Notch-mediated patterning and cell fate allocation of pancreatic progenitor cells

Abstract

Early pancreatic morphogenesis is characterized by the transformation of an uncommitted pool of pancreatic progenitor cells into a branched pancreatic epithelium that consists of 'tip' and 'trunk' domains. These domains have distinct molecular signatures and differentiate into distinct pancreatic cell lineages. Cells at the branched tips of the epithelium develop into acinar cells, whereas cells in the trunk subcompartment differentiate into endocrine and duct cells. Recent genetic analyses have highlighted the role of key transcriptional regulators in the specification of these subcompartments. Here, we analyzed in mice the role of Notch signaling in the patterning of multipotent pancreatic progenitor cells through mosaic overexpression of a Notch signaling antagonist, dominant-negative mastermind-like 1, resulting in a mixture of wild-type and Notch-suppressed pancreatic progenitor cells. We find that attenuation of Notch signaling has pronounced patterning effects on multipotent pancreatic progenitor cells prior to terminal differentiation. Relative to the wild-type cells, the Notch-suppressed cells lose trunk marker genes and gain expression of tip marker genes. The Notch-suppressed cells subsequently differentiate into acinar cells, whereas duct and endocrine populations are formed predominantly from the wild-type cells. Mechanistically, these observations could be explained by a requirement of Notch for the expression of the trunk determination gene Nkx6.1. This was supported by the finding of direct binding of RBP-jκ to the Nkx6.1 proximal promoter.

Fig. 1.

Notch signaling is required for endocrine differentiation. (A) The Notch transcriptional complex and truncated Maml1 (dnMAML1) lead to a dominant-negative effect on the expression of Notch target genes. (B) Transgenic overexpression of dnMAML1. Pdx1 promoter-driven expression of tTA results in transcriptional activation of the dnMAML1-IRES-nEGFP mRNA. Presence of doxycycline prevents tTA-mediated expression of the transgene. (C,D) Mid-gut dissection of wild-type (Wt) and dnMAML1;tTA double transgenic (DTG) embryos at E18.5 (C). Under fluorescent light (D), green fluorescence of the DTG pancreas in C is visible. (E-H) Immunofluorescence staining for pancreatic differentiation markers in wild-type (E,G) and DTG (F,H) pancreas at E18.5. (I,J) Morphometric quantification of the exocrine tissue based on amylase (I), the endocrine compartment based on endocrine-specific gene expression and the duct cells marked by the duct-specific lectin DBA (J). Values are mean ± s.d. ^*, P<0.05; ^**, P<0.005; n.s., not statistically significant. n=3 for all analyses in I,J. Amy, amylase; Ghr, ghrelin; Glu, glucagon; Ins, insulin. Scale bar: 50 μm.

Fig. 2.

Acinar cell-specific expression of the dnMAML1 transgene. (A-D) Immunofluorescence staining of amylase and insulin (A,B) and of the ductal markers Hnf1β and DBA lectin (C,D) in wild-type (A,C) and DTG (B,D) E18.5 pancreas. (B,D) The expression of EGFP (green) indicates the distribution of transgene expression relative to that of pancreatic differentiation markers. Relative expression levels of EGFP to differentiation markers are: amylase⁺; GFP⁺/amylase⁺=0.69±0.10; insulin⁺; GFP⁺/insulin⁺=0.048±0.031; Hnf1β⁺; GFP⁺/Hnf1β⁺=0. (E) Quantification of proliferation rates of E13.5 Pdx1⁺ wild-type and DTG pancreatic cells, Nkx6.1⁺ wild-type and DTG pancreatic cells, and transgene-negative (GFP^–) or transgene-positive (GFP⁺) epithelial cells in the DTG pancreas based on phospho-histone H3 (pHH3) immunofluorescence staining. n=3. Values are mean ± s.d. n.s., not statistically significant. Scale bar: 50 μm.

Fig. 3.

