Proteomic and functional analysis of NCS-1 binding proteins reveals novel signaling pathways required for inner ear development in zebrafish
© Petko et al; licensee BioMed Central Ltd. 2009
Received: 17 November 2008
Accepted: 25 March 2009
Published: 25 March 2009
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-1 (NCS-1) is required for semicircular canal formation. The function of NCS-1 in regulating semicircular canal formation has not yet been elucidated.
We initiated a multistep functional proteomic strategy to identify neuronal calcium sensor-1 (NCS-1) binding partners (NBPs) that contribute to inner ear development in zebrafish. By performing a Y2H screen in combination with literature and database searches, we identified 10 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-1, while morpholino-mediated knockdown experiments demonstrated an essential role for arf1, pi4kβ, dan, and pink1 in semicircular canal formation.
Based on their functional profiles, the hypothesis is presented that Ncs-1a/Pi4kβ/Arf1 form a signaling pathway that regulates secretion of molecular components, including Dan and Bmp4, that are required for development of the vestibular apparatus. A second set of NBPs, consisting of Pink1, Hint2, and Slc25a25, 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-1 in mitochondrial function via its association with several mitochondrial proteins.
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 [1–3].
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 (reviewed in ). In zebrafish, as well as other vertebrates, the semicircular canal system consists of three non-sensory components, the anterior, posterior, and lateral canal ducts (reviewed in ) 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 45 hpf with the development of three epithelial outpocketings: one anterior protrusion, one posterior protrusion, and one lateral protrusion . At 60 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 72 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 [8–10]. For example, a mutation in the jekyll gene leads to interrupted ear columns (epithelial pillars which form the hubs of the semicircular canals) 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 dfna5, 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 Bmp4 (bone morphogenetic protein 4) 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 (osteonectin) that functions in the genetic network regulating pharyngeal and inner ear development , tmie (transmembrane inner ear), a gene coding for a protein of unknown function that is important for normal hearing , and the Na, K-ATPase α subunit gene α1a.2 .
We have recently found that in zebrafish, the gene encoding neuronal calcium sensor-1a (ncs-1a) is required for semicircular canal formation . NCS-1 is the mammalian ortholog of Drosophila frequenin, a protein critical for synaptic transmission within the Drosophila nervous system . In mammalian cells, NCS-1 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 D2 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-1 in regulating semicircular canal formation has not been elucidated.
To gain a better understanding of the role of NCS-1 in regulating inner ear development, we initiated a proteomic screen to identify NCS-1 binding partners (NBPs). 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/Arf1 form a signaling pathway that is required for semicircular canal formation. A second set of NBPs, consisting of pink1, hint2, and slc25a25, are destined for localization in mitochondria. Using antisense morpholinos, we show that pink1 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-1 Binding Proteins (NBPs)
Identification, cloning, and function of NBPs
mRNA Accession Number (riboprobe NTs)
ADP-ribosylation factor 1
Phosphatidylinositol 4-kinase beta
Vesicle associated membrane protein 2/Synaptobrevin 2
Histidine triad protein
Mitochondrial AMP-lysine hydrolase
Ca2+-dependent mitochondrial carrier
Mitochondrial ATP-Mg/Pi carrier
Classical transient receptor potential-1
Classical transient receptor potential-5
Immuno-precipitation and GST-pulldown 
Inositol 1,4,5 trisphosphate receptor
Ca2+ release channel
Differential screening-selected gene aberrant in neuroblastoma
Extracellular BMP antagonist
By using BLAST searches of the zebrafish EST database and genomic sequences available from GenBank and the Zebrafish Sequencing Group at the Sanger Institute http://www.ensembl.org/ 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 1. Zebrafish ESTs were obtained from ATCC (Manassas, VA). In cases where ESTs contained partial sequences, full-length ORFs (open reading frames) were generated by RT-PCR. Alignments of the predicted amino acid sequences of human NBPs and their zebrafish orthologs are presented in Additional File 1.
