Comparison of VGF and trk mRNA distributions in the developing and adult rat nervous systems

Molecular Brain Research 49 Ž1997. 307–311

Short communication

Comparison of VGF and trk mRNA distributions in the developing and adult rat nervous systems Susan E. Snyder, Ji Li, Stephen R.J. Salton

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Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY 10029-6574, USA Accepted 17 June 1997

Abstract The Õgf gene encodes a neuronal secretory-peptide precursor that is rapidly induced by neurotrophic growth factors and by depolarization in vitro. To gain insight into the possible functions and regulation of VGF in vivo, we characterized the expression of VGF messenger RNA ŽmRNA. by in situ hybridization, during development and in adult brain, and directly compared it to the distributions of mRNAs encoding neurotrophin receptors TrkA, TrkB, and TrkC. Overlap in VGF and trk mRNA expression exists, and though no trk mRNA was exclusively colocalized with VGF message, the best correlation was found between the distributions of the VGF and trk B kinase mRNAs. q 1997 Elsevier Science B.V. Keywords: Hybridization, in situ; Neurotrophin; TrkA; TrkB; TrkC; VGF

Specific neurotrophins, and probably a subset of gene products that are regulated by them, contribute to the formation of synaptic connections during development w3x and following injury w8x, and also participate in the normal functioning of the adult brain. Understanding of neurotrophin actions has been aided by study of PC12 pheochromocytoma cells w5x which respond to neurotrophic growth factors by differentiating into sympathetic neuron-like cells. The Õgf gene encodes a secreted peptide precursor that is rapidly and relatively selectively induced in PC12 cells by nerve growth factor ŽNGF. w10,12x. VGF is synthesized by neurons in both the peripheral ŽPNS. and central nervous systems ŽCNS. w12,15–17x, and in the brain VGF expression is regulated by manipulations which affect neuronal activity andror neurotrophin expression, including intraocular tetrodotoxin injection, cortical injury, and seizure w7,13x. To investigate whether NGF, brain-derived neurotrophic factor ŽBDNF. and neurotrophin-3 ŽNT-3. might regulate VGF expression in vivo as they do in vitro w2,6,12x, we compared the distributions of the Trk neurotrophin receptor mRNAs with that of VGF. )

Corresponding author. Box 1065, Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029-6574, USA. Fax: q1 Ž212. 996-9785; E-mail: [email protected] 0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 2 1 6 - 7

Timed-pregnant, postnatal, or adult Sprague–Dawley rats Žthe day after the evening of conception being embryonic day 0 wE0x, and the day of birth being postnatal day 0 wP0x. were killed by CO 2 narcosis, and brains or whole embryos were embedded, frozen, and sectioned. In situ hybridization w14 x was performed using a VGF w 35 Sxriboprobe which corresponds to part of the 3X end of the VGF coding region w11x. Parallel hybridizations on adjacent sections were done using previously described trk probes w14x complementary to mRNA regions encoding Trk receptor extracellular domains Ždetect both full-length and noncatalytic truncated splice-variant trk mRNAs w9x. and a trk B kinase probe Ždetects only mRNA encoding full-length, catalytic forms of the receptor.. Appropriate trk or VGF sense-strand control riboprobes were hybridized to adjacent sections in parallel to antisense probes and produced no specific hybridization signal ŽFig. 1A.. In the E13.5 embryo ŽFig. 1, panels B–E., VGF and all three trk mRNAs were expressed strongly in the developing dorsal root ganglia ŽDRGs.. Moderate-high VGF mRNA expression was found in developing cranial ganglia we.g., trigeminal ŽV., facial ŽVII., and vestibular ŽVIII.x, sympathetic ganglia Žwhere trk B was also expressed., and the ventrolateral mantle layer of the developing spinal cord, with low levels in the remainder of the neuroepithelium of the brain and spinal cord ŽFig. 1B, Fig. 2A, and data not shown..

