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Fig. 3
Targeted mutation of stmn4 increased stmn1b expression and caused mild precocious neuronal differentiation in dorsal midbrain.
(a) A partial genomic structure of stmn4 shows the Exon 3 targeting gRNA site and sequence (PAM site in red). The two BfaI sites (arrowheads) and the sites complementary to the forward (F) and the reverse (R) primers (arrows) used for cloning for sequencing and restriction digestion assay are shown. (b) The sequence flanking the deletion site stmn4 mutants (stmn4Δ3-12) are shown. The deleted nucleotides are denoted by dashes. The cut sites of Bfa1 are indicated by arrowheads. (c) Restriction digestion by BfaI. The genomic DNAs from tail fins of wild type (+/+), heterozygous (+/−) or homozygous (−/−) stmn4Δ5 were isolated to be used as templates for PCR using the forward and reverse primers indicated in (a). The amplicons were digested and run in agarose gels and stained. A representative gel image is shown. The sizes in bp for selected DNA marker bands and digested fragments are shown. (d) Two stmn4 mutant alleles (stmn4Δ4 and stmn4Δ5) have a premature stop codons that resulted in truncated Stmn4 proteins as shown lacking all known functional domains. (e) Partial activation of precocious elavl3 expression in dorsal midbrain of F0 Tg(Huc:Kaede) embryos by stmn4 CRISPR/Cas9 compared to that of control embryos (Ctrl). (f) The % of premature elavl3 expression was determined in wildtype (C), stmn4Δ4 (Δ4) and stmn4Δ5 (Δ5) embryos-injected with indicated concentration of stmn4 tMO2. (g) Relative expression of stmn1b, stmn2a and stmn3 were measured in wildtype (C), morphant (M), stmn4Δ4 (Δ4) and stmn4Δ5 (Δ5) embryos at 12 and 18 hpf by qPCR. Data was analyzed by one-way ANOVA and presented as mean +/− s.e.m. *P < 0.05, **P < 0.005, ***P < 0.0005.