BACKGROUND
the vestibular system of the vertebrate inner ear is a highly complex set of structures that relays information about motion and spatial orientation to the brain.
the structural and functional properties of the vestibular apparatus are highly conserved among all vertebrate species.
the semicircular canals, one subdivision of the vestibular system, function as sensors of angular acceleration.
these structures enhance survival by implementing postural and visual stabilization during motion in a three-dimensional environment  <cit> .
zebrafish has emerged as an important model system for the study of ear development.
zebrafish represent an especially attractive system for analyzing otogenesis, owing to the fact that the ear can be easily visualized during early embryogenesis, and that many mutations in ear development have been described in zebrafish that appear to resemble those that cause human hearing and balance disorders .
in zebrafish, as well as other vertebrates, the semicircular canal system consists of three non-sensory components, the anterior, posterior, and lateral canal ducts  which are positioned in three different planes.
sensory regions lie at the end of each canal and detect differential flow of fluid through the non-sensory structures during angular rotation of the head.
the formation of the semicircular canals in zebrafish begins at  <dig> hpf with the development of three epithelial outpocketings: one anterior protrusion, one posterior protrusion, and one lateral protrusion  <cit> .
at  <dig> hpf, the lateral protrusion bifurcates into an anterior branch and a posterior branch that fuse with the anterior and posterior protrusions, respectively  <cit> .
shortly after the formation of the horizontal hub, a ventral protrusion develops at the bottom of the vesicle and fuses with the lateral protrusion.
finally, a thin septum arises from the dorsal wall forming a dorsolateral partition.
at  <dig> hpf, the four hubs are completely formed and appear as a cross shaped structure that spans the lumen of the otic vesicle.
little is currently known, however, regarding the underlying molecular mechanisms that govern the development of this intricate structure.
mutagenesis screens in zebrafish have uncovered several types of mutants with semicircular canal defects  <cit> .
for example, a mutation in the jekyll gene leads to interrupted ear columns  and malformations of pharyngeal cartilage  <cit> .
the jekyll gene encodes ugdh, an enzyme required for the production of several components of the extracellular matrix including hyaluronic acid  <cit> .
localized secretion of hyaluronic acid has been proposed to drive propulsion of the non-sensory epithelium during canal morphogenesis, a process shown to be necessary for proper semicircular canal formation in xenopus embryos  <cit> .
the zebrafish ortholog of the human deafness gene dfna <dig>  was subsequently shown to be necessary for ugdh expression and proper semicircular canal and jaw development  <cit> .
another zebrafish mutant, gallery, displays only a lateral semicircular canal protrusion and an immature anterior protrusion  <cit> .
although the gallery gene has not yet been identified, the defects in this mutant are believed to be caused by overexpression of bmp <dig>  in the presumptive cristae.
several other genes required for proper semicircular canal formation in zebrafish have recently been identified.
these include sparc, a gene encoding a matricellular glycoprotein  that functions in the genetic network regulating pharyngeal and inner ear development  <cit> , tmie , a gene coding for a protein of unknown function that is important for normal hearing  <cit> , and the na, k-atpase α subunit gene α1a.
<dig>  <cit> .
we have recently found that in zebrafish, the gene encoding neuronal calcium sensor-1a  is required for semicircular canal formation  <cit> .
ncs- <dig> is the mammalian ortholog of drosophila frequenin, a protein critical for synaptic transmission within the drosophila nervous system  <cit> .
in mammalian cells, ncs- <dig> has been shown to promote exocytosis from dense core vesicles in both neurons and neuroendocrine cells  <cit> , and plays a key role in g-protein coupled receptor desensitization via a direct interaction with the d <dig> dopamine receptor  <cit> .
in zebrafish, morpholino knockdown of ncs-1a blocks normal development of the non-sensory portion of the semicircular canals  <cit> .
however, the biochemical function of ncs- <dig> in regulating semicircular canal formation has not been elucidated.
to gain a better understanding of the role of ncs- <dig> in regulating inner ear development, we initiated a proteomic screen to identify ncs- <dig> binding partners .
using yeast two-hybrid and bioinformatic approaches, we identified a cohort of human nbps and their zebrafish orthologs.
we examined expression of ten nbps and identified seven that are expressed in the developing zebrafish inner ear.
based on their functional profiles, the hypothesis is presented that ncs-1a/pi4kβ/arf <dig> form a signaling pathway that is required for semicircular canal formation.
a second set of nbps, consisting of pink <dig>  hint <dig>  and slc25a <dig>  are destined for localization in mitochondria.
using antisense morpholinos, we show that pink <dig> is also required for proper development of the semicircular canals.
our findings reveal a cohort of novel intracellular nbps.
ncs-1/nbp interactions are likely to contribute to the molecular mechanisms that regulate the development of the vestibular apparatus.
RESULTS
identification of ncs- <dig> binding proteins
to identify ncs- <dig> binding proteins , we used full-length human ncs- <dig> as bait in a y2h screen of an adult human brain cdna library.
candidate ncs- <dig> binding partners are listed in table  <dig>  based on their functional properties we chose to pursue dan , a secreted bmp antagonist that has previously been shown to play a role in semicircular canal formation  <cit> ; vamp <dig>  a synaptosomal protein involved in exocytosis  <cit> ; and the mitochondrial proteins hint <dig>  slc25a <dig> , and pink <dig>  a mitochondrial kinase which has been found to be mutated in inherited forms of parkinson's disease  <cit> .
additional interacting proteins identified in the y2h screen were not examined further in this study.
by conducting literature and protein database searches, we also identified several previously characterized nbps  whose potential role in ear development was analyzed.
these include the adp-ribosylation protein arf <dig>  <cit> , pi4kβ  <cit> , the inositol  <dig> , <dig> triphosphate receptor ip3r  <cit> , and trpc  subtypes  <dig>  <cit>  and  <dig>  <cit> .
by using blast searches of the zebrafish est database and genomic sequences available from genbank and the zebrafish sequencing group at the sanger institute  we identified zebrafish orthologs of each of the ten human nbps.
the sources and accession numbers for each of the zebrafish orthologs of the human nbps are listed in table  <dig>  zebrafish ests were obtained from atcc .
in cases where ests contained partial sequences, full-length orfs  were generated by rt-pcr.
alignments of the predicted amino acid sequences of human nbps and their zebrafish orthologs are presented in additional file  <dig>
expression of nbps in zebrafish inner ear
we used whole-mount in situ hybridization to analyze mrna expression for each of the  <dig> nbps  during zebrafish otogenesis.
the otic expression profile of each candidate nbp gene at  <dig> hpf and  <dig> hpf is shown in fig.
