1 Department of Neurology, University Clinics Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, ... following nervous tissue injury (Logan et al.
Cell Tissue Res (1997) 287:471–480
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© Springer-Verlag 1997
Fibroblast growth factor-2 (FGF-2) and FGF-receptor (FGFR-1) immunoreactivity in embryonic spinal autonomic neurons Christian Stapf1, Gabriele Lück2, Mehdi Shakibaei2, Dieter Blottner2 1 Department of Neurology, University Clinics Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany 2 Institute for Anatomy, University Clinics Benjamin Franklin, Freie Universität Berlin, Königin-Luise-Strasse 15, D-14195 Berlin-Dahlem, Germany
&misc:Received: 8 May 1996 / Accepted: 23 October 1996
&p.1:Abstract. The development of the nervous system appears to be under the control of multiple growth factors, neurotrophins and cytokines, which may be expressed either continuously or transiently throughout defined stages of cellular generation, proliferation or differentiation. Fibroblast growth factor (FGF) cytokines and their receptors are abundantly expressed in the embryonic nervous system but their localization at autonomic levels in the fetal spinal cord has not yet been detailed. Immunoreactivity to FGF-2, probably the best characterized member of the FGF family (FGF-1 to FGF-10) and of one of its high affinity receptors, FGFR-1, was found in autonomic neurons at embryonic day E14, the peak day of generation and proliferation in the common ventral motoneuron pool. It was also continuously present throughout the investigated subsequent stages (E15 to postnatal day P30). Immunogold electron microscopy revealed the cytoplasmic localization of FGF-2 and FGFR1 in intermediolateral neurons, the major group of sympathetic preganglionic neurons in the spinal cord. In these neurons, immunocytochemistry from E14 onwards showed the co-distribution of both markers at the period of axonal outgrowth to peripheral targets, e.g. the adrenal medulla. Our findings suggest autocrine and/or paracrine actions of FGF-2 for sympathetic preganglionic development but do not support its role as a target-derived neurotrophic factor for autonomic neuron development. &kwd:Key words: FGF-2 – FGF receptor-1 – Autonomic nervous system – Sympathetic preganglionic neurons – Spinal cord – Development – Rat (Wistar)
This paper is dedicated to Professor Andreas Oksche on the occasion of his 70th birthday, in recognition of outstanding contributions to neuroscience and to the internationalisation of science The work in our laboratory is supported by the German Research Foundation (SFB 174, Projects B1 and Bl 259/3–1) Correspondence to: D. Blottner (Tel.: +49–30–838–3843; Fax: +49–30–838–3806; E-mail: blottner@zedat.fu-berlin.de)&/fn-block:
Introduction Soluble growth factors, neurotrophins and cytokines play important roles in the development of the vertebrate nervous system (Araujo et al. 1990; Bartlett et al. 1994; Davies 1994). Two of probably the best characterized cytokines in the nervous system, fibroblast growth factors 1 and 2 (FGF-1 and FGF-2, respectively; previously known as acidic and basic FGF), are prototypic members of the multifunctional FGF-gene family with important functions as proliferation and differentiation factors for mesenchymal and neuroectodermal-derived cell types (Baird 1994). In nervous tissue, FGF-1 and FGF-2 and their receptors have been found in defined neuronal populations during development (Caday et al. 1990; Ernfors et al. 1990; Kalcheim and Neufeld 1990; Despres et al. 1991; Grothe et al. 1991; Hondermarck et al. 1992; Weise et al. 1993; Gómez-Pinilla et al. 1994), adulthood (Emoto et al. 1989; Kato et al. 1992; Asai et al. 1993) or following nervous tissue injury (Logan et al. 1992; Koshinaga et al. 1993). Administration of FGF-2 in vivo promotes neural regeneration in various axotomy models (reviewed in Blottner and Baumgarten 1994), thus establishing the neurotrophic potential of FGF for defined peripheral and central neurons (Westermann et al. 1990; Unsicker et al. 1992). Furthermore, intriguing roles for growth factors or cytokines, such as the control of transmitter phenotype or neuronal survival, have been reported regarding the peripheral autonomic system (Coughlin 1984; Purves et al. 1988; Rao and Landis 1993), whereas the presence and cellular distribution of FGF has not yet been detailed at autonomic levels in the spinal cord. Putative functions of FGF2 and its receptor(s) may be reflected by the spatiotemporal patterns with respect to the marked topographical rearrangement during formation of the cytoarchitecture of the spinal autonomic system, as given by the sympathetic preganglionic nuclei of intermediate layers of the developing spinal cord (Markham et al. 1991; Phelps et al. 1991, 1993). In order to study the cellular and subcellular distribution of FGF at
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spinal autonomic levels, FGF-2 and one of its high-affinity receptors, FGF-receptor type-1 (FGFR-1; also termed flg in the literature), were immunolocalized by antigenspecific antibodies in the intermediolateral (IML)-cell column, the major spinal autonomic nuclei, from embryonic to early postnatal stages of the newborn rat.