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Fig. 1. In situ hybridization analysis of VGF and trk mRNA distributions in the developing and adult rat. Comparison of VGF ŽB, F, J, N., trkA ŽC, G, K, O., trk B ŽD, H, L, P., and trkC ŽE, I, M, Q. mRNA expression in the rat embryo ŽE13.5, A–E., fetal head ŽE19.5, F–I., postnatal brain ŽP10, J–M., and adult brain ŽN–Q.. ŽA. demonstrates lack of specific hybridization of control VGF sense-strand riboprobe. All sections are parasagittal except the adult sections which were cut horizontally. Dark field emulsion and inverted film autoradiograms are shown. Abbreviations: bs, brainstem; Cbm, cerebellum; Cx, cortex; DRGs, dorsal root ganglia; fb, forebrain; hb, hindbrain; Hippo, hippocampal formation; OB, olfactory bulb; S, septum; Str, striatum; TriG, trigeminal ganglion. Scale bars Žin A. s 1 mm; Žin B–E. s 1 mm; Žin F–I. s 2 mm; Žin J–M. s 2 mm; Žin N–Q. s 2 mm.

By E19.5, VGF mRNA was expressed at moderate to high levels in much of the brain, particularly in the thalamus, hypothalamus, and brainstem, in the pituitary, and continued to be found in cranial, sympathetic, and sensory ganglia ŽFig. 1F–I, and data not shown.. trkA message ŽFig. 1G. was seen in a few cells at the base of the forebrain, and in the trigeminal and cervical sympathetic ganglia, while trk B and trkC mRNAs ŽFig. 1H and I. were detectable not only in these ganglia, but throughout the brain. By P10, the distribution of VGF mRNA overlapped extensively with those of the trks, yet corresponded directly with none ŽFig. 1J–M.. VGF signal was strongest in several thalamic nuclei, but was clearly expressed throughout the brain, including the neocortex, hypothalamus and brainstem. In comparison, trkA was expressed in a limited population of cells in the striatum, basal forebrain, and

brainstem, while trk B and trkC mRNAs were more widely distributed. In adult female brain ŽFig. 1N–Q., although VGF mRNA was found throughout, expression varied from very high levels in several hypothalamic nuclei to very low levels in the cerebellum. The distributions of all three trk mRNAs overlapped in distinct regions with that of VGF, but each showed differences as well. Although trkA ŽFig. 1O. and VGF mRNAs were detected in the medial septal nucleus, the vertical limb of the diagonal band, in scattered cells throughout the striatum, and several brainstem nuclei, trkA expression was severely restricted compared to that of VGF. While trk B and VGF distributions overlapped, trk B was also expressed in many regions not expressing much VGF, such as white matter, the dentate gyrus, and cerebellum ŽFig. 1P.. trkC too showed regions of similarity and dissimilarity with VGF expression ŽFig. 1Q.. Both

S.E. Snyder et al.r Molecular Brain Research 49 (1997) 307–311

messages were broadly expressed in cortex, striatum, and olfactory bulb, yet in the cerebellum and dentate granule layer there was extremely strong trkC hybridization and only low levels of VGF, while there was a lack of trkC in thalamic nuclei where VGF levels were high. Expression of VGF and trk mRNAs is shown at higher magnification in Fig. 2. Panels A–D show hybridization to transverse sections through the E13.5 spinal cord and DRGs. Although all four genes were expressed in the developing DRGs, in spinal cord VGF was only seen in the ventrolateral portion of the basal plate, part of the mantle layer which also expressed trk B and trkC mRNA, while trkA signal was restricted to a narrow band in the

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ventromedial portion of the developing cord. In P10 brain, VGF was synthesized in areas not expressing trkA but containing trkB and trkC mRNA, as in the hippocampal formation ŽE–H., where VGF mRNA was abundant in the CA fields including the dentate hilus, in scattered cells of the stratum oriens and apical molecular layers, and in the subiculum, and was found at lower levels in the dentate granule layer. In P10 thalamus ŽI–L., VGF hybridization was strongest in the anteroventral nucleus ŽAV. and ventral group, while trkA was undetectable, trk B was found at fairly uniform levels throughout, and trkC was high in AV but low in the ventral group. Adult basal ganglia ŽM–P. contained diffuse, low trk B signal in the globus

