C-type natriuretic peptide (CNP) is the ancestral and most highly conserved member of the mammalian natriuretic peptide family. Although broadly expressed throughout the periphery (bone, gonads, kidneys), CNP is the major natriuretic peptide of the brain and CNS and is suspected to influence neuroendocrine function. Our recent studies using fetal human pituitary samples has shown that the gene encoding CNP, NPPC, is expressed early on in pituitary development but a comprehensive understanding of CNP expression in utero remains to be established. Employing the versatile Danio rerio (Zebrafish) as an excellent vertebrate model of development, we sought to establish the spatio-temporal expression of components of the natriuretic peptide system throughout development. Zebrafish embryos were captured at time points ranging from three hpf up to 120 hpf prior to extraction of total RNA. Additionally, adult Zebrafish were euthanized and total RNA was extracted from both head and body separately. RT-PCR enabled us to screen for the expression of nppa (ANP), nppb (BNP), nppc2, cnp3, nppc4, nppcl (CNPs), npr1a, nprA (GC-A), nprB (GC-B) and nprC (clearance receptor gene). All these genes were expressed, but clear temporal differences between the four distinct CNP transcripts were observed. Digoxigenin-labelled RNA probes were synthesised from all cloned PCR products prior to in situ hybridization using embryos captured at 24, 48, 72, 96 and 120 hpf. The four CNP genes each demonstrated individual spatial expression, yet were all concentrated in the head and notochord during development with the GC-B receptor nprb, more restricted in its expression. In silico phylogenetic analysis of the four CNP genes demonstrated evolutionary conservation was maintained between these transcripts in Zebrafish and conserved across vertebrate species. These data reveal the presence of an intact natriuretic peptide system in developing Zebrafish, and will facilitate a reverse genetics approach to identify their functional roles within neuroendocrine development.
Declaration of funding: Wellcome Trust Project Grant (WT093257MA)
RVC PhD Studentship.