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A tyrosine kinase screen of mouse vestibular maculae
Hearing Research 136 (1999) 100^104
A tyrosine kinase screen of mouse vestibular maculae James O. Pickles *, Walter R.A. van Heumen, Christina Claxton Vision, Touch and Hearing Research Centre, Department of Physiology and Pharmacology, University of Queensland, Brisbane, Qld. 4072, Australia Received 29 December 1998; received in revised form 25 May 1999; accepted 7 June 1999
Abstract Receptor tyrosine kinases allow extracellular signals to influence intracellular events, while other tyrosine kinases are involved in intracellular signalling. They may therefore be involved in the development, maintenance and repair of the sensory epithelia of the inner ear, since these are believed to be affected by inter- and intracellular signalling. In order to analyse possible tyrosine kinases expressed in sensory areas of the inner ear, a reverse transcription polymerase chain reaction screen of microdissected sensory epithelia was undertaken, using primers targeted at conserved sequences in tyrosine kinase domains. Tissue was taken from the maculae of the mouse vestibular organs, and consisted mainly of hair cells and their supporting cells. Of 80 clones sequenced, 49 coded for tyrosine kinases, and 11 for other known molecules. Further analysis of one of the sequences, for FGF receptor 4, showed a novel variant, expressed in the inner ear and elsewhere, with a variation in the intracellular domain which suggests differential activation of known signalling pathways. Other clones coded for tyrosine kinases expected to be involved in cell surface and intracellular signalling. The technique forms a powerful tool for analysing a range of the tyrosine kinases expressed, and provides a starting point for the analysis of cell-cell signalling in the inner ear. ß 1999 Elsevier Science B.V. All rights reserved. Key words: Tyrosine kinase; PCR screen; FGF receptor; IGF-1 receptor; Hair cell; Vestibular maculae; Mouse
1. Introduction Hair cells are arranged in a precise and complex array within the cochlear and vestibular sensory epithelia. Interactions between hair cells and their supporting cells are thought to be important in setting up and maintaining the pattern of cells in the cochlear sensory epithelium, and for initiating a repair response in the adjacent supporting cells after hair cell damage. In the organ of Corti the supporting cells expand to ¢ll the gap in the reticular lamina (e.g. Raphael and Altschuler, 1991). However, in vestibular organs, the supporting cells are triggered to produce new hair cells (Forge et al., 1993 ; Warchol et al., 1993). Given the role of receptor tyrosine kinases in cell interactions, it is likely that these molecules are involved in the initial development of the sensory epithelia, as well as in the normal maintenance of the epithelia, and in their response to damage. Tyrosine kinases are also involved in intracel-
* Corresponding author. Tel.: +61 (7) 3365-4125; Fax: +61 (7) 3365-4522; E-mail: [email protected]
lular signalling, and in this role too are likely to take part. Analyses of mRNAs coding for speci¢c tyrosine kinases can be undertaken with polymerase chain reaction primers speci¢cally targeted at selected candidate sequences (as published earlier by e.g. Lee and Cotanche, 1996 ; Sa¡er et al., 1996 ; Pickles and van Heumen, 1997). However a wider search for tyrosine kinases can be undertaken by means of a degenerate screen (e.g. Lai and Lemke, 1991; Robinson et al., 1996), which has the advantage that it is capable of revealing unanticipated or novel kinases. The screen takes advantage of the fact that tyrosine kinases express conserved motifs, a set number of peptides apart within the kinase domain (e.g. Lai and Lemke, 1991 ; Robinson et al., 1996). Degenerate polymerase chain reaction (PCR) primers targeted at these regions produce ampli¢cation products with only a small range of sizes, permitting the bands to be readily excised from gels, thus allowing further puri¢cation followed by cloning and sequencing. The tyrosine kinase screen was undertaken in microdissected sensory maculae, from neonatal mouse saccules and utricles. The neonatal mouse inner ear expresses
0378-5955 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 9 ) 0 0 1 1 4 - 8
