1 MRC Centre for Developmental Neurobiology, New Hunts House, Guys Hospital, Kings College London, London Bridge, London SE1 1UL, UK
2 The 2nd Research Deapartment, Central Technology Laboratory, Asahi Kasei Corporation, 2-1 Samejima, Fuji, Shizuoka, Japan 416-8501
*Authors for correspondence (e-mail: kalpana.patel{at}kcl.ac.uk; adrian.pini{at}kcl.ac.uk)
Accepted September 19, 2001
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SUMMARY |
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Key words: Chemorepulsion, Axon guidance, Slit, Lateral olfactory tract, Spinal motor axon, Rat
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INTRODUCTION |
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In vertebrates, three Slit genes (Itoh et al., 1998; W. Yuan et al., 1999) and three Robo genes (Brose et al., 1999; Li et al., 1999; Sundaresan et al., 1998; S. Yuan et al., 1999) have been identified. In vitro, Slit1 and Slit2 can cause chemorepulsion of olfactory tract axons (Ba-Charvet et al., 1999; Li et al., 1999; W. Yuan et al., 1999) while Slit2 causes chemorepulsion of spinal motor (Brose et al., 1999), hippocampal (Ba-Charvet et al., 1999) and retinal ganglion cell axons (Erskine et al., 2000; Niclou et al., 2000; Ringstedt et al., 2000). Additionally, Slit1 and Slit2 repel migrating neuronal precursors in vitro (Hu, 1999; Wu et al., 1999; Zhu et al., 1999) and Slit2 can promote the formation of axon collaterals (Wang et al., 1999).
Since the septum expresses Slit1 and Slit2 (Ba-Charvet et al., 1999; Brose et al., 1999; Li et al., 1999; W. Yuan et al., 1999) which cause chemorepulsion of olfactory tract axons (Ba-Charvet et al., 1999; Li et al., 1999; W. Yuan et al., 1999) and the floor plate expresses Slit2 (Brose et al., 1999; W. Yuan et al., 1999) which causes chemorepulsion of spinal motor neurons (Brose et al., 1999), it has been proposed that these Slits may be developmental guidance cues. However, there are two observations that are at odds with the hypothesis that Slit1 and Slit2 mediate both axon guidance and neuronal migration. First, Hu and Rutishauser (Hu and Rutishauser, 1996) demonstrated that the activity secreted by the neonatal caudal septum for migrating olfactory interneurone-precursors must be different from the embryonic septal activity that causes chemorepulsion of olfactory tract axons because the caudal septum does not cause axonal chemorepulsion. Secondly, expression of Slits within the septum (Ba-Charvet et al., 1999; Li et al., 1999; Wu et al., 1999) far outlasts the limited period, E14.5-E17, during which the septum secretes chemorepulsive activity for olfactory tract axons (Pini, 1993; Hu and Rutishauser, 1996).
We have used a soluble receptor for Slit proteins in an attempt to block the activities secreted from the septum and floor plate that cause chemorepulsion of olfactory tract and spinal motor axons respectively. We report here that chemorepulsion of both olfactory tract and spinal motor axons by the septum and floor plate are unaffected by soluble Robo/Fc constructs and conclude that other signalling mechanisms are more likely to predominate during development.
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MATERIALS AND METHODS |
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Cloning of recombinant Slits and Robo1
The complete open reading frames for human Slit1, Slit2 and Slit3 were amplified by PCR, and subcloned into pcDNA3.1/his-myc vector to allow expression of C-terminus epitope-tagged proteins. Similarly, the extracellular domain of human Robo1 and Robo2 were amplified by PCR and subcloned into the pIGplus vector to allow expression of soluble Robo/Fc chimeras. The sequence and orientation of the constructs were confirmed by DNA sequencing.
Cells
A CHO cell line, expressing polyoma large T antigen (CHOP), was obtained from Dr J. Dennis (Ontario, Canada) and grown in Dulbeccos modification of Eagles medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 20 mg/ml L-proline, 100 U/ml penicillin and 100 µg/ml streptomycin. 9E10 hybridoma cells (obtained from the American Type Culture Collection, Rockville, MD), secreting an antibody to the c-myc epitope, were grown in RPMI-1640 supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin and 10% fetal calf serum. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.
