DEVELOPMENTAL LOCALIZATION OF SEMAPHORIN H MESSENGER RNAACTING AS A COLLAPSING FACTOR ON SENSORY AXONS IN THE MOUSE

BRAIN

N. MIYAZAKI,*† T. FURUYAMA,‡ T. SAKAI,*§ S. FUJIOKA,* T. MORI,* Y. OHOKA,* N. TAKEDA,† T. KUBO†and S. INAGAKI*k

*Group of Neurobiology, School of Allied Health Sciences, †Department of Otorhinolaryngology, Osaka University, Faculty of Medicine,Yamadaoka 1-7, Suita-shi, Osaka 565-0871, Japan

‡Department of Molecular Genetic Research, National Institute for Longevity Sciences, Morioka-cho, Ohbu-shi 474, Japan

§Department of Oral and Maxillo-Facial Surgery, Osaka University Dental School, Suita-shi, Osaka 565-0871, Japan

Abstract—Semaphorins/collapsins, a family of genes with a semaphorin domain conserved from insects through to mammals, arebelieved to be involved in axon guidance during neuronal development. We report the expression patterns of mouse semaphorinmessenger RNAs. Among secreted semaphorins, mouse semaphorin H is structurally most similar to semaphorin III/D, the firstsemaphorin identified as a collapsing factor for sensory axons. However, its expression patterns apparently differ from those ofsemaphorin III/D. The messenger RNAs are distributed in the brain widely but unevenly during development, in particular, in themain olfactory bulb, hippocampus and pontine nucleus. In the trunk, the expression level is high in mesodermal tissues surroundingthe dorsal root ganglia, while it is low in the spinal cord. Moreover, we examined whether this molecule has activity to collapsegrowth cones of sensory neurons, as well as semaphorin III/D. Mouse semaphorin H collapsed growth cones of sensory neurons ofthe dorsal root ganglion in a dose-dependent manner, and anti-neuropilin antibodies inhibited this activity.

Taken together, these results suggest that mouse semaphorin H can function as a chemorepellent to guide sensory peripheralnerves, most likely via neuropilin as a receptor.q 1999 IBRO. Published by Elsevier Science Ltd.

Key words: semaphorin H, mouse, growth cone collapse, brain, dorsal root ganglion.

Axon pathfinding during neural development depends on bothattractive and repulsive guidance signals (reviewed byTessier-Lavigne and Goodman25). Chick collapsin-1 (coll-1)and its mammalian homologue, semaphorin III/D (semaIII),were identifiedin vitro as a collapsing chemorepulsive factorfor sensory growth cones, and shown to be involved in axonguidance.2,15,17,20,24Semaphorins/collapsins are a large familyof structurally distinct secreted and transmembrane proteinscharacterized by the presence of a conserved semaphorindomain of about 500 amino acids.25

To study the mechanism of neural network forming, wetried to clone several novel semaphorins, mouse semaphorinF (M-semaF)12, M-semaG8 and M-semaH, from mouse brain.M-semaH, a secreted semaphorin, is structurally similar tosemaIII. However, there is little data available on the distri-bution of its expression, and no data on its biological activ-ity.5,6 In order to explore the function of M-semaH in thebrain, we studied the expression pattern of M-semaHmRNA in developing mouse embryos and brains. Usingnorthern blot analysis, signals were detectable by embryonicday (E) 10.5. At the same stage,in situ hybridization histo-chemistry showed expression in the anlage of several brainareas and the acoustic ganglion. The distribution patternswere significantly different between M-semaH and semaIII,suggesting different roles of these semaphorins in the CNS.

