Mehdizadeh T , Majumdar HD , Ahsan S , Tavares ALP , Moody SA .

DE026434 NIDCR NIH HHS, DE022065 NIDCR NIH HHS, R01 DE026434 NIDCR NIH HHS

Xla Wt + six1 MO (Fig. 1A)
Xla Wt + six1 MO (Fig. 1B)
Xla Wt + six1 MO (Fig. 1C)
Xla Wt + six1 MO (Fig. 1D)
Xla Wt + six1 MO (Fig. 1E)
Xla Wt + six1 MO (Fig. 1F)
Xla Wt + six1-150 (Fig. 2EG)
Xla Wt + six1-150 (Fig. 2KM)
Xla Wt + six1-150 (Fig. 2NP)
Xla Wt + six1-150 (Fig. 3A (red))
Xla Wt + six1-400 (Fig 2B)
Xla Wt + six1-400 (Fig. 2HJ)
Xla Wt + six1-400 (Fig. 3B (red))
Xla Wt + six1-EnR (Fig. 2IJ)
Xla Wt + six1-EnR (Fig. 2OP)
Xla Wt + six1-R110W (Fig. 3B (black))
Xla Wt + six1-V17E (Fig. 3A (black))
Xla Wt + six1-VP16 (Fig 2CD)
Xla Wt + six1-VP16 (Fig. 2FG)
Xla Wt + six1-VP16 (Fig. 2LM)
Xla Wt + six1-W122R (Fig. 3B (pink))

