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Referee Comments: Referee 1

Posted by PLOS_ONE_Group on 12 May 2008 at 18:23 GMT

Referee 1's review:

N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication, the manuscript has been revised in light of these comments and to address other editorial requirements.

Defect in MyoVIIA is responsible for hearing impairment in both human and mouse. In a previous study, Todi et al. have shown that also in Drosophila, defective MyoVIIA causes hearing impairment due to Johnston's Organ (JO) disorganization. In this study, Todi and colleagues further examined in detail the role of this myosin during developmental stages.

NOMP-A, the only known component of the CAP structure was shown to be targeted to the dendritic caps independently of MyoVIIA (Todi et al. 2005). Flies expressing GFP-tagged NompA are thus analyzed by confocal microscopy to follow the formation of the dendritic cap in the absence of functional MyoVIIA. Anomalies in GFP-NompA distribution were already detected at 18 hrs APF. Disarrayed dendritic caps were observed both in the developing pupae, and in cultured antennal discs. As previously shown in adult flies (todi et al. 2005), no effect is observed on the forming cell-cell junctions too.
Also, the authors investigate the MyoVIIA involvement in the maintenance of JO organization during adulthood, using flies with the Gal4-UAS system and a ubiquitously expressed, temperature sensitive tubulin-Gal80ts. In permissive conditions, lower MyoVIIA levels lead to detachment of scolopidia. Recording from flies under these conditions, however, showed no difference, indicating that low levels of ck/MyoVIIA does not affect JO transduction.

Overall, this manuscript reports significant observations about the critical role of MyoVIIA during early development of the Drosophila auditory organ. Although the data do not highlight the mechanisms involved, either during development or in adults, I believe that the present study is suitable for publication in PLOS one.

I have some questions that the authors, perhaps, can answer.

- A detailed expression pattern of MyoVIIA in the developing puppae would help draw a clear picture on its developmental action. How its expression in neurons and/or scolopale cells correlates with the initiation of the observed anomalies? Is it right that loss of MyoVIIA function is embryonic lethal, and that only few Drosophila survive to reach adulthood? This should be mentioned and discussed in the manuscript.

- A previous study had shown that a balance between the activities of myosin II and MyoVIIA is important in regulating wing hair number (Winter et al. Cell 2001). Is it possible to address this issue in the JO? Is there any indication for genetic interactions between ck/MyoVIIA and components of the signal transduction pathway defined in the study of Winter et al. (e.g. Drok, MRLC, Fz/Dsh Polarity Signaling, ...), and how this may affect JO disorganization.

- The authors discussed the role of Drosophila MyoVIIA compared to the mammalian ortholog, myosin VIIa. Several myosin VIIa interacting partners have been identified in human and/or mouse. Some of these may have orthologous proteins in Drosophila. What the authors can tell through comparison between sequences of the myosin VIIa-ligands against the Drosophila genome?

- Page 6 (lines 3-4): «This indicates either that scolopidial apical attachment is not formed in mutants, or that it immediately fails to be maintained»
Could these attachments be visualized by EM? EM analysis in flies at 18-22 hrs APF can thus be used to confirm or not the formation of these attachment.
Would it be possible to use F-actin (and microtubule) disrupting drugs in the culture system to determine the part actin cytoskeleton plays in this process?

- Fig. 2A: if the authors can use higher magnifications of the confocal images (showing 2, 3 cells), that will be easier for the reader to follow changes in NompA distribution pattern.

- Fig. 4: The western blot: please indicate the molecular weights. The MyoVIIA band is huge? It will be more meaningful if the authors could use protein extracts from microdissected JO only, and not from entire fly body (if MyoVIIA is widely distributed in Drosophila, one cannot be sure the reduction in the western blot band refers to MyoVIIA in JO).