1 Departments of Pathology and Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, Box 8118, St Louis, MO 63110, USA
2 Department of Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, Box 8116, St Louis, MO 63110, USA
3 Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8113, St Louis, MO 63110, USA
*Author for correspondence (e-mail: jeff{at}pathbox.wustl.edu)
Accepted July 26, 2001
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SUMMARY |
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Key words: Ret, Knockout mouse, Sympathetic neuron, Axon growth, Axon guidance, Migration
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INTRODUCTION |
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Neurotrophic factors play essential roles in the proper development of a wide variety of neuronal populations. Neurotrophins are a family of neurotrophic factors that are required for the survival of sensory and sympathetic neurons during development (Snider and Wright, 1996). Accumulating evidence has also revealed the importance of these factors in promoting nerve growth. For example, injection of nerve growth factor (NGF) to the brain leads to the abnormal increase of sympathetic axon ingrowth in this region (Menesini Chen et al., 1978). In line with these classical experiments, tissue engineered to express high levels of NGF or NT3 harbor increased sympathetic and sensory fibers (Albers et al., 1996; Albers et al., 1994; Hassankhani et al., 1995). In slice cultures, sensory neurons project axons towards neurotrophin-impregnated beads that are placed in ectopic positions (Tucker et al., 2001). Furthermore, sensory neurons in mice with disruption of Ngf or TrkA (Ntrk1 Mouse Genome Informatics) fail to innervate the target properly, even when cell death of these neurons is prevented by deficiency of the pro-apoptotic gene, Bax (Patel et al., 2000). Despite these observations, however, neurotrophins do not appear to be physiologically required for initial sympathetic axonal growth. First, recent genetic evidence has revealed both NGF and NT3 influence sympathetic neurons only late in development (Francis et al., 1999), presumably exerting their effects via TRKA receptors (Wyatt et al., 1997). Second, expression of TrkA is barely detectable in sympathetic neuron precursors early in development (Wyatt et al., 1997). Finally, previous genetic evidence has demonstrated that initial projections of sympathetic axons occur normally in TrkA-deficient (TrkA/) embryos (Fagan et al., 1996).
The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), a newly identified family of neurotrophic factors, includes GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN) (Baloh et al., 1998; Kotzbauer et al., 1996; Lin et al., 1993; Milbrandt et al., 1998). GFLs signal through a receptor complex composed of a signaling subunit, the RET tyrosine kinase, and a binding subunit, the GFR (GDNF family receptor
) family which are glycosylphosphatidylinositol (GPI) linked cell surface proteins. To date, there are four members of GFR
family, GFR
1, GFR
2, GFR
3 and GFR
4, which serve as preferential receptors for GDNF, NRTN, ARTN and PSPN, respectively (Baloh et al., 1997; Buj-Bello et al., 1997; Jing et al., 1996; Jing et al., 1997; Klein et al., 1997; Sanicola et al., 1997; Suvanto et al., 1997; Treanor et al., 1996).
Although recent genetic experiments have established GFLs as neurotrophic factors essential for proper development of enteric, sympathetic, parasympathetic, sensory and motoneurons (Cacalano et al., 1998; Enomoto et al., 1998; Enomoto et al., 2000; Heuckeroth et al., 1999; Laurikainen et al., 2000; Marcos and Pachnis, 1996; Moore et al., 1996; Pichel et al., 1996; Rossi et al., 1999; Rossi et al., 2000; Sanchez et al., 1996; Schuchardt et al., 1994; Airaksinen et al., 1999; Baloh et al., 2000; Rosenthal, 1999), the biological roles of the GFLs and their receptors in sympathetic ganglion development have been enigmatic. In mice, it has been reported that RET deficiency leads to elimination of all SCG neurons by birth (Durbec et al., 1996). In contrast to the profound deficit in the SCG of Ret/ mice, neonatal mice lacking GDNF or GFR3, a cognate receptor for ARTN, only harbor a decrease of 30% of SCG neurons (Moore et al., 1996; Nishino et al., 1999). Moreover, no sympathetic deficits are found in Nrtn/ and GFR
2/ mice (Heuckeroth et al., 1999; Rossi et al., 1999). It is also unclear why RET deficiency affects only the SCG, given the high level of Ret expression in sympathetic precursors throughout the entire primitive sympathetic chain (Durbec et al., 1996). To address these questions, we have re-examined the development of sympathetic ganglia in Ret/ mice. Our analysis has revealed that RET deficiency affects proper migration as well as initiation/promotion of axonal growth of neurons and their precursors throughout the entire sympathetic ganglia. During periods of sympathetic precursor migration and axonal projection, ARTN, but not other GFLs, is expressed in various blood vessels. ARTN induces profuse neurite outgrowth from sympathetic ganglion explants and has an ability to direct growing axons. Our analysis identifies RET and ARTN as the receptor and ligand essential for an early phase of sympathetic neuron differentiation that occurs in close association with blood vessels.
