C-type natriuretic peptide (CNP) alters development and growth in Zebrafish (Dario rerio) and mice (Mus musculus): a role for the anterior pituitary?

C-type natriuretic peptide (CNP/ Nppc) and guanylyl cyclase-B (GC-B/ Npr2) are bidirectional regulators of growth; activating/over-expression mutations of NPPC/Nppc or NPR2/Npr2 in humans/mice leads to over-growth, whereas inactivating/knock-down mutations cause short-stature and dwarfism. Previous studies implicate roles for cGMP and CNP in acting as growth hormone secretagogues, and several pharmacological treatments for short-stature syndromes now use CNP analogs. However, the effect of CNP on growth during development has not been investigated in depth. Here, we use pharmacological and CRISPR approaches in Zebrafish, and generate pituitary-specific Nppc ^-/- mice, to determine the effect of CNP on developmental growth. Bioinformatics, in situ hybridization and multiplex PCR revealed four genes encoding CNP in Zebrafish, with nppcl profoundly upregulated during development (by 45.7±2.8-fold by 120h post-fertilization). Treatment of zebrafish larvae with exogenous CNP for up to 72hpf surprisingly caused impaired growth and development, potentially due to an increase in apoptosis (as determined by TUNEL). Gene expression assays revealed that exogenous CNP caused down-regulation of endogenous nppcl expression (by 0.43±0.04 fold by 48hpf). CRISPR knockdown of nppcl caused a similar disrupted growth phenotype to that seen with exogenous CNP treatment. Transcripts for the pituitary genes somatolactin and tshβ were affected by exogenous CNP treatment and nppcl CRISPR knockdown. Finally, male pituitary-specific Nppc ^-/- mice are significantly hypomorphic compared to wildtype mice, and both males and females displayed gonadal phenotypes consistent with impaired fertility. These findings reveal CNP can influence growth characteristics during development, that may influence growth and development in later life.