BACKGROUND
hearing and balance are essential for animal communication and locomotion.
auditory and vestibular disorders limit the perception of sound and spatial orientation.
in humans, such disorders detract from the quality of life through the impact they have on other activities, such as social interaction, education, and mobility.
diminished senses of hearing and balance frequently result from abnormalities in the organs of the inner ear.
the world health organization  estimates that hearing impairment and deafness impact over  <dig> million people, making sensorineural hearing loss a prevalent sensory disorder in humans worldwide  <cit> .
the incidence of vestibular disorders is more difficult to determine because of diagnostic challenges.
balance disabilities may reflect the abundance of vestibular disorders such as ménière’s disease, labyrinthitis, benign paroxysmal positional vertigo  and vestibular neuritis  <cit> .
in the united states, the incidence of ménière’s disease is estimated to increase by about  <dig>  persons each year  <cit> .
excessive ambient noise, aging populations, exposure to ototoxic drugs, and the inheritance of genetic mutations are believed to contribute to the prevalence of hearing and balance disorders.
understanding how environmental and genetic factors directly impact the function of the inner ear is therefore critical to the treatment and alleviation of auditory and vestibular problems  <cit> .
the senses of hearing and balance depend on the conversion of mechanical stimuli into neural signals by the auditory and vestibular endorgans of the inner ear  <cit> .
the endorgans contain sensory epithelia that comprise mechanoreceptor sensory hair cells and supporting cells  <cit> .
damage to endorgan tissue, such as injury to hair cells and the eighth cranial nerve, can cause sensorineural hearing loss and vestibular disorders  <cit> .
current understanding of inner ear biology stems from research that has focused on genetics, determining molecular elements required for hair cell function and regeneration, endorgan development, and identifying ototoxic factors and molecular targets for therapeutic treatments  <cit> .
although the inner ear endorgans of mammals and non-mammals are morphologically distinct, mechanosensory hair cells share physiological and structural similarities across species  <cit> .
cross-species comparisons of mammals , reptiles , birds, amphibians, and fish have collectively defined our current understanding of the processes of hair cell mechanotransduction, regeneration and transdifferentiation  <cit> .
genetic analysis has provided insight into the hereditary basis of deafness in humans and mice  <cit> .
large-scale transcriptome analysis tools such as cdna libraries and microarrays have been used to identify inner ear genes in human, mouse, chicken, rat, and zebrafish  <cit> .
outcomes of these investigations have established cross-species similarities in the genetic profile of the inner ear.
physiological and anatomical investigations of the class amphibia have been seminal to our understanding of the cellular basis of auditory and vestibular processing.
in particular, studies on the process of mechanotransduction in amphibian hair cells have formulated the framework for elucidating the biophysical details of hair cell mechanoreception  <cit> .
moreover, amphibians  have been shown to regenerate or transdifferentiate hair cells after trauma and therefore are a useful model for inner ear research  <cit> .
outcomes of experiments with amphibian genera such as rana , hyla , and the african clawed frog, xenopus , have contributed to our knowledge of peripheral sound reception and otoacoustic emissions  <cit>  as well as sensory endorgan development  <cit> .
in contrast to the emphasis on amphibians as model organisms for investigations of hair cell electrophysiology and mechanotransduction, amphibians have been underutilized as models for analysis of global gene expression in the inner ear.
this omission may be partially attributed to the novelty of transcriptional profiling and similar large-scale genetic analyses as tools for uncovering inner ear function in any species  <cit> .
although inner ear genes have been characterized individually in amphibians and other species  <cit> , large-scale transcriptome analysis has unprecedented potential to significantly advance the field of inner ear genetics  <cit> .
among amphibians, the genus xenopus offers unique opportunities for genetic investigations of inner ear structure and function due to the availability of a sequenced genome  <cit> , and the thorough characterization of developmental stages  <cit> .
furthermore, xenopus is well suited to genetic analysis because methods that enable the production of thousands of transgenic embryos are well established  <cit> .
online resources specific to xenopus, such as xendb and xenbase, facilitate cross-species genetic analysis  <cit> .
in addition, transcriptional profiling with microarrays has been used for large-scale analysis of xenopus gene expression to investigate early embryonic development, non-inner ear organ specific expression, and limb regeneration  <cit> .
genetic findings from such large-scale approaches can be contextualized by the aforementioned physiological studies of amphibian hair cell function.
transcriptional profiling of xenopus inner ear endorgans can potentially identify gene families and expression patterns that typify functional inner ear tissue.
to this end, we used microarray analysis to ascertain the genetic basis of xenopus auditory and vestibular sensation.
we profiled rna isolated from the inner ears of juvenile animals, a developmental age where all anatomical structures are fully formed, and animals are in the initial stages of postmetamorphic life  <cit> .
the affymetrix genechip® xenopus laevis genome array version  <dig>  was used for the identification of key molecular components of the x. laevis inner ear.
the unknown biological function of many x. laevis probe set identifiers  on the genechip®, a drawback that stems in part from the unsequenced x. laevis genome, prompted our use of extensive manual curation efforts to augment the functional significance of the array data.
in order to relate prior knowledge of genes with predicted inner ear function to the x. laevis inner ear transcriptome, we focused our x. laevis genechip® annotation efforts on five inner ear gene categories: genes that encode ion channels , ion transporters , and transcription factors ; genes found in inner ear tissue ; and genes with mutations that cause deafness .
sequence similarity mapping, semantic keyword querying and the xenenhance relational database  <cit>  enabled linkage of the more informative official gene symbols from the hugo gene nomenclature committee  to a subset of xl-psids on the x. laevis genechip®  <cit> .
throughout this paper we use the hgnc nomenclature to refer to genes of interest.
we approached our analysis of the x. laevis inner ear transcriptome by examining the intensity levels and functional classification of xl-psids.
as expected, xl-psids with high intensity values corresponded to genes that are predominantly involved in housekeeping and maintenance functions common to many cell and tissue types.
intensity analysis also highlighted the prevalence of xl-psids with no known annotation or protein counterpart.
our comparison of the inner ear gene categories revealed that the transcription factor gene category was characterized by the lowest xl-psid intensity value distribution of all five categories.
our manual curation efforts enabled us to evaluate whether genes associated with inner ear function in other organisms  were potentially represented either in the x. tropicalis genome or on the x. laevis genechip®.
sequence similarity alignments revealed that the majority of hgnc protein sequences from the inner ear gene categories have counterparts in the x. tropicalis genome and on the x. laevis genechip®.
taken together, the results of our transcriptome analysis demonstrate that xenopus is a viable model organism for auditory and vestibular research, and support the inclusion of xenopus in cross-species comparisons.
our results also have uncovered potential gene targets that, through transgenic approaches, have the potential to reveal genetic elements of inner ear function and dysfunction.
