Sonic hedgehog guides commissural axons along ... - Semantic Scholar

© 2005 Nature Publishing Group http://www.nature.com/natureneuroscience

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Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord Dimitris Bourikas1,4, Vladimir Pekarik3,4, Thomas Baeriswyl1, Åsa Grunditz2, Rejina Sadhu1, Michele Nardó1 & Esther T Stoeckli1 Dorsal commissural axons in the developing spinal cord cross the floor plate, then turn rostrally and grow along the longitudinal axis, close to the floor plate. We used a subtractive hybridization approach to identify guidance cues responsible for the rostral turn in chicken embryos. One of the candidates was the morphogen Sonic hedgehog (Shh). Silencing of the gene SHH (which encodes Shh) by in ovo RNAi during commissural axon navigation demonstrated a repulsive role in post-commissural axon guidance. This effect of Shh was not mediated by Patched (Ptc) and Smoothened (Smo), the receptors that mediate effects of Shh in morphogenesis and commissural axon growth toward the floor plate. Rather, functional in vivo studies showed that the repulsive effect of Shh on postcommissural axons was mediated by Hedgehog interacting protein (Hip).

During the development of neural circuits, growing axons respond to both attractive and repulsive guidance cues to navigate to their target cells, where they establish synaptic contacts. Guidance cues act either over distance to outline the target, or locally at choice points to specify the pathway taken by extending axons1,2. One of the bestunderstood model systems for axonal pathfinding is the navigation of axons from commissural neurons located in the dorsal spinal cord3,4. These axons extend toward the floor plate in response to the chemoattractant netrin-1 (refs. 3–6). Recently, the morphogen Shh has been identified as an additional chemoattractant for commissural axons7. In vivo experiments demonstrated a requirement for the cell adhesion molecules axonin-1, expressed on commissural axons, and NrCAM, expressed on floor-plate cells, for commissural axons to enter the floor plate and cross the midline8,9. F-spondin has been shown to restrict the turning angle of commissural axons into the longitudinal axis at the contralateral floor-plate border without affecting the direction of the turn10. Guidance cues directing postcommissural axons rostrally instead of caudally remained elusive until recently, when the presence of a wnt4 gradient was demonstrated along the longitudinal axis of the spinal cord, attracting postcommissural axons toward higher Wnt4 concentrations at more rostral levels11. In contrast to the candidate-based approach taken to describe the role of Wnt4 (ref. 11), we carried out a screen based on subtractive hybridization to identify guidance cues for postcommissural axons. In the screen, we focused on genes expressed in the floor plate at the time when commissural axons turn into the longitudinal axis. The floor plate is the source of the morphogen Shh that is involved in patterning the spinal cord during early stages of development12–15. At later

stages, the floor plate is the source of trophic and tropic factors, such as the chemoattractant netrin-1 (refs. 5,6). Because axons turn into the longitudinal axis in close contact with the floor-plate surface, a role of floor plate–associated cues in directing commissural axons along the longitudinal axis of the spinal cord seemed very likely. Using in vitro experiments and in ovo RNAi, a loss-of-function approach that allowed us to silence candidate genes in a temporally and spatially controlled manner in vivo16–18, we identified Shh as a guidance cue for postcommissural axons. Notably, the effect of Shh was not mediated by its well-characterized receptors Ptc and Smo but rather by Hip that is expressed transiently by commissural neurons. RESULTS A candidate guidance cue for postcommissural axons We decided to screen for differentially expressed floor-plate genes as candidate guidance cues directing commissural axons along the longitudinal axis of the spinal cord based on two observations. First, commissural axons turn in close contact with the floor-plate border (Fig. 1). Second, commissural axons do not turn into the longitudinal axis in the absence of a floor plate in mouse19,20, chick21, zebrafish22 and frog23. At the lumbosacral level of the spinal cord, in which we carried out all our analyses, dorsal commissural axons have turned into the longitudinal axis at stage 25, and, therefore, concentrations of the putative guidance cue(s) were expected to be the highest. At stage 20, when commissural axons have just started to grow in the dorsal spinal cord and have not yet reached the floor plate, we expected the guidance cue(s) not to be expressed or to be expressed only at low levels. Because of the limited amount of starting material, we used a

1University of Zurich, Institute of Zoology, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. 2Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. 3Current address: Cardiff School of Biosciences, Biomedical Building, Museum Avenue, P.O. Box 911, Cardiff CF10 3US, UK. 4These authors contributed equally to this work. Correspondence should be addressed to E.T.S. ([email protected]).

