Nkx6.1 controls a gene regulatory network required for establishing and maintaining pancreatic Beta cell identity

Abstract

All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin(+) cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)-derived insulin(+) cells, our study has significant implications for developing cell replacement therapies.

Figure 1. Forced Nkx6.1 expression favors the beta cell fate choice.

(A, B) Schematic of the transgenes for conditional Nkx6.1 misexpression and cell lineage tracing; Triangles, loxP sites; Ovals, internal ribosomal entry site (IRES). (C–L) Immunofluorescence staining of pancreata from Ngn3-Cre;Z/EG and Ngn3-Cre;Nkx6.1^OE mice at postnatal day (P) 2 for GFP together with each of the five endocrine hormones. The insets display higher magnification images. Arrowheads point to GFP⁺ cells expressing each of the five hormones in Ngn3-Cre;Z/EG mice and insulin, but not glucagon, somatostatin, pancreatic polypeptide, or ghrelin in Ngn3-Cre;Nkx6.1^OE mice. Quantification of hormone⁺GFP⁺ (M) or Ki67⁺GFP⁺ (N) co-positive cells as a percentage of all GFP-expressing cells in pancreata of Nkx6.1^f/−;Ngn3-Cre;Z/EG and Ngn3-Cre;Z/EG mice at P2 (n = 4). Forced expression of Nkx6.1 in endocrine precursors favors a beta cell fate choice over all other non-beta endocrine cell fate choices. Horm, hormones; Ins, insulin; Glc, glucagon; Som, somatostatin; PP, pancreatic polypeptide; Ghr, ghrelin; endo, endocrine. Scale bar = 50 µm. Error bars represent S.E.M; *p<0.05, **p<0.01, ***p<0.001.

Figure 2. Nkx6.1 is required for beta cell specification downstream of Ngn3.

(A, B) Schematic of the alleles and transgenes for Nkx6.1 inactivation and lineage tracing; Triangles, loxP sites. Immunofluorescence staining of pancreata at e15.5 (C, D) or postnatal day (P) 2 (E–P). Recombined, GFP⁺ cells are restricted to the endocrine compartment (antibody against the pan-endocrine marker Chromogranin A, Chga) in control (E) and Nkx6.1^f/−;Ngn3-Cre;Z/EG mice (F). The insets show higher magnifications and arrowheads point to GFP⁺ cells expressing Ngn3 (C, D) or hormones (G–P). Quantification of hormone⁺GFP⁺ (Q), Ki67⁺GFP⁺ (R), or TUNEL⁺GFP⁺ (S) co-positive cells as a percentage of all GFP-expressing cells in pancreata of Nkx6.1^f/−;Ngn3-Cre;Z/EG and Ngn3-Cre;Z/EG mice at P2 (n = 4). Loss of Nkx6.1 in endocrine precursors favors alternative, non-beta endocrine cell fate choices over beta cell fate. Horm, hormones; Ins, insulin; Glc, glucagon; Som, somatostatin; PP, pancreatic polypeptide; Ghr, ghrelin; endo, endocrine. Scale bar = 50 µm. Error bars represent S.E.M; *p<0.05, **p<0.01.

Figure 3. Nkx6.1 controls Pdx1 and MafB expression.

Immunofluorescence staining of pancreata at e15.5 shows no difference in Pax4 expression (A, B) in recombined, GFP⁺ cells between Nkx6.1^f/−;Ngn3-Cre;Z/EG embryos and control Ngn3-Cre;Z/EG embryos, while MafB (C, D) and Pdx1 (E, F) expression is reduced in Nkx6.1-deficient, GFP⁺ cells compared to control embryos. Ins, insulin. Scale bar = 50 µm.

Figure 4. Loss of Nkx6.1 in endocrine precursors results in activation of non-beta endocrine genes.

