Fig. 2

Knockdown of flcn in zebrafish embryos. a Schematic from the UCSC genome browser showing the binding sites of the ATG and splice site morpholinos used to knockdown the flcn gene (yellow box) (b) Electrophoresis gel showing; amplified flcn exon 1 and flcn exon 2 transcripts in wild type (WT), 100 μM of mismatch morpholino injected (sp2 MM) and 100 μM of flcn splice 2 morpholino injected (sp2 MO) embryos and; amplified flcn exon 1 to intron transcripts in 100 μM of flcn splice 2 morpholino injected embryos (sp2 MO) with no transcript in wild type (WT) and 100 μM of mismatch morpholino injected (sp2 MM) embryos (c) Box-whisker plots of flcn qPCR. Results, ΔC_t, indicate expression relative to uninjected embryos. Boxes denote interquartile range with central horizontal line showing median expression. Whiskers extend to 1.5 x interquartile range. MO = morpholino, MM = mismatch. d Prim-5 stage 100 μM control splice 2 mismatch morpholino injected embryo. e Prim-5 stage 100 μM control splice 2 mismatch morpholino injected embryo stained with Acridine Orange f Prim-5 stage 100 μM flcn splice 2 morpholino injected embryo (g) Prim-5 stage 100 μM flcn splice 2 morpholino injected embryo stained with Acridine Orange (h) Long-pec stage 100 μM control splice 2 mismatch morpholino injected embryo. i Long-pec stage 100 μM flcn splice 2 morpholino injected embryo. j Proportion of embryos that are normal (Red), show mild hydrocephalus, larger yolk and thinner yolk extension (blue) or severe hydrocephalus, enlarged yolk and very thin yolk extension (green) when injected with combinations of 100 μM flcn splice 2 morpholino and 50 pg/μl, 100 pg/μl or 200 pg/μl of flcn RNA. With increasing concentrations of RNA, there was as significant decrease in the proportion of severe hydrocephalus and corresponding increase in the proportion of normal phenotype (ordinal logistic regression, n = 3 where n is the number of independent injections at each concentration and p = 0.027)