Transcriptomic Analysis Identifies Candidate Genes for Differential Expression during Xenopus laevis Inner Ear Development.

Background: The genes involved in inner ear development and maintenance of the adult organ have yet to be fully characterized. Previous genetic analysis has emphasized the early development that gives rise to the otic vesicle. This study aimed to bridge the knowledge gap and identify candidate genes that are expressed as the auditory and vestibular sensory organs continue to grow and develop until the systems reach postmetamorphic maturity. Methods: Affymetrix microarrays were used to assess inner ear transcriptome profiles from three Xenopus laevis developmental ages where all eight endorgans comprise mechanosensory hair cells: larval stages 50 and 56, and the post-metamorphic juvenile. Pairwise comparisons were made between the three developmental stages and the resulting differentially expressed X. laevis Probe Set IDs (Xl-PSIDs) were assigned to four groups based on differential expression patterns. DAVID analysis was undertaken to impart functional annotation to the differentially regulated Xl-PSIDs. Results: Analysis identified 1510 candidate genes for differential gene expression in one or more pairwise comparison. Annotated genes not previously associated with inner ear development emerged from this analysis, as well as annotated genes with established inner ear function, such as oncomodulin, neurod1, and sp7. Notably, 36% of differentially expressed Xl-PSIDs were unannotated. Conclusions: Results draw attention to the complex gene regulatory patterns that characterize Xenopus inner ear development, and underscore the need for improved annotation of the X. laevis genome. Outcomes can be utilized to select candidate inner ear genes for functional analysis, and to promote Xenopus as a model organism for biomedical studies of hearing and balance.

RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays.

The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high-quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis and Illumina-Solexa sequencing (RNA-Seq) of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for transient transfection of Xenopus kidney cell lines with fluorescent protein fusion constructs.

RNA-Seq and microarray analysis of the Xenopus inner ear transcriptome discloses orthologous OMIM(®) genes for hereditary disorders of hearing and balance.

BACKGROUND: Auditory and vestibular disorders are prevalent sensory disabilities caused by genetic and environmental (noise, trauma, chemicals) factors that often damage mechanosensory hair cells of the inner ear. Development of treatments for inner ear disorders of hearing and balance relies on the use of animal models such as fish, amphibians, reptiles, birds, and non-human mammals. Here, we aimed to augment the utility of the genus Xenopus for uncovering genetic mechanisms essential for the maintenance of inner ear structure and function. RESULTS: Using Affymetrix GeneChip(®) X. laevis Genome 2.0 Arrays and Illumina-Solexa sequencing methods, we determined that the transcriptional profile of the Xenopus laevis inner ear comprises hundreds of genes that are orthologous to OMIM(®) genes implicated in deafness and vestibular disorders in humans. Analysis of genes that mapped to both technologies demonstrated that, with our methods, a combination of microarray and RNA-Seq detected expression of more genes than either platform alone. CONCLUSIONS: As part of this study we identified candidate scaffold regions of the Xenopus tropicalis genome that can be used to investigate hearing and balance using genetic and informatics procedures that are available through the National Xenopus Resource (NXR), and the open access data repository, Xenbase. The results and approaches presented here expand the viability of Xenopus as an animal model for inner ear research.

Probing the Xenopus laevis inner ear transcriptome for biological function.

BACKGROUND: The senses of hearing and balance depend upon mechanoreception, a process that originates in the inner ear and shares features across species. Amphibians have been widely used for physiological studies of mechanotransduction by sensory hair cells. In contrast, much less is known of the genetic basis of auditory and vestibular function in this class of animals. Among amphibians, the genus Xenopus is a well-characterized genetic and developmental model that offers unique opportunities for inner ear research because of the amphibian capacity for tissue and organ regeneration. For these reasons, we implemented a functional genomics approach as a means to undertake a large-scale analysis of the Xenopus laevis inner ear transcriptome through microarray analysis. RESULTS: Microarray analysis uncovered genes within the X. laevis inner ear transcriptome associated with inner ear function and impairment in other organisms, thereby supporting the inclusion of Xenopus in cross-species genetic studies of the inner ear. The use of gene categories (inner ear tissue; deafness; ion channels; ion transporters; transcription factors) facilitated the assignment of functional significance to probe set identifiers. We enhanced the biological relevance of our microarray data by using a variety of curation approaches to increase the annotation of the Affymetrix GeneChip(®) Xenopus laevis Genome array. In addition, annotation analysis revealed the prevalence of inner ear transcripts represented by probe set identifiers that lack functional characterization. CONCLUSIONS: We identified an abundance of targets for genetic analysis of auditory and vestibular function. The orthologues to human genes with known inner ear function and the highly expressed transcripts that lack annotation are particularly interesting candidates for future analyses. We used informatics approaches to impart biologically relevant information to the Xenopus inner ear transcriptome, thereby addressing the impediment imposed by insufficient gene annotation. These findings heighten the relevance of Xenopus as a model organism for genetic investigations of inner ear organogenesis, morphogenesis, and regeneration.

Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes.

The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel ß1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology.

RNA isolation from Xenopus inner ear sensory endorgans for transcriptional profiling and molecular cloning.

The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for transient transfection of Xenopus kidney cell lines with GFP fusion constructs.