expression and localisation of dynamin and syntaxin during neural development and neuromuscular...

Expression and Localisation of Dynaminand Syntaxin During Neural Developmentand Neuromuscular Synapse Formation

P.G. NOAKES,1 D. CHIN,1 S.S. KIM,2 S. LIANG,2 AND W.D. PHILLIPS,2*1Department of Physiology & Pharmacology, University of Queensland,

4072 Brisbane, Australia2Department of Physiology, University of Sydney, Sydney, NSW 2006 Australia

ABSTRACTThe expression and subcellular localisation of dynamin and syntaxin were examined

during the periods of motor neuron development and neuromuscular synaptogenesis in themouse embryo. Both dynamin and syntaxin could be detected by immunoblotting in the spinalcord at embryonic day 10 (E10; 2 days before axon outgrowth) and at all subsequent agesexamined. Reverse transcription and polymerase chain reaction (RT-PCR) identified lowlevels of all three carboxy-terminal splicing forms of dynamin I in spinal cord from as early asE10. During the period of maturation of spinal neurons, from E10 to the first postnatal day(P0), the short carboxy-terminal splicing form of dynamin I (dynamin I*b) was up-regulated,as was dynamin III, relative to dynamin II mRNA. Syntaxin immunostaining becamecolocalized with the synaptic vesicle protein, SV2, at neuromuscular synapses within 12 hoursof the commencement of synapse formation and throughout subsequent development. Incontrast, dynamin, which is important for activity-dependent synaptic vesicle recycling and,thus, sustained neurotransmission, could not be detected at most newly formed synapses untilseveral days after synapse formation. The delayed appearance of dynamin at the synapse,thus, heralds the neonatal development of robust synaptic transmission at the neuromuscularjunction. J. Comp. Neurol. 410:531–540, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: spinal cord; skeletal muscle; embryogenesis; gene expression; endocytosis

The fine control of voluntary muscle contraction dependsin part on the ability of the muscle action potential to echoexcitation of the motor neuron at frequencies of up to 100Hz (Burke et al., 1970). To this end, the adult skeletalneuromuscular junction is highly specialised to couplenerve activation, vesicle exocytosis and recycling of synap-tic vesicle membrane. The regulated exocytosis of acetylcho-line from specialised transmitter release sites is depen-dent on interactions of a group of presynaptic membraneproteins, including syntaxin and SNAP-25, with synapticvesicle proteins comprising synaptobrevin/VAMP and syn-aptotagmin (Sudhof, 1995; Martin, 1997). Syntaxin I, a,33-kDa protein attached to the cytoplasmic face of thepresynaptic membrane by means of its carboxy-terminal(C-terminal) hydrophobic domain, can bind to the synapticvesicle–associated protein synaptobrevin/VAMP in an in-teraction that is potentiated by the binding of anotherpresynaptic membrane protein, SNAP-25 (Bennett et al.,1992; Chapman et al., 1994). This complex is thought to beessential to prime vesicles for Ca21-evoked transmitterexocytosis. Cleavage of any of its component proteins bysequence-specific Clostridial endoproteases blocks electri-

cally evoked synaptic vesicle exocytosis (Martin, 1997;Montecucco and Schiavo, 1994).

Once the synaptic vesicle has fused with the presynapticmembrane, it is rapidly and efficiently salvaged by acalcium-regulated endocytotic mechanism, thus ensuringa continuing supply of synaptic vesicles for sustainedneurotransmission (von Gersdorff and Mathews, 1994).Vesicle retrieval is thought to be mediated by cytoplasmicproteins such as dynamin I, the activity of which seems tobe regulated by cytoplasmic Ca21 (McClure and Robinson,1996). Dynamin is thought to form a constricting collararound the invaginated membrane resulting in vesicle

Grant sponsor: National Health and Medical Research Council (Austra-lia); Grant sponsor: Australian Research Council; Grant sponsor: Rama-ciotti Foundation; Grant sponsor: University of Queensland Foundation;Grant sponsor: Faculty of Medicine, University of Sydney.

*Correspondence to: W.D. Phillips, Department of Physiology (F13),University of Sydney, Camperdown NSW 2006 Australia.E-mail: [email protected]

Received 1 October 1998; Revised 16 February 1999; Accepted 4 March1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 410:531–540 (1999)

r 1999 WILEY-LISS, INC.

budding (Hinshaw and Schmid, 1995; Takei et al., 1995;Sweitzer and Hinshaw, 1998). The role of dynamin I insynaptic vesicle recycling has been most convincinglydemonstrated in Drosophila, which contains a single dyna-min gene. A temperature-dependent dynamin mutantcalled shibire is paralysed when the temperature is raisedso as to inactivate dynamin within its cells. Studies withthese flies suggest that dynamin-dependent, Ca21-regu-lated retrieval of synaptic vesicle membrane occurs within30 seconds of transmitter release at sites immediatelyadjacent to the active zones of exocytosis (Koenig andIkeda, 1996).