Expression of NBPs in Zebrafish Inner Ear
At 48 hpf, ncs-1a transcripts can be visualized in the semicircular canal protrusions, and in the fully formed semicircular canal hubs at 72 hpf. We first analyzed expression of arf1, pi4kβ, and vamp2, NBP genes encoding proteins involved in exocytosis and intracellular trafficking. arf1 transcripts were expressed at 48 hpf in the epithelial protrusions of the semicircular canals but were not present in the semicircular canal hubs at 72 hpf, suggesting that arf1 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 48 hpf in the semicircular canal protrusions and in the fully formed semicircular canal hubs at 72 hpf. vamp2 mRNA was undetectable in the inner ear at either time point, indicating that vamp2 is unlikely to play a role in otogenesis. Three NBP genes, pink1, hint2, and slc25a25, each encoding a mitochondrial protein, showed very similar expression patterns. Transcripts of each gene were detected at 48 hpf in the semicircular canal protrusions and the anterior macula, and at 72 hpf in the semicircular canal hubs.
Amongst the genes encoding calcium channels, trpc1 transcripts were detectable at 48 hpf and 72 hpf in the semicircular canal protrusions and hubs, respectively. Transcripts of the other two calcium channel genes, trpc5 and ip3r, were undetectable in the inner ear at either 48 hpf or 72 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 48 hpf and 72 hpf.
Taken together, our data indicate that seven NBP genes including arf1, pi4kβ, trpc1, dan, pink1, hint2, and slc25a25, 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
Knockdowns of NBPs Involved in Exocytosis and Secretion
At 72 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 (Fig. 3L, N), failure of the pillars to fuse (Fig. 3P), as well as the presence of disorganized cell masses in the otic vesicle (Fig. 3L, N). For all genes tested, injection of a second, independent MO (either the ATG-MO or UTR-MO) phenocopied the ear defects generated by the primary MO (data not shown). 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 arf1, pi4kβ, and dan genes is required for semicircular canal formation in zebrafish.
ncs-1a, pi4kβ, and arf1 Functionally Interact to Regulate Formation of Semicircular Canals
pink1 is Required for Semicircular Canal Formation
PINK1 was initially implicated as an NBP in a Y2H screen (Table 1), while the interaction of zebrafish pink1 with NCS-1 was verified via pulldown with an NCS-1-GST fusion protein (Fig. 2). Whole-mount in situ hybridization analysis revealed expression of pink1 mRNA sequences in the epithelial pillars and hubs of the semicircular canals during zebrafish otogenesis (Fig. 1). To directly determine whether pink1 expression was necessary for ear development, we knocked down pink1 mRNA translation with a pink1- ATG MO and examined the effect on semicircular canal formation.
Ncs-1 is a calcium sensing molecule that is required for semicircular canal formation in zebrafish . To better understand of the role of NCS-1 in ear development, we employed a multistep functional proteomic strategy to identify molecular partners of NCS-1 that contribute to the development of the vestibular apparatus. First, by performing a Y2H screen in combination with literature and database searches, we identified 10 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 (vamp2, trpc5, and ip3r) 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-1. Finally, MO-mediated knockdowns confirmed that four selected NBPs were essential for semicircular canal formation in zebrafish.
The specificity of the phenotypes obtained by MO knockdown is supported by a number of considerations. First, morpholinos targeted against pi4kβ, arf1, 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, arf1, 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, arf1, and dan morphants until 48 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.
Dan, Bmp4, and Hyaluronic acid represent secreted molecules that play critical roles in semicircular canal formation [13–15, 23, 43] 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 [23, 44–48]. Bmp4 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 [15, 43]. 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-1. Although the significance of this interaction is currently unknown, it is possible that the Dan/NCS-1 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-1 (Frq1p) can recruit Pik1p (yeast ortholog of PI4kβ) from the endoplasmic reticulum to the trans-Golgi network .
Hyaluronic acid (HA) 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 (HAS) 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, Arf1, or Pi4kβ inhibits HA secretion.
