- Email: [email protected]
Segregation of the receptor EphA7 from its tyrosine kinase-negative isoform on neurons in adult mouse brain
Molecular Brain Research 74 Ž1999. 231–236 www.elsevier.comrlocaterbres
Short communication
Segregation of the receptor EphA7 from its tyrosine kinase-negative isoform on neurons in adult mouse brain T. Ciossek b
a,)
, A. Ullrich a , E. West b, J.H. Rogers
b,1
a Max-Planck-Institut for Biochemistry, 82152 Martinsried, Germany Department of Physiology, UniÕersity of Cambridge, Downing St., Cambridge CB2 3EG, UK
Accepted 7 September 1999
Abstract The EphA7 gene encodes not only a typical receptor tyrosine kinase ŽTK q . but also an isoform lacking the tyrosine kinase domain ŽTK y .. We have made antibodies to localise EphA7 TK q and TK y isoforms in mouse brain. The TK y isoform was not detectable prenatally, despite reported expression of the TK y mRNA in the embryo. However, both TK q and TK y isoforms showed striking distributions in adult brain. TK q receptor immunoreactivity was strong in neuropil throughout most of the telencephalon, probably on fine arborisations from neurons which expressed EphA7 during development Žin cerebral cortex, hippocampus, and striatum.. In contrast, TK y receptor immunoreactivity was conspicuous on cell bodies and proximal dendrites of a limited number of neuronal types, some of which carried EphA7 TK q receptor on their axons. This suggests that the TK y receptor, acting as a dominant negative antagonist, may ensure that the TK q receptor only responds to signals encountered by the growing extremities of axons or dendrites. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Eph receptor; Corticospinal tract; Hippocampus; Motor cortex; Purkinje cell
The receptor EphA7 Žformerly Mdk1rEbkrEhk3. is widely expressed in developing tissues and especially in developing brain w2,7,13,20x. In both developing and adult brain, its expression pattern by in situ hybridisation ŽISH. overlaps with those of other Eph receptors, notably in the major cell layers of cerebellum and hippocampus and cerebral cortex. All EphA receptors including EphA7 bind the family of cell-attached ephrin-A ligands w4,5,8–10,22x. Therefore, EphA7 probably cooperates with other EphA receptors in signalling. However it differs from the others in having a second isoform lacking the tyrosine kinase domain, produced by alternative RNA splicing w2,13,17,20x. The kinase-negative ŽTK y . form binds ligand almost as well as the kinase-positive ŽTK q . form w4x. By analogy with other TK y variants of receptor tyrosine kinases, it probably acts as a dominant negative antagonist w1,6,19,21x. ISH has provided evidence that the two forms are colo-
) Corresponding author. DeveloGen, Rudolf Wissell Str. 28, D-37079 Goettingen, Germany. Fax: q49-551-505-5886; e-mail: [email protected] 1 Also corresponding author. Fax: q44-1223-333840; e-mail: [email protected].
calised, with the TK q mRNA being predominant in the embryo but the TK y mRNA becoming more abundant in adult brain w13x.
Fig. 1. Reaction of the antibodies with EphA7 TKq and TKy isoforms by immunoprecipitation. Ž1, 2. HEK293 cells were untransfected Ž1. or transiently transfected Ž2. with the full-length cDNA ŽMDK1.; as in Ref. w2x, they were labelled with 35 S-methionine, lysed, immunoprecipitated with Ab-TKq, electrophoresed, blotted, and autoradiographed. The doublet at 120 and 114 kDa corresponds to EphA7 TKq, with and without glycosylation w2x. Ž3, 4. HEK293 cells were untransfected Ž1. or transiently transfected Ž2. with the cDNA for the TKy isoform ŽMDK1-T1., then processed as above with Ab-TKy. The doublet at approximately 70 and 80 kDa corresponds to EphA7 TKy, with and without glycosylation.
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 2 8 5 - 5
232
T. Ciossek et al.r Molecular Brain Research 74 (1999) 231–236
Therefore, it is important to know whether EphA7 mRNAs, as located by ISH, are translated to produce the TK q or the TK y forms at various stages and locations of brain development, and where each isoform is located on the neurons. To answer these questions, we have now produced antibodies to localise EphA7 TK q and TK y immunoreactivity. The antiserum against the EphA7 TK q isoform ŽAbTK q . was raised in rabbit against a fusion protein of glutathione-S-transferase with the last 111 amino acids of the mouse EphA7 receptor tyrosine kinase sequence ŽGSTct-EphA7. w2x. This portion covers the C-terminal SAM homology domain w16x. Similar fusions were made with EphA5, EphB3, and EphB4 w3x and used for absorption controls. By western blots and immunoprecipitations with various Eph receptors transiently overexpressed from HEK
293 cells w2,3x, Ab-TKq was shown to react with EphA7 ŽFig. 1.. It also showed some cross-reaction with some other Eph receptors; but in immunohistochemistry, this made at most a minor contribution, as the staining reported here was consistent with the ISH patterns, and different from the patterns we have seen with antibodies against EphA4 w12x and EphA5 ŽSanta Cruz. Žalthough there is much overlap.. Specificity was checked by absorption controls, which showed that immunoreactivity in brain was specific for EphA7, with two exceptions: Ž1. possible limited cross-reaction with other Eph receptors where coexpressed Že.g., in Purkinje cells.. Staining in such areas was weakened by pre-absorption with other GST-ct-Eph fusion proteins, but not eliminated unless pre-absorbed with the immunogen GST-ct-EphA7. Ž2. Irrelevant crossreaction with a GST epitope, restricted to glia in adult
Fig. 2. Adult forebrain, in parasagittal sections. Scale bar s 0.5 mm. ŽA. Stained with Ab-TKq , pre-absorbed with fusion protein GST-ct-EphB4ŽMdk2. to eliminate cross-reactions. ŽThis pre-absorption prevented some staining of glia, which would not be clearly visible at this scale, but the remaining staining of fibres and neuropil is essentially as seen with unabsorbed antibody. Pre-absorption with immunogen GST-ct-EphA7ŽMdk1. removed almost all staining; data not shown.. Positive regions include: all fibre layers of hippocampus Žvery strong; Hip.; corpus callosum Žventral half; cc.; anterior commissure Žposterior branch; acp.; neuropil throughout cortex, striatum ŽStr., nucleus accumbens ŽAcb., etc.; substantia nigra pars reticulata ŽSNR. and striatonigral tract Žsn.. There is also weak staining in parts of thalamus ŽTha.. ŽB. Stained with Ab-TKy . Cell bodies are stained in hippocampus ŽHip. and cortex Žpyramidal cells; pyr.. Cells andror fibres are stained in some nuclei of thalamus including anterodorsal ŽAD., anteroventral ŽAV., and reticular ŽRt..