Rodents express three closely related dynamin genes,the specific functions of which have not been fully defined(Nakata et al., 1993; Robinson et al., 1993; Sontag et al.,1994). Dynamin I is expressed predominantly in brain andspinal cord, dynamin III most strongly in the testis,whereas dynamin II is ubiquitously expressed. The threerat dynamins are most closely related at their amino-termini and diverge at their C-termini. Two alternativesplicing sites have been identified in each of these dy-namins (Robinson et al., 1993; van der Bliek et al., 1993;Sontag et al., 1994; Cook et al., 1996). For dynamins I andIII, splice sites are found in the mid-region between theGTPase and pleckstrin homology domains and at theproline-rich C-terminus. Alternative RNA splicing at theC-terminus gives rise to small differences in polypeptidesequence, molecular weight, and possibly function.

In the case of dynamin I, the most extensively studiedisoform, the proline-rich C-terminus is involved in protein-protein interactions within the nerve terminal. For ex-ample, this region interacts with amphiphysin (I and II)which in turn interacts with nerve terminal proteins suchas synaptojanin and adaptin protein 2 (Wang et al., 1995;Leprince et al., 1997; Ramjaun et al., 1997). These interac-tions are thought to be critical for dynamin I’s targeting tothe plasma membrane at a late stage in endocytosis(McClure and Robinson, 1996). Furthermore, microinjec-tion of a C-terminal dynamin I peptide or amphiphysin’sSH3 domain into living nerve terminals inhibited synapticvesicle endocytosis (Shupliakov et al., 1997).

Alternative splicing of the C-terminus of mouse dyna-min I gives rise to three cDNAs that encode uniqueC-terminal peptide sequences. The first two of thesesplicing forms to be described have predicted molecularweights of 96.1 and 97.3 and have been referred to in otherspecies as dynamin 1*b and 1*a, respectively. The up-stream splicing site, represented here by a wild-cardsymbol (*), does not affect the predicted molecular weightof the protein (Robinson et al., 1993; van der Bliek et al.,1993). A third C-terminal splicing form of 97.5 kDa has sofar been described only in mouse and will be referred tohere as the 21 amino acid C-terminal form (see Fig. 3A).

In the present study, we have examined the expressionand distribution of syntaxin and dynamin in the embry-onic spinal cord and muscle to test whether they partici-pate in the earliest stages of neuromuscular synapseformation. The first neurons to be generated in the spinalcord are the somatic motor neurons, and this generationoccurs during the last half of embryogenesis in the mouse(Nornes and Carry, 1978). The formation of synapticconnections between motor neurons and muscle cells be-gins at embryonic day (E) 13.5 (Bennett and Pettigrew,1974; Dennis et al., 1981; Noakes et al., 1993). Conse-quently our study is based on the developmental period

from E10 through to postnatal day (P) 0. Our resultssuggest that syntaxin functions at newly formed synapsesbut that dynamin I and dynamin III may play a role in thelater embryonic and postnatal stages of synaptic matura-tion.

MATERIALS AND METHODS

Animals

Embryos were obtained from pregnant C57BL6 femalemice (Jackson Laboratories, Bar Harbor, ME). Mice werekilled by overdose of Nembutal (30 mg; Boehringer Ingel-heim, Ridgefield, CT) followed by cervical dislocation.Embryos were killed by decapitation. The day on which avaginal plug appeared was called E0. The head-to-rumplength of each embryo was found to correspond to itsdevelopmental age according to Theiler (1989). For eachdevelopmental stage examined, at least three embryosfrom separate litters were examined. These procedureswere approved by the animal care and ethics committees ofthe authors’ institutions.

Antibodies and immunoblotting

Rabbits were immunised with a glutathione-S-transfer-ase-rat syntaxin IA fusion protein (p35; Bennett et al.,1992) that was gel purified from the bacterial extract. Thespecificity of antisyntaxin antiserum for syntaxin I wastested by probing immunoblots of total membrane prepara-tions (post-nuclear P100 pellets) from rat and mousebrain, mouse spleen and mouse heart (Fig. 1). Membraneprotein concentrations were determined by the BradfordMethod (Bio-Rad, Richmond, CA). Membrane proteinsextracted in 1% sodium dodecyl sulphate (SDS) in thepresence of reducing agents were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitro-cellulose membrane. After preblocking overnight at 4°Cwith phosphate buffered saline (PBS, pH 7.4) containing5% non-fat dried milk powder (blotto), membranes wereincubated for 2.5 hours at room temperature in a 1:1,600dilution of antisyntaxin antiserum diluted in Tris saline(TS; 0.9% NaCl, 10 mM Tris, pH 8) containing 2.5% blotto.After washing for 30 minutes in three changes of TScontaining 0.05% Tween 20, membranes were incubatedfor 1.5 hours in affinity purified alkaline phosphate-conjugated goat anti-rabbit IgG (Silenius, Melbourne,Australia). Membranes were then washed twice in TS,0.05% Tween 20, then once in TS. Finally bands werevisualised by using a nitro blue tetrazolium/bromo-4-chloro-indolyl phosphate formulation (Sigma, St. Louis, MO).

