ArxE mice have almost complete loss of α-cell fate by P14 with a concomitant decrease in total endocrine mass, but no change in β- and δ-cell mass.

<p>(<b>A–H</b>): P14 pancreatic sections were stained for glucagon (green) and insulin (red; A–B), somatostatin (Sst; red; C–D), PP (red; E–F), and ghrelin (Ghr; red; G–H). Scale bar denotes 50 µm. (<b>I</b>): Quantification of endocrine hormone mass including total endocrine mass (ChrgA), insulin, somatostatin, PP, and ghrelin displayed as fold change in ArxE mice (white bar) relative to control (black bar). (<b>J</b>): Analysis of glucagon mass over time starting at E15.5 and ending at P14 in control (black bar) and ArxE (white bar) pancreata. Resulting p value is listed. (*) denotes significance where p<0.05. Error bars represent standard error of the mean (I, J). For all analysis 4–5 animals per group were analyzed with all ArxE mice being males and control mice consisting of male and female mice.</p

ArxE mice are able to specify a subset of α-cells at E15.5.

<p>(<b>A–F</b>): Control and ArxE E15.5 pancreatic sections were stained for glucagon (green) and insulin (red; A–B), somatostatin (Sst; red; C–D), and ghrelin (Ghr; red; E–F). Scale bar denotes 50 µm. (<b>G</b>): Quantification of total endocrine (ChrgA) and β-, δ-, and α-cell mass in control (black bar) and ArxE (white bar) pancreata. (<b>H</b>): Quantification of transcript levels for each endocrine hormone in control (black bar) and ArxE pancreata (white bar) at E15.5 using qRT-PCR. All results are graphed as fold change relative to littermate controls ± standard error of the mean. Significance is denoted with (*) when p≤0.05. All analysis consists of 4–5 animals per group.</p

ArxE mice are able to correctly specify a subset of α-cells, but α-cells are gradually lost through apoptosis.

<p>Model demonstrating normal proliferation, but increased apoptosis in ArxE mice. Normal proliferation during embryonic time points maintains the α-cell lineage by replacing cells lost to apoptosis. However, proliferation slows during the neonatal stage leading to loss of the α-cell lineage.</p

Pancreatic α-Cell Specific Deletion of Mouse Arx Leads to α-Cell Identity Loss

<div><p>The specification and differentiation of pancreatic endocrine cell populations (α-, β-, δ, PP- and ε-cells) is orchestrated by a combination of transcriptional regulators. In the pancreas, <i>Aristaless-related homeobox</i> gene (<i>Arx</i>) is expressed first in the endocrine progenitors and then restricted to glucagon-producing α-cells. While the functional requirement of <i>Arx</i> in early α-cell specification has been investigated, its role in maintaining α-cell identity has yet to be explored. To study this later role of <i>Arx</i>, we have generated mice in which the <i>Arx</i> gene has been ablated specifically in glucagon-producing α-cells. Lineage-tracing studies and immunostaining analysis for endocrine hormones demonstrate that ablation of <i>Arx</i> in neonatal α-cells results in an α-to-β-like conversion through an intermediate bihormonal state. Furthermore, these <i>Arx</i>-deficient converted cells express β-cell markers including <i>Pdx1, MafA,</i> and <i>Glut2</i>. Surprisingly, short-term ablation of <i>Arx</i> in adult mice does not result in a similar α-to-β-like conversion. Taken together, these findings reveal a potential temporal requirement for <i>Arx</i> in maintaining α-cell identity.</p></div

Loss of Arx in glucagon⁺ cells results in the appearance of a glucagon⁺insulin⁺ population.

<p><b>(A–H):</b> P5 control and GKO pancreata were stained for glucagon (green), insulin (red; A, B), somatostatin (Sst; red; C, D), PP (red; E, F), and Ghrelin (red; G, H). Glucagon⁺insulin⁺ cells are the only bihormonal population unique to GKO animals (B, D, F). Glucagon/ghrelin coexpressing cells are both found in control and GKO animals (G, H). Male and female control and GKO animals (n≥3) were sex-matched for all analyses. Scale bar denotes 25 µm. (<b>I–L</b>): Quantitative PCR analysis examining glucagon (I), insulin (J), somatostatin (K), and PP (L) gene expression in P5 control and GKO animals. Control mRNA level was set at one fold ± standard error of the mean. For all GKO and control groups, at least 3 biologic replicates were performed.</p

Arx is specifically ablated in YFP⁺ α-cells of GKO;Rosa-YFP mice.

