Fig 1.
Different experimental time lines used for the beta-cell-specific Pax6 ablation.
Age at tamoxifen induction and analysis time point post-induction is indicated. After each timeline, the respective figure number is also mentioned.
Fig 2.
Beta-cell-specific ablation of Pax6.
Double immunofluorescence staining of pancreatic cryosections from 2 month old mice at 4 days (a-d) or 4 weeks (g-j) after tamoxifen induction. Pax6 expression is lost from a majority of insulin+ cells (c, d). Ablation of Pax6 is specific to YFP labeled cells (i, j) as YFP- insulin+ cells continue to express Pax6 (arrows i, j). Quantification of Pax6- insulin+ (e) and YFP+ insulin+ (f) cells in the pancreata of 2 month old mice at 4 days after tamoxifen induction (n = 3). Error bars represent SEM; *p<0.05.
Fig 3.
Expression of beta-cell-related factors in the islets of beta-cell-specific Pax6 KO mice.
Double immunofluorescence staining of pancreatic cryosections from 2.5 month old mice at 2 weeks after tamoxifen induction (a-h) or 3.5 month old mice at 6 weeks after tamoxifen induction (i, j). Expression of MafA and Glut2 is lost (e, f), expression of Pdx1 is maintained (g), and that of Nkx6.1 is maintained at 2 weeks (h) but sharply reduced at 6 weeks (j) post-tamoxifen induction in the islets of beta-cell-specific Pax6 KO pancreata.
Fig 4.
Inverse relationship of ghrelin and insulin expression in the pancreata of beta-cell-specific Pax6 KO mice.
Quantification of ghrelin+ and insulin+ cells in the islets of beta-cell-specific Pax6 KO mice (Control = RIP-CreER;Pax6+/+, KO = RIP-CreER;Pax6fl/fl) injected at 1.5 month of age and analysed at different indicated time points post tamoxifen induction (n = 3). Error bars represent SEM; *p<0.05.
Fig 5.
Gradual decrease in the population of insulin expressing cells and a proportional augmentation in the population of ghrelin expressing cells in the islets of beta-cell-specific Pax6 KO mice.
Immunofluorescence staining of pancreatic cryosections from 4 week old mice at 7 days after tamoxifen induction (a-d and g-j) and 9 week old mice at 6 weeks after tamoxifen induction (e, f and k, l). (a, b) In the control islets, all of the YFP+ cells express insulin. (c, d) At 7 days after tamoxifen induction few YFP+ Pax6-deficient cells are negative for insulin expression. (e, f) At 6 weeks after tamoxifen induction a majority of the YFP+ Pax6-deficient cells are negative for insulin expression. (g, h) Ghrelin+ cells are not detected in the control islets. (i, j) At 7 days after tamoxifen induction few YFP+ Pax6-deficient cells express ghrelin. (k, l) At 6 weeks after tamoxifen induction a majority of the YFP+ Pax6-deficient cells express ghrelin.
Fig 6.
Co-expression of ghrelin with insulin, iAPP, and PC1/3 in the islets of beta-cell-specific Pax6 KO mice.
Double immunofluorescence staining of pancreatic cryosections from 2.5 month old mice at 2 weeks after tamoxifen induction. Ghrelin+ cells are not detected in the control islets (a-c). In the beta-cell-specific Pax6 KO islets, ghrelin expression is up regulated (d-f) and some of the ghrelin+ cells co-express insulin (d). Additionally, these ghrelin+ cells co-express iAPP (e) and PC1/3 (f).
Fig 7.
Expression of beta-cell-related transcription factors in the ghrelin+ cell population of beta-cell-specific Pax6 KO islets.
Double immunofluorescence staining of pancreatic cryosections from 2.5 month old mice at 2 weeks after tamoxifen induction. Ghrelin expression is not detected in the control islets (a-c). In beta-cell-specific Pax6 KO islets, ghrelin expression is up regulated and ghrelin+ cells are negative for Pax6 (d) but positive for Pdx1 (e), and Nkx6.1 (f).
Fig 8.
Ghrelin cells originate from beta-cells in the beta-cell-specific Pax6 KO islets.
Double immunofluorescence staining of pancreatic cryosections from 4 month old mice at 6 weeks after tamoxifen induction (a-f) and 6 month old mice at 4.5 months after tamoxifen induction (g-i). Ghrelin expression is not detected in the control islets (a-c). In beta-cell-specific Pax6 KO islets, ghrelin expression is up regulated and that of insulin down regulated in the YFP labeled Pax6-deficient cells (d-f). Most of the ghrelin+ cells have lost the expression of insulin but some are still insulin+ (arrows d-f). Some YFP+ cells in the KO islets are negative for both insulin and ghrelin (arrowhead d-f). At 4.5 months after tamoxifen induction many of the YFP+ cells are negative for both insulin and ghrelin (arrowheads g-i).
Fig 9.
Expression of Rfx6 and Pdx1 in the islets of beta-cell-specific Pax6 KO mice.