The remaining antibodies used in this study were asfollows. Rabbit anti-rat dynamin I (DP-4.1), a polyclonalantiserum, was a gift of Dr. Phil Robinson (Powell andRobinson, 1995). Mouse anti-Ommata electric organ-SV2monoclonal (Buckley and Kelly, 1985; Developmental Hy-bridoma Bank, Iowa) was used to detect synaptic vesicles.A cocktail of affinity-purified rabbit anti-bovine 200-kDaneurofilament (Sigma) and rabbit anti-human synaptophy-sin (Dako, Clostrup, Denmark) antibodies was used todetect the full extent of motor axons and their terminalendings. Finally, rhodamine conjugated a-bungarotoxin(Rh-a-Btx; Molecular Probes, Eugene, OR) was used todetect acetylcholine receptors (AChRs) in developing

532 P.G. NOAKES ET AL.

muscle. Affinity purified, fluorescently labelled secondaryantibodies were obtained from Silenius (Australia) andSigma.

Immunofluorescent staining

Cryostat sections of embryonic mouse intercostal oradult diaphragm musculature were double labelled withRh-a-Btx (1:200) in combination with one of the followingprimary antibodies: antisyntaxin (1:500), anti-SV2 (1:20),antineurofilament (1:200) plus antisynaptophysin (1:50)cocktail, or antidynamin I (1:100). The primary antibodyincubation was overnight at 4°C. After washing threetimes with PBS over at least 30 minutes, sections wereincubated with affinity purified, fluorescein-conjugatedsecondary antibody for 1 hour at room temperature. Rhoda-mine a-bungarotoxin was included in both the primaryand secondary antibody incubation steps. All antibodiesand Rh-aBtx were diluted in 1% bovine serum albumin(BSA) in PBS. Sections were examined on either anOlympus BX60 fluorescence microscope that used a 1003NA 1.3 oil immersion objective, or a Zeiss-Axiophot, Bio-Rad MRC 600MS confocal microscope, that used 1003 NA1.3 oil immersion lens. The fraction of Rh-aBtx-stainedAChR clusters that displayed colocalised patches of stain-ing for each of the above proteins was scored (by eye) byswitching back and forth between the two fluorescencechannels.

All immunostaining protocols were accompanied bystaining controls. Controls included the following: preim-mune serum control for antisyntaxin I or dynamin I;coincubation with either syntaxin I or dynamin I peptidewith the respective antibodies to block staining and checkfor specificity; omission of the primary antibody; deletionof both primary and secondary antibodies. The possibilitythat optical cross-bleed could lead to false-positive resultswas tested on control sections where either Rh-aBtx or theprimary antibody (antisyntaxin, or antidynamin) was de-leted from the incubation.

Processing of digital images

Figures 4, 5, and 6 were constructed from digital imagesexported from the Bio-Rad MRC 600MS laser scanningconfocal microscope. Images were the Kalman average offive laser scans. Files in Bio-Rad file format were con-verted to TIFF files. Without further enhancement, thedigital images were then photographed onto Kodak Plus Xfilm by using a Lasergraphics image writer. Negatives ofeach panel were then photographically enlarged ontoIlford Multigrade paper for construction of plates. Figure 2was constructed by first scanning the original immunoblotby using a UMAX - UC 1260 scanner, with a resolution of700 dots per inch. The resulting digitized image file wasopened with Adobe Photoshop and the contrast was in-creased so as to show all bands present on the blot. Theimage was then labelled. No further alterations wereperformed. It was then printed on an Epson Stylus color600 ink jet printer that used Epson photographic-qualitypaper.

RT-PCR and Southern blotting

RNA was prepared from spinal cords by a modification ofthe acid-phenol method of Chomczynski and Sacchi (1987).