<p>P5 pancreatic sections were stained for glucagon (blue), Arx (red), and YFP (green). (<b>A</b>): Arx is expressed in all glucagon⁺ cells in control;Rosa-YFP pancreata. A subset of glucagon⁺Arx⁺ cells is YFP⁺. (<b>B</b>): In GKO;Rosa-YFP animals, there is a subset of glucagon⁺ cells that express YFP. These YFP⁺ cells have lost Arx expression. Scale bar represents 25 µm. (<b>C</b>): Quantitative analysis of Arx and YFP expressing cells within glucagon⁺ population in P5 animals. Over 500 total glucagon⁺ cells were counted with three mice per group used. Error bars represent standard error of the mean with <i>p-value</i> indicated. N.S: not significant. (<b>D</b>): Quantitative PCR analysis for <i>Arx</i> mRNA in total pancreata at P5 and islets from P21 control and GKO animals. Control mRNA level was set at one fold ± standard error of the mean. Male and female control and GKO animals (n≥3) were sex-matched for all analyses.</p

Lineage tracing studies demonstrate that Arx ablated α-cells become glucagon⁺insulin⁺ at P5 then insulin expressing at P21.

<p>(<b>A–D</b>): Triple immunostaining for glucagon (red), insulin (blue), and YFP (green) in control;Rosa-YFP and GKO;Rosa-YFP pancreata at P5 and P21. YFP⁺ cells in P5 or P21 control;Rosa-YFP animals are positive for glucagon (A and C;^▪). “*” denote insulin cells that are negative for glucagon or YFP expression (A and C; *) Glucagon⁺insulin⁺YFP⁺ cells are found in P5 GKO animals (B; ^▪), but rarely in controls (A). YFP⁺ cells are positive for glucagon in control P21 pancreata (C; ^▪) but insulin⁺ in P21 GKO pancreata (D; ^▪). (<b>E</b>): Schematic outlining cell populations resulting from lineage-tracing and immunostaining analysis. (<b>F, G</b>): Quantification of hormone expression in YFP⁺ cells at P5 (F) and P21 (G). At P5 and P21, over a total of 10,000 cells were counted from 3–5 animals per group. Out of the 10,000 cells counted, approximately 1,000 cells were YFP⁺. Each category was calculated and presented as a percentage of total YFP⁺ cells per animal and then averaged. Error bars are denoted as standard error of the mean with significance (p≤0.05) between each color denoted with “*”, “^▪”, and “•”. Male and female GKO mice (n≥3) were used for all analysis and compared to their sex-matched controls. Scale bar represents 25 µm.</p

YFP⁺ cells in GKO animals express markers of mature β-cells at P5 and P21.

<p>(<b>A–F</b>): Control;Rosa-YFP and GKO;Rosa-YFP P5 pancreata were stained for insulin (blue), YFP (green), Glut2 (A,B,red), MafA (C,D,red), Pdx1 (E,F,brown). YFP⁺ cells in GKO animals are insulin⁺Glut2⁺ (B;^▪ ), insulin⁺MafA⁺ (D; ←) and insulin⁺Pdx1⁺ (<b>F; ←</b>). In control animals, the majority of YFP⁺ do not express β-cell markers (A,C,E). The YFP⁺ cells seen in exocrine tissue (panels E and F) is background due to the combined IHC, IF staining method used and not true signal. (<b>G–L</b>): Quantification of percentage of YFP⁺ cells that express or do not express Glut2 at P5 (G) and P21 (H), MafA at P5 (I) and P21 (J), and Pdx1 at P5 (K) and P21 (L). Over 200 YFP⁺ cells were counted for each stage with 3–5 animals per group. Error bars represent standard error of the mean with significance between each cell population (p≤0.05) denoted as “*” and “^▪”. Male and female GKO mice (n≥3) were used for all analysis and compared to their sex-matched controls. Scale bar represents 25 µm.</p

Short-term complete ablation of Arx in adult mice does not result in a loss of α-cells or changes in endocrine cell populations.

<p>(A): Diagram outlining experimental design. (<b>B–C</b>): Arx (red) is expressed in glucagon (green) cells in control (B,B’; arrows) but lost in IKO (C,C’;arrows) animals. Asterisks (*) mark autoflourescent blood cells and are non-specific staining. (<b>D–I</b>): Control and IKO pancreata were stained for glucagon (green), insulin (red; D,E), somatostatin (Sst; red; F,G), and PP (red; H,I). No significant colocalization of glucagon with other hormones was seen in control or IKO mice. Male control and IKO mice were used for analysis though female control and IKO mice produced similar results. Scale bar denotes 75 µm. (<b>J</b>): Endocrine cell number quantification for insulin, glucagon, somatostatin, and PP in control and IKO animals (over 10,000 cells were counted from 3 animals per group). (<b>K–L</b>): Quantitative PCR analysis of gene expression in control and IKO islets. Results are displayed as fold change relative to control with error bars representing the standard error of the mean. For all analysis n = 3.</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