Published online 30 January 2005; doi:10.1038/nn1396

NATURE NEUROSCIENCE VOLUME 8 | NUMBER 3 | MARCH 2005

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Figure 1 Commissural axons turn into the longitudinal axis of the spinal cord in close contact with the contralateral floor-plate border. (a) Spinal cords were dissected and cut along the roof plate. (b) The trajectory of commissural axons in a control embryo was visualized by application of Fast DiI into the area of the cell body. The floor plate is indicated by dashed lines in b. Bar, 100 µm.

PCR-based approach for subtractive hybridization producing cDNA fragments with a length between several hundred and more than 1,000 base pairs that could be used directly to synthesize digoxigenin (DIG)labeled in situ probes. For further analysis, we selected those clones whose expression was predominantly restricted to the floor plate during the relevant time window, stages 23–26. One clone was identified as containing the gene encoding F-spondin10. For several clones we did not find any matches in the NCBI data bank using the BLAST program, presumably because we had sequence information only from the 3′-untranslated region (UTR). But full-length sequences of our candidate clones were not required for further analysis, as the isolated cDNA fragments could be used not only for the generation in situ probes but directly for functional analyses using in ovo RNAi17. Functional analysis of candidates by in ovo RNAi The injection of dsRNA derived from the cDNA fragments of candidate genes into the central canal of the spinal cord, in combination with in ovo electroporation, resulted in specific downregulation of the targeted genes16–18. Using this assay, we found that one of our candidate genes indeed interfered with the decision of postcommis-

sural axons to turn rostrally (Fig. 2). We did not detect any effects on axon growth and pathfinding toward and into the floor plate area; however, most axons lingered at the exit site from the floor plate. The majority of the growth cones did not have the bias to grow in the rostral direction or even pointed caudally. Relatively few axons extended along the longitudinal axis. Among those, many erroneously turned caudally instead of rostrally. A total of 29 embryos were treated with dsRNA from the identified clone. On average, nine injection sites were analyzed in each spinal cord. An abnormal phenotype (stalling with lack of rostral bias, caudal turn of axons, or both) was seen in 90% of the embryos and at least 78% of the injection sites per embryo. In age-matched control embryos (n = 27), commissural axons did not linger at the floor-plate exit site (average of nine injection sites per spinal cord). Even when commissural axons were analyzed in younger control embryos, axons never showed the lingering morphology seen in experimental embryos that is characterized by enlarged growth cones and/or orientation in any direction other than rostral. Therefore, we concluded that we had identified a guidance cue providing an instructive signal for the growth along the longitudinal axis. In its absence, commissural axons either stalled at the floor-plate exit site or randomly chose in which direction to turn. Shh identified as guidance cue for postcommissural axons To identify the candidate gene that was shown to interfere with the rostral turn of postcommissural axons, we used the cDNA fragment from our screen as a probe to search a cDNA library derived from E14 chicken brain. The resulting cDNA was sequenced and found to encode Shh. The role of Shh as guidance cue for postcommissural axons was confirmed by in ovo RNAi with a second, nonoverlapping fragment

Figure 2 Shh directs postcommissural axons rostrally. (a–d) Images of four chick embryos in which SHH function was abolished by in ovo RNAi. The phenotype reflecting the lack of SHH function was found with two independent fragments of SHH that were used for dsRNA production. One fragment was derived from the 3′-UTR of SHH (a and b) and the other from the fragment encoding the N-terminal part of Shh (c and d). Axons stalled at the contralateral floor-plate border or even turned caudally (arrows in a and c). Axons showed no difference in their growth toward, into, and across the floor plate as compared to controls (compare to Fig. 1b), but they did not turn rostrally along the contralateral floor-plate border. The majority of the axons stalled at the floor-plate exit site (a,b,d). Growth cones clearly lacked the rostral bias that was seen in control embryos analyzed at earlier stages (data not shown) and explored movement in all possible directions (b). In some cases, axons initially turned caudally but corrected their pathway by forming loops (arrowheads in d). Corrections of initial pathway choices were never seen in control embryos, in which axons were already biased in the rostral direction upon floor-plate exit. The floor plate is indicated by dashed lines. Rostral direction is toward the top in all panels. Bar, 100 µm in a,c, 30 µm in b,d.

