Arx and Nkx2.2 compound deficiency redirects pancreatic alpha- and beta-cell differentiation to a somatostatin/ghrelin co-expressing cell lineage

Abstract

Background: Nkx2.2 and Arx represent key transcription factors implicated in the specification of islet cell subtypes during pancreas development. Mice deficient for Arx do not develop any alpha-cells whereas beta- and delta-cells are found in considerably higher numbers. In Nkx2.2 mutant animals, alpha- and beta-cell development is severely impaired whereas a ghrelin-expressing cell population is found augmented.Notably, Arx transcription is clearly enhanced in Nkx2.2-deficient pancreata. Hence in order to precise the functional link between both factors we performed a comparative analysis of Nkx2.2/Arx single- and double-mutants but also of Pax6-deficient animals.

Results: We show that most of the ghrelin+ cells emerging in pancreata of Nkx2.2- and Pax6-deficient mice, express the alpha-cell specifier Arx, but also additional beta-cell related genes. In Nkx2.2-deficient mice, Arx directly co-localizes with iAPP, PC1/3 and Pdx1 suggesting an Nkx2.2-dependent control of Arx in committed beta-cells. The combined loss of Nkx2.2 and Arx likewise results in the formation of a hyperplastic ghrelin+ cell population at the expense of mature alpha- and beta-cells. Surprisingly, such Nkx2.2-/-Arx- ghrelin+ cells also express the somatostatin hormone.

Conclusions: Our data indicate that Nkx2.2 acts by reinforcing the transcriptional networks initiated by Pax4 and Arx in early committed beta- and alpha-cell, respectively. Our analysis also suggests that one of the coupled functions of Nkx2.2 and Pax4 is to counteract Arx gene activity in early committed beta-cells.

Figure 1

Expression of Arx and Brn4 in E16.5 pancreata of wild type, Nkx2.2^-/- and Pax6^-/- mice. (A, B) Arx is expressed in wild type glucagon⁺ cells. (A, inset) Moreover, Arx⁺/glucagon^- cells are detectable at this stage. (B, C, inset) Arx or Brn4 can be detected in a subset of ghrelin⁺ cells. (D, inset) In Nkx2.2-mutant pancreata some of the remaining glucagon⁺ cells do not express Arx. (E, inset, F) Most Nkx2.2-deficient ghrelin⁺ cells express Arx but only rarely Brn4. (G, H) Arx is expressed in pancreata of Pax6^-/- mice and co-localizes with most ghrelin⁺ cells. Scattered Arx⁺/ghrelin^- and ghrelin⁺/Arx^- cells can be detected. (I, inset) Brn4 is expressed in a subset of ghrelin⁺ cells and labels a ghrelin^- cell population in Pax6-deficient mice. Counterstaining with DAPI. (J-L) The percentages of Arx⁺/glucagon⁺, Arx⁺/ghrelin⁺ and Brn4⁺/ghrelin⁺ cells in pancreata of wild type (WT), Nkx2.2^-/- (N2.2) and Pax6^-/- (P6) mice within the total glucagon⁺ and ghrelin⁺ cell populations, respectively, are depicted. (M) Quantitative RT-PCR of Arx, Pax6 and Nkx2.2 mRNA in wild type, Nkx2.2^-/-, Pax6^-/- and Arx^- E16.5 pancreas. (N) Quantification of Arx⁺ cells at E16.5 in pancreata of wild type, Nkx2.2^-/- and Pax6^-/- mice (n = 3 pancreas per genotype). Single-factor ANOVA coupled to Newman-Keuls test was applied and p-values < 0,05 were assessed as statistically significant compared to wild type. Statistical significance is achieved with the assumption of a normal distribution. Error bars represent SEM. (original magnification: 400×, Insets magnification: 4×).

