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Figures
- Fig. 1.
BOS/BOR mutations and their transcriptional effects. (A) Amino acid alignment of the N-terminal region of Xenopus laevis Six1, human SIX1 and Drosophila Sine oculis (SO) shows a high level of identity across species; human and frog are 100% identical in this region; differences from fly are in white. The sequence shown begins with the six domain (SD), which contains six α-helices (blue bars), and ends with the homeodomain (HD, black bar). Amino acid substitutions/deletions that have been reported in human BOS/BOR patients are indicated with arrows; red arrows indicate the four mutations that were examined in this study. (B) Expression of Six1+Eya1 caused a significant ∼7-fold increase in luciferase activity when compared to activity of control vector (P<0.0001), Six1WT alone (P<0.0001) or Eya1 alone (P<0.0001). Each mutant plus Eya1 failed to significantly induce luciferase activity relative to control (V17E, P=0.27122; R110W, P=0.99999; W122R, P=0.99764; Y129C, P=0.99947) or in the absence of Eya1 (V17E, P=0.99988; R110W, P=0.99999; W122R, P=0.99999; Y129C, P=0.99999). Experiments were repeated five independent times and subjected to a one-way ANOVA with Tukey post hoc multiple comparisons test. Bars=mean±s.d. (C,I,O). HEK293T cells transfected with only Myc-Eya1 show both cytoplasmic (arrowheads) and nuclear localization. (D,J,P) Cells co-transfected with both Six1WT-FLAG and Myc-Eya1 show nuclear colocalization of both proteins. (E,K,Q) Those transfected with both V17E-FLAG and Myc-Eya1 showed nuclear colocalization and some cytoplasmic Eya1 (arrowheads). Those transfected with (F,L,R) R110W-FLAG and Myc-Eya1, (G,M,S) W122R-FLAG and Myc-Eya1, or (H,N,T) Y129C-FLAG and Myc-Eya1 showed exclusive nuclear colocalization. Scale bars: 10 µm.
- Fig. 2.
Changes in neural border and neural crest gene expression. (A,B) Both Six1WT-150 and Six1WT-400 reduce the neural border expression of msx1 on the injected side (indicated by asterisks, pink lineage tracer). (C) R110W either did not change the msx1 domain (left embryo) or caused it to be broader (right embryo, red bar) compared to the control side (black bar). (D) The expression domain size of msx1 on the Six1-mutant-injected side was compared to the control side of the same embryo and scored as reduced (blue), broader (red), broader but fainter (green) or unchanged (yellow). Phenotypes are expressed as frequencies and the sample size is within each bar (white numbers); experiments were repeated a minimum of three times. Frequencies for Six1 mutants were compared to those for embryos injected with Six1 WT mRNA; V17E was compared to Six1WT-150, and the others were compared to Six1WT-400. Significant differences between mutant and WT frequencies were assessed by the Chi-squared test (*P<0.05). (E) Six1WT-150 could either broaden (left embryo) or reduce (right embryo) the foxd3 domain. (F) V17E could either broaden (left) or reduce (right) the foxd3 domain. (G) Six1WT-400 could either broaden (left) or reduce (right) the foxd3 domain. (H,I) W122R (H) and Y129C (I) predominantly broadened the foxd3 domain. (J) Quantitation of foxd3 neural crest (NC) phenotypes, as in D. (K) Six1WT-400 broadened the anterior neural plate (np) domain (green bar) of zic2, but reduced its neural crest (nc) domain (compare to black bars and blue bar). (L) W122R caused both the anterior neural plate domain (green bar) and neural crest domain (red bar) of zic2 to broaden (compare to black and blue bars on control side). (M) Quantitation of zic2 neural crest phenotypes, as in D. (N) Six1WT-400 reduced both the neural crest and otic placode (oto) domains of sox9. (O) Y129C broadened both the neural crest (red bar) and otic placode (green dashed lines) domains of sox9. (P) Quantitation of sox9 neural crest phenotypes, as in D. Scale bars: 300 μm.
- Fig. 3.
Changes in PPE and cranial placode gene expression. (A) Six1WT-150 expanded the sox11 PPE domain [between arrows on control (left) side of the same embryo]. (B) V17E predominantly reduced the sox11 PPE domain [between arrows on control (left) side of the same embryo]. (C) Six1WT-400 (right) reduced the sox11 PPE domain [between arrows on control (left) side of the same embryo]. (D) W122R predominantly broadened the sox11 PPE domain [between arrows on control (left) side of the same embryo]. (E) Six1WT-400 reduced the irx1 placode domain [between arrows on control (left) side of the same embryo]. (F) W122R either caused irx1 PPE domain (between arrows in leftmost, control image) to be broader but fainter (left embryo) or simply broader (red dashed line and red arrow in right embryo, compared to black dashed line and black arrow on control side). (G) R110W expanded sox9 expression in the otic placode (between arrows) compared to control (left) side. Asterisks in A-G indicate the injected side. Scale bars: 300 µm. (H-J) Quantitation of sox11 (H), irx1 (I) and sox9 (J) cranial placode (PL) phenotypes, as in Fig. 2D. *P<0.05.
- Fig. 4.
Frequencies of otic vesicle gene expression changes. (A) sox9. (B) irx1. (C) tbx1. (D) dlx5. (E) otx2. (F) pax2. Quantitation as described in Fig. 2D. *P<0.05.