T. Ciossek et al.r Molecular Brain Research 74 (1999) 231–236
233
Fig. 2 Žcontinued..
brain Žespecially Bergmann glia in the cerebellum; only a minor contribution in the forebrain.. This was eliminated by pre-absorption with other GST fusion proteins, which was done in all data reported here. The antiserum for detection of the TK y isoform ŽAbTK y . was raised in rabbit against a synthetic peptide Žsequence: CSLVTNEHLSVL. representing the C-terminus of the TK y form of EphA7 in mouse w2x and rat w20x. The antiserum was shown to react with EphA7 TK y in immunoprecipitation and western blotting ŽFig. 1. as well as in immunohistochemistry. No potentially cross-reacting receptors are known. The immunising peptide was also used for absorption controls, and eliminated all staining. MFI mice were perfused with 4% paraformaldehyde. Immunohistochemistry was performed on sections by standard techniques, with antiserum diluted 1:2500 or 1:3000, followed by peroxidase-coupled anti-Žrabbit IgG. ŽVector Labs.. Staining of the developing nervous system with Ab-TK q was widespread w14x. Ab-TKy stained nothing in embryos, and very little in neonatal brains. However, both antisera gave strong and striking patterns of staining in the adult brain ŽFig. 2.. Table 1 lists six major brain regions that were EphA7positive in development, both by ISH and by EphA7 TK q
immunoreactivity on their developing axonal projections w14x. It indicates that these neuronal groups continued to express EphA7 TK q andror TK y immunoreactivity in different subcellular locations in the adult. These neuronal groups had all been reported as positive by ISH in the adult, though only the hippocampus gave strong ISH in all surveys. Ab-TKq stained neuropil strongly through most of the adult telencephalon, as well as in the hypothalamus and substantia nigra reticulata ŽFig. 2A.. This immunoreactivity was probably in axonal andror dendritic fields of neurons which expressed EphA7 mRNA and Ab-TKq immunoreactivity during development, viz. the cortex, hippocampus, and striatum w14x. Thus the hippocampus displayed strong staining throughout all its fibre fields, but little or no staining in the pyramidal cell layer ŽFig. 3B.. However, there was only weak TK q immunoreactivity in the adult thalamus and cerebellum, and none in mammillary nuclei, although these regions had expressed EphA7 earlier w14,15x. Weaker staining of neuropil was seen in some regions of midbrain and hindbrain. Ab-TKy , in contrast, conspicuously stained cell bodies and proximal dendrites of a small number of neuronal types. These include the pyramidal cells of motor cortex, piriform cortex, and hippocampus ŽFig. 3C,D., which car-
234
Brain region of origin
ISH ŽE14.5 to P1.
Neuronal cell bodies
TKy IR Žadult.
Target regions
TKq IR Žadult.
ISH Žadult. wReferencesx
Neocortex
q q
y q y
Neocortex Spinal cord grey matter Entorhinal cortex
qq Žy. qq
q w2,20x
Allocortex Žentorhinal, piriformrolfactory
Non-motor cortex Motor cortex pyram. cells Ento. cortex. pyram. cells
q w2,20x
Pirif. cortex. pyram. cells
q
Piriform cortex Most of telencephalon Hippocampus
qq qq qqq
qqq w2,7,13,20x
Lat. septal nuc. Striatum Globus pallidus Substantia nigra reticulata Interpeduncular nuclei Cerebellar nuclei
qqq qq y qq Žq. Žy.