RESULTS
data normalization and distribution
the microarray cel file raw data were preprocessed using the genechip robust multichip analysis  summarization method.
the distribution of xl-psid intensity values for the normalized data ranged from  <dig> - <dig>  .
box plots of triplicate x. laevis inner ear  arrays illustrate the similarity between replicates for both pre- and post-normalized data .
mva plots demonstrate the benefit of normalization and illustrate the same trend between the replicates as seen with box plots .
as with the box plots, mva plots of pre-normalized data  showed an asymmetrical distribution of data and greater inter-chip variation than normalized data .
the interquartile range  values were very low for normalized data  and much less than for pre-normalized data .
similarity among the replicate arrays was demonstrated by the analysis of the inter-chip and intra-chip averages and standard deviations  for normalized xl-psid intensity values.
the average xl-psid intensity values for the individual chips were almost identical; the inter-chip sd was  <dig> % of the inter-chip average xl-psid intensity value of  <dig> .
the individual intra-chip sds also were of comparable magnitude and ranged from  <dig>  to  <dig>  .
in our analysis of the x. laevis inner ear transcriptome we excluded the control xl-psids , a procedure that raised the inter-chip average xl-psid intensity from  <dig>  to  <dig>  .
all xl-psids
control xl-psids
experimental
xl-psids
xl-psids with
“p”/”m” gcos calls
xl-psids with “a” gcos calls in all  <dig> xie chips
gcrma intra-chip averages are the calculated average intensity of the normalized xl-psids on a given chip .
gcrma inter-chip averages are computed as the average intensity of all average xl-psid intensity values for all three chips.
data are presented as average + sd.
genes that correspond to xl-psid consensus sequences can be amplified with rt-pcr fromx.
laevisinner ear rna
primers were designed against consensus sequences for eight xl-psids with varied intensity levels : gene name, gene symbol , gata binding protein  <dig>  gata <dig> ; clusterin, clu ; profilin  <dig>  pfn <dig> ; six homeobox  <dig>  six <dig> ; matrilin  <dig>  matn <dig> ; peripheral myelin protein  <dig>  pmp <dig> ; chloride channel, voltage-sensitive ka, clcnkb ; and sodium channel, non-voltage-gated  <dig>  beta subunit, scnn1b .
rt-pcr products were obtained with all eight primer pairs.
figure 2a shows the amplification bands for gata <dig>  clu, pfn <dig>  and six <dig>  dna sequencing confirmed the predicted identity of all rt-pcr products.
the eight consensus sequences corresponded to genes associated with ion transport, the extracellular matrix, hearing impairment, and deafness.
sequence similarity between xenopus inner ear cdna library clones and xl-psid consensus sequences
the blastn algorithm was used to find homology between  <dig> clones from two xenopus inner ear cdna libraries  and xl-psid consensus sequences .
the number of blastn derived pairwise alignments in each similarity group  based on expect values  are shown in table  <dig>  we noted that in some instances more than one cdna library clone aligned with the same xl-psid.
consequently, the number of cdna/xl-psid affirmative pairwise alignments  was greater than the number of target xl-psids  on the x. laevis genechip®.
the five xl-psids with multiple cdna clone alignments represent hemoglobin, gamma g ; ferritin light chain ; ribosomal protein s <dig> ; an unknown sequence; and cytochrome c oxidase subunit va .
xl-psids were aligned to xenopus laevis  and xenopus tropicalis  inner ear library clone sequences using the blastn algorithm.
pairwise alignments were sorted into similarity groups based on e-value.
xl-psids with multiple pairwise alignments to cdna library clones were counted once.
four xl-psids aligned to two te cdna clones, and one xl-psid aligned to eight te cdna clones.
when we analyzed the intensity distribution of the  <dig> xl-psids with affirmative pairwise alignments with  <dig> inner ear cdna clones, we noted that  <dig> % of the cdna clones mapped to xl-psids with average intensity levels greater than four .
the seven cdna clones that mapped to xl-psids with average intensity values under four represented unknown sequences, the bmp <dig> gene, spondin  <dig> , and prolyl 4-hydroxylase, beta polypeptide.
we also found that more than 98% of all experimental xl-psids that were designated “a” by the affymetrix gcos software  had average intensity levels below four .
based on these observations, we expect that an xl-psid intensity value greater than or equal to four is likely to represent an expressed inner ear sequence.
xl-psid intensity analysis with decile groupings and functional characterization
as a prelude to functional analysis, we rank ordered the xl-psids  based on their average intensity values.
the ten xl-psids with the highest intensity values were: hemoglobin, gamma a, hbg1; ribosomal protein s <dig>  rps27; ferritin , fth1; ubiquitin b, ubb; ribosomal protein s <dig>  rps13; solute carrier family  <dig> , member  <dig>  slc11a2; ribosomal protein s <dig>  rps20;  <dig> unknown sequence; ribosomal protein s <dig>  rps14; and hypothetical protein mgc114621/ribosomal protein , rplp <dig>
we partitioned the xl-psids into deciles by two methods, equal number of xl-psids  and equal range of average intensity values .
for equal tally deciles, the variation of xl-psid average intensity values were low and comparable  for all the deciles except for the 10th .
in contrast to equal tally deciles,  <dig>  of the xl-psids in the equal intensity deciles were grouped in the 10th decile .
xl-psids were divided into decile groupings either by number  or intensity ; 10th decile corresponds to the highest intensity levels.
in the equal tally distribution , each decile comprises an equal number  of xl-psids.
in the equal intensity distribution , each decile comprises xl-psids within an equal intensity range .
the number of xl-psids without annotation after david queries is listed in the orphan xl-psid column.
we focused our functional analysis on the 10th deciles, which comprise xl-psids with the highest average intensity values in both instances.
the database for annotation, visualization and integrated discovery , kegg and sp-pir terms.
as shown on table 4a the most common functional annotation for the 10th equal tally decile was the go term “cellular processes” .
david analysis also classified the top 10% of xl-psids into other annotation categories, including “biosynthetic processes”, “gene expression”, “translation”, “non-membrane-bounded organelle”, and “structural molecule activity”.
the most common functional annotations for the 10th equal intensity decile  were: “non-membrane-bounded organelle”, “intracellular non-membrane-bounded organelle”, and “translation”.
we observed that some xl-psids were clustered in multiple functional categories.
moreover, david reported an “orphan”  status for  <dig> % of the xl-psids in the 10th equal tally decile and  <dig> % of the xl-psids in the 10th equal intensity decile .
we also noted a similar trend in both decile groupings; the number of orphan xl-psids within a decile decreased as the intensity values of their decile increased.
in comparison to david,  <dig> %  of the xl-psids in the 10th equal tally decile and 10% of the xl-psids in the 10th equal intensity decile  were without gene annotation based on the annotation file provided by the vendor .
the functional clusters with the highest david enrichment scores are shown for xl-psids in the 10th equal tally  and the 10th equal intensity  decile.
david assigned  <dig> ids to the 10th equal tally decile, resulting in  <dig> annotation clusters.
david assigned  <dig> ids to the 10th equal intensity decile, resulting in  <dig> annotation clusters.
assigning inner ear gene categories to xl-psids
as a prelude to analyzing the inner ear transcriptome, we identified xl-psids on the x. laevis genechip® with a probable role in the maintenance and function of auditory and vestibular inner ear endorgans.
we selected five gene categories for intensity analysis: inner ear tissue , deafness , ion channels , ion transport , and transcription factors .
the venn diagram in figure  <dig> shows the overlap between the five inner ear gene categories .
several approaches were used to assign these gene categories to xl-psids .
the number of xl-psids in all inner ear gene categories  and in the 10th deciles are reported together with compilation and analysis methods for each gene group.
the table reports the number of symbols for hgnc proteins that formed affirmative pairwise alignments with xl-psid consensus sequences through sequence similarity mapping .
