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First published online 1 June 2005
doi: 10.1242/dev.01870

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The simultaneous loss of Arx and Pax4 genes promotes a somatostatin-producing cell fate specification at the expense of the - and ß-cell lineages in the mouse endocrine pancreas

¹ Max-Planck Institute for Biophysical Chemistry, Department of Molecular Cell Biology, Am Fassberg, 37077 Göttingen, Germany
² Hagedorn Research Institute, Department of Developmental Biology, Niels Steensensvej 6, DK-2820 Gentofte, Denmark
³ DIBIT, San Raffaele Scientific Institute, Via Olgenittina 58, I-20132 Milano, Italy
⁴ Centre de Biologie du Développement, UMR-5547 CNRS-Université P. Sabatier, 118 Route de Narbonne, F-31062 Toulouse, Cedex 04, France

View larger version (90K): [in a new window]   Fig. 1. Increase in the population of somatostatin- or PP-producing cells at the expense of insulin- and glucagon-expressing cells in Arx/Pax4 double-mutant pancreas. Sections of P2 mice were examined for pancreatic hormones in wild-type (A-I), Arx/Pax4 double-heterozygous (J-R) and Arx/Pax4 double-deficient (S-Z2) animals by co-immunofluorescence. Islets were stained with antibodies directed against insulin (A-C,J-L,S-U), and antibodies recognizing either glucagon- (D,M,V), somatostatin- (E,N,W) or PP-producing cells (F,O,X), and sections were counter-stained with DAPI (merged in G-I,P-R,Y-Z2). Note the loss of the insulin- (A-C,S-U) and glucagon- (D,V) producing cell population, and the dramatic increase of the somatostatin- (E,W) or PP- (F,X) expressing cell numbers in the double mutants. The simultaneous lack of a single Arx and Pax4 allele does not provoke any significant endocrine alteration when compared with wild-type animals (A-I,J-R).

View larger version (25K): [in a new window]   Fig. 2. Quantification of the phenotypic changes in hormone-producing cell populations following the mono- or bi-allelic loss of Arx and/or Pax4. P2-independent pancreata estimated to be of the same size were serially sectioned. n, number of pancreata analyzed for each genotype. Every tenth section was stained as indicated and the numbers of positive cells were counted and compared with the total islet cell content (estimated on adjacent sections using a mixture of antibodies raised against the different endocrine hormones). Data are shown as percentage±s.e.m. of hormone-positive cells contributing to the total endocrine population. On average, the lack of one Arx and/or Pax4 allele does not alter the endocrine cell content. Overall, the islet-cell alterations observed in Arx- and Arx-Pax4+/-, or in Pax4-/- and Pax4-/- Arx+/-, appear to be similar. However, the loss of a single Pax4 allele in Arx- Pax4+/- animals results in a significant reduction of the ß-cell content together with an increase of the -cell population when compared with Arx mutants. Note, in mice depleted in Arx and Pax4, the loss of the insulin- and glucagon-expressing cell populations, and the substantial increase in the numbers of somatostatin- or PP-producing cells. It should be underlined that the total endocrine cell content is not statistically modified in all of the genotypes analyzed. Multiple comparisons of the data obtained for each endocrine population in each genotype were processed with a single-factor ANOVA coupled to Newman-Keuls test using the wild-type genotype as a reference (*P<0.05, **P<0.01). A similar study was performed comparing subtype-specific cell numbers between Arx-Pax4+/- mice and Arx mutants (P<0.05).

View larger version (74K): [in a new window]   Fig. 3. Lack of - and ß-cell fate specification resulting in promotion of a somatostatin-producing cell destiny in Arx/Pax4 double mutant embryos. Co-staining of E12 (A,B), E15 (C-F), E18 (G-N,Q-X) and P0 (O,P) pancreas. The genotypes examined and the different antibody combinations used are indicated. A quantification of the endocrine modifications between the two genotypes, estimated using Student's t-test, is provided in percent under each set of pictures (n3, P<0.05; U, unchanged). (A,B) At E12, no alteration in the hormone-expressing cell numbers could be detected between wild type and double mutants. (C-L) A loss of mature glucagon- (C,D) and insulin- (C-J) expressing cells, and a dramatic increase in the somatostatin-producing cell content (G,H,K,L) is already obvious as soon as cells begin to express hormone; the number of Ngn3+ cells is not modified (C-F). Importantly, in embryos, the number of PP-expressing cells is not altered by Arx/Pax4 co-depletion (I-L). The supernumerary somatostatin-producing cells found in the double mutants do not express the PP hormone at E18 (K,L). (M-X) In Arx- and Pax4-deficient pancreas, the expression of the ß-cell-specific transcription factors Nkx6.1 and Glut2, is dramatically reduced (N,P; compare with wild type in M,O). Interestingly, the number of ghrelin-expressing cells is also reduced (R, compare with Q) and these cells produce neither somatostatin nor insulin. (S,T) Similarly, the expression of Nkx2.2 appears to be severely diminished in the double mutants; it is found in the remaining PP-cells, but is excluded from the somatostatin-producing cells (U,V). Finally, Pax6 labelling can be seen in all endocrine cells in the double mutants (W,X).

