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Developmental expression of serum response factor in the rat central nervous system
Developmental Brain Research 138 (2002) 81–86 www.elsevier.com / locate / bres
Research report
Developmental expression of serum response factor in the rat central nervous system Janet L. Stringer a,b , *, Narasimhaswamy S. Belaguli c , Dinakar Iyer d , Robert J. Schwartz e , Ashok Balasubramanyam d,e a
Department of Pharmacology, Baylor College of Medicine, Houston, TX, USA Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA c Department of Surgery, Baylor College of Medicine, Houston, TX, USA d Department of Medicine, Baylor College of Medicine, Houston, TX, USA e Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA b
Accepted 25 July 2002
Abstract Serum response factor (SRF), a transcription factor known to be essential for early embryonic development as well as post-natal regulation of both cellular proliferation and myogenic differentiation, is expressed broadly in neurons within the adult mammalian central nervous system (CNS). The function of SRF within the developing CNS is not well established, but it is likely to play an important role in neuraxial development and neuronal function, since many of its known target genes (e.g., c-fos) and transcriptional partners (e.g., Elk-1) are also highly expressed in neurons. Immunohistochemical survey of the post-natal developing rat brain revealed a progressive increase in SRF immunoreactivity in neurons of the cerebral and cerebellar cortices, and in selective subcortical regions from birth (P0) through post-natal day 28 (P28). SRF immunoreactivity stabilized from P28 into adulthood. A few loci, such as the nucleus of cranial nerve VII, showed the reverse expression pattern (strong immunoreactivity at P0–P7, declining by P28). The developmental expression pattern of SRF overlaps significantly with that of myotonic dystrophy protein kinase, a potential upstream regulator, and of the LIM-only genes Lmo1, Lmo2 and Lmo3, whose products belong to a family of proteins known to be strong positive regulators of SRF’s transcriptional activity. These data suggest that SRF has a significant function in the early post-natal development of the CNS. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Cortex; Hippocampus; Post-natal; Transcription factor
1. Introduction Serum response factor (SRF) is an evolutionarily conserved transcription factor that plays a central role in early embryonic development by regulating the expression of immediate early genes associated with cellular proliferation (e.g., c-fos) [24,25] and muscle terminal differentiation marker genes (e.g., alpha-actins) [7,10,14]. SRF acts
*Corresponding author. Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Tel.: 11-713798-7937; fax: 11-713-798-3145. E-mail address: [email protected] (J.L. Stringer).
by binding as a homodimer to serum response elements (SREs, or ‘CArG boxes’) located in the regulatory promoter regions of target genes [24]. Both the binding of SRF to its cognate SREs as well as its transcriptional activity are regulated by numerous intracellular signaling pathways, including those involving growth factors [14], myotonic dystrophy protein kinase [13], Rho kinases [28] and LIMonly proteins [9]. SRF activity is further modified by coactivator proteins that participate in its nuclear transcriptional complex, including Ets proteins (e.g., Elk-1) in the context of the c-fos promoter [23], and Nkx2.5 and GATA4 in the context of the cardiac alpha-actin promoter [20]. Mice with homozygous deletion of SRF manifest a very early and lethal developmental block, with absence of
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a primitive streak, lack of mesoderm formation, and arrest of ectoderm and endoderm at a primitive stage [1]. Based on localization studies, SRF has been proposed to play a role in post-replicative neuronal gene expression, specifically in response to activation of the NMDA subtype of glutamate receptors in the hippocampus [2,29]. In the adult rat brain, SRF immunoreactivity (SRF-IR) has been described as exclusively nuclear and present in the majority of neurons in the cortex, caudate-putamen, amygdala and hippocampus [11]. SRF-IR was absent in the globus pallidus, thalamus, hypothalamus and mesencephalon. Scattered neurons in the medulla and dorsal horn of the spinal cord were SRF-IR positive. In the cerebellum, immunoreactivity was found only in the granule cells. Glial staining was notably absent except for a moderate stain in white matter tracts. A key unanswered question is whether SRF is involved in the early development or maturation of the nervous system. Therefore, the aim of the present study was to identify the expression pattern of SRF protein in the rat central nervous system during post-natal development.
3. Results
2. Materials and methods
3.2. SRF expression in the adult rat central nervous system ( CNS)
Rats were anesthetized with urethane (1.2 g / kg) and perfused through the heart with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. Brains were removed and stored overnight in 4% paraformaldehyde. They were then equilibrated in 15% followed by 30% sucrose in PBS. Sections (35 mm) were cut on a freezing microtome and stored in PBS with 0.05% azide. Two adult rats (150–175 g) were used for this study. The brain from one animal was sectioned horizontally and the brain from the other was sectioned coronally. Two litters of rat pups were utilized for the developmental study. Four pups were sacrificed on the day of birth (P0), four on P7, and two each on P14, P21 and P28. At each age, half of the brains were sectioned horizontally and half were sectioned coronally. For immunohistochemistry, anti-SRF polyclonal antibody [SRF (G-20): sc-335] was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). This antibody recognizes a peptide (amino acids 486–505) within the carboxy-terminal domain of human SRF, and is known to recognize murine SRF with high specificity [4]. Brain sections were incubated overnight with this antibody at a dilution of 1:2500. Staining was visualized by avidin– biotin complex assay (Elite Kit, Vector Laboratories, Burlingame, CA, USA), using diaminobenzidine with nickel enhancement (Vector Laboratories). At least six sections from each brain were stained for SRF-IR. Sections from the adult brain were used for the primary (without primary antibody) and secondary (without secondary antibody) controls. A few sections from the adult brains were counterstained with neutral red.
3.1. Cellular localization of SRF-IR in the brain SRF-IR appeared to be almost exclusively within the nuclei of neurons in both adult brain and P0–P28 brains. There was no staining of proximal dendrites, which would suggest cytoplasmic localization, nor was there any significant staining of astrocytes in either the adult or the developing brain. Despite the absence of dendritic staining, extra-nuclear localization of SRF in selected areas was suggested by SRF-IR in some axonal fiber tracts. Sections through the hippocampus revealed staining of the mossy fiber pathway from the dentate gyrus to the CA3 region (Fig. 1B). This band of staining was moderate in intensity and ended abruptly at the border of CA3 and CA2 / 1. Mossy fiber staining appeared between P14 and P21 (Fig. 2E and F), consistent with the time course of development of this pathway. Axonal fiber bundle staining was also present in a few other sites (stratum lacunosum-moleculare, base of the cortex), but less distinctly than in the mossy fiber tract.