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ARTICLES Figure 3 Neither Ptc nor Smo are expressed by commissural neurons when axons turn into the longitudinal axis. (a–c) Transverse sections of stage 23 spinal cords shown at low magnification. (d–f) Transverse sections at high magnification. Sections were used for in situ hybridization to demonstrate expression of Axonin-1 (a,d), Smo (b,d) and Ptc (c,f). Commissural axons at the lumbosacral level of the spinal cord cross the floor plate at stage 23. By stage 24 they have reached the contralateral floor-plate border and turn rostrally into the longitudinal axis. During that time commissural neurons in the dorsolateral spinal cord expressed axonin-1 (arrow in a,d) but they did not express Smo (open arrow in b,e) or Ptc (open arrow in c,f). The expression domains of both Smo and Ptc retracted to the ventricular zone. Bar, 200 µm in a–c, 100 µm in d–f.

of dsRNA that was derived from the N-terminal part of the SHH cDNA (Fig. 2c,d). The phenotypes obtained by in ovo RNAi using the two different dsRNA fragments were indistinguishable with respect to both quality and penetrance. As shown before with the dsRNA derived from the 3′-UTR of SHH, we injected embryos at stages 18–19 (n = 10) and analyzed them at stages 25–26. Eight of the ten embryos showed the abnormal phenotype at 92.5% of the injection sites (range, 78–100%; average, ten injection sites per spinal cord). Blocking Shh confirms its role in axon guidance As a different means to induce SHH loss-of-function phenotypes, we used function-blocking antibodies. In contrast to previous in vivo assays

Figure 4 Hip is expressed transiently by commissural neurons during the time when their axons turn into the longitudinal axis. (a–c) Hip expression was probed by in situ hybridization in transverse spinal cord sections at stage 23 (a), stage 24 (b) and stage 26 (c). At stage 23, when commissural axons are crossing the floor plate, Hip was not expressed by commissural neurons (open arrow in a). At this stage, its expression in the spinal cord was restricted to two areas of the ventricular zone. At stage 24, when commissural axons have reached the contralateral border of the floor plate and turned into the longitudinal axis, commissural neurons transiently expressed Hip (arrow in b). At stage 26, when commissural axons have extended along the longitudinal axis for some distance, Hip was downregulated to very low levels (open arrow in c). Bar, 200 µm in a, 300 µm in b,c.


at the protein level8,9,24, we did not only inject purified antibodies but also grafted hybridoma cells (clone 5E1) directly into the central canal of the spinal cord. The interference with SHH function at the protein level resulted in the same phenotype that was observed after silencing SHH by in ovo RNAi (Supplementary Fig. 1 online). Commissural axons were no longer instructed to turn rostrally after midline crossing and therefore stalled at the floor-plate exit site or chose randomly to grow either rostrally or caudally. The phenotype was observed in eight of ten embryos injected with 5E1 antibodies (average of eight injection sites (range, 5–10), 90% showing the indicated phenotype). Sixteen embryos received a graft of 5E1 hybridoma cells. As a control, hybridoma cells producing an antibody against c-Myc (9E10 cells) or against P0, an epitope not expressed in the spinal cord (1E8 cells), were used (n = 5 and 4 embryos, respectively). None of the control embryos showed either stalling or caudal turns at any of the injection sites (9 or 10 injection sites per spinal cord). Shh has a direct effect on postcommissural axons Shh is well known for its effect on spinal cord patterning13,25,26. It acts in a graded manner to establish different populations of neural progenitors, as defined by the expression of homeodomain transcription factors that are either repressed (class I) or induced (class II) by Shh. To exclude the possibility that the patterning of the spinal cord was changed by our interference with SHH function starting at stage 18–19, we analyzed the expression pattern of sample class I and class II genes and compared them to control embryos. We did not detect any differences in spinal cord patterning with respect to PAX7 (class I)25 or ISL1 and NKX2.2 (class II)27 (Supplementary Fig. 2 online). Therefore, we concluded that SHH did not affect commissural axon guidance indirectly by changing cell differentiation or the patterning of the spinal cord. However, downregulation of SHH in an earlier time window (before stage 14) using the same dsRNA fragment did result in a decrease (at lower thoracic levels; data not shown) or even in the absence of ISL1 and NKX2.2 expression (at lumbosacral levels; Supplementary Fig. 3 online). When SHH was downregulated by in ovo RNAi at stage 8, even HNF3β, a marker of floor-plate cells known to be a target of Shh signaling, was reduced, in line with the notion that Shh has a role in floor plate induction as well as patterning of the spinal cord along the dorsoventral axis25. Shh’s effect on axon guidance not mediated by Ptc and Smo To obtain further evidence for a direct effect of Shh on commissural axon guidance, we tried to identify its receptor on commissural neurons. In both invertebrates and vertebrates Ptc and Smo have been identified as coreceptors for Shh, mediating its inducing activities28,29. Upon binding of Shh, Ptc releases and derepresses Smo30,31, which, in turn, is responsible for signaling32. To test whether Ptc and Smo were mediating Shh’s effect on axon guidance, we looked at their expression pattern in the developing chicken spinal cord (Fig. 3). Throughout the relevant time window