Hippocampus
qq
Hippocampus Žpyram. cells and dentate gyrus.
q
Striatum
q
Striatum
y
Habenula Cerebellar cortex
qq q
Habenula Purkinje cells
Žq. q
q w2,13x
q w2,13x q w2,13,20x
T. Ciossek et al.r Molecular Brain Research 74 (1999) 231–236
Table 1 Neurons and their fibres expressing EphA7 isoforms in mouse brain For each major neuronal population positive by ISH, this table lists its major targetŽs., and whether they show EphA7 immunoreactivity ŽIR. on a scale from Žy. through Žq. Žweak. to qqq Žstrongly positive.. See Ref. w14x for table of EphA7 TKq IR on the growing axonal projections. Columns 1 and 2: Major brain regions which express EphA7 mRNA in development ŽE14.5 to postnatal day 1; Refs. w2,7x.. Columns 3 and 4: EphA7 TK-IR on cell bodies and proximal dendrites in these regions in adult Žand see below.. Columns 5 and 6: EphA7 TKq IR in neuropil of these same regions and their target fields Žwhere their axons terminate., in adult. Column 7: EphA7 mRNA reported in neuronal cell bodies in these regions in adult. TK y IR in adult brain is also conspicuous on cell bodies and proximal dendrites of the following neurons: rostral prefrontal cortex pyramidal cells, retrosplenial gyrusrsubiculum pyramidal cells, globus pallidus, ventral pallidum and nuclei of the diagonal band, nucleus isthmi magnocellularis pars compacta, red nucleus, mesencephalic trigeminal nucleus, scattered cells in midbrain, giant cells of reticular formation, cerebellar nuclei, prepositus hypoglossal nucleus. Also in the thalamus, there are also numerous very faintly stained neurons with positive dendrites in the following thalamic nuclei: anteromedial, anteroventral, dorsal lateral geniculate, laterodorsal, lateroposterior, mediodorsal, reuniens, reticular, ventrolateral, ventromedial, ventroposterior medial and lateral, zona incerta. Some other thalamic nuclei contain positive fibres, notably anterodorsal, parafascicular, and centrolaminar nuclei. Other thalamic nuclei, including the midline nuclei, are negative for TKy IR. In addition, in transverse sections of all regions of cortex and parts of hippocampus, Ab-TKy gives a diffuse ‘blotchy’ stain on a scale of ; 50 mm, which may represent domains of neuropil. We have also stained some transverse sections of rat brains with Ab-TKy and confirm the same distribution as in mouse, plus conspicuous cell staining in the substantia nigra reticulata.
T. Ciossek et al.r Molecular Brain Research 74 (1999) 231–236
235
Fig. 3. Adult brain, in parasagittal sections at high power. Scale bar s 100 mm. ŽA–D. TK q and TK y receptors in motor cortex ŽA, C. and hippocampus ŽB, D.. ŽA, B. Ab-TKq Žpre-absorbed with GST-ct-EphA5ŽEhk1. to eliminate glial cross-reaction., staining dense neuropil but no cell bodies. ŽC, D. Ab-TKy , staining cell bodies and proximal dendrites of pyramidal cells and a few interneurons. In hippocampus, CA2–CA3 pyramidal cell layer is marked with U , and two Ab-TKy IR interneurons are arrowed. ŽE–G. TK y receptors in hindbrain, stained with Ab-TKy . Note strong staining of cell bodies and proximal dendrites. ŽE. Cerebellar nuclei, ŽF. cerebellar cortex ŽPurkinje cells are stained., ŽG. mesencephalic trigeminal nucleus.
ried EphA7 TK q receptor on their axons both earlier and in the adult ŽTable 1.; and also the Purkinje cells and mesencephalic trigeminal neurons ŽFig. 3F,G., which carried EphA7 TK q receptor on their axons earlier but not in the adult w14x. Similar TK y receptor immunoreactivity was seen on the cell bodies and proximal dendrites in
several other nuclei where we have no evidence for EphA7 TK q expression in development Že.g., Fig. 3E; Table 1 footnote.. This expression pattern of the TK y isoform was more complex than expected. It was consistent with ISH results w13x in showing co-expression and an increasing ratio of
236
T. Ciossek et al.r Molecular Brain Research 74 (1999) 231–236
TK y to TK q isoform from embryo to adult. However, in embryos no TK y immunoreactivity was detected at all, suggesting translational or post-translational regulation. Conversely in adult brain, TK y immunoreactivity was present on several neuronal groups that were not reported positive by ISH. Some of these smaller neuronal groups may simply have been missed during development. But the lack of ISH signal may also suggest that expression is at a very low level with little turnover of the TK y receptor in the adult brain. In summary, the EphA7 TK q receptor is expressed on distal parts of axons and at least some dendrites, and is generally not detectable on the cell bodies in regions of origin, implying that it is efficiently transported along neurites. This pattern resembles that of the EphB2 receptor, which is expressed particularly in growth cones w11x and in adult forebrain synaptic membranes w18x. The wide distribution of EphA7 TK q immunoreactivity in neuropil, and especially in the dendritic and axonal fields of the most highly expressing cells of the hippocampus, suggests a role in synapses where the EphA4 receptor has also recently been located w12x. In contrast, the TK y variant is synthesised only after birth, and is localised to cell bodies and proximal dendrites of neurons, notably on some neurons which also express or expressed the TK q form. It could act simply as a binding partner for ligand if any is encountered near cell bodies, perhaps promoting cell adhesion. Alternatively, it could act as a dominant-negative antagonist. If so, its localisation on cell bodies and proximal dendrites may ensure that the EphA7 TK q receptor expressed by the same neurons only responds to signals encountered by the axonal or dendritic terminals.
Acknowledgements We thank Pauline Whiting and Sheila Barton for mice. Part of this work was supported by Sugen.
