Myosin VIIA, important for human auditory function, is necessary for Drosophila auditory organ development

Background

Myosin VIIA (MyoVIIA) is an unconventional myosin necessary for vertebrate audition [1]–[5]. Human auditory transduction occurs in sensory hair cells with a staircase-like arrangement of apical protrusions called stereocilia. In these hair cells, MyoVIIA maintains stereocilia organization [6]. Severe mutations in the Drosophila MyoVIIA orthologue, crinkled (ck), are semi-lethal [7] and lead to deafness by disrupting antennal auditory organ (Johnston''s Organ, JO) organization [8]. ck/MyoVIIA mutations result in apical detachment of auditory transduction units (scolopidia) from the cuticle that transmits antennal vibrations as mechanical stimuli to JO.

Principal Findings

Using flies expressing GFP-tagged NompA, a protein required for auditory organ organization in Drosophila, we examined the role of ck/MyoVIIA in JO development and maintenance through confocal microscopy and extracellular electrophysiology. Here we show that ck/MyoVIIA is necessary early in the developing antenna for initial apical attachment of the scolopidia to the articulating joint. ck/MyoVIIA is also necessary to maintain scolopidial attachment throughout adulthood. Moreover, in the adult JO, ck/MyoVIIA genetically interacts with the non-muscle myosin II (through its regulatory light chain protein and the myosin binding subunit of myosin II phosphatase). Such genetic interactions have not previously been observed in scolopidia. These factors are therefore candidates for modulating MyoVIIA activity in vertebrates.

Conclusions

Our findings indicate that MyoVIIA plays evolutionarily conserved roles in auditory organ development and maintenance in invertebrates and vertebrates, enhancing our understanding of auditory organ development and function, as well as providing significant clues for future research.     

Molecular evolutionary analysis of seminal receptacle sperm storage organ genes of Drosophila melanogaster

Sperm storage organs are common and broadly distributed among animal taxa. However, little is known about how these organs function at the molecular level. Additionally, there is a paucity of knowledge about the evolution of genes expressed in these organs. This investigation is an evolutionary expressed sequence tag (EST) study of genes expressed in the seminal receptacle, one of the sperm storage organs in Drosophila. The incidence of positive selection is higher for the seminal receptacle genes than Drosophila reproductive genes as a whole, but lower than genes associated with the spermatheca, a second type of Drosophila sperm storage organ. By identifying overrepresented classes of proteins and classes for which sperm storage function is suggested by the nature of the proteins, candidate genes were discovered. These candidates belong to protein classes such as muscle contraction, odorant binding and odorant receptor, protease inhibitor and immunity.     

Molecular evolution and population genetic analysis of candidate female reproductive genes in Drosophila

Molecular analyses in several taxa have consistently shown that genes involved in reproduction are rapidly evolving and subjected to positive selection. The mechanism behind this evolution is not clear, but several proposed hypotheses involve the coevolution between males and females. In Drosophila, several male reproductive proteins (Acps) involved in male-male and male-female interactions show evidence of rapid adaptive evolution. What has been missing from the Drosophila literature is the identification and analysis of female reproductive genes. Recently, an evolutionary expressed sequence tag analysis of Drosophila female reproductive tract genes identified 169 candidate female reproductive genes. Many of these candidate genes still await further molecular analysis and independent verification of positive selection. Our goal was to expand our understanding of the molecular evolution of Drosophila female reproductive genes with a detailed polymorphism and divergence study on seven additional candidate female reproductive genes and a reanalysis of two genes from the above study. We demonstrate that 6 candidate female genes of the 9 genes surveyed show evidence of positive selection using both polymorphism and divergence data. One of these proteins (CG17012) is modeled to reveal that the sites under selection fall around and within the active site of this protease, suggesting potential differences between species. We discuss our results in light of potential function as well as interaction with male reproductive proteins.     

