Expression of usherin in the anthozoan Nematostella vectensis.
Usher Syndrome is a human genetic disorder characterized by deafness and gradual loss of vision from retinitis pigmentosa. There are three clinical subtypes of Usher Syndrome that differ in the onset and severity of the loss of hearing. In Usher Syndrome I there is profound congenital hearing loss accompanied by a loss of vestibular function. In Usher Syndrome II, the most common subtype, the congenital hearing loss is less severe and vestibular function is spared. Usher Syndrome III is rare and is characterized by a progressive hearing loss. Each subtype has been linked to mutations in genes that are expressed in the sensory cells of the inner ear, where they appear to play roles in the organization of stereocilia and the mechanosensory transduction of sound (Saihan et al, 2009). One of the genes responsible for Usher Syndrome II encodes the protein usherin (Eudy et al, 1998; Bhattacharya et al, 2002). In vertebrates, usherin exists as one of two alternatively spliced variants (Adato et al, 2005b). The first is an extracellular matrix protein composed of a laminin N-terminal (Lam NT) domain, a series of 10 epidermal growth-factor-like (EGF-like) repeats, and four fibronectin-type III (FN3) domains. The second is a much larger (more than 570 kDa) transmembrane protein. It includes not only the regions found in the extracellular matrix variant but also two laminin G (Lam G) domains and 29 additional FN3 domains. The intracellular domain of this variant is able to bind to the PDZ module-containing proteins harmonin and whirlin, which are proposed to anchor usherin to the actin-based cytoskeleton via myosin VII (Adato et al, 2005a). Mutations in harmonin, whirlin, and myosin VII can also result in Usher Syndrome. The extracellular matrix variant of mammalian usherin is found in a subset of basement membranes (Pearsall et al, 2002); and the transmembrane variant, which is the form that is mutated in Usher Syndrome, is expressed almost exclusively in the inner ear and retina (van Wijk et al., 2004).
As part of earlier studies of the origins of the extracellular matrix protein tenascin (Tucker et al., 2006; Tucker and Chiquet-Ehrismann, 2009), we examined the genome of the estuarine anthozoan Nematostella vectensis Stephenson (1935) for genes predicted to encode EGF-like and FN3 domains. Though we were unable to find cnidarian orthologs of tenascin, we did discover a predicted protein with a domain organization identical to that found in the amino half of vertebrate usherin. Here we describe that predicted protein as well as an adjacent predicted gene that, when considered together, encode a single predicted protein with the same general domain organization as the transmembrane variant of vertebrate usherin. The expression of this protein in N. vectensis was confirmed by RT-PCR and in situ hybridization. Finally, we examined representative metazoan genomes and found highly conserved usherin genes throughout the deuterostome lineage as well as in some protostomes.
Materials and Methods
Sexually mature individuals of Nematostella vectensis descended from Cadet Hand's strains CH2 and CH6 (Hand and Uhlinger, 1992) were obtained from Carol Vines at the University of California, Davis, Bodega Marine Laboratory. Details of raising and breeding N. vectensis can be found in Darling et al. (2005). In brief, the anemones were maintained in 1/3 ASW (Instant Ocean), pH 8.0, at room temperature and fed Artemia nauplii and fragments of hardboiled egg yolk. About 24 to 48 h after transfer into freshly made 1/3 ASW, the females deposit egg masses that are fertilized externally. Gastrulae and planulae were removed from the egg jelly by using fresh 2% cysteine (Sigma), pH 8.0, prior to making poly A RNA or fixation for in situ hybridization (see below); juvenile polyps were raised exclusively on emulsified hardboiled egg yolk prior to processing, to avoid contamination with Artemia genes.