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ARTICLES Figure 5 Hip is the receptor that mediates the effect of Shh on postcommissural axons. (a–d) Images of open-book preparations from four different embryos in which HIP function was abolished by in ovo RNAi. Loss of HIP function resulted in the same phenotype as loss of SHH function (compare to Fig. 2). Commissural axons did not turn rostrally along the contralateral floor-plate border. Most of them stalled at the floor-plate exit site (a) but some of them grew caudally (arrows in b–d). As seen after perturbation of SHH function, some axons corrected their aberrant initial pathway to grow rostrally by forming loops (arrowheads in d). Loop formation and pathway corrections were never observed in control-injected embryos (compare Fig. 1b). Rostral is toward the top in all panels. The floor plate is indicated by dashed lines in a–c. Bar, 100 µm in a–c, 50 µm in d.

of Shh on postcommissural axon guidance was not mediated by Ptc and Smo.

during which commissural axons cross the midline and turn rostrally along the contralateral floor-plate border, neither PTC nor SMO are expressed by commissural neurons, suggesting that they are not required for SHH function in postcommissural axon guidance. Because expression levels of Ptc are influenced by Shh28,32, we also compared PTC expression by in situ hybridization between control embryos and embryos treated with SHH dsRNA (Supplementary Fig. 4 online). No changes in PTC or SMO expression in commissural neurons were detected at stage 25. Additional evidence for a receptor other than the complex of Ptc and Smo was obtained in functional experiments using cyclopamine, a small molecular inhibitor of Smo33. The injection of cyclopamine between stages 19 and 20 did not cause any changes in the turning behavior of commissural axons after midline crossing (n = 16; data not shown). Furthermore, downregulation of SMO by in ovo RNAi did not interfere with commissural axon guidance (n = 16 embryos; Supplementary Fig. 4 online). Therefore, we concluded that the effect

Shh’s effect on postcommissural axons is mediated by Hip Vertebrates, unlike invertebrates, express an additional receptor for Shh, Hip. Hip is a transmembrane protein that has been shown to bind directly to all vertebrate hedgehog proteins: Indian, Desert and Sonic hedghogs34. Because its distribution as well as functional evidence excluded Ptc as the Shh receptor involved in axon guidance, we analyzed the expression of HIP during the time window of commissural axon navigation (Fig. 4). HIP was regulated very dynamically and expressed by relatively few cells at any given time. Commissural neurons express HIP very briefly at stage 24, which is the time point at which axons have reached the contralateral floor-plate border and turn into the longitudinal axis. The expression of HIP in the spinal cord was not changed in response to silencing of SHH (Supplementary Fig. 5 online). Direct evidence for an involvement of Hip as mediator of the Shh signal was found by in ovo RNAi. Perturbation of HIP function in commissural axons resulted in the same turning phenotype as seen after downregulation of SHH (Fig. 5; n = 19 embryos, phenotype at 95% of the injection sites). Thus, we concluded that Hip was the receptor that mediated the effect of Shh on postcommissural axons. Graded SHH expression suggests a repulsive mechanism A graded distribution of a guidance cue could explain the rostral turn of postcommissural axons at the lumbosacral level of the embryonic

Figure 6 SHH is expressed in a gradient along the rostro-caudal axis of the lumbosacral spinal cord. (a,b) In situ hybridization reveals a graded expression of SHH in an open-book preparation of a stage 25 spinal cord with higher levels caudally. High (a) and low (b) magnification of the same spinal cord is shown. (c) In situ hybridization in open-book preparation with corresponding sense probe. Rostral is at the top of all panels. Only the lumbosacral level of the spinal cord is shown. The expression of Shh in open-book preparations is consistent with the expression of SHH in transverse sections (Supplementary Fig. 6 online). Bar, 500 µm.

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Figure 7 Postcommissural axons avoid high levels of Shh in vivo. (a–d) 5E1 staining of sections taken from different spinal cord levels. Shh was expressed ectopically in a spatially controlled manner in one half of the spinal cord at thoracic (a) and upper lumbosacral levels (b,c). No ectopic Shh expression was found at caudal lumbosacral levels (d). (e,f) Postcommissural axons encountering high levels of Shh stalled and did not leave the floor plate. (g,h) Axons crossing more caudally, where the ectopic Shh expression was decreasing, showed a lack of rostral bias or even turned caudally (arrows). Very few, if any, axons managed to extend rostrally (open arrow, g). (i) Summary of Shh expression and behavior of commissural axons at different spinal cord levels. At upper lumbosacral levels (top), commissural axons encountered high concentrations of ectopically expressed Shh and did not leave the floor-plate. In the transition zone between ectopic and endogenous Shh expression (middle), commissural axons encountered either no or a reversed Shh gradient and, therefore, responded with either stalling or caudal turns. At caudal lumbosacral levels (bottom), pathfinding was not affected, as axons were exposed only to the endogenous Shh gradient. (j) As a control, we traced commissural axons from the electroporated side of the spinal cord. Encountering high Shh before midline crossing, that is, before Hip is expressed, did not change their pathfinding behavior. Floor plate indicated by dashed lines in e–h and j. Rostral direction is toward the top in e–j. Bar, 100 µm in e–h,j.

