Jagged1 is a competitive inhibitor of Notch signaling in the embryonic pancreas

Abstract

Pancreatic endocrine cells originate from precursors that express the transcription factor Neurogenin3 (Ngn3). Ngn3 expression is repressed by active Notch signaling. Accordingly, mice with Notch signaling pathway mutations display increased Ngn3 expression and endocrine cell lineage allocation. To determine how the Notch ligand Jagged1 (Jag1) functions during pancreas development, we deleted Jag1 in foregut endoderm and examined postnatal and embryonic endocrine cells and precursors. Postnatal Jag1 mutants display increased Ngn3 expression, alpha-cell mass, and endocrine cell percentage, similar to the early embryonic phenotype of Dll1 and Rbpj mutants. However, in sharp contrast to postnatal animals, Jag1-deficient embryos display increased expression of Notch transcriptional targets and decreased Ngn3 expression, resulting in reduced endocrine lineage allocation. Jag1 acts as an inhibitor of Notch signaling during embryonic pancreas development but an activator of Notch signaling postnatally. Expression of the Notch modifier Manic Fringe (Mfng) is limited to endocrine precursors, providing a possible explanation for the inhibition of Notch signaling by Jag1 during mid-gestation embryonic pancreas development.

Figure 1

Jagged1 is initially expressed throughout the pancreatic epithelium but gradually becomes limited to ducts. (A) Primers for Jag1, Jag2, Dll1 and TBP amplify with equal efficiency. Not all of icons can be seen because they lie on top of each other. (B) qRT-PCR on total pancreas RNA for Jag1, Jag2 and Dll1 (normalized to TBP) indicate that Jag1 is expressed at much higher levels than the other Notch ligands throughout pancreas development (n=4). (C–R) Indirect immunofluorescence, shown both as single channel images of Pdx1, glucagon, DBA lectin, or insulin (column 1) plus Jag1 (column 2), the nuclear marker DAPI (column three) and merged images (column 4). (C–F) At E12.5, immunostaining indicates that Jag1 (D) is expressed in all Pdx1-expressing cells (C), which mark the undifferentiated pancreatic epithelium. (G–J) Jag1 (H) is expressed in differentiated α-cells (glucagon expression, G) at E14.5, the predominant endocrine cell at this timepoint. (K–N) At E18.5, Jag1 (L) is expressed in ducts labeled with DBA lectin (K). (O–R) Jag1 (P) is also expressed in differentiated islets at E18.5 (insulin, O). For all images, bar = 25 µm except insets, where bar = 50 µm.

Figure 2

Conditional ablation of Jag1 in the pancreatic epithelium. (A) qRT-PCR analysis of Jag1 mRNA levels (relative to TBP) in total pancreas (n=4–7). (B) Western blot on P8 pancreata confirms that Jag1 levels are decreased in Jag1^loxP/loxP;Foxa3-Cre⁺ mutants. α-tubulin is used as a loading control. (C–J) Immunofluorescence confirms deletion of Jag1 in pancreatic epithelium by E18.5. (C–F) At E18.5, Jag1 co-labels with the epithelial, transmembrane E-cadherin in control pancreas islet clusters. (G–J) In contrast, E18.5 Jag1^loxP/loxP;Foxa3-Cre⁺ animals no longer display Jag1 in E-cadherin-expressing islet clusters.

Figure 3

The α-cell compartment is expanded and the exocrine compartment reduced in the postnatal pancreas of Jag1^loxP/loxP;Foxa3-Cre⁺ mice. Indirect immunofluorescence detection at P21 of insulin and glucagon (A,B), insulin and somatostatin (C,D) or insulin and PPY (E,F) in control (A,C,E) and mutant (B,D,F) mice (bar = 50 µm). (G) Mutants display increased α-cell mass with no change β-cell mass. (n=4). (H) mRNA levels for the α-cell hormone glucagon and the α-cell transcription factors Arx and Brn4 are increased in the pancreas of P12 Jag1^loxP/loxP;Foxa3-Cre⁺ mutants while the expression of insulin mRNA is not changed (n=4). (*p<0.05).