Reverse transcription was primed with random hexanucleo-tides, and PCR was carried out with 1 µg of RNA accordingto Perkin-Elmer Cetus (Cetus Corp, Norwalk, CT). Twomicrolitres (one-tenth) of the cDNA preparation was usedas the template for each PCR amplification reaction.Dynamin I primers were designed to flank the 38 alterna-tive splicing site. The sense primer corresponds to nucleo-tides 654–675 of GenBank sequence L29457 (a partialcDNA for mouse dynamin I). The antisense primer corre-sponds to nucleotides 743–766 of the same cDNA. Theseprimers recognise the three mouse dynamin I cDNAslisted in GenBank (Fig. 3A). A nomenclature for thealternative splicing forms of dynamin I has been proposed(Robinson et al., 1993; van der Bliek et al., 1993; Sontag etal., 1994). Dynamin I*a and I*b represent the previouslypublished long (20 amino acid) and short (7 amino acid)inserts at the 38 splice site, respectively. The asterisk is awild-card symbol indicating that the upstream splicingsite (which may contain either an ‘‘a’’ or ‘‘b’’ insert) is notspecified. Splicing at this upstream site is not distin-guished by our primers. The third C-terminal splicingform is referred to here simply as the 21 amino acidC-terminal form. Dynamin II was amplified with primersbinding to nucleotides 334–355 (sense) and 2995–3018(antisense) of mouse dynamin II (GenBank: L31398).Dynamin III was amplified by using primers designed torecognise nucleotides 335–355 (sense) and 631–652 (anti-sense) of rat dynamin III (GenBank: D14076). The primersfor dynamin II and III do not flank splicing sites, therefore,do not distinguish between possible alternative splicingforms. All dynamin reactions were amplified for 40 cycles.Actin primers corresponded to nucleotides 46–67 of thecoding strand and 614–636 of the antisense stand of mouseb-actin (Alonso et al., 1986). Actin reactions were amplifiedfor 30 cycles. PCR products were separated by electropho-resis on a 3% agarose gel and were visualised by ethidiumbromide fluorescence.

Southern blot analysis was carried out according toManiatis et al. (1989). Bands were transferred overnightto nylon membrane and probed with a 32P end-labelledoligonucleotide (58-AGCCGATCGGGTCAG-38) that recog-nises a sequence within the amplified fragment common toall three reported C-terminal alternative splicing forms ofmouse dynamin I. The developed autoradiograph wascontact printed onto photographic paper to produce awhite-on-black image of the bands. The dynamin IIIfragment was sequenced on an ABI PRISM (model 377,version 3.0) automatic sequencer (Sydney University andPrince Alfred Macromolecular Analysis Centre).

RESULTS

Expression of syntaxin and dynaminisoforms in the developing spinal cord

Syntaxin protein was detected by immunoblotting withrabbit antiserum raised against syntaxin IA (antisyntaxin;see Materials and Methods section). Antisyntaxin detecteda doublet of bands in rodent brain and spinal cord (Figs. 1,2) of approximately 33 kDa. The same doublet was alsostained by HPC1, a monoclonal antibody specific for syn-taxin I (data not shown; Inoue and Akagawa, 1993). Inoueand coworkers ascribed this doublet of bands to rodentsyntaxin IA and IB, the closely related products of two

DYNAMIN AND SYNAPSE FORMATION 533

neurally expressed genes. Other members of the syntaxingene family are more divergent in sequence (Bennett et al.,1993; Bock et al., 1996; Advani et al., 1998; Tang et al.,1998; Wong et al., 1998). Heart and spleen, which expressthe more ubiquitous syntaxins, did not produce a bandwith our antisyntaxin antiserum, even with 20-fold greaterloading of membrane protein (Fig. 1, lanes 3 and 4,respectively). Controls consisting of preimmune serum orserum preabsorbed with the immunogen produced nobands (Fig. 1, lanes 5 and 6, respectively). Although theseresults suggest that the antisyntaxin antibody is specificfor syntaxin I, we cannot definitively exclude the possibil-ity that it cross-reacts with more recently cloned membersof the syntaxin gene family.

Syntaxin could be detected (albeit weakly) in homoge-nates of the neural tube at E10 (Fig. 2, top panel). This isat the stage when the first motor neurons are born (Nornesand Carry, 1978). At E12, immunofluorescent stainingrevealed prominent antisyntaxin staining throughout theventral portion of the spinal cord including the territory ofthe immature motor neurons (data not shown). By E16–E18, both syntaxin bands became more prominent, corre-sponding to the age when axonal branching and synapseformation within the skeletal muscle reaches its peak(Dennis et al., 1981; Noakes et al., 1993). Thus, syntaxinexpression is detectable in the spinal cord from the earlieststages of neurogenesis, and well before the formation ofsynapses between the motor neurons and target musclecells begins.

Dynamin I expression was followed by using previouslycharacterised antibodies against rat dynamin I (Powelland Robinson, 1995). A single dynamin I band of about 96kDa (the predicted size for dynamin I) could be detected as

early as E10 (Fig. 2, bottom). The period from E15 to E18was marked by the presence of several closely spacedbands straddling the 96-kDa marker band. Between E15and P0, the slightly faster migrating of these bandsbecame progressively stronger, whereas the slower migrat-ing bands waned (Fig. 2, bottom).

