (Lower Right) served as an internal control. (E) At E18.5, TUNEL⁺ cells are observed at the same frequency in both wild-type and bigenic intestines, predominantly at villus tips (arrowheads). (F and G) Loss of Pdx1 and NotchIC transgene expression in the bigenic intestinal epithelium. At E15.5, most duodenal epithelial cells in wild-type mice express Pdx1 (left). By contrast, most cells in the duodenal epithelium of bigenic embryos have lost Pdx1 expression (right). At E18.5, the NotchIC transgene is expressed throughout the cyst epithelium of bigenic embryos (G Center arrowheads) but is detectable in only a few isolated cells in the intestine (G Right). NotchIC expression is below the level of detection in wild-type intestine by in situ hybridization at this stage (G Left).

Fig. 4.

The intestinal effects of Notch activation are reversible. (A) Scheme for temporal regulation of the NotchIC transgene. Each red bar indicates the time window during which the transgene was expressed (i.e., no tetracycline), corresponding to panels B–E. Silencing of the transgene was achieved by the addition of tetracycline (vertical bar). Embryos were killed at E18.5 (arrow) and examined grossly and histologically. A section of bigenic intestine stained with H&E is included for reference. (B–E) Whole mount and H&E staining of E18.5 intestines (bracketed areas) from embryos in which NotchIC has been repressed starting at E14.5 (n = 2; B and B′), E13.5 (n = 4; C and C′), E12.5 (n = 2; D and D′), or E11.5 (n = 3; E and E′). Recovery of pancreatic development (yellow arrowheads) occurs with transgene silencing at E13.5 or earlier (C–E), whereas recovery of intestinal morphogenesis is observed at all stages (B′–E′). In some dissections, a portion of the liver was left in situ (*).