Endocrine Abstracts

The family of calcium/calmodulin-dependent protein kinases (CaMK) are major effectors of calcium binding proteins in virtually all systems. Recently, CaMKs have been implicated in regulating pituitary and hypothalamic gene transcription to control the expression of gonadotrophin hormones and adrenocorticotrophin in the pituitary, and as a pivotal protein in the satiety pathway. Despite these observations, the spatial and temporal expression of the CaMKs is poorly documented and the role of these kinases in endocrine development is unknown. Previous studies have implicated a role for CaMK-II in pituitary gene expression (1). Therefore, in this study, we have taken advantage of the highly versatile zebrafish as a physiological model in which to examine the expression and function of CaMKII in neuroendocrine tissues. Considerable bioinformatic screening of the zebrafish database revealed five different isoforms of CaMKII, specifically IIα, IIβ, IIγ, IIδa and IIδb. Oligonucleotide primers were designed to specifically amplify each of these isoforms in zebrafish. Total RNA was extracted from a range of zebrafish embryos at different time points (between 24 and 72 hpf), and converted to cDNA. PCR was performed on a gradient block using specific amplification conditions for each isoform. Transcripts corresponding to every CaMKII isoform were detected in zebrafish, as confirmed by dideoxy DNA sequencing of PCR products following pGEMT cloning. Preliminary immunohistochemistry, using antibodies specific for CaMKII or phospho-CaMKII, indicated expression of these proteins within the presumptive hypothalamus and pituitary regions of the developing zebrafish. In summary, we have successfully cloned all CaMKII isoforms expressed in the zebrafish and have localised expression of some of these isoforms to major neuroendocrine regions in developing embryos. These data strongly implicate the CaMKII proteins in the development of the hypothalamus and anterior pituitary. Supported by a Small Project Grant from the Society for Endocrinology.

1. Fowkes et al. Endocrine Abstracts 2002 4 P69.