Materials and methods
6 h at 37°C with goat anti-mouse IgG coupled to 5-nm colloidal gold or goat anti-rabbit IgG coupled to 1-nm gold (Amersham, Braunschweig, Germany; diluted 1:50 in BSA-gelatine), rinsed in buffer and fixed in 2.5% (w/v) buffered glutaraldehyde. The detectability of the gold particles was improved by silver enhancement according to Danscher (1981). The sections were dehydrated in a graded ethanol series and flat-embedded in Araldite. Semithin sections (1 µm) were inspected by light microscopy. Areas detected by light microscopy as having neurons labelled by colloidal gold were ultrathin-sectioned, double-stained with lead citrate and uranyl acetate, and examined and photographed with a Zeiss EM10 electron microscope.
Animal stages and tissue preparation Rat fetuses from embryonic day 14 (E-14) to E-21 (n=4 for each stage) were obtained from a group of 15 pregnant Wistar rats (day 0 was determined by a positive vaginal clot following mating overnight) under deep chloral hydrate anaesthesia (3.5%, 1 ml per 100 gm b.wt., i.p.). After birth, the pups remained with their mothers. At postnatal day 0 (P0) to P30, pups/young animals were separated from the litter, anaesthetized with choral hydrate and perfused via the heart with cold Bouin fixative. The vertebrate column was dissected and postfixed overnight in the same fixative. The excised cord tissue was then rinsed in buffer, dehydrated in a graded ethanol series (70%, 90%, 95%, 100%, v/v) followed by routine embedding in paraffin wax (Paraplast). Serial paraffin sections (6 mm thick) were cut according to an established protocol (Stapf et al. 1995) and mounted onto protein-coated slides. Animal care followed the European Council Guidelines (86/609 EEC) and experiments were approved by the local government in accordance with German law regarding the protection of animals.
Western-blot analysis Western-blotting for the FGF-2 and FGFR-1 proteins was performed according to a routine protocol. Two young adult rats were given an anaesthetic overdose and their spinal cords were rapidly dissected, homogenized and washed in 60 mM sodium phosphate buffer (pH 7.6). After centrifugation, the pellet was resuspended in lysis buffer (100 mM sodium phosphate, 25 mM NaCl, 0.5% Pepstatin A, 0.1% Chymostatin, 0.1 mM phenylmethane sulphonyl-fluoride) in an ultrasonic bath on ice for 60 min. The eluate was separated by 15% SDS polyacrylamide gel electrophoresis for 45 min., electroblotted onto nitrocellulose (100 V, 1 h) and incubated overnight at 4°C with the monoclonal anti-bovine FGF-2 antibody (1:100 in 60 mM sodium phosphate, pH 7.6) or with the polyclonal anti-FGFR-1 antibody (1:100, same buffer). The immunoreactive bands were detected by an alkaline phosphatase reaction (Vectastain, Vector Laboratories, Burlingame, Calif., USA) and photodocumented.
