- Email: [email protected]
Expression of mRNAs coding for the transforming growth factor-β receptors in brain regions of euthyroid and hypothyroid neonatal rats and in adult brain
Developmental Brain Research 99 Ž1997. 61–65
Research report
Expression of mRNAs coding for the transforming growth factor-b receptors in brain regions of euthyroid and hypothyroid neonatal rats and in adult brain T.A. Slotkin ) , X-F. Wang, H.S. Symonds, F.J. Seidler Department of Pharmacology, Duke UniÕersity Medical Center, Box 3813, Durham, NC 27710, USA Accepted 29 October 1996
Abstract The TGF-b family of peptides has been postulated to play a role in control of the cell cycle but also may act in the developing brain to influence neuronal differentiation and survival. Because reception of TGF-b signals requires the simultaneous expression of all three known receptor subtypes, we examined two neonatal rat brain regions in which neurogenesis has been largely completed. mRNA coding for all three receptors was detectable in both the forebrain and brainstem but only the type II receptor in brainstem showed a difference from adult levels of expression. Animals given perinatal PTU treatment to achieve congenital cretinism did not show significant differences in expression of any of the receptor subtypes in either of the regions, despite the fact that the treatment is known to cause anomalies of neuronal differentiation. These results indicate that regions in which neurons are undergoing axonogenesis and synaptogenesis rather than neurogenesis, nevertheless express the mRNAs coding for TGF-b receptors and are thus likely to be receptive to trophic signals mediated through TGF-b . However, synthesis and release of TGF-b , rather than receptor expression per se, is more likely to be the major point for regulation of signaling. The potential roles of TGF-b in developmental events outside of the cell cycle, such as synaptogenesis and apoptosis, need to be examined. Keywords: Propylthiouracil, effects on TGF-b receptor expression; Thyroid hormone, role in development of TGF-b receptor mRNA; Transforming growth factor-b receptors, in developing brain
1. Introduction TGF-b defines a family of peptide hormones that functions to modulate cell growth, development, and differentiation w2x. Although TGF-b was first discovered by its ability to induce the proliferation of rat kidney cells in culture w23x, it functions primarily as an inhibitor of growth for a variety of cells including ones of lymphoid, hematopoietic, epithelial, endothelial, neural, and fibroblast lineages w15x. The growth inhibitory effect of TGF-b is thought to be the molecular basis for its regulation of immune responses, development, and cellular differentiation; however, TGF-b also regulates transcription of a wide variety of genes implicated in other processes such as
Abbreviations: PTU, propylthiouracil; TGF-b , transforming growth factor-b ) Corresponding author. Fax: q1 Ž919. 684-8197. E- mail: [email protected]
wound healing, cellular adhesion, and extracellular matrix deposition w2,15x. In light of the multiplicity of genes activated by TGF-b , it is not surprising that its role in mammalian cell development is complex and as yet, ill-defined. To some extent, the effects of TGF-b depend upon the state of differentiation of the cell andror the presence of other trophic factors. Thus, despite its ability to arrest the cell cycle in many cell systems, fetal cells may give mitogenic responses to TGF-b w27x. In nervous system maturation, potential roles of TGF-b have been postulated for both proliferative phases of cell development w30x and for postmitotic phases w9,11x. Indeed, TGF-b may participate in glial–neuronal interactions that influence postmitotic neuronal survival w9–11,16x. Because TGF-b may be co-released along with neurotransmitters such as the catecholamines w12x, the possibility also exists that exposure of target cells to this trophic factor may surge during synaptogenesis, a postmitotic event in neurodevelopment. Accordingly, the present study addresses the issue of whether cells in brain regions undergoing axonogenesis and synaptogenesis, rather than neurogene-
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T.A. Slotkin et al.r DeÕelopmental Brain Research 99 (1997) 61–65
sis, are likely targets for TGF-b . We have chosen the brainstem and forebrain of the 2-day-old postnatal rat, regions in which neurogenesis has largely been completed w22,24x and have studied the elaboration of message coding for all known TGF-b receptor subtypes. The reception of the TGF-b signal by target cells requires the expression of three transmembrane receptors, type I, type II, and type III, all of which have been cloned w32x. Actual signaling by TGF-b occurs through a heteromeric complex consisting of the type I and type II receptor w31x, whereas the type III receptor, although not directly required to transmit TGF-b signals across the plasma membrane, acts to present ligand to the signaling receptors w14,32x. Thus, if TGF-b communicates with cells at this stage of development, we should detect significant amounts of mRNA for all three receptors. We have also determined whether receptor expression is developmentally regulated in two different settings: comparing the developing brain Žneonates. to the mature brain Žadults., and comparing normal neonates to hypothyroid neonates using a well-established model of congenital cretinism that is known to compromise brain cell differentiation w7,13,17,20,21,26x.
