The HGn8H insertion disrupted the synembryn-like gene. (A) The structure of the T2KHG insertion in the synbl locus in the HG8H enhancer trap line. Arrowheads indicate positions and directions of primers. Blue and purple boxes indicate exons of the synbl and rfx4 genes, respectively. White boxes indicate 5′ and 3′ UTRs. (B-E) The pigment and edema phenotype in HGn8H embryos at day 5. Dorsal and side views of heterozygous (B,C) and homozygous (D,E) embryos. (F) Summary of the link between embryonic lethality and GFP expression. (G) Examples of the genotype analysis by PCR using synbl-f and synbl-r (top) and control PCR using r2 and Tol2-OUT (bottom). M, DNA size marker; N, no DNA; P, positive control. (H) Summary of the genotype analysis. (I) RT-PCR analysis of wild-type, heterozygous and homozygous embryos using synbl-f and r2 (left). Control RT-PCR using ef1α-f and ef1α-r primers (right). (J,K) GFP expression pattern in 24 hpf heterozygous embryos. GFP fluorescence (J) and whole-mount in situ hybridization using the gfp probe (K). (L,M) Whole-mount in situ hybridization of 24 hpf wild-type embryos using the synbl (L) and rfx4 (M) probes. (N) A phylogenic analysis of vertebrate synembryn homologs. (O,P) Whole-mount in situ hybridization of 24 hpf wild-type embryos using the synbl (O) and ric8a (P) probes. (Q-V) Rescue of the pigment phenotype by forskolin. Pigmentation of heterozygous (Q,T), homozygous (R,U) and synbl-MO-injected wild-type (S,V) embryos at day 5. Embryos were soaked in 1 μM (T,U) and 2 μM (V) forskolin.
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