Figure 1.
ArxE mice are able to specify a subset of α-cells at E15.5.
(A–F): 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. (G): Quantification of total endocrine (ChrgA) and β-, δ-, and α-cell mass in control (black bar) and ArxE (white bar) pancreata. (H): 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.
Figure 2.
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.
(A–H): 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. (I): 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). (J): 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.
Figure 3.
Specified α-cells in ArxE mice do not misexpress β-cell specific transcription factors at E15.5.
(A): Quantification of transcript levels for α- and β-cell specific transcription factors graphed as fold change in ArxE pancreata (white bar) relative to littermate control (black bar). Error bars represent standard error of the mean. Significance when p≤0.05, is denoted with (*). (B–G): Control and ArxE E15.5 pancreatic sections were stained for glucagon (green) and Pdx1 (red; B–C), Glut2 (red; D–E), and MafA (red; F–G). Five specimens were analyzed for each group and representative images taken. (*) denotes autofluorescence of red blood cells. Scale bar denotes 50 µm.
Figure 4.
There is a drastic increase in the percentage of apoptotic glucagon+ cells in ArxE mice, but no apparent change in proliferation.
(A–B): Control and ArxE E15.5 pancreatic sections were stained for glucagon (green) and Ki67 (red). Scale bar denotes 50 µm. (C): qRT-PCR analysis for two markers of proliferation, Ki67 and Birc5, in E15.5 control (black bar) and ArxE pancreata (white bar). Results are graphed as fold change relative to littermate control ± the standard error of the mean. (D–E): Control and ArxE E15.5 pancreatic sections were stained for glucagon (green) and TUNEL (red). Arrow points to TUNEL+ cell. Scale bar denotes 10 µm. (F): Quantification of the percentage TUNEL+glucagon+ cells over total counted glucagon+ cells in control and ArxE E15.5 pancreatic sections. All glucagon+ cells in five different pancreatic sections from both control (black bar) and ArxE (white bar) mice were counted and determined to be TUNEL positive or negative. The percentage of TUNEL+glucagon+ cells was calculated and graphed as ± standard error of the mean. (*) denotes significance where p≤0.05. Between 4 and 5 animals were examined per group for each analysis.
Figure 5.
ArxE mice are able to correctly specify a subset of α-cells, but α-cells are gradually lost through apoptosis.
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.