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high levels of growth factors (e.g. Luo et al., 1993), and it is therefore likely that the corresponding receptors are also expressed. While the target tissue was not in a pathological state, we expect the signalling pathways that are able to detect damage will be present, as well as those involved in the maintenance of the sensory epithelium. 2. Materials and methods Neonate mice (postnatal day 0: P0) were killed by decapitation, the heads bisected, and the temporal bones removed under ice-cold Hank's balanced salt solution. The vestibular areas were removed, the saccules and utricles opened, and the sensory epithelia plus some adjacent supporting structures removed. Histology of tissue removed in an identical way, and estimated over all sections taken through the tissues, revealed that between 50 and 70% of the tissue volume consisted of the hair cells plus their intervening supporting cells, the remainder consisting mainly of basal ¢broblasts and fragments of lateral walls of the organs. The microdissected sensory areas were lysed in bu¡ered guanidium thiocyanate and N-lauroyl sarcosine. Poly-A+ RNA was extracted with oligo-dT cellulose (Pharmacia Quick-Prep Micro mRNA Puri¢cation Kit). Seven to fourteen sensory areas were used for each of three separate extractions. Following reverse transcription with random hexamers as primers, 1/30th of the product volume was PCR ampli¢ed with degenerate primers. One of two di¡erent upper (forward) primers were used, either (in 5P to 3P order) CCA (CT)CG (AGCT)GA (CT)(CT)T (AGCT)GC or AA(AG) GT(AGCT) (AGT)(GCT)(AGCT) GA(CT)TT (CT)GG, in combination with a single lower (reverse) primer: (AC)C(AG) (AT)A(AGCT) (GC)(ACT)C CA(AGCT). In amino acid single-letter code, the two upper primers were targeted at either the conserved region HRDLAAR found in protein tyrosine kinases, or at the K(V/I)(A/S/T)DFG region common to protein tyrosine and serine/threonine kinases. The single lower primer was targeted at the conserved kinase region DVW(S/A)(F/Y) (Hanks et al., 1988). Reaction conditions were: annealing 1 min at 42³C, extending 2 min at 60³C, melting 1 min at 94³C, for 35 cycles. Following gel puri¢cation, 20% of the PCR products were reampli¢ed in a new reaction using the same conditions, for either 20 or 35 cycles. Following analysis on an agarose gel, visible bands were obtained at the expected lengths (approximately 200 bp with ¢rst upper primer, 150 bp with second upper primer). Because one of the reaction conditions (55 cycles total ampli¢cation) produced only weakly visible bands on the gel, it is likely that the reaction had not saturated in this con-
101
dition. In this condition the di¡erent products would not compete for substrate, and the ampli¢cation of one product would be unlikely to suppress the ampli¢cation of others. The bands were cut out, cloned into a pGEM-T vector (Promega, Madison, WI, USA) and sequenced using the Dye Deoxy Terminator Cycle Sequencing Kit (Perkin Elmer, Australia) and an automatic DNA sequencer (Model 373A Applied Biosystems Division, Australia). Control material ampli¢ed without reverse transcription did not produce visible bands, indicating no contamination by genomic DNA. The MPK-11 variant of FGFR4 described below was further examined by amplifying fresh cDNA from neonatal (P0) mouse brain, using an upstream primer AAA TGG ATG GCT CCA GAG GCG with its 3P end on the MPK-11 speci¢c G at position 2102 of Genbank MMFGFR4M, in combination with a degenerate downstream primer AA(AC) GG(GC) TC(AG) TG(AG) (GC)(AT)G AA(AG) AC(AC) GAG with 3P end at position 2459. The product was cloned and sequenced as above. In order to detect the full known range of FGF receptors and their Ig (immunoglobinlike) III domain splice variants, separate PCR reactions were run on the utricular and saccular material, using primers speci¢cally targeted at the di¡erent Ig III domains, with primers and ampli¢cation conditions as described by Pickles et al. (1998). 3. Results and discussion Of the 80 clones sequenced, 49 contained inserts coding for tyrosine kinases or occasionally serine/threonine kinases, as shown by homology with other kinase sequences in the relatively conserved stretch between the two primer regions (Table 1). The remainder of the clones contained inserts that either had homology to known non-kinase sequences (11) or had no homology to any sequences in the Genbank or EMBL databases and did not contain any tyrosine kinase-like motifs. No completely novel tyrosine kinases were found. The two di¡erent upper primers did not identi¢ably appear to target di¡erent populations of tyrosine kinases, nor did the di¡ering reaction conditions (55 vs. 70 total number of cycle ampli¢cation). Because 30^50% of the material analysed lay outside the sensory epithelium proper, it should be borne in mind that some of the sequences analysed may have been expressed partly or exclusively in these other areas. The greatest number of clones (16) coded for FGF receptor 4. The sequence was a slight variant called MPK-11 (Gilardi-Hebenstreit et al., 1992). Compared with the standard FGF receptor 4 sequence (Genbank MMFGFR4M), MPK-11 has a G instead of A at posi-
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Table 1 The deduced amino acid sequences of the di¡erent clones found with the degenerate PCR primers, showing only the region between the most closely spaced pair of primers
The amino acids are shown as single-letter codes. The amino acids targeted by the primers are shown at the bottom. Where a sequence codes for the same amino acid as FGF receptor 4, the symbol is replaced by a dot. Dashes show gaps introduced to produce maximum alignment between sequences. The primers are shown in the correct alignment with the other sequences; the cloned sequences are not shown within the primer regions.