References w1x E. Amaya, T.J. Musci, M.W. Kirschner, Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos, Cell 66 Ž1991. 256–270. w2x T. Ciossek, B. Millauer, A. Ullrich, Identification of alternatively spliced mRNAs encoding variants of MDK1, a novel RTK expressed in the murine nervous system, Oncogene 9 Ž1995. 97–108. w3x T. Ciossek, M.M. Lerch, A. Ullrich, Cloning, characterization and differential expression of MDK2 and MDK5, two novel RTKs of the eckreph family, Oncogene 11 Ž1995. 2085–2095. w4x T. Ciossek, A. Ullrich, Identification of Elf-1 and B61 as high affinity ligands for the RTK MDK1, Oncogene 14 Ž1997. 35–43.
w5x U. Drescher, R. Klein, C. Stuermer, A. Faissner, F.G. Rathjen ŽEds.., Special issue: molecular bases of axonal growth and pathfinding, Cell Tissue Res. 290 Ž2. Ž1997.. w6x F.F. Eide, E.R. Vining, B.L. Eide, K. Zang, X.-Y. Wang, L.F. Reichardt, Naturally occurring truncated trkB receptors have dominant inhibitory effects on BDNF signalling, J. Neurosci. 16 Ž1996. 3123–3129. w7x J. Ellis, Q. Liu, M. Breitman, N.A. Jenkins, D.J. Gilbert, N.G. Copeland, H.V. Tempest, S. Warren, E. Muir, H. Schilling, F.A. Fletcher, S.F. Ziegler, J.H. Rogers, Embryo Brain Kinase: a novel gene of the ephrelk receptor tyrosine kinase family, Mech. Dev. 52 Ž1995. 319–341. w8x Eph Nomenclature Committee, Unified nomenclature for Eph family receptors and their ligands, the ephrins, Cell 90 Ž1997. 403–404. w9x J.G. Flanagan, P. Vanderhaeghen, The ephrins and Eph receptors in neural development, Annu. Rev. Neurosci. 21 Ž1998. 309–345. w10x N. Gale, S.J. Holland, D.M. Valenzuela, A. Flenniken, L. Pan, T.E. Ryan, M. Henkemeyer, K. Strebhardt, H. Hirai, D.G. Wilkinson, T. Pawson, S. Davis, G.D. Yancopoulos, Eph receptors and ligands comprise two major specificity subclasses, and are reciprocally compartmentalized during embryogenesis, Neuron 17 Ž1996. 9–19. w11x M. Henkemeyer, L.E.M. Marengere, J. McGlade, J.P. Olivier, R.A. Conlon, D.P. Holmyard, K. Letwin, T. Pawson, Immunolocalization of the Nuk receptor tyrosine kinase suggests roles in segmental patterning of the brain and axonogenesis, Oncogene 9 Ž1994. 1001– 1014. w12x M.E. Martone, J.A. Holash, A. Bayardo, E.B. Pasquale, M.H. Ellisman, Immunolocalization of the receptor tyrosine kinase EphA4 in the adult rat central nervous system, Brain Res. 771 Ž1997. 238–250. w13x T. Mori, A. Wanaka, A. Taguchi, K. Matsumoto, M. Tohyama, Localization of novel RTK genes of the eph family, MDK1 and its splicing variant, in the developing mouse nervous system, Mol. Brain Res. 34 Ž1995. 154–160. w14x J.H. Rogers, T. Ciossek, A. Ullrich, E. West, M. Hoare, E.M. Muir, Distribution of the receptor EphA7 and its ligands in development of the mouse nervous system, submitted to Mol. Brain Res., 1999. w15x J.H. Rogers, T. Ciossek, P. Menzel, E.B. Pasquale, Eph receptors and ephrins demarcate cerebellar lobules before and during their formation, Mech. Dev. Ž1999. in press. w16x J. Schultz, C.P. Ponting, K. Hofmann, P. Bork, SAM as a protein interaction domain involved in developmental regulation, Protein Sci. 6 Ž1997. 249–253. w17x A.H. Talukder, T. Muramatsu, N. Kaneda, A novel truncated variant form of EbkrMDK1 receptor tyrosine kinase is expressed in embryonic mouse brain, Cell Struct. Funct. 22 Ž1997. 477–485. w18x R. Torres, B.L. Firestein, H. Dong, J. Staudinger, E.N. Olson, R.L. Huganir, D.S. Bredt, N.W. Gale, G.D. Yancopoulos, PDZ proteins bind, cluster and synaptically colocalize with Eph receptors and their ephrin ligands, Neuron 21 Ž1998. 1453–1463. w19x H. Ueno, H. Colbert, J.A. Escobedo, L.T. Williams, Inhibition of PDGFb signal transduction by coexpression of a truncated receptor, Science 252 Ž1991. 844–848. w20x D.M. Valenzuela, E. Rojas, J.A. Griffiths, D.L. Compton, M. Gisser, N.Y. Ip, M. Goldfarb, G.D. Yancopoulos, Identification of full-length and truncated forms of Ehk-3, a novel member of the Eph RTK family, Oncogene 9 Ž1995. 1573–1580. w21x Q. Xu, G. Alldus, N. Holder, D.G. Wilkinson, Expression of truncated Sek-1 RTK disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain, Development 121 Ž1995. 4005–4016. w22x R. Zhou, The Eph family receptors and ligands, Pharmacol. Ther. 77 Ž1998. 151–181.