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MATERIALS AND METHODS |
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Histological analysis
Immunohistochemical detection for RET, Phox2a, Phox2b, caspase 3, TH, TUNEL and BrdU and Phox2b double labeling, as well as in situ hybridization are described elsewhere (Enomoto et al., 1998; Enomoto et al., 2000). The antibodies for GFP (Abcom), class III ß tubulin (TUJ1: BAbCO) and neurofilament (2H3: Developmental Studies Hybridoma Bank) were used at 1:200, 1:2000 and 1: 200 dilution, respectively. GFP signals of TGM could also be detected directly under fluorescent microscopy. For estimation of ganglion volume, ganglion area of the SCG was measured in every fifth section of consecutive parasagittal sections using Image Analysis Software. The volume was calculated by multiplying the total ganglion area by the distance between two sections (30 µm). Neuronal cell death was quantified as number of cells with pyknotic nuclei per 1000 counted neurons. At least 2000 neurons were counted on randomly selected parasagittal sections of every ganglion for each genotype (n=3).
For whole-mount TOH immunohistochemistry, 4% paraformaldehyde-fixed embryos or newborn mouse tissues were dehydrated by methanol series (50-80%) and incubated overnight in 20% dimethylsulfoxide (DMSO)/80% methanol solution containing 3% H2O2 to quench endogenous peroxidase activity. Tissues were then re-hydrated, blocked overnight in blocking solution (4% BSA/1% Triton X-100 in PBS) and incubated for 48-72 hours at 4°C with sheep anti-TH antibodies (1:200 in blocking solution). The signal was detected using diaminobenzidine after successive treatment of the tissues with horseradish peroxidase (HRP)-conjugated anti-sheep Ig antibodies. Tissues were re-fixed, dehydrated by methanol series and cleared with benzyl benzoate/benzyl alcohol (2:1 mixture) to allow visualization of staining inside the tissue.
For GFP and activated caspase 3 double staining, anti-GFP and anti-activated caspase 3 antibodies (Cell Signaling) were used at 1: 12800 and 1: 100 dilutions, respectively. A more detailed protocol for the double labeling using antibodies raised in the same species is described elsewhere (Shindler and Roth, 1996).
Explant culture of mouse sympathetic ganglion
Sympathetic ganglia (SCG and STG) were dissected out from E13.5 mouse embryos. Ganglia were cut into pieces of appropriate size and cultured in 1 mg/ml type I collagen (rat) in DMEM/F12 media containing 5% horse serum. For neurite outgrowth assay, GDNF, NRTN, ARTN or NT-3 was applied at concentrations ranging from 5 to 100 ng/ml. For chemotaxis assay, a NRTN- or ARTN- impregnated heparin bead was placed at a 300-400 µm distance from the explant in a collagen gel. For both assays, explants were cultured for 40 hours in 5% CO2 at 37°C. The SCG and STG were examined separately, and almost identical results were obtained from both cultures. Representative results from the STG cultures of three independent experiments are described in the text. To quantify axonal growth, axons were visualized by immunostaining with anti-Class III ß tubulin antibodies and axon-covering area was measured using NIH Image software.
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RESULTS |
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Recent genetic evidence has shown that induction of Ret gene expression is dependent on Phox2b (Pattyn et al., 1999), which begins to be expressed immediately after the formation of the primary sympathetic chain (Ernsberger et al., 2000). Phox2b is also required for induction and maintenance of Phox2a and Mash1 expression in sympathetic cells, respectively (Pattyn et al., 1999). These transcription factors act in concert to regulate expression of lineage-specific and pan-neuronal markers including TH, intermediate neurofilament (NF160), SCG10 and class III ß tubulin (Guillemot et al., 1993; Pattyn et al., 1999; Sommer et al., 1995). Therefore, we examined expression of these transcription factors and neuronal markers in Ret/ sympathetic cells during embryonic days 10.5-11.5 (E10.5-11.5). No difference was observed in the expression of these molecules between Ret/ and wild-type embryos (data not shown). Therefore, RET does not appear to be essential for the commitment of sympathetic lineage or determination of neuronal cell fate.