Alternative RNA splicing at the C-terminus gives rise todynamin I isoforms that differ slightly in molecular weight(van der Bliek et al., 1993; Sontag et al., 1994). In the caseof the mouse, three C-terminal splice variants have beenreported in GenBank. The predicted protein products ofthese mRNAs would have distinctive C-termini of 7 (1*b),20 (1*a), and 21 amino acids (Fig. 3A). Two of these splicevariants (1*a, and 1*b) correspond to previously character-ised human and rat C-terminal variants (Robinson et al.,1993; van der Bliek et al., 1993). To see whether changes indynamin I splicing might explain the differences in appar-ent molecular weight of the protein seen on immunoblots(Fig. 2), cDNAs were prepared from spinal cord andamplified by RT-PCR. Oligonucleotide primers flankingthe alternatively spliced C-terminal region were designedto distinguish C-terminal splicing forms according to thesize of the amplified fragment. Southern blotting with aprobe specific to dynamin I identified PCR products withsizes corresponding to those expected for dynamin I*b(146-bp band), dynamin I*a (113-bp band), and the 21amino acids C-terminal form (680-bp band; Fig. 3C). Oneother band, of approximately 500 bp, was also detected(Fig. 3C). This band may represent an, as yet uncloned,

Fig. 1. Characterisation of antisyntaxin antibodies by immunoblot-ting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Antiserum raised against a fusion protein containing recombinant ratsyntaxin IA detected the expected doublet of bands at about 33 kDa incrude membrane preparations from rat brain (lane 1; 0.3 µg mem-brane protein) and mouse brain (lane 2; 0.3 µg). No reactivity wasdetected for crude membranes from heart (lane 3; 6 µg) or spleen (lane4; 15 µg). Controls, in which brain membranes (0.3 µg) were probedwith antiserum that had been preabsorbed with immunogen (lane 5)or with preimmune serum (lane 6) were negative.

Fig. 2. Immunoblotting analysis reveals changes in syntaxin anddynamin protein expression in the embryonic spinal cord. Top: Syn-taxin could be detected in homogenates of neural tube at embryonicday (E) 10 and at all subsequent ages of spinal cord developmentexamined (E12 to postnatal day 0, P0). The far left lane shows adultbrain membrane (Ad) for comparison. The E16 homogenate revealedadditional, faster migrating bands. Together with the ,33-kDa dou-blet, these faster migrating bands disappeared when the antiserumwas preabsorbed with immunogen (not shown), thus, they may arisefrom partial proteolysis of syntaxin. Bottom: Dynamin bands migrat-ing in the vicinity of the 96-kDa marker were detected with antidyna-min I antiserum at the neural tube stage and at each subsequent stageof development (E10–P0). Several bands of slightly differing mobilitycould be detected straddling the 96-kDa marker. The period E15–E18was marked by a transition toward the more rapidly migrating band(lower band at E15, E16). No band was detected if antidynamin Iantibody was deleted from the primary incubation (Contr.).


alternative splicing form. The 500-bp band was not furtherinvestigated.

Although we have not attempted to quantitate thevarious mRNAs, changes in the intensity of the bandsrelative to each other provide an indication of developmen-tal changes in the relative levels of the different splicingforms of dynamin I. At E10, all the dynamin I bands wereweak. They can be seen only with the greater sensitivityafforded by Southern blotting (Fig. 3C). However, thedynamin I*b band (146 bp) was strongly up-regulated inthe later period of embryogenesis relative to both dynaminI*a (113-bp band) and the 21 amino acid C-terminal form(680-bp band; Fig. 3C). By P0, dynamin I*b (146-bp band)was predominant and was the only dynamin I splicing

form clearly visible by the less-sensitive technique ofethidium bromide staining (Fig. 3B).

Dynamin I*b has a slightly lower predicted molecularweight (96.1 kDa) than either dynamin I*a (97.3 kDa) orthe 21 amino acid C-terminal splicing form (97.5 kDa).Hence, the increase in mRNA for dynamin I*b relative tothe other splicing forms may help to explain the increasedprominence of the fastest migrating dynamin protein bandduring later stages of embryogenesis (Fig. 2, bottom). Itremains possible, however, that the differences in electro-phoretic mobility of dynamin protein represent some, asyet uncharacterised, posttranslational modifications.