2. Methods 2.1. Animals and treatments Adult male or timed pregnant Sprague-Dawley rats ŽZivic-Miller Laboratories, Allison Park, PA. were shipped by climate-controlled truck Žtransit time, 12 h. and were housed individually, with a 12-h light–dark cycle and free access to food and water. To achieve perinatal hypothyroidism, pregnant rats were given daily subcutaneous injections of 20 mgrkg of PTU ŽSigma Chemical Co., St. Louis, MO. dissolved in 0.1 N NaOH and titrated with HCl to a final pH of 9.3, starting at gestational day 17 Žfive days before birth. and continued through postnatal day 1; pups also received PTU on postnatal day 1. Control dams and pups received equivalent volumes of alkaline saline vehicle Ž1 mlrkg. on the same schedules. With this regimen, thyroid hormone levels are severely reduced w19x, resulting in the slowing of brain cell development characteristic of congenital cretinism w6,7,18,20,21x. For each experiment, animals of both sexes were selected from several different cages. All experimental treatments and protocols involving live animals were approved by the Institutional Animal Care and Use Committee of Duke University Medical Center and conformed to guidelines set out in the NIH Guide for the Care and Use of Laboratory Animals. Control and PTU-treated 2-day-old rats and adult rats were decapitated and the brains were rapidly dissected: blunt cuts were made through the cerebellar peduncles, whereupon the cerebellum Žincluding the flocculi. was removed; a cut was then made rostral to the thalamus to
separate the forebrain from the brainstem. This dissection, which follows the natural dissection planes of the rat brain, includes the basal ganglia, hippocampal formation and neocortex within the area designated as ‘forebrain;’ the region designated as ‘brainstem’ includes the midbrain, colliculi, pons and medulla oblongata Žbut not cervical spinal cord., as well as the thalamus Žwhich is ordinarily not considered to be part of this region.. Tissues were frozen immediately in liquid nitrogen and maintained at y458C until assayed. Total cellular RNA was isolated using the guanidinium thiocyanate-CsCl method w4x. In brief, tissues were homogenized ŽPolytron, Brinkmann Instruments, Westbury, NY. in at least 5 volumes of 4 M guanidine thiocynate, 100 mM Tris-HCl, 1% b-mercaptoethanol, 0.5% Antifoam-A ŽpH 7.5.. Sodium lauryl sarcosine was then added to a final concentration of 0.5% and the homogenate was layered onto a cushion of 5.7 M CsCl, 10 mM EDTA ŽpH 7.5. and sedimented at 260,000 = g for 4 h. The resultant pellet was resuspended in 10 mM Tris, 1 mM EDTA, 0.1% SDS, pH 7.5, precipitated in 95% ethanol, and the RNA stored at y808C. The concentration of RNA was determined by spectrophotometry at 260 nm, the purity was verified by ratio of optical densities at 260 nm and 280 nm Žwhich was always 2.0., and the integrity of the RNA was confirmed by ethidium bromide staining of the 28S and 18S ribosomal bands after gel electrophoresis. 2.2. Probe constructs and RNase protection assays The rat TGF-b receptor type I cDNA was isolated from the urogenital ridge of 14.5–15-day fetal Sprague-Dawley rats w3,8x. 370 Base pairs of RI cDNA Žbetween nucleotides 519 and 889. were cloned into the BamHI site of pBs-SKq ŽStratagene, La Jolla, CA.. The cDNA encoding the rat type II receptor, originally isolated by hybridization from a rat pituitary gland cDNA library w28x, was obtained from W.W. Vale ŽSalk Institute, La Jolla, CA.. A PstIrXbaI, 286 base pair fragment of the type II cDNA Žnucleotides 1059–1345. was cloned into corresponding sites of pBs-SKq . Nucleotides 1581–1771 of the type III rat receptor cDNA w29x were subcloned into the BamHI site of pBs-SKq . Riboprobes were constructed by linearizing the receptor subclones for use as templates for incorporation of dUTP Ž a 32 P. with T7 RNA polymerase Žtype I. or T3 RNA polymerase Žtypes II and III.. A control rat b-actin riboprobe ŽAmbion Inc., Austin, TX. was constructed according to company protocol. RNase protection assays were performed using the RPAII Ribonuclease Protection Assay kit ŽAmbion.. Briefly, 1 = 10 5 cpm of labeled probes Žreceptor type I, II, or III and rat b-actin. were incubated overnight at 558C with 10 m g of RNA. Hybridized samples were digested with an RNaseArRNase T1 mixture. After precipitation, protected fragments were separated on a 6% denaturing polyacrylamide electrophoresis gel apparatus and quanti-
T.A. Slotkin et al.r DeÕelopmental Brain Research 99 (1997) 61–65
63
tated using a Molecular Dynamics Phosphorimager ŽSunnyvale, CA.. The density of bands corresponding to each TGF-b receptor subtype mRNA was normalized to the density of the b-actin band and was then corrected for the relative specific activities of each probe. Data are presented as means and standard errors, with statistical significance Ž P - 0.05 or better. determined by Fisher’s Protected Least Significant Difference.