Quantitative genetic analysis of among-population variation in sperm and female sperm-storage organ length in Drosophila mojavensis

In Drosophila, sperm length and the length of the females' primary sperm-storage organ have rapidly coevolved through post-copulatory sexual selection. This pattern is evident even among geographic populations of Drosophila mojavensis. To understand better these traits of potential importance for speciation, we performed quantitative genetic analysis of both seminal receptacle length and sperm length in two divergent populations. Parental strains, F1, F1 reciprocal (F1r), F2, F2r, backcross and backcross reciprocal generations were used in a line-cross (generation means) analysis. Seminal receptacle length is largely an autosomal additive trait, whereas additivity, dominance and epistasis all contributed to the means of sperm length. Either an X-chromosome or a Y-chromosome effect was necessary for models of sperm length to be significant. However, the overall contributions from the X and Y chromosomes to sperm length was small.     

A clonal genetic screen for mutants causing defects in larval tracheal morphogenesis in Drosophila

The initial establishment of the tracheal network in the Drosophila embryo is beginning to be understood in great detail, both in its genetic control cascades and in its cell biological events. By contrast, the vast expansion of the system during larval growth, with its extensive ramification of preexisting tracheal branches, has been analyzed less well. The mutant phenotypes of many genes involved in this process are probably not easy to reveal, as these genes may be required for other functions at earlier developmental stages. We therefore conducted a screen for defects in individual clonal homozygous mutant cells in the tracheal network of heterozygous larvae using the mosaic analysis with a repressible cell marker (MARCM) system to generate marked, recombinant mitotic clones. We describe the identification of a set of mutants with distinct phenotypic effects. In particular we found a range of defects in terminal cells, including failure in lumen formation and reduced or extensive branching. Other mutations affect cell growth, cell shape, and cell migration.     

Neuroprosthetic hearing with auditory brainstem implants.

The auditory brainstem implant (ABI) does provide auditory sensations, recognition of environmental sounds and aid in spoken communication in about 300 patients worldwide. It is no more an investigative device but widely accepted for the treatment of patients who have lost hearing due to bilateral tumors of the hearing nerve who transmits the acoustic information from the cochlea to the brain. Most of the implanted patients are completely deaf when the implant is switched off. In contrast to cochlear implants, only few of the implanted patients achieve open-set speech recognition without the help of visual cues. On average, the ABI improves communicative functions like speech recognition at about 30% when compared to lip-reading only. The task for the next years is to improve the outcome of ABI further by developing new less invasive operative approaches as well as new hardware and software for the ABI device.     

Beaked whale auditory evoked potential hearing measurements

Several mass strandings of beaked whales have recently been correlated with military exercises involving mid-frequency sonar highlighting unknowns regarding hearing sensitivity in these species. We report the hearing abilities of a stranded juvenile beaked whale (Mesoplodon europaeus) measured with auditory evoked potentials. The beaked whale’s modulation rate transfer function (MRTF) measured with a 40-kHz carrier showed responses up to an 1,800 Hz amplitude modulation (AM) rate. The MRTF was strongest at the 1,000 and 1,200 Hz AM rates. The envelope following response (EFR) input–output functions were non-linear. The beaked whale was most sensitive to high frequency signals between 40 and 80 kHz, but produced smaller evoked potentials to 5 kHz, the lowest frequency tested. The beaked whale hearing range and sensitivity are similar to other odontocetes that have been measured.     

Developmental and genetic mosaic analysis of Drosophila m-dy mutants: Tissue foci for behavioral and morphogenetic defects

Mutants of the Drosophila miniature-dusky (m-dy) gene complex display morphogenetic phenotypes (miniature or dusky) caused by a change in the size and/or shape of the epidermal cells comprising the adult wing. In addition to a dusky phenotype, certain Andante-type mutants also exhibit lengthened circadian periods for two different behavioral rhythms. If the latter phenotype results from a direct effect on the circadian pacemaker, the Andante function should be required within the brain. In order to define the tissues that require the morphogenetic and behavioral functions, we have carried out a genetic mosaic analysis. This study demonstrates that normal wing morphogenesis is entirely dependent on the genotype of wing cells. Furthermore, temperature-shift experiments with a temperature-sensitive dy mutant indicate that the morphogenetic function is required during adult development, and after the cessation of wing epidermal cell proliferation. At this time in development, a columnar epithelium in the developing wing becomes flattened into the mature wing blade, and we postulate that the cell-size defect of m-dy mutants results from an alteration of this mor-phogenetic process. In contrast to the wing mor-phogenesis phenotype, the characterization of locomotor activity in mosaic adults revealed a strong correlation between the head genotype and the Andante circadian-period phenotype. This result indicates that neural tissues mediate the rhythm function. Thus, the behavioral and morphogenetic functions require gene expression in distinct tissues. Furthermore, the behavioral results are consistent with a requirement for Andante function within circadian pacemaker neurons. © 1995 Wiley-Liss, Inc.     