Although we have focused here on dynamin I, two otherdynamin genes have been identified in rodents (Nakata etal., 1993; Sontag et al., 1994). Oligonucleotide primersdesigned to specifically amplify codons 860–963 of mousedynamin II yielded the expected fragment size of 312 bp(Fig. 3B, Dyna II). The identity of this band was confirmedby the pattern of restriction fragments produced by diges-tion with the restriction endonuclease, HaeIII (data notshown). Like the PCR product for b-actin, which served asa control, the dynamin II band changed little between E10,E13, and P0, consistent with the view that dynamin II is aubiquitously expressed housekeeping protein. Mouse dyna-min III has not yet been cloned, but primers designed toamplify codons 68–174 of rat dynamin III yielded a band ofthe expected size (318 bp) from P0 spinal cord. Sequencingof this fragment showed that the predicted amino acidsequence was more than 98% identical to rat dynamin III(codons 73–167; excluding primer sequence). There wasonly one amino acid substitution, at codon 118 (valine toisoleucine). No dynamin III band was seen at E10, butstrong bands appeared at E13 and P0 (Fig. 3B, Dyna III).Thus, both dynamin III and dynamin I*b seem to bestrongly and selectively up-regulated in the spinal cordduring the later stages of embryonic development relativeother isoforms of dynamin.

Localisation of syntaxin and dynamin atnewly formed synapses in muscle

Acetylcholine receptors (AChRs) become clustered post-synaptically within a few hours of nerve-muscle contactand, thereby, serve as a marker of newly formed neuromus-cular synapses (Chow and Cohen, 1983; Noakes et al.,1993). Transverse sections of adult muscle revealed AChRclusters labelled with rhodamine-a-bungarotoxin (Rh-aBtx) with corresponding antisyntaxin (FITC) immuno-staining (Fig. 4A,B). AChR clusters could first be detectedin the developing intercostal musculature at E13.5–E14(Fig. 4E). No AChR clusters could be found in embryos atE12 or E13 (Fig. 4G). Double labelling with antibody toN-CAM confirmed the presence of premuscle cells at thisage (Fig. 4H). This finding confirms that, although pre-muscle cells are present at E12–E13, the first synapsesformed in this tissue at E13.5–E14 (Noakes et al., 1993). Acocktail of antineurofilament and antisynaptophysin dem-onstrated that nerve terminals were present and coloca-lised with the newly formed AChR clusters by E14 (Fig.6E,F). Our results, thus, are consistent with previousstudies in which the first synapses in the intercostalmusculature appeared at E13.5–E14 (Noakes et al., 1993).

Many of the newly formed AChR clusters present at E14already had colocalized (presumably presynaptic) antisyn-taxin immunostaining (Fig. 4E,F). Antisyntaxin stainingadjacent to AChR clusters was a characteristic feature of

Fig. 3. Changes in the expression of dynamin mRNA during spinalcord development. Dynamin isoforms were detected by reverse tran-scription and polymerase chain reaction (RT-PCR). PCR fragmentsseparated by agarose gel electrophoresis were visualised by ethidiumbromide staining (B) and Southern blotting (C). Oligonucleotideprimers were designed to specifically amplify either dynamin I, II, orIII, or b-actin. Dynamin II primers detected the predicted band size(312 bp) throughout the embryonic period (Dyna II). Primers forb-actin (used as a control to test for RNA integrity) yielded a strongband of the predicted size at each age examined (591 bp; Actin).Primers specific for dynamin III detected the predicted band of 318 bponly at embryonic day (E) 13 and postnatal day (P) 0 (Dyna III).A: Primers for dynamin I were designed to detect all three alterna-tively spliced forms of dynamin I as outlined (and see text). After 40cycles of amplification, ethidium bromide staining detected dynaminI*b (146-bp PCR band) in spinal cord at E13 and P0. Southern blottingthat used an oligonucleotide probe specific for dynamin I confirmed theidentity of the 146-bp dynamin I*b band and revealed the presence ofweaker bands corresponding to dynamin I*a (113 bp) and the 21 aminoacid C-terminal splicing form (680 bp; C). An additional, unidentifiedband of ,500 bp may represent an, as yet uncloned, alternativesplicing form. The dynamin I*b isoform was up-regulated between E10and P0 relative to the other dynamin I splicing forms.


Fig. 4. Syntaxin is present at the neuromuscular synapse from theearliest stages of synapse formation. Double fluorescence labelling ofacetylcholine receptor (AChR) clusters with rhodamine-a-bunga-rotoxin (Rh-a-Btx; A,C,E,G,I) and antisyntaxin (B,D,F,L) in the adultdiaphragm muscle (A,B) and intercostal musculature at embryonicdays 17 (C,D,I–L), 14 (E,F), and 13 (G,H). Antisyntaxin staining wascolocalised with most AChR clusters from the first day on which AChRclusters appeared in the developing musculature (E,F). Most embryos

examined at E13 displayed no AChR clusters (G), despite the presenceof the anti–N-CAM-stained premuscle cell cells (H) within the presump-tive muscle fields. Only weak staining was observed in the fluoresceinisothiocyanate (syntaxin) channel when antisyntaxin was preab-sorbed with the immunogen fusion-protein (J compared with I).Similarly, when Rh-a-Btx was deleted from the staining solution, nostaining was seen in the rhodamine channel (K compared with L).Scale bar 5 10 µm in I (applies to A–L).