Immunohistochemistry From each pre- and postnatal stage, deparaffinized sections were incubated either with a monoclonal anti-bovine FGF-2 antibody (1:250 in 100 mM TRIS-HCl, pH 7.6, 0.5% Triton X-100, 2% preimmune serum) or with polyclonal anti-FGFR-1 antibody (1:300 in the same buffer) for 18 h in a moist chamber at 4°C (antibodies from Upstate Biotechnology, Frankfurt). The latter antibody was raised against an external region peptide of chicken FGFR. Visualization of the primary antibodies was performed by incubation with fluorescently labelled secondary antibodies (Cy3 anti-rabbit or anti-mouse IgG; Jackson ImmunoResearch Laboratories, West Grove, Pa.), for 1 h (1:300) at room temperature. For controls, the primary antibody was omitted or was preabsorbed with the relevant antigen (1 µg FGF-2 or 2.5 µg chicken FGFR protein, 6 h on a shaker), sections being subsequently treated according to the above immunocytochemical protocol. The controls were always negative.
Results Western-blot analysis Western-blot analyses show that extracts from young adult rat spinal cords contain several immunoreactive FGF-2 species. Anti-FGF-2 immunostains three bands at 23 kDa, 21 kDa and 18 kDa (Fig. 1). They correspond to well-known molecular weight forms of the FGF-2 protein (Florkiewicz and Sommer 1989; Gonzalez et al. 1995). The polyclonal anti-FGFR-1 antibody used in our study immunostains two expected bands at 105 kDa and 116 kDa and a faintly detectable band at 155 kDa, corresponding to the different isoforms of FGFR-1 protein previously described (Gonzalez et al. 1995; Stachowiak et al. 1996).
Immunogold electron microscopy At P0 and P30 (n=3 for each stage), dissected cords (without meninges) were perfusion-fixed in 4% paraformaldehyde, 0.25% glutaraldehyde, mounted on the stage of a Vibratome tissue cutter (Oxford Instruments, Cambridge, UK) and thick-sectioned (100 µm) in the longitudinal plane. The sections were transferred to an incubation buffer consisting of 0.5% (w/v) sodium borohydride, 0.1% (w/v) glycine in 60 mM sodium phosphate, pH 7.3, for 20 min. This step was followed by incubation in 0.5% (w/v) bovine serum albumin, 0.1% (w/v) gelatine (BSA-gelatine) for 30 min. Sections were incubated overnight with anti-FGF-2, antiFGFR-1 (1:250 in BSA-gelatine) or with antigen-preabsorbed antibodies according to the protocol described above. After several rinses with BSA-gelatine on a shaker, sections were incubated for
Developmental studies The distribution patterns of FGF-2 and FGFR-1 immunoreactivity were studied in paraffin-sections in longitudinal planes through the lower (Th7-L1) thoracolumbar spinal cord at prenatal stages E14–E21 and postnatal stages P0, P8 and P30 (Figs. 2, 3). In the autonomic spinal cord areas of intermediate layers VI, VII and X, both antibodies marked the IML-cell column, the main preganglionic sympathetic nuclei located in a rostrocaudal position at the white/grey matter boundary (Fig. 2a,
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FGF – 2
FGFR – 1
23 21 18 116 105
kDa
kDa
Fig. 1. Western-blot analysis of rat spinal cord extracts. AntiFGF-2 immunostains three bands at 23 kDa, 21 kDa (only faintly) and 18 kDa, which correspond to different well-known isoforms of FGF-2 (left). Anti-FGFR-1 immunostains two expected bands at 105 kDa and 116 kDa, corresponding to the FGFR-1 protein (right). A faintly stained band is also detectable at 155 kDa (Gonzalez et al. 1995)&ig.c:/f
3a). Immunoreactivity of FGF-2 was detectable in the cytosol of these IML cells, whereas their nuclei remained non-stained (Figs. 2b, 3b). Fluorescently stained cells were determined as being neurons by distinct morphological criteria, i.e. their topographical distribution and somal size, aspects that were clearly different from non-neuronal cells, e.g. astrocytes of the spinal grey matter. Neurons positive for FGF-2 and FGFR-1 can be detected from embryonic stage E14 onwards in spinal cord sections cut serially at longitudinal planes (Fig. 4). The immunostaining patterns for FGF-2 and FGFR-1 probably reflect the changing cellular topography known to occur during development. At stage E14, a densely packed column of immunostained neurons is observed at the white/grey matter boundary (Fig. 4a, e), reminiscent of the known common ventral pool of somatic and autonomic neurons. At subsequent stages, viz. E16–E21 (Figs. 4 b-c, f-g), groups of immunostained neurons are found at the white/grey matter boundary of intermediate spinal cord layers, thus probably reflecting preganglionic neurons of the IML column, which has formed by marked dorsolateral translocation of defined subpopulations originating from the common ventral neuron pool during development of the spinal cord.
