Cell-specific, developmental, and hormonal regulation of corticotropin-releasing hormone gene expression in transgenic mice.

Abstract

The expression of the stress neuropeptide corticotropin releasing hormone (CRH) was examined by in situ hybridization histochemistry. CRH is expressed in many neuronal groups within the adult mouse brain, with the highest levels in the paraventricular nucleus (PVN), inferior olivary nucleus, and Barrington's nucleus. Glucocorticoid regulation of CRH mRNA occurs specifically in the PVN, consistent with the expected role of CRH in the hypothalamic-pituitary-adrenal axis. CRH mRNA is differentially regulated in various regions of the developing mouse brain. It is also expressed prenatally in lung and celiac ganglion although the role of CRH in ontogeny has not yet been defined. In an effort to identify the cis-acting DNA sequences important for cell-specific expression of CRH in vivo, transgenic mice were generated with varying amounts of the CRH gene and flanking DNA fused to a $\beta$-galactosidase reporter gene. Expression was assayed by histochemical staining in adult and developing mice. Results demonstrated that elements that repress expression in inappropriate peripheral tissues are located within the CRH gene and 2 kb of 3$\sp\prime$ flanking DNA. Important regulatory elements for expression in major CRH neuronal groups are located between 8.7 and 0.5 kb 5$\sp\prime$ to the transcription start site of the CRH gene. However, the observation of ectopic neuronal expression suggested that additional DNA sequences were necessary for appropriate CRH expression. A 30 kb rat CRH transgene was efficiently reconstituted by homologous recombination between three overlapping fragments injected into fertilized mouse eggs. Expression of this transgene was similar to 16 kb transgenes, suggesting that additional regulatory elements probably exist outside of a 30 kb genomic region surrounding the CRH gene. Finally, the chicken lysozyme locus control region (LCR) was coinjected with a previously examined CRH transgene to test the efficacy of a heterologous LCR to insulate the transgene from position effects. This LCR did not improve the cell-specificity of transgene expression, demonstrating that it is not a generic insulator element. These studies illustrate that CRH gene expression is controlled by multiple interacting transcriptional regulatory elements including some at a large distance from the gene.