tion 2102, and T instead of C at position 2114, neither of which lead to a predicted di¡erence at the protein level. Further analysis of this sequence using new primers designed to speci¢cally anneal to FGF receptor 4 sequences with the variant G at position 2102 and using new material (see Section 2) con¢rmed upon sequencing that the T to C substitution at position 2114 was indeed present. This con¢rmed the identity of the original MPK-11 sequence, and showed that it was not an ampli¢cation artefact resulting from the high degree of ampli¢cation used in the original tyrosine kinase screen. While the majority of these clones were otherwise identical to the published FGFR4 (Stark et al., 1991), a minority of clones coded for a novel splice variant in which some of the sequence corresponding to intron 17 was expressed, predicting a premature stop codon (van Heumen et al., submitted for publication). This results in a predicted deletion of the tyrosine at position 760. On the basis of homology with FGF receptor 1, phosphorylation of the tyrosine at this position signals via the phosphatidylinositol biphosphate signalling pathway, unlike the other tyrosines in the kinase region which signal via the MAP kinase pathway (Mohammadi et al., 1991). Thus di¡erential downstream signalling by the FGF receptor 4 could be ac-
complished by di¡erential splicing in the C-terminal region. In comparison, only one clone coded for FGF receptor 2. This must not be taken as any indication of the relative expression of the two receptors, because the distributions are highly a¡ected by the relative ampli¢cation e¤ciencies of the PCR process, which are highly sequence-dependent. Separate ampli¢cations of the starting material with primers speci¢c for all the known FGF receptors and their Ig III (third immunoglobinlike domain) splice variants, showed that in the starting material all receptors and all Ig IIIb and c splice variants were expressed (Fig. 1). As shown by Ornitz et al. (1996), the di¡erent Ig III splice variants of the di¡erent FGF receptors have di¡erent sensitivities to the di¡erent FGFs. The present results suggest that both FGF1 and FGF2 could be e¡ective in stimulating the FGF receptors of the utricle and saccule. There have been previous reports of FGF receptor expression in the inner ear. In the mammal, FGF receptor 3 expression is critical for normal development of the organ of Corti, particularly in the region of the outer hair cells and Deiters' cells (Colvin et al., 1996). Expression of FGF receptor 1 mRNA was shown in rat utricles by Sa¡er et al. (1996), the present results sug-
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Fig. 1. RT-PCR for FGF receptor Ig III isoforms, from mouse saccules and utricles (upper gel). The lower gel shows control ampli¢cations of non-reverse transcribed material, and shows no ampli¢cation products; nor did negative controls where no material was used. FGF receptors (FGFR1^4) and their b and c Ig III variants are marked below each lane. All FGF receptor Ig III isoforms are present in the upper gel, as shown by the presence of bands of correct size in each lane, although FGFR1b and FGFR3b bands are relatively weak compared with the others. FGF receptor 4 does not have a splice variant in the Ig III region. Markers: 100 base-pair intervals.
gesting that this expression is dominated by the IIIc variant. In the rat cochlea, acoustic trauma enhances FGF receptor 3 expression (Pirvola et al., 1995), while in the chick, acoustic and ototoxic damage enhance FGF receptor 1 expression in the supporting cells (Lee and Cotanche, 1996; Pickles and van Heumen, 1997). In the chick, FGF1 and FGF2 levels rise after damage (Umemoto et al., 1995; Pickles and van Heumen, 1997). These results suggest that FGFs are involved in the response to trauma, and, particularly in the mammal, in initial development. Messenger RNA coding for another growth factor receptor, the IGF-1 receptor, was also found, as had previously been shown by Sa¡er et al. (1996) in rat utricles. Sa¡er et al. showed that the level of mRNA was reduced in damaged epithelia. This was consistent with preferential expression in hair cells, which would account for a reduction in levels as the cells were lost. A further group of tyrosine kinases found in the screen are likely to be involved in cell-cell signalling. Focal adhesion kinase, which, as the name implies, is