An efficient genetic screen in Drosophila to identify nuclear-encoded genes with mitochondrial function

We conducted a screen for glossy-eye flies that fail to incorporate BrdU in the third larval instar eye disc but exhibit normal neuronal differentiation and isolated 23 complementation groups of mutants. These same phenotypes were previously seen in mutants for cytochrome c oxidase subunit Va. We have molecularly characterized six complementation groups and, surprisingly, each encodes a mitochondrial protein. Therefore, we believe our screen to be an efficient method for identifying genes with mitochondrial function.     

Sound-evoked radial strain in the hearing organ

The hearing organ contains sensory hair cells, which convert sound-evoked vibration into action potentials in the auditory nerve. This process is greatly enhanced by molecular motors that reside within the outer hair cells, but the performance also depends on passive mechanical properties, such as the stiffness, mass, and friction of the structures within the organ of Corti. We used resampled confocal imaging to study the mechanical properties of the low-frequency regions of the cochlea. The data allowed us to estimate an important mechanical parameter, the radial strain, which was found to be 0.1% near the inner hair cells and 0.3% near the third row of outer hair cells during moderate-level sound stimulation. The strain was caused by differences in the motion trajectories of inner and outer hair cells. Motion perpendicular to the reticular lamina was greater at the outer hair cells, but inner hair cells showed greater radial vibration. These differences led to deformation of the reticular lamina, which connects the apex of the outer and inner hair cells. These results are important for understanding how the molecular motors of the outer hair cells can so profoundly affect auditory sensitivity.     

A mosaic genetic screen for genes necessary for Drosophila mushroom body neuronal morphogenesis

Neurons undergo extensive morphogenesis during development. To systematically identify genes important for different aspects of neuronal morphogenesis, we performed a genetic screen using the MARCM system in the mushroom body (MB) neurons of the Drosophila brain. Mutations on the right arm of chromosome 2 (which contains approximately 20% of the Drosophila genome) were made homozygous in a small subset of uniquely labeled MB neurons. Independently mutagenized chromosomes (4600) were screened, yielding defects in neuroblast proliferation, cell size, membrane trafficking, and axon and dendrite morphogenesis. We report mutations that affect these different aspects of morphogenesis and phenotypically characterize a subset. We found that roadblock, which encodes a dynein light chain, exhibits reduced cell number in neuroblast clones, reduced dendritic complexity and defective axonal transport. These phenotypes are nearly identical to mutations in dynein heavy chain Dhc64 and in Lis1, the Drosophila homolog of human lissencephaly 1, reinforcing the role of the dynein complex in cell proliferation, dendritic morphogenesis and axonal transport. Phenotypic analysis of short stop/kakapo, which encodes a large cytoskeletal linker protein, reveals a novel function in regulating microtubule polarity in neurons. MB neurons mutant for flamingo, which encodes a seven transmembrane cadherin, extend processes beyond their wild-type dendritic territories. Overexpression of Flamingo results in axon retraction. Our results suggest that most genes involved in neuronal morphogenesis play multiple roles in different aspects of neural development, rather than performing a dedicated function limited to a specific process.     

Structural and genetic studies of the proliferation disrupter genes of Drosophila simulans and D. melanogaster

To examine whether structural and functional differences exist in the proliferation disrupter (prod) genes between Drosophila simulans and D. melanogaster, we analyzed and compared both genes. The exon–intron structure of the genes was found to be the same – three exons were interrupted by two introns, although a previous report suggested that only one intron existed in D. melanogaster. The prod genes of D. simulans and D. melanogaster both turn out to encode 346 amino acids, not 301 as previously reported for D. melanogaster. The numbers of nucleotide substitutions in the prod genes was 0.0747 ±  per synonymous site and 0.0116 ± 0.0039 per replacement site, both comparable to those previously known for homologous genes between D. simulans and D. melanogaster. Genetic analysis demonstrated that D. simulans PROD can compensate for a deficiency of D. melanogaster PROD in hybrids. The PRODs of D. simulans and D. melanogaster presumably share the same function and a conserved working mechanism. The prod gene showed no significant interaction with the lethality of the male hybrid between these species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of Johnston's organ in Drosophila