RNA isolation and polymerase chain reaction
Specimens of N. vectensis at representative stages of development were homogenized by repeated passage though a 24G needle in the disruption buffer of the RNeasy Mini Kit (Qiagen). PolyA RNA purification was then carried out following the kit manufacturer's instructions. Four primer pairs (Eurofins MWG/Operon) selected using Primer3 (Rozen and Skaletsky, 1998) were then used to make cDNA and PCR products from the RNA template by using the Super Script III One Step RT-PCR Kit (Invitrogen) and 40 cycles of PCR using 94 [degrees] C melting (15 s), 55 [degrees] C annealing (30 s), and 64 [degrees] C extension (1 min to 2.5 min) cycles: nN2 forward (5'-aacggcgagctctatttgtg-3'), nN2 reverse (5'-ttactgatggcacggtcgta-3'), nEGF forward (5'-ggagaaaagtgcgaaagctg-3'), nEGF reverse (5'-ctgctttacatcg-gcattga-3''), nFN3 forward (5'-cccgctacgagatttacgag-3'), nFN3 reverse (5'-tgcggaactggtacagagtg-3'), nFN3C forward (5'-cttcatcatccgcgtctaca-3'), and nFN3C reverse (5'-tgaaccagatgctgcgatag-3') primers. RT-PCR fragments joining the two predicted usherin-like proteins were amplified using the nFN3 forward primer (see above) and nFN31ink2 forward (5'-gtcactgccaaagctttcaa-3') with nFN31ink reverse (5'-tcactgaggcaactctctgg-3') primers. For positive controls, a primer pair known to span an intron in an N. vectensis alpha tubulin gene was used (forward, 5'-gccagctttttcatcct-gag-3'; reverse, 5'-agcttggatttcttgccgta-3'). PCR products were separated and visualized on a 1% agarose gel with ethidium bromide. PCR products were then subcloned into pCRII TOPO (Invitrogen), and the plasmids were purified with the QIAprep Spin Miniprep Kit (Qiagen) and sequenced by Davis Sequencing using M13 reverse and/or forward primers.
In situ hybridization
The nN2A Nematostella usherin plasmid was selected to generate probes for in situ hybridization. The plasmid was linearized with appropriate restriction enzymes and purified with phenol: chloroform prior to making antisense and control sense digoxygenin-labeled RNA probes, using reagents and methods supplied by Boehringer-Mannheim. A collection of planulae and juvenile polyps were fixed in ice-cold 3.7% paraformaldehyde (Fisher)/0.2% gluteraldehyde (Sigma) in 1/3 ASW for 90 s, then postfixed in ice-cold 3.7% paraformaldehyde in 1/3 ASW for 15 min prior to storage in 100% methanol (Fisher) at--20 [degrees]C. In situ hybridization was carried out following a previously published general protocol (Martindale et al., 2004), supplemented with a detailed protocol kindly provided by M. Q. Martindale (University of Hawaii at Manoa). In brief, samples were rehydrated into phosphate buffered saline (PBS; Sigma) with 0.5% Tween-20 (PTw; BioRad); digested with 0.01 mg/ml proteinase K (Sigma); quenched in 2 mg/ml glycine (Sigma); rinsed in 1% triethanolamine (Sigma) and acetic anhydride (Fisher); washed in PTw; refixed in 3.7% paraformaldehyde in PTw; rinsed in PTw; and prehybridized in 50% formamide (Sigma), 5X SSC (Sigma), 50 [micro]g/ml heparin (Grade 1A, Sigma), 0.1% Tween-20, 20% SDS (Fisher), and 100 [micro]g/ml salmon sperm DNA (Sigma) overnight at 55 [degrees]C or 62 [degrees]C. Specimens were then hybridized overnight at 55 [degrees] C or 62 [degrees] C (with identical results) in prehybridization buffer including 1 ng/ml sense or antisense probe. Following extensive washes in 2X SSC and 0.05X SSC, followed by washes in PBS/Triton X-100, the specimens were blocked using Boehringer-Mannheim blocking solution and incubated overnight in 1:4000 anti-digoxygenin (Boehringer-Mannheim) at 4 [degrees] C. An NBT/BCIP (Boehringer-Mannheim)-based color reaction was carried out following PTw rinses, and the reaction was stopped in five changes of PTw. A Nikon Eclipse E800 photomicroscope was used to photograph whole-mount specimens. Some specimens were then cryoprotected overnight in 20% sucrose in PBS, embedded in OCT mounting medium (Fisher), frozen and sectioned at 15 [micro]m, and collected on pre-subbed slides (Fisher). After being rinsed in PBS, sections were counterstained in Hoechst 33258 (Molecular Probes), coverslipped, and photographed as described above.
