Myt1 and Ngn3 form a feed-forward expression loop to promote endocrine islet cell differentiation

Abstract

High levels of Ngn3 expression in pancreatic progenitor cells are both necessary and sufficient to initiate endocrine differentiation. While it is clear that the Notch-Hes1-mediated signals control the number of Ngn3-expressing cells in the developing pancreas, it is not known what factors control the level of Ngn3 expression in individual pancreatic cells. Here we report that Myt1b and Ngn3 form a feed-forward expression loop that regulates endocrine differentiation. Myt1b induces glucagon expression by potentiating Ngn3 transcription in pancreatic progenitors. Vice versa, Ngn3 protein production induces the expression of Myt1. Furthermore, pancreatic Myt1 expression largely, but not totally, relies on Ngn3 activity. Surprisingly, a portion of Myt1 expressing pancreatic cells express glucagon and other alpha cell markers in Ngn3 nullizygous mutant animals. These results demonstrate that Myt1b and Ngn3 positively regulate each other's expression to promote endocrine differentiation. In addition, the data uncover an unexpected Ngn3 expression-independent endocrine cell production pathway, which further bolsters the notion that the seemingly equivalent endocrine cells of each type, as judged by hormone and transcription factor expression, are heterogeneous in their origin.

Figure 1. Derivation of mouse lines that ectopically express Myt1b and Ngn3

(A) Pancreatic Myt1b expression (red) at E10.5. Wild type (WT), which showed identical Myt1 expression pattern as Pdx1^tTA/+ pancreas, and Pdx1^tTA/tTA (labeled as tTA/tTA) pancreata were used as controls. The presence of Myt1b^tet2 (labeled as Myt1b) led to Myt1b ectopic expression in the presence of tTA. Pdx1 (green) or glucagon staining (green) were used to mark the position of the pancreatic buds. Note that Myt1 antibodies only stain frozen, lightly fixed sections, on which the Pdx1 signal appears diffuse. (B) Quantification of Myt1b ectopic expression in control and Myt1b; Pdx1^tTA/+ pancreata. The number of Myt1b⁺ cells is normalized against the number of Pdx1⁺ cells. (C) Ngn3 expression (green) in WT and Ngn3^tet8; Pdx1^tTA (labeled as Ngn3; tTA) pancreata. White broken lines, pancreatic region. Note the apparent thickening of pancreatic epithelium that ectopically expresses Ngn3. All panels used identical scales, bar = 20 μm.

Figure 2. Ectopic Myt1b expression induces glucagon expression

(A) The relative number (mean ± SEM) of glucagon-expressing cells in E11.5 embryos (tTA = Pdx1^tTA. Myt1b = Myt1b^tet2). (B–E) Myt1 and glucagon (gluc) production in representative sections of E11.5 pancreata. Scale bars = 20 μm.

Figure 3. Myt1b ectopic expression induces the expression of α cell markers

Sections of E12.5 pancreata. Pdx1^tTA/tTA pancreata were used as controls. (A) Co-expression of glucagon and PC1/3 in Myt1b induced endocrine cells. (B and C) MafB and Nkx6.1 expression in pancreata that ectopically express Myt1b, respectively. Each sample contains two single fluorescence channels and one merge. tTA/tTA = Pdx1^tTA/tTA. 1b;tTA/tTA = Myt1b^tet2; Pdx1^tTA/tTA. White arrows in B point to blood cells. All panels used identical scales, bar = 20 μm.