*hgnc symbols were linked to more than one xl-psid, resulting in a number that is higher than the number xl-psids.
mapping iet, df and ic inner ear gene categories to xl-psids
to assess the utility of the x. laevis genechip® in inner ear array studies, we evaluated whether genes associated with inner ear function in xenopus and other organisms  were arrayed on the chip.
to this end, we used sequence similarity mapping with the tblastn algorithm to determine whether hgnc human protein sequences from the iet, df, and ic gene lists aligned with xl-psid consensus sequences .
the top blast pairwise alignment was used to assign putative function to the xl-psid consensus sequence .
hgnc human protein sequences  formed affirmative pairwise alignment with  <dig> xl-psid consensus sequences.
we noted that in some instances a single xl-psid aligned with multiple human protein sequences .
xl-psid intensity analysis of inner ear gene categories
as shown in figure 4a, the histogram of average intensity values for all experimental xl-psids on the microarray was characterized by an asymmetrical left-skewed distribution.
since approximately  <dig> % of xl-psids were scored with average intensity levels above four , we estimated that two-thirds of the x. laevis genechip® could be used to detect xenopus inner ear transcripts.
the histograms of average intensity values for each of the five gene categories also showed an asymmetrical left-skewed distribution.
the majority of xl-psids were scored with average intensity levels greater than or equal to “four” in all gene categories except “transcription factor” .
inner ear tissue genes
approximately  <dig> % of genes from the iet list were linked by affirmative pairwise alignments to xl-psids  with intensities ranging from  <dig>  to  <dig> .
average intensities above four were detected from approximately 68% of iet/xl-psids .
we noted that about  <dig> % of the iet/xl-psids were clustered in the top two equal tally deciles .
the range of intensities  for iet/xl-psids was greatest in the 10th decile.
when the iet/xl-psids were grouped into equal intensity deciles, the 5th decile contained the most iet/xl-psids with intensities that ranged from  <dig> - <dig>  .
the iet genes linked to the  <dig> xl-psids with the highest intensities are listed in table 6a .
the xl-psids with the  <dig> highest intensity values are listed for each inner ear gene category.
the corresponding hgnc symbols or gene symbols are listed for all categories.
in , two pairs of xl-psids correspond to the same hgnc symbol and have probes derived from the same consensus sequence.
one xl-psid in  did not have a hgnc symbol due to the lack of sequence similarity to a human orthologue.
human deafness genes
approximately 71% of df genes were linked by affirmative pairwise alignments to xl-psids  with average intensities from  <dig> - <dig> .
figure 4c shows that  <dig> % of df/xl-psids had average intensities greater than four.
df/xl-psids were predominantly in the 5th, 9th, and 10th equal tally deciles .
whereas, when df/xl-psids were grouped into equal intensity deciles, the distribution was mostly in the 3rd, 4th, and 5th deciles .
the df genes linked to the  <dig> xl-psids with the highest intensities are listed in table 6b and represent various cellular functions.
ion channel genes
approximately 69% of ic genes were linked by affirmative pairwise alignments to sequences for  <dig> xl-psids  that ranged in intensity from  <dig>  to  <dig> .
the small number of xl-psids relative to the number of genes  is partially due to the fact that many ion channel pore subunits aligned to the same xl-psid.
as a group, the ic/xl-psids have lower average intensity levels than both iet/xl-psids and df/xl-psids .
only  <dig> % of ic/xl-psids had average intensity values greater than four .
the ic genes linked to xl-psids with the highest intensities were voltage-dependent anion-selective channels, glutamate receptors, and subunits from k+ and na+ channels .
ion transport genes
the  <dig> xl-psids that represent it and ic genes on the x. laevis genechip®  had intensities distributed from  <dig> - <dig> .
approximately 59% of it/xl-psids had average intensity values greater than four; most were in the 9th and 10th equal tally deciles .
when it/xl-psids were grouped into equal intensity deciles, the 1st and 4th deciles contained the most it/xl-psids .
it/xl-psids with the highest intensities were mostly subunits for sodium/potassium/hydrogen transporting atpases .
putative transcription factors
for this category of genes,  <dig> % of the identified ptf/xl-psids have average intensity values above four .
in contrast to iet/xl-psids, df/xl-psids, and it/xl-psids intensity values distributions, the majority of ptf/xl-psids are in the 1st equal tally decile  as opposed to the 10th.
however, when grouped into equal intensity deciles, the 1st and 4th deciles contained the most ptf/xl-psids .
the ptf/xl-psids with the highest intensities are listed in table 6e.
trends in xl-psid intensity distributions for inner ear gene categories
we compared the distribution of xl-psid intensity values for the four gene categories in order to ascertain potential differences in the relative expression levels of inner ear genes based on functional classification.
we observed that the ptf category, with the largest number of xl-psids , was characterized by the lowest intensity value distribution of all the gene categories.
moreover, a larger percentage of the ptf/xl-psids  have gcos absent calls as compared with xl-psids in the other categories .
we further noted that the largest proportions of df/xl-psids , it/xl-psids , and iet/xl-psids  were found in the 10th equal tally decile .
manual curation efforts improved x. laevis genechip® annotation
we implemented david analysis to assess whether or not manual curation improved the annotation results for iet/xl-psids , the largest category with manually-linked hgnc symbols.
to this end, we compared the outcomes of david analyses for iet/xl-psids to that of iet/hgnc symbols .
the iet category of  <dig> iet/hgncs was represented by  <dig> david ids, with one orphan iet/hgnc.
david grouped inner ear specific go terms  into two iet/hgnc functional annotation clusters.
in contrast, for the corresponding  <dig> iet/xl-psids, no inner ear specific go terms were recovered from analysis of the david annotation clusters.
furthermore, the number of ids that david associated with iet/hgncs  was greater than the number of ids that david associated with the iet/xl-psid counterparts .
moreover, the number of orphan iet/xl-psids  was greater than the single iet/hgnc orphan.
taken together, these findings suggest that manual curation of the x. laevis genechip® by assignment of hgnc symbols to the xl-psids improved annotation.
identification of putative human inner ear orthologues in the xenopus tropicalis genome
in order to determine the extent to which xenopus is a practical model organism for auditory and vestibular research, we used the products of our manual curation efforts to evaluate whether genes associated with inner ear function in other organisms  were present within the x. tropicalis genome.
to this end, we used the blastp algorithm to determine whether hgnc human protein sequences from the iet, df, and ic gene lists aligned with curated x. tropicalis  <dig>  predicted proteins from the joint genome institute  x. tropicalis sequencing project .
the average e-value for hgnc/xt <dig>  predicted protein mappings  were lower than the average e-value for hgnc/xl-psid mappings .
moreover, the number and percentage of affirmative pairwise alignments between hgnc human protein and x. tropicalis  <dig>  predicted protein sequences  exceeded the number of affirmative pairwise alignments between hgnc human protein sequences and xl-psids .
these sequence similarity alignments demonstrate that more orthologues with high similarity to human proteins from all three gene lists were identified in the xenopus genome than on the x. laevis genechip® .
discussion
microarray technology for transcriptional profiling of inner ear endorgans
limited access to human inner ear rna mandates the use of model organisms, such as xenopus laevis for transcriptome analysis.
cellular and molecular investigations of the inner ear are challenged by the size and location of the organ.
the human cochlea measures almost 1 cm in size while the entire xenopus inner ear is about one third of that size; both are encased by the temporal bone, the densest bone in the body.
we overcame the constraints imposed by the inaccessibility of the inner ear through implementation of rigorous surgical procedures that yielded high quality rna from a small amount of inner ear tissue.
reproducibility between our arrayed biological replicates was evidenced by the similar intra-chip sds for xl-psid intensity values and the low inter-chip sds.
these results illustrate the efficacy of our protocols in restricting biological and technical variation between replicates that may be introduced by experimental procedures such as tissue isolation, rna extraction, and array hybridization.
the usefulness of inner ear genetics as an approach to develop treatments for inner ear disorders can be heightened through analysis of the relative expression of genes within the transcriptome.
data normalization is a critical step in achieving this objective.
box plots and mva plots of normalized data showed low inter-chip variability as compared to plots constructed with pre-normalized data, demonstrating the efficacy of the normalization method.
we elected to use data normalized with gcrma because this data normalization method performs well with regard to accurate detection of low abundance transcripts and precision  <cit> .
this data normalization method also is recommended when the objectives are to identify differentially expressed genes, or to estimate relative gene expression.
the ability to assess whether microarray intensity values are predictors of verifiable gene expression facilitates the interpretation of microarray data.