	Figure 1. Six1 is required for otic gene expression. Top: Six1 is predicted to act upstream of the investigated genes. If correct, then the expression domain and/or intensity should be reduced (red arrow) when Six1 is knocked down. Those genes with an asterisk were shown to have Six1 bound to an enhancer in mouse E10.5 otic vesicles [42]. We found that the otic placode ((A), left image) or otic vesicle ((A), right image, (B–F)) expression of eya2 (A), prdm1 (B), spry1 (C), tspan13 (D), zbtb16 (E) and pa2g4 (F) was reduced on the MO-mediated knock-down side of each embryo (red arrow) compared to the control side (black arrow) of the same embryo. A, left image and B are frontal views; A, right image and C-F are side views. Dorsal is to the top.
	Figure 2. Increased Six1 alters otic gene expression. Transcripts encoding wild-type Six1 (Six1WT, either 150 or 400 pg), activating Six1 (VP16, 100 pg) or repressive Six1 (EnR, 100 pg) were microinjected into blastomeres that contribute to otic structures on one side of embryos containing endogenous levels of Six1. (A) The effects on otic vesicle gene expression (blue) were compared between embryos injected with Six1WT mRNA (+Six1WT) versus those injected with either Six1VP16 (+Six1VP16) or Six1EnR (+Six1EnR) mRNAs. If Six1 acts as a repressor (top row), then additional Six1WT should reduce gene expression, Six1VP16 should either cause no change or increase it, and Six1EnR should also reduce it. If Six1 acts as an activator (bottom row), then additional Six1WT should increase gene expression, Six1VP16 should also increase it, and Six1EnR should cause either no change or reduce it. (B–P) Gene expression was assayed by ISH for eya2 (B,C), prdm1 (E,F), spry1 (H,I), tspan13 (K,L) and pa2g4 (N,O). The control, uninjected side of each embryo is on the left and the mRNA-injected side of the same embryo is on the right. Otic gene expression on the control side is indicated by black arrows, and that on the mRNA-injected side by red arrows. In C and F, the width of the otic vesicle is indicated by a black (control) or red (injected) bar. Frequencies of the effects (blue, decreased expression; yellow, increased expression; grey, no change) are indicated by bar graphs (D,G,J,M,P). For eya2 (D) and prdm1 (G), the frequencies for Six1VP16 were significantly different from both Six1WT-150 and SixWT-400 (*, p < 0.0001). For tspan13 (M), the frequencies for Six1VP16 were significantly different from Six1WT-150 (*, p < 0.05), but not from SixWT-400. For pa2g4 (P), the frequencies for Six1VP16 were significantly different from SixWT-400 (*, p < 0.0001), and the frequencies for Six1EnR were significantly different from SixWT-150 (*, p < 0.01). White numbers inside each bar denotes the number of embryos analyzed.
	Figure 3. Gene expression changes in whole head samples assessed by qPCR. (A). Levels of expression in whole heads collected from uninjected, sibling-matched embryos (controls, blue bars), and embryos injected with mRNA encoding either Six1WT (red bars) or V17E (black bars). Significant differences from control (p < 0.05) are indicated by an asterisk; significant differences from Six1WT (p < 0.05) are indicated by #. (B). Levels of expression in whole heads collected from uninjected, sibling-matched embryos (controls, blue bars), and embryos injected with mRNA encoding either Six1WT (red bars), R110W (black bars), W122R (purple bars) or Y129C (orange bars). Significant differences from control (p < 0.05) are indicated by an asterisk. Significant differences from Six1WT (p < 0.05) are indicated by #. Data are from three independent samples run in duplicate.
	Figure 4. Otic gene expression is altered by Six1 mutants as assessed by ISH. (A): Transcripts encoding wild-type Six1 (Six1WT) or BOR variants were microinjected into blastomeres that contribute to otic structures on one side of embryos containing endogenous levels of Six1. The effects on otic vesicle gene expression (blue) were compared between embryos injected with Six1WT (+Six1WT) versus those injected with a BOR variant (+BOR variant). Since Six1WT reduced each otic target gene, if the BOR variant also reduced it at the same frequency, then the variant has the same activity as SixWT. If the BOR variant caused no change in expression, then it has lost activity. If the BOR variant increased expression, then it has the opposite activity of SixWT. (B). Percentages of embryos in which eya2 otic expression was decreased (blue), increased (yellow) or not changed (grey) after microinjection of 150 pg of Six1WT, V17E, R110W, W122R or Y129C mRNAs. Numbers inside bars denote number of embryos analyzed. Lower line and asterisks indicate comparison of Six1WT frequencies to those of each mutant. Upper line and asterisks indicate comparison of V17E frequencies to those of the other three mutants. * p < 0.01; **, p < 0.001; ***, p < 0.0001 comparison by chi-square test. (C). Percentages of embryos in which eya2 otic expression was altered after microinjection of 400 pg of Six1WT, R110W, W122R or Y129C mRNAs. There were no significant differences between the different groups. Labeling as in (B). (D). Percentages of embryos in which prdm1 otic expression was altered after microinjection of 150 pg of Six1WT, V17E, R110W, W122R or Y129C mRNAs. Labeling as in (B). (E). Percentages of embryos in which prdm1 otic expression was altered after microinjection of 400 pg of Six1WT, R110W, W122R or Y129C mRNAs. Labeling as in (B). (F). Percentages of embryos in which spry1 otic expression was altered after microinjection of 150 pg of Six1WT, V17E, R110W, W122R or Y129C mRNAs. Labeling as in (B). (G). Percentages of embryos in which spry1 otic expression was altered after microinjection of 400 pg of Six1WT, R110W, W122R or Y129C mRNAs. Labeling as in (B). (H). Percentages of embryos in which tspan13 otic expression was altered after microinjection of 150 pg of Six1WT, V17E, R110W, W122R or Y129C mRNAs. Labeling as in (B). (I). Percentages of embryos in which tspan13 otic expression was altered after microinjection of 400 pg of Six1WT, R110W, W122R or Y129C mRNAs. There were no significant differences between the different groups. Labeling as in (B). (J). Percentages of embryos in which zbtb16 otic expression was altered after microinjection of 150 pg of Six1WT, V17E, R110W, W122R or Y129C mRNAs. Labeling as in (B). (K). Percentages of embryos in which zbtb16 otic expression was altered after microinjection of 400 pg of Six1WT, R110W, W122R or Y129C mRNAs. Labeling as in (B). (L). Percentages of embryos in which pa2g4 otic expression was altered after microinjection of 150 pg of Six1WT, V17E, R110W, W122R or Y129C mRNAs. Labeling as in (B). (M). Percentages of embryos in which pa2g4 otic expression was altered after microinjection of 400 pg of Six1WT, R110W, W122R or Y129C mRNAs. Labeling as in (B).
	Graphical abstract

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