<dig>  while additional file  <dig> shows expression of these genes at  <dig>   <dig> and  <dig> hpf in the head region.
genes were grouped according to their known functional properties  or intracellular localization , and their otic expression profiles compared with that of the ncs-1a gene .
all of the genes investigated were expressed within the brain at each time point examined, while some were detected at high levels in additional structures including branchial arches , somites , and heart .
similarly, ncs-1a expression was detected throughout the brain and in the posterior somites and pharyngeal pouches  <cit> .
at  <dig> hpf, ncs-1a transcripts can be visualized in the semicircular canal protrusions, and in the fully formed semicircular canal hubs at  <dig> hpf.
we first analyzed expression of arf <dig>  pi4kβ, and vamp <dig>  nbp genes encoding proteins involved in exocytosis and intracellular trafficking.
arf <dig> transcripts were expressed at  <dig> hpf in the epithelial protrusions of the semicircular canals but were not present in the semicircular canal hubs at  <dig> hpf, suggesting that arf <dig> contributes to early events in semicircular canal formation.
expression of pi4kβ mrna, on the other hand, was very similar to the expression profile seen for ncs-1a mrna.
pi4kβ transcripts were detected at  <dig> hpf in the semicircular canal protrusions and in the fully formed semicircular canal hubs at  <dig> hpf.
vamp <dig> mrna was undetectable in the inner ear at either time point, indicating that vamp <dig> is unlikely to play a role in otogenesis.
three nbp genes, pink <dig>  hint <dig>  and slc25a <dig>  each encoding a mitochondrial protein, showed very similar expression patterns.
transcripts of each gene were detected at  <dig> hpf in the semicircular canal protrusions and the anterior macula, and at  <dig> hpf in the semicircular canal hubs.
amongst the genes encoding calcium channels, trpc <dig> transcripts were detectable at  <dig> hpf and  <dig> hpf in the semicircular canal protrusions and hubs, respectively.
transcripts of the other two calcium channel genes, trpc <dig> and ip3r, were undetectable in the inner ear at either  <dig> hpf or  <dig> hpf.
expression of dan, a gene encoding a secreted protein involved in bmp signaling, was similar to that of the pi4kβ gene and was present in the semicircular canals at both  <dig> hpf and  <dig> hpf.
taken together, our data indicate that seven nbp genes including arf <dig>  pi4kβ, trpc <dig>  dan, pink <dig>  hint <dig>  and slc25a <dig>  are expressed in the semicircular canals during zebrafish embryogenesis and are likely to contribute to the formation of the vestibular apparatus.
validation of ncs-1/nbp interactions
we used pulldown techniques to confirm the interaction between ncs- <dig> and several of the nbps expressed in ear.
lysates from bacteria expressing s-tagged zebrafish proteins arf <dig> , dan , pi4kβ , pink <dig> , or hint <dig>  were tested for the ability to associate with an ncs-1-gst fusion protein containing full-length  human ncs- <dig>  in this context it is important to note that zebrafish ncs1a and human ncs- <dig> differ at only  <dig> of  <dig> positions  <cit> .
pulldowns were not performed for slc25a <dig> or trpc <dig>  as shown in fig.
<dig>  a western blot containing lysate from bacteria expressing s-tagged arf <dig> produced an immunoreactive band of ~ <dig> kda when probed with an anti-s-protein antibody.
this band corresponds to the expected size of the arf <dig> fragment encoded by the cdna construct.
the same band was detected by pulldown after the bacterial lysate was incubated with the ncs-1-gst fusion protein, but not when the lysate was incubated with gst-containing beads alone.
we next used the gst-pulldown assay to test whether ncs- <dig> also interacted with dan, pi4kβ, hint <dig>  and pink <dig> .
lysates from bacteria expressing s-tagged fragments of dan , pi4kβ , hint <dig> , and pink <dig> , each produced an immunoreactive band of the expected size .
in each case, the same band was detected by pulldown after incubation with the ncs-1-gst fusion protein .
virtually no immunoreactivity was detected when the lysates were incubated with beads containing gst alone .
these results support the validity of the interaction between ncs- <dig> and arf <dig>  dan, pi4kβ, pink <dig>  and hint <dig>
knockdowns of nbps involved in exocytosis and secretion
to gain an understanding of the function of nbps during otogenesis, we used morpholino antisense oligonucleotides to knock down translation of selected nbp mrnas in developing zebrafish.
our initial experiments involved knocking down translation of arf <dig> and pi4kβ mrnas, encoding nbps involved in exocytosis, and dan mrna, encoding a secreted nbp known to play a role in semicircular canal formation  <cit> .
we generated a set of non-overlapping morpholinos  targeted against either the initiating methionine  or the 5'-utr  of pi4kβ, arf <dig>  and dan mrnas , and microinjected the mos into one cell-stage embryos.
representative examples of the nbp knockdowns are shown in fig.
<dig>  in wild type embryos, saccular and utricular otoliths are present at  <dig> hpf, and the semicircular canals protrusions are clearly visible .
by  <dig> hpf, the epithelial pillars have fused to form the semicircular canal hubs exhibiting the characteristic cruciform pattern .
microinjection of mos against each of the three nbp genes produced noticeable effects on inner ear development.
mos targeted against either pi4kβ , arf <dig> , or dan , produced morphants with a shortened body axis, reduced eye size, and curved tails.
these defects were most pronounced in dan morphants , even at low doses  of the dan mo.
at  <dig> hpf, semicircular canal protrusions were missing in > 90% of pi4kβ , arf <dig> , and dan  morphants .
analysis of the developing ear through  <dig> hpf revealed that each of the nbp morphants exhibited normal otic placode induction and otolith formation compared to control embryos.
at  <dig> hpf, we observed that many of the morphants had begun to develop semicircular canals.
however, the canals exhibited a range of aberrant morphologies including missing protrusions , failure of the pillars to fuse , as well as the presence of disorganized cell masses in the otic vesicle .
for all genes tested, injection of a second, independent mo  phenocopied the ear defects generated by the primary mo .
in general, higher doses of the secondary mo were required to achieve effects comparable to those produced by the primary mo on semicircular canal formation.
together, these mo knockdown experiments provide compelling evidence that in addition to ncs-1a, expression of arf <dig>  pi4kβ, and dan genes is required for semicircular canal formation in zebrafish.
ncs-1a, pi4kβ, and arf <dig> functionally interact to regulate formation of semicircular canals
ncs- <dig>  arf <dig>  and pi4kβ have been shown to functionally interact in pc <dig> cells to regulate neuronal secretion  <cit> .
we wished to determine whether these three genes functionally interact within the zebrafish inner ear to regulate semicircular canal development.
to test this idea, we co-injected subeffective doses of ncs-1a-atg  plus arf1-utr  mos, ncs-1a-atg plus pi4kβ-atg  mos, and arf1-utr plus pi4kβ-atg mos, and examined the effect on semicircular canal formation.
the results are shown in fig.