Hearing is a specialized mechanosensory modality that is refined during evolution to meet the particular requirements of different organisms. In the fruitfly, Drosophila, hearing is mediated by Johnston's organ, a large chordotonal organ in the antenna that is exquisitely sensitive to the near-field acoustic signal of courtship songs generated by male wing vibration. We summarize recent progress in understanding the molecular genetic determinants of Johnston's organ development and discuss surprising differences from other chordotonal organs that likely facilitate hearing. We outline novel discoveries of active processes that generate motion of the antenna for acute sensitivity to the stimulus. Finally, we discuss further research directions that would probe remaining questions in understanding Johnston's organ development, function and evolution.     

A gain-of-function screen for genes that affect the development of the Drosophila adult external sensory organ

The Drosophila adult external sensory organ, comprising a neuron and its support cells, is derived from a single precursor cell via several asymmetric cell divisions. To identify molecules involved in sensory organ development, we conducted a tissue-specific gain-of-function screen. We screened 2293 independent P-element lines established by P. Rorth and identified 105 lines, carrying insertions at 78 distinct loci, that produced misexpression phenotypes with changes in number, fate, or morphology of cells of the adult external sensory organ. On the basis of the gain-of-function phenotypes of both internal and external support cells, we subdivided the candidate lines into three classes. The first class (52 lines, 40 loci) exhibits partial or complete loss of adult external sensory organs. The second class (38 lines, 28 loci) is associated with increased numbers of entire adult external sensory organs or subsets of sensory organ cells. The third class (15 lines, 10 loci) results in potential cell fate transformations. Genetic and molecular characterization of these candidate lines reveals that some loci identified in this screen correspond to genes known to function in the formation of the peripheral nervous system, such as big brain, extra macrochaetae, and numb. Also emerging from the screen are a large group of previously uncharacterized genes and several known genes that have not yet been implicated in the development of the peripheral nervous system.     

Incorporating genetic analyses into birth defects cluster investigations: strategies for identifying candidate genes

BACKGROUND: Incorporating genetic analyses into birth defect cluster investigations may increase understanding of both genetic and environmental risk factors for the defect. Current constraints of most birth defect cluster investigations make candidate gene selection the most feasible approach. Here, we describe strategies for choosing candidate genes for such investigations, which will also be applicable to more general gene-environment studies. METHODS: We reviewed publicly available web-based resources for selection of candidate genes and identification of risk factors, as well as publications on different strategies for candidate gene selection. RESULTS: Candidate gene selection requires consideration of available gene-disease databases, previous epidemiological studies, animal model research, linkage and expression studies, and other resources. We describe general considerations for utilizing available resources, as well as provide an example of a search for candidate genes related to gastroschisis. CONCLUSIONS: Available web resources could facilitate selection of candidate genes, but selection of optimal candidates will still require a strong understanding of genetics and the pathogenesis of the defect, as well as careful consideration of previous epidemiological studies.     

A mosaic genetic screen for Drosophila neoplastic tumor suppressor genes based on defective pupation

The Drosophila neoplastic tumor suppressor genes (TSGs) coordinately control cell polarity and proliferation in epithelial and neuronal tissues. While a small group of neoplastic TSG mutations have been isolated and their corresponding genes cloned, the regulatory pathways that normally prevent inappropriate growth remain unclear. Identification of additional neoplastic TSGs may provide insight into this question. We report here the design of an efficient screen for isolating neoplastic TSG mutations utilizing genetically mosaic larvae. This screen is based on a defective pupation phenotype seen when a single pair of imaginal discs is homozygous for a neoplastic TSG mutation, which suggests that continuously proliferating cells can interfere with metamorphosis. Execution of this screen on two chromosome arms led to the identification of mutations in at least seven new neoplastic TSGs. The isolation of additional loci that affect hyperplastic as well as neoplastic growth indicates the utility of this screening strategy for studying epithelial growth control.