In the adult, SRF-IR was seen throughout the cortex, in layers II–VI (Fig. 2C). Staining was more intense in the piriform cortex and less intense in the entorhinal cortex. SRF-IR was present in neurons throughout the caudateputamen, but notably absent in the globus pallidus (Fig. 1A). In the hippocampus, SRF-IR was present predominantly within the principal cell layers, although some hilar neurons were also stained. Few, if any, interneurons in the CA1 or CA3 areas showed SRF-IR. There was a clear distinction in the intensity of staining within the principal cells of the hippocampus: the granule cells of the dentate gyrus and pyramidal cells in CA1 were the most intensely stained, while the pyramidal cells in CA3 were more lightly stained (Fig. 1B). There was intense SRF-IR in the granule cells of the cerebellum and staining was also present within the nuclei of Purkinje cells (Fig. 1C). The neurons in the amygdala were positive for SRF-IR, although there were some differences between the various amygdala nuclei. For the most part the thalamus was very lightly stained or negative for SRF-IR. There was moderate intensity of SRF-IR in the hypothalamus, and some neurons in the lateral septum, nucleus accumbans, olfactory tubercle, mamillary nuclei, red nucleus, and spinal cord were stained. There was also light staining of SRF in the nuclei of some cranial nerves (CNs): III, VII (Fig. 3 adult), and possibly IV.
3.3. Developmental expression of SRF in the CNS In general, developmental changes in SRF-IR were
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consistent across different CNS regions. SRF staining was light at birth (P0), and increased gradually in intensity until P21. Intensity of SRF-IR remained stable from P21 to the adult stage. This was true in the caudate-putamen, cerebellum, amygdala, septum, and cortex. There were a few exceptions to this general pattern. In the cortex (Fig. 2A–C) a band of staining was present at the base of the cortical layers at P7 (arrow in Fig. 2A), which was not present in brains from older animals. In the hippocampus (Fig. 2D–F), staining intensity increased in all principal cell layers between P0 and P7. By P14, SRF-IR was less intense in CA3 compared to the granule cells of the dentate gyrus and pyramidal cells of CA1, and remained lighter in CA3 in subsequent stages. This appeared to be due to a continued increase in SRF-IR in CA1 and the dentate gyrus, rather than a decrease in immunoreactivity in CA3. Finally, in the nucleus of CNVII the staining intensity was greatest at P7, decreased by P14, and remained lighter in subsequent stages through adulthood (Fig. 3).
4. Discussion
Fig. 1. SRF-IR in the adult. Three areas of SRF-IR in the adult rat are shown. The top photograph shows a coronal section through the basal ganglia and demonstrates the striking border between the positively stained neurons in the caudate-putamen (CP) and the absence of staining in the globus pallidus (GP). The middle photograph shows a horizontal section through an adult brain at the level of the ventral hippocampus. The CA1, CA3 and dentate gyrus (DG) regions are indicated. The mossy fiber pathway that is moderately stained is bordered by the CA3 cell layer and the dashed line (arrow). The bottom photograph shows SRF-IR in the granule cell layer of a single folium in the cerebellum. Along the edge of the granule cell layer is a row of positively stained cells that appear to be Purkinje cells (arrow points to one Purkinje cell). Magnification was 1003 and the calibration bar in the middle represents 1 mm for all three photographs.
The widespread expression of SRF in the rat CNS and its dynamic expression pattern in selected cortical and subcortical regions during early post-natal maturation suggest a potentially important role for SRF in CNS development and function. However, data on the function of SRF in the CNS are sparse. SRF has been shown to be essential for at least one neuronal activity, that of gene expression resulting from calcium influx via the NMDA receptor [2,29]. In neurons, but not in glial cells, signaling from the NMDA receptor to c-fos gene transcription requires SRF and a serum response element (SRE) in the c-fos promoter [2,29]. Calcium influx via activated NMDA receptors increases the binding of SRF to the SRE [15,17]. These data indicate that in addition to its role as a growth factor activated transcription factor and as a myogenic transcription factor, SRF also functions in the CNS as a calcium-responsive factor targeted by a glutamate-NMDA signaling pathway. In the mouse embryo, SRF is expressed in all germ cell layers at an early stage (embryonic day 7.5). Shortly thereafter, SRF immunostaining becomes more circumscribed with highest expression being apparent in the myocardium and myotome [1]. In the adult rat brain, SRF has been localized in an earlier study to essentially the same regions as the present study [11], except for the observation of SRF-IR in the mossy fiber pathway. SRF immunostaining has been described as being located exclusively within cell nuclei, consistent with its function as a transcription factor. A conundrum in this regard is that at least one of its putative direct regulators, DMPK, is thought to have an exclusively cytoplasmic location. However, recent evidence indicates that SRF is also present in the cytoplasm during early myogenic differentia-
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Fig. 2. Developmental changes in the expression of SRF-IR in the cortex and hippocampus. On the left side (A–C) are shown representative sections of the cortex from a horizontal section just outside the ventral hippocampus and extending more anteriorly, at stages P7, P14 and P28. In the P7 rat brains, a denser layer of positively stained cells was present at the base of the cortex (arrow). This layer of cells was not seen in the older age groups. SRF-IR was present in layers II–VI of the cortex at every age after P7. On the right side (D–F), are shown representative horizontal sections of the hippocampus at the same stages. The CA1, CA3 and dentate gyrus (DG) regions of the hippocampus are labeled in F. At P7 there is approximately equal SRF-IR in the principal cells of the hippocampus. By P14, SRF-IR is clearly less in the CA3 region than in the dentate gyrus and in CA1. By P28 the moderately stained mossy fiber pathway is clearly visible between the dashed line and the CA3 cell layer. Magnification was 1003 for all photographs and the calibration bar represents 1 mm.