3. Results Representative lanes for the mRNAs corresponding to each of the TGF-b receptor subtypes appear in Fig. 1. In each case, the band for the appropriate receptor message was readily discernible, although the probes showed several additional bands; because the location of these bands would interfere with measurement of other subtypes, each subtype was quantitated from a separate lane. For the quantitation shown below, although relative units are given, the readings for each probe were corrected for the different specific activities so that they represent relative molar concentrations. All three receptor subtype mRNAs were detected in neonatal and adult rats in both of the brain regions ŽFig. 2.. The type I receptor showed the lowest expression and had virtually the same concentration regardless of age. In contrast, the type II receptor showed higher expression in neonatal brainstem with a further three-fold increase apparent in adults. The forebrain also showed higher expression of type II receptor mRNA than the type I, but displayed no developmental change. For the type III receptor, expres-
Fig. 2. Relative molar amounts of mRNAs coding for TGF-b receptors in 2-day-old and adult rat brain regions. Data represent means and standard errors of the number of determinations in parentheses. Asterisk denotes significant difference ŽFisher’s Protected Least Significant Difference. between groups.
Fig. 1. Representative lanes of gels for detection of mRNAs coding for TGF-b receptor subtypes. The left lane contains a sample from the forebrain of a 2-day-old PTU-treated rat; the middle lane contains a sample from adult forebrain; the right lane contains a sample from the brainstem of a 2-day-old control rat. The b-actin band is shown at the exposure used for detecting TGF-b subtypes Žleft. and at lower exposure Žright..
sion in the neonatal brainstem was as high as for the type II receptor but did not demonstrate any increase from the neonatal stage to adulthood Žif anything, values declined slightly.. In the forebrain, neonatal values of the type III receptor were midway between those of the type I and type II receptors and again, only small, non-significant differences were seen between neonates and adults. Despite the virtually complete ablation of thyroid hormone levels caused by the PTU regimen used here w19x, the hypothyroid animals did not exhibit significant deficits of TGF-b receptor expression for any of the subtypes in
64
T.A. Slotkin et al.r DeÕelopmental Brain Research 99 (1997) 61–65
Fig. 3. Effects of PTU-induced perinatal hypothyroidism on expression of TGF-b receptor mRNAs. Data represent means and standard errors of the number of determinations in parentheses.
either of the two brain regions ŽFig. 3.. Values were virtually unchanged for the type I receptor, were slightly increased Žbrainstem. or decreased Žforebrain. for the type II receptor, and were slightly increased in both regions for the type III receptor.
4. Discussion Our results indicate that expression of all three TGF-b receptor subtypes occurs in neonatal rat brain regions that have largely completed their major phases of neurogenesis. Thus, postmitotic cells are probably capable of manufacturing the receptors necessary to receive TGF-b signals,
conditions that are required if TGF-b is to play a role in subsequent phases of neuronal differentiation and survival w9–11,16x. Since TGF-b signaling requires all three receptors, these results confirm and expand previous work demonstrating selective expression of individual subtypes during proliferative phases of neurodevelopment w30x. It is of particular interest that only one subtype, the type II receptor, showed a changing developmental profile and even then, in only one region. Similar results have been found in the developing heart and lung, where expression of the type I and III receptors remains fairly constant whereas the type II receptor undergoes a major maturational increase w5,33x. Apparently, two of the receptors are expressed constitutively whereas developmental changes concern primarily the type II receptor. One puzzling feature was the absence of effects of perinatal PTU treatment on expression of any of the receptors and particularly of the type II receptor, which undergoes the major ontogenetic increase. If receptor expression is a major control point for regulation of the response of developing cells to TGF-b , then cretinism, which causes gross delays in cell differentiation, should have affected receptor expression. These findings raise the possibility that, although receptor expression by target cells is required for responsiveness, the production and release of TGF-b may be relatively more important in determining the actual point of transmission of trophic signals than is the absolute magnitude of receptor expression. Similar conclusions have been reached for development of classical neurotransmitter receptors, which appear well before the ingrowth of the nerve terminals that supply the neurotransmitter. In support of this interpretation, the production of mRNAs coding for the TGF-b peptide family Žas distinct from the receptors. undergoes surges during development and during the injuryrrepair cycle in postmitotic neurons w1,10,16,25x. The possibility that TGFb may be co-released along with classical neurotransmitters w11,12x would provide a mechanism whereby synaptogenesis promotes the survival of targeted neurons, whereas those that fail to receive innervation would undergo apoptosis. Future work should thus address the role of TGF-b in such processes, concentrating on the ability of developing neurons to produce and release this trophic factor.