However, despite the intact expression of these differentiation-associated molecules, Ret/ sympathetic neuronal precursors display severe deficits in axonal projection. For example, at E10.5, when some sympathetic precursors in wild-type embryos begin to extend long processes as revealed by anti-Class III ß tubulin antibodies (Fig. 4A), very few cells were observed to extend long processes in Ret/ embryos (Fig. 4D; n=3). Impaired axonal projection of sympathetic cells in Ret/ mice became more obvious in the SCG, STG and prevertebral ganglia during the period from E12.5 to E13.5 (Fig. 4B-F, data not shown, n=3). The SCG and STG in Ret/ embryos also failed to undergo morphogenetic alteration to form their characteristic ganglion shape (Fig. 4E,F).
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Importantly, although the cell number in the SCG primordia is decreased and the axonal projection of the STG and SCG is severely impaired, no increased cell death was detected in both ganglia of Ret/ embryos from E10.5-E13.5, as judged by nuclear morphology or activated caspase 3 immunohistochemistry (not shown). Furthermore, proliferating population in the sympathetic precursors at E11.5 identified by BrdU and Phox2b double-labeling was comparable (approx. 40% of the total precursors were doubly labeled) between Ret/ embryos and their wild-type littermates. While BrdU labeling may not detect subtle differences in cell proliferation, RET deficiency does not appear to have a major impact on proliferation. Thus, we conclude that the reduced cell number in the SCG primordia of Ret/ embryos is primarily caused by a failure in rostral migration of the sympathetic precursors, rather than increased cell death or impaired proliferation of these cells.
Finally, we observed that RET deficiency affects the proper pathfinding of growing sympathetic axons. At E15.5, profuse nerve fibers from the SCG project rostrally along the internal carotid artery to form the internal carotid nerve in wild-type embryos (Fig. 4M, arrow). Although these nerve fibers were recognizable in most Ret/ embryos by E15.5, these nerve bundles were thin. Moreover, in some Ret/ embryos (two out of four examined), these nerve bundles projected caudally, instead of rostrally (Fig. 4N, arrows), indicating that axon projection is mis-routed. Formation of the sympathetic trunk from the STG was not yet observed at this period in Ret/ embryos. Collectively, in sympathetic neuron development, RET is required primarily for migration as well as axonal growth and guidance of sympathetic neuronal precursors.
RET does not directly support the survival of sympathetic neurons
Although the analysis of sympathetic chain at early embryonic stages has revealed the primary requirement of RET for differentiation of sympathetic precursors, not for their survival, it remained unclear why all sympathetic ganglion of Ret/ embryos exhibit dramatic reduction in size at birth. To answer this question, we examined SCG and STG neurons in later development. This analysis revealed retarded differentiation of neurons in these ganglia of Ret/ embryos. For example, many sympathetic cells contained little cytoplasm and displayed neuroblast-like morphology at E16.5 (STG shown in Fig. 5A,B). Indeed, Phox2b and BrdU staining showed the presence of proliferating sympathetic neuronal precursors in the SCG and STG of Ret/ embryos, which was not detected in their wild-type or heterozygous counterparts (Fig. 5C,D). In addition, more apoptotic figures were observed in the STG, but not the SCG, of Ret/ embryos at E16.5 (Fig. 5G), an observation that is confirmed by increased numbers of cells that stain for activated caspase 3 (Fig. 5E,F). Moreover, a significant increase in neuronal cell death in Ret/ embryos was observed in both the SCG and STG at P0 (Fig. 5G). Thus, in Ret/ animals, sympathetic neuronal differentiation is severely delayed, and neuronal cell death increases during E16.5-P0.
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Artemin is a vascular-derived factor that promotes and attracts sympathetic axonal growth
While the analysis above has underscored the primary importance of RET signaling for inducing differentiation of early sympathetic precursors, it remained unclear which factors activate RET during the period of early sympathetic differentiation. Previous expression studies have shown that neither GDNF nor NRTN is expressed at the site of sympathetic ganglion formation (Golden et al., 1999). Examination of E11.5 GDNF/ embryos using whole-mount TH immunohistochemistry revealed no discernible deficits in the sympathetic chain in these animals (data not shown). Furthermore the SCG was found in the normal location in newborn mice lacking both GDNF and NRTN (data not shown). Thus, it is unlikely that GDNF or NRTN is responsible for in vivo activation of RET in early differentiating sympathetic precursors. As migration of the SCG is affected by GFR3 deficiency (Nishino et al., 1999), and GFR
3 is expressed in the primary sympathetic chain (data not shown), we examined the expression pattern of ARTN, a specific ligand for GFR
3 in rat embryos, by in situ hybridization.