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Fig. 3. SRF-IR in the neurons of CNVII. Horizontal sections from a P7 animal and an adult are shown at higher magnification. Neurons (cluster of three neurons at arrow) belonging to the mesencephalic nucleus of CNVII are darkly stained in the very young brain. Intensity of immunostaining decreases gradually with increasing age. The neurons are larger, but still lightly stained in the adult (arrow). Magnification of the photographs was 2003 and the calibration bar represents 200 mm.
tion [5,8,10], and the present data suggest that SRF may have an extranuclear location in axonal fibers. The presence of SRF in axons will need to be confirmed and its function in this location determined. It is interesting to correlate the developmental and adult expression patterns of SRF in the CNS to that of its known or putative regulators and co-factors in transcriptional activity. p160 ROCK, a member of the Rho GTPaseassociated kinase family, is a known upstream activator of SRF in its transcription of myogenic genes [27], and has recently been shown to be essential for embryogenesis, including neuronal development [28]. Treatment of neurulating chick embryos with a specific inhibitor of Rho kinases blocked formation of the brain and neural tube, among other early morphogenetic abnormalities [28]. These data suggest a key role for Rho kinases, possibly acting via SRF, in early CNS differentiation. There are no precise localization studies of Rho kinases in the CNS. However, there are published data on the localization, in both the developing and adult mammalian CNS, of myotonic dystrophy protein kinase (DMPK), which is related to the Rho kinases and which we have also shown to be a key regulator of SRF’s transcriptional activity [13]. The data suggest that there is a significant, though not perfect, overlap between the localization of SRF in the CNS and that of its regulator, DMPK [3,19]. Elk-1, which associates with SRF to activate the transcription of many immediate early genes in a ternary complex on the SREs of their promoters, is widely expressed throughout the CNS in both cytoplasm and nuclei of neuronal cell bodies, but not in glia [22], in a manner similar to SRF. Recently, Vanhoutte et al. have
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described short Elk-1, a brain-specific isoform of Elk-1, that has an exclusively nuclear localization in cortical tissues and inhibits the transactivating function of Elk-1 at the SRE [26], thus potentially blocking the expression of key genes involved in cellular proliferation. It would be of great interest to understand the developmental expression patterns of these Elk-1 isoforms, since their expression and activity in the CNS might be expected to be coordinated with those of SRF to regulate the switch from neuronal proliferation to differentiation. Recent studies on the role of SRF in myogenic differentiation have demonstrated the importance of interactions between several proteins in assembling an optimal transcriptional complex at regulatory gene promoter regions. The muscle tissue-restricted expression of differentiation marker genes is regulated by combinatorial and synergistic interactions among SRF, members of the Nkx family of homeobox genes such as Nkx2.5, Nkx3.1 and Nkx3.2, members of the GATA family of zinc-finger DNA-binding proteins such as GATA4 and GATA6, and LIM-only factors such as CRP1 / 2 / 3 [9,21]. Since related Nkx factors (Nkx2.1, Nkx2.2) [6,16] as well as GATA factors (GATA2, GATA3) [18] have essential functions in neural development, a possible interaction among these factors and SRF may be important in the establishment and maintenance of the neuronal gene expression program. Perhaps most interesting and immediately relevant to the developmental expression of SRF, three LIM-only genes (Lmo1, Lmo2, and particularly Lmo3) have expression patterns that overlap strikingly with that of SRF protein in the hippocampus (with a lesser expression in CA3 compared to CA1 and the dentate gyrus), caudate-putamen and cerebral cortex [12]. In conclusion, we have described a unique pattern of expression of SRF in the developing rat CNS. The expression pattern and localization of SRF overlap with those of its key regulators and gene targets. These data are consistent with a role for SRF in early brain development and in neuronal signaling pathways (e.g., via NMDA receptors) leading to gene expression. They set the stage for further studies into the function of SRF in the mammalian CNS.
Acknowledgements This study was supported by NIH grant NS39941 to J.L.S.; and a Chao Scholar Award and a grant from The Methodist Hospital Foundation to A.B.
References [1] S. Arsenian, B. Weinhold, M. Oelgeschlager, U. Ruther, A. Nordheim, Serum response factor is essential for mesoderm formation during mouse embryogenesis, EMBO J. 17 (1998) 6289–6299.
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[2] H. Bading, D.D. Ginty, M.E. Greenberg, Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways, Science 260 (1993) 181–186. [3] A. Balasubramanyam, D. Iyer, J.L. Stringer, C. Beaulieu, A. Potvin, A.M. Neumeyer, J. Avruch, H.F. Epstein, Developmental changes in expression of myotonic dystrophy protein kinase in rat central nervous system, J. Comp. Neurol. 394 (1998) 309–325. [4] N.S. Belaguli, W. Zhou, T.