Spot pattern of leopard Danio is caused by mutation in the zebrafish connexin41.8 gene

Abstract

Leopard, a well-known zebrafish mutant that has a spotted skin pattern instead of stripes, is a model for the study of pigment patterning. To understand the mechanisms underlying stripe formation, as well as the spot variation observed in leopard, we sought to identify the gene responsible for this phenotype. Using positional cloning, we identified the leopard gene as an orthologue of the mammalian connexin 40 gene. A variety of different leopard alleles, such as leo(t1), leo(tq270) and leo(tw28), show different skin-pattern phenotypes. In this manuscript we show that the mutation in allele leo(t1) is a nonsense mutation, whereas alleles leo(tq270) and leo(tw28) contain the missense mutations I202F and I31F, respectively. Patch-clamp experiments of connexin hemichannels demonstrated that the I202F substitution in allele leo(tq270) disrupted the channel function of connexin41.8. These results demonstrate that mutations in this gene lead to a variety of leopard spot patterns.

Figure 1

Stripe patterns of zebrafish. (A) Wild-type (WT) zebrafish with normal horizontal stripes. (B,D,F) Zebrafish heterozygous for leo^t1, leo^tw28 or leo^tq270. (C,E,G) Zebrafish homozygous for leo^t1, leo^tw28 or leo^tq270. The inset boxes in each image show magnified images of stripes or spots on the skin of the fish.

Figure 2

Zebrafish connexin41.8 as a leopard gene. (A) Map of the leopard region. The yellow arrowhead indicates the microsatellite marker z9704. The red bar indicates the bacterial artificial chromosome (BAC) clone zK53o8, which includes the critical region for leopard. The black bars show the BAC clones located around this region. Green arrowheads indicate single nucleotide polymorphism marker sites. Each site was named as shown above the arrowhead. The numbers below the green arrowheads refer to the number of recombination events (supplementary information online). This region includes two genes, bcl9 and Cx41.8. Cx41.8 is indicated by a red arrowhead. (B) Schematic of zebrafish connexin41.8 showing predicted structural motifs and mutations detected in leopard alleles.

Figure 3

Patch-clamp analysis of Cx41.8 hemichannels expressed in HeLa cells. (A) Establishment of conditions for patch-clamp analysis with HeLa cells transfected with the wild-type (WT) Cx41.8 cDNA. I_m responses recorded in solution with no added Ca²⁺ (upper), with 2 mM Ca²⁺ (middle) and with 200 μM carbenoxolone (lower) in the bath solution. (B) Superimposed I_m traces from HeLa cells expressing WT, leo^tw28 or leo^tq270 connexin41.8, and untransfected HeLa cells (negative control). (C) Bar graph of WT (N=4), mutant leo^tw28 (N=7) and mutant leo^tq270 (N=8), compared with untransfected HeLa cells (N=8, negative control). Data represent the means±s.e.m. Each value was compared with that of WT. ^*P<0.005.

Figure 4

Cx41.8 expression in adult tissues. Reverse transcription–PCR was performed with RNA samples isolated from the indicated adult fish tissues. M indicates molecular size marker. Total RNAs were reverse-transcribed with oligo-d(T)₁₈ primer, and the resultant complementary DNAs were amplified by PCR using specific primers for (A) Cx41.8 and (B) β-actin.