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concentrated at focal adhesions, is tyrosine-phosphorylated in response to components of the extracellular matrix. These include ¢bronectin which uses the integrins as an intermediate in the signalling pathway (Hanks et al., 1992). A second tyrosine kinase found in the screen and also thought to be activated by integrins is the proto-oncogene c-Abl. Here, cell adhesion to ¢bronectin triggers the recruitment of c-Abl from the nucleus to the focal adhesions leading to activation of the kinase (e.g. Lewis and Schwartz, 1998). These receptor tyrosine kinases could be involved in interactions between hair cells and their supporting cells, and between both cell types and the basement membranes. Other sequences found include Tyk-2, which is one of the four known Janus kinase (JAK) tyrosine kinases, and is a non-receptor tyrosine kinase which forms a complex with a number of cytokine receptor subunits, to phosphorylate themselves as well as the receptors (e.g. Carter-Su and Smit, 1998), in£uencing intracellular events. C-fes may be involved in vesicle transport (Haigh et al., 1996). Tyro-3 (also called rse, brt and sky) is strongly expressed in the central nervous system, and is concentrated in the synaptosome fraction (Lai et al., 1994; Schulz et al., 1995). The structure contains extracellular motifs common to neural cell adhesion molecules such as NCAM, as well as the intracellular tyrosine kinase domain. It is therefore likely that it is involved in extracellular recognition at the hair-cell/vestibular nerve synapse. RYK is a mammalian homologue of the Drosophila linotte gene, which is thought to be involved in axonal guidance (Moreau-Fauvarque et al., 1998). It is therefore likely to assist in directing the innervation within the organ. 4. Conclusion The present technique is a powerful method for sampling a group of important genes that are expressed in a given tissue. The present paper shows that it can successfully be used on very small amounts of starting tissue, appropriate for sensory areas of the inner ear. While no completely novel tyrosine kinases were found, it permitted the discovery of a novel splice variant of FGF receptor 4, with implications for di¡erential activation of downstream signalling pathways. It is highly likely that the present screen only found a minority of the tyrosine kinases expressed in the tissue. The detection and distribution of the di¡erent sequences is likely to re£ect the intrinsic e¤ciency of primer annealing and ampli¢cation of the di¡erent sequences found, as well as their relative abundance in the test tissue.
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Acknowledgements Supported by the Garnett-Passe and Rodney Williams Memorial Foundation. References Carter-Su, C., Smit, L.S., 1998. Signalling via JAK tyrosine kinases: growth hormone receptor as a model system. Recent Prog. Horm. Res. 53, 61^82. Colvin, J.S., Bohne, B.A., Hording, G.W., McEwen, D.G., Ornitz, D.M., 1996. Skeletal overgrowth and deafness in mice lacking ¢broblast growth factor receptor 3. Nat. Genet. 12, 390^397. Forge, A., Li, L., Corwin, J.T., Nevill, G., 1993. Ultrastructural evidence for hair cell regeneration in the mammalian inner ear. Science 259, 1616^1619. Gilardi-Hebenstreit, P., Nieto, M.A., Frain, M., Mattei, M.G., Chestier, A., Wilkinson, D.G., Charnay, P.G., 1992. An Eph-related receptor protein tyrosine kinase gene segmentally expressed in the developing mouse hindbrain. Oncogene 7, 2499^2506. Haigh, J., McVeigh, J., Greer, P., 1996. The fps/fes tyrosine kinase is expressed in myeloid, vascular endothelial, epithelial, and neuronal cells and is localized in the trans-Golgi network. Cell Growth Di¡er. 7, 931^944. Hanks, S.K., Quinn, A.M., Hunter, T., 1988. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 42^52. Hanks, S.K., Calab, M.B., Harper, M.C., Patel, S.K., 1992. Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to ¢bronectin. Proc. Natl. Acad. Sci. USA 89, 8487^8491. Lai, C., Lemke, G., 1991. An extended family of protein-tyrosine kinase genes di¡erentially expressed in the vertebrate nervous system. Neuron 6, 691^704. Lai, C., Gore, M., Lemke, G., 1994. Structure, expression, and activity of Tyro-3, a neural adhesion-related tyrosine kinase. Oncogene 9, 2567^2578. Lee, K.H., Cotanche, D.A., 1996. Potential role of bFGF and retinoic acid in the regeneration of chicken cochlear hair cells. Hear. Res. 94, 1^13. Lewis, J.M., Schwartz, M.A., 1998. Integrins regulate the association and phosphorylation of paxillin by c-Abl. J. Biol. Chem. 273, 14225^14230. Luo, L., Koutnouyan, H., Baird, A., Ryan, A.F., 1993. Acidic and basic FGF mRNA expression in the adult and developing rat cochlea. Hear. Res. 69, 182^193.