-H. Trinh, M.W. Majesky, R.J. Schwartz, Dominant negative murine serum response factor: alternative splicing within the activation domain inhibits transactivation of serum response factor binding targets, Mol. Cell. Biol. 19 (1999) 4582– 4591. [5] S. Beqaj, S. Jakkaraju, R.R. Mattingly, D. Pan, L. Schuger, High RhoA activity maintains the undifferentiated mesenchymal cell phenotype, whereas RhoA down-regulation by laminin-2 induces smooth muscle myogenesis, J. Cell Biol. 156 (2002) 893–903. [6] J. Briscoe, L. Sussel, P. Serup, D. Hartigan-O’Connor, T.M. Jessell, J.L. Rubenstein, J. Ericson, Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signaling, Nature 398 (1999) 622–627. [7] C.L. Browning, D.E. Culberson, I.V. Aragon, R.A. Fillmore, J.D. Croissant, R.J. Schwartz, W.E. Zimmer, The developmentally regulated expression of serum response factor plays a key role in the control of smooth muscle-specific genes, Dev. Biol. 194 (1998) 18–37. [8] B. Camoretti-Mercado, H.-W. Liu, A.J. Halayko, S.M. Forsythe, J.W. Kyle, B. Li, Y. Fu, J. McConville, P. Kogut, J.E. Vieira, N.M. Patel, M.B. Hirshenson, E. Fuchs, S. Sinha, J.M. Miano, M.S. Parmacek, J.K. Burkhardt, J. Solway, Physiological control of smooth musclespecific gene expression through regulated nuclear translocation of serum response factor, J. Biol. Chem. 275 (2000) 30387–30393. [9] D.F. Chang, W. Roberts, J. Marx, M.S. Moore, X.R. Dong, M.W. Majesky, R.J. Schwartz, N.S. Belaguli, Vascular smooth muscle LIM-only proteins are potent cardiovascular transcription co-factors, Cell, submitted for publication. [10] J.D. Croissant, J.H. Kim, G. Eichele, L. Goering, J. Lough, R. Prywes, R.J. Schwartz, Avian serum response factor expression restricted primarily to muscle cell lineages is required for alphaactin gene transcription, Dev. Biol. 177 (1996) 250–264. [11] T. Herdegen, A. Blume, T. Buschmann, E. Georgakopoulos, C. Winter, W. Schmid, T.F. Hsieh, M. Zimmermann, P. Gass, Expression of activating transcription factor-2, serum response factor and cAMP/ Ca response element binding protein in the adult rat brain following generalized seizures, nerve fiber lesion and ultraviolet irradiation, Neurosci. 81 (1997) 199–212. [12] G.L. Hinks, B. Shah, S.J. French, L.S. Campos, K. Staley, J. Hughes, M.V. Sofroniew, Expression of LIM protein genes Lmo1, Lmo2, and Lmo3 in adult mouse hippocampus and other forebrain regions: differential regulation by seizure activity, J. Neurosci. 17 (1997) 5549–5559. [13] D. Iyer, N.S. Belaguli, M. Fluck, B. Rowan, L. Wei, N.L. Weigel, F.W. Booth, H.F. Epstein, R.J. Schwartz, A. Balasubramanyam, Regulation of serum response factor by myotonic dystrophy protein kinase (DMPK), in: Proceedings of the 3rd International Conference on Myotonic Dystrophy, Kyoto, 2001, p. 27. [14] F.E. Johansen, R. Prywes, Serum response factor: transcriptional regulation of genes induced by growth factors and differentiation, Biochim. Biophys. Acta 1242 (1995) 1–10.
[15] C.M. Johnson, C.S. Hill, S. Chawla, R. Treisman, H. Bading, Calcium controls gene expression via three distinct pathways that can function independently of the Ras / mitogen-activated protein kinases (ERKs) signalling cascade, J. Neurosci. 17 (1997) 6189– 6202. [16] B.J. Lee, G.J. Cho, R.B. Norgren Jr., M.P. Junier, D.F. Hill, V. Tapia, M.E. Costa, S.R. Ojeda, TTF-1, a homeodomain gene required for diencephalic morphogenesis, is postnatally expressed in the neoroendocrine brain in a developmentally regulated and cell-specific fashion, Mol. Cell. Neurosci. 1 (2001) 107–126. [17] T.A. Morris, N. Jafari, A.c. Rice, O. Vaconcelos, R.J. DeLorenzo, Persistent increased DNA-binding and expression of serum response factor occur with epilepsy-associated long-term plasticity changes, J. Neurosci. 19 (1999) 8234–8243. [18] I. Pata, M. Studer, J.J. van Doorninck, J. Briscoe, S. Kuuse, J.D. Engel, F. Grosveld, A. Karis, The transcription factor GATA3 is a downstream effector of Hoxb1 specification in rhombomere 4, Development 126 (1999) 5523–5531. [19] Y.C.N. Pham, N.T. Man, L.T. Lam, G.E. Morris, Localization of myotonic dystrophy protein kinase in human and rabbit tissues using a new panel of monoclonal antibodies, Hum. Mol. Genet. 7 (1998) 1957–1965. [20] J.L. Sepulveda, N. Belaguli, V. Nigam, C.Y. Chen, M. Nemer, R.J. Schwartz, GATA-4 and Nkx-2.5 coactivate Nkx-2 DNA binding targets: role for regulating early cardiac gene expression, Mol. Cell. Biol. 18 (1998) 3405–3415. [21] J.L. Sepulveda, S. Vlahopoulos, D. Iyer, N. Belaguli, R.J. Schwartz. Combinatorial expression of GATA4, Nkx2.5 and serum response factor directs early cardiac gene activity, J. Biol. Chem., in press. [22] V. Sgambato, P. Vanhoutte, C. Pages, M. Rogard, R. Hipskind, M.J. Besson, J. Caboche, In vivo expression and regulation of Elk-1, a target of the extracellular-related kinase signaling pathway, in the adult rat brain, J. Neurosci. 18 (1998) 214–225. [23] P.E. Shaw, Ternary complex formation over the c-fos serum response element: p62TCF exhibits dual component specificity with contacts to DNA and an extended structure in the DNA-binding domain of p67SRF, EMBO J. 11 (1992) 3011–3019. [24] R. Treisman, The SRE: a growth factor responsive transcriptional regulator, Semin. Cancer Biol. 1 (1990) 47–58. [25] R. Treisman, Regulation of transcription by MAP kinase cascades, Curr. Opin. Cell Biol. 8 (1996) 205–215. [26] P. Vanhoutte, J.L. Nissen, B. Brugg, B. Della Gaspera, M.-J. Besson, R.A. Hipskind, J. Caboche, Opposing roles of Elk-1 and its brainspecific isoform short Elk-1, in nerve growth factor-induced PC12 differentiation, J. Biol. Chem. 276 (2001) 5189–5196. [27] L. Wei, W. Zhou, J.D. Croissant, F.E. Johansen, R. Prywes, A. Balasubramanyam, R.J. Schwartz, RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation, J. Biol. Chem. 273 (1998) 30287–30294. [28] L. Wei, W. Roberts, L. Wang, M. Yamada, S. Zhang, Z. Zhao, S.A. Rivkees, R.J. Schwartz, K. Imanaka-Yoshida, Rho kinases play an obligatory role in verebrate embryonic organogenesis, Development 128 (2001) 2953–2962. [29] Z. Xia, H. Dudek, C.K. Miranti, M.E. Greenberg, Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase / ERK-dependent mechanism, J. Neurosci. 16 (1996) 5425–5436.