(A–L) Immunofluorescence staining of pancreata from Ngn3-Cre;Z/EG and Nkx6.1^f/−;Ngn3-Cre;Z/EG mice at postnatal day (P) 2 shows reduced Pdx1 (A, B), absence of MafA (C, D), and ectopic expression of Arx (E, F), Brn4 (G, H), glucagon (Glc; I, J), and somatostatin (Som; K, L) in Nkx6.1-deficient, recombined, insulin⁺GFP⁺ cells. Arrowheads point to insulin⁺ cells ectopically expressing non-beta endocrine markers. Ins, insulin. Scale bar = 50 µm.

Figure 5. Nkx6.1 and Pdx1 collectively stabilize beta cell identity.

(A–H) Immunofluorescence staining of pancreata from wild type, Nkx6.1^f/+;Ngn3-Cre, Pdx1^+/−, and Nkx6.1^f/+;Ngn3-Cre;Pdx1^+/− mice at postnatal day (P) 2 reveals occasional coexpression of insulin with glucagon but not with somatostatin in all genotypes (A–D; arrowheads and insets). Both Arx⁺ (E–H) and Arx⁻ (E′–H′) insulin⁺glucagon⁺ cells (arrowheads and insets) are found in all genotypes. (I) Quantification of the percentage of insulin⁺ cells co-expressing glucagon at P2 reveals significantly more insulin⁺glucagon⁺ cells in Nkx6.1^f/+;Ngn3-Cre;Pdx1 ^+/−, Nkx6.1^f/+;Ngn3-Cre, and Pdx1^+/− mice compared to wild type controls. In addition, Nkx6.1^f/+;Ngn3-Cre;Pdx1 ^+/− mice show more insulin⁺glucagon⁺ cells than either single heterozygous mutant (n = 3). (J) Quantification of insulin⁺ and glucagon⁺ cell numbers in P2 pancreata shows an increase in glucagon⁺ cells in Nkx6.1^f/+;Ngn3-Cre;Pdx1^+/− and Pdx1^+/− mice compared to Nkx6.1^f/+;Ngn3-Cre and wild type mice demonstrating that loss of a single Nkx6.1 allele does not significantly affect alpha cell numbers (n = 3). Ins, insulin; Glc, glucagon; Som, somatostatin. Scale bar = 50 µm. Error bars represent S.E.M; **p<0.01, N.S. = not significant.

Figure 6. Nkx6.1 and Isl1 function as antagonistic transcriptional regulators of the Arx Re1 enhancer.

Immunofluorescence staining of pancreata from Ngn3-Cre;Z/EG mice at e14.5 (A) and e16.5 (B) for Nkx6.1, Arx, and GFP shows that the majority of progeny of Ngn3-expressing cells (GFP⁺) co-express Arx and Nkx6.1 at e14.5 (arrowheads in A), while the Arx⁺ and Nkx6.1⁺ domains are distinct at e16.5 (arrowheads in B point to GFP⁺Arx⁺Nkx6.1⁻ cells). (C) Schematic of the Arx locus; asterisks indicate phylogenetically-conserved Nkx6.1 binding motifs and numbers indicate the distance from the transcriptional start site. Nkx6.1 binds to site C (Re1 element) in the Arx locus in chromatin from Min6 cells (D) and FACS-sorted GFP⁺ cells (E) from e15.5 pancreata of Neurog3 ^eGFP embryos analyzed by ChIP with antibodies against Nkx6.1 or control immunoglobulin G (IgG). Mouse glucagon promoter and intergenic primers were used as positive (+) and negative (−) controls, respectively. (F) Co-transfection of αTC1–6 cells with the Arx Re1 enhancer-luciferase construct, the CMV-Renilla expression construct, and with or without the CMV-Nkx6.1 expression construct. Lane one (M) represents basal luciferase expression of the minimal promoter. Luciferase activity was quantified relative to the expression of the minimal promoter. Activity of the Re1 enhancer is repressed by Nkx6.1. (G) Co-transfection of αTC1–6 cells with the Arx Re1 enhancer-luciferase construct, CMV-Renilla, and with different concentration of CMV-Nkx6.1 and CMV-Isl1, as indicated. Nkx6.1 prevents activation of the Arx Re1 enhancer by Isl1 in a dose-dependent manner (lanes 2–7). Luciferase activity was quantified relative to the expression of the Re1 enhancer. Increasing concentrations of Isl1 are not sufficient to restore baseline activity of the Re1 enhancer in the presence of 200 ng of CMV-Nkx6.1 (lanes 8–12). Scale bar = 50 µm. Error bars represent S.E.M; *p<0.05, **p<0.01, ***p<0.001.