<dig>  at a concentration of  <dig>  ng, neither the ncs-1a-atg mo nor the arf1-utr mo produced severe semicircular canal development defects, although a small percentage of fish  displayed no epithelial protrusions at  <dig> hpf.
100% of embryos injected with  <dig> ng of the pi4kβ-atg mo exhibited normal semicircular canal development at  <dig> hpf.
however, co-injection of subeffective doses of ncs-1a-atg plus arf1-utr mos caused an absence of epithelial protrusions in 53% of embryos at  <dig> hpf.
co-injection of ncs-1a-atg plus pi4kβ-atg mos caused an absence of epithelial protrusions in 40% of embryos, while co-injection of subeffective doses of arf1-utr plus pi4kβ-atg mos blocked outgrowth of epithelial protrusions in 44% of embryos.
at  <dig> hpf, < 7% of embryos injected with subeffective doses of either ncs-1a-atg, arf1-utr, or pi4kβ-atg mos alone exhibited abnormal semicircular canal hub formation.
abnormal semicircular canal hub formation was observed in 58% of embryos co-injected with subeffective doses of ncs-1a plus arf <dig> mos, in 38% of embryos co-injected with ncs-1a-atg plus pi4kβ-atg mos, and in 49% of embryos co-injected with subeffective doses of arf1-utr plus pi4kβ-atg mos.
co-injection of each pair of mos thus appears to amplify the semicircular canal defects observed when subeffective doses of either ncs-1a-atg, arf1-utr, or pi4kβ-atg mos are injected alone.
the functional interaction observed in this assay, in addition to the demonstrated physical interaction of their encoded proteins, suggests that ncs1a, arf <dig>  and pi4kβ may act in a common pathway leading to formation of the semicircular canal system.
pink <dig> is required for semicircular canal formation
pink <dig> was initially implicated as an nbp in a y2h screen , while the interaction of zebrafish pink <dig> with ncs- <dig> was verified via pulldown with an ncs-1-gst fusion protein .
whole-mount in situ hybridization analysis revealed expression of pink <dig> mrna sequences in the epithelial pillars and hubs of the semicircular canals during zebrafish otogenesis .
to directly determine whether pink <dig> expression was necessary for ear development, we knocked down pink <dig> mrna translation with a pink1-atg mo and examined the effect on semicircular canal formation.
a representative set of pink <dig> morphants is shown in fig.
<dig>  in control embryos at  <dig> hpf , otoliths are present and the epithelial protrusions of the semicircular canals are clearly visible.
in  <dig> hpf mo-injected embryos, otoliths appear to have developed normally, although the overall size of the otocyst is somewhat smaller compared to control embryos.
microinjection of either  <dig> ng  or  <dig> ng  of a pink1-atg mo produced morphants that were missing semicircular canal protrusions at  <dig> hpf in > 95% of embryos examined .
these results strongly support the idea that expression of the pink <dig> gene is required for semicircular canal formation in zebrafish.
discussion
ncs- <dig> is a calcium sensing molecule that is required for semicircular canal formation in zebrafish  <cit> .
to better understand of the role of ncs- <dig> in ear development, we employed a multistep functional proteomic strategy to identify molecular partners of ncs- <dig> that contribute to the development of the vestibular apparatus.
first, by performing a y2h screen in combination with literature and database searches, we identified  <dig> human nbps.
second, zebrafish orthologs of each of these nbps were identified using blast searches of the zebrafish est and genomic databases.
third, by investigating the expression profiles of nbps, we were able to deduce which candidate nbps were likely to interact with ncs-1a in the ear and contribute to semicircular canal formation.
we found that mrna sequences encoding seven of the putative zebrafish nbps were detected in the developing ear and exhibited an overlap in expression with that of the ncs-1a gene.
on the other hand, three nbp genes  were not expressed in the ear, and thus were eliminated from further analysis.
fourth, gst pulldown experiments were carried out to confirm that candidate zebrafish nbps also interacted with ncs- <dig>  finally, mo-mediated knockdowns confirmed that four selected nbps were essential for semicircular canal formation in zebrafish.
the strategy outlined above has proven to be a fruitful method for identifying novel genes that contribute to inner ear development.
using this approach, we show that two previously identified  and two novel  nbp genes are required for semicircular canal formation .
in the present study, we did not examine the role of hint <dig>  slc25a <dig>  or trpc <dig> using a morpholino knockdown approach.
further studies will be required to analyze the role of these and other nbps in ear development.
mutagenesis screens in zebrafish combined with positional cloning approaches have traditionally been used to identify genes that contribute to ear development  <cit> .
for example, the zebrafish dog-eared mutation was identified in a large-scale enu screen by abnormal morphology of the inner ear  <cit> .
the identity of this gene was then determined to be zebrafish eya <dig> via positional cloning  <cit> .
while mutagenesis screens provide an unbiased strategy for gene discovery, this approach is generally extremely labor intensive and time consuming.
our strategy, on the other hand, circumvents some of the limitations inherent in an unbiased screen, by rapidly identifying interacting partners of genes already known to regulate inner ear development.
this approach, in combination with the reverse genetic techniques available in zebrafish, should make it feasible to identify in a systematic fashion additional genes whose roles in ear development were previously unknown or overlooked.
the specificity of the phenotypes obtained by mo knockdown is supported by a number of considerations.
first, morpholinos targeted against pi4kβ, arf <dig>  and dan all caused similar phenotypes including vestibular malformation, shortened body axis, reduced head and eye size, and curved tails.
