Crispr-Induced Genetic Manipulations to C-Type Natriuretic Peptide Transcripts in Danio Rerio (Zebrafish) Larvae Results in Impaired Growth and Development

Presentation Number: LBF-086
Date of Presentation: March 6th, 2015

Andrew James Lessey*1, Samantha Mary Mirczuk1, Jordan Elise Read1, Stijn J Niessen2, Imelda Mary McGonnell1 and Robert C Fowkes1
1The Royal Veterinary College, London, United Kingdom, 2The Royal Veterinary College, North Mymms, United Kingdom


Human patients with mutations within NPPC or NPR2 genes (encoding C-type natriuretic peptide (CNP) and guanylyl cyclase-B (GC-B), respectively) display clinical signs associated with skeletal abnormalities, such as overgrowth or short stature. In mice, spontaneously occurring, as well as induced models of Nppc or Npr2 deletion result in profound achondroplasia, dwarfism and early death, with growth hormone deficiency present in some instances; thus, current pharmacological therapies to treat short stature have included long-acting CNP analogues. We have demonstrated the presence of NPPC and NPR2 transcripts in normal human fetal pituitaries, as well as human pituitary adenomas of differing origins, suggesting potential roles for CNP during development. Using the versatile Danio rerio (Zebrafish) as a model for vertebrate development, we previously reported that exposure of Zebrafish larvae to excessive exogenous CNP leads to altered pituitary gene expression and stunted growth. In the current study, we have used a reverse genetics approach to alter expression of two of the four genes that encode CNP in the Zebrafish (nppcl and nppc4). Here we employed the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas 9 system to efficiently facilitate genome editing of Zebrafish embryos in vivo. RNA-guided Cas9 endonuclease targeting nppcl or nppc4 was injected into wild type Zebrafish embryos at the one-cell stage. Mutant embryos were subsequently reared to either 24, 48 or 72 hours post fertilization (hpf) alongside uninjected controls and gDNA extracted. A T7 endonuclease I assay was used to demonstrate the induction of indels in the nppcl and nppc4 loci in Zebrafish. Gross morphological and biometric analyses of these mutants showed that disruption of either nppcl or nppc4 transcripts in vivo severely impaired embryonic development. Body length was significantly reduced in comparison to wild type controls (nppcl: p<0.01, nppc4: p<0.01) and eye diameter was significantly smaller (nppcl: p<0.05, nppc4: p<0.01). Mutant embryos also display impaired forebrain development, impaired locomotion and substantial cardiac oedema in comparison to wild type controls. Collectively, these data suggest that CNP in Zebrafish is crucial for normal embryonic development, specifically with regards to  skeletal growth, brain development and fluid homeostasis. Furthermore, the phenotypic similarity of nppcl and nppc4 mutants, without apparent compensation, requires further elucidation as to their specific developmental functions.


Nothing to Disclose: AJL, SMM, JER, SJN, IMM, RCF