Mohammadi, M., Honegger, A.M., Rotin, D., Fischer, R., Bellot, F., Li, W., Dionne, C.A., Jaye, M., Rubinstein, M., Schlessinger, J., 1991. A tyrosine-phosphorylated carboxy-terminal peptide of the ¢broblast growth factor receptor (£g) is a binding site for the SH2 domain of phospholipase C-Q1. Mol. Cell. Biol. 11, 5068^5078. Moreau-Fauvarque, C., Taillebourg, E., Boissoneau, E., Mesnard, J., Dura, J.M., 1998. The receptor tyrosine kinase gene linotte is required for neuronal pathway selection in the Drosophila mushroom bodies. Mech. Dev. 78, 47^61. Ornitz, D.M., Xu, J., Colvin, J.S., McEwen, D.G., MacArthur, C.A., Coulier, F., Gao, G., Goldfarb, M., 1996. Receptor speci¢city of the ¢broblast growth factor family. J. Biol. Chem. 271, 15292^ 15297. Pickles, J.O., van Heumen, W.R.A., 1997. The expression of messenger RNAs coding for growth factors, their receptors, and eph-class receptor tyrosine kinases in normal and ototoxically damaged chick cochleae. Dev. Neurosci. 19, 476^487. Pickles, J.O., Harter, C., Rebillard, G., 1998. Fibroblast growth factor receptor expression in outer hair cells of rat cochlea. NeuroReport 9, 4093^4095. Pirvola, U., Cao, Y., Oellig, C., Suoqiang, Z., Pettersson, R.F., Ylikoski, J., 1995. The site of action of neuronal acidic ¢broblast growth factor in the organ of Corti of the rat cochlea. Proc. Natl. Acad. Sci. USA 92, 9269^9273. Raphael, Y., Altschuler, R.A., 1991. Reorganization of cytoskeletal and junctional proteins during cochlear hair cell degeneration. Cell Motil. Cytosk. 18, 215^227. Robinson, D., He, F., Pretlow, T., Kung, H.J., 1996. A tyrosine kinase pro¢le of prostate carcinoma. Proc. Natl. Acad. Sci. USA 93, 5958^5962. Sa¡er, L.D., Gu, R., Corwin, J.T., 1996. An RT-PCR analysis of mRNA for growth factor receptors in damaged and control sensory epithelia of rat utricles. Hear. Res. 94, 14^23. Schulz, N.T., Paulhiac, C.I., Lee, L., Zhou, R., 1995. Isolation and expression analysis of tyro3, a murine growth factor receptor tyrosine kinase preferentially expressed in adult brain. Brain Res. Mol. Brain Res. 28, 273^280. Stark, K.L., McMahon, J.A., McMahon, A.P., 1991. FGFR-4, a new member of the ¢broblast growth factor family, expressed in the de¢nitive endoderm and skeletal muscle lineages of the mouse. Development 113, 641^651. Umemoto, M., Sakagami, M., Fukazawa, K., Ashida, K., Kubo, T., Senda, T., Yoneda, Y., 1995. Hair cell regeneration in the chick inner ear following acoustic trauma: ultrastructural and immunohistochemical studies. Cell Tissue Res. 281, 435^443. Warchol, M.E., Lambert, P.R., Goldstein, B.J., Forge, A., Corwin, J.T., 1993. Regenerative proliferation in inner ear sensory epithelia from adult guinea pigs and humans. Science 259, 1619^1622.
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A tyrosine kinase screen of mouse vestibular maculae James O. Pickles *, Walter R.A. van Heumen, Christina Claxton Vision, Touch and Hearing Research Centre, Department of Physiology and Pharmacology, University of Queensland, Brisbane, Qld. 4072, Australia Received 29 December 1998; received in revised form 25 May 1999; accepted 7 June 1999
Abstract Receptor tyrosine kinases allow extracellular signals to influence intracellular events, while other tyrosine kinases are involved in intracellular signalling. They may therefore be involved in the development, maintenance and repair of the sensory epithelia of the inner ear, since these are believed to be affected by inter- and intracellular signalling. In order to analyse possible tyrosine kinases expressed in sensory areas of the inner ear, a reverse transcription polymerase chain reaction screen of microdissected sensory epithelia was undertaken, using primers targeted at conserved sequences in tyrosine kinase domains. Tissue was taken from the maculae of the mouse vestibular organs, and consisted mainly of hair cells and their supporting cells. Of 80 clones sequenced, 49 coded for tyrosine kinases, and 11 for other known molecules. Further analysis of one of the sequences, for FGF receptor 4, showed a novel variant, expressed in the inner ear and elsewhere, with a variation in the intracellular domain which suggests differential activation of known signalling pathways. Other clones coded for tyrosine kinases expected to be involved in cell surface and intracellular signalling. The technique forms a powerful tool for analysing a range of the tyrosine kinases expressed, and provides a starting point for the analysis of cell-cell signalling in the inner ear. ß 1999 Elsevier Science B.V. All rights reserved. Key words: Tyrosine kinase; PCR screen; FGF receptor; IGF-1 receptor; Hair cell; Vestibular maculae; Mouse
1. Introduction Hair cells are arranged in a precise and complex array within the cochlear and vestibular sensory epithelia. Interactions between hair cells and their supporting cells are thought to be important in setting up and maintaining the pattern of cells in the cochlear sensory epithelium, and for initiating a repair response in the adjacent supporting cells after hair cell damage. In the organ of Corti the supporting cells expand to ¢ll the gap in the reticular lamina (e.g. Raphael and Altschuler, 1991). However, in vestibular organs, the supporting cells are triggered to produce new hair cells (Forge et al., 1993 ; Warchol et al., 1993). Given the role of receptor tyrosine kinases in cell interactions, it is likely that these molecules are involved in the initial development of the sensory epithelia, as well as in the normal maintenance of the epithelia, and in their response to damage. Tyrosine kinases are also involved in intracel-
