Bioelectric signaling regulates size in zebrafish fins

The amino acid sequence of zebrafish Kcnk5b was retrieved from Ensembl (http://www.ensembl.org) and used to search the PDB database with HHpred (http://toolkit.tuebingen.mpg.de/) [84]. The first hit in the search (human KCNK4, PDB ID: 3um7 [39] identity 36%, similarity 0.646; 22nd March 2012) was used to build the 3D model. The model was processed with MacPyMol (http://pymol.org).

kcnk5b was subcloned from pGEM-T Easy to pSGEM expression vector via SacII and SpeI sites. After linearization with NheI, cRNA was synthesized with Ambion mMessage mMachine (Invitrogen) and cleaned up with mRNeasy Mini Kit (Qiagen). X. laevis oocytes were injected as described previously [85] (kcnk5b single alleles: 4 ng wild type or mutant kcnk5b cRNA; co-injections of two kcnk5b alleles: 2 ng cRNA each, for a total of 4 ng per oocyte). Measurements were done from a holding potential of −80 mV with 0.5 s long pulses from −100 to +60 mV with increments of 20 mV. Recorded currents (n = 5–26) were averaged and normalized to the mean value recorded for oocytes injected with the wild type channel at +60 mV.

PCR mutagenesis was performed as described [86] using Pfu polymerase (Fermentas) (OSP-15 CCC TGA CGA CTG TCG CTG CAG CTG ACT ATG TGG CAG GGG C; OSP-16 CCT GCC ACA TAG TCA GCT GCA GCG ACA GTC GTC AGG GTG G, Tm = 70°C, 30 cycles) on pSGEM:kcnk5b(wt).

ef1a:DsRed vector. A ef1a:DsRed cassette generated with KOD Hot Start DNA Polymerase (Toyobo) (primers: TAA TTT AAA TAG ATC TTC GAG CAG GGG GAT CAT CTA ATC A; CTA GAT GGC CAG ATC TGC CCG GGA CTT GAT TAG GGT GAT GGT TCA CGT AGT G, Tm = 59°C, 30 cycles) from plasmid Ale237 (kind gift of Alessandro Mongera) was inserted in plasmid 587jk (kind gift of Dr. Jana Krauß) using BglII restriction sites through In-Fusion Advantage (Clontech) cloning according to manufacturer's protocol.

ef1a:DsRed; ef1a:kcnk5b wild type and mutant vectors. The ef1a promoter was amplified from plasmid Ale237 (primers: ATT AAT TCG AGC TCG GTA CCC CTC GAG CAG GGG GAT CAT CT; GAA CAA GCA AGC TGG GTA CCC CGG CCG TCG AGG AAT TCT TTG, Tm = 59°C, 30 cycles) and inserted into the pSGEM vector at the KpnI restriction site using In-Fusion Advantage (Clontech) cloning. The ef1a:kcnk5b cassette was amplified from the resulting plasmid as above (primer: AAA CCT AGG TCG AGC AGG GGG ATC ATC T; AAA CCT AGG ATG ACC ATG ATT ACG CCA AGC TAT), digested with AvrII and inserted into ef1a:DsRed vector using the SpeI restriction site.

Plasmids (5–20 ng/µl), Tol2 mRNA (25 ng/µl) and 20% (v/v) phenol red solution (Sigma- Aldrich, P0290-100ML) were injected into the zygote of 1-cell stage embryos under a dissecting microscope (Zeiss, Stemi 2000) using 275 Pa (40 psi) injecting pressure for 100 ms (World Precision Instruments, Pneumatic PicoPump PV820). Adults were analyzed with Zeiss, SteREO Discovery and Zeiss LSM 5 Live.

Transplantations were performed as previously described [82]. At mid blastula stage (1000 cell stage), about 20–40 cells were transplanted from the pfaudt30mh/+ donors into the recipient close to the yolk cell and chimeras were raised to adulthood.

Fluorescent dye experiments were performed by adapting described protocols [22], [87]. Briefly, wild type and mutant juvenile fish (STL = 16–18 mm) were incubated in fluorescent dye diluted 1∶2000 in fish water (stock solutions: DiSBAC2(3) (Bis-(1,3-Diethylthiobarbituric Acid)Trimethine Oxonol, Life Technologies): 1 mg/ml in DMSO) for 30 min in the dark, anesthetized with tricaine solution and placed on a custom-made chamber for confocal imaging. The chamber was obtained by removing the bottom of a 55 mm plastic dish and by replacing it through a round cover slip fastened with silicone. Fish were held in place with a tissue soaked in dye and imaged upon excitation at 561 nm. Unstained animals were imaged as a negative control. p-values from unpaired Student's t-test were obtained with Microsoft Excel.