Our yeast two-hybrid screen revealed interaction of NCS-1 with three proteins, HINT2, SLC25a25, and PINK1, that are destined to localize to mitochondria. The interaction of NCS-1 with Pink1 ( phosphatase/tensin homolog on chromosome 10 (PTEN)- induced putative kinase 1) is of particular interest. Mutations in the human PINK1 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 pink1 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 pink1 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 Pink1 and defects in inner ear development in mammalian systems. It is therefore possible that Pink1 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-1 with zebrafish Pink1 raises another interesting issue. NCS-1 does not possess a defined mitochondrial targeting sequence, and its expression in mitochondria has not been documented. In mammalian cells, NCS-1 is constitutively associated with the plasma membrane and Golgi complex . It will be important to determine whether NCS-1 serves to regulate Pink1 function, either within mitochondria or other cellular compartments, and whether the NCS-1/Pink1 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-1 interacts with several mitochondrial proteins, including Pink1, a gene that contributes to familial Parkinson's disease. These findings suggest a previously unrecognized role for NCS-1 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.
Yeast two-hybrid Screen
A yeast two-hybrid (Y2H) screen was performed as previously described  using human NCS-1 as bait to screen an adult human brain cDNA library. Full length human NCS-1 was subcloned into the yeast GAL4 DNA-binding domain expression vector pAS2-1 (Clontech, Palo Alto, CA), while the human brain cDNA library was subcloned into the GAL4 activation domain vector pACT2 (Clontech). Bait and prey plasmids were simultaneously co-transformed into yeast strain MaV103 . Positive clones were identified by growth on Leu-/Trp-/His-/Ura- selection plates. Protein interaction was assayed for by β-galactosidase activity via the nitrocellulose filter lift method . cDNAs were extracted from yeast colonies, sequenced, and subjected to a BLAST search against the human genome to determine the identity of the NCS-1 interacting cDNA clones.
Identification and Cloning of Zebrafish NBP Orthologs
Primers for cloning NBPs
RT-PCR Forward Primer
RT-PCR Reverse Primer
Whole-mount in situ Hybridization
48 and 72 hpf zebrafish embryos were preserved in 4% paraformaldehyde in PBS. Whole-mount in situ hybridization analysis was performed as previously described . Riboprobes were synthesized for the following genes (Table 1): arf1, dan, hint2, ip3r, pink1, pi4kβ, slc25a25, trpc1, trpc5, and vamp2.
Antisense morpholino oligonucleotides (MOs) were obtained from Gene Tools LLC (Philomath, OR). Two non-overlapping MOs were targeted against either the initiating methionine or the 5' untranslated region of pi4kβ, arf1, ncs-1a, dan, and pink1 mRNAs. The sequence for each morpholino is listed in Table 2. MOs were resuspended in 1× Danieau buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5 mM HEPES, pH 7.6), and microinjected directly into the yolk sac of single cell embryos.
Glutathione S-transferase Pulldown
An NCS-1-GST (glutathione S-transferase) fusion protein was constructed by fusing amino acids 1–190 of human NCS-1 to GST in the expression vector pGEX-4T-1 (Amersham Biosciences, Piscataway, NJ). The NCS-1-GST-fusion protein was induced in Escherichia coli strain BL21 (DE3) using the ZYP-5052 auto-induction media described previously [58, 59], then purified on glutathione sepharose beads (Amersham) according to manufacturer's instructions. cDNAs encoding zebrafish arf1 (amino acids 1–180), pi4kβ (residues 1–423), dan (residues 1 – 183), pink1 (residues 1–574), and hint2 (residues 1–161) were subcloned into the pET30C expression vector containing an S-tag. Constructs were induced using auto-induction media as described above. NCS-1-GST fusion protein was used to pull down NBPs from bacterial lysates as previously described . Eluted proteins were separated by SDS-PAGE and transferred to a PVDF (polyvinylidene fluoride) filter. The filter was probed with a horseradish peroxidase conjugated S-tag antibody (1:5000 dilution, Santa Cruz Biotechnology). Immunoreactivity was detected by enhanced chemiluminescence with an ECL Plus kit (Amersham).
NCS-1 binding protein
hours post fertilization
yeast 2 hybrid
expressed sequence tag
reverse transcriptase polymerase chain reaction
open reading frame
sodium dodecyl sulfate polyacrylamide gel electrophoresis.
We thank Joe Bednarczyk (Molecular Genetics Core Facility, Penn State College of Medicine) for DNA sequencing. This project was funded by an NIH Conte Center Grant (MH068789) and a grant from the Pennsylvania Department of Health using Tobacco Settlement Funds.
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