Mechanosensory hair cells in the inner ear are susceptible to a wide variety of environmental insults. However, the large amount of variation in hearing and balance problems resulting from environmental or age-related challenges among normal individuals is neither well documented nor well understood. There is even large variance among individuals with the A1555G mutation in the mitochondrial 12S rRNA that increases susceptibility to neomycin toxicity 
. We hypothesize that alterations in unidentified components of the network of cellular pathways involved in cell death and cell survival would confer resistance to ototoxic compounds. The absence of secondary phenotypes in some of our mutants supports the idea that variation affecting drug response can exist without other outward manifestation. Identification of the human orthologs of these genes may provide candidates involved in the variability underlying human hearing and balance disorders.
Our data suggest that hair cell death after neomycin treatment can involve multiple signaling pathways. Several mutations confer only partial protection against neomycin exposure. Although in some cases this might result from mutations that cause only partial loss of function, in the case of sentinel
we suspect that the mutation is a functional null. The mutation introduces a stop codon early in the coding sequence and before the highly conserved regions. In addition, mRNA levels are reduced in sentinel
mutants, suggesting nonsense-mediated decay. Together these observations suggest that gene function is completely lost, while protection against hair cell loss is only partial. Similarly, only partial protection is observed after treatment with maximal doses of PROTO-1 or PROTO-2. The idea that there are several possible responses to aminoglycosides is consistent with our previous observed variations in ultrastructural changes after aminoglycoside exposure 
mutation also genetically distinguishes between aminoglycoside-induced and cisplatin-induced death; mutant animals are resistant to neomycin but still sensitive to cisplatin. Both aminoglycoside and cisplatin exposure have been proposed to result in oxidative stress 
, raising the possibility that ototoxic compounds share similar mechanisms. If such shared mechanisms occur, the sentinel
gene product must act upstream of these events. Treatment with PROTO-1 also offered no protection against cisplatin, suggesting that its cellular target acts specifically during aminoglycoside toxicity.
Inactivation of sentinel and treatment with PROTO-1 similarly alter the response of hair cells to neomycin treatment. Both modulators offer only partial protection against neomycin, offer no protection against cisplatin, and do not affect entry of FM1-43 or labeled aminoglycoside. Together these results suggest they work in common pathways. To test this idea, we performed epistasis experiments treating wildtype and mutant animals. While the effects of sentinel and PROTO-1 are not additive, there is a small but significant increase in protection when combined, suggesting that they may be accessing different cellular pathways to promote cell survival. Understanding similarities and differences among possible pathways will await the identification of the cellular targets of PROTO-1.
The identification of the sentinel
gene highlights one strength of forward genetic screening, as it would be difficult or impossible to choose this gene a priori
as a candidate regulator of mechanosensory hair cell death. No functional information is known about any of the sentinel
orthologs. The only functional domain of note, the C2 domain, has been associated with calcium regulation and interaction with phospholipid membranes in signaling proteins such as protein kinase C or membrane trafficking proteins like Synaptotagmin 
. However, the function of this domain has been demonstrated in only a few of the many proteins that contain it. Intriguingly, the D. melanogaster ortholog CG18631 was identified in a comparative bioinformatics screen as being associated with compartmentalized cilia-bearing organisms suggesting it may have a role in regulation of cilia 
. Other members of this group include molecules related to intraflagellar transport (IFT) proteins and Bardet-Biedl syndrome (BBS)-related proteins associated with auditory function. In addition, the C. elegans K07G5.3 ortholog is enriched in ciliated neurons by SAGE analysis and localizes to ciliated sensory neurons 
. Hair cells of the zebrafish lateral line and inner ear are also ciliated, bearing a microtubule-based kinocilium in addition to the actin-based stereocilia either throughout life (lateral line and vestibular epithelia) or during development (auditory epithelia/cochlea). However, the broad distribution of sentinel
mRNA and lack of hair cell functional defects in mutants suggest that the gene product does not have a role specific to hair cells.
In addition to identifying possible therapeutic approaches, unbiased small molecule screening may reveal new molecular pathways that regulate hair cell death. This approach has been taken previously in a small molecule screen for compounds affecting zebrafish blood development; by identifying several compounds that affected prostaglandin metabolism, PGE2 was revealed as a regulator of haematopoiesis 
. PROTO-1 and PROTO-2 are related benzothiophene carboxamides, suggesting that they may have the same molecular targets. Other benzothiophene carboxamides have previously been identified as HIV inhibitors, having effects on casein kinase, calcineurin and p53 
. Further work will be needed to determine whether any of these pathways modulate hair cell death.
Perhaps the most important contribution here is the suggestion that our screens can serve as templates for other research programs to identify other gene-drug interactions. Individuals respond remarkably differently to environmental exposures and drug treatment in most disease conditions. Efforts to understand population variation have centered on epidemiological and pharmacogenomic approaches 
. However there are only a few cases in which the genes responsible for this phenotypic variability have been identified, such as for VKORC1-warfarin response or PON1-organophosphate toxicity 
. Genetic analysis may provide a systematic method to identify new molecules involved in cellular responses to drugs or disease.