Lipofection
CHOP cells were transfected with plasmid DNA using Lipofectamine Plus reagent (Life Technologies, Scotland) according to the manufacturers instructions. To make Robo/Fc-conditioned medium, the lipofection medium was replaced after 3 hours with complete DMEM and then allowed to become conditioned for 5 days. Medium was harvested and clarified before use in explant cultures. To make aggregates, cells were re-suspended in 1.5% low-melting agarose at 3x107 cells/ml and 15 µl droplets of the cell suspension were allowed to set before being trimmed into blocks of around 400 µm2 for use in explant co-cultures. For each experiment, expression of Slits and Robo/Fc proteins were confirmed by western blotting using monoclonal antibody 9E10 and anti-human immunoglobulin (Fc-specific) antibody respectively.
Explant cultures
The olfactory bulbs and septa from E15 embryos were dissected into pieces of around 200-400 µm2 using fine tungsten needles. Olfactory bulb explants were co-cultured with either E15 septal explants or CHOP cell aggregates expressing Slit1, Slit2 or Slit3 in rat tail collagen gels (Lumsden and Davies, 1983). Cultures were incubated at 37°C in complete DMEM in a humidified atmosphere containing 5% CO2. Embryonic spinal motor neurons were grown from E12 basal plate explants that were co-cultured with E12 floor plate explants as described previously (Guthrie and Pini, 1995). After 24-48 hours, the cultures were examined by phase contrast microscopy. For quantitative analysis, axons of the proximal and distal segments were counted to determine whether outgrowth was radial or asymmetrical. For qualitative analysis, scoring was always carried out blind by two independent observers. In order to test the effects of soluble Robo, either Robo1/Fc, Robo2/Fc or mock-conditioned medium was added to the co-cultures.
Immunoprecipitation
48 hours after lipofection, conditioned medium was harvested and clarified by centrifugation. Immunoprecipitation of Slits was carried out at 4°C for 1 hour by incubating 1 ml of conditioned medium with monoclonal antibody 9E10. The immune complexes were precipitated with Protein-G Sepharose (Pharmacia) and then separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. The membranes were then probed with monoclonal antibody 9E10 and anti-mouse immunoglobulin-horseradish peroxidase (HRP) conjugate using diaminobenzidine (DAB)/nickel as substrate (Vector Laboratories).
Similarly, Robo1/Fc and Robo2/Fc were detected in the medium using Protein-G Sepharose for immunoprecipitation and following separation by SDS-PAGE and transfer, the blots were probed with an anti-human immunoglobulin (Ig)-biotin conjugate, streptavidin/HRP and DAB/nickel substrate.
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RESULTS |
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Soluble Robo/Fc abolishes chemorepulsion of olfactory tract axons by Slit2 but not by the septum
Slit2 has been shown to cause growth-cone collapse and chemorepulsion of olfactory bulb axons in vitro and has recently been proposed as a candidate for the septal activity that causes chemorepulsion of olfactory tract axons (Ba-Charvet et al., 1999; Li et al., 1999). We have tested this hypothesis as follows. First, we demonstrated that soluble Robo1/Fc was able to block chemorepulsion of olfactory tract axons by Slit2-expressing CHOP cell aggregates. In the absence of soluble Robo1/Fc, Slit2-expressing aggregates caused robust chemorepulsion of olfactory tract axons in 86% of cases (44 of 51 explants). The mean number of axons emerging from the proximal segment was 9.60±1.68 (mean ± standard error of the mean, s.e.m.) and from the distal half was 31.70±3.35 (Table 1; Table 2; Fig. 2A). However, in the presence of soluble Robo1/Fc, chemorepulsion of these axons occurred in only 13% of cases (6 out of 48 explants; P<0.001, 2 test). The remainder showed radial outgrowth (Fig. 2B) with 30.20±6.56 axons emerging from the proximal half and 33.80±2.62 axons emerging from the distal half of the explant (Table 2; P<<0.01,
2 test). Thus, our soluble Robo1/Fc chimera both binds Slit2 and neutralises its effect in co-culture.
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Since Robo2 is expressed in olfactory bulb (Ba-Charvet et al., 1999) and Slits bind Robo2 with higher affinity (Brose et al., 1999), we carried out co-culture experiments to determine whether chemorepulsion of olfactory tract axons by the septum could be inhibited by the presence of soluble Robo2/Fc. We found that Robo2/Fc could inhibit chemorepulsion of olfactory tract axons in co-culture with Slit2-expressing CHOP cells (Fig. 2C) resulting in 21.25±2.32 and 28.75±3.50 axons emerging from the proximal and distal halves of the explant (Table 2; 0<0.05, 2 test). In contrast chemorepulsion of olfactory tract axons by the septum was unaffected by the presence of Robo2/Fc (Fig. 2F) since 6.20±1.69 and 32.60±1.42 axons emerged from the proximal and distal halves of the explant (Table 2; P>0.5,
2 test).