Short communication
Segregation of the receptor EphA7 from its tyrosine kinase-negative isoform on neurons in adult mouse brain T. Ciossek b
a,)
, A. Ullrich a , E. West b, J.H. Rogers
b,1
a Max-Planck-Institut for Biochemistry, 82152 Martinsried, Germany Department of Physiology, UniÕersity of Cambridge, Downing St., Cambridge CB2 3EG, UK
Accepted 7 September 1999
Abstract The EphA7 gene encodes not only a typical receptor tyrosine kinase ŽTK q . but also an isoform lacking the tyrosine kinase domain ŽTK y .. We have made antibodies to localise EphA7 TK q and TK y isoforms in mouse brain. The TK y isoform was not detectable prenatally, despite reported expression of the TK y mRNA in the embryo. However, both TK q and TK y isoforms showed striking distributions in adult brain. TK q receptor immunoreactivity was strong in neuropil throughout most of the telencephalon, probably on fine arborisations from neurons which expressed EphA7 during development Žin cerebral cortex, hippocampus, and striatum.. In contrast, TK y receptor immunoreactivity was conspicuous on cell bodies and proximal dendrites of a limited number of neuronal types, some of which carried EphA7 TK q receptor on their axons. This suggests that the TK y receptor, acting as a dominant negative antagonist, may ensure that the TK q receptor only responds to signals encountered by the growing extremities of axons or dendrites. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Eph receptor; Corticospinal tract; Hippocampus; Motor cortex; Purkinje cell
The receptor EphA7 Žformerly Mdk1rEbkrEhk3. is widely expressed in developing tissues and especially in developing brain w2,7,13,20x. In both developing and adult brain, its expression pattern by in situ hybridisation ŽISH. overlaps with those of other Eph receptors, notably in the major cell layers of cerebellum and hippocampus and cerebral cortex. All EphA receptors including EphA7 bind the family of cell-attached ephrin-A ligands w4,5,8–10,22x. Therefore, EphA7 probably cooperates with other EphA receptors in signalling. However it differs from the others in having a second isoform lacking the tyrosine kinase domain, produced by alternative RNA splicing w2,13,17,20x. The kinase-negative ŽTK y . form binds ligand almost as well as the kinase-positive ŽTK q . form w4x. By analogy with other TK y variants of receptor tyrosine kinases, it probably acts as a dominant negative antagonist w1,6,19,21x. ISH has provided evidence that the two forms are colo-
) Corresponding author. DeveloGen, Rudolf Wissell Str. 28, D-37079 Goettingen, Germany. Fax: q49-551-505-5886; e-mail: [email protected] 1 Also corresponding author. Fax: q44-1223-333840; e-mail: [email protected].
calised, with the TK q mRNA being predominant in the embryo but the TK y mRNA becoming more abundant in adult brain w13x.
Fig. 1. Reaction of the antibodies with EphA7 TKq and TKy isoforms by immunoprecipitation. Ž1, 2. HEK293 cells were untransfected Ž1. or transiently transfected Ž2. with the full-length cDNA ŽMDK1.; as in Ref. w2x, they were labelled with 35 S-methionine, lysed, immunoprecipitated with Ab-TKq, electrophoresed, blotted, and autoradiographed. The doublet at 120 and 114 kDa corresponds to EphA7 TKq, with and without glycosylation w2x. Ž3, 4. HEK293 cells were untransfected Ž1. or transiently transfected Ž2. with the cDNA for the TKy isoform ŽMDK1-T1., then processed as above with Ab-TKy. The doublet at approximately 70 and 80 kDa corresponds to EphA7 TKy, with and without glycosylation.
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 2 8 5 - 5
232
T. Ciossek et al.r Molecular Brain Research 74 (1999) 231–236
Therefore, it is important to know whether EphA7 mRNAs, as located by ISH, are translated to produce the TK q or the TK y forms at various stages and locations of brain development, and where each isoform is located on the neurons. To answer these questions, we have now produced antibodies to localise EphA7 TK q and TK y immunoreactivity. The antiserum against the EphA7 TK q isoform ŽAbTK q . was raised in rabbit against a fusion protein of glutathione-S-transferase with the last 111 amino acids of the mouse EphA7 receptor tyrosine kinase sequence ŽGSTct-EphA7. w2x. This portion covers the C-terminal SAM homology domain w16x. Similar fusions were made with EphA5, EphB3, and EphB4 w3x and used for absorption controls. By western blots and immunoprecipitations with various Eph receptors transiently overexpressed from HEK
293 cells w2,3x, Ab-TKq was shown to react with EphA7 ŽFig. 1.. It also showed some cross-reaction with some other Eph receptors; but in immunohistochemistry, this made at most a minor contribution, as the staining reported here was consistent with the ISH patterns, and different from the patterns we have seen with antibodies against EphA4 w12x and EphA5 ŽSanta Cruz. Žalthough there is much overlap.. Specificity was checked by absorption controls, which showed that immunoreactivity in brain was specific for EphA7, with two exceptions: Ž1. possible limited cross-reaction with other Eph receptors where coexpressed Že.g., in Purkinje cells.. Staining in such areas was weakened by pre-absorption with other GST-ct-Eph fusion proteins, but not eliminated unless pre-absorbed with the immunogen GST-ct-EphA7. Ž2. Irrelevant crossreaction with a GST epitope, restricted to glia in adult
Fig. 2. Adult forebrain, in parasagittal sections. Scale bar s 0.5 mm. ŽA. Stained with Ab-TKq , pre-absorbed with fusion protein GST-ct-EphB4ŽMdk2. to eliminate cross-reactions. ŽThis pre-absorption prevented some staining of glia, which would not be clearly visible at this scale, but the remaining staining of fibres and neuropil is essentially as seen with unabsorbed antibody. Pre-absorption with immunogen GST-ct-EphA7ŽMdk1. removed almost all staining; data not shown.. Positive regions include: all fibre layers of hippocampus Žvery strong; Hip.; corpus callosum Žventral half; cc.; anterior commissure Žposterior branch; acp.; neuropil throughout cortex, striatum ŽStr., nucleus accumbens ŽAcb., etc.; substantia nigra pars reticulata ŽSNR. and striatonigral tract Žsn.. There is also weak staining in parts of thalamus ŽTha.. ŽB. Stained with Ab-TKy . Cell bodies are stained in hippocampus ŽHip. and cortex Žpyramidal cells; pyr.. Cells andror fibres are stained in some nuclei of thalamus including anterodorsal ŽAD., anteroventral ŽAV., and reticular ŽRt..