Research report
Developmental expression of serum response factor in the rat central nervous system Janet L. Stringer a,b , *, Narasimhaswamy S. Belaguli c , Dinakar Iyer d , Robert J. Schwartz e , Ashok Balasubramanyam d,e a
Department of Pharmacology, Baylor College of Medicine, Houston, TX, USA Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA c Department of Surgery, Baylor College of Medicine, Houston, TX, USA d Department of Medicine, Baylor College of Medicine, Houston, TX, USA e Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA b
Accepted 25 July 2002
Abstract Serum response factor (SRF), a transcription factor known to be essential for early embryonic development as well as post-natal regulation of both cellular proliferation and myogenic differentiation, is expressed broadly in neurons within the adult mammalian central nervous system (CNS). The function of SRF within the developing CNS is not well established, but it is likely to play an important role in neuraxial development and neuronal function, since many of its known target genes (e.g., c-fos) and transcriptional partners (e.g., Elk-1) are also highly expressed in neurons. Immunohistochemical survey of the post-natal developing rat brain revealed a progressive increase in SRF immunoreactivity in neurons of the cerebral and cerebellar cortices, and in selective subcortical regions from birth (P0) through post-natal day 28 (P28). SRF immunoreactivity stabilized from P28 into adulthood. A few loci, such as the nucleus of cranial nerve VII, showed the reverse expression pattern (strong immunoreactivity at P0–P7, declining by P28). The developmental expression pattern of SRF overlaps significantly with that of myotonic dystrophy protein kinase, a potential upstream regulator, and of the LIM-only genes Lmo1, Lmo2 and Lmo3, whose products belong to a family of proteins known to be strong positive regulators of SRF’s transcriptional activity. These data suggest that SRF has a significant function in the early post-natal development of the CNS. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Cortex; Hippocampus; Post-natal; Transcription factor
1. Introduction Serum response factor (SRF) is an evolutionarily conserved transcription factor that plays a central role in early embryonic development by regulating the expression of immediate early genes associated with cellular proliferation (e.g., c-fos) [24,25] and muscle terminal differentiation marker genes (e.g., alpha-actins) [7,10,14]. SRF acts
*Corresponding author. Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Tel.: 11-713798-7937; fax: 11-713-798-3145. E-mail address: [email protected] (J.L. Stringer).
by binding as a homodimer to serum response elements (SREs, or ‘CArG boxes’) located in the regulatory promoter regions of target genes [24]. Both the binding of SRF to its cognate SREs as well as its transcriptional activity are regulated by numerous intracellular signaling pathways, including those involving growth factors [14], myotonic dystrophy protein kinase [13], Rho kinases [28] and LIMonly proteins [9]. SRF activity is further modified by coactivator proteins that participate in its nuclear transcriptional complex, including Ets proteins (e.g., Elk-1) in the context of the c-fos promoter [23], and Nkx2.5 and GATA4 in the context of the cardiac alpha-actin promoter [20]. Mice with homozygous deletion of SRF manifest a very early and lethal developmental block, with absence of
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a primitive streak, lack of mesoderm formation, and arrest of ectoderm and endoderm at a primitive stage [1]. Based on localization studies, SRF has been proposed to play a role in post-replicative neuronal gene expression, specifically in response to activation of the NMDA subtype of glutamate receptors in the hippocampus [2,29]. In the adult rat brain, SRF immunoreactivity (SRF-IR) has been described as exclusively nuclear and present in the majority of neurons in the cortex, caudate-putamen, amygdala and hippocampus [11]. SRF-IR was absent in the globus pallidus, thalamus, hypothalamus and mesencephalon. Scattered neurons in the medulla and dorsal horn of the spinal cord were SRF-IR positive. In the cerebellum, immunoreactivity was found only in the granule cells. Glial staining was notably absent except for a moderate stain in white matter tracts. A key unanswered question is whether SRF is involved in the early development or maturation of the nervous system. Therefore, the aim of the present study was to identify the expression pattern of SRF protein in the rat central nervous system during post-natal development.
3. Results
2. Materials and methods
3.2. SRF expression in the adult rat central nervous system ( CNS)
Rats were anesthetized with urethane (1.2 g / kg) and perfused through the heart with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. Brains were removed and stored overnight in 4% paraformaldehyde. They were then equilibrated in 15% followed by 30% sucrose in PBS. Sections (35 mm) were cut on a freezing microtome and stored in PBS with 0.05% azide. Two adult rats (150–175 g) were used for this study. The brain from one animal was sectioned horizontally and the brain from the other was sectioned coronally. Two litters of rat pups were utilized for the developmental study. Four pups were sacrificed on the day of birth (P0), four on P7, and two each on P14, P21 and P28. At each age, half of the brains were sectioned horizontally and half were sectioned coronally. For immunohistochemistry, anti-SRF polyclonal antibody [SRF (G-20): sc-335] was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). This antibody recognizes a peptide (amino acids 486–505) within the carboxy-terminal domain of human SRF, and is known to recognize murine SRF with high specificity [4]. Brain sections were incubated overnight with this antibody at a dilution of 1:2500. Staining was visualized by avidin– biotin complex assay (Elite Kit, Vector Laboratories, Burlingame, CA, USA), using diaminobenzidine with nickel enhancement (Vector Laboratories). At least six sections from each brain were stained for SRF-IR. Sections from the adult brain were used for the primary (without primary antibody) and secondary (without secondary antibody) controls. A few sections from the adult brains were counterstained with neutral red.
3.1. Cellular localization of SRF-IR in the brain SRF-IR appeared to be almost exclusively within the nuclei of neurons in both adult brain and P0–P28 brains. There was no staining of proximal dendrites, which would suggest cytoplasmic localization, nor was there any significant staining of astrocytes in either the adult or the developing brain. Despite the absence of dendritic staining, extra-nuclear localization of SRF in selected areas was suggested by SRF-IR in some axonal fiber tracts. Sections through the hippocampus revealed staining of the mossy fiber pathway from the dentate gyrus to the CA3 region (Fig. 1B). This band of staining was moderate in intensity and ended abruptly at the border of CA3 and CA2 / 1. Mossy fiber staining appeared between P14 and P21 (Fig. 2E and F), consistent with the time course of development of this pathway. Axonal fiber bundle staining was also present in a few other sites (stratum lacunosum-moleculare, base of the cortex), but less distinctly than in the mossy fiber tract.