We ruled out the possibility that the addition of Robo/Fc could lead to an increase in axon outgrowth by culturing olfactory bulb explants in the absence and presence of Robo/Fc. We found that addition of either Robo1/Fc or Robo2/Fc did not affect axonal outgrowth from olfactory bulb explants (Fig. 2G-I).
Since we have demonstrated that the Robo1/Fc and Robo2/Fc constructs block chemorepulsion of olfactory tract axons mediated by Slit2 but not by the septum, it is unlikely that this protein is the major determinant of chemorepulsion mediated by the septum.
Slit1 and Slit3 cause chemorepulsion of olfactory tract axons which is abolished in the presence of soluble Robo/Fc
However, it is possible that Robo1/Fc and Robo2/Fc do not bind Slit1, which is known to cause chemorepulsion of olfactory tract axons (W. Yuan et al., 1999) (although data not shown) and whose mRNA is also expressed in the septum (Ba-Charvet et al., 1999). We tested this possibility by setting up co-cultures of Slit1-expressing cell aggregates and olfactory bulb explants in the presence of soluble Robo1/Fc or Robo2/Fc. Slit1 caused chemorepulsion of olfactory tract axons in 90% of cases (22 out of 24 explants; Fig. 3A) with 10.0±1.73 and 28.00±3.98 axons emerging from the proximal and distal halves (Table 1; Table 2) whereas in the presence of Robo1/Fc, chemorepulsion occurred in only 8% of cases (1 out of 13 explants) leading to radial axon-outgrowth (Fig. 3B) with 23.75±2.00 and 30.25±2.98 axons emerging from the proximal and distal halves (Table 1; Table 2; P<0.05, 2 test). Similar studies showed that Slit1-mediated chemorepulsion of olfactory tract axons could be inhibited by Robo2/Fc (data not shown).
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Soluble Robo1/Fc does not abolish chemorepulsion of embryonic spinal motor neurons mediated by the floor plate
The floor plate mediates chemorepulsion of spinal motor neurons in vitro (Guthrie and Pini, 1995) and at E12, all three Slits are expressed by the floor plate (Brose et al., 1999; W. Yuan et al., 1999). In vitro, Slit2 causes chemorepulsion of embryonic spinal motor neurons (Brose et al., 1999) raising the possibility that Slit2 or another member of Slit family may contribute to the chemorepulsive activity of the floor plate. We tested this hypothesis by conducting co-culture experiments with E12 floor plate and basal plate explants in the presence and absence of soluble Robo1/Fc. Again, chemorepulsion of spinal motor axons by the floor plate was demonstrated in 87% of cases (13 out of 15 explants; Fig. 4A) which was not blocked by soluble Robo1/Fc in 90% of cases (9 out of 10 explants; Fig. 4B). Data are summarised in Table 1.
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DISCUSSION |
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This suggests that either endogenous Slits are not the key mediators or are minor components of the septal chemorepulsive activity. It is, however, possible that Slits may mediate effects on very early olfactory tract axons since Slit2 is expressed when the first mitral and tufted cells are born at around E14 in the rat (Ba-Charvet et al., 1999; Bayer, 1983), but this remains to be determined. In any event, between E14.5 and E17, when the majority of olfactory tract axons are developing, chemorepulsion by the septum is not abolished in vitro by blocking the functioning of Slits. Hirata et al. (Hirata et al., 2001) have reported that RoboN (the hemagglutinin-tagged extracellular domain of Robo) did not affect formation of the lateral olfactory tract and that an apparently normal pathway developed in the absence of the septum in organotypic cultures. However, in the absence of axon counts it is difficult to know whether the tract that developed in the absence of the septum contained equivalent numbers of axons. Those axons deriving from the lateral aspects of the olfactory bulb might be expected to arrive in the tract in the absence of chemorepulsion mediated by the septum. Furthermore, these experiments do not address the possibility that lateral olfactory tract axons never innervate the septum because they are actively prevented from doing so by chemorepulsion.