T. Ciossek et al.r Molecular Brain Research 74 (1999) 231–236
233
Fig. 2 Žcontinued..
brain Žespecially Bergmann glia in the cerebellum; only a minor contribution in the forebrain.. This was eliminated by pre-absorption with other GST fusion proteins, which was done in all data reported here. The antiserum for detection of the TK y isoform ŽAbTK y . was raised in rabbit against a synthetic peptide Žsequence: CSLVTNEHLSVL. representing the C-terminus of the TK y form of EphA7 in mouse w2x and rat w20x. The antiserum was shown to react with EphA7 TK y in immunoprecipitation and western blotting ŽFig. 1. as well as in immunohistochemistry. No potentially cross-reacting receptors are known. The immunising peptide was also used for absorption controls, and eliminated all staining. MFI mice were perfused with 4% paraformaldehyde. Immunohistochemistry was performed on sections by standard techniques, with antiserum diluted 1:2500 or 1:3000, followed by peroxidase-coupled anti-Žrabbit IgG. ŽVector Labs.. Staining of the developing nervous system with Ab-TK q was widespread w14x. Ab-TKy stained nothing in embryos, and very little in neonatal brains. However, both antisera gave strong and striking patterns of staining in the adult brain ŽFig. 2.. Table 1 lists six major brain regions that were EphA7positive in development, both by ISH and by EphA7 TK q
immunoreactivity on their developing axonal projections w14x. It indicates that these neuronal groups continued to express EphA7 TK q andror TK y immunoreactivity in different subcellular locations in the adult. These neuronal groups had all been reported as positive by ISH in the adult, though only the hippocampus gave strong ISH in all surveys. Ab-TKq stained neuropil strongly through most of the adult telencephalon, as well as in the hypothalamus and substantia nigra reticulata ŽFig. 2A.. This immunoreactivity was probably in axonal andror dendritic fields of neurons which expressed EphA7 mRNA and Ab-TKq immunoreactivity during development, viz. the cortex, hippocampus, and striatum w14x. Thus the hippocampus displayed strong staining throughout all its fibre fields, but little or no staining in the pyramidal cell layer ŽFig. 3B.. However, there was only weak TK q immunoreactivity in the adult thalamus and cerebellum, and none in mammillary nuclei, although these regions had expressed EphA7 earlier w14,15x. Weaker staining of neuropil was seen in some regions of midbrain and hindbrain. Ab-TKy , in contrast, conspicuously stained cell bodies and proximal dendrites of a small number of neuronal types. These include the pyramidal cells of motor cortex, piriform cortex, and hippocampus ŽFig. 3C,D., which car-
234
Brain region of origin
ISH ŽE14.5 to P1.
Neuronal cell bodies
TKy IR Žadult.
Target regions
TKq IR Žadult.
ISH Žadult. wReferencesx
Neocortex
q q
y q y
Neocortex Spinal cord grey matter Entorhinal cortex
qq Žy. qq
q w2,20x
Allocortex Žentorhinal, piriformrolfactory
Non-motor cortex Motor cortex pyram. cells Ento. cortex. pyram. cells
q w2,20x
Pirif. cortex. pyram. cells
q
Piriform cortex Most of telencephalon Hippocampus
qq qq qqq
qqq w2,7,13,20x
Lat. septal nuc. Striatum Globus pallidus Substantia nigra reticulata Interpeduncular nuclei Cerebellar nuclei
qqq qq y qq Žq. Žy.