since each of these genes is expressed throughout the brain and jaw during embryonic development, defects in head formation were not unexpected.
these defects are not likely due to off-target effects, since for each of the genes tested, two independent non-overlapping mos produced identical phenotypes.
second, injection of buffer alone did not produce morphant phenotypes.
third, a mo targeted against otopetrin, a gene previously shown to be required for otolith formation  <cit> , blocked development of otoliths but did not affect semicircular canal development.
fourth, injection of subeffective doses of pi4kβ, arf <dig>  and ncs-1a mos alone did not produce inner ear, jaw, or brain defects.
co-injection of subeffective doses of mos against a pair of genes was synergistic and phenocopied the ear, jaw, and brain defects obtained using an effective dose of each of mo alone.
finally, brain and ear development appear normal in pi4kβ, arf <dig>  and dan morphants until  <dig> hpf, indicating that disruption of early patterning events is not likely to have caused vestibular phenotypes or developmental delay.
together, these results support the idea that the phenotypes produced by nbp knockdowns are specific, and do not result from non-specific cytotoxic or off-target gene effects.
our double mo knockdown studies, together with the pulldown experiments, indicate that ncs- <dig>  arf <dig> and pi4kβ form a small interaction network  that is likely to have functional significance for development of the zebrafish semicircular canal system.
we now propose a model in which these three proteins regulate the targeted secretion of molecules, such as dan, that are required for formation of the semicircular canals .
this model is based on the following lines of evidence.
pi4kβ is the enzyme responsible for the synthesis of phosphatidylinositol 4-phosphate, a lipid species that regulates trafficking of cargo from the golgi apparatus to the plasma membrane in yeast and mammalian cells  <cit> .
pi4kβ has also been shown to regulate secretion in both pc <dig> cells and pancreatic β cells  <cit> .
ncs- <dig>  <cit>  and arf <dig>  <cit>  are known activators of pi4kβ, however a direct interaction between ncs- <dig> and arf <dig> has been shown to antagonize the enzymatic activity of pi4kβ  <cit> .
bidirectional control of pi4kβ activation/inhibition may thus serve to regulate the trafficking of molecules involved in inner ear development from the trans-golgi network to the plasma membrane during otogenesis.
dan, bmp <dig>  and hyaluronic acid represent secreted molecules that play critical roles in semicircular canal formation  <cit>  and may require an ncs-1a/arf1/pi4kβ pathway for secretion.
bmps are expressed in the developing vertebrate inner ear and participate in axial patterning of the otic vesicle and differentiation of the sensory epithelium  <cit> .
bmp <dig> is secreted from the apical membrane of cells located in the cristae of the otic vesicle and its regulated expression is essential for proper development of the non-sensory portion of the semicircular canals  <cit> .
dan is a bmp antagonist that plays a key role in the patterning of the chick inner ear, and is also secreted from the apical membrane  <cit> .
in chick, loss of dan leads to structural deformities in the semicircular canals and the endolymphatic duct and sac.
the mo knockdown experiments we performed show that dan also contributes to semicircular canal formation in zebrafish.
we have also demonstrated a direct interaction between dan and ncs- <dig>  although the significance of this interaction is currently unknown, it is possible that the dan/ncs- <dig> interaction may be required to deliver dan to the golgi apparatus.
this idea is supported by previous studies which showed that the yeast ortholog of ncs- <dig>  can recruit pik1p  from the endoplasmic reticulum to the trans-golgi network  <cit> .
hyaluronic acid  is an extracellular matrix molecule that is secreted from the basolateral membrane of otic epithelium during semicircular canal formation and is thought to drive the outgrowth of the developing protrusions  <cit> .
hyaluronan synthases  are the enzymes responsible for ha synthesis and secretion at the plasma membrane  <cit> .
it is possible that trafficking of has from the golgi apparatus to the plasma membrane may also be regulated by pi4kβ activity.
since ha secretion from epithelial pillars in zebrafish inner ear can be monitored histochemically  <cit> , it should be possible to test this hypothesis by determining whether knockdown of ncs-1a, arf <dig>  or pi4kβ inhibits ha secretion.
our yeast two-hybrid screen revealed interaction of ncs- <dig> with three proteins, hint <dig>  slc25a <dig>  and pink <dig>  that are destined to localize to mitochondria.
the interaction of ncs- <dig> with pink <dig> - induced putative kinase 1) is of particular interest.
mutations in the human pink <dig> gene are the second most common cause of autosomal recessive parkinson's disease  <cit> , and are also implicated in sporadic cases of the disease  <cit> .
mo knockdown studies we performed indicate that pink <dig> morphants exhibit severe brain abnormalities as well as defects in development of the semicircular canal system.
interestingly, a recent study in zebrafish also showed that pink <dig> morphants display severe defects in brain morphology, as well an impaired escape response  <cit> .
in this context, it is important to consider that no direct link has been established between mutations in pink <dig> and defects in inner ear development in mammalian systems.
it is therefore possible that pink <dig> has evolved to perform a critical role in brain and ear development in zebrafish, while mammals may have developed compensatory mechanisms that regulate development of these organs.
the interaction of ncs- <dig> with zebrafish pink <dig> raises another interesting issue.
ncs- <dig> does not possess a defined mitochondrial targeting sequence, and its expression in mitochondria has not been documented.
in mammalian cells, ncs- <dig> is constitutively associated with the plasma membrane and golgi complex  <cit> .
it will be important to determine whether ncs- <dig> serves to regulate pink <dig> function, either within mitochondria or other cellular compartments, and whether the ncs-1/pink <dig> interaction contributes to the etiology of parkinson's disease.
CONCLUSIONS
in this study, we show that several new and previously identified nbps appear to play an important role in the development of the zebrafish inner ear.
we suggest a model in which ncs-1a/arf1/pi4kβ interactions may form a signaling pathway that mediates trafficking and export of secreted molecules that are required for semicircular canal formation.
we also made an unexpected observation that ncs- <dig> interacts with several mitochondrial proteins, including pink <dig>  a gene that contributes to familial parkinson's disease.
these findings suggest a previously unrecognized role for ncs- <dig> in mitochondrial function via its association with mitochondrial proteins, and reveal a new and potentially important signaling pathway involved in development of the vestibular apparatus.