* Corresponding author. Tel.: +61 (7) 3365-4125; Fax: +61 (7) 3365-4522; E-mail: [email protected]
lular signalling, and in this role too are likely to take part. Analyses of mRNAs coding for speci¢c tyrosine kinases can be undertaken with polymerase chain reaction primers speci¢cally targeted at selected candidate sequences (as published earlier by e.g. Lee and Cotanche, 1996 ; Sa¡er et al., 1996 ; Pickles and van Heumen, 1997). However a wider search for tyrosine kinases can be undertaken by means of a degenerate screen (e.g. Lai and Lemke, 1991; Robinson et al., 1996), which has the advantage that it is capable of revealing unanticipated or novel kinases. The screen takes advantage of the fact that tyrosine kinases express conserved motifs, a set number of peptides apart within the kinase domain (e.g. Lai and Lemke, 1991 ; Robinson et al., 1996). Degenerate polymerase chain reaction (PCR) primers targeted at these regions produce ampli¢cation products with only a small range of sizes, permitting the bands to be readily excised from gels, thus allowing further puri¢cation followed by cloning and sequencing. The tyrosine kinase screen was undertaken in microdissected sensory maculae, from neonatal mouse saccules and utricles. The neonatal mouse inner ear expresses
0378-5955 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 9 ) 0 0 1 1 4 - 8
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J.O. Pickles et al. / Hearing Research 136 (1999) 100^104
high levels of growth factors (e.g. Luo et al., 1993), and it is therefore likely that the corresponding receptors are also expressed. While the target tissue was not in a pathological state, we expect the signalling pathways that are able to detect damage will be present, as well as those involved in the maintenance of the sensory epithelium. 2. Materials and methods Neonate mice (postnatal day 0: P0) were killed by decapitation, the heads bisected, and the temporal bones removed under ice-cold Hank's balanced salt solution. The vestibular areas were removed, the saccules and utricles opened, and the sensory epithelia plus some adjacent supporting structures removed. Histology of tissue removed in an identical way, and estimated over all sections taken through the tissues, revealed that between 50 and 70% of the tissue volume consisted of the hair cells plus their intervening supporting cells, the remainder consisting mainly of basal ¢broblasts and fragments of lateral walls of the organs. The microdissected sensory areas were lysed in bu¡ered guanidium thiocyanate and N-lauroyl sarcosine. Poly-A+ RNA was extracted with oligo-dT cellulose (Pharmacia Quick-Prep Micro mRNA Puri¢cation Kit). Seven to fourteen sensory areas were used for each of three separate extractions. Following reverse transcription with random hexamers as primers, 1/30th of the product volume was PCR ampli¢ed with degenerate primers. One of two di¡erent upper (forward) primers were used, either (in 5P to 3P order) CCA (CT)CG (AGCT)GA (CT)(CT)T (AGCT)GC or AA(AG) GT(AGCT) (AGT)(GCT)(AGCT) GA(CT)TT (CT)GG, in combination with a single lower (reverse) primer: (AC)C(AG) (AT)A(AGCT) (GC)(ACT)C CA(AGCT). In amino acid single-letter code, the two upper primers were targeted at either the conserved region HRDLAAR found in protein tyrosine kinases, or at the K(V/I)(A/S/T)DFG region common to protein tyrosine and serine/threonine kinases. The single lower primer was targeted at the conserved kinase region DVW(S/A)(F/Y) (Hanks et al., 1988). Reaction conditions were: annealing 1 min at 42³C, extending 2 min at 60³C, melting 1 min at 94³C, for 35 cycles. Following gel puri¢cation, 20% of the PCR products were reampli¢ed in a new reaction using the same conditions, for either 20 or 35 cycles. Following analysis on an agarose gel, visible bands were obtained at the expected lengths (approximately 200 bp with ¢rst upper primer, 150 bp with second upper primer). Because one of the reaction conditions (55 cycles total ampli¢cation) produced only weakly visible bands on the gel, it is likely that the reaction had not saturated in this con-
101
dition. In this condition the di¡erent products would not compete for substrate, and the ampli¢cation of one product would be unlikely to suppress the ampli¢cation of others. The bands were cut out, cloned into a pGEM-T vector (Promega, Madison, WI, USA) and sequenced using the Dye Deoxy Terminator Cycle Sequencing Kit (Perkin Elmer, Australia) and an automatic DNA sequencer (Model 373A Applied Biosystems Division, Australia). Control material ampli¢ed without reverse transcription did not produce visible bands, indicating no contamination by genomic DNA. The MPK-11 variant of FGFR4 described below was further examined by amplifying fresh cDNA from neonatal (P0) mouse brain, using an upstream primer AAA TGG ATG GCT CCA GAG GCG with its 3P end on the MPK-11 speci¢c G at position 2102 of Genbank MMFGFR4M, in combination with a degenerate downstream primer AA(AC) GG(GC) TC(AG) TG(AG) (GC)(AT)G AA(AG) AC(AC) GAG with 3P end at position 2459. The product was cloned and sequenced as above. In order to detect the full known range of FGF receptors and their Ig (immunoglobinlike) III domain splice variants, separate PCR reactions were run on the utricular and saccular material, using primers speci¢cally targeted at the di¡erent Ig III domains, with primers and ampli¢cation conditions as described by Pickles et al. (1998). 