For example, semaIII mRNA is expressed in spinal motorneurons and most cranial motor neurons, while M-semaHexpression was weak in motor neurons.9 In the olfactorybulb, a high level of expression of semaIII was found in mitralcells of the accessory olfactory bulb, whereas the expressionlevel of M-semaH was low in the accessory olfactory bulb,9

although it was high in the main olfactory bulb.SemaIII repels axons of sensory and sympathetic

neurons.15,20,22 SemaIII also repels most axons of bothventrally and dorsally projecting motor neurons, such asspinal motor and abducens, trigeminal motor, facial andglossopharyngeal axons, except for oculomotor and trochlearaxons.26 Mice homozygous for a targeted mutation in thesemaIII gene showed severe abnormality of the peripheralnerve projection involving the spinal, trigeminal, facial,vagus, accessory and glossopharyngeal nerves, while no orlittle abnormality was found in the CNS, suggesting importantroles for semaIII in the axon guidance of peripheral sensory,motor and autonomic neurons.2,24 Much less is known aboutthe other semaphorins. Recently, the effects of some secretedsemaphorins, such as semaphorin A (semA), coll-2 and sema-phorin E (semE)/coll-3, on sensory and sympathetic axonswere studied. In explant cultures, semA and semE/coll-3 actas chemorepellents on specific populations of axons.1,18

Repulsive effects of semA and semE/coll-3 are restricted tosympathetic axons, and coll-2 has no such effect on eithersensory or sympathetic axons, while semaIII repels a widerange of sensory and sympathetic axons. Chemorepellentactivities for sensory and sympathetic axons differ amongsecreted semaphorins. Thus, in the present study, we exam-ined whether M-semaH acts as a repulsive factor on theseaxons. A part of this study was published previously.21

Localization of M-semaH mRNA in the brain 401

401

NeuroscienceVol. 93, No. 1, pp. 401–408, 1999Copyrightq 1999 IBRO. Published by Elsevier Science Ltd

Printed in Great Britain. All rights reserved0306-4522/99 $20.00+0.00PII: S0306-4522(99)00134-7

Pergamon

kTo whom correspondence should be addressed.Abbreviations: AP, alkaline phosphatase; coll, collapsin; DRG, dorsal root

ganglion; E, embryonic day; GAPDH, glyceraldehyde 3-phosphate dehy-drogenase; M-sema, mouse semaphorin; M-semaH–AP, AP-fused M-semaH; NGF, nerve growth factor; PCR, polymerase chain reaction;semA, semaphorin A; semaIII, semaphorin III/D; semE, semaphorin E.

EXPERIMENTAL PROCEDURES

Isolation of complementary DNA clones

cDNA was amplified by using polymerase chain reaction (PCR)with first-strand cDNA synthesized by reverse transcriptase frommRNA of ICR mouse E14 embryos, as described previously.8,12 PCRfragments were used as a probe to screen a mouse brain library primedwith oligo(dT) (Stratagene).8 Sequencing was carried out using ABI373A DNA sequencer and Taq dye primer cycle or Taq dye terminatorcycle sequencing kit (Applied Biosystems). The final sequence wasconfirmed from both strands. Sequence analysis or comparison wasdone through BLAST on the National Center of Biotechnology Infor-mation. The C1 clone containing the entire coding region of M-semaHwas used as a probe and for construction of vectors.

Northern blot hybridization

Northern blot hybridization was performed as described.12

Poly(A)1 RNA was prepared from ddY mouse embryos and postnatalbrains (Quick prep micro mRNA purification kits, Pharmacia).Poly(A)1 RNA (2 mg/lane) was separated on formaldehyde gels andblotted onto nylon membranes (Hybond N, Amersham). Hybridizationwas performed with [32P]dCTP (NEN)-labelled cDNA probe for apartial 1731-bp fragment of M-semaH in hybridization buffer contain-ing 50% formamide (Nakarai) at 428C overnight. After washing to afinal stringency of 0.1×standard saline citrate/0.1% sodium dodecylsulfate at 608C, the filters were analysed using a BAS-2000 imageanalyser (Fuji-Film). cDNA probe for rat glyceraldehyde 3-phosphatedehydrogenase (GAPDH) was also hybridized to normalize the mRNAfrom different tissues.11