IML
Fig. 2a, b. Immunofluorescence micrographs of FGF-receptor (FGFR-1) immunoreactivity in the thoracolumbar spinal cord of rat at prenatal stage E21. a FGFR-1-like expression in the IML
column of the right lateral horn (arrow). b Higher magnification of FGFR-1-positive IML neurons with non-stained nuclei and cytosolic immunoreactivity (arrow). Bar: 100 µm&ig.c:/f
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IML
Fig. 3a, b. Immunofluorescence micrographs of FGF-2 immunoreactivity in the thoracolumbar spinal cord of rat at prenatal stage E21. a Right IML column (arrow). b Cytoplasmic FGF-2 immunoreactivity and non-stained nuclei in the IML neurons. Bar: 100 µm&ig.c:/f
Subcellular localization
Discussion
The subcellular localization of FGF-2 and FGFR-1 in IML neurons was further examined by pre-embedding immunogold electron microscopy. Immunogold-positive neurons were identified by the presence of silver-enhanced gold particles detectable in IML perikarya as black dotted reaction products in sections (1 µm thick) at the light-microscopic level (Fig. 5a, b). At the ultrastructural level (Fig. 5c, d), immunogold-labelled antigenic sites of both FGF-2 and FGFR-1 were detectable in the cytoplasm and in the nucleus, corresponding to the formerly described distribution of various isoforms of FGF2 and FGFR-1 in different cell fractions (Renko et al. 1990; Maher 1996; Stachowiak et al. 1996). Immunogold particles were absent from the relevant structures in ultra-thin sections of FGF-2 and FGFR-1 antigen pre-absorption controls (Fig. 6).
This study shows immunohistochemical and immunoelectron-microscopical evidence for the cellular and subcellular distribution and coexistence of FGF-2 and one of its high-affinity receptors, FGFR-1, at autonomic levels of the developing and perinatal spinal cord of the rat. In order to prevent disparate patterns of FGF and receptor distribution in various cellular types of nervous tissue (e.g. neurons, glia, endothelia), adequate blocking experiments and optimal fixation conditions have been used to achieve reliable data by FGF immunocytochemistry (Hanneken and Baird 1992). In addition, the antibodies used in the experiments have been further characterized by Western-blot analysis. Throughout the pre- and postnatal stages investigated (E14–P30), we have been able to document the coexistence of FGF2 and FGFR-1 expression in the perikarya
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Fig. 4a-h. Developmental patterns of FGFR-1 and FGF-2 proteinlike expression in the IML column in the thoracolumbar spinal cord of the rat at pre- and early postnatal stages E14–P8. a-d, e-h
FGFR-1 and FGF-2 immunoreactivity, respectively, at stages E14 (a, e), E16 (b, f), E18 (c, g) and P8 (d, h) shown in longitudinal planes. Bar: 20 µm&ig.c:/f
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Fig. 5a-d. Subcellular distribution of FGF-2 and FGFR-1 in the IML column at P8. a, b IML neurons positive for FGF-2 and FGFR-1, respectively, can be identified by their black dotted (i.e. silver-enhanced gold particles) cell bodies in 1-µm-thick sections by light microscopy. Methylene blue/Azur-II counterstain. c, d By electron microscopy, immunogold particles (detectable as irregu-
larly shaped, electron-dense particles, because of silver enhancement) are localized preferentially in the cytoplasm of IML neurons positive for FGF-2 and FGFR-1, respectively (dashed line, nucleus/cytoplasm border). Immunogold particles also mark nuclear structures but to a much lower intensity than those in the cytoplasm. N, Nucleus. Bars: 10 µm (a, b), 0.5 µm (c, d)&ig.c:/f
of sympathetic preganglionic neurons of the IML column, the major autonomic cell group in the spinal cord. The presence of FGF-2 cytokine and FGFR-1 in the development of sympathetic preganglionic neurons thus implies important roles for the structural and functional organization and maturation of autonomic neurons in the spinal cord.