Effect of dnMAML1 on pancreatic epithelial patterning. (A-P) Fluorescence visualization of transgene-derived nEGFP expression relative to immunofluorescence staining of Ptf1a (A-C), Nkx6.1 (E-G), Hnf1β (I-K) or Sox9 (M-O). Quantitative analysis of the percentage of nEGFP cells that are positive or negative for Ptf1a (D), Nkx6.1 (H), Hnf1β (L) or Sox9 (P). Sox9 expression was additionally categorized as high (Sox9hi) or medium (Sox9me) level. White arrows indicate GFP⁺ cells that are negative for a given pancreatic marker; arrowheads indicate EGFP⁺ cells that are positive for pancreatic markers; yellow arrows indicate EGFP⁺ cells that express medium level of Sox9. (Q) Isolation of E13.5 EGFP-positive and EGFP-negative pancreatic epithelial cells by flow cytometry. Dissociated embryonic pancreas tissue was stained with APC-conjugated anti-E-cadherin prior to sorting. (R) x-axis (FITC) indicates GFP intensity and y-axis (APC-A) indicates intensity of epithelial staining by APC-conjugated E-cadherin. (S) Quantitative RT-PCR analysis of pancreatic progenitor markers in EGFP-positive epithelial cells expressed as fold increase or decrease relative to EGFP^– epithelial cells. EGFP⁺ cells have 790.8±71.6-fold more dnMAML1 than EGFP^– cells. Values are mean ± s.d. Scale bar: 50 μm.

Fig. 4.

Suppression of Nkx6.1 expression by dnMAML1 precedes its effect on Ptf1a. Fluorescence visualization of the transgene-derived nEGFP relative to immunofluorescence staining of (A-C) Hes1, (E-G) Ptf1a, (I-K) Nkx6.1, (M-O) Hnf1β and (Q-S) Sox9. Arrows indicate EGFP⁺ cells that are negative, and arrowheads indicate EGFP⁺ cells that are positive, for a given pancreatic marker gene. Quantitative analysis of the percentage of EGFP⁺ cells that are positive or negative for (D) Hes1, (H) Ptf1a, (L) Nkx6.1, (P) Hnf1β and (T) Sox9. Values are mean ± s.d. Scale bar: 50 μm.

Fig. 5.

Nkx6.1 is a direct target of Notch. (A-C) Immunofluorescence staining of Nkx6.1 (A) and Hes1 (B) in E12.5 wild-type pancreas. (C) Overlay of A and B. (D,E) ChIP-Seq analysis on the Nkx6.1 locus (D) and Hes1 locus (E) with anti-RBP-jκ antibodies on E12.5, E15.5 and E17.5 pancreatic chromatin (red tracks), anti-Ptf1a antibody on E15.5 pancreatic chromatin (blue tracks), anti-RNA polymerase II antibody on E15.5 pancreatic chromatin (yellow tracks), as well as input tracks. Arrows (D,E) indicate RBP-jκ occupancy of the Nkx6.1 and Hes1 loci, respectively. Scale bar: 50 μm.

Fig. 6.

Suppression of Notch signaling through dnMAML1 does not lead to premature endocrine cell differentiation. Immunofluorescence staining of Pdx1 and insulin in (A) E11.5 and (C) E12.5 wild-type pancreas and in the presence of transgene-derived nEGFP expression in (B) E11.5 and (D) E12.5 DTG embryos. Immunofluorescence staining of Pdx1 and glucagon in (E) E11.5 and (G) E12.5 wild-type pancreas and in the presence of transgene-derived nEGFP in DTG embryos at (F) E11.5 and (H) E12.5. Scale bar: 50 μm.

Fig. 7.

Model of Notch-mediated patterning of multipotent pancreatic progenitor cells into tip and trunk domains. (A) At E12.5, the pancreatic epithelium is organized into tip and trunk domains, and Pdx1 is expressed throughout all pancreatic cells, whereas Ptf1a is restricted to the tip domain marking pro-acinar cells. Although the majority of Notch-deficient cells are localized to the tip domain, a few remain in the trunk (arrows). (B) By E14.5, almost all Notch-deficient cells have resolved into the tip domain. (C) This suggests a dynamic process of Notch-mediated patterning of MPCs, in which the loss of Notch within a multipotent progenitor leads to a tip fate and localization. MPC, multipotent pancreatic progenitor cell; TipPC, tip domain; TrPC, trunk domain. Scale bar: 50 μm.