they grew through the area of high Shh before they expressed HIP, but encountered a normal Shh gradient upon floor-plate exit, that is, when they expressed HIP (Fig. 7j).

chicken spinal cord. Depending on the mechanism, higher expression of an attractive cue would be expected rostrally, whereas a repellent cue should be expressed at higher levels caudally. As shown in open-book preparations, the expression of SHH was higher caudally (Fig. 6 and Supplementary Fig. 6 online), suggesting that Shh was providing a repellent signal for commissural axons that was mediated by Hip. SHH gain-of-function consistent with a repulsive role To provide evidence for a repulsive activity of Shh on postcommissural axons, we used in ovo electroporation to selectively express SHH in the thoracic and upper lumbosacral levels on one side of the spinal cord (n = 23 embryos; Fig. 7). In areas with high Shh expression, commissural axons mostly did not leave the floor plate and stalled at the exit site (Fig. 7e,f). Some axons encountering a reversed gradient upon floor-plate exit—that is, axons turning slightly caudally of the electroporated area—showed the expected pathfinding errors: they stalled or turned caudally to avoid high concentrations of Shh (n = 20; Fig. 7g,h). As a control, commissural axons ipsilateral to the ectopic SHH expression were traced. As expected, no effect on their turning behavior was detected, as


Shh’s repulsive activity confirmed by in vitro experiments Further evidence for a repulsive role of Shh on postcommissural axons was found in an explant assay similar to the one described previously for rat tissue35. We cultured spinal cord explants from stage 24–25 embryos in collagen gels in the presence of beads soaked in Shh or control beads soaked in bovine serum albumin (Fig. 8). To identify postcommissural axons, we labeled dorsal commissural neurons with the dye DiI in open-book preparations of dissected spinal cords before cutting and culturing explants (Fig. 8a). Unlabeled axons, therefore, represent predominantly motor axons (which grow well in collagen gels), unlabeled postcommissural axons from more ventral areas of the spinal cord, or dorsal postcommissural axons that were not labeled with DiI. No DiIlabeled axons were found contacting Shh-treated beads (Fig. 8b–d). Control beads were neither attractive nor repulsive for postcommissural axons (Fig. 8e,f). Most often (89.5% of the explants; n = 19), axons did not grow out on the explant side facing a Shh bead, or they turned away from the bead as soon as they entered the collagen gel (Fig. 8b,c). In one case the Shh bead was placed 0.9 mm away from the edge of the explant (Fig. 8d). In this situation axons started to grow toward the bead but stalled or turned approximately 300 µm away from the bead. Taken together, our results demonstrate that postcommissural axons avoid territories with high Shh both in vivo and in vitro, indicating that Shh acts as a repellent for postcommissural axons. The repulsive activity of Shh is mediated by Hip, in contrast to its attractive effect that is mediated by Ptc and Smo7. DISCUSSION Receptor switch at the midline for response to Shh Using in ovo RNAi, a technique recently developed in our lab to specifically silence candidate genes during commissural axon pathfinding17,18, we identified Shh as a guidance cue that directs these axons rostrally along the longitudinal axis of the spinal cord after they have crossed the midline. In contrast to its earlier effects, such as the induction of specific cell populations in the spinal cord14 and the chemoattractive effect7 that were mediated by Ptc and Smo, Shh uses a different receptor, Hip, to mediate its effect on postcommissural axons.

