Comprehensive spatio-temporal expression profiling reveals a complete natriuretic peptide system in the developing Zebrafish (Danio rerio)

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 142-166-Hypothalamus-Pituitary Development & Biology
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-163
Andrew James Lessey*, Samantha Mary Mirczuk, Imelda Mary McGonnell and Robert C Fowkes
The Royal Veterinary College, London, United Kingdom
C-type natriuretic peptide (CNP) is the major natriuretic peptide of the brain and central nervous system. Suspected to influence neuroendocrine development and function, our recent studies using fetal human pituitary samples has shown that the gene encoding CNP, NPPC, is expressed early in pituitary development(1) however, a comprehensive understanding of CNP expression in utero remains to be established. Employing 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. RNA was extracted from embryos at time points ranging from 3 to 120hrs post fertilization (hpf), and from adult Zebrafish head and body separately. Expression was examined by RT-PCR, that indicated nppa (ANP), nppb (BNP), nppc2, cnp3, nppc4, nppcl (CNPs), npr1a, nprA (GC-A), nprB (GC-B) and nprC (clearance receptor) were expressed in Zebrafish, 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 120hpf. 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. To investigate the effects of CNP on pituitary gene expression, Zebrafish embryos were treated with 10μM CNP from 0hpf and harvested at either 48 or 72hpf. Total RNA was extracted and simultaneous expression of Zebrafish cga (alpha-subunit), gh1 (growth hormone), pomca (pro-opiomelanocortin), prl (prolactin), and slβ (somatolactin) and housekeepers elfα and 18S genes were examined and quantified using a multiplex real time-PCR assay and GenomeLab GeXP analysis system.  This prelimary data shows that CNP has a regulatory role for pituitary gene expression within the developing embryo and points to a key role for CNP in pituitary function during development. Overall, these data reveal the presence of an intact natriuretic peptide system in developing Zebrafish, and will facilitate a reverse genetics approach to identify the functional roles of CNP within neuroendocrine development.

(1) Thompson et al., Endocr Relat Cancer. 2012 Jul; 19(4):497-508.

Nothing to Disclose: AJL, SMM, IMM, RCF

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Sources of Research Support: Wellcome Trust Project Grant 093257Royal Veterinary College PhD Studentship