22-P005 Mechanisms of primitive endoderm specification – From ES cells to preimplantation mouse development

Gonadotropin releasing hormone (GnRH) is a multifunctional decapeptide that possess endocrine as well as neuromodulatory roles and is present in all vertebrates. Endocrine GnRH cells play a crucial role in the establishment of the hypothalamic–pituitary– gonadal axis in many species. Recent studies have found that the prokineticin2 (pk2) and prokineticin receptor2 (pkr2) genes are involved in the development of olfactory bulb and reproductive system and mutations in either of these genes result in a phenotype similar to Kallmann syndrome. Using zebrafish as a model system we are investigating the role of pk2 and pkr2 genes in the development of endocrine GnRH cells. Using specific primers we cloned genes from the zebrafish genome coding for predicted proteins having portions of high identity with pk2 and pkr2 genes from Mus musculus. We found two mouse homologous of pkr2 gene on the zebrafish genome with a 69% (putative pkr2a; XP_001342503) and 62% (putative pkr2b; XP_001338684) identity, respectively. Also, we found one homologous of pk2 gene with a 65% (putative pk2; XP_001342503) of identity. PCR revealed that ppkr2a, ppkr2b and ppk2 genes are expressed starting as early as 24hpf and that the receptors are more highly expressed than the ligand. Knockdown of ppk2 and ppkr2a proteins caused a reduction of endocrine GnRH cells and isotocin cells compared with control. Currently we are exploring the spatial expression of these genes with antisense digoxigenin labeled specific probes on adult zebrafish brains slices.