Characterization of brn1.2 and corticotropin-releasing hormone genes in zebrafish

Abstract

The zebrafish (Danio rerio), a tropical fresh water fish originally found in the rivers of India and Bangladesh has become a popular vertebrate model system over the last decade. The rapid sequencing of the zebrafish genome together with the latest advances in forward and reverse genetics has made this model organism more fascinating as it can be used to decipher the genetic mechanisms involved in the vertebrate development.

Corticotropin-releasing hormone (CRH) regulates the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. Alteration in the production of CRH can lead to Cushing s syndrome, anxiety-related disorders and other neurodegenerative disorders such as Alzheimers s disease, Parkinson s dementia and Huntington s disease. In mammals it was reported that a POU class III gene by name Brn-2 is essential for the development of specific neuroendocrine cell types. Brn-2 can also regulate the transcription of corticotropin-releasing hormone by binding to its promoter. Corroborating the above fact, knockout studies in mice have shown that deletion of Brn-2 affects the development of corticotropin-releasing hormone producing neurons in addition to oxytocin and vasopressin synthesizing neurons.

In order to understand the genetic mechanisms involved in the development of zebrafish neuroendocrine hypothalamus, we attempted to characterize the developmental expression profile of brn1.2 and crh in zebrafish. Zebrafish crh gene has not been characterized before and only a partial sequence of brn 1.2 was reported earlier.

We screened the zebrafish EST database for brn1.2 and crh through a blast search and identified EST s for crh and brn1.2 that contained their full ORF. We used non-quantitative RT-PCR to analyze embryonic and post-embryonic expression pattern of crh. Expression of crh mRNA was detected from 1dpf and it remained stable through 5 dpf. RT-PCR analysis of the tissues revealed strong expression of crh mRNA in the brain, eye, gills and weak expression in the testis. Spatial expression pattern of both the genes were analyzed by whole mount in situ hybridization. Expression of crh was detected in different parts of the embryonic brain including telencephalon, hypothalamus, posterior tuberculum, thalamus, preoptic area, epiphysis, midbrain tegmentum, hindbrain and in the retina. Localization of CRH transcripts in the preoptic area is consistent with its role as a hypophysiotropin in zebrafish.

Zebrafish POU class III genes include brn1.2, zp47, zp12, zp23 and zp50. zp47 and brn1.2 are found to be the direct homologs of the mammalian Brn-2 gene. Expression of brn1.2 begins at tailbud stage. From 5-somite stage on, brn1.2 is detected in the midbrain, hindbrain and weakly in the spinal cord. At 24hpf brn1.2 is widely expressed in the diencephalon, midbrain and hindbrain but not detected in the telencephalon. At later stages of development brn 1.2 transcripts were found in the hypothalamus, preoptic area, ventral thalamus, dorsal thalamus, pretectum, tectum, cerebellum and hindbrain. Additionally, brn 1.2 was also detected in the otic vesicle at 48hpf.

In this study we have described the developmental expression pattern of brn1.2 and crh. Future studies will include a complete analysis of the function of brn1.2 in the specification of the hypothalamic neurons and also identification of other candidate genes that are required for the development these neurons.