Acknowledgements Supported by USPHS HD-09713 and DK-45746.
References w1x Akhurst, R.J., Fitzpatrick, D.R., Fowlis, D.J., Gatherer, D., Millan, F.A. and Slager, H., The role of TGF-b s in mammalian development and neoplasia, Mol. Reprod. DeÕ. 32 Ž1992. 127–135.
T.A. Slotkin et al.r DeÕelopmental Brain Research 99 (1997) 61–65 w2x Barnard, J.A., Lyons, R.M. and Moses, H.L., The cell biology of transforming growth factor b , Biochim. Biophys. Acta 1032 Ž1990. 79–87. w3x Bassing, C.H., Yingling, J.M., Howe, D.J., Wang, T., He, W.W., Gustafson, M.L., P. Shah. Donahoe, P.K. and Wang, X.-F., A transforming growth factor b type I receptor that signals to activate gene expression, Science 263 Ž1994. 87–89. w4x Chirgwin, S.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolation of RNA from living tissues using a modified guanidine thiocyanatercesium chloride method, Biochemistry 18 Ž1979. 5294–5299. w5x Engelmann, G.L. and Grutkoski, P.S., Coordinate TGF-b receptor gene expression during rat heart development, Cell. Mol. Biol. Res. 40 Ž1994. 93–104. w6x Francis, G.L. and Rennert, O.M., Correlation of plasma propylthiouracil levels with inhibition of thyroid hormone synthesis in the rat, Metabolism 29 Ž1980. 410–414. w7x Grave, G.D., Thyroid Hormones and Brain DeÕelopment, Raven Press, New York, 1977. w8x He, W.W., Gustafson, M.L., Hirobe, S. and Donahoe, P.K., Developmental expression of four novel serinerthreonine kinase receptors homologous to the actinrtransforming growth factor-b type II receptor family, DeÕ. Dynam. 196 Ž1993. 133–142. w9x Henderson, C.E., Role of neurotrophic factors in neuronal development, Curr. Opinion Neurobiol. 6 Ž1996. 64–70. w10x Klempt, N.D., Sirimanne, E., Gunn, A.J., Klempt, M., Singh, K., Williams, C. and Gluckman, P.D., Hypoxia-ischemia induces transforming growth factor b 1 mRNA in the infant rat brain, Mol. Brain Res. 13 Ž1992. 93–101. w11x Krieglstein, K., Suter-Crazzolara, C. and Unsicker, K., Development of mesencephalic dopaminergic neurons and the transforming growth factor-b superfamily, J. Neural Transm. 46 Suppl. Ž1995. 209–216. w12x Krieglstein, K. and Unsicker, K., Bovine chromaffin cells release a transforming growth factor-beta-like molecule contained within chromaffin granules, J. Neurochem. 65 Ž1995. 1423–1426. w13x Lauder, J.M., The effects of early hypo- and hyperthyroidism on the development of rat cerebellar cortex. III. Kinetics of cell proliferation in the external granular layer, Brain Res. 126 Ž1977. 31–51. w14x Lopez-Casillas, F., Wrana, J.L. and Massague, J., Betaglycan presents ligand to the TGF beta signaling receptor, Cell 73 Ž1993. 1436–1444. w15x Massague, J., The transforming growth factor-b family, Annu. ReÕ. Cell Biol. 6 Ž1990. 597–641. w16x McNeill, H., Williams, C., Guan, J., Dragunow, M., Lawlor, P., Sirimanne, E., Nikolics, K. and Gluckman, P., Neuronal rescue with transforming growth factor-b1 after hypoxic-ischaemic brain injury, Neuroreport 5 Ž1994. 901–904. w17x Patel, A.J., Rabie, ´ A., Lewis, P.D. and Balazs, ´ R., Effect of thyroid deficiency on postnatal cell formation in the rat brain: a biochemical investigation, Brain Res. 104 Ž1976. 33–48. w18x Pracyk, J.B., Lappi, S.E. and Slotkin, T.A., Role of thyroid hormone in the development of beta adrenergic control of ornithine decarbox-