In situ hybridization

ddY mice purchased from SLC (Hamamatsu, Japan) were used. Theday of detection of the vaginal plug was designated E0.5. Embryos andneonates were anaesthetized on ice and cut into sections of 15mmthickness on a cryostat. The sections were processed forin situ hybri-dization with antisense and sense RNA probes (676-bp fragments)labelled with [35S]UTP-labelled RNA probes, as describedpreviously.12

Expression vectors and transfection

To generate placental alkaline phosphatase (AP)-fused M-semaHprotein (M-semaH–AP), the entire coding region of M-semaH inAP-tag1 vector4,7 was induced into NIH3T3 cells by calcium phos-phate co-precipitation following the procedures described by Flanaganand Leder.7 To exclude the possibility that the activity of M-semaHis derived from tagged AP, AP protein secreted from NIH3T3 cellstransfected with AP-tag4 vector was used as a control. Amounts of M-semaH–AP and control AP were calculated by measuring AP activitiesphotometrically with p-nitrophenyl phosphate (optical density at405 nm), as described by Flanagan and Leder.7 Two-day culturemedium supernatant containing M-semaH–AP and AP was ultra-filtrated and concentrated, respectively, by centrifugation using cen-tricon-100 (Amicon). The ultrafiltrated medium containing either M-semaH–AP or control AP proteins was used for the collapse assay. Inorder to generate more secreted AP protein for dose–response curves,293T cells (ATCC CRL 1573) were transfected with AP-tag4, and APprotein was concentrated in the same way by ultrafiltration.

Mouse semaphorin H antiserum production

Anti-M-semaH antibodies were produced by immunizing rabbitswith a 6-histidine-tagged M-semaH protein that was produced inE.coli. The bacterial expression construct was made by PCR amplifica-tion of a fragment encoding amino acids 702–766 of M-semaH andinserted into the Sal1 and Not1 sites of pET32a (Novagen). Expressedprotein was purified by immobilized nickel–chelate affinity chromato-graphy, and separated by sodium dodecyl sulfate–polyacrylamide gelelectrophoresis. Rabbits were immunized with a mixture of separatedgel extract and complete Freund’s adjuvant, and boosted every two tothree weeks. M-semaH antibodies were used for the detection of M-semaH–AP in immunoblot experiments.

Collapse assay

Collapse assays were carried out following the procedure of Raperand Kapfhammer.23 In brief, dissected dorsal root ganglia (DRGs)from mouse E12.5 embryos were placed on laminin (40mg/ml)-coatedeight-well chamber slides (Lab-Tek) and incubated at 378C inDulbecco’s modified Eagle medium/5% fetal calf serum mediumcontaining nerve growth factor (NGF 7.5S, 50 ng/ml). After 12–24 h, the medium of the explants was gently replaced respectivelywith the same medium, M-semaH–AP medium and control APmedium. After 1 h, the explants were fixed with 10% formalin inphosphate-buffered saline and stained with concanavalin A–fluores-cein isothiocyanate.14 Superior sympathetic ganglia from E13.5 wereused for the assay. Fixed preparations could be scored quantitatively.In untreated cultures, about 80% of extending neurites possessedrecognizable growth cones at their tips. These had lamellipodia andfilopodia. The neurites exposed to medium with collapsing activity hadno lamellipodia and a few filopodia.

To establish whether the collapsing activity of M-semaH–APmedium was derived from M-semaH–AP protein, DRG explantswere incubated with M-semaH–AP medium absorbed by anti-AP anti-body–agarose (Sigma, 8B6). To clarify whether the effects of M-semaH–AP were induced via neuropilin as a receptor, anti-neuropilinantibodies were added to the explants 30 min before incubation withM-semaH–AP medium.10

For dose–response curves, three independent experiments wereperformed. In each experiment, 150–300 neurite tips were scored foreach concentration. The values of the percentage of cells showingcollapsed growth cones in response to M-semaH were corrected bytaking into account that 16.3% (average from five explants) ofuntreated explants undergo collapse.