in particular, it is useful to identify an intensity value, or threshold, above which pcr methods are likely to independently validate gene expression.
inner ear cdna library clones represent expressed sequences and thus are a useful tool for assessing whether expressed sequences are detected in the array.
the combination of gcos absent calls and intensity levels of cdna clones represented on the x. laevis genechip®, led us to predict that an xl-psid intensity value greater than or equal to four is likely to represent an expressed sequence that can be confirmed by rt-pcr.
as expected, rt-pcr experiments with inner ear rna confirmed the presence of 100% of eight genes that mapped to xl-psids with average intensity levels above six.
annotation curation expands the biological relevance of the microarray data
transcriptome analysis is facilitated by genomic sequence data and quality gene annotation for the species of interest.
the quality and the extent of annotation have been acknowledged as particular impediments to the advancement of transcriptomics  <cit> .
for this reason, the national human genome research institute  initiated the encyclopedia of dna elements , a project aimed at the functional annotation of all elements in the human genome  <cit> .
the utility of the x. laevis genechip® is reduced by the number of arrayed genes  with no known biological function, a limitation that stems in part from the unsequenced x. laevis genome.
we enhanced the biological relevance of the data by using a variety of computational strategies to link xl-psids to hgnc official gene symbols.
the linkage of hgnc symbols to xl-psids exploited the detailed annotations of human genes as compared to other species.
sequence similarity mapping and semantic keyword querying facilitated the assignment of putative functions important for inner ear processes.
our x. laevis genechip® annotation efforts focused on five gene categories relevant for inner ear function: inner ear tissue , deafness , ion channels , ion transporters  and transcription factors .
no single tool is sufficiently robust to assign function to genes from a species such as x. laevis, whose genome has not been sequenced.
in order to impart biological function to our microarray data, it was mandatory to combine many curation approaches .
we found that the best approach involved combining reading the scientific research literature, keyword and nucleotide database queries, and functional annotation clustering with data-mining tools from the david bioinformatics resource.
david was useful for providing information about groups of xl-psids through functional clustering.
for example, david analysis of the 10th deciles  revealed many go categories associated with genes commonly found in all tissues and not exclusive to the inner ear, and identified the orphan xl-psids with no known annotation.
gene groups facilitate the analysis of trends in the x. laevis inner ear transcriptome
through transcriptional profiling of the inner ear, we aimed to garner a comprehensive perspective of an understudied organ.
the implementation of gene lists and decile groupings facilitated the analysis of the inner ear transcriptome by restricting our focus to a subset of xl-psids culled from known inner ear genes and from xl-psids with intensity values in the top 10%.
the combination of these two approaches allowed us to identify patterns in the relative intensities of xl-psids, to compare xenopus inner ear genes to the known inner ear genes of other species, and to gain insight about the contribution of genes with no known function to the inner ear transcriptome.
trends in the intensity values for xl-psids were extricated through histogram and decile analysis.
we observed that the distribution of xl-psid intensities in the histograms for categories with predicted inner ear function resembled the cumulative histogram for all xl-psids.
we also noticed that all gene categories are represented in all equal tally and all equal intensity deciles .
average intensity values were as follows: cumulative xl-psids,  <dig> ; iet/xl-psids,  <dig> ; df/xl-psids,  <dig> ; ic/xl-psids,  <dig> ; it/xl-psids,  <dig> ; ptf/xl-psids,  <dig> .
we interpret the similarities between xl-psid distributions for the inner ear transcriptome and the gene categories as indicating that the gene categories are representative of the whole inner ear transcriptome.
this outcome is interesting because the inner ear research that formed the basis for our selection of gene categories was rich in the science of mechanosensory hair cells whose numbers comprise very few cells of the inner ear.
for example, the auditory hair cells in a juvenile xenopus animal total approximately  <dig>  <cit> ; whereas, cochlear hair cells of the human inner ear number approximately  <dig>   <cit> .
nevertheless, the gene categories captured data that encompassed a full range of xl-psid intensity values.
the utility of xenopus as a model organism for inner ear research is supported by the commonality we observed between the x. laevis transcriptome and the outcomes of gene analyses for other species traditionally used for auditory and vestibular research.
many of the common genes are ion channels and transporters, transcription factors, gap junction proteins, cytoskeletal proteins, and structural proteins that have been implicated in inner ear function in humans and mice  <cit> .
various genes common to the x. laevis inner ear and those of other species are associated with deafness, including structural proteins ; collagen, type i, alpha  <dig> ; and tectorin alpha ), all of which have corresponding df/xl-psid intensity levels above four.
of the  <dig> ion channel genes identified in both human cochlear and mouse organ of corti cdna libraries by gabashvili et al.
<cit> , nine were represented on the x. laevis genechip®.
moreover, seven of these nine genes corresponded to xl-psids with intensity values above four .
ion transporters that play a role in k+ cycling and maintenance of endolymph in the cochlea of human, mouse, and rat  <cit>  were also represented on the x. laevis genechip® by xl-psids with high intensities .
additionally, transcription factors implicated in hair cell regeneration in the chicken inner ear, such as jun d proto-oncogene , ccaat/enhancer binding protein c/ebp, gamma , and paired box  <dig>   <cit>  were identified as ptf/xl-psids with intensities above four in the x. laevis inner ear.
the bone morphogenetic protein bmp <dig> <cit> , which is important for cochlea and sensory organ development in mouse and chicken, was also detected in xenopus .
the prevalence of similar genes identified in both the human cochlea and xenopus inner ear support the notion that physiological processes essential for inner ear function are shared between the two species.
it was notable that xl-psids with intensities in the top 1% , sparc; eukaryotic translation elongation factor  <dig> alpha  <dig>  eef1a1) correspond to the most abundant transcripts identified in a human fetal cochlear cdna library .
clusterin  was found in human perilymph with high protein concentrations  <cit> ; currently the function of this glycoprotein in the inner ear is unknown.
taken together, the identification of xl-psids from all five gene categories with high intensity values supports the use of x. laevis to advance our understanding of the genes critical for inner ear function.
moreover, previously uncharacterized genes are now found to have a putative function in the xenopus inner ear.
focusing our attention on xl-psids with the highest intensity values uncovered the genes that are predominant in the juvenile x. laevis inner ear transcriptome.
our analysis of genes associated with the top  <dig> xl-psids , as well as david analysis of the 10th decile, revealed that xl-psids with the highest intensity values in the x. laevis inner ear are linked to cellular maintenance functions, especially “housekeeping”.
these cellular maintenance genes were represented in greater numbers in comparison to genes specific to inner ear function such as iet/xl-psids and df/xl-psids .
these findings are consistent with observations by other researchers who have noted that genes influential in other tissue types  are highly expressed in the inner ear  <cit> .
finally, our david analysis of the xenopus inner ear transcriptome revealed that 13% of the xl-psids in the 10th equal tally decile are “orphans” and have no annotation.
analysis of the highest xl-psid intensity values highlighted the predominance of xl-psids without gene titles in the affymetrix annotation file ; 12% of the  <dig> most highly expressed xl-psids and  <dig> % of the  <dig> xl-psids in the 10th equal tally decile fell into this category.
taken together, these results imply that the roles of many genes important for inner ear function have yet to be defined.
as functional characterization of genomes expands through the use of interdisciplinary approaches and cross-species analysis, knowledge of the genetic elements essential to inner ear function and dysfunction is expected to increase.