3. Results and discussion Of the 80 clones sequenced, 49 contained inserts coding for tyrosine kinases or occasionally serine/threonine kinases, as shown by homology with other kinase sequences in the relatively conserved stretch between the two primer regions (Table 1). The remainder of the clones contained inserts that either had homology to known non-kinase sequences (11) or had no homology to any sequences in the Genbank or EMBL databases and did not contain any tyrosine kinase-like motifs. No completely novel tyrosine kinases were found. The two di¡erent upper primers did not identi¢ably appear to target di¡erent populations of tyrosine kinases, nor did the di¡ering reaction conditions (55 vs. 70 total number of cycle ampli¢cation). Because 30^50% of the material analysed lay outside the sensory epithelium proper, it should be borne in mind that some of the sequences analysed may have been expressed partly or exclusively in these other areas. The greatest number of clones (16) coded for FGF receptor 4. The sequence was a slight variant called MPK-11 (Gilardi-Hebenstreit et al., 1992). Compared with the standard FGF receptor 4 sequence (Genbank MMFGFR4M), MPK-11 has a G instead of A at posi-
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Table 1 The deduced amino acid sequences of the di¡erent clones found with the degenerate PCR primers, showing only the region between the most closely spaced pair of primers
The amino acids are shown as single-letter codes. The amino acids targeted by the primers are shown at the bottom. Where a sequence codes for the same amino acid as FGF receptor 4, the symbol is replaced by a dot. Dashes show gaps introduced to produce maximum alignment between sequences. The primers are shown in the correct alignment with the other sequences; the cloned sequences are not shown within the primer regions.
tion 2102, and T instead of C at position 2114, neither of which lead to a predicted di¡erence at the protein level. Further analysis of this sequence using new primers designed to speci¢cally anneal to FGF receptor 4 sequences with the variant G at position 2102 and using new material (see Section 2) con¢rmed upon sequencing that the T to C substitution at position 2114 was indeed present. This con¢rmed the identity of the original MPK-11 sequence, and showed that it was not an ampli¢cation artefact resulting from the high degree of ampli¢cation used in the original tyrosine kinase screen. While the majority of these clones were otherwise identical to the published FGFR4 (Stark et al., 1991), a minority of clones coded for a novel splice variant in which some of the sequence corresponding to intron 17 was expressed, predicting a premature stop codon (van Heumen et al., submitted for publication). This results in a predicted deletion of the tyrosine at position 760. On the basis of homology with FGF receptor 1, phosphorylation of the tyrosine at this position signals via the phosphatidylinositol biphosphate signalling pathway, unlike the other tyrosines in the kinase region which signal via the MAP kinase pathway (Mohammadi et al., 1991). Thus di¡erential downstream signalling by the FGF receptor 4 could be ac-
complished by di¡erential splicing in the C-terminal region. In comparison, only one clone coded for FGF receptor 2. This must not be taken as any indication of the relative expression of the two receptors, because the distributions are highly a¡ected by the relative ampli¢cation e¤ciencies of the PCR process, which are highly sequence-dependent. Separate ampli¢cations of the starting material with primers speci¢c for all the known FGF receptors and their Ig III (third immunoglobinlike domain) splice variants, showed that in the starting material all receptors and all Ig IIIb and c splice variants were expressed (Fig. 1). As shown by Ornitz et al. (1996), the di¡erent Ig III splice variants of the di¡erent FGF receptors have di¡erent sensitivities to the di¡erent FGFs. The present results suggest that both FGF1 and FGF2 could be e¡ective in stimulating the FGF receptors of the utricle and saccule. There have been previous reports of FGF receptor expression in the inner ear. In the mammal, FGF receptor 3 expression is critical for normal development of the organ of Corti, particularly in the region of the outer hair cells and Deiters' cells (Colvin et al., 1996). Expression of FGF receptor 1 mRNA was shown in rat utricles by Sa¡er et al. (1996), the present results sug-
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Fig. 1. RT-PCR for FGF receptor Ig III isoforms, from mouse saccules and utricles (upper gel). The lower gel shows control ampli¢cations of non-reverse transcribed material, and shows no ampli¢cation products; nor did negative controls where no material was used. FGF receptors (FGFR1^4) and their b and c Ig III variants are marked below each lane. All FGF receptor Ig III isoforms are present in the upper gel, as shown by the presence of bands of correct size in each lane, although FGFR1b and FGFR3b bands are relatively weak compared with the others. FGF receptor 4 does not have a splice variant in the Ig III region. Markers: 100 base-pair intervals.
