myo7aa−/− neuromastsexposed to L-type voltage-gated calcium channel agonists show a Ctbp2 distribution which more closely resembles that in wild-type larvae. (A-D,K-N) Representative maximum-intensity projection (z-stack top-down image) of MI1 neuromasts from untreated and treated 5 dpf wild-type larvae (A-D) and untreated and treated myo7aa−/− larvae (K-N). Nuclei were labeled with DAPI (blue), staining for Ctbp2 is shown in red. Scale bars: 10 µm. (I) The majority of 5 dpf untreated wild-type ribbon-containing cells show three Ctbp2 puncta, with a statistical significance of wild-type larvae treated with 5 µM (±)-Bay K 8644 showing one, two, three or four puncta and those treated with 125 µM (R)-Baclofen showing two puncta. There was no statistically significant change upon incubation with 250 µM Nefiracetam. (S) The majority of 5 dpf untreated myo7aa−/− ribbon-containing cells have two Ctbp2 puncta. Upon incubation with 5 µM (±)-Bay K 8644, 250 µM Nefiracetam or 125 µM (R)-Baclofen the majority of hair cells have three Ctbp2 puncta, with a statistical significance of three puncta in all treatment groups. (E-H,O-R) Representative maximum intensity projection (z-stack top-down image) of MI1 neuromasts from 5 dpf wild-type larvae untreated and treated (E-H) and myo7aa−/− larvae untreated and treated (O-R). Nuclei were labeled with DAPI (blue), staining for MAGUK is shown in green. Scale bars: 10 µm. (J) There is no statistical significance in the proportion of one, two, three, four, five or six+ MAGUK puncta between untreated wild-type larvae and any of the treatment groups – except the occurrence of four puncta in response to treatment with 5 µM (±)-Bay K 8644. (T) There is no statistical significance in the proportion of one, two, three, four, five or six+ MAGUK puncta between untreated myo7aa−/− and any of the treatment groups. All error bars are 95% confidence intervals. Experiments for which Ctbp2 staining was carried out in wild-type animals were replicated twice for 5 µM (±)-Bay K 8644, three times for 250 µM Nefiracetam and twice for 125 µM (R)-Baclofen; MAGUK-labeling experiments were conducted twice for 5 µM (±)-Bay K 8644, three times for 250 µM Nefiracetam and three times for 125 µM (R)-Baclofen. Experiments for which Ctbp2 staining was carried out in myo7aa−/− mutants were replicated twice for 5 µM (±)-Bay K 8644, twice for 250 µM Nefiracetam and three times for 125 µM (R)-Baclofen; MAGUK-labeling experiments were conducted twice for 5 µM (±)-Bay K 8644, three times for 250 µM Nefiracetam and three times for 125 µM (R)-Baclofen.
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