Figure 2

Co-detection of ghrelin and insulin, iAPP, PC1/3 or Pdx1 in pancreata of wild type, Nkx2.2^-/- and Pax6^-/- mice at E17.5. (A-D) Wild type ghrelin⁺ cells do not express insulin, PC1/3 or Pdx1. However, scattered ghrelin⁺ cells express iAPP (Arrow). (E-H) No insulin reactive cells are detectable in Nkx2.2-deficient pancreata. The majority of ghrelin⁺ cells express iAPP and low levels of Pdx1. Scattered iAPP⁺/ghrelin^- (F) and Pdx1⁺/ghrelin^- cells (H, Arrows) are detectable. (G) PC1/3 is associated with a subpopulation of ghrelin-expressing cells in Nkx2.2-deficient pancreata. (I-L) In pancreata deficient for Pax6 the population of ghrelin⁺ cells does not express insulin or iAPP except in very rare instances (Arrows in I+J). Almost all Pax6-deficient ghrelin⁺ cells express PC1/3 but none produce Pdx1. Counterstaining with DAPI. (M-P) The percentages of insulin⁺/ghrelin⁺, iAPP⁺/ghrelin⁺, PC1/3⁺/ghrelin⁺ and Pdx1⁺/ghrelin⁺ cells in wild type (WT), Nkx2.2^-/- (N2.2) and Pax6^-/- (P6) mice within the total ghrelin⁺ cell population are depicted. Pictures were taken using confocal microscopy.

Figure 3

Co-detection of Arx and Pdx1, iAPP or PC1/3 in pancreata of wild type and Nkx2.2^-/- mice at E17.5. (A-C) Wild type Arx⁺ cells do not express Pdx1 (A) or PC1/3 (C, inset). Most Arx⁺ cells do not express iAPP (B), except in rare instances (Arrowhead). (D-F) The majority of Arx⁺ cells in Nkx2.2^-/- pancreata express Pdx1^low (Arrows), iAPP (E, inset) and PC1/3 (F, inset). Counterstaining with DAPI. Pictures were taken using confocal microscopy (Insets magnification: 4×).

Figure 4

Expression of endocrine hormones in pancreata of wild type, Arx^-, Nkx2.2^-/- and Nkx2.2^-/-Arx^- newborns. Insulin (A-D), glucagon (E-H), pancreatic polypeptide (I-L), somatostatin (M-P) and ghrelin (Q-T) immunofluorescence-detection at P0. (D, H) Note that both beta- and alpha-cells lack in Nkx2.2^-/-Arx^- double-mutant mice (original magnification: 200×). (U-X) Co-detection of ghrelin and somatostatin in P0 pancreata of wild type, Arx^-, Nkx2.2^-/- and Nkx2.2^-/-Arx^- mice. Somatostatin⁺ cells and ghrelin⁺ cells label distinct cell populations in wild type (U), Arx- (V) and Nkx2.2-deficient (W) pancreata. (X) However, in Nkx2.2^-/-Arx^- double mutant mice a population of ghrelin⁺/somatostatin⁺ co-positive cells is detectable besides single-hormone-expressing ghrelin⁺ or somatostatin⁺ cells. Pictures in U-X were taken using confocal microscopy. Counterstaining with DAPI. (Y) Quantification of hormone producing cells (without PP-cells) in pancreata of wild type, Arx^-, Nkx2.2^-/- and Nkx2.2^-/-Arx^- mice at P0. Bars represent percentage of fluorescent cell area per total pancreatic area. n = 3 pancreas per genotype. Single-factor ANOVA coupled to Newman-Keuls test was applied and p-values < 0,05 were assessed as statistically significant compared to wild type. Statistical significance is achieved with the assumption of a normal distribution. Error bars represent SEM.

Figure 5

Expression of Pdx1, Pax6 and iAPP in pancreata of wild type and Nkx2.2^-/-Arx^- newborns. (A) In wild type pancreata, a subpopulation of delta-cells expresses low levels of Pdx1 (Arrow) while the bulk does not. (B, C) Wild type delta-cells express Pax6 and iAPP. (D, inset) Notably, in Nkx2.2^-/-Arx^- mice almost all somatostatin⁺ cells display low levels of Pdx1. (E, F, insets) Nkx2.2^-/-Arx^- somatostatin⁺ cells are immunoreactive for iAPP and low levels of Pax6. (G) Wild type epsilon-cells do not express Pdx1. (H, I) Pax6 or iAPP can be detected in a subset of wild type ghrelin⁺ cells (Arrows); a few ghrelin⁺ cells are negative for Pax6 or iAPP (Arrowheads). (J, K) In Nkx2.2^-/-Arx^- pancreata, the majority of ghrelin⁺ cells are found positive for low levels of Pdx1 and Pax6; scattered ghrelin⁺ cells do not express Pax6 (K, Arrowhead). (L, inset) Islet amyloid polypeptide is detectable in the majority of ghrelin⁺ cells; some are negative for iAPP (Arrowhead). Pictures in D, E, J and K have been captured using a 2-fold exposure time compared to respective control images in order to allow for a clear detection of Pdx1 and Pax6 (original magnification 800×). C, F, I and L are counterstained with DAPI. (M-N) Confocal images of E17.5 wild type and Nkx2.2/Arx double-mutant pancreata. Similar to the expression patterns in newborns, Nkx2.2/Arx double-deficient ghrelin⁺ cells express somatostatin, Pdx1^low and iAPP at E17.5.