 the vestibular system of the vertebrate inner ear is a highly complex set of structures that relays information about motion and spatial orientation to the brain. the structural and functional properties of the vestibular apparatus are highly conserved among all vertebrate species. the semicircular canals, one subdivision of the vestibular system, function as sensors of angular acceleration. these structures enhance survival by implementing postural and visual stabilization during motion in a three-dimensional environment. zebrafish has emerged as an important model system for the study of ear development. zebrafish represent an especially attractive system for analyzing otogenesis, owing to the fact that the ear can be easily visualized during early embryogenesis, and that many mutations in ear development have been described in zebrafish that appear to resemble those that cause human hearing and balance disorders. in zebrafish, as well as other vertebrates, the semicircular canal system consists of three non-sensory components, the anterior, posterior, and lateral canal ducts which are positioned in three different planes. sensory regions lie at the end of each canal and detect differential flow of fluid through the non-sensory structures during angular rotation of the head. the formation of the semicircular canals in zebrafish begins at hpf with the development of three epithelial outpocketings: one anterior protrusion, one posterior protrusion, and one lateral protrusion. at hpf, the lateral protrusion bifurcates into an anterior branch and a posterior branch that fuse with the anterior and posterior protrusions, respectively. shortly after the formation of the horizontal hub, a ventral protrusion develops at the bottom of the vesicle and fuses with the lateral protrusion. finally, a thin septum arises from the dorsal wall forming a dorsolateral partition. at hpf, the four hubs are completely formed and appear as a cross shaped structure that spans the lumen of the otic vesicle. little is currently known, however, regarding the underlying molecular mechanisms that govern the development of this intricate structure. mutagenesis screens in zebrafish have uncovered several types of mutants with semicircular canal defects. for example, a mutation in the jekyll gene leads to interrupted ear columns and malformations of pharyngeal cartilage. the jekyll gene encodes ugdh, an enzyme required for the production of several components of the extracellular matrix including hyaluronic acid. localized secretion of hyaluronic acid has been proposed to drive propulsion of the non-sensory epithelium during canal morphogenesis, a process shown to be necessary for proper semicircular canal formation in xenopus embryos. the zebrafish ortholog of the human deafness gene dfna was subsequently shown to be necessary for ugdh expression and proper semicircular canal and jaw development. another zebrafish mutant, gallery, displays only a lateral semicircular canal protrusion and an immature anterior protrusion. although the gallery gene has not yet been identified, the defects in this mutant are believed to be caused by overexpression of bmp in the presumptive cristae. several other genes required for proper semicircular canal formation in zebrafish have recently been identified. these include sparc, a gene encoding a matricellular glycoprotein that functions in the genetic network regulating pharyngeal and inner ear development, tmie, a gene coding for a protein of unknown function that is important for normal hearing, and the na, k-atpase α subunit gene α1a.. we have recently found that in zebrafish, the gene encoding neuronal calcium sensor-1a is required for semicircular canal formation. ncs- is the mammalian ortholog of drosophila frequenin, a protein critical for synaptic transmission within the drosophila nervous system. in mammalian cells, ncs- has been shown to promote exocytosis from dense core vesicles in both neurons and neuroendocrine cells, and plays a key role in g-protein coupled receptor desensitization via a direct interaction with the d dopamine receptor. in zebrafish, morpholino knockdown of ncs-1a blocks normal development of the non-sensory portion of the semicircular canals. however, the biochemical function of ncs- in regulating semicircular canal formation has not been elucidated. to gain a better understanding of the role of ncs- in regulating inner ear development, we initiated a proteomic screen to identify ncs- binding partners. using yeast two-hybrid and bioinformatic approaches, we identified a cohort of human nbps and their zebrafish orthologs. we examined expression of ten nbps and identified seven that are expressed in the developing zebrafish inner ear. based on their functional profiles, the hypothesis is presented that ncs-1a/pi4kβ/arf form a signaling pathway that is required for semicircular canal formation. a second set of nbps, consisting of pink hint and slc25a are destined for localization in mitochondria. using antisense morpholinos, we show that pink is also required for proper development of the semicircular canals. our findings reveal a cohort of novel intracellular nbps. ncs-1/nbp interactions are likely to contribute to the molecular mechanisms that regulate the development of the vestibular apparatus.  identification of ncs- binding proteins to identify ncs- binding proteins, we used full-length human ncs- as bait in a y2h screen of an adult human brain cdna library. candidate ncs- binding partners are listed in table based on their functional properties we chose to pursue dan, a secreted bmp antagonist that has previously been shown to play a role in semicircular canal formation; vamp a synaptosomal protein involved in exocytosis; and the mitochondrial proteins hint slc25a, and pink a mitochondrial kinase which has been found to be mutated in inherited forms of parkinson's disease. additional interacting proteins identified in the y2h screen were not examined further in this study. by conducting literature and protein database searches, we also identified several previously characterized nbps whose potential role in ear development was analyzed. these include the adp-ribosylation protein arf, pi4kβ, the inositol, triphosphate receptor ip3r, and trpc subtypes and. by using blast searches of the zebrafish est database and genomic sequences available from genbank and the zebrafish sequencing group at the sanger institute we identified zebrafish orthologs of each of the ten human nbps. the sources and accession numbers for each of the zebrafish orthologs of the human nbps are listed in table zebrafish ests were obtained from atcc. in cases where ests contained partial sequences, full-length orfs were generated by rt-pcr. alignments of the predicted amino acid sequences of human nbps and their zebrafish orthologs are presented in additional file expression of nbps in zebrafish inner ear we used whole-mount in situ hybridization to analyze mrna expression for each of the nbps during zebrafish otogenesis. the otic expression profile of each candidate nbp gene at hpf and hpf is shown in fig. while additional file shows expression of these genes at and hpf in the head region. genes were grouped according to their known functional properties or intracellular localization, and their otic expression profiles compared with that of the ncs-1a gene. all of the genes investigated were expressed within the brain at each time point examined, while some were detected at high levels in additional structures including branchial arches, somites, and heart. similarly, ncs-1a expression was detected throughout the brain and in the posterior somites and pharyngeal pouches. at hpf, ncs-1a transcripts can be visualized