Opposing actions of Arx and Pax4 in endocrine pancreas development

Abstract

Genes encoding homeodomain-containing proteins potentially involved in endocrine pancreas development were isolated by combined in silico and nested-PCR approaches. One such transcription factor, Arx, exhibits Ngn3-dependent expression throughout endocrine pancreas development in alpha, beta-precursor, and delta cells. We have used gene targeting in mouse embryonic stem cells to generate Arx loss-of-function mice. Arx-deficient animals are born at the expected Mendelian frequency, but develop early-onset hypoglycemia, dehydration, and weakness, and die 2 d after birth. Immunohistological analysis of pancreas from Arx mutants reveals an early-onset loss of mature endocrine alpha cells with a concomitant increase in beta-and delta-cell numbers, whereas islet morphology remains intact. Our study indicates a requirement of Arx for alpha-cell fate acquisition and a repressive action on beta-and delta-cell destiny, which is exactly the opposite of the action of Pax4 in endocrine commitment. Using multiplex reverse transcriptase PCR (RT-PCR), we demonstrate an accumulation of Pax4 and Arx transcripts in Arx and Pax4 mutant mice, respectively. We propose that the antagonistic functions of Arx and Pax4 for proper islet cell specification are related to the pancreatic levels of the respective transcripts.

Figure 1.

Arx is expressed in normal mouse pancreas, but not in that of Ngn3-deficient animals. (A,B) Whole-mount in situ hybridization of an E9.5 mouse embryo. Sagittal section (A) and enlarged view of the pancreatic area (B), revealing Arx transcripts in the pancreas anlage (arrowhead in B). (C-E) When analyzed by in situ hybridization, pancreas sections display widespread Arx expression at E12.5 (C), E14.5 (D), and E16.5 (E). (F-I) Detection of glucagon and Arx expression by in situ hybridization in E14.5 wild-type (F,G) and Ngn3 homozygous mutant animals (H,I). Ngn3 mutant pancreas lacks Arx expression. (RT-PCR) RT-PCR analysis performed on mRNA from E14.5 wild-type or Ngn3 mutant animals (left and middle lanes, respectively). RT-PCR controls were performed with primers designed from the glucose-6-phosphate dehydrogenase (G6PDH) and the TATA-box-binding protein (TBP) reference genes; positive controls with primers from the amylase gene and negative controls were assayed with water instead of DNA template (right lane). RT-PCR results confirm a lack of glucagon and Arx expression in Ngn3 mutant mice.

Figure 2.

Disruption of the Arx gene by homologous recombination and genotype analysis of heterozygous intercrosses. (A) Maps of the wild-type Arx locus, targeting vector, and disrupted Arx allele. Exons are indicated as black rectangles with encoded protein motifs underneath. The 1.3-kb 5′ external probe used for Southern blotting is shown at left. (B) BamHI, (H) HindIII, (I) EcoRI, (II) HincII, (V) EcoRV, (X) XhoI, (TK) Herpes simplex virus thymidine kinase gene. (B) Southern analysis of EcoRI-digested DNA from three male ES clones, Arx being located on the X chromosome, either the 10.5-kb wild-type allele (+) or the 5.7-kb mutated allele (-) is detected. (C) Genotyping and sex characterization by PCR of a litter from an intercross of heterozygous mice. (Top gel) Sex determination using Sry gene-derived primers producing a 350-bp band in the case of male genomic DNA. (Bottom gel) Three genotyping primers (represented by green and red arrows in A) were used to generate the wild-type (258-bp) and mutant (470-bp) allele bands. (D) Photograph of 2-day-old wild-type (left) and Arx mutants (right) with the corresponding transverse sections of neural tubes assayed by in situ hybridization with an Arx-cDNA-derived-probe (bottom). Arx-deficient animals are smaller and dehydrated and die at this stage. The loss of Arx expression in mutant mice demonstrates that Arx is inactivated. (E) Blood glucose analysis in the offspring of F1 hybrids 12 h (P12h), 24 h (P24h), and 48 h (P48h) after birth indicates that Arx-disrupted animals exhibit severe hypoglycemia that plunges shortly before death (p48h-SBD). Values are means ± S.E. of the mean (S.E.M.) and are representative of at least 20 animals. Differences between glucose levels were statistically significant by Student's t-test (^***) at P48h between wild-type/heterozygous and mutant animals (p < 0.001).

Figure 3.

Arx expression assessed by LacZ staining demonstrates an increase in the number of β-galactosidase-producing cells within the islet of Langerhans in Arx-deficient mice. (A,B) Hematoxylin-eosin staining of 2-day-old pancreas sections reveals no morphological discrepancy between heterozygous (A) and mutant (B) Arx mice. (C,D) Whole-mount LacZ staining of Arx-deficient embryos (C) and enlarged view (D) reveals β-galactosidase expression in the pancreas as early as E10.5, as well as in additional hitherto unmentioned domains. Pancreas sections of heterozygous (E,G,I,K) and mutant (F,H,J,L) Arx animals were examined for β-galactosidase activity by X-Gal staining at different developmental stages as follows: E10,5 (E,F, outlined), E12.5 (G,H), E16.5 (I,J), and P2 (K,L). β-galactosidase-positive cells present a similar distribution at early embryonic stages in wild-type and mutant pancreas. After E16.5, the content of β-galactosidase-producing cells is increased in mutant animals as compared with littermates. Each picture is representative of at least eight animals from different litters.

Figure 4.

