The functional unit of the endocrine pancreas is the islet of Langerhans. Islets are nested within the exocrine tissue of the pancreas and are composed of alpha-, beta-, delta- and gamma-cells. beta-Cells produce insulin and form the core of the islet, whereas alpha-, delta- and gamma-cells are arranged at the periphery of the islet and secrete glucagon, somatostatin and a pancreatic polypeptide, respectively. Little is known about the molecular and genetic factors regulating the lineage of the different endocrine cells. Pancreas development is known to be abolished in Pdx1-mutant mice and Pax4 mutants lack insulin-producing beta-cells. Here we show that the paired-box gene Pax6 is expressed during the early stages of pancreatic development and in mature endocrine cells. The pancreas of Pax6 homozygous mutant mice lack glucagon-producing cells, suggesting that Pax6 is essential for the differentiation of alpha-cells. As mice lacking Pax4 and Pax6 fail to develop any mature endocrine cells, we conclude that both Pax genes are required for endocrine fate in the pancreas.

To delete genes specifically from mammary tissue using the Cre-lox system, we have established transgenic mice expressing Cre recombinase under control of the WAP gene promoter and the MMTV LTR. Cre activity in these mice was evaluated by three criteria. First, the tissue distribution of Cre mRNA was analyzed. Second, an adenovirus carrying a reporter gene was used to determine expression at the level of single cells. Third, tissue specificity of Cre activity was determined in a mouse strain carrying a reporter gene. In adult MMTV-Cre mice expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. Expression of WAP-Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Analysis of transgenic mice carrying both the MMTV-Cre and the reporter transgenes revealed recombination in every tissue. In contrast, recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally observed in the brain. These results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.

Mouse embryonic stem (ES) cells differentiate into cells of all three primary germ layers including endodermal cells that produce insulin in vitro.We show that constitutive expression of Pax4 (Pax4 ؉ ), and to a lesser extent Pdx1 (Pdx1 ؉ ), affects the differentiation of ES cells and significantly promote the development of insulin-producing cells. In Pax4 overexpressing R1 ES cells, isl-1, ngn3, insulin, islet amyloid polypeptide, and glucose transporter 2 (Glut-2) mRNA levels increase significantly. The number of nestinexpressing (nestin؉) cells also increases. Constitutive Pax4 expression combined with selection of nestin؉ cells and histotypic culture conditions give rise to spheroids containing insulin-positive granules typical of embryonal and adult ␤ cells. In response to glucose, Pax4 ؉ and wild-type ES-derived cells release insulin. Transplantation of these cells into streptozotocin-treated diabetic mice results in a normalization of blood glucose levels. We conclude that constitutive expression of Pax4 in combination with histotypic cultivation facilitates ES cell differentiation into the pancreatic lineage, which leads to the formation of islet-like spheroid structures that produce increased levels of insulin.

The mechanism by which the β-cell transcription factor Pax4 influences cell function/mass was studied in rat and human islets of Langerhans. Pax4 transcripts were detected in adult rat islets, and levels were induced by the mitogens activin A and betacellulin. Wortmannin suppressed betacellulin-induced Pax4 expression, implicating the phosphatidylinositol 3-kinase signaling pathway. Adenoviral overexpression of Pax4 caused a 3.5-fold increase in β-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively. Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient. Bcl-xL activity resulted in altered mitochondrial calcium levels and ATP production, explaining impaired glucose-induced insulin secretion in transduced islets. Infection of human islets with an inducible adenoviral Pax4 construct caused proliferation and protection against cytokine-evoked apoptosis, whereas the mutant was less effective. We propose that Pax4 is implicated in β-cell plasticity through the activation of c-myc and potentially protected from apoptosis through Bcl-xL gene expression.