Figure 4. Myt1b induces glucagon expression through Ngn3

Hemizygous pancreata containing Pdx1^tTA and other mutations were utilized (tTA = Pdx1^tTA. Myt1b = Myt1b^tet2). (A) The relative number of Ngn3⁺ cells in Myt1b; Pdx1^tTA/+ pancreata over that of wild type control littermates at E10.25 (n = 7. P = 0.015). (B) The average number of Ngn3⁺ cells in Pdx1^−/− pancreata that ectopically express Myt1b at E10.25. (C) Immunofluorescence showing Ngn3 production in pancreata that ectopically express Myt1b. Two stages, E9.5 and E10.25, are shown. Note the presence of glucagon⁺Ngn3⁺ cells (white arrows) in pancreata with ectopic Myt1 expression. Red, Ngn3. Green, glucagon. (D–F) The production and genotyping strategies of a novel Ngn3 null allele (Ngn3⁻). (D) The structure of the Ngn3 wild type, floxed (Ngn3^F) and null allele. A Pme I site was introduced in the 5′end of the Ngn3 coding cDNA in the Ngn3^F allele. Also note that the Ngn3⁻ allele produces CreER^™. (E) Southern blot identifies four targeted ES cell clones, producing a 8.1 kb wild type and a 6.4 kb mutant band when digested with XbaI + PmeI and blotted with P1 probe. The numbers (45, 722, and 723) and small arrows indicate the position of oligos used for PCR-based genotyping (F). (G) Myt1b-induced glucagon expression in pancreata with or without Ngn3 at E12.5. Note the rare glucagon⁺ cells (green, white arrowhead) in Ngn3^−/−mutant pancreas. Red, Myt1. All panels utilized identical scales, bar = 20 μm.

Figure 5. Ngn3^−/− mutant pancreas maintains Myt1 and glucagon expression

Glucagon (green), Myt1 or MafB (red) expression at three stages, E9.5, E12.5, and E13.5. (A–D) Ngn3^−/−pancreata. (E-H) Wild type pancreata. White arrowheads point to glucagon⁺Myt1⁺ cells in Ngn3^−/− pancreata. All panels used identical scales, bars = 10μm.

Figure 6. Ectopic Ngn3 expression induces Myt1 expression

(A and B) Myt1 and Ngn3 expression in pancreata that ectopically express Ngn3. (C) Genomic pair-wise alignment of Myt1 promoters from different species. Grey peaks indicate conserved regions. The pink column indicates the first exon. The 1.8 kb Myt1b promoter region used for reporter assays is marked by a light green bar on top. (D and E) Myt1 promoter activity assays in 3T3 and P19 cell lines. In each cell line, mock transfected, Ngn3, and a mutant Ngn3 (N89D) expressing vectors were co-transfected with the respective luciferase reporter plasmid. * denotes that the differences between control and experimental groups were statistically significant. Scale bars = 10μm.

Figure 7. Transactivating properties of Myt1 is required for endocrine differentiation

(A) A scheme showing the structures of Myt1-EnR and Myt1-Vp16. cDNA encoding zinc fingers 2 and 3 of Myt1b was cloned in frame with the engrailed repressive domain (Myt1-EnR) or the VP16 activation domain (Myt1-VP16) to obtain the desired open reading frames. (B–G) Endocrine marker expression in chicken endodermal cells electroporated with different DNA constructs, all shown as confocal image projections. (B) A chicken embryo electroporated with EGFP alone. Note that electroporated cells (green) could be observed in the dorsal part of the gut tube while glucagon-expressing cells (red) could only be detected in the dorsal pancreas. (C and D) Results of Ngn3 and Myt1-VP16 co-expression. Note the expression of glucagon (C) and Pax6 (D) in electroporated cells (green). The endogenous pancreas in C is outside of the view. (E) An embryo electroporated with mouse Ngn3 expressing plasmid alone. Note glucagon expression outside the pancreatic bud (white broken lines). (F and G) Embryos with Ngn3 and Myt1-EnR co-expression. Note the delamination of electroporated cells with or without Myt1-EnR production outside the endogenous pancreas (white broken lines, F). Also note the near absence of Pax6⁺ cells in the cells that ectopically express Ngn3 and Myt1-EnR (G). In B, C, E, and F, Foxa2 expression (grey color) highlights the gut tube. Bars = 20 μm.

Figure 8. A model for Myt1-Ngn3 interaction

Notch inactivation may induce the expression of Myt1 and Ngn3. The presence of both Myt1 and Ngn3 promotes endocrine differentiation synergistically.