Hippocampus
Hippocampus Žpyram. cells and dentate gyrus.
q
Striatum
q
Striatum
y
Habenula Cerebellar cortex
qq q
Habenula Purkinje cells
Žq. q
q w2,13x
q w2,13x q w2,13,20x
T. Ciossek et al.r Molecular Brain Research 74 (1999) 231–236
Table 1 Neurons and their fibres expressing EphA7 isoforms in mouse brain For each major neuronal population positive by ISH, this table lists its major targetŽs., and whether they show EphA7 immunoreactivity ŽIR. on a scale from Žy. through Žq. Žweak. to qqq Žstrongly positive.. See Ref. w14x for table of EphA7 TKq IR on the growing axonal projections. Columns 1 and 2: Major brain regions which express EphA7 mRNA in development ŽE14.5 to postnatal day 1; Refs. w2,7x.. Columns 3 and 4: EphA7 TK-IR on cell bodies and proximal dendrites in these regions in adult Žand see below.. Columns 5 and 6: EphA7 TKq IR in neuropil of these same regions and their target fields Žwhere their axons terminate., in adult. Column 7: EphA7 mRNA reported in neuronal cell bodies in these regions in adult. TK y IR in adult brain is also conspicuous on cell bodies and proximal dendrites of the following neurons: rostral prefrontal cortex pyramidal cells, retrosplenial gyrusrsubiculum pyramidal cells, globus pallidus, ventral pallidum and nuclei of the diagonal band, nucleus isthmi magnocellularis pars compacta, red nucleus, mesencephalic trigeminal nucleus, scattered cells in midbrain, giant cells of reticular formation, cerebellar nuclei, prepositus hypoglossal nucleus. Also in the thalamus, there are also numerous very faintly stained neurons with positive dendrites in the following thalamic nuclei: anteromedial, anteroventral, dorsal lateral geniculate, laterodorsal, lateroposterior, mediodorsal, reuniens, reticular, ventrolateral, ventromedial, ventroposterior medial and lateral, zona incerta. Some other thalamic nuclei contain positive fibres, notably anterodorsal, parafascicular, and centrolaminar nuclei. Other thalamic nuclei, including the midline nuclei, are negative for TKy IR. In addition, in transverse sections of all regions of cortex and parts of hippocampus, Ab-TKy gives a diffuse ‘blotchy’ stain on a scale of ; 50 mm, which may represent domains of neuropil. We have also stained some transverse sections of rat brains with Ab-TKy and confirm the same distribution as in mouse, plus conspicuous cell staining in the substantia nigra reticulata.
T. Ciossek et al.r Molecular Brain Research 74 (1999) 231–236
235
Fig. 3. Adult brain, in parasagittal sections at high power. Scale bar s 100 mm. ŽA–D. TK q and TK y receptors in motor cortex ŽA, C. and hippocampus ŽB, D.. ŽA, B. Ab-TKq Žpre-absorbed with GST-ct-EphA5ŽEhk1. to eliminate glial cross-reaction., staining dense neuropil but no cell bodies. ŽC, D. Ab-TKy , staining cell bodies and proximal dendrites of pyramidal cells and a few interneurons. In hippocampus, CA2–CA3 pyramidal cell layer is marked with U , and two Ab-TKy IR interneurons are arrowed. ŽE–G. TK y receptors in hindbrain, stained with Ab-TKy . Note strong staining of cell bodies and proximal dendrites. ŽE. Cerebellar nuclei, ŽF. cerebellar cortex ŽPurkinje cells are stained., ŽG. mesencephalic trigeminal nucleus.
ried EphA7 TK q receptor on their axons both earlier and in the adult ŽTable 1.; and also the Purkinje cells and mesencephalic trigeminal neurons ŽFig. 3F,G., which carried EphA7 TK q receptor on their axons earlier but not in the adult w14x. Similar TK y receptor immunoreactivity was seen on the cell bodies and proximal dendrites in
several other nuclei where we have no evidence for EphA7 TK q expression in development Že.g., Fig. 3E; Table 1 footnote.. This expression pattern of the TK y isoform was more complex than expected. It was consistent with ISH results w13x in showing co-expression and an increasing ratio of
236
T. Ciossek et al.r Molecular Brain Research 74 (1999) 231–236
TK y to TK q isoform from embryo to adult. However, in embryos no TK y immunoreactivity was detected at all, suggesting translational or post-translational regulation. Conversely in adult brain, TK y immunoreactivity was present on several neuronal groups that were not reported positive by ISH. Some of these smaller neuronal groups may simply have been missed during development. But the lack of ISH signal may also suggest that expression is at a very low level with little turnover of the TK y receptor in the adult brain. In summary, the EphA7 TK q receptor is expressed on distal parts of axons and at least some dendrites, and is generally not detectable on the cell bodies in regions of origin, implying that it is efficiently transported along neurites. This pattern resembles that of the EphB2 receptor, which is expressed particularly in growth cones w11x and in adult forebrain synaptic membranes w18x. The wide distribution of EphA7 TK q immunoreactivity in neuropil, and especially in the dendritic and axonal fields of the most highly expressing cells of the hippocampus, suggests a role in synapses where the EphA4 receptor has also recently been located w12x. In contrast, the TK y variant is synthesised only after birth, and is localised to cell bodies and proximal dendrites of neurons, notably on some neurons which also express or expressed the TK q form. It could act simply as a binding partner for ligand if any is encountered near cell bodies, perhaps promoting cell adhesion. Alternatively, it could act as a dominant-negative antagonist. If so, its localisation on cell bodies and proximal dendrites may ensure that the EphA7 TK q receptor expressed by the same neurons only responds to signals encountered by the axonal or dendritic terminals.
Acknowledgements We thank Pauline Whiting and Sheila Barton for mice. Part of this work was supported by Sugen.
