Mechanisms of Corticotropin-Releasing Factor (CRF) Gene Regulation in the Frog Xenopus laevis.

Abstract

The vertebrate hypothalamic-pituitary-adrenal/interrenal (HPA/HPI) axis plays a central role in integrating the stress response and maintaining homeostasis. My dissertation research focused on understanding the regulatory mechanisms of the key regulator of the stress axis, the corticotropin-releasing factor (CRF). I took a comparative/evolutionary approach to this problem, and utilized the South African clawed frog Xenopus laevis as a model system. I found that the general distribution of CRF expression in the frog brain are highly conserved with other vertebrates. Similar to mammals, exposure of juvenile frogs to a stressor caused rapid activation of CRF neurons in the anterior preoptic area (POA), which is homologous to the mammalian paraventricular nucleus (PVN) and the primary site of neuroendocrine control of the HPA/HPI axis. The CRF neurons in the limbic structure the medial amygdala (MeA) and bed nucleus of the stria terminalis (BNST) were also activated following exposure to the stressor. I found that the structures and sequences of the CRF genes are highly conserved among tetrapods. Using a comparative genomic approach, I identified putative transcription factor binding sites in the proximal promoters of the frog CRF genes. I then tested the functionality of these sites by cell transfection, in vitro binding assays, chromatin precipitation (ChIP) assays, and in vivo electroporation-mediated gene transfer. I showed that a conserved cAMP response element (CRE) in the CRF promoter mediates gene regulation by the PKA pathway in vitro, and stressor-dependent activation in vivo. I also found that the distribution of the glucocorticoid receptor (GR) in the frog CNS is highly conserved with mammals. Similar to mammals, I showed that glucocorticoids down-regulate CRF expression in the POA; whereas they up-regulate it in the MeA and BNST. Overall, my research showed that the cell-specific expression within the CNS, stressor-dependent activation, feedback regulation, and transcriptional regulatory mechanisms of CRF genes are highly conserved among the tetrapods. My discoveries suggest that the basic regulatory mechanisms and neuronal circuits of the CRF system arose before the divergence of the amphibian and amniote lineages, and have been conserved by strong positive selection.