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ylase in rat heart and kidney, J. Pharmacol. Exp. Ther. 256 Ž1991. 757–766. Pracyk, J.B., Seidler, F.J., McCook, E.C. and Slotkin, T.A., Pituitary-thyroid axis reactivity to hyper- and hypothyroidism in the perinatal period: ontogeny of regulation and long-term programming of responses, J. DeÕ. Physiol. 18 Ž1992. 105–109. Pracyk, J.B. and Slotkin, T.A., Thyroid hormone regulates ontogeny of b-adrenergic receptors and adenylate cyclase in rat heart and kidney: effects of propylthiouracil-induced perinatal hypothyroidism, J. Pharmacol. Exp. Ther. 261 Ž1992. 951–958. Rastogi, R.B. and Singhal, R.L., Effect of neonatal hypothyroidism and delayed L-triiodothyronine treatment on behavioural activity and norepinephrine and dopamine biosynthetic systems in discrete regions of rat brain, Psychopharmacology 62 Ž1979. 287–293. Reinis, S. and Goldman, J.M., The DeÕelopment of the Brain: Biological and Functional PerspectiÕes, Charles C. Thomas, Springfield, IL, 1980. Roberts, A.B., Anzano, M.B., Lamb, L.C., Smith, J.M. and Sporn, M.B., New class of transforming growth factors potentiated by epidermal growth factor, Proc. Natl. Acad. Sci. USA 78 Ž1981. 5339–5343. Rodier, P.M., Structural-functional relationships in experimentally induced brain damage, Prog. Brain Res. 73 Ž1988. 335–348. Runyan, R.B., Potts, J.D. and Weeks, D.L., TGF-b 3-mediated tissue interaction during embryonic heart development, Mol. Reprod. DeÕ. 32 Ž1992. 152–159. Schalock, R.L., Brown, W.J. and Smith, R.L., Neonatal hypothyroidism: behavioral, thyroid hormonal and neuroanatomical effects, Physiol. BehaÕ. 19 Ž1977. 489–491. Teruel, T., Valverde, A.M., Benito, M. and Lorenzo, M., Transforming growth factor b 1 induces mitogenesis in fetal rat brown adipocytes, J. Cell. Physiol. 166 Ž1996. 577–584. Tsuchida, K., Lewis, K.A., Mathews, L.S. and Vale, W.W., Molecular characterization of rat transforming growth factor-beta type II receptor, Biochem. Biophys. Res. Commun. 191 Ž1993. 790–795. Wang, X.-F., Lin, H.Y., Ng-Eaton, E., Downward, J., Lodish, H.F. and Weinberg, R.A., Expression cloning and characterization of the TGF-b type III receptor, Cell 67 Ž1991. 797–805. Wang, Y.Q., Sizeland, A., Wang, X.F. and Sassoon, D., Restricted expression of type-II TGF-beta receptor in murine embryonic development suggests a central role in tissue modeling and CNS patterning, Mech. DeÕ. 52 Ž1995. 275–289. Wrana, J.L., Attisano, L., Wieser, R., Ventura, F. and Massague, J., Mechanism of activation of the TGFb receptor, Nature 370 Ž1994. 341–347. Yingling, J.M., Wang, X.-F. and Bassing, C.H., Signaling by the transforming growth factors-b receptors, Biochim. Biophys. Acta 1242 Ž1995. 115–136. Zhao, Y. and Young, S.L., Expression of transforming growth factor-beta type II receptor in rat lung is regulated during development, Am. J. Physiol. – Lung Cell. Mol. Physiol. 13 Ž1995. L419–L426.