FGF-2 and FGFR-1 in spinal autonomic neuron development In the rat, spinal autonomic neurons are generated synchronously with somatic motoneurons in the common ventral motoneuron pool between stages E10 and E12 (Phelps et al. 1991). At E13, the peak day of cell prolif-
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Fig. 6a, b. Electron micrographs of IML neurons from control experiments. Immunogold particles are absent from relevant structures in ultra-thin sections incubated with antigen-preabsorbed FGF-2 (a) and FGFR-1 (b) antibodies followed by the routine im-
munogold-staining protocol (arrow, silver-enhanced immunogold particle with non-specific extracellular localization). N, Nucleus. Bar: 0.5 µm&ig.c:/f
eration (Barber et al. 1991), autonomic neurons separate from this common pool and start to migrate to their final positions at intermediate spinal cord layers to form periodic cell clusters with rostrocaudally and mediolaterally oriented dendritic bundles, which constitute the known architecture of the main sympathetic nuclei of the thoracolumbar embryonic and postnatal spinal cord (Markham et al. 1991; Phelps et al. 1984, 1991, 1993). In the present study, FGF-2 and FGFR-1 immunoreactivity has been detected in groups of spinal neurons from E14 onwards, the day of separation of autonomic neurons from the common ventral neuron pool of both somatic and autonomic neurons. Both markers are present at subsequent stages of autonomic neuron translocation up until their final dorsolateral positions in the intermediate grey at E18 (Phelps et al. 1991). The spatiotemporal relationship between FGF-2 and FGFR-1 expression and the period of cell proliferation and migration at spinal autonomic areas (between E12 to E18) suggest autocrine and/or paracrine actions of this cytokine for sympathetic preganglionic neuron development.
choline acetyltransferase (ChAT), thus determining the cholinergic phenotype of both somatic and autonomic (i.e. sympathetic preganglionic) neurons (Barber et al. 1991; Phelps et al. 1991). In addition to being cholinergic, spinal autonomic neurons co-express nitric oxide synthase (NOS) and its catalytic NADPH-diaphorase activity in the pre- and postnatal rat (Anderson 1992; Blottner and Baumgarten 1992a, Wetts and Vaughn 1994; Wetts et al. 1995). In the IML of adult rat spinal cord, coexistence of FGF-2 and FGFR-1 with ChAT and NADPH-diaphorase suggests mutual actions of the FGF2 cytokine and the novel messenger nitric oxide (NO) in the spinal autonomic system (Stapf et al. 1995). NADPH-diaphorase is either transiently expressed in subsets of ventrolateral somatic motoneurons (from E15 to birth) or first expressed late in development in superficial laminae I-II dorsal horn cells, which reach their final locations at E21. The preganglionic autonomic neurons however continuously express the markers ChAT and NADPH-diaphorase from development to adulthood (Wetts and Vaughn 1994; Stapf et al. 1995; Wetts et al. 1995). This indicates that the cholinergic or nitroxergic phenotype of spinal autonomic neurons is not determined, for example, by trophic factors derived from target organs in development. Likewise, NADPH-diaphorase is maintained in axotomized IML neurons by treatment with FGF in vivo
Functional aspects in autonomic neuron development Between stages E13 and E14, spinal ventral neurons start to express the neurotransmitter-synthetizing enzyme,
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(Blottner and Baumgarten 1992b), suggesting an even closer relationship between FGF and the synthesis of key molecules in autonomic neurons. A functional link between the co-existence of FGF-2 and FGFR-1 and the differentiation or maintenance of the cholinergic and/or nitroxergic/nitrergic phenotype of spinal autonomic neurons in development, however, cannot be inferred from this study and needs further investigation. Is FGF-2 a neurotrophic factor for autonomic neurons in development? In the mature central and peripheral nervous systems, neuronal survival and transmitter phenotype are regulated in vivo amongst other things by target-derived and retrogradely transported neurotrophic factors, nerve growth factor being