CONCLUSIONS
the genus xenopus affords unique opportunities for inner ear research because of its utility as a developmental model for genetic investigations as well as the amphibian capacity for regeneration of mechanosensory hair cells and neural tissue.
while amphibians have furthered our understanding of inner ear hair cell mechanotransduction and physiology, the inner ear transcriptome of amphibians is not comparably well-characterized.
for this reason, we implemented microarray transcriptional profiling for large-scale analysis of the x. laevis inner ear transcriptome.
we heightened the functional significance of our analysis by targeting groups of genes considered essential for inner ear function.
we overcame challenges faced by investigators working with organisms with unsequenced genomes through informatics approaches that significantly enhanced the annotation of the x. laevis genechip®.
our results suggest that the xenopus inner ear transcriptome comprises genes that share significant sequence similarity with genes associated with non-syndromic deafness in other species , as well as a high abundance of xl-psids with no known annotation .
we propose that the aforementioned putative mammalian orthologues and unknown xl-psids identified in this study represent ideal targets for functional analysis through genetic approaches.
our findings provide a resource that can be used by the xenopus community for shared research enterprises such as xendb  <cit> , xenbase  <cit>  and the recently established national xenopus resource at the marine biological laboratory  <cit>  that produces transgenic xenopus.
taken together, our results support the implementation of xenopus as a viable model for inner ear research, especially for investigation of hair cell regeneration, morphogenesis, and organogenesis.

 hearing and balance are essential for animal communication and locomotion. auditory and vestibular disorders limit the perception of sound and spatial orientation. in humans, such disorders detract from the quality of life through the impact they have on other activities, such as social interaction, education, and mobility. diminished senses of hearing and balance frequently result from abnormalities in the organs of the inner ear. the world health organization estimates that hearing impairment and deafness impact over million people, making sensorineural hearing loss a prevalent sensory disorder in humans worldwide. the incidence of vestibular disorders is more difficult to determine because of diagnostic challenges. balance disabilities may reflect the abundance of vestibular disorders such as ménière’s disease, labyrinthitis, benign paroxysmal positional vertigo and vestibular neuritis. in the united states, the incidence of ménière’s disease is estimated to increase by about persons each year. excessive ambient noise, aging populations, exposure to ototoxic drugs, and the inheritance of genetic mutations are believed to contribute to the prevalence of hearing and balance disorders. understanding how environmental and genetic factors directly impact the function of the inner ear is therefore critical to the treatment and alleviation of auditory and vestibular problems. the senses of hearing and balance depend on the conversion of mechanical stimuli into neural signals by the auditory and vestibular endorgans of the inner ear. the endorgans contain sensory epithelia that comprise mechanoreceptor sensory hair cells and supporting cells. damage to endorgan tissue, such as injury to hair cells and the eighth cranial nerve, can cause sensorineural hearing loss and vestibular disorders. current understanding of inner ear biology stems from research that has focused on genetics, determining molecular elements required for hair cell function and regeneration, endorgan development, and identifying ototoxic factors and molecular targets for therapeutic treatments. although the inner ear endorgans of mammals and non-mammals are morphologically distinct, mechanosensory hair cells share physiological and structural similarities across species. cross-species comparisons of mammals, reptiles, birds, amphibians, and fish have collectively defined our current understanding of the processes of hair cell mechanotransduction, regeneration and transdifferentiation. genetic analysis has provided insight into the hereditary basis of deafness in humans and mice. large-scale transcriptome analysis tools such as cdna libraries and microarrays have been used to identify inner ear genes in human, mouse, chicken, rat, and zebrafish. outcomes of these investigations have established cross-species similarities in the genetic profile of the inner ear. physiological and anatomical investigations of the class amphibia have been seminal to our understanding of the cellular basis of auditory and vestibular processing. in particular, studies on the process of mechanotransduction in amphibian hair cells have formulated the framework for elucidating the biophysical details of hair cell mechanoreception. moreover, amphibians have been shown to regenerate or transdifferentiate hair cells after trauma and therefore are a useful model for inner ear research. outcomes of experiments with amphibian genera such as rana, hyla, and the african clawed frog, xenopus, have contributed to our knowledge of peripheral sound reception and otoacoustic emissions as well as sensory endorgan development. in contrast to the emphasis on amphibians as model organisms for investigations of hair cell electrophysiology and mechanotransduction, amphibians have been underutilized as models for analysis of global gene expression in the inner ear. this omission may be partially attributed to the novelty of transcriptional profiling and similar large-scale genetic analyses as tools for uncovering inner ear function in any species. although inner ear genes have been characterized individually in amphibians and other species, large-scale transcriptome analysis has unprecedented potential to significantly advance the field of inner ear genetics. among amphibians, the genus xenopus offers unique opportunities for genetic investigations of inner ear structure and function due to the availability of a sequenced genome, and the thorough characterization of developmental stages. furthermore, xenopus is well suited to genetic analysis because methods that enable the production of thousands of transgenic embryos are well established. online resources specific to xenopus, such as xendb and xenbase, facilitate cross-species genetic analysis. in addition, transcriptional profiling with microarrays has been used for large-scale analysis of xenopus gene expression to investigate early embryonic development, non-inner ear organ specific expression, and limb regeneration. genetic findings from such large-scale approaches can be contextualized by the aforementioned physiological studies of amphibian hair cell function. transcriptional profiling of xenopus inner ear endorgans can potentially identify gene families and expression patterns that typify functional inner ear tissue. to this end, we used microarray analysis to ascertain the genetic basis of xenopus auditory and vestibular sensation. we profiled rna isolated from the inner ears of juvenile animals, a developmental age where all anatomical structures are fully formed, and animals are in the initial stages of postmetamorphic life. the affymetrix genechip® xenopus laevis genome array version was used for the identification of key molecular components of the x. laevis inner ear. the unknown biological function of many x. laevis probe set identifiers on the genechip®, a drawback that stems in part from the unsequenced x. laevis genome, prompted our use of extensive manual curation efforts to augment the functional significance of the array data. in order to relate prior knowledge of genes with predicted inner ear function to the x. laevis inner ear transcriptome, we focused our x. laevis genechip® annotation efforts on five inner ear gene categories: genes that encode ion channels, ion transporters, and transcription factors; genes found in inner ear tissue; and genes with mutations that cause deafness. sequence similarity mapping, semantic keyword querying and the xenenhance relational database enabled linkage of the more informative official gene symbols from the hugo gene nomenclature committee to a subset of xl-psids on the x. laevis genechip®. throughout this paper we use the hgnc nomenclature to refer to genes of interest. we approached our analysis of the x. laevis inner ear transcriptome by examining the intensity levels and functional classification of xl-psids. as expected, xl-psids with high intensity values corresponded to genes that are predominantly involved in housekeeping and maintenance functions common to many cell and tissue types. intensity analysis also highlighted the prevalence of xl-psids with no known annotation or protein counterpart. our comparison of the inner ear gene categories revealed that the transcription factor gene category was characterized by the lowest xl-psid intensity value distribution of all five categories. our manual curation efforts enabled us to evaluate whether genes associated with inner ear function in other organisms were potentially represented either in the x. tropicalis genome or on the x. laevis genechip®. sequence similarity alignments revealed that the majority of hgnc protein sequences from the inner ear gene categories have counterparts in the x. tropicalis genome and on the x. laevis genechip®. taken together, the results of our transcriptome analysis demonstrate that xenopus is a viable model organism for auditory and vestibular research, and support the inclusion of xenopus in cross-species comparisons. our results also have uncovered potential gene targets that, through transgenic approaches, have the potential to reveal genetic elements of inner ear function and dysfunction.  