developing synapses at each subsequent stage examined(Fig. 4C–F). Furthermore, counts from sections throughthe intercostal musculature at E13.5, E14, E15, E17,confirmed that antisyntaxin staining became concentratedat the majority of AChR clusters within 0.5 days of the firstappearance of AChR clusters (Table 1). Double labellingwith Rh-aBtx and antibodies to either SV2 or synaptophy-sin confirmed that synaptic vesicle proteins were alsoconcentrated at sites of AChR clustering (Fig. 6). Likesyntaxin, SV2 became concentrated at newly formed syn-apses (Table 1).

Antidynamin staining was also found to be concentratedat AChR clusters on adult intercostal muscles (Fig. 5A,B).However, unlike staining for syntaxin and SV2, antidyna-min staining only became concentrated at most neuromus-cular synapses by E17 (Fig. 5C,D). Antidynamin stainingwas only occasionally detected with AChR clusters at E14.Most AChR clusters at stages E13.5 to 15 displayed noantidynamin staining (Fig. 6A,B; Table 1). This was de-spite the presence of the nerves stained with antineurofila-ment/antisynaptophysin, syntaxin, and SV2 at these primi-tive synapses (Figs. 4E,F, 6C–F).

DISCUSSION

Coupling of vesicle exocytosis and recapture is necessaryfor the development of a fully functional chemical synapse.Two molecules thought to be essential components of themachinery of synaptic vesicle exocytosis and recapture,respectively, are syntaxin I and dynamin I (Montecuccoand Schiavo, 1994; McClure and Robinson, 1996). The goalof this study was to determine the expression and subcellu-lar localisation of these molecules during the periods ofmotor neuron development and subsequent neuromuscu-lar synaptogenesis in the mouse embryo. We found thatdynamin I and III were up-regulated together with syn-taxin in the spinal cord during the period of synaptogen-esis. Surprisingly, our immunofluorescent studies showedthat the localisation of antidynamin I staining at neuromus-cular synapses occurred several days later in synapticmaturation than syntaxin and SV2.

Expression of dynamin mRNAs

The isoforms of dynamin responsible for synaptic vesiclerecapture at the neuromuscular synapse have not been

unequivocally identified. Dynamin I is expressed stronglyand exclusively in the adult nervous system, making it thefocus of numerous studies (McClure and Robinson, 1996;Urrutia et al., 1997). Several dynamin I cDNAs have beencloned and are thought to be derived from alternative RNAsplicing (Robinson et al., 1993; van der Bliek et al., 1993).Alternative splicing occurs at a site near the middle of thecoding sequence and at a second site at the C-terminus.The C-terminal splicing site is of particular interest be-cause of its proximity to the proline-rich binding sites forvarious SH3 domain containing proteins (Ringstad et al.,1997). These proline-rich binding sites are implicated inthe targeting and endocytotic function of dynamin I (Oka-moto et al., 1997; Shupliakov et al., 1997; Urrutia et al.,1997). Our results provide clues as to which of theseisoforms may be most important for development of syn-apses.

By using sensitive and specific techniques, Southernblotting of RT-PCR products, we detected the presence ofboth dynamin I*a and I*b mRNAs in the spinal cord asearly as E10 (Fig. 3C). Southern blotting also detected twoother dynamin I bands. The first of these (,680 bp)presumably represents a previously cloned splicing formwith a unique 21 amino acid C-terminus (A. Stief and H.van der Putten, 1994; GenBank accession number L31397).The second, a band of approximately 500 bp, did notcorrespond to any of the annotated mouse dynamin Isplicing forms so far listed in GenBank. E10 is when thefirst formed motor neurons appear in the ventral horn ofthe spinal cord in mouse (Nornes and Carry, 1978). Duringthe later stages of embryogenesis, the mRNA for dynaminI*b, was strongly up-regulated relative to the other splicingforms (Fig. 3B,C). Thus, dynamin I*b, with its uniqueseven amino acid C-terminus, may be important for synap-tic maturation.

Dynamin III expression was originally thought to beexclusive to the testes (Nakata et al., 1993). Recently,however, Cook and coworkers (1996) have shown that it isup-regulated in the developing postnatal rat brain. Here,we show that up-regulation of dynamin III occurs in thespinal cord during embryonic development. Dynamin IIImRNA was up-regulated between E10 and E13, relative todynamin II and b-actin. This expression pattern is alsocoincident with neuronal differentiation and synapse for-mation (Nornes and Carry, 1978; Stanfield, 1992; Auclairet al., 1993; Kudo et al., 1993), consistent with a role fordynamin III in central and/or peripheral synapse forma-tion.

