Expression of slc12a7a/kcc4a and its paralog slc12a7b/kcc4b in fin mesenchyme. A, B) In situ hybridization of slc12a7a/kcc4a in cross sections of adult caudal fins of wild-type (A) and heterozygous schleier mutants (B). Inset, signal from control in situ probes on serial sections. C) Quantitative PCR measurement of slc12a7a/kcc4a transcript in wild-type and heterozygous schleier caudal fin tissue. Gene expression was normalized to the geometric mean of three reference genes; n = 4 samples per genotype; 4 adult fins per sample. Error bars indicate SEM; ns, not significant using Student’s t-test. C, D) In situ hybridization of the paralogue slc12a7b/kcc4b in cross sections of adult caudal fins of wild-type (D) and heterozygous schleier mutants (E). Inset, signal from control in situ probes on serial sections. F) Quantitative PCR measurement of slc12a7b/kcc4b transcript in wild-type and heterozygous schleier caudal fin tissue. Gene expression was normalized to the geometric mean of three reference genes; n = 4 samples per genotype; 4 adult fins per sample. Error bars indicate SEM; *, p < 0.01 using Student’s t-test. G-L), Immunofluorescence of antibodies against Kcc4 in adult caudal fins. G, J) Experimental (primary plus secondary antibody) on wild-type and heterozygous schleier fin sections. Arrow, fin epidermis; arrowhead, fin mesenchyme. H, K), DAPI counterstaining of wild-type and heterozygous schleier fin sections. I, L) Signal from control sections hybridized with secondary antibody only. M, N) Number of slc12a7a/kcc4a and slc12a7b/kcc4b transcripts in wild-type caudal fin, measured in fragments per kilobase of transcript per million mapped reads (FPKM). Data were obtained from www.zfregeneration.org (Nieto-Arellano and Sanchez-Iranzo, 2019). O) Endpoint RT-PCR on cDNA synthesized from wild-type (WT) and schleier heterozygous caudal fin RNA. No RT, no reverse transcriptase control.
|