View larger version (49K): [in a new window]   Fig. 4. Expression of the PP hormone in somatostatin-producing cells in Arx-/Pax4-/- animals after the onset of feeding. Pancreas was isolated 12 hours after birth either from normally fed pups (P12h-fed; A-F) or from starved animal separated from their mother after delivery (P12h-not fed; G-L). Every tenth section of wild-type and Arx/Pax4 double mutant pancreas (indicated on the left side) was labelled with anti-somatostatin (A,B,G,H) and anti-PP (C,D,I,J) antibodies, and the number of marked cells was quantified; merged pictures of the different staining are presented in E,F and K,L. A quantification of the endocrine modifications between the two genotypes, estimated using Student's t-test, is provided in percent under each set of pictures (n3, P<0.05; U, unchanged). In normally fed P12h Arx/Pax4 double mutants, note the more than fivefold increase in PP production by the excess somatostatin-producing cells, when compared with wild type (E,F). Importantly, in starved animals, the content in PP-marked cells was found to be normal when compared with controls (K,L).

View larger version (49K): [in a new window]   Fig. 5. Arx and Pax4 mutually inhibit the transcription of one another through a direct interaction. (A) A sequence analysis of a 50 kb region centered on the Arx locus was performed, comparing Mus musculus with Homo sapiens (H/M), Rattus norvegicus (M/R) or Danio rerio (M/D). Despite an overall high similarity, comparison with the zebrafish sequence reveals the presence of only a few highly homologous zones, in the homeobox region and also in a domain located 11 to 14 kb downstream of the Arx gene (highlighted in red in the alignment). No obvious similarity was found upstream of the Arx gene. The mouse Arx locus is represented underneath the result of the alignment (exons are shown as black rectangles), as are the three typical -helices of the homeodomain. The NheI-EcoRV 9.7 kb region encompassing the 3' conserved region was further used for the generation of transgenic animals using the ß-galactosidase reporter gene lacZ. (B) ß-Gal staining of such animals demonstrates a recapitulation of the Arx expression pattern at E10.5 (see Collombat et al., 2003). The pattern of labelling in the pancreas corresponds perfectly to endogenous Arx expression (arrowhead; P.C., unpublished). (C) A search for Pax4-binding sites (P4BS) within the 9.7 kb region demonstrated that a labelled 37 bp DNA fragment, located 8.5 kb downstream of the NheI site (red rectangle in A), can efficiently interact with Pax4, as evidenced by EMSA analysis (lane 3). A competition assay with increasing concentrations of unlabelled P4BS further demonstrates the specificity of the interaction (lanes 4-6). Lane 1 shows the Pax4-binding-site consensus used as control (C). (D) The specificity of the interaction Pax4-P4BS was validated by ChIP analysis. No binding was detected in COS-cells (lanes 2-4), whereas, in ß-cells (lanes 5-7), PCR and sequencing reactions confirmed that Pax4 binds to P4BS (lane 7; P, P4BS-specific primers; C, control primers). (E) A reporter assay study indicates that Pax4 interacts with P4BS and acts as a transcriptional inhibitor. (F) Representation of the Pax4 locus, as well as the encoded homeodomain and the SmaI-NcoI 0.9 kb Pax4 enhancer region. (G) This 0.9 kb region was subdivided into five overlapping domains that were tested for recognition by the Arx protein. The 400 to 600 bp region (ArBS) can interact with Arx (blue rectangle in F,G). By means of EMSA and competition assay, Arx was found to preferentially interact with ArBS (lanes 2 and 3-5, respectively). (H) Using ChIP assay on isolated pancreas, an Arx-ArBS interaction is observed in E14 pancreas (lanes 5-7) but not in hind limb (lanes 2-4). (I) A reporter assay analysis suggests that Arx can bind to ArBS and acts as a transcription inhibitor. P, minimal promoter; Luc, luciferase gene. Results are presented as mean of luciferase activity ±s.e.m. Statistically significant differences of reporter activity, comparing cells transfected with the indicated reporters alone or with a Pax4-encoding vector (n=3), were estimated using Student's t-test (***P<0.001).

View larger version (47K): [in a new window]   Fig. 6. Model of endocrine subtype specification during pancreatic development. (A) An endocrine precursor cell initially expresses both Arx and Pax4, most probably in an inactive form. In a first round of competitive fate allocation, an unknown factor determines which factor will predominate: if it is Arx, the -cell fate will be specified (with Arx inhibiting Pax4 expression), whereas Pax4 will induce ß-/-cell lineages through the inhibition of Arx transcription (1). In the case of Pax4 prevalence, the resulting ß-/-cell precursor is poised to undergo a second round of fate allocation (2). In this second event, Pax4 seemingly induces the ß-cell fate at the expense of the -cell lineage. A hypothetical `factor X' is envisioned to have an opposite function, promoting the -cell fate to the detriment of ß-cell specification (3). (B-D) Fate changes in the case of Arx (B), Pax4 (C), or combined Arx/Pax4 (D) deficiency. See main text for details. For the purpose of simplification, exocrine cell and PP-cell development are not represented.

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