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Figure 2: Changes in neural border and neural crest gene expression. (A, B) Both Six1- 150 and Six1-400 reduce the neural border expression of msx1 on the injected side (*, 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 embryos injected with Six1 WT mRNA; V17E was compared to Six1-150, and the others were compared to Six1-400. Significant differences between mutant and WT frequencies were assessed by the Chi-squared test (*, p<0.05). (E) Six1-150 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) Six1- 400 could either broaden (left) or reduce (right) the foxd3 domain. W122R (H) and Y129C
(I) predominantly broadened the foxd3 domain. (J) Quantitation of foxd3 neural crest (NC) phenotypes, as in (D). (K) Six1-400 broadened the anterior neural plate domain (thin green bar) of zic2, but reduced its neural crest domain (compare to black bars [neural plate, np] and blue bar [neural crest, nc]). (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 (NC) phenotypes, as in (D). (N) Six1-400 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 outline) domains of sox9. (P) Quantitation of sox9 neural crest (NC) phenotypes, as in (D). |
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Figure 3: Changes in PPE and cranial placode gene expression. (A) Six1-150 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) Six1-400 (right) reduced the sox11 PPE domain (between arrows on control [left] side of the same embryo). (D) W122R predominantly broadened the sox11 PPE domain. (E) Six1-400 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 left,
control image) to be broader but fainter (left embryo) or simply broader (red outline and red arrow on right embryo). (G) R110W expanded sox9 expression in the otic placode (between arrows) compared to control (left) side. (H) Quantitation of sox11 cranial placode (PL) phenotypes, as in Fig. 2D. (I) Quantitation of irx1 cranial placode (PL) phenotypes, as in Fig. 2D. (J) Quantitation of sox9 cranial placode (PL) phenotypes, as in Fig. 2D. *, p<0.05. |
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Figure 5: Examples of changes in otic vesicle gene expression. (A) Six1-150 reduced the otic expression of sox9 (red arrow) compared to control side (black arrow) of same embryo. (B) V17E had a similar effect. (C) Six1-400 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). (E) Y129C reduced irx1 otic expression. (F) W122R reduced tbx1 otic expression. (G) Six1-150 reduced dlx5 otic expression. (H) V17E also reduced dlx5 otic expression. (I) Six1-400 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 this Y129C larva, pax2 expression was reduced in the otic vesicle on the injected side (red arrow) compared to the control side (black arrow). (L) The otic vesicle volumes of SixWT, mutant Six1 and control side of the same larva were calculated (Table S1). Because larvae were different sizes, mean experimental volumes were plotted as percent change compared to mean control volumes (+/- s.e.m.) (two-tailed t-test, *, p<0.05). Six1-150 and R110W caused a significant increase in otic vesicle volume compared to the control side of the same embryo,
whereas V17E and Six1-400 caused a significant decrease. Experiments were replicated three times and the number of tadpoles analyzed noted within each bar. |
