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Fig. 5. Dorsal-ventral patterning of the otic vesicle requires proper expression levels of Sobp. (A-I) In situ hybridization for six1 (A-C), dlx5 (D-F) and pax2 (G-I) at larval stages. Images in each box are the control and injected sides of the same embryo, and are representative of the most frequent phenotype for CRISPR (A,D,G) and sobp mRNA (B,E,H). Images for p.R651X mRNA represent the less frequently observed phenotype (C,F,I). Insets show a higher magnification of the otic vesicle. Yellow dotted boxes denote the areas contained in the insets. Sobp knockdown leads to decreased otic expression of dlx5 (D) and pax2 (G), whereas six1 expression is unchanged (A). Increased Sobp causes a decrease in six1 expression (B) and increased expression with a variable domain size of dlx5 (E) and pax2 (H). Less frequently, increased p.R651X Sobp expression caused a decrease in six1 (C) and dlx5 (F) and increased expression of pax2 (I). (J-L) Frequencies of changes in gene expression illustrated in A-I. The number in each bar denotes the sample size. (M) qPCR analysis of whole larval heads after CRISPR (∼25% decrease in sobp mRNA) shows significant decrease in dlx5 (∼34%) and pax2 (∼22%) mRNAs, whereas changes in six1 are not significant. (N) qPCR analysis of whole larval heads injected with sobp mRNA (∼2.5-fold increase) shows a significant increase in pax2 (∼1.4 fold); changes in six1 and dlx5 are not significant. (O) qPCR analysis of whole larval heads injected with p.R651X mRNA (∼10-fold increase) shows no significant changes in six1, dlx5 or pax2. ns, not significant; *P<0.05, **P<0.01. qPCR experiments were repeated at least four independent times. Error bars represent s.d. with symbols depicting individual data points. |