Immunofluorescence staining of pancreatic cryosections from 6 month old mice at 4.5 month after tamoxifen induction. Expression of Rfx6 (e, f) and Pdx1 (g, h) is maintained in the YFP+ Pax6-deficient cells of the beta-cell-specific Pax6 KO islets.
Fig 10.
Pax6-deficient beta-cells do not proliferate.
Immunofluorescence staining of pancreatic cryosections from 7 week old mice at 4 weeks after tamoxifen induction. Ki67+ YFP+ cells are detected in the control islets (arrows a, b) but not in the beta-cell-specific Pax6 KO islets (c, d). On the other hand, Ki67+ YFP- cells are detected in both the control and KO islets (arrowhead a-d). Quantification of Ki67+ YFP+ cells (e) in the islets of 7 week old mice at 4 weeks after tamoxifen induction (n = 3). Error bars represent SEM; *p<0.05.
Fig 11.
Expression of C-peptide, PC1/3, and PC2 in the islets of beta-cell-specific Pax6 KO mice.
Double immunofluorescence staining of pancreatic cryosections from 2 month old mice at 4 weeks after tamoxifen induction. In beta-cell-specific Pax6 KO islets, C-peptide expression is lost from majority of the YFP+ Pax6-deficient cells (d). However, PC1/3 (e) and PC2 (f) expression is maintained in the YFP+ Pax6-deficient cells.
Fig 12.
Expression of proSAAS and 7B2 in the islets of beta-cell-specific Pax6 KO mice.
Double immunofluorescence staining of pancreatic cryosections from 4 month old mice at 6 weeks after tamoxifen induction. In the control islets, expression of proSAAS and 7B2 is low in the YFP labeled beta-cells (a-d). In beta-cell-specific Pax6 KO islets, expression of both proSAAS and 7B2 is highly up regulated in the YFP+ Pax6-deficient cells (e-h).
Fig 13.
Loss of Glut2 expression due to direct and/or indirect effect of Pax6 ablation in the islets of beta-cell-specific Pax6 KO mice.
Double immunofluorescence staining of pancreatic cryosections from 2 month old mice at 4 weeks after tamoxifen induction. In the control islets, expression of Glut2 is detected in both YFP+ and YFP- insulin+ cells (a-c). In the beta-cell-specific Pax6 KO islets, Glut2 expression is lost in the YFP labeled Pax6-deficient cells as well as in a majority of the YFP- insulin+ cells (arrows d-f). Rarely, some YFP- insulin+ cells do express Glut2 in the KO islets (arrowhead d-f).
Fig 14.
Expression of GLP-1 receptor in the islets of beta-cell-specific Pax6 KO mice.
Double immunofluorescence staining of pancreatic cryosections from 4 month old mice at 6 weeks after tamoxifen induction. GLP-1 receptor is expressed in both YFP+ and YFP- insulin+ cells in the control islets (a-d). In beta-cell-specific Pax6 KO islets, GLP-1 receptor expression is lost from the YFP labeled Pax6-deficient cells (arrowheads e-h) but maintained in the YFP- insulin+ cells (arrows e-h).
Fig 15.
Development of hyperglycemia and resulting beta-cell regeneration in the beta-cell-specific Pax6 KO mice.
Measurement of blood glucose level in mice after tamoxifen induction at 3 weeks (a) or 1.5 month (b) (n = 5). Increase in blood glucose level is observed at either age but a slight decrease in the long term is only seen in young mice (a). Quantification of insulin+ YFP-, YFP+ insulin-, and insulin+ YFP+ cells in the islets of 13 week old mice at 10 weeks after tamoxifen induction (c) and 6 month old mice at 4.5 month after tamoxifen induction (d) (n = 3). Number of insulin+ YFP- cells is increased at either age indicating some beta-cell regeneration. Due to the loss of insulin expression in YFP labeled Pax6-deficient cells, a significant increase in the number of YFP+ insulin- and a decrease in the number of YFP+ insulin+ cells is also observed in the KO islets. (Control = RIP-CreER;R26-YFP;Pax6+/+, Pax6 KO = RIP-CreER;R26-YFP;Pax6fl/fl). Error bars represent SEM; *p<0.05.
Fig 16.
Alpha-cell-specific ablation of Pax6.
Double immunofluorescence staining of pancreatic cryosections from 1 month old mice. In control islets, all the YFP+ cells express Pax6 and glucagon (a-c). In alpha-cell-specific Pax6 KO islets, most of the YFP+ cells are negative for Pax6 and have lost the expression of glucagon as well (d-f). YFP- glucagon+ cells in the KO islets express Pax6 (arrowheads d-f). Rarely YFP+ glucagon+ Pax6- cells are also found in the KO islets (arrows d-f). Quantification of YFP+ glucagon+ cells in relation to total glucagon+ cells in 1 month old non-inducible alpha-cell-specific control (Glu-Cre;R26-YFP;Pax6+/+) mice, and in Dox-inducible alpha-cell-specific control (Glu-rtTA;TetO-Cre;R26-YFP;Pax6+/+) mice at 4 months of age following 6 weeks of Doxycycline treatment (g) (n = 3). Nearly 68% and 75% of the glucagon+ cells are labeled with YFP in non-inducible and Dox-inducible alpha-cell-specific control mice, respectively. Error bars represent SEM.