in the semicircular canal protrusions, and in the fully formed semicircular canal hubs at hpf. we first analyzed expression of arf pi4kβ, and vamp nbp genes encoding proteins involved in exocytosis and intracellular trafficking. arf transcripts were expressed at hpf in the epithelial protrusions of the semicircular canals but were not present in the semicircular canal hubs at hpf, suggesting that arf contributes to early events in semicircular canal formation. expression of pi4kβ mrna, on the other hand, was very similar to the expression profile seen for ncs-1a mrna. pi4kβ transcripts were detected at hpf in the semicircular canal protrusions and in the fully formed semicircular canal hubs at hpf. vamp mrna was undetectable in the inner ear at either time point, indicating that vamp is unlikely to play a role in otogenesis. three nbp genes, pink hint and slc25a each encoding a mitochondrial protein, showed very similar expression patterns. transcripts of each gene were detected at hpf in the semicircular canal protrusions and the anterior macula, and at hpf in the semicircular canal hubs. amongst the genes encoding calcium channels, trpc transcripts were detectable at hpf and hpf in the semicircular canal protrusions and hubs, respectively. transcripts of the other two calcium channel genes, trpc and ip3r, were undetectable in the inner ear at either hpf or hpf. expression of dan, a gene encoding a secreted protein involved in bmp signaling, was similar to that of the pi4kβ gene and was present in the semicircular canals at both hpf and hpf. taken together, our data indicate that seven nbp genes including arf pi4kβ, trpc dan, pink hint and slc25a are expressed in the semicircular canals during zebrafish embryogenesis and are likely to contribute to the formation of the vestibular apparatus. validation of ncs-1/nbp interactions we used pulldown techniques to confirm the interaction between ncs- and several of the nbps expressed in ear. lysates from bacteria expressing s-tagged zebrafish proteins arf, dan, pi4kβ, pink, or hint were tested for the ability to associate with an ncs-1-gst fusion protein containing full-length human ncs- in this context it is important to note that zebrafish ncs1a and human ncs- differ at only of positions. pulldowns were not performed for slc25a or trpc as shown in fig. a western blot containing lysate from bacteria expressing s-tagged arf produced an immunoreactive band of ~ kda when probed with an anti-s-protein antibody. this band corresponds to the expected size of the arf fragment encoded by the cdna construct. the same band was detected by pulldown after the bacterial lysate was incubated with the ncs-1-gst fusion protein, but not when the lysate was incubated with gst-containing beads alone. we next used the gst-pulldown assay to test whether ncs- also interacted with dan, pi4kβ, hint and pink. lysates from bacteria expressing s-tagged fragments of dan, pi4kβ, hint, and pink, each produced an immunoreactive band of the expected size. in each case, the same band was detected by pulldown after incubation with the ncs-1-gst fusion protein. virtually no immunoreactivity was detected when the lysates were incubated with beads containing gst alone. these results support the validity of the interaction between ncs- and arf dan, pi4kβ, pink and hint knockdowns of nbps involved in exocytosis and secretion to gain an understanding of the function of nbps during otogenesis, we used morpholino antisense oligonucleotides to knock down translation of selected nbp mrnas in developing zebrafish. our initial experiments involved knocking down translation of arf and pi4kβ mrnas, encoding nbps involved in exocytosis, and dan mrna, encoding a secreted nbp known to play a role in semicircular canal formation. we generated a set of non-overlapping morpholinos targeted against either the initiating methionine or the 5'-utr of pi4kβ, arf and dan mrnas, and microinjected the mos into one cell-stage embryos. representative examples of the nbp knockdowns are shown in fig. in wild type embryos, saccular and utricular otoliths are present at hpf, and the semicircular canals protrusions are clearly visible. by hpf, the epithelial pillars have fused to form the semicircular canal hubs exhibiting the characteristic cruciform pattern. microinjection of mos against each of the three nbp genes produced noticeable effects on inner ear development. mos targeted against either pi4kβ, arf, or dan, produced morphants with a shortened body axis, reduced eye size, and curved tails. these defects were most pronounced in dan morphants, even at low doses of the dan mo. at hpf, semicircular canal protrusions were missing in > 90% of pi4kβ, arf, and dan morphants. analysis of the developing ear through hpf revealed that each of the nbp morphants exhibited normal otic placode induction and otolith formation compared to control embryos. at hpf, we observed that many of the morphants had begun to develop semicircular canals. however, the canals exhibited a range of aberrant morphologies including missing protrusions, failure of the pillars to fuse, as well as the presence of disorganized cell masses in the otic vesicle. for all genes tested, injection of a second, independent mo phenocopied the ear defects generated by the primary mo. in general, higher doses of the secondary mo were required to achieve effects comparable to those produced by the primary mo on semicircular canal formation. together, these mo knockdown experiments provide compelling evidence that in addition to ncs-1a, expression of arf pi4kβ, and dan genes is required for semicircular canal formation in zebrafish. ncs-1a, pi4kβ, and arf functionally interact to regulate formation of semicircular canals ncs- arf and pi4kβ have been shown to functionally interact in pc cells to regulate neuronal secretion. we wished to determine whether these three genes functionally interact within the zebrafish inner ear to regulate semicircular canal development. to test this idea, we co-injected subeffective doses of ncs-1a-atg plus arf1-utr mos, ncs-1a-atg plus pi4kβ-atg mos, and arf1-utr plus pi4kβ-atg mos, and examined the effect on semicircular canal formation. the results are shown in fig. at a concentration of ng, neither the ncs-1a-atg mo nor the arf1-utr mo produced severe semicircular canal development defects, although a small percentage of fish displayed no epithelial protrusions at hpf. 100% of embryos injected with ng of the pi4kβ-atg mo exhibited normal semicircular canal development at hpf. however, co-injection of subeffective doses of ncs-1a-atg plus arf1-utr mos caused an absence of epithelial protrusions in 53% of embryos at hpf. co-injection of ncs-1a-atg plus pi4kβ-atg mos caused an absence of epithelial protrusions in 40% of embryos, while co-injection of subeffective doses of arf1-utr plus pi4kβ-atg mos blocked outgrowth of epithelial protrusions in 44% of embryos. at hpf, < 7% of embryos injected with subeffective doses of either ncs-1a-atg, arf1-utr, or pi4kβ-atg mos alone exhibited abnormal semicircular canal hub formation. abnormal semicircular canal hub formation was observed in 58% of embryos co-injected with subeffective doses of ncs-1a plus arf mos, in 38% of embryos co-injected with ncs-1a-atg plus pi4kβ-atg mos, and in 49% of embryos co-injected with subeffective doses of arf1-utr plus pi4kβ-atg mos. co-injection of each pair of mos thus appears to amplify the semicircular canal defects observed when subeffective doses of either ncs-1a-atg, arf1-utr, or pi4kβ-atg mos are injected alone. the functional interaction observed in this assay, in addition to the demonstrated physical interaction of their encoded proteins, suggests that ncs1a, arf and pi4kβ may