Silic et al., 2020 - Potassium Channel-Associated Bioelectricity of the Dermomyotome Determines Fin Patterning in Zebrafish. Genetics   215(4):1067-1084 Full text @ Genetics

Figure 1

Fins are elongated in the adult zebrafish mutant Dhi2059. (A) WT fish with normal-sized fins. (B and C) Both one- and two-copy transallelic mutants of Dhi2059 possessed elongated paired and median fins. Bars, 50 mm in (A–C). (D) The lengths of caudal fins in Dhi2059 mutants are significantly longer compared to WT siblings. Fish were generated by kcnj13Dhi2059/+ incrossing. For each group, n = 40 (20 males and 20 females). * P < 0.0001 by ordinary one-way ANOVA test (Tukey’s multiple comparisons) based on mean difference and SD. (E and F) Normal fin ray segments in WT fish. (G and H) In the Dhi2059 mutant, the fin ray segments of the caudal fins are increased. The number of fin ray joints are barely noticeable. Black/white arrowheads indicate natural ray joints. Red arrows indicate the healing scars of broken bone structures. (E–H) Tg (col2a1a: EGFP-CXXC) labeled WT and Dhi2059 mutant fish. Bars, 25 mm in (E–H). (I) Allometric growth of caudal fins in kcnj13Dhi2059/+ mutants (n = 39; k = 1.5) compared to isometric WT siblings (n = 23, k = 1.0), reflected by linear regression analysis on the lengths of caudal fins and standard body lengths (measured from the tips of the snouts to the posterior ends of the last vertebrae). k = allometric coefficient. The slopes are significantly different, P = 0.002, F-test of general linear model analysis. All fish were generated by incrossing of kcnj13Dhi2059/+ fish. WT, wild-type.

Figure 2

Viral insertion was identified in the kcnj13 gene locus and mRNA. (A) Schematic of the zebrafish kcnj13 gene denoting the position of viral insertions in the hi2059 mutant. The viral DNA randomly inserted into the fifth exon of the gene. The horizontal black arrow indicates the protein-coding area. (B) Extended majority-rule consensus phylogenetic tree for the Bayesian analysis of KCNJ13 proteins. Numbers at each node indicate pp values based on 20 million replicates. Branch lengths are proportional to means of the pp densities for their expected replacements per site. (C) The diagram of the viral insertion in the kcnj13 mRNA. The approximate positions of PCR primers are indicated with purple/blue arrows. Green lines indicate broken exon 5, and blue lines indicate the remaining exons. (D) Confirmation of chimera viral sequence and kcnj13 mRNA by RT-PCR. The expected sizes of PCR products are 5708 bp (Lacz-129F + kcnj13-462R) and 4509 bp (kcnj13-U74F + LacZ-2486R), respectively. The smaller bands in lane 4 are nonspecific amplified bands. kcnj13-U74F and kcnj13-462R are targeted at the beginning of exons 5 and 7, respectively. CDS, coding DNA sequence; LTR, long terminal repeat; mRNA, messenger RNA; pp, posterior probability; UTR, untranslated region.

Figure 3

Kcnj13 is ectopically expressed in somites of the Dhi2059 mutant. Whole-mount in situ hybridizations of zebrafish embryos at the stages from 15S to 48 hpf were performed. WT embryos (A, D–F, and J). kcnj13Dhi2059/+ fish embryos (B, C, G–I, and K). (A) Kcnj13 is expressed at the pnd in WT embryos. (B) In the kcnj13Dhi2059/+ mutant, kcnj13 is expressed in the somites at the 15S stage. (C) Dorsal view of the embryo in (B). (D–F) At the 24-hpf stage, kcnj13 is continually expressed in the pronephric ducts of WT embryos. (D) Lateral view. (E) Ventral view of the 24-hpf embryos. The dashed line indicates the approximate position of the transverse section in (F). (G–I) The expression of kcnj13 in the kcnj13Dhi2059/+ mutant at the 24-hpf stage. The kcnj13 mRNA is also expressed in the somites in addition to pnds. (H) Ventral view of the same embryo in (G). The dashed line indicates the position of the transverse section in (I). (I) Transverse section revealed that kcnj13 is expressed in the dm. (J) At the 48-hpf stage, kcnj13 is continually expressed in pnds in WT fish embryos, and it is also expressed in melanocytes (arrowheads). (K) In the kcnj13Dhi2059/+ fish embryos, kcnj13 expression is similar to WT embryos at 48 hpf. The somite expression is no longer detectable. Bar, 250 μm (A–E, G, H, J, and K). Bar, 100 μm (F and I). 15S, 15-somite; dm, dermomyotome; hpf, hours postfertilization; mRNA, messenger RNA; n, notochord; nt, neural tube; pnd, pronephric ducts; so, somite, WT, wild-type.