data normalization and distribution the microarray cel file raw data were preprocessed using the genechip robust multichip analysis summarization method. the distribution of xl-psid intensity values for the normalized data ranged from -. box plots of triplicate x. laevis inner ear arrays illustrate the similarity between replicates for both pre- and post-normalized data. mva plots demonstrate the benefit of normalization and illustrate the same trend between the replicates as seen with box plots. as with the box plots, mva plots of pre-normalized data showed an asymmetrical distribution of data and greater inter-chip variation than normalized data. the interquartile range values were very low for normalized data and much less than for pre-normalized data. similarity among the replicate arrays was demonstrated by the analysis of the inter-chip and intra-chip averages and standard deviations for normalized xl-psid intensity values. the average xl-psid intensity values for the individual chips were almost identical; the inter-chip sd was % of the inter-chip average xl-psid intensity value of. the individual intra-chip sds also were of comparable magnitude and ranged from to. in our analysis of the x. laevis inner ear transcriptome we excluded the control xl-psids, a procedure that raised the inter-chip average xl-psid intensity from to. all xl-psids control xl-psids experimental xl-psids xl-psids with “p”/”m” gcos calls xl-psids with “a” gcos calls in all xie chips gcrma intra-chip averages are the calculated average intensity of the normalized xl-psids on a given chip. gcrma inter-chip averages are computed as the average intensity of all average xl-psid intensity values for all three chips. data are presented as average + sd. genes that correspond to xl-psid consensus sequences can be amplified with rt-pcr fromx. laevisinner ear rna primers were designed against consensus sequences for eight xl-psids with varied intensity levels: gene name, gene symbol, gata binding protein gata; clusterin, clu; profilin pfn; six homeobox six; matrilin matn; peripheral myelin protein pmp; chloride channel, voltage-sensitive ka, clcnkb; and sodium channel, non-voltage-gated beta subunit, scnn1b. rt-pcr products were obtained with all eight primer pairs. figure 2a shows the amplification bands for gata clu, pfn and six dna sequencing confirmed the predicted identity of all rt-pcr products. the eight consensus sequences corresponded to genes associated with ion transport, the extracellular matrix, hearing impairment, and deafness. sequence similarity between xenopus inner ear cdna library clones and xl-psid consensus sequences the blastn algorithm was used to find homology between clones from two xenopus inner ear cdna libraries and xl-psid consensus sequences. the number of blastn derived pairwise alignments in each similarity group based on expect values are shown in table  we noted that in some instances more than one cdna library clone aligned with the same xl-psid. consequently, the number of cdna/xl-psid affirmative pairwise alignments was greater than the number of target xl-psids on the x. laevis genechip®. the five xl-psids with multiple cdna clone alignments represent hemoglobin, gamma g; ferritin light chain; ribosomal protein s; an unknown sequence; and cytochrome c oxidase subunit va. xl-psids were aligned to xenopus laevis and xenopus tropicalis inner ear library clone sequences using the blastn algorithm. pairwise alignments were sorted into similarity groups based on e-value. xl-psids with multiple pairwise alignments to cdna library clones were counted once. four xl-psids aligned to two te cdna clones, and one xl-psid aligned to eight te cdna clones. when we analyzed the intensity distribution of the xl-psids with affirmative pairwise alignments with inner ear cdna clones, we noted that % of the cdna clones mapped to xl-psids with average intensity levels greater than four. the seven cdna clones that mapped to xl-psids with average intensity values under four represented unknown sequences, the bmp gene, spondin, and prolyl 4-hydroxylase, beta polypeptide. we also found that more than 98% of all experimental xl-psids that were designated “a” by the affymetrix gcos software had average intensity levels below four. based on these observations, we expect that an xl-psid intensity value greater than or equal to four is likely to represent an expressed inner ear sequence. xl-psid intensity analysis with decile groupings and functional characterization as a prelude to functional analysis, we rank ordered the xl-psids based on their average intensity values. the ten xl-psids with the highest intensity values were: hemoglobin, gamma a, hbg1; ribosomal protein s rps27; ferritin, fth1; ubiquitin b, ubb; ribosomal protein s rps13; solute carrier family, member slc11a2; ribosomal protein s rps20; unknown sequence; ribosomal protein s rps14; and hypothetical protein mgc114621/ribosomal protein, rplp we partitioned the xl-psids into deciles by two methods, equal number of xl-psids and equal range of average intensity values. for equal tally deciles, the variation of xl-psid average intensity values were low and comparable for all the deciles except for the 10th. in contrast to equal tally deciles, of the xl-psids in the equal intensity deciles were grouped in the 10th decile. xl-psids were divided into decile groupings either by number or intensity; 10th decile corresponds to the highest intensity levels. in the equal tally distribution, each decile comprises an equal number of xl-psids. in the equal intensity distribution, each decile comprises xl-psids within an equal intensity range. the number of xl-psids without annotation after david queries is listed in the orphan xl-psid column. we focused our functional analysis on the 10th deciles, which comprise xl-psids with the highest average intensity values in both instances. the database for annotation, visualization and integrated discovery, kegg and sp-pir terms. as shown on table 4a the most common functional annotation for the 10th equal tally decile was the go term “cellular processes”. david analysis also classified the top 10% of xl-psids into other annotation categories, including “biosynthetic processes”, “gene expression”, “translation”, “non-membrane-bounded organelle”, and “structural molecule activity”. the most common functional annotations for the 10th equal intensity decile were: “non-membrane-bounded organelle”, “intracellular non-membrane-bounded organelle”, and “translation”. we observed that some xl-psids were clustered in multiple functional categories. moreover, david reported an “orphan” status for % of the xl-psids in the 10th equal tally decile and % of the xl-psids in the 10th equal intensity decile. we also noted a similar trend in both decile groupings; the number of orphan xl-psids within a decile decreased as the intensity values of their decile increased. in comparison to david, % of the xl-psids in the 10th equal tally decile and 10% of the xl-psids in the 10th equal intensity decile were without gene annotation based on the annotation file provided by the vendor. the functional clusters with the highest david enrichment scores are shown for xl-psids in the 10th equal tally and the 10th equal intensity decile. david assigned ids to the 10th equal tally decile, resulting in annotation clusters. david assigned ids to the 10th equal intensity decile, resulting in annotation clusters. assigning inner ear gene categories to xl-psids as a prelude to analyzing the inner ear transcriptome, we identified xl-psids on the x. laevis genechip® with a probable role in the maintenance and function of auditory and vestibular inner ear endorgans. we selected five gene categories for intensity analysis: inner ear tissue, deafness, ion channels, ion transport, and transcription factors. the venn diagram in figure  shows the overlap between the five inner ear gene categories. several approaches were used to assign these gene categories to xl-psids. the number of xl-psids in all inner ear gene categories and in the 10th deciles are reported together with compilation and analysis methods for each gene group. the table reports the number of symbols for hgnc proteins that formed affirmative pairwise alignments with xl-psid consensus sequences through sequence similarity mapping. *hgnc symbols were linked to more than one xl-psid, resulting in a number that is higher than the number xl-psids. mapping iet, df and ic inner ear gene categories to xl-psids to assess the utility of the x. laevis genechip® in inner ear array studies, we evaluated whether genes associated with inner ear function in xenopus and other organisms were arrayed on the chip. to this end, we used sequence similarity mapping with the tblastn algorithm to determine whether hgnc human protein sequences from the iet, df, and ic gene lists aligned with xl-psid consensus sequences. the top blast pairwise alignment was used to assign putative function to the xl-psid consensus sequence. hgnc human protein sequences formed affirmative pairwise alignment with xl-psid consensus sequences. we noted that in some instances a single xl-psid aligned with multiple human protein sequences. xl-psid intensity analysis of inner ear gene categories as shown in figure 4a, the histogram of average intensity values for all experimental xl-psids on the microarray was characterized by an asymmetrical left-skewed distribution. since approximately % of xl-psids were scored with average intensity levels above four, we estimated that two-thirds of the x. laevis genechip® could be used to detect xenopus inner ear transcripts. the histograms of average intensity values for each of the five gene categories also showed an asymmetrical left-skewed distribution. the majority of xl-psids were scored with average intensity levels greater than or equal to “four” in all gene categories except “transcription factor”. inner ear tissue genes approximately % of genes from the iet