Bioelectric-calcineurin signaling module regulates allometric growth and size of the zebrafish fin

The fifth and terminal exon of zebrafish kcnk5b encodes the last transmembrane domain and cytoplasmic C-terminus. We designed guide RNAs (gRNA) tiling this exon to screen for sufficiency of deletions in this region to affect fin growth in mosaic injected animals. The ChopChop online was used to design gRNAs to limit predicted off-target gRNA cutting49,50. The pool of 5 distinct gRNAs were simultaneously injected to blanket the start of the exon. These gRNAs were assembled according to Gangon et al.51. Briefly, oligos containing the gRNA target were annealing to a universal oligo containing the tracrRNA and SP6 promoter. The annealed oligo ends were then filled in with T4 polymerase for 20 minutes at 12 °C. gRNA was synthesized from this oligo template using Ambion MEGAscript SP6 Kit. For transcription efficiency, the first two bases of each gRNA were changed to ‘GG’ as there is evidence that these bases have less effect on Cas9 cutting efficiency or off-target binding than mutations closer to the PAM site52–54. gRNAs were injected into single cell zebrafish embryos at a concentration of 50 ng/μl blanket and 300 ng/μl Cas9. To screen for deletion efficiency, the target exon was amplified from pools of three 24 hour embryos and the resulting amplicons were heated to 95 °C and cooled at −0.1 °C per second to form heteroduplexes. Following heteroduplex PCR, a T7 endonuclease digestion for 30 minutes at 37 °C in NEB Buffer 2 was used to generate deletions in the presence of Cas9-induced indels. Primers for colony PCR cloning of the exon 5 screen: 5′-GGCAAATCAAACTGGTTAGTCC-3′, 5′-CGCTGTAGTCCTCGACCTTC-3′. gRNA sequences: 5′-ATCACTTTGTTTCCATACAG-3′, 5′-GACAAAGAATCTGTAGAGAG-3′, 5′-GGATCTACCTGGGCCTTGCT-3′, 5′-TTGGAACGTGCATATGGTGG-3′, 5′-CGTCATGGTGGGCAGCCTG-3′.

The diversity of forms in Nature is frequently derived from variation in the proportions of organs and tissues. The ability of organs and tissues to attain proper proportions is generally unknown. Recent work has shown that inhibition of the activity of calcineurin modulates size of fins in zebrafish. Here, we show that the action of a potassium channel, Kcnk5b is required for growth caused by calcineurin inhibition. Importantly, we show that these factors specify fin growth independently from inherent cues of size and positional identity of the fin. Our work shows that this signaling node acts as a molecular rheostat, able to integrate bioelectric and calcium-mediated signaling systems to dial relative growth rates guiding ultimate fin size.