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Terms:
Stage Range: 14-19 somites to Long-pec
PHENOTYPE:
Fish:
Observed In:
Stage Range: 14-19 somites to Prim-5

Figure 4

Dhi2059 long-finned phenotype was only able to be rescued by a kcnj13 loss-of-function mutation in an allelic-specific manner. (A) Illustration for principle of the genetic rescue experiment. Purple dots represent WT kcnj13 gene expression in both endogenous domains and ectopic expression in somites. The blue dots represent the loss-of-function kcnj13 endogenous expression by the jaguar (Kcnj13G157E/) allele, which is driven by an intact promoter. The orange dots represent the overlapped expression. The green lines indicate pronephric ducts. (B) WT control fish. (C) Morphology of heterozygous jaguar mutant. (D) Morphology of homozygous jaguar mutant. (E) Morphology of kcnj13Dhi2059/+ mutant. (F and G) Morphology of double heterozygous jaguar and Dhi2059 mutants, kcnj13Dhi2059/G157E. (F) Male. (G) Female. Note, the caudal fin broke due to its large size before imaging. Although pigmentation patterns were altered, the long-finned phenotype still remained in kcnj13Dhi2059/G157E mutants. (H) Quantitative comparison between kcnj13Dhi2059/+ mutant and kcnj13Dhi2059/G157E mutants. WT (n = 20); G157E, jaguar (G157E, n = 15); Dhi2059 (n = 19); Dhi2059; G157E (n = 21). * P < 0.0001 by unpaired Student’s t-test. (I) Location of CRISPRs against kcnj13 coding region. CRISPR and important functional domains are annotated with colored arrows. (J–L) illustration and morphology of CRISPR-induced insertion/deletion mutation in Dhi2019 mutants. All fish shown are injected F0 adults. (J) CRISPR mutation is located on the WT allele (orange bar) of kcnj13Dhi2059/+. (K) CRISPR mutation is located on the virial inserted allele (orange bar) of kcnj13Dhi2059/+. (L) CRISPR mutations are located on both virial inserted alleles (orange bars) of kcnj13Dhi2059/Dhi2059. (M–N) F3 generation adult fish with CRISPR mutant that linked with Dhi2059 inserted kcnj13 allele. (M) Morphology of heterozygous kcnj13hi2059-CRmutant. (N) Morphology of homozygous kcnj13hi2059-CR/hi2059-CR mutant. Long fins are completely rescued in both conditions. Bars, 5 mm. CR, CRISPR mutant; CRISPR, clustered regularly interspaced short palindromic repeats; gRNA, guide RNA; WT, wild-type.

Figure 5

Transient ectopic expression of kcnj13 by pax3a promoter phenocopies Dhi2059 elongated fins. (A) Schematic illustration of a construct (pax3a:kcnj13-IRES-EGFP) used for Tol2 transgenesis. A 5.4-kbp-long pax3a promoter drives kcnj13 and independent EGFP expression. (B–E) Representatives of F1 generation of pax3a:kcnj13-IRES-EGFP transgenic fish. (B) EGFP expressed in the somites of a 12S-stage fish embryo. White arrowheads indicate the somites. (B’) Bright-field image of the same embryos in (B). (C) EGFP expressed in the somites and dermomyotome of a 24-hpf-stage fish embryo. White arrows indicated the dermomyotome. (C’) Bright-field image of the same embryos in (C). (D) EGFP expression disappears from somites in a 48-hpf-stage zebrafish embryo. Only autofluorescence is visible. (E) Representative gross morphology of an adult Tg(pax3a:kcnj13-IRES-EGFP). Elongation of the fins is similar to the Dhi2059 mutant. (F–I) Representatives of F3 generation of pax3a:kcnj13-IRES-EGFP transgenic fish. (F). A dark-EGFP fish embryo. (G) A bright-EGFP fish embryo. (H) An adult dark-EGFP transgenic fish. The fin is slightly elongated compared to WT. (I) An adult bright-EGFP transgenic fish. (J) Comparison of the ratio of caudal fin over standard body length among no-EGFP (n = 16), dark-EGFP (n = 11), and bright-EGFP (n = 10) adult transgenic fish. *) P < 0.0001 by pairwise Student’s t-test. Bars, 200 μm (B–D, F, and G), Bars, 5 mm (E, H, and I). 12S, 12-somite; e, eye; EGFP, enhanced GFP; IRES, internal ribosome entry site; WT, wild-type.