Fig. 2. High magnification comparison of VGF and trk mRNA distributions in the developing and adult rat. Dark-field emulsion autoradiograms comparing VGF ŽA, E, I, M, Q., trkA ŽB, F, J, N, R., trk B ŽC, G, K, O, S., and trkC ŽD, H, L, P, T. mRNA expression at higher magnification in transverse sections through E13.5 spinal cord and DRGs ŽA–D., hippocampus ŽE–H. and thalamus ŽI–L. in parasagittal section at P10, and the striatum ŽM–P. and septal region ŽQ–T. in horizontal section in adulthood. Abbreviations: alar, alar plate; AV, anteroventral nucleus of the thalamus; basal, basal plate; DG, dentate gyrus; DRG, dorsal root ganglion; fx, fornix; GP, globus pallidus; LS, lateral septum; mantle, mantle layer; MS, medial septum; nbM, nucleus basalis magnocellularis; pvn, periventricular nucleus of the thalamus; Str, striatum; V, ventral nuclei of the thalamus; vz, ventricular zone. Scale bars Žin A–D. s 100 m m; Žin E–H. s 250 m m; Žin I–L. s 500 m m; Žin M–P. s 100 m m; Žin Q–T. s 200 m m.

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Fig. 3. Comparison of VGF and trk B kinase mRNA distributions. Film autoradiograms show VGF ŽA. and trk B kinase ŽB. mRNA expression in horizontal sections of adult rat brain. Scale bar s 2 mm.

pallidus, while trkC and VGF probes labeled scattered cells, and trkA was not expressed. In striatum and nucleus basalis magnocellularis ŽnbM. all four mRNAs were expressed: dense striatal trk B signal was noted while trkA labeled the fewest cells; however, in the nbM VGF and trkA displayed the most prominent labeling of the four probes. In the septal region ŽQ–T. trk B displayed high but diffuse expression including labeling of the ependyma and fornix. VGF and trkC were both expressed throughout the septum, but in nearby thalamic nuclei VGF expression was much higher than that of trkC. trkA was expressed in fewer cells, and restricted to the medial septal nucleus and anterior paraventricular thalamic nuclei. Since the trk B probe above hybridizes to mRNAs encoding truncated non-catalytic isoforms, expressed primarily by astrocytes, and full-length kinase forms, expressed exclusively by neurons w1,4x, we compared the VGF hybridization pattern with that produced by a probe specific for the kinase formŽs. of TrkB Ž trk BK probe.. Though differences in distribution were seen particularly in the cerebellum and in thalamic and brainstem nuclei, overall, VGF and trkBK expression patterns were quite similar ŽFig. 3.. Taken together our data support the following general relationships between VGF and trk expression: trkA expressing regions were a subset of VGF expressing ones; while the patterns of VGF and trkC mRNA expression overlapped, some areas with high VGF expressed little trkC and vice versa; VGF expressing areas seemed to be a subset of those expressing trkB, and correlated particularly well with the neuronal sites of trkB expression. Recent work has shown that in addition to being NGF-inducible, VGF is highly inducible by BDNF and NT-3 Žendogenous ligands of TrkB and TrkC respectively. in primary CNS neuronal cultures w2x. In addition to demonstrating widespread distribution of VGF mRNA in the embryonic, neonatal and adult rat nervous systems, this

study provides the first direct comparison of VGF mRNA expression with that of the neurotrophin receptors, TrkA, TrkB, and TrkC. A role for BDNF and NT-3 in the induction of VGF in vivo, in the CNS, would therefore be consistent with in vitro studies w2x and the in situ hybridization data presented here. Although p75 Žlow affinity NGF receptor. and VGF distributions do not overlap in the septum w16x, we did find trkA mRNA in areas expressing VGF, suggesting that NGF may regulate VGF in these small populations of neurons. Should NGF, BDNF and NT-3 induce Õgf gene expression in vivo as robustly as they do in vitro, and given that trk B message is found in all regions expressing VGF, and trkC mRNA in many of them, then VGF expression in the brain may be regulated by BDNF, neurotrophin-4r5 Žwhich also binds TrkB., andror NT-3. This could be one of several mechanisms that might contribute to VGF induction following CNS injury.

Acknowledgements This work was supported by grants from the National Institutes of Health ŽAG 10676., The Dysautonomia Foundation, and Pew Scholar and Irma T. Hirschl Career Scientist Awards to S.R.J.S. Portions of this work have been presented previously in abstract and dissertation form. We thank Drs. R. Streck, P. Hof, and V. Friedrich Jr. for helpful discussions and technical advice.