Figure 7. Loss of Nkx6.1 in beta cells causes beta-to-delta cell conversion.

Immunofluorescence staining of pancreata from Nkx6.1^f/+;RIP-Cre;R26-YFP and Nkx6.1^f/−;RIP-Cre;R26-YFP mice at 6 weeks of age shows Nkx6.1 (A) and insulin (B) expression in YFP⁺ cells of Nkx6.1^f/+;RIP-Cre;R26-YFP control mice, but loss of Nkx6.1 (F) and insulin (G) in YFP⁺ cells of Nkx6.1^f/−;RIP-Cre;R26-YFP mice. The insets display higher magnification images. YFP⁺ cells do not express glucagon (C, H) and rarely express pancreatic polypeptide (E, J; insets, arrowheads) in either genotype. While YFP⁺ cells are somatostatin⁻ in Nkx6.1^f/+;RIP-Cre;R26-YFP mice (D; insets, arrowheads), YFP-labeled cells are mostly somatostatin⁺ in Nkx6.1^f/−;RIP-Cre;R26-YFP mice (I; insets, arrowheads), suggesting beta-to-delta cell conversion. Arx expression is similar in both genotypes and absent from lineage-labeled YFP⁺ cells (K, M; inset, arrowhead), showing that loss of Nkx6.1 in beta cells does not activate Arx. Pdx1⁺somatostatin⁺ cells are found in both genotypes (L, N; insets, arrowheads), but express YFP only in Nkx6.1^f/−;RIP-Cre;R26-YFP mice (L; inset, arrowhead). (O) Quantification of the percentage of hormone⁺YFP⁺ cells relative to all hormone⁺ cells for each islet cell type shows reduced numbers of insulin⁺YFP⁺ cells and increased numbers of in somatostatin⁺YFP⁺ cells in Nkx6.1^f/−;RIP-Cre;R26-YFP mice compared to Nkx6.1^f/+;RIP-Cre;R26-YFP mice at 6 weeks (n = 3). Wks, weeks; Ins, insulin; Glc, glucagon; PP, pancreatic polypeptide; Som, somatostatin; Horm, hormones. Scale bar = 50 µm. Error bars represent S.E.M; ***p<0.0001.

Figure 8. Model of Nkx6.1 function in endocrine precursor cells.

(A) Expression of Nkx6.1 results in allocation of precursors from all non-beta endocrine lineages to the beta cell lineage. Deletion of Nkx6.1 in endocrine precursors has the opposite effect. When Nkx6.1 is deleted in beta cells, beta cells convert into delta cells, but not into alpha or pancreatic polypeptide (PP)-producing cells. (B) Our study suggests that in endocrine precursors, Nkx6.1 and Isl1 compete for repression and activation, respectively, of the alpha cell fate determinant Arx. We also demonstrate that the expression of Pdx1 in endocrine precursors depends on Nkx6.1. In conjunction with previous studies, showing repression of Pdx1 and Nkx6.1 by Arx and activation of Nkx6.1 by Pdx1 , our data support a model whereby cross-repression between Arx and Nkx6.1 confers alpha versus beta cell precursor identity. In beta cell precursors, Nkx6.1 expression is reinforced by Pdx1, which is repressed by Arx in alpha cell precursors.