References w1x E. Amaya, T.J. Musci, M.W. Kirschner, Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos, Cell 66 Ž1991. 256–270. w2x T. Ciossek, B. Millauer, A. Ullrich, Identification of alternatively spliced mRNAs encoding variants of MDK1, a novel RTK expressed in the murine nervous system, Oncogene 9 Ž1995. 97–108. w3x T. Ciossek, M.M. Lerch, A. Ullrich, Cloning, characterization and differential expression of MDK2 and MDK5, two novel RTKs of the eckreph family, Oncogene 11 Ž1995. 2085–2095. w4x T. Ciossek, A. Ullrich, Identification of Elf-1 and B61 as high affinity ligands for the RTK MDK1, Oncogene 14 Ž1997. 35–43.
w5x U. Drescher, R. Klein, C. Stuermer, A. Faissner, F.G. Rathjen ŽEds.., Special issue: molecular bases of axonal growth and pathfinding, Cell Tissue Res. 290 Ž2. Ž1997.. w6x F.F. Eide, E.R. Vining, B.L. Eide, K. Zang, X.-Y. Wang, L.F. Reichardt, Naturally occurring truncated trkB receptors have dominant inhibitory effects on BDNF signalling, J. Neurosci. 16 Ž1996. 3123–3129. w7x J. Ellis, Q. Liu, M. Breitman, N.A. Jenkins, D.J. Gilbert, N.G. Copeland, H.V. Tempest, S. Warren, E. Muir, H. Schilling, F.A. Fletcher, S.F. Ziegler, J.H. Rogers, Embryo Brain Kinase: a novel gene of the ephrelk receptor tyrosine kinase family, Mech. Dev. 52 Ž1995. 319–341. w8x Eph Nomenclature Committee, Unified nomenclature for Eph family receptors and their ligands, the ephrins, Cell 90 Ž1997. 403–404. w9x J.G. Flanagan, P. Vanderhaeghen, The ephrins and Eph receptors in neural development, Annu. Rev. Neurosci. 21 Ž1998. 309–345. w10x N. Gale, S.J. Holland, D.M. Valenzuela, A. Flenniken, L. Pan, T.E. Ryan, M. Henkemeyer, K. Strebhardt, H. Hirai, D.G. Wilkinson, T. Pawson, S. Davis, G.D. Yancopoulos, Eph receptors and ligands comprise two major specificity subclasses, and are reciprocally compartmentalized during embryogenesis, Neuron 17 Ž1996. 9–19. w11x M. Henkemeyer, L.E.M. Marengere, J. McGlade, J.P. Olivier, R.A. Conlon, D.P. Holmyard, K. Letwin, T. Pawson, Immunolocalization of the Nuk receptor tyrosine kinase suggests roles in segmental patterning of the brain and axonogenesis, Oncogene 9 Ž1994. 1001– 1014. w12x M.E. Martone, J.A. Holash, A. Bayardo, E.B. Pasquale, M.H. Ellisman, Immunolocalization of the receptor tyrosine kinase EphA4 in the adult rat central nervous system, Brain Res. 771 Ž1997. 238–250. w13x T. Mori, A. Wanaka, A. Taguchi, K. Matsumoto, M. Tohyama, Localization of novel RTK genes of the eph family, MDK1 and its splicing variant, in the developing mouse nervous system, Mol. Brain Res. 34 Ž1995. 154–160. w14x J.H. Rogers, T. Ciossek, A. Ullrich, E. West, M. Hoare, E.M. Muir, Distribution of the receptor EphA7 and its ligands in development of the mouse nervous system, submitted to Mol. Brain Res., 1999. w15x J.H. Rogers, T. Ciossek, P. Menzel, E.B. Pasquale, Eph receptors and ephrins demarcate cerebellar lobules before and during their formation, Mech. Dev. Ž1999. in press. w16x J. Schultz, C.P. Ponting, K. Hofmann, P. Bork, SAM as a protein interaction domain involved in developmental regulation, Protein Sci. 6 Ž1997. 249–253. w17x A.H. Talukder, T. Muramatsu, N. Kaneda, A novel truncated variant form of EbkrMDK1 receptor tyrosine kinase is expressed in embryonic mouse brain, Cell Struct. Funct. 22 Ž1997. 477–485. w18x R. Torres, B.L. Firestein, H. Dong, J. Staudinger, E.N. Olson, R.L. Huganir, D.S. Bredt, N.W. Gale, G.D. Yancopoulos, PDZ proteins bind, cluster and synaptically colocalize with Eph receptors and their ephrin ligands, Neuron 21 Ž1998. 1453–1463. w19x H. Ueno, H. Colbert, J.A. Escobedo, L.T. Williams, Inhibition of PDGFb signal transduction by coexpression of a truncated receptor, Science 252 Ž1991. 844–848. w20x D.M. Valenzuela, E. Rojas, J.A. Griffiths, D.L. Compton, M. Gisser, N.Y. Ip, M. Goldfarb, G.D. Yancopoulos, Identification of full-length and truncated forms of Ehk-3, a novel member of the Eph RTK family, Oncogene 9 Ž1995. 1573–1580. w21x Q. Xu, G. Alldus, N. Holder, D.G. Wilkinson, Expression of truncated Sek-1 RTK disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain, Development 121 Ž1995. 4005–4016. w22x R. Zhou, The Eph family receptors and ligands, Pharmacol. Ther. 77 Ž1998. 151–181.