References w1x C.A. Altar, J.A. Siuciak, P. Wright, N.Y. Ip, R.M. Lindsay, S.J. Wiegand, In situ hybridization of trkB and trkC receptor mRNA in rat forebrain and association with high-affinity binding of w 125 IxBDNF, w 125 IxNT-4r5 and w 125 IxNT-3, Eur. J. Neurosci. 6 Ž1994. 1389–1405. w2x A. Bonni, D.D. Ginty, H. Dudek, M.E. Greenberg, Serine 133-phosphorylated CREB induces transcription via a cooperative mechanism that may confer specificity to neurotrophin signals, Mol. Cell. Neurosci. 6 Ž1995. 168–183. w3x R.J. Cabelli, A. Hohn, C.J. Shatz, Inhibition of ocular dominance column formation by infusion of NT-4r5 or BDNF, Science 267 Ž1995. 1662–1666. w4x J. Frisen, V.M. Verge, K. Fried, M. Risling, H. Persson, J. Trotter, T. Hokfelt, D. Lindholm, Characterization of glial trkB receptors: differential response to injury in the central and peripheral nervous systems, Proc. Natl. Acad. Sci. USA 90 Ž1993. 4971–4975. w5x L.A. Greene, A.S. Tischler, Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, Proc. Natl. Acad. Sci. USA 73 Ž1976. 2424–2428. w6x A. Levi, J.D. Eldridge, B.M. Paterson, Molecular cloning of a gene sequence regulated by nerve growth factor, Science 229 Ž1985. 393–395. w7x A. Lombardo, S.A. Rabacchi, F. Cremisi, T. Pizzorusso, M.C. Cenni, R. Possenti, G. Barsacchi, L. Maffei, A developmentally regulated nerve growth factor-inducible gene, VGF, is expressed in geniculocortical afferents during synaptogenesis, Neuroscience 65 Ž1995. 997–1008.

S.E. Snyder et al.r Molecular Brain Research 49 (1997) 307–311 w8x F. Longo, D. Holtzman, M. Grimes, W. Mobley, Nerve growth factor: actions in the peripheral and central nervous systems, in: S. Loughlin, J. Fallon ŽEds.., Neurotrophic Factors, Academic Press, San Diego, CA, 1993, pp. 209–256. w9x D.S. Middlemas, R.A. Lindberg, T. Hunter, trkB, a neural receptor protein-tyrosine kinase: evidence for a full-length and two truncated receptors, Mol. Cell. Biol. 11 Ž1991. 143–153. w10x R. Possenti, J.D. Eldridge, B.M. Paterson, A. Grasso, A. Levi, A protein induced by NGF in PC12 cells is stored in secretory vesicles and released through the regulated pathway, EMBO J. 8 Ž1989. 2217–2223. w11x S.R.J. Salton, Nucleotide sequence and regulatory studies of VGF, a nervous system-specific mRNA that is rapidly and relatively selectively induced by nerve growth factor, J. Neurochem. 57 Ž1991. 991–996. w12x S.R.J. Salton, D.J. Fischberg, K.-W. Dong, Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells, Mol. Cell. Biol. 11 Ž1991. 2335–2349.

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w13x S.E. Snyder, H.-W. Cheng, K.D. Murray, P.J. Isackson, T.H. McNeill, S.R.J. Salton, The mRNA encoding VGF, a neuronal peptide precursor, is rapidly regulated in the rat central nervous system by neuronal activity, seizure and lesion, Neuroscience, in press. w14x S.E. Snyder, J. Li, P.E. Schauwecker, T.H. McNeill, S.R.J. Salton, Comparison of RPTPz r b , phosphacan, and trkB mRNA expression in the developing and adult rat nervous system, and induction of RPTPz r b and phosphacan mRNA following brain injury, Mol. Brain Res. 40 Ž1996. 79–96. w15x S.E. Snyder, R.D. Streck, J.E. Pintar, S.R.J. Salton, Distribution of VGF mRNA in the postnatal rat brain, Soc. Neurosci. Abstr. 18 Ž1992. 1291. w16x A.N. van den Pol, K. Bina, C. Decavel, P. Ghosh, VGF expression in the brain, J. Comp. Neurol. 347 Ž1994. 455–469. w17x A.N. van den Pol, C. Decavel, A. Levi, B. Paterson, Hypothalamic expression of a novel gene product, VGF: immunocytochemical analysis, J. Neurosci. 9 Ž1989. 4122–4137.