the synapsin ( syn ) family consists of three neuronal phosphoproteins encoded in mammals by distinct genes ( i.e. , syni , ii , and iii ) .
although the biology of syni and ii has been extensively studied , the functions of syniii are still largely uncharacterized . in  vitro studies
show that syniii is involved in axonal elongation and growth - cone formation during early neurodevelopment ( feng et  al . , 2002 ; ferreira et  al . , 2000
) . accordingly , syniii is the earliest expressed syn isoform during development ( porton et  al .
moreover , single - nucleotide polymorphisms in syniii have been linked to neurodevelopmental disorders ( i.e. , schizophrenia ; chen et  al . , 2009 ) .
similar to the other family members , syniii is a substrate for protein kinases ( pks ) ( cesca et  al . , 2010 ) .
remarkably , many pathways that are essential for the migration and lamination of cortical neurons during brain development involve pks and lead to the phosphorylation of specific substrates ( ayala et  al . , 2007 ) .
thus , the developmental expression of syniii , its role in neuronal developmental processes in  vitro , and its phosphorylation profile suggest that it may be a downstream effector in neuronal migration . here , we showed that syniii is involved in neocortical development in  vivo ; specifically , both the knockdown ( kd ) of syniii and  its genetic deletion lead to defective radial migration and orientation of layer ii / iii pyramidal neurons ( pns ) .
proper development requires syniii phosphorylation by cyclin - dependent kinase-5 ( cdk5 ) , putting syniii downstream of the semaphorin-3a ( sema3a)-signaling cascade .
to investigate the role of syniii in cortical development , we first confirmed its expression in the rat brain cortex at developmental stages ( supplemental results ; figures s1a 
subsequently , we designed two short - hairpin ( sh)rnas against syniii  but not syni / ii ( figures s2a and s2b)to examine the effects of syniii kd on the radial migration of newly generated cortical pns in  vivo . using in utero electroporation ( iue ) at embryonic day 17 ( e17 ) , we expressed active shrnas ( shrna no . 1 and shrna no .
2 ) or a control scrambled shrna vector ( shrnascr ; figures s2a and s2b ) in  a subpopulation of neural progenitors that would normally migrate to layer ii / iii of the somatosensory cortex ( dal maschio et  al . , 2012 ) .
we analyzed the radial migration of layer ii / iii pns derived from shrna progenitors at e21 and postnatal day 7 ( p7 ) ( the first and last time points during the peak of syniii expression , respectively ) as well as at p14 ( the first time point at which syniii expression begins to be endogenously downregulated ; see figures s1a and s1b ) . at e21 ,
control shrnascr cells were primarily found in the cortical plate ( cp ) and intermediate zone ( iz ) , whereas only few  cells remained in the ventricular zone / subventricular zone ( vz / svz ) ( figures 1a and 1b ) .
the migrating pns located at the iz expressed syniii at e21 ( figures s1 g and s1h ) .
interestingly , we observed a significant delay in radial migration when either shrna no . 1 or no .
2 was electroporated ( figures 1a and 1b ) . as the effect on migration was larger in shrna no .
1 experiments , we performed all subsequent experiments with this construct ( shrna onward ) . at p7 , almost all shrnascr cells reached cortical layer ii / iii , whereas many shrna cells were misplaced in deep layers ( figures 1c and 1i ) .
the severity of misplacement was proportional to the severity of syniii downregulation ( supplemental results ; figure  s2e ) . to investigate whether the effect of syniii kd was long lasting , we examined neuronal migration at p14 , when endogenous expression of syniii is low ( see figures s1a and s1b ) .
we found that syniii shrna cortices exhibited ectopic cells at p7 ( figures 1c , 1h , and 1i ) and at p14 ( figures s3d and s3e ) .
syniii downregulation did not affect neuronal identity , proliferation , or radial glia ( rg ) scaffold organization ( supplemental results ; figures s2c , s2d , and s2f ) .