In the adult, SRF-IR was seen throughout the cortex, in layers II–VI (Fig. 2C). Staining was more intense in the piriform cortex and less intense in the entorhinal cortex. SRF-IR was present in neurons throughout the caudateputamen, but notably absent in the globus pallidus (Fig. 1A). In the hippocampus, SRF-IR was present predominantly within the principal cell layers, although some hilar neurons were also stained. Few, if any, interneurons in the CA1 or CA3 areas showed SRF-IR. There was a clear distinction in the intensity of staining within the principal cells of the hippocampus: the granule cells of the dentate gyrus and pyramidal cells in CA1 were the most intensely stained, while the pyramidal cells in CA3 were more lightly stained (Fig. 1B). There was intense SRF-IR in the granule cells of the cerebellum and staining was also present within the nuclei of Purkinje cells (Fig. 1C). The neurons in the amygdala were positive for SRF-IR, although there were some differences between the various amygdala nuclei. For the most part the thalamus was very lightly stained or negative for SRF-IR. There was moderate intensity of SRF-IR in the hypothalamus, and some neurons in the lateral septum, nucleus accumbans, olfactory tubercle, mamillary nuclei, red nucleus, and spinal cord were stained. There was also light staining of SRF in the nuclei of some cranial nerves (CNs): III, VII (Fig. 3 adult), and possibly IV.
3.3. Developmental expression of SRF in the CNS In general, developmental changes in SRF-IR were
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consistent across different CNS regions. SRF staining was light at birth (P0), and increased gradually in intensity until P21. Intensity of SRF-IR remained stable from P21 to the adult stage. This was true in the caudate-putamen, cerebellum, amygdala, septum, and cortex. There were a few exceptions to this general pattern. In the cortex (Fig. 2A–C) a band of staining was present at the base of the cortical layers at P7 (arrow in Fig. 2A), which was not present in brains from older animals. In the hippocampus (Fig. 2D–F), staining intensity increased in all principal cell layers between P0 and P7. By P14, SRF-IR was less intense in CA3 compared to the granule cells of the dentate gyrus and pyramidal cells of CA1, and remained lighter in CA3 in subsequent stages. This appeared to be due to a continued increase in SRF-IR in CA1 and the dentate gyrus, rather than a decrease in immunoreactivity in CA3. Finally, in the nucleus of CNVII the staining intensity was greatest at P7, decreased by P14, and remained lighter in subsequent stages through adulthood (Fig. 3).
4. Discussion
Fig. 1. SRF-IR in the adult. Three areas of SRF-IR in the adult rat are shown. The top photograph shows a coronal section through the basal ganglia and demonstrates the striking border between the positively stained neurons in the caudate-putamen (CP) and the absence of staining in the globus pallidus (GP). The middle photograph shows a horizontal section through an adult brain at the level of the ventral hippocampus. The CA1, CA3 and dentate gyrus (DG) regions are indicated. The mossy fiber pathway that is moderately stained is bordered by the CA3 cell layer and the dashed line (arrow). The bottom photograph shows SRF-IR in the granule cell layer of a single folium in the cerebellum. Along the edge of the granule cell layer is a row of positively stained cells that appear to be Purkinje cells (arrow points to one Purkinje cell). Magnification was 1003 and the calibration bar in the middle represents 1 mm for all three photographs.
The widespread expression of SRF in the rat CNS and its dynamic expression pattern in selected cortical and subcortical regions during early post-natal maturation suggest a potentially important role for SRF in CNS development and function. However, data on the function of SRF in the CNS are sparse. SRF has been shown to be essential for at least one neuronal activity, that of gene expression resulting from calcium influx via the NMDA receptor [2,29]. In neurons, but not in glial cells, signaling from the NMDA receptor to c-fos gene transcription requires SRF and a serum response element (SRE) in the c-fos promoter [2,29]. Calcium influx via activated NMDA receptors increases the binding of SRF to the SRE [15,17]. These data indicate that in addition to its role as a growth factor activated transcription factor and as a myogenic transcription factor, SRF also functions in the CNS as a calcium-responsive factor targeted by a glutamate-NMDA signaling pathway. In the mouse embryo, SRF is expressed in all germ cell layers at an early stage (embryonic day 7.5). Shortly thereafter, SRF immunostaining becomes more circumscribed with highest expression being apparent in the myocardium and myotome [1]. In the adult rat brain, SRF has been localized in an earlier study to essentially the same regions as the present study [11], except for the observation of SRF-IR in the mossy fiber pathway. SRF immunostaining has been described as being located exclusively within cell nuclei, consistent with its function as a transcription factor. A conundrum in this regard is that at least one of its putative direct regulators, DMPK, is thought to have an exclusively cytoplasmic location. However, recent evidence indicates that SRF is also present in the cytoplasm during early myogenic differentia-
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Fig. 2. Developmental changes in the expression of SRF-IR in the cortex and hippocampus. On the left side (A–C) are shown representative sections of the cortex from a horizontal section just outside the ventral hippocampus and extending more anteriorly, at stages P7, P14 and P28. In the P7 rat brains, a denser layer of positively stained cells was present at the base of the cortex (arrow). This layer of cells was not seen in the older age groups. SRF-IR was present in layers II–VI of the cortex at every age after P7. On the right side (D–F), are shown representative horizontal sections of the hippocampus at the same stages. The CA1, CA3 and dentate gyrus (DG) regions of the hippocampus are labeled in F. At P7 there is approximately equal SRF-IR in the principal cells of the hippocampus. By P14, SRF-IR is clearly less in the CA3 region than in the dentate gyrus and in CA1. By P28 the moderately stained mossy fiber pathway is clearly visible between the dashed line and the CA3 cell layer. Magnification was 1003 for all photographs and the calibration bar represents 1 mm.