list were linked by affirmative pairwise alignments to xl-psids with intensities ranging from to. average intensities above four were detected from approximately 68% of iet/xl-psids. we noted that about % of the iet/xl-psids were clustered in the top two equal tally deciles. the range of intensities for iet/xl-psids was greatest in the 10th decile. when the iet/xl-psids were grouped into equal intensity deciles, the 5th decile contained the most iet/xl-psids with intensities that ranged from -. the iet genes linked to the xl-psids with the highest intensities are listed in table 6a. the xl-psids with the highest intensity values are listed for each inner ear gene category. the corresponding hgnc symbols or gene symbols are listed for all categories. in, two pairs of xl-psids correspond to the same hgnc symbol and have probes derived from the same consensus sequence. one xl-psid in did not have a hgnc symbol due to the lack of sequence similarity to a human orthologue. human deafness genes approximately 71% of df genes were linked by affirmative pairwise alignments to xl-psids with average intensities from -. figure 4c shows that % of df/xl-psids had average intensities greater than four. df/xl-psids were predominantly in the 5th, 9th, and 10th equal tally deciles. whereas, when df/xl-psids were grouped into equal intensity deciles, the distribution was mostly in the 3rd, 4th, and 5th deciles. the df genes linked to the xl-psids with the highest intensities are listed in table 6b and represent various cellular functions. ion channel genes approximately 69% of ic genes were linked by affirmative pairwise alignments to sequences for xl-psids that ranged in intensity from to. the small number of xl-psids relative to the number of genes is partially due to the fact that many ion channel pore subunits aligned to the same xl-psid. as a group, the ic/xl-psids have lower average intensity levels than both iet/xl-psids and df/xl-psids. only % of ic/xl-psids had average intensity values greater than four. the ic genes linked to xl-psids with the highest intensities were voltage-dependent anion-selective channels, glutamate receptors, and subunits from k+ and na+ channels. ion transport genes the xl-psids that represent it and ic genes on the x. laevis genechip® had intensities distributed from -. approximately 59% of it/xl-psids had average intensity values greater than four; most were in the 9th and 10th equal tally deciles. when it/xl-psids were grouped into equal intensity deciles, the 1st and 4th deciles contained the most it/xl-psids. it/xl-psids with the highest intensities were mostly subunits for sodium/potassium/hydrogen transporting atpases. putative transcription factors for this category of genes, % of the identified ptf/xl-psids have average intensity values above four. in contrast to iet/xl-psids, df/xl-psids, and it/xl-psids intensity values distributions, the majority of ptf/xl-psids are in the 1st equal tally decile as opposed to the 10th. however, when grouped into equal intensity deciles, the 1st and 4th deciles contained the most ptf/xl-psids. the ptf/xl-psids with the highest intensities are listed in table 6e. trends in xl-psid intensity distributions for inner ear gene categories we compared the distribution of xl-psid intensity values for the four gene categories in order to ascertain potential differences in the relative expression levels of inner ear genes based on functional classification. we observed that the ptf category, with the largest number of xl-psids, was characterized by the lowest intensity value distribution of all the gene categories. moreover, a larger percentage of the ptf/xl-psids have gcos absent calls as compared with xl-psids in the other categories. we further noted that the largest proportions of df/xl-psids, it/xl-psids, and iet/xl-psids were found in the 10th equal tally decile. manual curation efforts improved x. laevis genechip® annotation we implemented david analysis to assess whether or not manual curation improved the annotation results for iet/xl-psids, the largest category with manually-linked hgnc symbols. to this end, we compared the outcomes of david analyses for iet/xl-psids to that of iet/hgnc symbols. the iet category of iet/hgncs was represented by david ids, with one orphan iet/hgnc. david grouped inner ear specific go terms into two iet/hgnc functional annotation clusters. in contrast, for the corresponding iet/xl-psids, no inner ear specific go terms were recovered from analysis of the david annotation clusters. furthermore, the number of ids that david associated with iet/hgncs was greater than the number of ids that david associated with the iet/xl-psid counterparts. moreover, the number of orphan iet/xl-psids was greater than the single iet/hgnc orphan. taken together, these findings suggest that manual curation of the x. laevis genechip® by assignment of hgnc symbols to the xl-psids improved annotation. identification of putative human inner ear orthologues in the xenopus tropicalis genome in order to determine the extent to which xenopus is a practical model organism for auditory and vestibular research, we used the products of our manual curation efforts to evaluate whether genes associated with inner ear function in other organisms were present within the x. tropicalis genome. to this end, we used the blastp algorithm to determine whether hgnc human protein sequences from the iet, df, and ic gene lists aligned with curated x. tropicalis predicted proteins from the joint genome institute x. tropicalis sequencing project. the average e-value for hgnc/xt predicted protein mappings were lower than the average e-value for hgnc/xl-psid mappings. moreover, the number and percentage of affirmative pairwise alignments between hgnc human protein and x. tropicalis predicted protein sequences exceeded the number of affirmative pairwise alignments between hgnc human protein sequences and xl-psids. these sequence similarity alignments demonstrate that more orthologues with high similarity to human proteins from all three gene lists were identified in the xenopus genome than on the x. laevis genechip®. discussion microarray technology for transcriptional profiling of inner ear endorgans limited access to human inner ear rna mandates the use of model organisms, such as xenopus laevis for transcriptome analysis. cellular and molecular investigations of the inner ear are challenged by the size and location of the organ. the human cochlea measures almost 1 cm in size while the entire xenopus inner ear is about one third of that size; both are encased by the temporal bone, the densest bone in the body. we overcame the constraints imposed by the inaccessibility of the inner ear through implementation of rigorous surgical procedures that yielded high quality rna from a small amount of inner ear tissue. reproducibility between our arrayed biological replicates was evidenced by the similar intra-chip sds for xl-psid intensity values and the low inter-chip sds. these results illustrate the efficacy of our protocols in restricting biological and technical variation between replicates that may be introduced by experimental procedures such as tissue isolation, rna extraction, and array hybridization. the usefulness of inner ear genetics as an approach to develop treatments for inner ear disorders can be heightened through analysis of the relative expression of genes within the transcriptome. data normalization is a critical step in achieving this objective. box plots and mva plots of normalized data showed low inter-chip variability as compared to plots constructed with pre-normalized data, demonstrating the efficacy of the normalization method. we elected to use data normalized with gcrma because this data normalization method performs well with regard to accurate detection of low abundance transcripts and precision. this data normalization method also is recommended when the objectives are to identify differentially expressed genes, or to estimate relative gene expression. the ability to assess whether microarray intensity values are predictors of verifiable gene expression facilitates the interpretation of microarray data. in particular, it is useful to identify an intensity value, or threshold, above which pcr methods are likely to independently validate gene expression. inner ear cdna library clones represent expressed sequences and thus are a useful tool for assessing whether expressed sequences are detected in the array. the combination of gcos absent calls and intensity levels of cdna clones represented on the x. laevis genechip®, led us to predict that an xl-psid intensity value greater than or equal to four is likely to represent an expressed sequence that can be confirmed by rt-pcr. as expected, rt-pcr experiments with inner ear rna confirmed the presence of 100% of eight genes that mapped to xl-psids with average intensity levels above six. annotation curation expands the biological relevance of the microarray data transcriptome analysis is facilitated by genomic sequence data and quality gene annotation for the species of interest. the quality and the extent of annotation have been acknowledged as particular impediments to the advancement of transcriptomics. for this reason, the national human genome research institute initiated the encyclopedia of dna elements, a project aimed at the functional annotation of all elements in the human genome. the utility of the x. laevis genechip® is reduced by the number of arrayed genes with no known biological function, a limitation that stems in part from the unsequenced x. laevis genome. we enhanced the biological relevance of the data by using a variety of computational strategies to link xl-psids to hgnc official gene symbols. the linkage of hgnc symbols to xl-psids exploited the detailed annotations of human genes as compared to other species. sequence similarity mapping and semantic keyword querying facilitated the assignment of