Research report
Expression of mRNAs coding for the transforming growth factor-b receptors in brain regions of euthyroid and hypothyroid neonatal rats and in adult brain T.A. Slotkin ) , X-F. Wang, H.S. Symonds, F.J. Seidler Department of Pharmacology, Duke UniÕersity Medical Center, Box 3813, Durham, NC 27710, USA Accepted 29 October 1996
Abstract The TGF-b family of peptides has been postulated to play a role in control of the cell cycle but also may act in the developing brain to influence neuronal differentiation and survival. Because reception of TGF-b signals requires the simultaneous expression of all three known receptor subtypes, we examined two neonatal rat brain regions in which neurogenesis has been largely completed. mRNA coding for all three receptors was detectable in both the forebrain and brainstem but only the type II receptor in brainstem showed a difference from adult levels of expression. Animals given perinatal PTU treatment to achieve congenital cretinism did not show significant differences in expression of any of the receptor subtypes in either of the regions, despite the fact that the treatment is known to cause anomalies of neuronal differentiation. These results indicate that regions in which neurons are undergoing axonogenesis and synaptogenesis rather than neurogenesis, nevertheless express the mRNAs coding for TGF-b receptors and are thus likely to be receptive to trophic signals mediated through TGF-b . However, synthesis and release of TGF-b , rather than receptor expression per se, is more likely to be the major point for regulation of signaling. The potential roles of TGF-b in developmental events outside of the cell cycle, such as synaptogenesis and apoptosis, need to be examined. Keywords: Propylthiouracil, effects on TGF-b receptor expression; Thyroid hormone, role in development of TGF-b receptor mRNA; Transforming growth factor-b receptors, in developing brain
1. Introduction TGF-b defines a family of peptide hormones that functions to modulate cell growth, development, and differentiation w2x. Although TGF-b was first discovered by its ability to induce the proliferation of rat kidney cells in culture w23x, it functions primarily as an inhibitor of growth for a variety of cells including ones of lymphoid, hematopoietic, epithelial, endothelial, neural, and fibroblast lineages w15x. The growth inhibitory effect of TGF-b is thought to be the molecular basis for its regulation of immune responses, development, and cellular differentiation; however, TGF-b also regulates transcription of a wide variety of genes implicated in other processes such as
Abbreviations: PTU, propylthiouracil; TGF-b , transforming growth factor-b ) Corresponding author. Fax: q1 Ž919. 684-8197. E- mail: [email protected]
wound healing, cellular adhesion, and extracellular matrix deposition w2,15x. In light of the multiplicity of genes activated by TGF-b , it is not surprising that its role in mammalian cell development is complex and as yet, ill-defined. To some extent, the effects of TGF-b depend upon the state of differentiation of the cell andror the presence of other trophic factors. Thus, despite its ability to arrest the cell cycle in many cell systems, fetal cells may give mitogenic responses to TGF-b w27x. In nervous system maturation, potential roles of TGF-b have been postulated for both proliferative phases of cell development w30x and for postmitotic phases w9,11x. Indeed, TGF-b may participate in glial–neuronal interactions that influence postmitotic neuronal survival w9–11,16x. Because TGF-b may be co-released along with neurotransmitters such as the catecholamines w12x, the possibility also exists that exposure of target cells to this trophic factor may surge during synaptogenesis, a postmitotic event in neurodevelopment. Accordingly, the present study addresses the issue of whether cells in brain regions undergoing axonogenesis and synaptogenesis, rather than neurogene-
0165-3806r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 6 . 0 0 1 9 9 - X
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T.A. Slotkin et al.r DeÕelopmental Brain Research 99 (1997) 61–65
sis, are likely targets for TGF-b . We have chosen the brainstem and forebrain of the 2-day-old postnatal rat, regions in which neurogenesis has largely been completed w22,24x and have studied the elaboration of message coding for all known TGF-b receptor subtypes. The reception of the TGF-b signal by target cells requires the expression of three transmembrane receptors, type I, type II, and type III, all of which have been cloned w32x. Actual signaling by TGF-b occurs through a heteromeric complex consisting of the type I and type II receptor w31x, whereas the type III receptor, although not directly required to transmit TGF-b signals across the plasma membrane, acts to present ligand to the signaling receptors w14,32x. Thus, if TGF-b communicates with cells at this stage of development, we should detect significant amounts of mRNA for all three receptors. We have also determined whether receptor expression is developmentally regulated in two different settings: comparing the developing brain Žneonates. to the mature brain Žadults., and comparing normal neonates to hypothyroid neonates using a well-established model of congenital cretinism that is known to compromise brain cell differentiation w7,13,17,20,21,26x.