The analysis revealed a strong correlation between the site of Artn mRNA expression and sympathetic neuron development. Artn mRNA was expressed at high levels in the wall of the dorsal aorta and its dorsal proximity at E12.5 (Fig. 6A, arrow), around the period of primitive sympathetic chain formation in the rat. At E14.5, high levels of Artn mRNA were detected in many blood vessels, including the dorsal aorta, celiac, superior mesenteric (Fig. 6B), inferior mesenteric and vertebral arteries (Fig. 6C, data not shown). At E16.5, whereas expression of Artn in the dorsal aorta became almost undetectable (Fig. 6D, arrow), Artn expression persisted in the small peripheral branches of the celiac and mesenteric arteries (Fig. 6D; arrowheads, 6E,F; arrows). Modest expression of Artn was also detected in the SCG and the internal carotid artery (data not shown). Artn expression was also observed in the esophagus and the stomach, but not in the digestive tract distal to the stomach (data not shown). Importantly, sympathetic axons grow towards or along these blood vessels during these periods and deficits in sympathetic innervation in Ret/ mice are found at sites expressing Artn.
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DISCUSSION |
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RET signaling is required by all sympathetic ganglia
It has previously been reported that RET deficiency affects only the SCG, but not other sympathetic ganglia, eliminating all SCG neuronal precursors by E12 in mice (Durbec et al., 1996). As the enteric neurons are completely depleted in the gut distal to the stomach in Ret/ mice, it has been proposed that the SCG and those enteric neurons derive from a commonly shared precursor pool. Our present study has shown that RET deficiency does not eliminate all SCG neurons, but instead leads to formation of the SCG in an aberrant, more caudal location. We speculate that the aberrantly located SCG was misinterpreted as being absent in the previous report. The presence of the SCG in Ret/ mice indicates that precursors for the SCG and those enteric neurons are distinct at least in GFL dependency. Furthermore, our results have shown that RET deficiency influences precursor migration and their axonal projections quite uniformly throughout the entire sympathetic nervous system. Thus, it is reasonable to conclude that SCG precursors are more similar to precursors of the more caudal sympathetic ganglia, rather than to precursors of enteric neurons. This conclusion is consistent with the previous lineage studies showing that enteric and sympathetic precursors are derived from distinct levels of the neural crest (Le Douarin and Teillet, 1973; Le Douarin, 1986).
Biological role of RET signaling bridges neural commitment and neuronal survival in sympathetic neuron development
Present analysis demonstrates that RET signaling is primarily required for axonal growth and migration of sympathetic precursors, but not for their survival or proliferation. Despite the profound migration and innervation deficits in Ret/ embryos, the primary sympathetic chain formation and expression of adrenergic as well as pan-neuronal phenotypes occur essentially normally in these embryos, indicating that RET signaling is not essential for the generation or determination of neuronal identity of sympathetic neurons. This raises the question as to whether RET is regulating migration and axon projection through activating expression of other molecules yet to be identified, or, alternatively, whether RET signaling is directly linked to the cell locomotive machinery (see below).
It is noteworthy that, in Ret/ embryos, the innervation and migration deficits of sympathetic precursors precede the abnormal cell death that occurs both in the SCG and STG during the perinatal period. Paradoxically, this abnormal cell death only becomes evident after Ret expression is confined to a small subpopulation of neurons in these ganglia. Indeed our analysis demonstrated that the Ret-expressing population is not selectively dying in the absence of RET. Because of the severe depletion of sympathetic fibers, these sympathetic neurons are likely to be dying because of deprivation of the target-derived survival-promoting factors such as NGF. Collectively, our present study, together with previous in vivo evidence, provides new insight into sympathetic neuron development. Generation of the primitive chain, commitment to sympathetic lineage and expression of catecholaminergic traits requires Neuregulin signaling, BMPs, Mash1 and Phox2 proteins (Britsch et al., 1998; Guillemot et al., 1993; Pattyn et al., 1999; Schneider et al., 1999). Phox2b induces RET expression in sympathetic neuronal precursors (Pattyn et al., 1999). The activation of RET in sympathetic neuron precursors allows these cells to initiate axonal growth and/or migrate to the proper location, steps that are crucial for successive maturation and reaching a source of NGF for survival later in development.