putative functions important for inner ear processes. our x. laevis genechip® annotation efforts focused on five gene categories relevant for inner ear function: inner ear tissue, deafness, ion channels, ion transporters and transcription factors. no single tool is sufficiently robust to assign function to genes from a species such as x. laevis, whose genome has not been sequenced. in order to impart biological function to our microarray data, it was mandatory to combine many curation approaches. we found that the best approach involved combining reading the scientific research literature, keyword and nucleotide database queries, and functional annotation clustering with data-mining tools from the david bioinformatics resource. david was useful for providing information about groups of xl-psids through functional clustering. for example, david analysis of the 10th deciles revealed many go categories associated with genes commonly found in all tissues and not exclusive to the inner ear, and identified the orphan xl-psids with no known annotation. gene groups facilitate the analysis of trends in the x. laevis inner ear transcriptome through transcriptional profiling of the inner ear, we aimed to garner a comprehensive perspective of an understudied organ. the implementation of gene lists and decile groupings facilitated the analysis of the inner ear transcriptome by restricting our focus to a subset of xl-psids culled from known inner ear genes and from xl-psids with intensity values in the top 10%. the combination of these two approaches allowed us to identify patterns in the relative intensities of xl-psids, to compare xenopus inner ear genes to the known inner ear genes of other species, and to gain insight about the contribution of genes with no known function to the inner ear transcriptome. trends in the intensity values for xl-psids were extricated through histogram and decile analysis. we observed that the distribution of xl-psid intensities in the histograms for categories with predicted inner ear function resembled the cumulative histogram for all xl-psids. we also noticed that all gene categories are represented in all equal tally and all equal intensity deciles. average intensity values were as follows: cumulative xl-psids,; iet/xl-psids,; df/xl-psids,; ic/xl-psids,; it/xl-psids,; ptf/xl-psids,. we interpret the similarities between xl-psid distributions for the inner ear transcriptome and the gene categories as indicating that the gene categories are representative of the whole inner ear transcriptome. this outcome is interesting because the inner ear research that formed the basis for our selection of gene categories was rich in the science of mechanosensory hair cells whose numbers comprise very few cells of the inner ear. for example, the auditory hair cells in a juvenile xenopus animal total approximately; whereas, cochlear hair cells of the human inner ear number approximately. nevertheless, the gene categories captured data that encompassed a full range of xl-psid intensity values. the utility of xenopus as a model organism for inner ear research is supported by the commonality we observed between the x. laevis transcriptome and the outcomes of gene analyses for other species traditionally used for auditory and vestibular research. many of the common genes are ion channels and transporters, transcription factors, gap junction proteins, cytoskeletal proteins, and structural proteins that have been implicated in inner ear function in humans and mice. various genes common to the x. laevis inner ear and those of other species are associated with deafness, including structural proteins; collagen, type i, alpha; and tectorin alpha ), all of which have corresponding df/xl-psid intensity levels above four. of the ion channel genes identified in both human cochlear and mouse organ of corti cdna libraries by gabashvili et al., nine were represented on the x. laevis genechip®. moreover, seven of these nine genes corresponded to xl-psids with intensity values above four. ion transporters that play a role in k+ cycling and maintenance of endolymph in the cochlea of human, mouse, and rat were also represented on the x. laevis genechip® by xl-psids with high intensities. additionally, transcription factors implicated in hair cell regeneration in the chicken inner ear, such as jun d proto-oncogene, ccaat/enhancer binding protein c/ebp, gamma, and paired box were identified as ptf/xl-psids with intensities above four in the x. laevis inner ear. the bone morphogenetic protein bmp, which is important for cochlea and sensory organ development in mouse and chicken, was also detected in xenopus. the prevalence of similar genes identified in both the human cochlea and xenopus inner ear support the notion that physiological processes essential for inner ear function are shared between the two species. it was notable that xl-psids with intensities in the top 1%, sparc; eukaryotic translation elongation factor alpha eef1a1) correspond to the most abundant transcripts identified in a human fetal cochlear cdna library. clusterin was found in human perilymph with high protein concentrations; currently the function of this glycoprotein in the inner ear is unknown. taken together, the identification of xl-psids from all five gene categories with high intensity values supports the use of x. laevis to advance our understanding of the genes critical for inner ear function. moreover, previously uncharacterized genes are now found to have a putative function in the xenopus inner ear. focusing our attention on xl-psids with the highest intensity values uncovered the genes that are predominant in the juvenile x. laevis inner ear transcriptome. our analysis of genes associated with the top xl-psids, as well as david analysis of the 10th decile, revealed that xl-psids with the highest intensity values in the x. laevis inner ear are linked to cellular maintenance functions, especially “housekeeping”. these cellular maintenance genes were represented in greater numbers in comparison to genes specific to inner ear function such as iet/xl-psids and df/xl-psids. these findings are consistent with observations by other researchers who have noted that genes influential in other tissue types are highly expressed in the inner ear. finally, our david analysis of the xenopus inner ear transcriptome revealed that 13% of the xl-psids in the 10th equal tally decile are “orphans” and have no annotation. analysis of the highest xl-psid intensity values highlighted the predominance of xl-psids without gene titles in the affymetrix annotation file; 12% of the most highly expressed xl-psids and % of the xl-psids in the 10th equal tally decile fell into this category. taken together, these results imply that the roles of many genes important for inner ear function have yet to be defined. as functional characterization of genomes expands through the use of interdisciplinary approaches and cross-species analysis, knowledge of the genetic elements essential to inner ear function and dysfunction is expected to increase.  the genus xenopus affords unique opportunities for inner ear research because of its utility as a developmental model for genetic investigations as well as the amphibian capacity for regeneration of mechanosensory hair cells and neural tissue. while amphibians have furthered our understanding of inner ear hair cell mechanotransduction and physiology, the inner ear transcriptome of amphibians is not comparably well-characterized. for this reason, we implemented microarray transcriptional profiling for large-scale analysis of the x. laevis inner ear transcriptome. we heightened the functional significance of our analysis by targeting groups of genes considered essential for inner ear function. we overcame challenges faced by investigators working with organisms with unsequenced genomes through informatics approaches that significantly enhanced the annotation of the x. laevis genechip®. our results suggest that the xenopus inner ear transcriptome comprises genes that share significant sequence similarity with genes associated with non-syndromic deafness in other species, as well as a high abundance of xl-psids with no known annotation. we propose that the aforementioned putative mammalian orthologues and unknown xl-psids identified in this study represent ideal targets for functional analysis through genetic approaches. our findings provide a resource that can be used by the xenopus community for shared research enterprises such as xendb, xenbase and the recently established national xenopus resource at the marine biological laboratory that produces transgenic xenopus. taken together, our results support the implementation of xenopus as a viable model for inner ear research, especially for investigation of hair cell regeneration, morphogenesis, and organogenesis. 
the senses of hearing and balance depend upon mechanoreception, a process that originates in the inner ear and shares features across species. 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. 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. in addition, annotation analysis revealed the prevalence of inner ear transcripts represented by probe set identifiers that lack functional characterization. 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. the use of gene categories facilitated the assignment of functional significance to probe set identifiers. in contrast, much less is known of the genetic basis of auditory and vestibular function in this class of animals. 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. we identified an abundance of targets for genetic analysis of auditory and vestibular function.
 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.  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 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.  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. amphibianauditorydeafnesshearingmicroarrayorganvestibular 