The expression patterns of robo receptors on olfactory tract axons are as yet unresolved. In some studies, robo1 but not robo2 was found in the olfactory bulb whereas in other studies the converse appeared to be the case (Ba-Charvet et al., 1999; Li et al., 1999). We have, therefore, tested the effects of soluble Robo2/Fc on chemorepulsion of olfactory tract axons. Again, as with Robo1/Fc, we found that chemorepulsion of olfactory tract axons by the septum was unaffected by the presence of either Robo2/Fc or a combination of the two Robos (n=4; data not shown).
We conclude that while the Slits are very effective chemorepulsive molecules for olfactory tract and spinal motor axons in vitro (Ba-Charvet et al., 1999; Brose et al., 1999; Li et al., 1999; W. Yuan et al., 1999), they may not be responsible for the chemorepellent activities described elsewhere (Pini, 1993; Guthrie and Pini, 1995; Hu and Rutishauser, 1996). Other observations are consistent with our conclusions on the contribution of Slits in chemorepulsion of olfactory tract axons. The most relevant are those of Hu and Rutishauser (Hu and Rutishauser, 1996) who demonstrated the presence of a repellent migratory factor, for subventricular precursor cells, emanating from the caudal septum. In contrast to the chemorepulsive activity that is present during E14.5-E17 (Pini, 1993; Hu and Rutishauser, 1996), the septal-derived migratory factor is present throughout embryogenesis and in the postnatal period up to P7 (Hu and Rutishauser, 1996). Slit1 and Slit2 have recently been proposed as candidate septal-derived migratory factors (Hu, 1999; Wu et al., 1999) because of their ability to cause asymmetric migration of neuronal precursors from subventricular zone explants in vitro. Crucially, soluble Robo neutralises the effects of endogenous Slit in whole-mount telencephalon studies consistent with its function as a migratory factor for olfactory bulb interneurone precursors (Wu et al., 1999).
Further evidence that Slits may not be the major chemorepellents for olfactory tract axons comes from expression data. Both Slit1 and Slit2 are expressed at E15 and at E18 in the septum (Ba-Charvet et al., 1999), yet the septal-derived activity declines from E14.5 and is absent at E18 (Pini, 1993). Secondly, the expression of Slit1 (Ba-Charvet et al., 1999; W. Yuan et al., 1999) in neocortex is inconsistent with our observation that E14.5 but not E18/P0 neocortex causes chemorepulsion of olfactory tract axons (Coutinho, 1999) although Slit1 is expressed at both these developmental stages (W. Yuan et al., 1999). This suggests that although many classes of axons are susceptible to chemorepulsion by Slits in vitro, this is not necessarily indicative of a developmental function.
Our observations also demonstrate that chemorepulsion of spinal motor axons by the floor plate is not blocked by the presence of soluble Robo1/Fc and thus we also suggest that Slits may not be major determinants for axon guidance of spinal motor neurons. However, the contribution of Slits as migratory factors secreted from the septum and acting on olfactory bulb interneurone precursors is consistent with all of the available data.
We do not dogmatically exclude a contribution of Slits, but our results suggest that Slit/Robo interactions are not the dominant factor in chemorepulsion of olfactory tract and spinal motor axons. It is possible that having been bound by the soluble Robo/Fc, Slits could still bind to some, as yet, unidentified receptor but this remains speculation. In addition, redundancy between different Slits is an unlikely explanation since the Robo/Fc constructs we use actually bind all three Slits. Thus, one explanation for our results is that there is redundancy between Slits and other as yet unidentified guidance molecules. They are unlikely to be secreted semaphorins because these either do not cause chemorepulsion of olfactory tract axons in vitro or, if they do, they are not expressed in the septum (DeCastro et al., 1999; Hu and Rutishauser, 1996; Li et al., 1999). It is also unlikely that netrins are strong candidates since neither netrin1- nor netrin2-expressing cells cause chemorepulsion of olfactory tract axons (Hu and Rutishauser, 1996; Li et al., 1999) and olfactory tract development is normal in netrin1-deficient animals (Serafini et al., 1996). Our results provide data consistent with the existence of an unidentified guidance molecule whose activity is expected to be dominant over that of Slit/Robo interactions.
The generation of mice deficient in Slit1 or Slit2 should help to resolve the contribution of Slit in the guidance of olfactory tract and spinal motor axons.
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ACKNOWLEDGMENTS |
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