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ARTICLES A modulatory role of Shh on axon growth during later stages of development was first described for retinal ganglion cell axons36. Further evidence for a role in axon growth was provided in a turning assay using spinal cord explants, which demonstrated that Shh can attract commissural axons toward the floor plate7. In both cases, the effect of Shh on axon growth was mediated by Ptc and Smo acting as coreceptors37,38. Notably, Smo and Ptc are no longer expressed by commissural axons once they have grown into the floor-plate area (Fig. 3). Therefore, it is not surprising that we did not find an effect on commissural axon turning into the longitudinal axis using cyclopamine or in ovo RNAi to downregulate SMO (Supplementary Fig. 4 online). In search for an alternative receptor for Shh, we turned our attention to Hip34. Analysis by in situ hybridization revealed a highly dynamic temporal and spatial control of HIP expression in the developing chicken spinal cord (Fig. 4). HIP is not expressed during the initial phase of commissural axon growth toward and across the floor plate but is transiently upregulated when commissural axons reach the contralateral floorplate border. After the commissural axons turn, when they extend along the contralateral floor-plate border, HIP is downregulated to barely detectable levels (Fig. 4c). Hip is a type I transmembrane protein but lacks an intracellular domain. Its last 22 amino acids are hydrophobic and have been suggested to form the transmembrane domain34. Thus, it is unclear how Hip transmits the Shh signal that results in commissural axons’ turn into the longitudinal axis. In analogy to Ptc and Smo, Hip could represent a component of a receptor complex that would consist of a Shh binding unit (Hip) and a signal-transmitting unit that has not yet been identified. Repulsion: a previously unknown activity of Shh The graded expression of Shh, with high levels in the caudal-most region of the spinal cord, suggests a repulsive signal (Fig. 6a,b and Supplementary Fig. 6 online). Evidence for a repellent activity of Shh on postcommissural axons was obtained in gain-of-function experiments in vivo. Because it is unclear how far Shh can diffuse through the tissue39, we decided to express Shh locally in one half of the spinal cord at low thoracic and upper lumbosacral levels. Tracing commissural axons in embryos with a reversed Shh gradient (high levels rostrally and lower levels caudally) resulted in their expected failure to turn rostrally (Fig. 7). The repellent activity of Shh on postcommissural axons was confirmed by in vitro analysis (Fig. 8), where postcommissural axons were repelled by beads soaked in Shh but not by control beads. Because Shh is a morphogen13 and can act as an attractant for commissural axons7, the temporal and spatial control of gene silencing that is possible with in ovo RNAi becomes extremely important. With experiments that interfered with Shh levels before commissural axons had reached the floor plate, we would not have been able to detect the involvement of Shh in commissural axon guidance. Because we blocked

SHH expression only after stage 18–19, however, either the residual Shh was sufficient to attract commissural axons to the floor plate or, alternatively, in vivo the presence of netrin was sufficient to counterbalance the decrease in Shh. Our in situ hybridization analysis indicated that the expression of SHH dropped transiently between stages 19 and 21 (Supplementary Fig. 7 online). Thus, the downregulation of SHH owing to gene silencing initiated at stage 18–19 would prevent the effect of Shh on axon guidance but not on axon attraction. Morphogens act as guidance cues for postcommissural axons The observation that Wnt4 affects postcommissural axon guidance along the longitudinal axis of the spinal cord in rat and mouse11 is of great interest in the context of our results. Shh has been shown to regulate the expression of secreted frizzled-related proteins (Sfrps)40. Sfrps, in turn, are potent inhibitors of the effect of Wnt4 on commissural axon guidance11. Thus, it is tempting to speculate that Shh, Sfrps and Wnt4 cooperate in longitudinal axon guidance. High levels of Shh would induce high levels of Sfrps in caudal segments of the spinal cord. Therefore a Wnt4 gradient with the opposite orientation (high rostral to low caudal levels) would be strengthened by inhibition through Sfrps at more caudal levels. Complementary expression patterns and competitive interactions of Wnt4 and Sfrp2 have been described in chick41,42 and mouse embryos40. Functional in vivo experiments will be required to test for a cooperation of Shh and Wnt4 in postcommissural axon guidance. CONCLUSION Silencing SHH by in ovo RNAi in a temporally and spatially controlled manner demonstrated the involvement of Shh in guidance of postcommissural axons along the longitudinal axis of the spinal cord. Notably, the morphogenic effects of Shh on spinal cord patterning and the chemoattractive effect of Shh on precommissural axons are mediated by the Ptc-Smo receptor complex, whereas the repulsive effect of Shh on postcommissural axon guidance along the longitudinal axis of the spinal cord is mediated by Hip. METHODS Subtractive hybridization screen. To search for candidate guidance cues that would direct postcommissural axons rostrally along the longitudinal axis of the spinal cord, we set up a screen for differentially expressed floor-plate genes (stage 26 versus 20) that was based on subtractive hybridization. For this purpose, we isolated mRNA from floor-plate cells dissected from stage 25–26 embryos43. Stage 20 was the earliest time point at which floor-plate cells could be obtained without contamination by motor neurons. The subtractive hybridization was carried out according to the manufacturer’s recommendation using the PCR-Select cDNA subtraction kit (Clontech). Using this approach we initially obtained several hundred clones, from which 400 were randomly picked and subjected to further analysis. We used forward and reverse hybridization to eliminate false-positive Figure 8 Shh acts as a repellent on postcommissural axons. Postcommissural axons were labeled with DiI in open-book preparation of the lumbosacral level of the spinal cord from stage 24 embryos. (a) Explants were cut as shown (blue dashed line) and cultured in collagen gels. (b–f) Beads soaked either in Shh (b–d) or in serum albumin (e,f) were positioned between 200 and 700 µm away from the explants. Postcommissural axons did not grow from the edge of explants facing Shh beads (b,c). Either axons did not leave the explant or they turned away from the bead as soon as they entered the collagen gel. The bead shown in d was positioned more than 900 µm away from the explant. In that case, axons left the explant also on the side facing the bead, but they either stalled (arrowhead) or turned away from the bead (arrow) at a distance of approximately 320 µm. The insert in d shows that the axons are, in fact, labeled dorsal postcommissural axons. Control beads coated with serum albumin did not affect the growth of postcommissural axons (e,f). Bar, 200 µm in d–f, 400 µm in b,c.