N. vectensis sequences encoding predicted proteins with an architecture similar to portions of vertebrate usherin were found using the program Superfamily 1.69 (Gough et al., 2001). In brief, the N. vectensis genome was searched for sequences encoding FN3 domains in combination with EGF-like domains. The carboxy-half of the usherin sequence was found the same way by searching for strings of FN3 domains. The genes encoding the predicted proteins were used for primer design. The same search parameters were also applied to identify usherin in the genomes of Homo sapiens, the domestic chicken Gallus gallus, the pufferfish Takifugu rubripes, the tunicate Ciona savignyi, the lancelet Branchiostoma floridae, the purple sea urchin Strongylocentrotus purpuratus, the limpet Lottia gigantea, and the polychaete Capitella sp. No usherin-like sequences could be found in this way in Drosophila melanogaster, Caenorhabditis elegans, or the branchiopod crustacean Daphnia pulex. Sequences were aligned using ClustalW (Thompson et al., 1994; full multiple alignment, default gap penalties, 1000 bootstraps). Domains were identified using SMART (Letunic et al., 2009), Pfam (Finn et al., 2010), or both
The Nematostella genome contains usherin-like sequences
The genome of Nematostella vectensis encodes a predicted protein (Nemvel: 199904) with a Lam NT domain, 10 EGF-like domains, four FN3 domains, and two Lam G domains followed by 12 more FN3 domains (Fig. 1A). This matches precisely the domain organization of the N-terminal two-thirds of the protein usherin in vertebrates. Further examination of the N. vectensis genome indicates that the adjacent gene encodes an open reading frame in the same orientation that is predicted to include 16 FN3 domains, a transmembrane domain, and a short intracellular domain (Nemvel:59990) (Fig. 1A). If expressed together (see below), the single predicted protein would have the same general domain organization as the transmembrane variant of vertebrate usherin.
[FIGURE 1 OMITTED]
Usherin is expressed in gastrulae, planulae, and polyps
To determine if these predicted proteins are expressed in N. vectensis, PCR primers designed from the nucleic acid sequences encoding both predicted proteins were used to amplify products from a template derived from RNA isolated from juvenile polyps. Products of the predicted sizes were amplified from regions corresponding to the Lam NT domain, EGF-like domains, and the FN3 domains of Nemvel: 199904 and Nemvel:59990 (Fig. 1B). The identity of nN2, nFN3, and nFN3C was confirmed by sequencing. The sequences of the first two cDNAs matched precisely with predicted sequences (GenBank accession numbers GU320063, GU320064), but the nFN3C sequence contained one mismatch at position 51 and a novel stretch of 96 nucleic acids starting at position 252. This novel stretch represents an exon predicted to be an intron, as it encodes an open reading frame with 44% similarity to an exon found in the corresponding FN3 domain of usherin from Branchio-stoma ftoridae (alignment not shown). This novel sequence has been submitted to GenBank (GU320065). To determine if splice variants of usherin are expressed in N. vectensis, and if this expression is developmentally regulated, RT-PCR was conducted using mRNA from different developmental stages and PCR primers that would amplify from both the extracellular matrix variant (if it exists in N. vectensis) and the long transmembrane variant (nN2), as well as a primer pair that would be specific for the transmembrane variant (nFN3). Both primer pairs amplified usherin products from cDNA from planulae and juvenile polyps (Fig. 1C). Both primer pairs amplified usherin products from the gastrula-derived cDNA as well, though the nFN3 product is only faintly visible. In the mature polyp, however, only the nN2 primer amplified the expected product. Thus, a long variant (and possibly a short variant) are expressed during development, but in the mature polyp only a putative short variant with the Lam NT domain but missing some central FN3 domains appears to be expressed.
In situ hybridization with an usherin-specific riboprobe (nN2A) confirms that usherins are expressed both in planulae and in juvenile polyps (Fig. 2A, C). Expression is seen throughout the planula larva, but is restricted to the column of the polyp. Planulae and polyps processed in parallel with a control sense probe were unlabeled (Fig. 2B). Frozen sections of juvenile polyps revealed that the hybridization signal seen in the column is limited to the ectoderm (Fig. 2D, E).