RET and ARTN are required for sympathetic axon guidance
The present study also establishes ARTN as a strong candidate responsible for RET activation in early sympathetic development. Expression of Artn is detected in a number of blood vessels including the dorsal aorta, mesenteric, celiac, vertebral and internal carotid arteries during periods of axonal projections by sympathetic neurons. Importantly, sites of Artn expression significantly overlap regions where innervation deficits are found in Ret/ mice. Consistent with the in vivo expression pattern, ARTN has a strong neurite outgrowth-promoting activity. Sympathetic neurons from E13.5 mouse embryos extend neurites in response to ARTN in a dose-dependent fashion, and the response is not suppressed even at high concentration of ARTN. This suggests that growing axons are capable of responding continuously to higher levels of ARTN in vivo. In this respect, temporal changes in the sites of Artn expression are particularly intriguing. Artn mRNA is initially detected in the dorsal aorta. As development proceeds, Artn expression in the dorsal aorta is downregulated, whereas expression in the peripheral branches of the mesenteric arteries persists. This movement of Artn expression towards the periphery would allow sympathetic growth cones to grow in a directed fashion, towards the site of highest ARTN expression.
Although RET deficiency severely affects the initiation of axonal growth in early embryos, axonal outgrowth is not completely halted. In fact, many sympathetic ganglia begin to project fibers late in development. The presence of abnormal trajectories of sympathetic fibers, which are occasionally seen in these late-projecting sympathetic fibers, confirms the critical requirement of RET signaling in axon guidance of sympathetic neurons (Song and Poo, 2001). We propose that ARTN-mediated RET activation provides a crucial guidance signal required for sympathetic axons to travel along blood vessels. The late onset of axonal projection may be potentiated by intrinsic mechanisms, or, alternatively, may reflect compensatory effects by other neurotrophic factors. One such candidate is NT3, which is expressed in blood vessels to support perinatal survival of sympathetic neurons (Francis et al., 1999). Thus, it would be interesting to examine whether RET and NT3 double mutation results in a more dramatic reduction and a greater abnormality in the trajectory of sympathetic nerves.
Common biological responses are induced by GFL-mediated RET signaling in development of the autonomic nervous system
While recent genetic evidence has established GFL-mediated RET signaling as crucial for proper development of a variety of neuronal populations including enteric, sensory, sympathetic, parasympathetic and motoneurons, the central biological process dependent on RET signaling has been unclear. We have recently conducted detailed analysis on development of parasympathetic neurons in Gdnf/, Nrtn/, GFR1/ or Ret/ animals and found that proper migration of parasympathetic neuronal precursors as well as target innervation of those neurons require RET signaling (Enomoto et al., 2000). Thus, together with the present study, RET activation by different ligands, in different types of cells, and at different developmental periods leads to two closely related biological responses: enhanced cell motility and axon outgrowth. Similar regulation of cell migration and axonal growth by a single molecule has been demonstrated in a number of axon guidance molecules including netrin, slit and Eph receptors (Chisholm and Tessier-Lavigne, 1999).
The mechanism by which RET controls cell migration and axon growth is unclear. One possibility is that RET activation is directly involved in regulating reorganization of cytoskeletal proteins such as actin or neurofilament. Indeed, multiple lines of evidence have shown that activation of RET signaling is capable of enhancing cell motility, which is associated with rapid actin reorganization (Tang et al., 1998; van Weering and Bos, 1997). It is also noteworthy that RET signaling mediated by GFLs requires recruitment of RET to lipid rafts and interaction with Src family kinases (SFKs) for efficient neurite outgrowth in vitro (Encinas et al., 2001; Tansey et al., 2000). SFKs are enriched in neuronal growth cones (Maness et al., 1988) and have been shown to play a crucial role in guidance and fasciculation of olfactory axons in vivo (Morse et al., 1998). Moreover, recent genetic evidence has demonstrated the critical requirement of the Src substrate, p190 GAP, for proper axonal projection of the anterior commissure and formation of the subcortical axons (Brouns et al., 2001), supporting further the importance of Src signaling in axon guidance and growth. Thus, it will be intriguing to determine the relationship between RET-mediated Src-activation and cytoskeletal protein organization. Finally RET signaling appears to be important not only in physiological but also pathological conditions, as GDNF significantly promotes regeneration of spinal nerves after injury (Ramer et al., 2000). Elucidation of mechanisms that underlie neurite outgrowth-promoting activity of RET will be a crucial step toward potential application of GFLs for treatment of neurodegenerative diseases and nerve or spinal cord injuries.
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ACKNOWLEDGMENTS |
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