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Terms:
Stage Range: 10-13 somites to Prim-5
PHENOTYPE:
Fish:
Observed In:
Stage: Adult

Figure 6

Generation of Kcnj13 mutants with different potassium conductance. (A) Protein sequence alignment of site-directed mutants. TM1, TM2, and P-loop domains are highlighted in green, brown, and blue, respectively. Altered amino acids are highlighted in yellow. (B) A computation-predicted three-dimensional structure of the Kcnj13 subunit. Mutated sites are indicated with arrows. (C) Conductance characterization of three Kcnj13 mutants compared to WT showing decreased conductance of Q153H (n = 12, P = 0.022) and increased conductance of M135R (n = 19, P = 0.020). The mutant T131A did not show a significant difference in conductance (n = 12, P > 0.9) compared to WT (n = 13). The current density was calculated by dividing current (pA) at each holding potential by the cell membrane capacitance (pF) to normalize the difference in the cell size. Statistical differences of mean values were calculated using a two-tailed Student’s t-test. (D) Replotted part of (C) with enlarged y-axis to show the difference between the T131A mutant and the WT in resting membrane potential (also see Figure S7). (E) Example recording traces from each mutant and WT channels. Holding potential was −80 mV and step size was 10 mV. P-loop, pore-forming; TM, transmembrane; WT, wild-type.

Figure 7

Long-finned phenotype is dependent on potassium conductance. (A–E) Overexpression of kcnj13 mimics Dhi2059 phenotypes. (A and B) Schematic illustration of constructs used for Tol2 transgenesis with a kcnj13-EGFP fusion construct or with independent EGFP. (C) WT noninjected fish. (D) Representative injected fish with actinb-kcnj13-EGFP construct. (E) Representative injected fish with actinb-kcnj13-IRES-EGFP construct. (F–H) Representative phenotypic changes of the three kcnj13 mutants. actinb-kcnj13-T131A-IRES-EGFP (F), actinb-kcnj13-Q153H-IRES-EGFP (G), and actinb-kcnj13-M135R-IRES-EGFP (H). Only M135R-, but not T131A- or Q153H-, injected fish developed long-fins, but all have pigmentation pattern disruption. Partial elongation of certain fins and pigmentation changes most likely result from the mosaic nature of Tol2 transgenesis. Bars, 5 mm. AmpR, ampicillin resistance, EGFP, enhanced GFP; IRES, internal ribosome entry site; WT, wild-type.

Figure 8

Transient ectopic expression of multiple potassium channel genes also induces long-finned phenotype. (A) WT adult control fish without any injection. (B) Representative injected adult F0 fish image of human KCNJ13. Note: a barbel is also elongated in this fish. (C) Representative injected adult F0 fish image of zebrafish kcnj10a. (D) Representative injected adult F0 fish image of zebrafish kcnj1b. (E) Representative injected adult F0 fish image of zebrafish kcnk9. (F) Representative injected adult F0 fish image of zebrafish kcna1a. Arrows indicate the potions of the elongated parts of fins. All the potassium channel genes are driven by the actinb promoter. The partial and variable elongated fins are due to the mosaic nature of Tol2 injection. Bars, 5 mm. WT, wild-type.

Figure 9

Model of kcnj13 regulation in the Dhi2059 mutant and fin patterning by bioelectricity. (A) Illustration of cell bioelectric dynamic properties, which may be contributed by potassium channels (K+); calcium channels (Ca++), chloride channels (Cl), solute carriers, connexins (cx), and Na+-K+ ATPases. (B–E) Two-stage bioelectric model of zebrafish fins. (B) The first stage of bioelectric regulation happens among the fin progenitor cells within a somite. The dashed lines indicate that the possible source of the second wave progenitors may come from the myotome or sclerotome. The bioelectric alteration of either the first-wave progenitor cells (orange dots) or the second-wave progenitor cells (orange or blue dots) cause fin patterning changes. (C) The second stage of bioelectric regulation happens within the fin anlagen. (D) The interaction of the fibroblasts and osteoblast eventually determine fin size and pattern. (E) Either attraction or repulsion occurs between fibroblasts and osteoblasts, depending on their dynamic bioelectric status. (F) A model of kcnj13 regulation by retroviral insertion in the Dhi2059 mutant. The Moloney murine leukemia virus is inserted within exon 5 of the kcnj13 allele in Dhi2059 fish. This viral insertion may negatively affect the melanocyte-specific enhancer and somite/dermomyotome-specific silencer, either through physical distance isolation or viral long terminal repeat regulation. Thus, this insertion leads to ectopic somite expression and slightly reduced expression in the melanocyte. This leads to cell bioelectricity change and subsequent patterning alterations in corresponding organs. DM, dermomyotome; dpf, days postfertilization; mRNA, messenger RNA; n, notochord; NT, Neural tube; Scl, sclerotome.

Acknowledgments:
ZFIN wishes to thank the journal Genetics for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Genetics