Mutations in SIX1 Associated with Branchio-oto-Renal Syndrome (BOR) Differentially Affect Otic Expression of Putative Target Genes

Abstract

Several single-nucleotide mutations in SIX1 underlie branchio-otic/branchio-oto-renal (BOR) syndrome, but the clinical literature has not been able to correlate different variants with specific phenotypes. We previously assessed whether variants in either the cofactor binding domain (V17E, R110W) or the DNA binding domain (W122R, Y129C) might differentially affect early embryonic gene expression, and found that each variant had a different combination of effects on neural crest and placode gene expression. Since the otic vesicle gives rise to the inner ear, which is consistently affected in BOR, herein we focused on whether the variants differentially affected the otic expression of genes previously found to be likely Six1 targets. We found that V17E, which does not bind Eya cofactors, was as effective as wild-type Six1 in reducing most otic target genes, whereas R110W, W122R and Y129C, which bind Eya, were significantly less effective. Notably, V17E reduced the otic expression of prdm1, whereas R110W, W122R and Y129C expanded it. Since each mutant has defective transcriptional activity but differs in their ability to interact with Eya cofactors, we propose that altered cofactor interactions at the mutated sites differentially interfere with their ability to drive otic gene expression, and these differences may contribute to patient phenotype variability.

Keywords: Xenopus; cranial placodes; neural crest; transcription.

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).