gesting that this expression is dominated by the IIIc variant. In the rat cochlea, acoustic trauma enhances FGF receptor 3 expression (Pirvola et al., 1995), while in the chick, acoustic and ototoxic damage enhance FGF receptor 1 expression in the supporting cells (Lee and Cotanche, 1996; Pickles and van Heumen, 1997). In the chick, FGF1 and FGF2 levels rise after damage (Umemoto et al., 1995; Pickles and van Heumen, 1997). These results suggest that FGFs are involved in the response to trauma, and, particularly in the mammal, in initial development. Messenger RNA coding for another growth factor receptor, the IGF-1 receptor, was also found, as had previously been shown by Sa¡er et al. (1996) in rat utricles. Sa¡er et al. showed that the level of mRNA was reduced in damaged epithelia. This was consistent with preferential expression in hair cells, which would account for a reduction in levels as the cells were lost. A further group of tyrosine kinases found in the screen are likely to be involved in cell-cell signalling. Focal adhesion kinase, which, as the name implies, is
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concentrated at focal adhesions, is tyrosine-phosphorylated in response to components of the extracellular matrix. These include ¢bronectin which uses the integrins as an intermediate in the signalling pathway (Hanks et al., 1992). A second tyrosine kinase found in the screen and also thought to be activated by integrins is the proto-oncogene c-Abl. Here, cell adhesion to ¢bronectin triggers the recruitment of c-Abl from the nucleus to the focal adhesions leading to activation of the kinase (e.g. Lewis and Schwartz, 1998). These receptor tyrosine kinases could be involved in interactions between hair cells and their supporting cells, and between both cell types and the basement membranes. Other sequences found include Tyk-2, which is one of the four known Janus kinase (JAK) tyrosine kinases, and is a non-receptor tyrosine kinase which forms a complex with a number of cytokine receptor subunits, to phosphorylate themselves as well as the receptors (e.g. Carter-Su and Smit, 1998), in£uencing intracellular events. C-fes may be involved in vesicle transport (Haigh et al., 1996). Tyro-3 (also called rse, brt and sky) is strongly expressed in the central nervous system, and is concentrated in the synaptosome fraction (Lai et al., 1994; Schulz et al., 1995). The structure contains extracellular motifs common to neural cell adhesion molecules such as NCAM, as well as the intracellular tyrosine kinase domain. It is therefore likely that it is involved in extracellular recognition at the hair-cell/vestibular nerve synapse. RYK is a mammalian homologue of the Drosophila linotte gene, which is thought to be involved in axonal guidance (Moreau-Fauvarque et al., 1998). It is therefore likely to assist in directing the innervation within the organ. 4. Conclusion The present technique is a powerful method for sampling a group of important genes that are expressed in a given tissue. The present paper shows that it can successfully be used on very small amounts of starting tissue, appropriate for sensory areas of the inner ear. While no completely novel tyrosine kinases were found, it permitted the discovery of a novel splice variant of FGF receptor 4, with implications for di¡erential activation of downstream signalling pathways. It is highly likely that the present screen only found a minority of the tyrosine kinases expressed in the tissue. The detection and distribution of the di¡erent sequences is likely to re£ect the intrinsic e¤ciency of primer annealing and ampli¢cation of the di¡erent sequences found, as well as their relative abundance in the test tissue.
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