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Fig. 3. SRF-IR in the neurons of CNVII. Horizontal sections from a P7 animal and an adult are shown at higher magnification. Neurons (cluster of three neurons at arrow) belonging to the mesencephalic nucleus of CNVII are darkly stained in the very young brain. Intensity of immunostaining decreases gradually with increasing age. The neurons are larger, but still lightly stained in the adult (arrow). Magnification of the photographs was 2003 and the calibration bar represents 200 mm.
tion [5,8,10], and the present data suggest that SRF may have an extranuclear location in axonal fibers. The presence of SRF in axons will need to be confirmed and its function in this location determined. It is interesting to correlate the developmental and adult expression patterns of SRF in the CNS to that of its known or putative regulators and co-factors in transcriptional activity. p160 ROCK, a member of the Rho GTPaseassociated kinase family, is a known upstream activator of SRF in its transcription of myogenic genes [27], and has recently been shown to be essential for embryogenesis, including neuronal development [28]. Treatment of neurulating chick embryos with a specific inhibitor of Rho kinases blocked formation of the brain and neural tube, among other early morphogenetic abnormalities [28]. These data suggest a key role for Rho kinases, possibly acting via SRF, in early CNS differentiation. There are no precise localization studies of Rho kinases in the CNS. However, there are published data on the localization, in both the developing and adult mammalian CNS, of myotonic dystrophy protein kinase (DMPK), which is related to the Rho kinases and which we have also shown to be a key regulator of SRF’s transcriptional activity [13]. The data suggest that there is a significant, though not perfect, overlap between the localization of SRF in the CNS and that of its regulator, DMPK [3,19]. Elk-1, which associates with SRF to activate the transcription of many immediate early genes in a ternary complex on the SREs of their promoters, is widely expressed throughout the CNS in both cytoplasm and nuclei of neuronal cell bodies, but not in glia [22], in a manner similar to SRF. Recently, Vanhoutte et al. have
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described short Elk-1, a brain-specific isoform of Elk-1, that has an exclusively nuclear localization in cortical tissues and inhibits the transactivating function of Elk-1 at the SRE [26], thus potentially blocking the expression of key genes involved in cellular proliferation. It would be of great interest to understand the developmental expression patterns of these Elk-1 isoforms, since their expression and activity in the CNS might be expected to be coordinated with those of SRF to regulate the switch from neuronal proliferation to differentiation. Recent studies on the role of SRF in myogenic differentiation have demonstrated the importance of interactions between several proteins in assembling an optimal transcriptional complex at regulatory gene promoter regions. The muscle tissue-restricted expression of differentiation marker genes is regulated by combinatorial and synergistic interactions among SRF, members of the Nkx family of homeobox genes such as Nkx2.5, Nkx3.1 and Nkx3.2, members of the GATA family of zinc-finger DNA-binding proteins such as GATA4 and GATA6, and LIM-only factors such as CRP1 / 2 / 3 [9,21]. Since related Nkx factors (Nkx2.1, Nkx2.2) [6,16] as well as GATA factors (GATA2, GATA3) [18] have essential functions in neural development, a possible interaction among these factors and SRF may be important in the establishment and maintenance of the neuronal gene expression program. Perhaps most interesting and immediately relevant to the developmental expression of SRF, three LIM-only genes (Lmo1, Lmo2, and particularly Lmo3) have expression patterns that overlap strikingly with that of SRF protein in the hippocampus (with a lesser expression in CA3 compared to CA1 and the dentate gyrus), caudate-putamen and cerebral cortex [12]. In conclusion, we have described a unique pattern of expression of SRF in the developing rat CNS. The expression pattern and localization of SRF overlap with those of its key regulators and gene targets. These data are consistent with a role for SRF in early brain development and in neuronal signaling pathways (e.g., via NMDA receptors) leading to gene expression. They set the stage for further studies into the function of SRF in the mammalian CNS.
Acknowledgements This study was supported by NIH grant NS39941 to J.L.S.; and a Chao Scholar Award and a grant from The Methodist Hospital Foundation to A.B.
References [1] S. Arsenian, B. Weinhold, M. Oelgeschlager, U. Ruther, A. Nordheim, Serum response factor is essential for mesoderm formation during mouse embryogenesis, EMBO J. 17 (1998) 6289–6299.
86
J.L. Stringer et al. / Developmental Brain Research 138 (2002) 81–86