act in a common pathway leading to formation of the semicircular canal system. pink is required for semicircular canal formation pink was initially implicated as an nbp in a y2h screen, while the interaction of zebrafish pink with ncs- was verified via pulldown with an ncs-1-gst fusion protein. whole-mount in situ hybridization analysis revealed expression of pink mrna sequences in the epithelial pillars and hubs of the semicircular canals during zebrafish otogenesis. to directly determine whether pink expression was necessary for ear development, we knocked down pink mrna translation with a pink1-atg mo and examined the effect on semicircular canal formation. a representative set of pink morphants is shown in fig. in control embryos at hpf, otoliths are present and the epithelial protrusions of the semicircular canals are clearly visible. in hpf mo-injected embryos, otoliths appear to have developed normally, although the overall size of the otocyst is somewhat smaller compared to control embryos. microinjection of either ng or ng of a pink1-atg mo produced morphants that were missing semicircular canal protrusions at hpf in > 95% of embryos examined. these results strongly support the idea that expression of the pink gene is required for semicircular canal formation in zebrafish. discussion ncs- is a calcium sensing molecule that is required for semicircular canal formation in zebrafish. to better understand of the role of ncs- in ear development, we employed a multistep functional proteomic strategy to identify molecular partners of ncs- that contribute to the development of the vestibular apparatus. first, by performing a y2h screen in combination with literature and database searches, we identified human nbps. second, zebrafish orthologs of each of these nbps were identified using blast searches of the zebrafish est and genomic databases. third, by investigating the expression profiles of nbps, we were able to deduce which candidate nbps were likely to interact with ncs-1a in the ear and contribute to semicircular canal formation. we found that mrna sequences encoding seven of the putative zebrafish nbps were detected in the developing ear and exhibited an overlap in expression with that of the ncs-1a gene. on the other hand, three nbp genes were not expressed in the ear, and thus were eliminated from further analysis. fourth, gst pulldown experiments were carried out to confirm that candidate zebrafish nbps also interacted with ncs- finally, mo-mediated knockdowns confirmed that four selected nbps were essential for semicircular canal formation in zebrafish. the strategy outlined above has proven to be a fruitful method for identifying novel genes that contribute to inner ear development. using this approach, we show that two previously identified and two novel nbp genes are required for semicircular canal formation. in the present study, we did not examine the role of hint slc25a or trpc using a morpholino knockdown approach. further studies will be required to analyze the role of these and other nbps in ear development. mutagenesis screens in zebrafish combined with positional cloning approaches have traditionally been used to identify genes that contribute to ear development. for example, the zebrafish dog-eared mutation was identified in a large-scale enu screen by abnormal morphology of the inner ear. the identity of this gene was then determined to be zebrafish eya via positional cloning. while mutagenesis screens provide an unbiased strategy for gene discovery, this approach is generally extremely labor intensive and time consuming. our strategy, on the other hand, circumvents some of the limitations inherent in an unbiased screen, by rapidly identifying interacting partners of genes already known to regulate inner ear development. this approach, in combination with the reverse genetic techniques available in zebrafish, should make it feasible to identify in a systematic fashion additional genes whose roles in ear development were previously unknown or overlooked. the specificity of the phenotypes obtained by mo knockdown is supported by a number of considerations. first, morpholinos targeted against pi4kβ, arf and dan all caused similar phenotypes including vestibular malformation, shortened body axis, reduced head and eye size, and curved tails. since each of these genes is expressed throughout the brain and jaw during embryonic development, defects in head formation were not unexpected. these defects are not likely due to off-target effects, since for each of the genes tested, two independent non-overlapping mos produced identical phenotypes. second, injection of buffer alone did not produce morphant phenotypes. third, a mo targeted against otopetrin, a gene previously shown to be required for otolith formation, blocked development of otoliths but did not affect semicircular canal development. fourth, injection of subeffective doses of pi4kβ, arf and ncs-1a mos alone did not produce inner ear, jaw, or brain defects. co-injection of subeffective doses of mos against a pair of genes was synergistic and phenocopied the ear, jaw, and brain defects obtained using an effective dose of each of mo alone. finally, brain and ear development appear normal in pi4kβ, arf and dan morphants until hpf, indicating that disruption of early patterning events is not likely to have caused vestibular phenotypes or developmental delay. together, these results support the idea that the phenotypes produced by nbp knockdowns are specific, and do not result from non-specific cytotoxic or off-target gene effects. our double mo knockdown studies, together with the pulldown experiments, indicate that ncs- arf and pi4kβ form a small interaction network that is likely to have functional significance for development of the zebrafish semicircular canal system. we now propose a model in which these three proteins regulate the targeted secretion of molecules, such as dan, that are required for formation of the semicircular canals. this model is based on the following lines of evidence. pi4kβ is the enzyme responsible for the synthesis of phosphatidylinositol 4-phosphate, a lipid species that regulates trafficking of cargo from the golgi apparatus to the plasma membrane in yeast and mammalian cells. pi4kβ has also been shown to regulate secretion in both pc cells and pancreatic β cells. ncs- and arf are known activators of pi4kβ, however a direct interaction between ncs- and arf has been shown to antagonize the enzymatic activity of pi4kβ. bidirectional control of pi4kβ activation/inhibition may thus serve to regulate the trafficking of molecules involved in inner ear development from the trans-golgi network to the plasma membrane during otogenesis. dan, bmp and hyaluronic acid represent secreted molecules that play critical roles in semicircular canal formation and may require an ncs-1a/arf1/pi4kβ pathway for secretion. bmps are expressed in the developing vertebrate inner ear and participate in axial patterning of the otic vesicle and differentiation of the sensory epithelium. bmp is secreted from the apical membrane of cells located in the cristae of the otic vesicle and its regulated expression is essential for proper development of the non-sensory portion of the semicircular canals. dan is a bmp antagonist that plays a key role in the patterning of the chick inner ear, and is also secreted from the apical membrane. in chick, loss of dan leads to structural deformities in the semicircular canals and the endolymphatic duct and sac. the mo knockdown experiments we performed show that dan also contributes to semicircular canal formation in zebrafish. we have also demonstrated a direct interaction between dan and ncs- although the significance