Figure 6

Pax4 expression in pancreata of wild type, Arx^-, Nkx2.2^-/- and Nkx2.2^-/-/Arx^- at E16.5. (A-D) Pax4-expressing cells are present in normal numbers at E16.5 in pancreata of wild type, Arx^-, Nkx2.2^-/- and Nkx2.2^-/-/Arx^- mice. Notably, no co-expression of Pax4 and ghrelin is detectable in wild type or mutant pancreata. Pictures were taken using confocal microscopy. (E) Quantification of Pax4⁺ cells at E16.5 in pancreata of wild type, Arx^-, Nkx2.2^-/- and Nkx2.2^-/-Arx^- mice (n = 3 pancreas per genotype). Single-factor ANOVA coupled to Newman-Keuls test was applied and p-values < 0,05 were assessed as statistically significant compared to wild type. Statistical significance is achieved with the assumption of a normal distribution. Error bars represent SEM.

Figure 7

Nkx6.1 expression is differentially dependent on Nkx2.2. (A-D) Nkx6.1 is broadly expressed in the pancreatic epithelium of wild type, Arx^-, Nkx2.2^-/- and Nkx2.2^-/-Arx^- mice at embryonic day 15.5. (E, F) In wild type and Arx-mutants, Nkx6.1 expression is maintained and detectable at P0. (G, H) However, in the absence of Nkx2.2 or Nkx2.2/Arx, a steady decrease in Nkx6.1⁺ cell numbers is detected from E15.5 on so that Nkx6.1⁺ cells are not detectable in pancreata of newborns. No co-expression of Nkx6.1 and ghrelin is detectable in wild type or mutant tissues at the investigated developmental time-points (original magnification 400×). (I-L) Nkx6.1⁺Sox9⁺ ductal/endocrine progenitors are present in all four genotypes at E15.5. In wild type and Arx-mutants Nkx6.1⁺Sox9^- cells are detected in comparable numbers. (K, L) However, in the absence of Nkx2.2 or Nkx2.2 and Arx no such cells are detectable. Pictures in I-L were taken using confocal microscopy. (M) Quantification of Nkx6.1⁺Sox9^- cell numbers in E15.5 pancreata of wild type, Arx^-, Nkx2.2^-/- and Nkx2.2^-/-Arx^- mice (n = 3 pancreas per genotype). Single-factor ANOVA coupled to Newman-Keuls test was applied and p-values < 0,05 were assessed as statistically significant compared to wild type. Statistical significance is achieved with the assumption of a normal distribution. Error bars represent SEM.

Figure 8

Schematic representation of transcription factor combinations guiding beta-cell differentiation. Following the loss of Nkx2.2 or Pax6, endocrine cells retain some beta-cell characteristics, such as the expression of iAPP, Pdx1 and/or PC1/3 (indicated as "β"). Rectangles describe transcription factor combinations required to maintain the embryonic beta-cell differentiation program pre- and post-insulin expression (light and dark green, respectively). Shortly after the activation of Ngn3, both Nkx2.2 and Pax4 are necessary for the expression of additional beta-cell specific TF's such as Nkx6.1 (not depicted). Concomitantly, this combination of TF's prevents the misexpression of Arx and ghrelin in committed beta-cells. In the absence of both Nkx2.2 and Arx, endocrine cells retain the same beta-cell features as noted in the absence of Nkx2.2 only, but then express ghrelin and somatostatin. Pax6 appears necessary for maintaining normal beta-cell identity at later stages of differentiation, probably shortly after insulin activation. At that stage Pax6 presumably takes over some of the functions previously exerted by Pax4, whose expression decreases.