RESULTS

Isolation of mouse semaphorin H

PCR fragments amplified from mouse brain by using fullydegenerate primers derived from conserved motifs of thecollapsin and grasshopper semaphorin I genes8,12 were usedas probes to screen several cDNA libraries of mouse brain,resulting in the isolation of one full-length clone and serialpartial clones encoding a semaphorin, M-semaH. The cDNAcontains an open reading frame of about 2.23 kb. The openreading frame starts with an ATG codon and matches theconsensus sequence of a strong translation initiator19 andends with a TGA stop codon. The cDNA predicts a proteinof 775 amino acids which consists of a semaphorin domain of523 amino acids, a type 2 immunoglobulin domain and basicregions at the C-terminus (Fig. 1). Twenty-three amino acidsdiffer between M-semaH (AF034744) and a transcript of M-semaH cloned from tumour cells by Christensenet al.5

(z93947). The semaphorin domain of M-semaH is 81% iden-tical to chick coll-5, most likely a chick homologue of M-semaH, and 53% identical to mouse semaIII/D. Among thesemaphorins, M-semaH is most similar to semaIII/D.

Expression of mouse semaphorin H messenger RNA duringdevelopment

To explore the expression of M-semaH mRNA, weperformed northern blot analysis andin situ hybridizationhistochemistry. Northern blot analysis revealed a transcriptof about 4.0 kb for M-semaH, and weak signals of about7.5 and 9 kb for M-semaH (Fig. 2). The 4.0- and 7.5-kb tran-scripts for M-semaH are consistent with the results ofChristensenet al.5 The signals for M-semaH could bedetected from whole embryos as early as E10.5 (Fig. 2).The expression levels in the brain appeared to be almostunchanged during development from E14.5 to postnatalstages. The expression level decreased slightly toward

N. Miyazaki et al.402

Localization of M-semaH mRNA in the brain 403

Fig. 1. Comparison of amino acid sequences of M-semaH from mouse brain cDNA library, M-semaH (z93947) cloned by Christensenet al., and human andchick homologues. The numbers on the right side of the sequence show the positions of amino acids. The predicted signal sequence is underlined. The sema

domain is indicated by double underlines. Asterisks indicate conserved cystein residues in the sema domain.

adulthood. These results show that M-semaH mRNA appearsat an early embryonic stage, is expressed during developmentand remains detectable in the adult brain.

In situ hybridization histochemistry revealed an extensivebut characteristic distribution of M-semaH mRNA duringdevelopment. At E11.5, the expression was restricted to thelower brainstem, such as the mesencephalon, metencephalonand myelencephalon (Fig. 3A, B). In the mesencephalon, theexpression was detected in the tegmental region just beneaththe neuroepithelium (Fig. 3A). In the metencephalon, thesignals were found in the lateral tegmental regions facingthe trigeminal ganglia (Fig. 3B). The signals were alsoexpressed in the neuroepithelium of the myelencephalon(Fig. 3A) and choroid plexus (Fig. 3B). The signals werenot detected in the trigeminal ganglia, but in the acousticganglia (Fig. 3B). At E16.5, the signals were found in variousnuclei throughout the brain, such as in the olfactory bulb,cortical plate, the primodium of the hippocampus, basal gang-lia, thalamus, mesencephalon, pons and medulla oblongata(Fig. 4A). These signals were not found in the control tissueshybridized with the sense probes (Fig. 4B). In the adult brain,the signals remained detectable in various nuclear regions,including the olfactory bulb, neocortex, hippocampus, basalnuclei, reticular thalamus and throughout the lower brainstem(Fig. 4C). The signals were also detected in the pontine

nucleus and cerebellum, among other regions. In the olfactorybulb, the signals were expressed in the mitral cell layer byE14.5, thereafter increasing in intensity, and were detected inthe adult in tufted cells of the glomerular and external plexi-form layers, as well as in the mitral cells, and in some sub-populations of granular cells of the main olfactory bulb. Thesignals in the neocortex increased as the animals grew, and inthe adult they were expressed in layers V and Vl of theneocortex. In the brainstem, the signals were detected invarious regions, such as the reticular formation and trigeminalsensory nuclei, but barely or not at all in the motor nuclei,such as the facial and hypoglossal nuclei.