the prototypic member of the family of neurotrophins (Thoenen 1991). Additionally, a considerable number of “non-neurotrophin” neurotrophic factors (neurotrophic cytokines or neurokines), such as ciliary neurotrophic factor, leukemia inhibitory factor, transforming growth factors beta and FGFs, have been found in the nervous system (Unsicker et al. 1992). Recently, the concept of neurotrophic factors has been reexamined with respect to their multifunctionality in proliferation and differentiation (Korsching 1993), their survival-promoting functions in embryonic neuronal death (Oppenheim 1991) and the switching of trophic-factor requirements in developing neural systems (Davies 1994). Thus, the system-specific functional significance of multiple neurotrophic factors must be taken into account for individual central or peripheral pathways, such as motory, sensory or autonomic routes. In the adult rat, FGFR mRNA can be detected in spinal cord neurons by in situ hybridization (Wanaka et al. 1991). In adult rat spinal cords, FGFR-1 mRNA, but not FGF-2 mRNA transcripts, are detectable in IML-tissue preparations and can be detected in IML neurons by in situ hybridization, suggestive of protein translocation in vivo (D. Blottner, C. Stapf, C. Meisinger, C. Grothe, in press). Likewise, FGFR-1 mRNA transcripts have been detected in embryonic spinal cord extracts by the highly sensitive RNAase protection assay (Grothe and Meisinger 1995). However, the sources of FGF-2 in spinal autonomic neuron development remain undetermined. FGF-2, nonetheless, is a heparin-binding protein (Burgess and Maciag 1989) binding to extracellular matrix heparan sulphate proteoglycans involved in neural signalling processes (Nurcombe et al. 1993). Therefore, one cannot exclude the possibility that FGFR-bearing IML neurons take up and sequester heparin-binding molecules, such as FGF or similar peptides, from extracellular matrix stores during development (Nurcombe et al. 1993; Wanaka et al. 1993; Stachowiak et al. 1994). This may account for the strong FGFR-1 and FGF-2 immunoreactivity found in these neurons from stage E14 onwards. Although mature axotomized IML neurons can be induced to survive and maintain ChAT/NADPH-diaphorase expression by local FGF administration in vivo (Blottner and Baumgarten 1992b), the role of FGF is
less clear for autonomic pathways, such as the IML-adrenal axis, during development. In the rat IML column, expression of the transmitter enzyme ChAT and NOS/NADPH-diaphorase can be detected at E13, several days before growing preganglionic axons reach their peripheral target areas (Barber et al. 1991; Wetts et al. 1995). Synapse formation takes place, as exemplified by preganglionic synapses on E15/16 rat adrenal medullary chromaffin cells (Daikoku et al. 1977). Recently, adult chromaffin cells have been shown to express FGF-2 and FGFR-1 mRNA (Meisinger et al. 1996a, b) and thus trophic mechanisms by, for example, target-derived retrogradely transported FGF-2, probably exist in the mature sympathoadrenal system (D. Blottner, C. Stapf, C. Meisinger, C. Grothe, in press). In this respect, FGF-2 may not be supplied, during development, to autonomic neurons from classical target cells by retrograde axonal transport but rather may act by other known mechanisms of neurotrophic cytokines, such as via autocrine or paracrine pathways (Baird 1994; Murphy et al. 1994). Consequently, neuronal proliferation, migration and differentiation appears to be ruled by intrinsic or extrinsic signal mechanisms in autonomic neuron development before trophic target-dependence is made possible by neurontarget connections. Similar mechanisms in early development have been reported for embryonic sensory neurons (Wright et al. 1992). As shown in this study, FGF-2 and FGFR-1 proteins are continuously expressed in spinal autonomic neurons during development. Therefore, a system-specific multiplicity of actions of FGF-2 must influence developing neural pathways; this is currently under investigation. &p.2:Acknowledgements. The technical expertise of Ms. R. Gießler and photographic work of Ms. U. Sauerbier are appreciated.
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