<i>Nkx6.1</i> is required for beta cell specification downstream of Ngn3.

<p>(A, B) Schematic of the alleles and transgenes for <i>Nkx6.1</i> inactivation and lineage tracing; Triangles, <i>loxP</i> 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 <i>Nkx6.1^f/−;Ngn3-Cre;Z/EG</i> 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 <i>Nkx6.1^f/−;Ngn3-Cre;Z/EG</i> and <i>Ngn3-Cre;Z/EG</i> mice at P2 (n = 4). Loss of <i>Nkx6.1</i> 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.</p

Nkx6.1 and Isl1 function as antagonistic transcriptional regulators of the <i>Arx Re1</i> enhancer.

<p>Immunofluorescence staining of pancreata from <i>Ngn3-Cre;Z/EG</i> 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 <i>Arx</i> 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 (<i>Re1</i> element) in the <i>Arx</i> locus in chromatin from Min6 cells (D) and FACS-sorted GFP⁺ cells (E) from e15.5 pancreata of <i>Neurog3</i>^eGFP embryos analyzed by ChIP with antibodies against Nkx6.1 or control immunoglobulin G (IgG). Mouse <i>glucagon</i> promoter and intergenic primers were used as positive (+) and negative (−) controls, respectively. (F) Co-transfection of αTC1–6 cells with the <i>Arx Re1</i> enhancer-luciferase construct, the <i>CMV-Renilla</i> expression construct, and with or without the <i>CMV-Nkx6.1</i> 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 <i>Re1</i> enhancer is repressed by Nkx6.1. (G) Co-transfection of αTC1–6 cells with the <i>Arx Re1</i> enhancer-luciferase construct, <i>CMV-Renilla</i>, and with different concentration of <i>CMV-Nkx6.1</i> and <i>CMV-Isl1</i>, as indicated. Nkx6.1 prevents activation of the <i>Arx Re1</i> enhancer by Isl1 in a dose-dependent manner (lanes 2–7). Luciferase activity was quantified relative to the expression of the <i>Re1</i> enhancer. Increasing concentrations of Isl1 are not sufficient to restore baseline activity of the <i>Re1</i> enhancer in the presence of 200 ng of <i>CMV-Nkx6.1</i> (lanes 8–12). Scale bar = 50 µm. Error bars represent S.E.M; *p<0.05, **p<0.01, ***p<0.001.</p

Loss of <i>Nkx6.1</i> in beta cells causes beta-to-delta cell conversion.

<p>Immunofluorescence staining of pancreata from <i>Nkx6.1^f/+;RIP-Cre;R26-YFP</i> and <i>Nkx6.1^f/−;RIP-Cre;R26-YFP</i> mice at 6 weeks of age shows Nkx6.1 (A) and insulin (B) expression in YFP⁺ cells of <i>Nkx6.1^f/+;RIP-Cre;R26-YFP</i> control mice, but loss of Nkx6.1 (F) and insulin (G) in YFP⁺ cells of <i>Nkx6.1^f/−;RIP-Cre;R26-YFP</i> 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 <i>Nkx6.1^f/+;RIP-Cre;R26-YFP</i> mice (D; insets, arrowheads), YFP-labeled cells are mostly somatostatin⁺ in <i>Nkx6.1^f/−;RIP-Cre;R26-YFP</i> 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 <i>Nkx6.1</i> in beta cells does not activate <i>Arx</i>. Pdx1⁺somatostatin⁺ cells are found in both genotypes (L, N; insets, arrowheads), but express YFP only in <i>Nkx6.1^f/−;RIP-Cre;R26-YFP</i> 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 <i>Nkx6.1^f/−;RIP-Cre;R26-YFP</i> mice compared to <i>Nkx6.1^f/+;RIP-Cre;R26-YFP</i> 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.</p

Forced <i>Nkx6.1</i> expression favors the beta cell fate choice.

<p>(A, B) Schematic of the transgenes for conditional <i>Nkx6.1</i> misexpression and cell lineage tracing; Triangles, <i>loxP</i> sites; Ovals, <i>internal ribosomal entry site</i> (IRES). (C–L) Immunofluorescence staining of pancreata from <i>Ngn3-Cre;Z/EG</i> and <i>Ngn3-Cre;Nkx6.1^OE</i> 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 <i>Ngn3-Cre;Z/EG</i> mice and insulin, but not glucagon, somatostatin, pancreatic polypeptide, or ghrelin in <i>Ngn3-Cre;Nkx6.1^OE</i> mice. Quantification of hormone⁺GFP⁺ (M) or Ki67⁺GFP⁺ (N) co-positive cells as a percentage of all GFP-expressing cells in pancreata of <i>Nkx6.1^f/−;Ngn3-Cre;Z/EG</i> and <i>Ngn3-Cre;Z/EG</i> mice at P2 (n = 4). Forced expression of <i>Nkx6.1</i> 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.</p

Loss of <i>Nkx6.1</i> in endocrine precursors results in activation of non-beta endocrine genes.

<p>(A–L) Immunofluorescence staining of pancreata from <i>Ngn3-Cre;Z/EG</i> and <i>Nkx6.1^f/−;Ngn3-Cre;Z/EG</i> 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 <i>Nkx6.1</i>-deficient, recombined, insulin⁺GFP⁺ cells. Arrowheads point to insulin⁺ cells ectopically expressing non-beta endocrine markers. Ins, insulin. Scale bar = 50 µm.</p