[2] H. Bading, D.D. Ginty, M.E. Greenberg, Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways, Science 260 (1993) 181–186. [3] A. Balasubramanyam, D. Iyer, J.L. Stringer, C. Beaulieu, A. Potvin, A.M. Neumeyer, J. Avruch, H.F. Epstein, Developmental changes in expression of myotonic dystrophy protein kinase in rat central nervous system, J. Comp. Neurol. 394 (1998) 309–325. [4] N.S. Belaguli, W. Zhou, T.-H. Trinh, M.W. Majesky, R.J. Schwartz, Dominant negative murine serum response factor: alternative splicing within the activation domain inhibits transactivation of serum response factor binding targets, Mol. Cell. Biol. 19 (1999) 4582– 4591. [5] S. Beqaj, S. Jakkaraju, R.R. Mattingly, D. Pan, L. Schuger, High RhoA activity maintains the undifferentiated mesenchymal cell phenotype, whereas RhoA down-regulation by laminin-2 induces smooth muscle myogenesis, J. Cell Biol. 156 (2002) 893–903. [6] J. Briscoe, L. Sussel, P. Serup, D. Hartigan-O’Connor, T.M. Jessell, J.L. Rubenstein, J. Ericson, Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signaling, Nature 398 (1999) 622–627. [7] C.L. Browning, D.E. Culberson, I.V. Aragon, R.A. Fillmore, J.D. Croissant, R.J. Schwartz, W.E. Zimmer, The developmentally regulated expression of serum response factor plays a key role in the control of smooth muscle-specific genes, Dev. Biol. 194 (1998) 18–37. [8] B. Camoretti-Mercado, H.-W. Liu, A.J. Halayko, S.M. Forsythe, J.W. Kyle, B. Li, Y. Fu, J. McConville, P. Kogut, J.E. Vieira, N.M. Patel, M.B. Hirshenson, E. Fuchs, S. Sinha, J.M. Miano, M.S. Parmacek, J.K. Burkhardt, J. Solway, Physiological control of smooth musclespecific gene expression through regulated nuclear translocation of serum response factor, J. Biol. Chem. 275 (2000) 30387–30393. [9] D.F. Chang, W. Roberts, J. Marx, M.S. Moore, X.R. Dong, M.W. Majesky, R.J. Schwartz, N.S. Belaguli, Vascular smooth muscle LIM-only proteins are potent cardiovascular transcription co-factors, Cell, submitted for publication. [10] J.D. Croissant, J.H. Kim, G. Eichele, L. Goering, J. Lough, R. Prywes, R.J. Schwartz, Avian serum response factor expression restricted primarily to muscle cell lineages is required for alphaactin gene transcription, Dev. Biol. 177 (1996) 250–264. [11] T. Herdegen, A. Blume, T. Buschmann, E. Georgakopoulos, C. Winter, W. Schmid, T.F. Hsieh, M. Zimmermann, P. Gass, Expression of activating transcription factor-2, serum response factor and cAMP/ Ca response element binding protein in the adult rat brain following generalized seizures, nerve fiber lesion and ultraviolet irradiation, Neurosci. 81 (1997) 199–212. [12] G.L. Hinks, B. Shah, S.J. French, L.S. Campos, K. Staley, J. Hughes, M.V. Sofroniew, Expression of LIM protein genes Lmo1, Lmo2, and Lmo3 in adult mouse hippocampus and other forebrain regions: differential regulation by seizure activity, J. Neurosci. 17 (1997) 5549–5559. [13] D. Iyer, N.S. Belaguli, M. Fluck, B. Rowan, L. Wei, N.L. Weigel, F.W. Booth, H.F. Epstein, R.J. Schwartz, A. Balasubramanyam, Regulation of serum response factor by myotonic dystrophy protein kinase (DMPK), in: Proceedings of the 3rd International Conference on Myotonic Dystrophy, Kyoto, 2001, p. 27. [14] F.E. Johansen, R. Prywes, Serum response factor: transcriptional regulation of genes induced by growth factors and differentiation, Biochim. Biophys. Acta 1242 (1995) 1–10.
[15] C.M. Johnson, C.S. Hill, S. Chawla, R. Treisman, H. Bading, Calcium controls gene expression via three distinct pathways that can function independently of the Ras / mitogen-activated protein kinases (ERKs) signalling cascade, J. Neurosci. 17 (1997) 6189– 6202. [16] B.J. Lee, G.J. Cho, R.B. Norgren Jr., M.P. Junier, D.F. Hill, V. Tapia, M.E. Costa, S.R. Ojeda, TTF-1, a homeodomain gene required for diencephalic morphogenesis, is postnatally expressed in the neoroendocrine brain in a developmentally regulated and cell-specific fashion, Mol. Cell. Neurosci. 1 (2001) 107–126. [17] T.A. Morris, N. Jafari, A.c. Rice, O. Vaconcelos, R.J. DeLorenzo, Persistent increased DNA-binding and expression of serum response factor occur with epilepsy-associated long-term plasticity changes, J. Neurosci. 19 (1999) 8234–8243. [18] I. Pata, M. Studer, J.J. van Doorninck, J. Briscoe, S. Kuuse, J.D. Engel, F. Grosveld, A. Karis, The transcription factor GATA3 is a downstream effector of Hoxb1 specification in rhombomere 4, Development 126 (1999) 5523–5531. [19] Y.C.N. Pham, N.T. Man, L.T. Lam, G.E. Morris, Localization of myotonic dystrophy protein kinase in human and rabbit tissues using a new panel of monoclonal antibodies, Hum. Mol. Genet. 7 (1998) 1957–1965. [20] J.L. Sepulveda, N. Belaguli, V. Nigam, C.Y. Chen, M. Nemer, R.J. Schwartz, GATA-4 and Nkx-2.5 coactivate Nkx-2 DNA binding targets: role for regulating early cardiac gene expression, Mol. Cell. Biol. 18 (1998) 3405–3415. [21] J.L. Sepulveda, S. Vlahopoulos, D. Iyer, N. Belaguli, R.J. Schwartz. Combinatorial expression of GATA4, Nkx2.5 and serum response factor directs early cardiac gene activity, J. Biol. Chem., in press. [22] V. Sgambato, P. Vanhoutte, C. Pages, M. Rogard, R. Hipskind, M.J. Besson, J. Caboche, In vivo expression and regulation of Elk-1, a target of the extracellular-related kinase signaling pathway, in the adult rat brain, J. Neurosci. 18 (1998) 214–225. [23] P.E. Shaw, Ternary complex formation over the c-fos serum response element: p62TCF exhibits dual component specificity with contacts to DNA and an extended structure in the DNA-binding domain of p67SRF, EMBO J. 11 (1992) 3011–3019. [24] R. Treisman, The SRE: a growth factor responsive transcriptional regulator, Semin. Cancer Biol. 1 (1990) 47–58. [25] R. Treisman, Regulation of transcription by MAP kinase cascades, Curr. Opin. Cell Biol. 8 (1996) 205–215. [26] P. Vanhoutte, J.L. Nissen, B. Brugg, B. Della Gaspera, M.-J. Besson, R.A. Hipskind, J. Caboche, Opposing roles of Elk-1 and its brainspecific isoform short Elk-1, in nerve growth factor-induced PC12 differentiation, J. Biol. Chem. 276 (2001) 5189–5196. [27] L. Wei, W. Zhou, J.D. Croissant, F.E. Johansen, R. Prywes, A. Balasubramanyam, R.J. Schwartz, RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation, J. Biol. Chem. 273 (1998) 30287–30294. [28] L. Wei, W. Roberts, L. Wang, M. Yamada, S. Zhang, Z. Zhao, S.A. Rivkees, R.J. Schwartz, K. Imanaka-Yoshida, Rho kinases play an obligatory role in verebrate embryonic organogenesis, Development 128 (2001) 2953–2962. [29] Z. Xia, H. Dudek, C.K. Miranti, M.E. Greenberg, Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase / ERK-dependent mechanism, J. Neurosci. 16 (1996) 5425–5436.