2. Methods 2.1. Animals and treatments Adult male or timed pregnant Sprague-Dawley rats ŽZivic-Miller Laboratories, Allison Park, PA. were shipped by climate-controlled truck Žtransit time, 12 h. and were housed individually, with a 12-h light–dark cycle and free access to food and water. To achieve perinatal hypothyroidism, pregnant rats were given daily subcutaneous injections of 20 mgrkg of PTU ŽSigma Chemical Co., St. Louis, MO. dissolved in 0.1 N NaOH and titrated with HCl to a final pH of 9.3, starting at gestational day 17 Žfive days before birth. and continued through postnatal day 1; pups also received PTU on postnatal day 1. Control dams and pups received equivalent volumes of alkaline saline vehicle Ž1 mlrkg. on the same schedules. With this regimen, thyroid hormone levels are severely reduced w19x, resulting in the slowing of brain cell development characteristic of congenital cretinism w6,7,18,20,21x. For each experiment, animals of both sexes were selected from several different cages. All experimental treatments and protocols involving live animals were approved by the Institutional Animal Care and Use Committee of Duke University Medical Center and conformed to guidelines set out in the NIH Guide for the Care and Use of Laboratory Animals. Control and PTU-treated 2-day-old rats and adult rats were decapitated and the brains were rapidly dissected: blunt cuts were made through the cerebellar peduncles, whereupon the cerebellum Žincluding the flocculi. was removed; a cut was then made rostral to the thalamus to
separate the forebrain from the brainstem. This dissection, which follows the natural dissection planes of the rat brain, includes the basal ganglia, hippocampal formation and neocortex within the area designated as ‘forebrain;’ the region designated as ‘brainstem’ includes the midbrain, colliculi, pons and medulla oblongata Žbut not cervical spinal cord., as well as the thalamus Žwhich is ordinarily not considered to be part of this region.. Tissues were frozen immediately in liquid nitrogen and maintained at y458C until assayed. Total cellular RNA was isolated using the guanidinium thiocyanate-CsCl method w4x. In brief, tissues were homogenized ŽPolytron, Brinkmann Instruments, Westbury, NY. in at least 5 volumes of 4 M guanidine thiocynate, 100 mM Tris-HCl, 1% b-mercaptoethanol, 0.5% Antifoam-A ŽpH 7.5.. Sodium lauryl sarcosine was then added to a final concentration of 0.5% and the homogenate was layered onto a cushion of 5.7 M CsCl, 10 mM EDTA ŽpH 7.5. and sedimented at 260,000 = g for 4 h. The resultant pellet was resuspended in 10 mM Tris, 1 mM EDTA, 0.1% SDS, pH 7.5, precipitated in 95% ethanol, and the RNA stored at y808C. The concentration of RNA was determined by spectrophotometry at 260 nm, the purity was verified by ratio of optical densities at 260 nm and 280 nm Žwhich was always 2.0., and the integrity of the RNA was confirmed by ethidium bromide staining of the 28S and 18S ribosomal bands after gel electrophoresis. 2.2. Probe constructs and RNase protection assays The rat TGF-b receptor type I cDNA was isolated from the urogenital ridge of 14.5–15-day fetal Sprague-Dawley rats w3,8x. 370 Base pairs of RI cDNA Žbetween nucleotides 519 and 889. were cloned into the BamHI site of pBs-SKq ŽStratagene, La Jolla, CA.. The cDNA encoding the rat type II receptor, originally isolated by hybridization from a rat pituitary gland cDNA library w28x, was obtained from W.W. Vale ŽSalk Institute, La Jolla, CA.. A PstIrXbaI, 286 base pair fragment of the type II cDNA Žnucleotides 1059–1345. was cloned into corresponding sites of pBs-SKq . Nucleotides 1581–1771 of the type III rat receptor cDNA w29x were subcloned into the BamHI site of pBs-SKq . Riboprobes were constructed by linearizing the receptor subclones for use as templates for incorporation of dUTP Ž a 32 P. with T7 RNA polymerase Žtype I. or T3 RNA polymerase Žtypes II and III.. A control rat b-actin riboprobe ŽAmbion Inc., Austin, TX. was constructed according to company protocol. RNase protection assays were performed using the RPAII Ribonuclease Protection Assay kit ŽAmbion.. Briefly, 1 = 10 5 cpm of labeled probes Žreceptor type I, II, or III and rat b-actin. were incubated overnight at 558C with 10 m g of RNA. Hybridized samples were digested with an RNaseArRNase T1 mixture. After precipitation, protected fragments were separated on a 6% denaturing polyacrylamide electrophoresis gel apparatus and quanti-
T.A. Slotkin et al.r DeÕelopmental Brain Research 99 (1997) 61–65
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tated using a Molecular Dynamics Phosphorimager ŽSunnyvale, CA.. The density of bands corresponding to each TGF-b receptor subtype mRNA was normalized to the density of the b-actin band and was then corrected for the relative specific activities of each probe. Data are presented as means and standard errors, with statistical significance Ž P - 0.05 or better. determined by Fisher’s Protected Least Significant Difference.
