Candidate transcription factors involved in pancreatic endocrine development have been isolated using insulin gene regulation as a paradigm. The cell-type restricted basic helix-loop-helix (bHLH) gene, BETA2/NeuroD, expressed in pancreatic endocrine cells, the intestine, and the brain, activates insulin gene transcription and can induce neurons to differentiate. To understand the importance of BETA2 in pancreatic endocrine cell differentiation, mice lacking a functional BETA2 gene were generated by gene targeting experiments. Mice carrying a targeted disruption of the BETA2 gene developed severe diabetes and died perinatally. Homozygous BETA2 null mice had a striking reduction in the number of insulin-producing ␤ cells and failed to develop mature islets. Islet morphogenesis appeared to be arrested between E14.5 and E17.5, a period characterized by major expansion of the ␤ cell population. The presence of severe diabetes in these mice suggests that proper islet structure plays an important role in blood glucose homeostasis. In addition, secretin-and cholecystokinin-producing enteroendocrine cells failed to develop in the absence of BETA2. The absence of these two pancreatic secretagogs may explain the abnormal cellular polarity and inability to secrete zymogen granules in pancreatic acinar exocrine cells. The nervous system appeared to develop normally, despite abundant expression of BETA2 in differentiating neurons. Thus, BETA2 is critical for the normal development of several specialized cell types arising from the gut endoderm.

Localized synthesis of insulin-like growth factors (IGFs) has been broadly implicated in skeletal muscle growth, hypertrophy and regeneration. Virally delivered IGF-1 genes induce local skeletal muscle hypertrophy and attenuate age-related skeletal muscle atrophy, restoring and improving muscle mass and strength in mice. Here we show that the molecular pathways underlying the hypertrophic action of IGF-1 in skeletal muscle are similar to those responsible for cardiac hypertrophy. Transfected IGF-1 gene expression in postmitotic skeletal myocytes activates calcineurin-mediated calcium signalling by inducing calcineurin transcripts and nuclear localization of calcineurin protein. Expression of activated calcineurin mimics the effects of IGF-1, whereas expression of a dominant-negative calcineurin mutant or addition of cyclosporin, a calcineurin inhibitor, represses myocyte differentiation and hypertrophy. Either IGF-1 or activated calcineurin induces expression of the transcription factor GATA-2, which accumulates in a subset of myocyte nuclei, where it associates with calcineurin and a specific dephosphorylated isoform of the transcription factor NF-ATc1. Thus, IGF-1 induces calcineurin-mediated signalling and activation of GATA-2, a marker of skeletal muscle hypertrophy, which cooperates with selected NF-ATc isoforms to activate gene expression programs.

The insulin gene is one of the best paradigms of tissue-specific gene expression. It is developmentally regulated and is expressed exclusively in the pancreatic 13-cell. This restricted expression is directed by a tissue-specific enhancer, within the promoter, which contains an E-box sequence. The insulin E-box binds an islet-specific protein complex, termed 3al. E-boxes bind proteins belonging to the basic helix-loop-helix (bHLH) family of transcription factors. The bHLH proteins function as potent transcriptional activators of tissue-specific genes by forming heterodimers between ubiquitous and cell-restricted family members. In addition, the cell-restricted bHLH members play an important role in specifying cell fate. To isolate the tissue-specific bHLH factor controlling insulin gene expression and study its role in islet cell differentiation, a modified yeast two-hybrid system was utilized to clone a novel bHLH factor, BETA2 ([f-cell _E-box trans-activator 2_), from a hamster insulin tumor (HIT) cell cDNA library. Northern analysis demonstrates that high-level expression of the BETA2 gene is restricted to pancreatic o~-and [~-cell lines. As expected of tissue-specific bHLH members, BETA2 binds to the insulin E-box sequence with high affinity as a heterodimer with the ubiquitous bHLH factor E47. More importantly, antibody supershift experiments clearly show that BETA2 is a component of the native insulin E-box-binding complex. Transient transfection assays demonstrate that the BETA2/E47 heterodimer synergistically interacts with a neighboring 13-cell-specific complex to activate an insulin enhancer. In contrast, other bHLH factors such as MyoD and E47, which can bind to the insulin E-box with high affinity, fail to do so. Thus, a unique, cooperative interaction is the basis by which the insulin E-box enhancer discriminates between various bHLH factors to achieve tissue-specific activation of the insulin gene.

Different patterns of motor nerve activity drive distinctive programs of gene transcription in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myo®ber subtypes. Recently, we proposed that the in¯uence of motor nerve activity on skeletal muscle ®ber type is transduced to the relevant genes by calcineurin, which controls the functional activity of NFAT (nuclear family of activated T cell) proteins. Here we demonstrate that calcineurin-dependent gene regulation in skeletal myocytes is mediated also by MEF2 transcription factors, and is integrated with additional calcium-regulated signaling inputs, speci®cally calmodulin-dependent protein kinase activity. In skeletal muscles of transgenic mice, both NFAT and MEF2 binding sites are necessary for properly regulated function of a slow ®ber-speci®c enhancer, and either forced expression of activated calcineurin or motor nerve stimulation up-regulates a MEF2-dependent reporter gene. These results provide new insights into the molecular mechanisms by which specialized characteristics of skeletal myo®ber subtypes are established and maintained.

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