- Fig. 5.
Examples of changes in otic vesicle gene expression. (A) Six1WT-150 reduced the otic expression of sox9 (red arrow) compared to the control side (black arrow) of same embryo. (B) V17E had a similar effect. (C) Six1WT-400 reduced sox9 otic expression. (D) W122R caused slightly darker otic expression of sox9 and what appeared to be a slightly larger otic vesicle (red bar) compared to the control side (black bar). (E) Y129C reduced irx1 otic expression. (F) W122R reduced tbx1 otic expression. (G) Six1WT-150 reduced dlx5 otic expression. (H) V17E also reduced dlx5 otic expression. (I) Six1WT-400 reduced the ventral otic expression of otx2. (J) R110W did the same. See Fig. 4 for frequencies. (K) Some larvae were sectioned to measure otic vesicle volume. In the shown Y129C larva, pax2 expression was reduced in the otic vesicle on the injected side (red arrow) compared to the control side (black arrow). hb, hind brain. (L) The otic vesicle volumes of SixWT, mutant Six1 and the control side of the same larva were calculated (Table S1). Because larvae were different sizes, mean experimental volumes were plotted as percentage change compared to mean control volumes (±s.e.m.) (two-tailed Student's t-test, *P<0.05). Six1WT-150 and R110W caused a significant increase in otic vesicle volume compared to the control side of the same embryo, whereas V17E and Six1WT-400 caused a significant decrease. Experiments were replicated three times and the number of tadpoles analyzed noted within each bar. Scale bars: 300 μm (A-J), 50 μm (K).
- Fig. 6.
Mutant Six1 proteins affect otic capsule and otolith volumes. (A) Alcian Blue-stained tadpole head in which the right side expressed V17E. The otic capsule (oto) on the injected side (red arrow) is not notably different from that on the control side (black arrow) in this individual. e, eye; g, gill cartilages. Similar results were seen for R110W and W122R. (B) Alcian Blue-stained tadpole head in which the right side expressed Y129C. The otic capsule on the injected side (red arrow) is much smaller than that on the control side (left). b, bubble in the mounting medium. (C) Vibratome section reveal the cartilaginous otic capsules on control (ctrl, black arrow) and injected (inj, red arrow) sides of a V17E tadpole (top) and W122R tadpole (bottom). hb, hind brain; n, notochord. (D) The otic cartilage volumes of mutant Six1 and control sides of the same tadpole were calculated (Table S2) and compared by a paired, two-tailed Student's t-test. Because tadpoles were different sizes, the mean experimental volumes were plotted as percentage change compared to mean control volumes (±s.e.m.). V17E, W122R and Y129C caused significant decreases in otic cartilage volume (*P<0.05). (E) Three-dimensional reconstruction of transverse sections collected using OCT. Otic vesicle is in gray with otoliths in white from frontal (leftmost) and dorsal (middle left) views. Four transverse sections (1-4) taken at the levels indicated on the dorsal view reveal internal structures: A, anterior canal; H, horizontal canal; O, otolith; P, posterior canal; S, saccule; U, utricle. (F) Otolith volumes of SixWT, mutant Six1 and control sides of the same tadpole were calculated from OCT images (Table S3) and compared by a paired, one-tailed Student's t-test. As in D, the mean experimental volumes were plotted as percentage change compared to mean control volumes (±s.e.m.). Six1WT-150, V17E and Y129C resulted in significantly smaller otolith volumes (*P<0.05). Experiments were replicated three times and the number of tadpoles analyzed noted within each bar in D and F. Scale bars: 100 μm (A,B), 70 μm (C,E).
- Fig. 7.
Mutant Six1 proteins affect inner ear luminal volumes. (A) Single confocal optical section through a phalloidin-stained tadpole inner ear showing the lumen and a single sensory patch containing hair cells (red arrow). (B) Image of same section showing outline of lumen in IMARIS software. (C) Dorsal view of a 3D reconstruction of the same inner ear, showing anterior (A), posterior (P) and horizontal (H) semicircular canals. (D) Ventral view of a 3D reconstruction of an inner ear, highlighting the different sensory end organs: A, anterior canal; H, horizontal canal; P, posterior canal; S, saccule; U, utricle. In this specimen, the amphibian papilla (am) also has differentiated, but since this was not a consistent feature at this developmental stage, it was not included in the volume measurements. (E) The inner ear luminal volumes of SixWT, mutant Six1 and control sides of the same tadpole were calculated from OCT images (Table S4) and compared by a paired, one-tailed Student's t-test. As in Fig. 6D, the mean experimental volumes were plotted as percentage change compared to mean control volumes (±s.e.m.). Significantly smaller volumes were detected for V17E and Y129C (*P<0.05). (F) The inner ear luminal volumes of SixWT, mutant Six1 and control sides of the same tadpole were calculated from confocal images (Table S5) and compared by a paired, one-tailed Student's t-test. As in Fig. 6D, the mean experimental volumes were plotted as percentage change compared to mean control volumes (±s.e.m.). Six1WT-400 caused a significant increase in luminal volume, whereas V17E, R110W and Y129C caused significantly smaller volumes (*P<0.05). Experiments were replicated three times and the number of tadpoles analyzed noted within each bar in E and F. Scale bars: 25 μm.
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