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clones. Finally, we selected 30 clones with a clear difference in expression level between the two stages. From these cDNA fragments we generated DIG-labeled in situ probes to characterize the expression pattern in the spinal cord. In ovo RNAi. Loss-of-function phenotypes of SHH, HIP, PTC and SMO were induced by in ovo RNAi16–18 as described earlier by injection of dsRNA followed by electroporation at stages 18–1917 (Supplementary Figs. 6 and 8 online). Embryos were injected with a solution containing dsRNA alone or mixed with a plasmid encoding yellow fluorescent protein (YFP) under the control of the ACTB (βactin) promoter. Embryos injected and electroporated with the plasmid encoding YFP were used as controls. No difference in the overall development of the embryo or in axon growth and guidance were found between control-injected and noninjected embryos, indicating that the manipulation of the embryos did not induce any nonspecific changes. Numbers of embryos (n) given for control groups include only the injected and electroporated embryos; untreated control embryos that were analyzed to exclude an effect of the treatment alone were not counted. Analysis of commissural axon growth and guidance. The analysis of commissural axon trajectories was carried out as described previously8,44. In brief, embryos were sacrificed between stages 25 and 26. The spinal cord was removed from the embryo, opened at the roof plate (open-book preparation) and fixed. The trajectories of commissural axons at the lumbosacral level of the spinal cord were visualized using the lipophilic dye Fast-DiI (5 mg ml–1 in methanol; Molecular Probes) applied to the cell bodies. Care was taken to specifically label the dorsal population of commissural neurons to avoid any confusion with more ventral populations of commissural neurons that show a different pathfinding behavior. Cyclopamine, an alkaloid shown to interfere with Shh signaling by binding directly to Smo, was dissolved either in 45% 2-hydroxypropyl-β-cyclodextrin (Sigma; n = 11 embryos) or, because solubility was limited, in DMSO. The stock solution of cyclopamine in DMSO was diluted in PBS to limit DMSO in the injected solution to 2% (n = 5 embryos). Control embryos were injected either with 45% 2-hydroxypropyl-β-cyclodextrin (n = 9 embryos) or 2% DMSO (n = 4 embryos). Preparation of dsRNA. dsRNA for in ovo RNAi was transcribed in vitro as described before17. The cDNA fragment of SHH obtained in the subtractive hybridization screen was extended with 5′-RACE using the FirstChoice RMLRACE kit (Ambion) according to the manufacturer’s recommendations. Primers used were 5′-TACTCAGACCCTGAAAATGGACG-3′ and 5′-GGTCAGTCATCAGAGTTACGTGC-3′. The PCR product was ligated into the pCRII-TOPO vector (Invitrogen). Sequencing of the resulting clones confirmed their identity as chicken SHH. The fragment that was originally used to produce dsRNA and in situ probes was from the 3′-UTR of SHH and did not overlap with the open reading frame. To confirm our results we also used a second, nonoverlapping fragment covering the N-terminal sequence of SHH for dsRNA production. DsRNA and in situ probes for PTC were obtained by RT-PCR using poly(A) RNA isolated from embryonic spinal cords (stages 25–28). The primer used for reverse transcription was 5′-AGCACCAATCCATTGAGAACTCCC-3′. For primary PCR we used 5′-TCGGGAGTTAAACTACACACGGC-3′ and 5′-CTACGGTTCTTATCTCCTATGGC-3′. Nested PCR products were obtained with 5′-ATGTACTCACAACAGAAGCACTCC-3′ and 5′-GCCAGCACCACCACAATGATCCC-3′. The final PCR product was cloned into pCRII–TOPO. Similarly, we obtained a fragment of the cDNA encoding Smo. The primers used were for RT-PCR 5′-AGTCCATGTGTGGGGACCGAAATC-3′, and for PCR the forward primers 5′-CGCGCTGCCCTACGCGCACACC-3′ and 5′ AGCTGCCCAGTCAGACCCTGTGCC-3′ with the reverse primers 5′-AGCGTCCCCTTCACCCCTAAATCC-3′ and 5′-CCAGTTTCTTCTCTCCTCCCATCC-3′. For HIP, the primers used were 5′-CAAGAATACCTGGCCTTGTAACTC-3′, 5′-TGCGCACACTGCTCACCTCATGCC-3′ and 5′-TCGACATGCTGGGTCACACTTTGC-3′. Hybridoma cell grafting. The hybridoma cell lines 5E1 (producing a function-blocking anti-Shh antibody), 1E8 and 9E10 were obtained from the Developmental Studies Hybridoma Bank (Univ. Iowa). The cell lines 9E10 and