Figure 4

Ngn3-eGFP is upregulated and Hes3 mRNA downregulated in Jag1 mutants. (A–B) At P0, brown Ngn3 staining is detected in only a few cells in control (A) but in many mutant cells (B). (C–D) Ngn3-eGFP expression is not detected in P7 control (C) but many eGFP⁺ cells can be detected in Jag1 deficient pancreas (D) (bars = 50 µm).

Figure 5

Acinar tissue is reduced due to postnatal cell death. (A) Pancreas weight is decreased at P21. (B) Total pancreatic epithelium is unchanged at E12.5, E16.5, or E18.5. (C–E) Acinar cell death, marked by TUNEL staining, is increased by approximately 6-fold in P0-P1 Jag1 mutants. (C) Quantification of TUNEL⁺ acinar cells as number of cells per acinar area (n=6–10). (D–E) Representative images of TUNEL staining for control (D) and mutant (E) pancreas. Arrows designate TUNEL-positive cells. (bars = 50 µm).

Figure 6

Paradoxical reduction of the endocrine lineage in E16.5 Jag1 mutant embryos. (A) qRT-PCR on E16.5 total pancreas mRNA. mRNA of transcriptional targets of Notch is increased and that for endocrine markers is reduced in E16.5 Jag1 mutants. (n=4; for Hes3, p=0.065). (B) Quantification of percentage of nuclei positive for Ngn3⁺ (representative images shown in C&D) indicates a smaller percentage of endocrine precursors within Jag1 mutants at E16.5 (n=4–5). (C,D) Representative images of indirect immunofluorescence detection of Ngn3 with DAPI counterstaining in control (C) and mutant (D) pancreas. The red signal in the upper righthand corner of the image is due to autofluorescent blood cells. (E–F) Indirect immunofluorscence of insulin and glucagon in control (E) and mutant (F) pancreas. (G) Quantification of insulin⁺ and glucagon⁺ cells as cells per square millimeter. Glucagon⁺ cells are reduced by approximately one-half in Jag1 mutants (n=10–14). (H) qRT-PCR for ngn3, ins, and glu mRNA shows that Jag1 mutants begin to recover endocrine mass by E18.5 (n=4) (I) Quantification of E18.5 α-and β-cells confirms that endocrine mass starts to recover by E18.5 (n=6). (J) Recovery of endocrine mass does not occur by proliferation of α- or β-cells (n=5–10). (*p<0.05; bar = 50 µm).

Figure 7

Manic Fringe is expressed exclusively in endocrine precursors of the fetal pancreas and causes Jag1 to act as a competitive inhibitor of Dll1. (A) In the pancreas, Mfng expression is limited to cells that express Ngn3-eGFP, i.e. endocrine precursors and their immediate descendants. Mfng expression was normalized to the mean of four housekeeping genes (TBP, GAPDH, HPRT, UBC) (B) Jag1 acts as a competitive inhibitor of Dll1-Notch signaling in NIH 3T3 cells expressing Manic Fringe. Representative Western blots and quantification (n=3 for lanes 1–2 and n=5) for Lanes 3–8). Control cells (lanes 1, 3, 5 & 7) or cells expressing Mfng (lanes 2, 4, 6 & 8) were plated on Jag1 ligand (lanes 3–4), Dll1 ligand (lanes 5–6), or a combination of both (lanes 7–8) and Notch activation was measured by immunoblotting for cleaved Notch1. All ligands activated Notch cleavage compared to uninduced cells (lanes 1&2), and the combination of Dll1, Jag1, and Mfng yielded low levels of Notch activation (*p<0.05). (C) Model: Manic Fringe promotes endocrine differentiation by causing Jag1 to act as a competitive inhibitor of Dll1 during pancreatogenesis. When Mfng expression is high, resulting in high levels of Notch glycosylation, Jag1 binds to but does not activate Notch, thus acting as a competitive inhibitor of Dll1. In this situation, Notch signaling decreases, Hes/Hey gene expression decreases, and Ngn3 is expressed. However, in Jag1 mutants, Dll1 has increased access to the Notch receptor, and glycosylation potentiates the cleavage of Notch when bound by Dll1, leading to relatively high Notch activation in cells that express Mfng. Active Notch signaling represses Ngn3 expression, resulting in fewer endocrine precursors and differentiated endocrine cells.