of this interaction is currently unknown, it is possible that the dan/ncs- interaction may be required to deliver dan to the golgi apparatus. this idea is supported by previous studies which showed that the yeast ortholog of ncs- can recruit pik1p from the endoplasmic reticulum to the trans-golgi network. hyaluronic acid is an extracellular matrix molecule that is secreted from the basolateral membrane of otic epithelium during semicircular canal formation and is thought to drive the outgrowth of the developing protrusions. hyaluronan synthases are the enzymes responsible for ha synthesis and secretion at the plasma membrane. it is possible that trafficking of has from the golgi apparatus to the plasma membrane may also be regulated by pi4kβ activity. since ha secretion from epithelial pillars in zebrafish inner ear can be monitored histochemically, it should be possible to test this hypothesis by determining whether knockdown of ncs-1a, arf or pi4kβ inhibits ha secretion. our yeast two-hybrid screen revealed interaction of ncs- with three proteins, hint slc25a and pink that are destined to localize to mitochondria. the interaction of ncs- with pink - induced putative kinase 1) is of particular interest. mutations in the human pink gene are the second most common cause of autosomal recessive parkinson's disease, and are also implicated in sporadic cases of the disease. mo knockdown studies we performed indicate that pink morphants exhibit severe brain abnormalities as well as defects in development of the semicircular canal system. interestingly, a recent study in zebrafish also showed that pink morphants display severe defects in brain morphology, as well an impaired escape response. in this context, it is important to consider that no direct link has been established between mutations in pink and defects in inner ear development in mammalian systems. it is therefore possible that pink has evolved to perform a critical role in brain and ear development in zebrafish, while mammals may have developed compensatory mechanisms that regulate development of these organs. the interaction of ncs- with zebrafish pink raises another interesting issue. ncs- does not possess a defined mitochondrial targeting sequence, and its expression in mitochondria has not been documented. in mammalian cells, ncs- is constitutively associated with the plasma membrane and golgi complex. it will be important to determine whether ncs- serves to regulate pink function, either within mitochondria or other cellular compartments, and whether the ncs-1/pink interaction contributes to the etiology of parkinson's disease.  in this study, we show that several new and previously identified nbps appear to play an important role in the development of the zebrafish inner ear. we suggest a model in which ncs-1a/arf1/pi4kβ interactions may form a signaling pathway that mediates trafficking and export of secreted molecules that are required for semicircular canal formation. we also made an unexpected observation that ncs- interacts with several mitochondrial proteins, including pink a gene that contributes to familial parkinson's disease. these findings suggest a previously unrecognized role for ncs- in mitochondrial function via its association with mitochondrial proteins, and reveal a new and potentially important signaling pathway involved in development of the vestibular apparatus. 
the semicircular canals, a subdivision of the vestibular system of the vertebrate inner ear, function as sensors of angular acceleration. zebrafish represent a particularly tractable model system for the study of inner ear development. we initiated a multistep functional proteomic strategy to identify neuronal calcium sensor- binding partners that contribute to inner ear development in zebrafish. by investigating the expression profiles of zebrafish nbp mrnas, we identified seven that were expressed in the developing inner ear and overlapped with the ncs-1a expression profile. based on their functional profiles, the hypothesis is presented that ncs-1a/pi4kβ/arf form a signaling pathway that regulates secretion of molecular components, including dan and bmp that are required for development of the vestibular apparatus. this is because the ear can be easily visualized during early embryogenesis, and both forward and reverse genetic techniques are available that can be applied to the discovery of novel genes that contribute to proper ear development. little is currently known, however, regarding the underlying molecular mechanisms that govern the development of this intricate structure. gst pulldown experiments confirmed that selected nbps interacted with ncs- while morpholino-mediated knockdown experiments demonstrated an essential role for arf pi4kβ, dan, and pink in semicircular canal formation. blast searches of the zebrafish est and genomic databases allowed us to clone zebrafish orthologs of each of the human nbps. our findings reveal a novel signalling pathway involved in development of the semicircular canal system, and suggest a previously unrecognized role for ncs- in mitochondrial function via its association with several mitochondrial proteins. by performing a y2h screen in combination with literature and database searches, we identified human nbps. we have previously shown that in zebrafish, the calcium sensing molecule neuronal calcium sensor- is required for semicircular canal formation. the function of ncs- in regulating semicircular canal formation has not yet been elucidated.
 the semicircular canals, a subdivision of the vestibular system of the vertebrate inner ear, function as sensors of angular acceleration. little is currently known, however, regarding the underlying molecular mechanisms that govern the development of this intricate structure. zebrafish represent a particularly tractable model system for the study of inner ear development. this is because the ear can be easily visualized during early embryogenesis, and both forward and reverse genetic techniques are available that can be applied to the discovery of novel genes that contribute to proper ear development. we have previously shown that in zebrafish, the calcium sensing molecule neuronal calcium sensor- is required for semicircular canal formation. the function of ncs- in regulating semicircular canal formation has not yet been elucidated.  we initiated a multistep functional proteomic strategy to identify neuronal calcium sensor- binding partners that contribute to inner ear development in zebrafish. by performing a y2h screen in combination with literature and database searches, we identified human nbps. blast searches of the zebrafish est and genomic databases allowed us to clone zebrafish orthologs of each of the human nbps. by investigating the expression profiles of zebrafish nbp mrnas, we identified seven that were expressed in the developing inner ear and overlapped with the ncs-1a expression profile. gst pulldown experiments confirmed that selected nbps interacted with ncs- while morpholino-mediated knockdown experiments demonstrated an essential role for arf pi4kβ, dan, and pink in semicircular canal formation.  based on their functional profiles, the hypothesis is presented that ncs-1a/pi4kβ/arf form a signaling pathway that regulates secretion of molecular components, including dan and bmp that are required for development of the vestibular apparatus. a second set of nbps, consisting of pink hint and slc25a are destined for localization in mitochondria. our findings reveal a novel signalling pathway involved in development of the semicircular canal system, and suggest a previously unrecognized role for ncs- in mitochondrial function via its association with several mitochondrial proteins. 