Mouse semaphorin H as a chemorepulsive molecule

Explanted DRGs extended axons with well-defined growthcones on laminin-coated chambers (Fig. 5A). Growth conesof NGF-dependent outgrowing axons collapsed in response toexposure to M-semaH–AP medium (Fig. 5B). To exclude thepossibility that tagged AP caused this collapse, growth coneswere exposed to control AP medium. Growth cones did notcollapse following exposure to control AP medium (Fig. 5C).Moreover, M-sema H–AP medium was absorbed with ananti-AP antibody in order to confirm that the collapsing activ-ity of M-semaH–AP medium was derived from M-semaH–AP protein. Absorption by an anti-AP antibody of the mediummarkedly reduced the activity of the M-semaH–AP medium(Fig. 5D). Immunoblot experiments showed that M-semaH–AP protein was detected by using anti-M-semaH antibodieson immunoprecipitation of the medium with an anti-AP anti-body (Fig. 5E). To clarify whether M-semaH induces growthcone collapse via neuropilin as a receptor, anti-neuropilinantibodies were added to DRG explants 30 min before incu-bation with M-semaH–AP medium. Anti-neuropilin neutral-izing antibodies inhibited growth cone collapse induced byM-semaH–AP (Fig. 5F), while preimmune antibodies did not.Next, to elucidate whether M-semaH can guide DRG growthconesin vivo, we examined the mRNA expression in the trunk(Fig. 5G). The expression was detected in the mesodermaland mesenchymal tissues surrounding the DRG and in itsaxon trajectory, and was particularly concentrated in thesclerotome lateral to the DRG (Fig. 5G). The expressionwas weak in the spinal cord, except for the floor plate (Fig.5G). Growth cones of sympathetic ganglionic neurons also

N. Miyazaki et al.404

Fig. 3. In situ hybridization histochemistry showing the expression of M-semaH mRNA in the brain of E11.5 embryo. (A, B) Dark-field photographs ofparasagittal sections which were hybridized with antisense probes. (A) The signals were seen in the tegmental region just beneath the neuroepithelium of themesencephalon (arrow), and in the neuroepithelium of the myelencephalon (arrowhead). (B) The signals were also found in the lateral regions of themetencephalon (double arrowheads) close to the trigeminal ganglia (dotted line). No signals were found in the trigeminal ganglia, while strong signals

were detected in the acoustic ganglion (arrow) and choroid plexus (arrowhead). Scale bars�300mm.

Fig. 2. Northern blot demonstrating M-semaH mRNA signals. Poly(A)1RNA (2mg), isolated from whole embryos at E10.5, heads of E12.5embryos and brains of E14.5, E16.5, postnatal day 0 and 7, and adultanimals, were hybridized to32P-labelled M-semaH cDNA probe. Hybridi-zation with GAPDH probe produced a positive 1.3-kb band in each lane(lower panel). The M-semaH mRNA is expected to be about 4.0 kb in size.The probe also detected two additional weak signals of about 7.5 and 9 kb.

collapsed in response to exposure to M-semaH–AP, but not inresponse to control AP (data not shown).