1E8, producing antibodies against c-Myc and P0, respectively, were used as controls. Cells grown in DMEM/F12 supplemented with 10% FCS were collected and resuspended in PBS for injection into the lumbar spinal cord of stage 20 embryos in ovo. A peroxidase-coupled secondary antibody (rabbit anti–mouse IgG; Cappel) was used to test for antibody production in situ. Comparable numbers of cells from the 1E8 or the 9E10 hybridoma cell lines were grafted in control embryos (n = 9). Ectopic expression of SHH. For gain-of-function experiments the open reading frame of chicken SHH was cloned into the pMES plasmid (kindly provided by C. Krull) using EcoRI and a plasmid derived from pIRES (Clontech). The CMV promoter of the pIRES plasmid was exchanged for the chicken ACTB promoter, and the IRES sequence was removed. SHH was inserted using NheI and SalI sites. To localize transfected cells, we coinjected a plasmid encoding YFP. In the pMES plasmid, the IRES sequence is followed by EGFP, allowing for direct detection of transfected cells. To achieve a reversal of the endogenous Shh gradient, we used either shorter electrodes (2-mm rather than 4-mm) or placed them more rostrally, such that cells at caudal levels of the lumbosacral region of the spinal cord were not transfected (Fig. 7a–d,i). The density of green cells was used to determine the electroporated area of the spinal cord and to distinguish between sites with high and low Shh expression. Staining and in situ hybridization. Antibodies recognizing Shh, Pax7, Isl1 and Nkx2.2 were obtained from the Developmental Studies Hybridoma Bank. Control and experimental embryos were sacrificed, fixed in 4% paraformaldehyde and cryoprotected in 25% sucrose. Sections 20 µm thick were stained as described previously using Cy3-conjugated goat anti–mouse IgG (Jackson Laboratories) as secondary antibody24. The expression of SHH, HIP, SMO and PTC was analyzed by in situ hybridization in transverse spinal cord sections45. In vitro assay. Open-book preparations of the lumbosacral spinal cord of stage 24–25 embryos43 were cultured in collagen gels essentially as described. However, we used the lipophilic dye Fast-DiI to label dorsal postcommissural axons rather than an anti–axonin-1 antibody, as axonin-1 (the ortholog of rat TAG-1) is expressed also by motoneurons of the lumbosacral spinal cord (Fig. 3a). As described previously for two-dimensional explant cultures of chicken commissural axons9, we used a serum-free medium to grow postcommissural axons in collagen gels. Heparin acrylic beads (Sigma) were soaked in either 0.5 mg ml–1 recombinant human Shh (R&D, with 25 mg ml–1 bovine serum albumin as carrier), or 25 mg ml–1 bovine serum albumin (Albumax, Invitrogen) for 1 h. Shh or control beads were positioned 200–700 µm from the explants. Cultures were grown for 24–36 h before inspection on an inverted microscope. Explants with only very few axons entering the collagen gels or explants in direct contact with the bead were not included in the analysis. A total of 19 explants with Shh and 20 explants with control beads, respectively, from three independent experiments were analyzed. Note: Supplementary information is available on the Nature Neuroscience website. ACKNOWLEDGMENTS We thank C. Krull for the pMES plasmid, M. Mielich for technical assistance and M. Gesemann for critical reading of the manuscript. This work was supported by grants from the Swiss National Science Foundation, the Human Frontier Science Program Organization, the Olga Mayenfisch Stiftung and the Ott Foundation. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Received 7 December 2004; accepted 10 January 2005 Published online at http://www.nature.com/natureneuroscience/

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