DEV012922 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Targeted disruption of the Ush1c gene. (A) Maps of the targeting construct, Ush1c wild-type and exon 1 knockout alleles. Constitutive and alternatively spliced exons are represented by white and light-gray boxes, respectively. Exon 1 of the Ush1c wild-type allele was replaced by homologous recombination with a construct containing both the neomycin (neo) selection cassette and Ush1c exon 1, flanked by loxP sites. The targeting construct was removed from the recombined allele using Cre recombinase, generating the Ush1c exon 1 knockout allele Ush1c^tm1Ugds (MGI: 3757683), here referred to as Ush1c⁻. (B) RT-PCR analysis of Ush1c expression in wild-type (+/+) and homozygous mutant (−/−) mouse inner ears and brains (n=6). Primers spanning the exon 2-3 junction (ex2-3F) and exon 8 (ex8R) were used, resulting in a 517 bp amplification product from wild-type tissues. Consistent with exon 1 containing the transcription start site for every known harmonin mRNA, we did not detect any Ush1c transcripts in inner ears and brains from Ush1c^−/− mice. The expression of Hgprt was analyzed as a control for primer sequences, see Verpy et al. (Verpy et al., 2000). (C) Cochlear whole-mounts of E18.5 wild-type and Ush1c^−/− mice were stained with phalloidin (red) and with the harmonin-H3 antibody, which recognizes the three classes of harmonin isoforms (green) (Reiners et al., 2003). An intense harmonin-H3 staining was detected at the stereocilia tips of Ush1c^+/+ hair cells, but was completely lost in Ush1c^−/− hair cells. Scale bars: 2 µm.

• Supplemental Figure S2 -

  Fig. S2. Normal localization of key PCP proteins in Ush1 mutant organs of Corti. Cochlear whole-mounts of E18.5 wild-type, Ush1c^−/−, Cdh23^v2J/v2J and Pcdh15^av3J/av3J mice were stained with antibodies to frizzled 3 (Fz3), Vangl2, or scribble 1 (Scrib1; green), and with phalloidin or for β-catenin (red) to reveal the cell apical compartment. To help in locating IHCs and OHCs, one cell of the inner pillar cell row, which lies on the medial side of three OHCs rows and on the lateral side of the IHC row, is marked with an asterisk in each panel. The three PCP proteins are present at similar locations in wild-type and Ush1 mutant hair cells. Fz3 and Vangl2 are detected on the medial side of the hair cell apices (arrowheads), whereas Scrib1 is present along the hair cell basolateral membrane. Scale bars: 5 µm.

• Supplemental Figure S3 -

  Fig. S3. Antibody specificity tests. Cochlear whole-mounts of P5 Myo7a^{4626SB/4626SB}, Ush1c^{dfcr2J/dfcr2J}, Cdh23^v2J/v2J and Pcdh15^av3J/av3J mice were stained with phalloidin (red) and with Myo7a-F1, harmonin-H1b, Cdh23-N1 or Pcdh15-cter antibodies (green). The specificity of each antibody is demonstrated by the absence of detectable signals in hair bundles of mice deficient for the target proteins. Scale bars: 2 µm.