M-semaH–AP induced growth cone collapse of DRGaxons in a dose-responsive manner, whereas control AP hadno apparent collapsing activity (Fig. 6). This dose dependency

effect of M-semaH–AP supports the idea that M-semaH canspecifically collapse DRG growth cones. Percentages ofcollapsed growth cones at each concentration were treatedstatistically and are listed in Fig. 6A. M-semaH–AP (4 nM)induced 72.6% growth cone collapse of mouse DRGs. The

Localization of M-semaH mRNA in the brain 405

Fig. 4. In situ hybridization histochemistry showing the expression of M-semaH mRNA at the late embryo stage and adulthood. Dark-field photographs ofparasagittal sections hybridized with antisense (A, C) and sense (B) probes. The signals were detected at E16.5 in the olfactory bulb, cortical plate, primodia ofthe hippocampus, reticular thalamic nucleus (indicated by an arrow) and various regions of the brainstem (A). No specific signals were detected in theadjacenttissue section hybridized with sense probes (B). In the adult brain, signals for M-semaH remained detectable throughout the brain (C). In particular, the signals

were prominent in the olfactory bulb, hippocampus and pontine nucleus (C). Scale bars�1 mm.

N. Miyazaki et al.406

Fig. 5. M-semaH causes growth cone collapse of NGF-sensitive sensory axons. DRGs from E12.5 embryos were explanted on laminin-coated chambers.Growth cones with lamellipodia and filopodia were observed in DRG explant cultures with NGF (50 ng/ml; A). These collapsed and lost lamellipodia inresponse to 1 nM of M-semaH-AP (B), while most growth cones did not collapse when incubated with 1 nM of AP (C). M-semaH–AP medium absorbed by ananti-AP antibody immobilized on beads markedly diminished growth cone collapsing activity (D). M-semaH–AP protein immunoprecipitated by anti-APantibody was detected by western analysis with an antibody against M-semaH (E). Anti-neuropilin antibodies (10mg/ml) inhibited the collapsing activity of M-semaH–AP (F). The expression of M-semaH mRNA was detected byin situhybridization histochemistry in the spinal cord and associated structures at E12.5(G). The mRNAs were detected in the mesenchymal and mesodermal tissues surrounding the DRG. The sclerotome (arrows) lateral to the DRG expresses

strong signals. No or weak expression was seen in the spinal cord except for the floor plate (arrowhead). Scale bars: (A–D, F)�50mm, (G)�200mm.

concentration that induced 50% maximal collapse (ec50) was0.91 nM.

DISCUSSION

The transcripts for M-semaH (z93947 and z93948) wererecently isolated from tumour cells by Christensenet al.5 Thatthe amino acid sequences of these transcripts differ may bebecause one transcript was isolated from tumour cells and theother from normal brain tissues, or due to sequence errors inz93947 and z93948.

Expression pattern of mouse semaphorin H messenger RNA inthe brain

It is important to expand our knowledge of the expressionpatterns of semaphorins, to predict their roles. Since, in aminoacid sequence, M-semaH is most similar to semaIII/coll-1, weinvestigated regional and developmental expression patternsof M-semaH mRNA and compared them to those of semaIIImRNA. The signals for M-semaH mRNA were detectable asearly as E10.5 by northern blotting.In situ hybridizationhistochemistry revealed the distribution patterns of M-semaH signals during development. The expression was

restricted to lower brainstem regions at E11.5, but thereafterdeveloped throughout the brain from forebrain to lower brain-stem. The distribution pattern of M-semaH mRNA differsfrom that of semaIII mRNA. For example, the expression ofM-semaH mRNA was weak in the accessory bulb, but strongin the main olfactory bulb, while semaIII expression wasmuch stronger in the accessory olfactory bulb than in themain olfactory bulb.9 Another example is in the motor nuclei.A large population of somatomotor neurons in the brainstemshowed prominent expression for semaIII,9 but no or weakexpression for M-semaH. In these regions, M-semaH mayfunction separately from semaIII, even if they have similarroles.