Fig 17.
Ghrelin+ cells originate from alpha-cells in the alpha-cell-specific Pax6 KO islets.
Double immunofluorescence staining of pancreatic cryosections from 1 month old mice. Ghrelin expression is not detected in the control islets (a-c). In alpha-cell-specific Pax6 KO islets ghrelin expression is up regulated in YFP labeled cells (d-f) and rarely ghrelin expression co-localizes with glucagon expression (arrows d-f).
Fig 18.
Expression of alpha-cell-related transcription factors in ghrelin+ cells of alpha-cell-specific Pax6 KO islets.
Double immunofluorescence staining of pancreatic cryosections from 1 month old mice. Ghrelin expression is not detected in the control islets (a-c). In alpha-cell-specific Pax6 KO islets ghrelin expression is up regulated in YFP labeled cells and ghrelin+ cells are negative for Pax6 (d) and MafB (e) expression but positive for Arx (f) expression.
Fig 19.
Expression of 7B2 and PC2 in alpha-cell-specific Pax6 KO islets.
Immunofluorescence staining of pancreatic cryosections from 1 month old mice. 7B2 expression is very low in the control islets (a, b). In alpha-cell-specific Pax6 KO islets, 7B2 expression is highly up regulated in YFP labeled Pax6-deficient cells (e, f). Compared to control islets PC2 expression is unchanged in the alpha-cell-specific Pax6 KO islets (c, d and g, h).
Fig 20.
Alpha-cell neogenesis in the alpha-cell-specific Pax6 KO islets.
Quantification of total glucagon+ cell population in non-inducible alpha-cell-specific Pax6 KO islets at 1 month, 3 months, and 8 months of age, and in Dox-inducible alpha-cell-specific Pax6 KO islets at 4 months of age following 6 weeks of Doxycycline treatment (a) (n = 3). Number of total glucagon+ cells is unchanged at the younger age but significantly reduced at 8 months of age in the KO islets. Quantification of glucagon+ YFP-, YFP+ glucagon-, and YFP+ glucagon+ cells in Dox-inducible alpha-cell-specific Pax6 KO islets at 4 months of age following 6 weeks of Doxycycline treatment (b) and in the non-inducible alpha-cell-specific Pax6 KO islets at 1 month, 3 months, and 8 months of age (c) (n = 3). Number of glucagon+ YFP- cells is significantly increased in the Dox-inducible KO islets as well as in the non-inducible KO islets at 1 month and 3 months of age but not at 8 months of age. Due to the loss of glucagon expression in YFP labeled Pax6-deficient cells, a significant increase in the number of YFP+ glucagon- and a decrease in the number of YFP+ glucagon+ cells is also observed at all stages and in both types of islets. (Non-inducible control = Glu-Cre;R26-YFP;Pax6+/+, Non-inducible Pax6 KO = Glu-Cre;R26-YFP;Pax6fl/fl, Dox-inducible control = Glu-rtTA;TetO-Cre;R26-YFP; Pax6+/+, Dox-inducible Pax6 KO = Glu-rtTA;TetO-Cre;R26-YFP;Pax6fl/fl). Error bars represent SEM; *p<0.05.
Fig 21.
Double immunofluorescence staining of pancreatic cryosections from mice that were injected with tamoxifen at 2 months of age and analysed at 1 week post tamoxifen induction. A nearly 100% ablation of Pax6 was confirmed in the islets of ubiquitous Pax6 KO mice (c, d).
Fig 22.
Variable effect of Pax6 ablation in the islets of adult-ubiquitous Pax6 KO mice.
Double immunofluorescence staining of pancreatic cryosections from mice that were injected with tamoxifen at 2 months of age and analysed at 1 week, 2 weeks, and 3 weeks post tamoxifen induction. Ghrelin+ cells are not detected in the control islets (a-d). In the ubiquitous Pax6 KO islets, ghrelin expression is up regulated (e-p). Ghrelin expression shows co-localization with that of glucagon (e, i, m) and insulin (f, j, n) but never with that of somatostatin (g, k, o) and PP (h, l, p). In ubiquitous Pax6 KO islets, a loss of glucagon (e, i, m) and insulin expression (f, j, n) was observed but not that of somatostatin (g, k, o) and PP (h, l, p). Furthermore, glucagon+ cells were the first ones to be affected in relation to the loss of glucagon expression and upregulation of ghrelin expression (e, i, m). (TI = Tamoxifen induction).
Fig 23.
Ghrelin expressing cells, in the islets of adult-ubiquitous Pax6 KO mice, can be differentially identified.
Double immunofluorescence staining of pancreatic cryosections from mice that were injected with tamoxifen at 2 months of age and analysed at 3 weeks post tamoxifen induction. Ghrelin+ cells are not detected in the control islets (a). In the ubiquitous Pax6 KO islets, ghrelin expression is up regulated and ghrelin+ cells may or may not express Pdx1 depending on the fact that they originally come from insulin+ or glucagon+ cells, respectively (b).