3. Results Representative lanes for the mRNAs corresponding to each of the TGF-b receptor subtypes appear in Fig. 1. In each case, the band for the appropriate receptor message was readily discernible, although the probes showed several additional bands; because the location of these bands would interfere with measurement of other subtypes, each subtype was quantitated from a separate lane. For the quantitation shown below, although relative units are given, the readings for each probe were corrected for the different specific activities so that they represent relative molar concentrations. All three receptor subtype mRNAs were detected in neonatal and adult rats in both of the brain regions ŽFig. 2.. The type I receptor showed the lowest expression and had virtually the same concentration regardless of age. In contrast, the type II receptor showed higher expression in neonatal brainstem with a further three-fold increase apparent in adults. The forebrain also showed higher expression of type II receptor mRNA than the type I, but displayed no developmental change. For the type III receptor, expres-
Fig. 2. Relative molar amounts of mRNAs coding for TGF-b receptors in 2-day-old and adult rat brain regions. Data represent means and standard errors of the number of determinations in parentheses. Asterisk denotes significant difference ŽFisher’s Protected Least Significant Difference. between groups.
Fig. 1. Representative lanes of gels for detection of mRNAs coding for TGF-b receptor subtypes. The left lane contains a sample from the forebrain of a 2-day-old PTU-treated rat; the middle lane contains a sample from adult forebrain; the right lane contains a sample from the brainstem of a 2-day-old control rat. The b-actin band is shown at the exposure used for detecting TGF-b subtypes Žleft. and at lower exposure Žright..
sion in the neonatal brainstem was as high as for the type II receptor but did not demonstrate any increase from the neonatal stage to adulthood Žif anything, values declined slightly.. In the forebrain, neonatal values of the type III receptor were midway between those of the type I and type II receptors and again, only small, non-significant differences were seen between neonates and adults. Despite the virtually complete ablation of thyroid hormone levels caused by the PTU regimen used here w19x, the hypothyroid animals did not exhibit significant deficits of TGF-b receptor expression for any of the subtypes in
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T.A. Slotkin et al.r DeÕelopmental Brain Research 99 (1997) 61–65
Fig. 3. Effects of PTU-induced perinatal hypothyroidism on expression of TGF-b receptor mRNAs. Data represent means and standard errors of the number of determinations in parentheses.
either of the two brain regions ŽFig. 3.. Values were virtually unchanged for the type I receptor, were slightly increased Žbrainstem. or decreased Žforebrain. for the type II receptor, and were slightly increased in both regions for the type III receptor.
4. Discussion Our results indicate that expression of all three TGF-b receptor subtypes occurs in neonatal rat brain regions that have largely completed their major phases of neurogenesis. Thus, postmitotic cells are probably capable of manufacturing the receptors necessary to receive TGF-b signals,
conditions that are required if TGF-b is to play a role in subsequent phases of neuronal differentiation and survival w9–11,16x. Since TGF-b signaling requires all three receptors, these results confirm and expand previous work demonstrating selective expression of individual subtypes during proliferative phases of neurodevelopment w30x. It is of particular interest that only one subtype, the type II receptor, showed a changing developmental profile and even then, in only one region. Similar results have been found in the developing heart and lung, where expression of the type I and III receptors remains fairly constant whereas the type II receptor undergoes a major maturational increase w5,33x. Apparently, two of the receptors are expressed constitutively whereas developmental changes concern primarily the type II receptor. One puzzling feature was the absence of effects of perinatal PTU treatment on expression of any of the receptors and particularly of the type II receptor, which undergoes the major ontogenetic increase. If receptor expression is a major control point for regulation of the response of developing cells to TGF-b , then cretinism, which causes gross delays in cell differentiation, should have affected receptor expression. These findings raise the possibility that, although receptor expression by target cells is required for responsiveness, the production and release of TGF-b may be relatively more important in determining the actual point of transmission of trophic signals than is the absolute magnitude of receptor expression. Similar conclusions have been reached for development of classical neurotransmitter receptors, which appear well before the ingrowth of the nerve terminals that supply the neurotransmitter. In support of this interpretation, the production of mRNAs coding for the TGF-b peptide family Žas distinct from the receptors. undergoes surges during development and during the injuryrrepair cycle in postmitotic neurons w1,10,16,25x. The possibility that TGFb may be co-released along with classical neurotransmitters w11,12x would provide a mechanism whereby synaptogenesis promotes the survival of targeted neurons, whereas those that fail to receive innervation would undergo apoptosis. Future work should thus address the role of TGF-b in such processes, concentrating on the ability of developing neurons to produce and release this trophic factor.
Acknowledgements Supported by USPHS HD-09713 and DK-45746.
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