Expression and localisation of dynamin andsyntaxin in the developing spinal cord and at

neuromuscular synapses

Immunoblotting detected dynamin I protein in develop-ing spinal cord from E10 onward (Fig. 2). At the earlystages (E10–E12), we observed a single dynamin I band.By E15, several dynamin I bands were found straddlingthe 96-kDa marker band. By P0, the fastest migratingband came to be dominant. Alternative splicing at thecentral site in dynamin I makes no difference in theprotein molecular weight (Robinson et al., 1993; van derBliek et al., 1993). However, C-terminal splicing of dyna-min I gives rise to dynamin I protein products that differ

TABLE 1. Colocalisation of Presynaptic Antigens With AcetylcholineReceptor Clusters During Neuromuscular Synapse Formation1

AgeSyntaxin/

aBtx SV2/aBtxSynaptophy/

aBtxDynamin I/

aBtx

Adult 35/38 77/83 126/126 144/169(n 5 1) (n 5 2) (n 5 3) (n 5 5)

% colocalised 92 93 99 85E17 88/110 222/227 137/140 261/378

(n 5 4) (n 5 4) (n 5 4) (n 5 5)% colocalised 80 98 98 69

E15 72/104 70/96 25/26 34/112(n 5 6) (n 5 4) (n 5 1) (n 5 5)

% colocalised 69 61 96 30E14 81/127 30/46 46/50 8/145

(n 5 6) (n 5 7) (n 5 4) (n 5 5)% colocalised 64 61 92 3

E13.5 1/40 1/37 4/7 0/54(n 5 1) (n 5 4) (n 5 4) (n 5 4)

% colocalised 3 3 57 0E13 No acetylcholine receptor clusters

1Counts of the fraction of rhodamine-a-bungarotoxin (Btx)–labelled acetylcholinereceptor clusters that displayed colocalized staining for syntaxin, SV2, synaptophysin/neurofilament and dynamin (see Materials and Methods section). E, embryonic day.


slightly in size: 96.1-kDa for dynamin 1*b; 97.3-kDa fordynamin 1*a, and 97.5-kDa for the 21 amino acid C-terminalsplicing form of dynamin I (see above). Thus, it is possible thatthe closely spaced bands seen in our immunoblots may repre-sent these different C-terminal splicing forms. Alternatively,these bands could represent posttranslational modifications toone or more of these dynamin I isoforms. For example,dynamin I is phosphorylated in nerve terminals by proteinkinase C (Robinson et al., 1993). Nevertheless, the parallelincreases in the dynamin I*b mRNAand the fastest-migratingdynamin I protein band favours the idea that dynamin I*bprotein levels follow the developmental up-regulation of theappropriately spliced mRNA.

The relationship between dynamin I and functionalmaturation of the synapse can be best gauged through

immunostaining of the developing neuromuscular syn-apse. The observations outlined here support the ideathat dynamin I is involved in synaptic maturation. Al-though dynamin I mRNA and protein were detected in thespinal cord as early as E10, the appearance of immunostain-ing for dynamin I at the neuromuscular junction laggedsome 72 hours behind that of other presynaptic antigenssuch as syntaxin and SV2 (see Table 1). Excitationaljunctional potentials can be detected as soon as nervemuscle contact is made but these early synapses respondpoorly to repetitive activation; robust synaptic transmis-sion develops only after birth (Bennett and Pettigrew,1974; Dennis et al., 1981). The delayed accumulation ofdynamin I at synapses may contribute to this functionalmaturation.

Fig. 5. Antidynamin I staining at synapses in adult (A,B,E–H) andembryonic day 17 (C,D) skeletal muscle. Double fluorescence labellingof acetylcholine receptor (AChR) clusters with Rh-a-Btx (A,C,E) andantidynamin (B,D,H) shows colocalisation of dynamin with AChRclusters. Control sections incubated with preimmune serum showed

no fluorescein isothiocyanate staining (F) compare with Rh-a-Btx (E).Antiserum absorbed with dynamin peptide (G) showed reduced antidy-namin staining (compare with nonabsorbed [H]). Scale bar 5 25 µm inD (applies to A–H).


ACKNOWLEDGMENTS

We thank Dr Mark Bennett for the expression plasmidfor syntaxin IA, Dr Phil Robinson for antidynamin antibod-ies, and Dr Nicholas Lavidis for his critical reading of themanuscript. P.G.N. and W.D.P. were supported by grantsfrom the National Health and Medical Research Council(Australia) and the Australian Research Council. P.G.N.was further supported by Ramaciotti Foundation and theUniversity of Queensland Foundation. S.L. was supportedby a postgraduate scholarship from the Faculty of Medi-cine, University of Sydney.

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