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Fig. 1. Sobp is expressed with Six1 in the cell nucleus and represses the transcriptional activation of Six1+Eya1 target genes. (A-D) In situ hybridization for sobp (A,C) and six1 (B,D). At neural plate stages (A,B), although sobp expression in the PPE overlaps with that of six1, its expression is more intense in the anterior domain (arrow), whereas six1 expression is more intense in the posterior domain (arrowhead). In transverse sections through the larval otic vesicle (C,D), sobp is expressed with six1 in the ventral-medial wall. sobp expression is more intense at the dorsal pole (arrowhead). D, dorsal; ep, epidermis; L, lateral; M, medial; np, neural plate; ppe, pre-placodal ectoderm; V, ventral. (E-K) Confocal images of HEK293T cells expressing HA-Sobp (green, E-G) and cells co-expressing HA-Sobp (green) and Six1-Flag (red) (H-K). Sobp is localized in the cell nuclei in both the absence and presence of Six1-Flag. Cell nuclei are stained with DAPI (blue, F,G,J,K). Scale bars: 5 μm. (L) HEK293T cells were co-transfected with combinations of HA-Sobp and/or Six1-Flag followed by multiplex fluorescence western blot detection for HA-Sobp (green) and Six1-Flag (red). Six1 was detected after HA-Sobp was immunoprecipitated with anti-HA magnetic beads (IP, left two rows). Right two rows show expression of the constructs prior to immunoprecipitation. (M) Graph depicting the luciferase activity of the pGL3-6xMEF3-luciferase reporter in HEK293T cells transfected with different combinations of constructs expressing Six1, Eya1 and/or Sobp. Data are normalized to Renilla expressed with a constitutive promoter. Luciferase activity is significantly induced (P<0.0001) by Six1+Eya1, whereas Sobp reduces this induction to levels indistinguishable from control (Six1+Eya1 versus Six1+Eya1+Sobp, P<0.0001; control versus Six1+Eya1+Sobp, P=0.2226). Six1 (P=0.9984), Sobp (P=0.9184) and Six1+Sobp (P=0.9184) did not cause any significant changes in luciferase activity compared with control. ns, not significant; *P<0.05, **P<0.01, ****P<0.0001. Experiments were repeated in duplicate at least three independent times. Error bars represent s.d. with circles depicting individual data points. |
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Fig. 4. Sobp is required for proper formation of the embryonic ectodermal domains at neural plate stages. (A) qPCR analysis of whole neural plate embryos injected with MO or after CRISPR shows that Sobp KD caused a significant decrease in the mRNA levels for foxd3 (MO, ∼33%; CRISPR, ∼20%) and six1 (MO, ∼40%; CRISPR, ∼32%) relative to uninjected control embryos, whereas changes in sox2 or krt12.4 were not significant. Levels of sobp mRNA verified reduced transcripts after CRISPR (MO, not significant; CRISPR, ∼50% decrease). (B) qPCR analysis of whole embryos injected with sobp mRNA shows that increasing Sobp (∼50-fold increase) significantly reduced mRNA levels for foxd3 (∼20%) and six1 (∼20%), and increased that of krt12.4 (∼1.3 fold), whereas sox2 levels were not significantly affected. (C) qPCR analysis of whole embryos injected with p.R651X mRNA shows that increased expression (∼80-fold increase) significantly reduced mRNA levels for six1 (∼25%) and increased that of krt12.4 (∼1.3 fold), whereas foxd3 and sox2 levels were not significantly affected. ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. qPCR experiments were repeated at least four independent times. Error bars represent s.d. with symbols depicting individual data points. (D-S) In situ hybridization for sox2 (neural plate, D,H,L,P), foxd3 (neural crest, E,I,M,Q), six1 (PPE, F,J,N,R) and krt12.4 (epidermis, G,K,O,S). Images are representative of the most frequent phenotype, except for krt12.4 in O and S. Knockdown of Sobp on one side in morphants (MO, D-G) or F0 crispants (CRISPR, H-K) reduced the intensity of sox2 expression concomitant with expansion of its domain, indicated by yellow lines (D,H). They also reduced expression of foxd3 (arrowheads, E,I), six1 (arrowheads, F,J) and krt12.4 (indicated by distance from the midline, yellow line, G,K). Increased expression of Sobp (L-O) or the p.R651X variant of sobp (P-S) on one side caused a decrease in the expression of foxd3 (arrowheads, M, Q) and six1 (arrowheads, N,R), whereas sox2 expression was unchanged (L,P). Although krt12.4 expression was unchanged in most embryos, we detected ectopic expression overlapping the lineage tracer (arrowheads, O,S). (T-X) Frequencies of changes in gene expression illustrated in D-S. The number in each bar denotes sample sizes. |
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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. |
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Fig. S1. In situ hybridization for sobp and six1 at larval stages. Whole-mount view of embryos sectioned in Fig. 1C (sobp) and D (six1) showing expression in the otic vesicle (arrowheads) |