[FIGURE 2 OMITTED]
The domain organization of Nematostella usherin is highly conserved
To determine if both predicted proteins illustrated in Figure 1A are in fact encoded on the same gene, two forward PCR primers (nFN3 forward and nFN31ink2 forward) from the C-terminal sequence of Nemvel:199904 were used in conjunction with a single reverse PCR primer (nFN31ink reverse) based on sequence found in the N-terminal FN3 of Nemvel:59990. Both RT-PCR reactions generated products of reasonable sizes (Fig. 3A). The first (nLinkL) is about 2.4K bp, and the second (nLinkS) is about 450 bp in length. The products were subcloned and sequenced; the sequences of both confirm that both predicted proteins are encoded on a single mRNA (Fig. 3B; see also GenBank GU320066, GU320067); that is, the 16th FN3 domain found at the C-terminus of Nemvel:199904 is adjacent to the FN3 domain found at the N-terminus of Nemvel:59990. The model of the resulting transmembrane form of usherin, based on cDNA and predicted sequences, is remarkably similar to the transmembrane variant of usherin found in vertebrates (Fig. 3C).
[FIGURE 3 OMITTED]
Usherins in other invertebrates
Searches based on domain organization were used to identify genes encoding usherin-like proteins in the genomes of chordates (including the tunicate Ciona savignyi and the lancelet Branchiostoma floridae), the echinoderm Strongylocentrotus purpuratus, as well as in the limpet Lottia gigantea and the polychaete Capitella (Table 1). The domain organization of the predicted usherins from each of these diverse species is remarkably similar (Table 1), as are the amino acid sequences of representative domains (Fig. 4). Note that usherins could not be identified in the genomes of Drosophila melanogaster, the crustacean Daphia pulex, or Caenorhabditis elegans using this method, or by BLAST-based sequence homology searches. Thus, usherin is another example of a gene lost in Ecdysozoa (see Putnam et al., 2007).
[FIGURE 4 OMITTED]
Table 1 Domain organization of predicted usherins Organism Protein ID or Domain organization(1) accession no. Nematostella Nemvel: 199904 LamNT + 10 EGF-like + 4 FN3 + 2 vectensis and 59990 LamG + 28 FN3 Homo sapiens 075445 LamNT + 10 EGF-like + 4 FN3 + 2 LamG + 29 FN3 Gallus gallus XM 419417 LamNT + 10 EGF-like + 4 FN3 + 2 LamG + 29 FN3 Takifugu rubripes UPI00006615D9 LamNT + 10 EGF-like + 4 FN3 + 2 LamG + 29 FN3 Ciona savignyi ENSCSAVT LamNT + 10 EGF-like + 4 FN3 + 2 00000003863 LamG + 27 FN3 Branchiostoma XM 002242043 LamNT + 10 EGF-like + 4 FN3 + 2 floridae and XM LamG + 29 FN3 002214611(2) Strongylocentrotus XP 793438 LamNT + 10 EGF-like + 4 FN3 + 2 purpuratus LamG 4+ 30 FN3 Lottia gigantea Lotgil: 18598 LamNT + 10 EGF-like + 4 FN3 + 2 LamG + 29 FN3 Capitella sp. Capcal: 4EGF-like + 4FN3 + 2 LamG + 219707 28FN3(3) (1) See text for guide to abbreviations; as determined by SMART and/or Pfam (see text for details). (2) XM 002242043 contains the C-terminal half of the protein, while XM 002214611 contains the overlapping N-terminal half of the protein. (3)'The Capitella sequence may be missing its N-terminus.
The predicted intracellular domain of usherin from N. vectensis is only 22 amino acids long, in contrast to the intracellular domain of human usherin, which has 141 amino acids. Though this may be an artifact of sequence prediction, the predicted intracellular domains of usherins from amphioxus and the limpet Lottia are similarly abbreviated. A highly conserved tyrosine residue that is predicted to be phosphorylated is found at residue 5692 in the ClustalWaligned sequences of usherins from N. vectensis, H. sapiens, G. gallus, B. floridae, S. purpuratus, and L. gigantea (Fig. 4). This is followed by a region with numerous conserved charged residues that may represent a protein-interaction domain.
In mammals, usherins exist in two alternatively spliced variants: a short extracellular matrix variant (Lam NT + 10 EGF-like + 4 FN3) and a long transmembrane variant (Lam NT + 10 EGF-like + 2 Lam G + 29 FN3) (Baux et al., 2007). The extracellular matrix variant is found in some basement membranes (Pearsall et al., 2002), while the transmembrane variant is expressed primarily in the hair cells of the inner ear (Adato et al., 2005b), where it participates in the formation of the ankle-link complex that anchors adjacent stereocilia to each other (Adato et al., 2005a, Michalski et al., 2007; see review by Petit and Richardson, 2009). Here we show that genes encoding a highly conserved (both domain organization and sequence) potential usherin are found not only in the deuterostome lineage but also in some protostomes (molluscs and annelids) and the anthozoan Nematostella vectensis. The expression of usherin in N. vectensis was confirmed with RT-PCR and in situ hybridization, and the conserved and organism-specific features of usherin were studied by sequence alignment.
