Embryonic endocrine pancreas and mature beta cells acquire alpha and PP cell phenotypes upon Arx misexpression

Abstract

Aristaless-related homeobox (Arx) was recently demonstrated to be involved in pancreatic alpha cell fate specification while simultaneously repressing the beta and delta cell lineages. To establish whether Arx is not only necessary, but also sufficient to instruct the alpha cell fate in endocrine progenitors, we used a gain-of-function approach to generate mice conditionally misexpressing this factor. Mice with forced Arx expression in the embryonic pancreas or in developing islet cells developed a dramatic hyperglycemia and eventually died. Further analysis demonstrated a drastic loss of beta and delta cells. Concurrently, a remarkable increase in the number of cells displaying alpha cell or, strikingly, pancreatic polypeptide (PP) cell features was observed. Notably, the ectopic expression of Arx induced in embryonic or adult beta cells led to a loss of the beta cell phenotype and a concomitant increase in a number of cells with alpha or PP cell characteristics. Combining quantitative real-time PCR and lineage-tracing experiments, we demonstrate that, in adult mice, the misexpression of Arx, rather than its overexpression, promotes a conversion of beta cells into glucagon- or PP-producing cells in vivo. These results provide important insights into the complex mechanisms underlying proper pancreatic endocrine cell allocation and cell identity acquisition.

Figure 1. Generation of animals conditionally misexpressing the Arx gene.

Schematic depicting the targeting vector before (top) and after (bottom) recombination of the 2 LoxP sites induced by the phage P1 Cre recombinase. β-Galactos., β-galactosidase. Inset: Visual examination of representative Cre-negative E10 embryo and adult pancreas under fluorescent light. Original magnification, ×40.

Figure 2. Conditional Arx misexpression restricted to the early pancreas results in a favoring of the glucagon- or PP-producing cell fates at the expense of the β and δ cell lineages.

Sections of 6-week-old mice were examined for the presence of pancreatic hormones in cArxOE (A, D, G, J, M, and P), cArxOE::Pax6Cre (B, E, H, K, N, and Q), and cArxOE::Pdx1Cre (C, F, I, L, O, and R) animals by immunofluorescence. (A–C) The uniform GFP expression found in cArxOE mice (A) is expectedly excluded from the islets in cArxOE::Pax6Cre animals (B) and missing in cArxOE::Pdx1Cre pancreatic tissue (C). Conversely, the β-galactosidase activity is clearly detected in cArxOE::Pax6Cre islets (B, inset) and cArxOE::Pdx1Cre pancreata (C, inset). (D–R) The Cre-mediated misexpression of Arx in Pax6 or Pdx1 expression domains promotes a loss of β cell (D–F) and δ cell (G–I) populations, concurrently with an increase in numbers of glucagon-producing (D–O) or PP-producing (J–L) cell numbers. Arx production is uniformly detectable in the endocrine tissue of cArxOE::Pax6Cre and cArxOE::Pdx1Cre mice (M–O). Note that Arx is present in glucagon-labeled cells, and, most likely, in PP-positive cells (insets in N and O). A coexpression of Arx and PP was demonstrated both in controls (P) and in double-transgenic animals (Q and R). Each picture is representative of 6–15 independent animals. Glu, glucagon; Ins, insulin; Som, somatostatin. Original magnification, ×40.

Figure 3. Quantification of the phenotypic alterations in hormone-producing cell contents following the targeted misexpression of Arx.

Six-week-old independent pancreata estimated to be of the same size were serially sectioned, every tenth section was stained as indicated, and the numbers of positive cells were counted and reported to the total islet cell content (estimated on adjacent sections with the use of a mixture of antibodies raised against the different endocrine hormones). Data are shown as the percentage ± SEM of hormone-positive cells contributing to the total endocrine population. On average, the misexpression of Arx during early pancreas genesis promotes a dramatic loss of β and δ cells, whereas the contents in glucagon- and PP-labeled cells are proportionally increased. *P < 0.05, **P < 0.01, ***P < 0.001. n ≥ 8.

Figure 4. Favoring of the glucagon- and PP-producing cell fates at the expense of the β and δ cell lineages during endocrine pancreas morphogenesis in cArxOE::Pax6Cre or cArxOE::Pdx1Cre pancreata.

(A–DD) Representative pancreatic sections costained with the indicated antibody combinations, at the indicated embryonic stages. A quantification of the endocrine modifications between the Cre-negative and Cre-positive mice, estimated by the 1-tailed Student’s t test, is provided in percent under each set of pictures in matching colors (n ≥ 3, P < 0.05). A clear loss of β (A–C) and δ (D–F and V–X) cells is evidenced in animals misexpressing Arx. Concurrently, an increase in glucagon (A–U and Y–DD) or PP (G–I) cell content occurs, with Arx being detected in most of the endocrine tissue (J–L), but also in exocrine cells in the case of cArxOE::Pdx1Cre (L, arrows). The numbers of cells labeled with the β cell–specific markers Nkx6.1 (M–O), Pdx1 (P–R), and Glut2 (S–U) are drastically reduced in Cre-positive mice. Similarly, the expression of CART, normally marking δ cells, is profoundly diminished (V–X), whereas the expression of the α cell–specific gene Brn4 is augmented (Y–AA). The content in cells positive for the endocrine marker Pax6 is not affected (BB–DD). Each picture is representative of 3–8 animals from different litters. U, unchanged. Original magnification, ×40.

Figure 5. Arx misexpression converts mature β cells into glucagon- or PP-producing cells.

(A–D) Representative staining and quantification of the endocrine cell content alterations found in tamoxifen-treated cArxOE::IndPdx1Cre pancreata with the use of the indicated antibodies. (E) Co-immunohistochemical detection of the glucagon (blue) and PP hormones (green) with the β-galactosidase enzyme (red). Note that glucagon- or PP-producing cells are positive for the β-galactosidase. Each picture is representative of 4–7 animals from different litters. Original magnification, ×40.