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 ␣, ␤-precursor, and ␦ 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 ␣ cells with a concomitant increase in ␤-and ␦-cell numbers, whereas islet morphology remains intact. Our study indicates a requirement of Arx for ␣-cell fate acquisition and a repressive action on ␤-and ␦-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.

The homeobox gene Arx is a novel positive regulator of embryonic myogenesis

“…These mice die shortly after birth and display retarded growth, probably due to an abnormal pancreas. 13 At the end of gestation (E19.5), no overt anomalies were observed in the growing muscle masses of mutant mice (data not shown) and adult heterozygous Arx þ /nlacZ mice appeared normal, although some were obese. Nevertheless, at E11.5, MyHC þ muscles in the limb anlagen appeared smaller in the mutant embryos ( Figure Arx synergizes with MyoD and Mef2C in the activation of muscle-specific transcription.…”

“…Arx has been shown to be important for testes, 12 central nervous system 12 and pancreas development. 13 Here, we show that Arx is predominantly expressed in embryonic myoblasts and primary fibers. Arx acts as positive modulator of myogenesis by synergizing with MyoD and Mef2C and establishing an activating loop with Myogenin.…”

“…Adult muscle could only be analyzed from Arx þ /nlacZ mice, because mutant Arx nlacZ males die perinatally. 13 Bar, 50 mm. BA, branchial arches; T, telencephalic vesicle; S, somites; first C1, cranial somite; T1, first thoracic somite; FL, forelimb; HL, hind limb; NT, neural tube; TA, tibialis anterior; Qu, quadriceps; G, gastrocnemius; IM, intercostal muscles; TM, temporalis muscle; BM, branchial muscles; R, ribs overexpressing cells showed reduced BrdU incorporation (32.274.7 versus 42.472.8% in the control cells), consistent with the observed enhanced differentiation state.…”

“…For evaluating the proliferative capacity, cells were incubated with 50 mm BrdU (Sigma) for 1 h, washed, fixed in a 95% ethanol/5% (v/v) acetic acid solution for 20 min and then treated with 1.5 M HCl for 10 min. Immunofluorescent staining was carried out using the following antibodies: rabbit polyclonal 33 and mouse monoclonal (MF20) antibody to all sarcomeric MyCHs; mouse anti-BrdU (Amersham, Buckinghamshire, UK); mouse anti-Myogenin (F5D); rabbit antiArx, 13 Quantitative real-time polymerase chain reaction. Total cellular RNA was isolated from cell populations using RNeasy RNA isolation kit (Qiagen, Hilden, Germany).…”

“…The mutant and transgenic animals and genotyping primers used in this work were published: Pax3 GFP , 5 Arx nlacZ , 13 Tg: 3F-nlacZ-2E, 37 MyoD À/À , 38 Myf5 GFPÀP , Myf5 loxP , Myf5 nlacZ . 7,39 In Tg:3F-nlacZ-2E mice, b-galactosidase expression is restricted to differentiated skeletal and cardiac muscle.…”