Figure 1

Figure 1

(A–C) Structural and molecular analyses of zebrafish C-type natriuretic peptides. (A) Multiple sequence alignments of published whole-natriuretic-peptide precursor sequences were aligned using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo). Evolutionary genetic analysis was performed on the resulting alignment using MEGA5 (http://megasoftware.net) and a phylogenetic tree created using the maximum-likelihood method and Tamura–Nei substitution model. Nonparametric bootstrap sampling was performed (1000 replicates) alongside and shown as a percentage at each corresponding node. Scale bar represents a genetic distance of 0.2. (B) Zebrafish C-type natriuretic peptide, CNP1 (nppc-like), CNP2 (nppc2-like), CNP 3, (cnp3-like) and CNP 4 (nppc4-like), amino acid sequences were obtained from NCBI Protein (http://www.ncbi.nlm.nih.gov/protein) and processing endopeptidase cleavage sites identified. Predicted mature zebrafish CNPs were aligned with human and murine CNP using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo) to assess structural similarities. Resulting sequence alignment with regions of high or absolute conservation highlighted in grey. Structural representation of known and predicted mature peptides. Amino acids that differ from human CNP are highlighted in red and calculated sequence homology with human CNP displayed as a percentage next to each zebrafish peptide. (C) Tupfel-long fin (TL) wild-type zebrafish embryos were captured at 6, 12, 24, 36, 48, 72, 96 and 120 h post-fertilization (hpf) along with adult zebrafish head (AH) and torso (AT) segments and harvested for total RNA extraction. cDNA was subsequently generated at 100 ng/µL for each developmental time point and used in PCR reactions with oligonucleotide primers designed against zebrafish nppc-like (CNP1) (product size: 602 bp), nppc2-like (CNP2) (332 bp), cnp3-like (CNP3) (557 bp), nppc4-like (CNP4) (379 bp) npra (GC-A) (352 bp), npr1a (GC-A) (640 bp), nprb (GC-B) (429 bp), nprc (NPR3) (748 bp), nppa (ANP) (602 bp), nppb (BNP) (332 bp) and β-actin (410 bp) alongside a no template control (ø). PCR products were electrophoresed on a 1.6% (w/v) agarose gel and visualized using ethidium bromide on a Bio-Rad ChemiDoc MP system (Bio-Rad, Hercules, CA, USA).