Increased δ- and β-cell populations at the expense of α cells in Arx-deficient mice. Single sections of 2-day-old mice were examined for the presence of pancreatic hormone in control (A-I) and Arx mutant (J-R) animals by coimmunofluorescence. (A-C,J-L) Insulin-expressing cells were localized in well-defined islets using 488-ALEXA secondary antibody. Codetection experiments were performed with 594-ALEXA secondary antibody to visualize either glucagon-producing cells (D,M), somatostatin-producing cells (E,N), or PP-producing cells (F,O), and sections were counter-stained with DAPI (G-I,P-R, in blue). (G-I,P-R) A picture combining codetection of β cells with other endocrine cells and counter-staining is provided in each case. Note the slight increase of the insulin-producing cell population (cf. A-C and J-L), the loss of glucagon-secreting cells (cf. D and M), and the increase in the number of somatostatin-expressing cells (cf. E and N) in Arx mutant animals. Arx deletion also leads to a modification of the spatial distribution of δ cells with an additional localization within the islet core (cf. E,H and N,Q). Each picture is representative of 8-20 animals from different litters.

Figure 5.

Alteration of mature endocrine cell-subtype specification following Arx inactivation. (A-D) Staining of E12.5 heterozygous (A,C) and mutant (B,D) Arx pancreata for insulin (A,B), glucagon (A-D), and β-galactosidase (C,D) proteins, counter-stained with DAPI (blue). β-galactosidase is not expressed in the early hormone-producing cells. There is no change in the number of insulin-expressing cells (often producing glucagon) following Arx deletion. (E-L) Sections of pancreas from E15.5 (E-J) and E18.5 (K,L) stained with anti-β-galactosidase (E,F,I-L), anti-glucagon (E,F), anti-Nkx6.1 (G,H), anti-DIG (G,H, shown in red), anti-insulin (I,J), or anti-somatostatin (K,L) antisera and counter-stained with DAPI (blue) in heterozygous (E,G,I,K) or mutant (F,H,J,L) animals except in G and H. (E,F) Glucagon-secreting cells coexpress β-galactosidase and are lacking in mutant pancreas. (G,H) β cells (stained with Nkx6.1 for technical reasons) do not express β-galactosidase in E15.5 mutant and control pancreas. (I,J) The β-cell population is increased in mutant islets. A residual presence of β-galactosidase protein is observed. (K,L) β-galactosidase staining is detected in δ cells, which are overrepresented in E18.5 Arx-deficient mice. (M-R) Endocrine differentiation appears normal following Arx inactivation. Sections of heterozygous (M,O,Q) and mutant (N,P,R) pancreas were stained with anti-Pax6 (M,N), anti-Ngn3 (O,P), or anti-Isl1 (Q,R) antisera together with anti-β-galactosidase antisera and counter-stained with DAPI (blue). (M,N) All β-galactosidase-labeled cells coexpress Pax6. Pax6-marked cells are normally represented in both genotypes. (O,P) Arrows show a few Arx- and Ngn3-labeled cells. Arx deletion does not alter the numbers of Ngn3-stained cells. (Q,R) Isl1-marked cells are exclusively found among the β-galactosidase-labeled population, but numerous cells are β-galactosidase⁺/Isl1^-, suggesting a function of Arx in nonmature cells. Similar numbers of Isl1⁺ cells are observed in control and Arx mutant pancreas. Each picture is representative of at least three animals from different litters.

Figure 6.

Increase of Arx and Pax4 transcript levels in the pancreas of Pax4- and Arx-deficient mice, respectively. Multiplex amplification of pancreatic mRNA from Pax-4 (A) and Arx (B) mutant mice at E14.5. The genotypes of control and mutant mice are indicated above the gel scans. Measurements of the intensity of radioactive Arx and Pax4 PCR products, after normalization with the coamplified TATA-box-binding protein (TBP) amplicon, are represented as graphs. Below are indicated the average normalized intensities in control and mutant mice as well as their relative difference; the Arx transcript content is 3.3-fold higher in E14.5 Pax4 mutant pancreas compared with control animals. Arx-deficient pancreas contains four times more Pax4 mRNA at the same embryonic stage. (C,D) An anti-Arx antibody recapitulates Arx expression pattern at E11.5 (sagittal section in C and enlarged picture in D) in the forebrain (FB) and the floor plate of the diencephalon (Fp). (E-G) Analysis of Arx (red) and β-galactosidase (green) expression in Pax4 heterozygous (E,F) and homozygous (G) pancreas. The average percentage of Arx⁺/β-galactosidase⁺ cells from three serially sectioned pancreata is indicated below each picture. (E) In Pax4 heterozygous pancreas, Arx is found colocalized with the β-galactosidase protein at E13.5 in 35% of endocrine cells. (F) At E14.5, only 8% of islet cells are Arx⁺/β-galactosidase⁺. (G) At the same stage, in Pax4 homozygous pancreas, this number increases with a codetection of Arx and β-galactosidase found in 72% of the endocrine cells.

Figure 7.

Schematic model representing the transcription factors implicated in the specification of the endocrine pancreas, on the basis of temporal expression and phenotypic results of specific gene deletions. Circles represent endocrine cells at particular developmental stages. The different transcription factors expressed in a particular cell type are indicated within the circles. Arrows represent different (hypothetical) endocrine differentiation steps. Loss-of-function mutant mice phenotypes demonstrate antagonistic endocrine-specifying activities for Arx and Pax4; Pax4 promotes β- and δ-cell fates, whereas Arx favors α-cell destiny. The question mark indicates a fate remaining to be elucidated. For the purpose of simplification, exocrine/ductal- and PP-cell development are not represented.