Mouse semaphorin H as a collapsing factor

In explant cultures, several semaphorins of the secretedtype, such as semA, semaIII/coll-1 and semaE/coll-3, actedas chemorepellents on specific populations of axons.1,18

Although semaIII/coll-1 repelled a wide range of sensoryand sympathetic axons, the effects of other semaphorins,such as semA and semE, were restricted to sympatheticaxons, and no effect of coll-2 was found on either sensoryor sympathetic ganglia. We found that M-semaH acts as acollapsing factor on sensory axons in a dose-dependentmanner. This finding may correspond to the result that theamino acid sequence of the semaphorin domain of M-semaHis most similar to that of semaIII. In this context, it is impor-tant to investigate the mRNA expression pattern of M-semaHin the trunk to compare M-semaH with semaIII. That M-semaH mRNA was expressed in mesodermal tissuessurrounding the DRG and in its axon trajectory supports theidea that M-semaH functionsin vivo as a chemorepellentwhen sensory peripheral axons outgrow toward their targets.The expression patterns of M-semaH and semaIII mRNA inthe mesodermal tissues appear to overlap. On the other hand,the weak expression in the spinal cord suggests that M-semaHhas no effect on central axons of DRGs. This contrasts withthe report that semaIII mRNAs are highly expressed in theventral region of the spinal cord and predicted semaIII to actas a collapsing factor on central axons of the DRG, as well asperipheral axons.9,22

Coll-1 induced about 100% growth cone collapse of chickDRGs,18,20 while M-semaH–AP induced a maximum 73%collapse of mouse DRGs, i.e. about 27% of NGF-dependentDRG neurons do not respond to M-semaH. It is conceivablethat there exist heterogeneous subpopulations of NGF-sensi-tive DRG cells. M-semaH may function as a fine filter throughwhich only subpopulations of NGF-sensitive DRG neuronscan pass through, while coll-1 blocks all NGF-sensitiveaxons.

The ec50 of coll-1 collapsing activity for DRG neurons(0.03 nM)18,20 is 30-fold lower than that of the M-semaH–AP activity (0.91 nM). This highec50 may be explained bytagged AP for M-semaH, and/or species differences in thesensitivity between mouse and chick. However, if it is indeeda property of M-semaH, then M-semaH would be expected toaffect sensory nerves much more locally than coll-1.

Possible receptors for mouse semaphorin H

Recently, neuropilin has been shown to be a receptor forsemaIII by using expression cloning.10,16 Antibodies to

Localization of M-semaH mRNA in the brain 407

Fig. 6. Dose-dependent curve showing collapsing activity of M-semaH–AP. (A) Data shown are the mean percentage of collapsed growthcones S.E. from three separate experiments. At least 150 neurite tipswere counted at each concentration. (B) The percentages of collapsedgrowth cones (meanS.E.) are plotted against the concentration of M-semaH–AP (filled circles) and control AP (open circles). M-semaH–APinduced growth cone collapse of DRGs in a dose-dependent manner, whilecontrol AP did not. The concentration that induced 50% maximal collapse

(ec50) was 0.91 nM.

neuropilin blockedin vitro semaIII chemorepulsive activ-ity.10,16 We also found that the collapsing activity of M-semaH is inhibited by anti-neuropilin antibody, suggestingthat M-semaH most likely causes sensory growth conecollapse via neuropilin as its receptor. Neuropilin expressionin embryonic DRG neurons also supports this possibility.13

However, some results require clarification. First, severalsecreted semaphorins, including coll-2, semE/coll-3 andsemA, have no effect on sensory ganglion cells, althoughthey can bind recombinant neuropilin with comparable affin-ity.1,3,6 Second, patterns of the binding sites on brain sections

differ among semaphorins.6 The functions and targets ofsecreted semaphorins still remain to be studied.

Acknowledgements—This work was supported by a Grant-in-Aid forInternational Scientific Research to S.I. and a Grant-in-Aid forEncouragement of Young Scientists to T.F. from the Ministry ofEducation Science and Culture. We thank J. G. Flanagan forproviding AP vectors and helpful instructions, M. Tessier-Lavignefor providing the antibody to neuropilin, K. Imaizumi and T. Takagifor providing GAPDH plasmids, and T. Kimura and H. Kobayashi forhelpful comments on collapse assays. We also thank N. Allen forreading the manuscript. GeneBank Acc. No. for M-semaH: AF034744.

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(Accepted1 March 1999)

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