Others have speculated that the stereocilia of the vertebrate cochlea evolved from the mechanosensory apparatuses of cnidarians (Watson et al., 1997; Thurm et al., 2004). These include the cnidocil complex, the trigger that initiates the changes in membrane permeability that result in the firing of a nematocyst (for a review, see Anderson and Bouchard, 2009). In the hydrozoan man-o'-war Physalia, where the ultrastructure of the cnidoblast has been studied in detail (Cormier and Hessinger, 1980), the cnidocil complex is composed of a large, central kinocilium surrounded by a ring of inward-leaning stereocilia. In anthozoans the mechanosensory apparatus of the cnidoblast is simpler: it lacks the central kinocilium and instead is formed from a cone-like arrangement of the stereocilia with or without a related flagellum (Schmidt and Moraw, 1982; Kass-Simon and Scappaticci, 2002). As usherin is important for the organization of stereocilia in the cochlea, it seemed likely that this highly conserved gene would be expressed in the cnidoblasts of N. vectensis. However, the cnidoblast-rich tentacles of the juvenile polyps do not express detectable levels of usherin transcripts, and the results of RT-PCR with primers that are specific for a part of the transcript corresponding to the large, transmembrane variant of vertebrate usherin suggest that the expression of the putative variant necessary for stereocilia organization is lost in mature polyps. It remains possible, however, that usherin expression coincides with the early phases of cnidoblast differentiation, as others have shown that cnidoblast precursors are found in the column of Hydra and the mature cells then migrate into the tentacles (e.g., see Fujisawa and Sugiyama, 1978; Engel et al., 2002). Ultrastructural evidence exists in anthozoans as well, that cnidocytes originate in the column and migrate to the tentacles (Schmidt and Moraw, 1982). If similar migration occurs in N. vectensis, then usherin protein may be present in the stereocilia cone complex even though the gene is no longer transcriptionally active. The loss of a large form of usherin in the mature polyp may be due to the onset of senescence. Future studies with antibodies to anthozoan usherins may resolve this issue.
There is some evidence that the intracellular domain of mammalian usherin interacts with the PDZ-containing proteins harmonin and whirlin, which are proposed to act as a link between usherin and microfilaments (Adato et al., 2005b). In contrast to the conserved extracellular domain of usherin, the domain responsible for this interaction is not present in the intracellular domains of predicted usherin from N. vectensis, B. floridae, or L. gigantea. Care must be taken not to conclude that additional intracellular domain sequence does not exist in these genomes. Interestingly, the genome of N. vectensis contains a gene predicted to encode a protein that is remarkably similar to vertebrate harmonin (Nemve1:239158). The presence of a tyrosine residue that is predicted to be phophorylated in the intracellular domains of all the usherins studied here suggests that a phylogenetically conserved action of usherin may be mediated by a tyrosine protein kinase.
If the usherin of N. vectensis is processed similarly to vertebrate usherin, with a small extracellular splice variant in addition to a large transmembrane splice variant (as supported but not proven by the RT-PCR results), then an important, phylogenetically conserved role for the form found in the extracellular matrix needs to be considered. Future studies should be directed to identifying sequences that may be unique to the small variant and that would permit the construction of variant-specific primers and probes, as well as to northern blotting to determine the number and sizes of usherin transcripts.
I thank N. Leachman for practical advice concerning RT-PCR and subcloning, C. Vines for providing mature Nematostella to start our own colony, M.Q. Martindale for helpful discussions and for providing a detailed in situ hybridization protocol, and T. Blankenship for assistance with photography.
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Received 29 April 2009; accepted 18 November 2009.
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
Abbreviations: EGF, epidermal growth factor; FN3, fibronectin-type 3; Lam G, laminin G domain; Lam NT, laminin N-terminal domain; PBS, phosphate buffered saline; PTw, PBS with Tween-20.
RICHARD P. TUCKER
Department of Cell Biology and Human Anatomy, University of California at Davis, Davis, California 95616-8643
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|Author:||Tucker, Richard P.|
|Publication:||The Biological Bulletin|
|Date:||Apr 1, 2010|
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