cell migration , orientation , and polarization share molecular mechanisms , and often the same proteins are involved in all three processes ( chen et  al . , 2008 ; polleux et  al . , 2000
to further explore the role of syniii in cortical development in  vivo , we investigated whether its kd affected the orientation of pns in the layer ii / iii .
control cells were typically characterized by an upright ( ur ) orientation with a pyramid - shaped cell body , one thick apical dendrite directed toward the pial surface , and one axon pointing to the ventricle .
interestingly , 20% of syniii shrna pns were misoriented in layer ii / iii ( figure  1d ; polleux et  al . , 2000 ) , 10% of pns were  inverted ( in ) , and 10% of pns were horizontal ( hor ) ( figures 1f and 1 g ) .
similar results were obtained at p14 ( figures s3a  s3c ) . given the common mechanisms involved in migration and orientation ( chen et  al . , 2008 ) , we expected a larger effect on the orientation of ectopic cells .
indeed , 1/3 of ectopic cells depleted of syniii were in ( figures 1e and 1h1j ) .
when we divided the cortical region where the ectopic cells were located ( from layer iv to layer vi ) in two equal parts , we found that , whereas 65% of the ectopic cells were located in the upper half , the in ectopic cells were evenly distributed in the two halves , suggesting that the cortical location and neuronal orientation were not causally linked ( figure  1k ) .
moreover , when we immunostained against the -tubulin centrosomal marker , we found that the centrosome colocalized with the apical dendrite regardless of cell orientation in both shrnascr and syniii shrna cells ( figure  s4a ) . whereas syniii regulated the morphological maturation of pns both in the axonal and dendritic compartments
, it  did not affect the basic electrophysiological proprieties of pns ( supplemental results ; figures s3f  s3l and s4c  s4e ) . finally , we investigated whether syniii kd also affected the orientation and migration of pns in deep cortical layers ( i.e. , iv / v ) by performing iue at earlier embryonic stages ( e15.5 ) . in p7 syniii
shrna - transfected brains , we found migration defects and significant cell misorientation similar to that observed in superficial layers ( figure  s4b ) . to exclude off - target effects ( de paula et  al . , 2007 ) , we generated a shrna - resistant syniii cdna with four silent mutations in the shrna target sequence ( rsyniii ; figure  2a ) .
after confirming rsyniii insensitivity to shrna ( figure  2b ) , we performed rescue experiments with rsyniii / shrna co - expression in e17 embryos .
the co - expression of rsyniii and shrna at a ratio of 0.5:1 rescued the delay in radial migration at e21 ( figures 2c and 2d ) as well as the number and orientation of ectopic cells ( figures 2e2 g ) ; however , the phenotype of hor and in cells in the cp at p7 was not rescued ( figures 2h and 2i ) .
it is possible that the lack of rescue in the orientation phenotype in the cp was due to the concentration gradient of syniii expression across the cortex , which shows the highest levels in the cp ( figures s1c and s1f ) . indeed , by increasing the syniii / shrna ratio to 1:1 , we achieved complete rescue of the orientation defect of layer ii / iii pns ( figures 2h and 2i ) . although syniii knockout ( ko ) mice display behavioral defects and changes in synaptic transmission , no gross brain abnormalities have been described ( feng et  al . , 2002 ; porton et  al . ,
nevertheless , no study has specifically investigated whether subtler developmental defects occur in the syniii ko somatosensory cortex . to address this issue , we first performed immunostaining with layer - specific markers ( i.e. , cux1 , layer ii / iii / iv ; ctip2 , layer v ; foxp2 , layer vi ) in somatosensory cortex slices of syniii ko and control littermates .
we did not observe any overt abnormality in cortical layering at p7 in syniii ko mice ( figure  s5 ) .
thus ,  we considered two possible scenarios : ( 1 ) given the high homology among syn isoforms , other members of the family , such as syni , which is also expressed at embryonic ages , may compensate for syniii s absence during development and ( 2 ) immunostaining with layer - specific markers may not be sufficiently sensitive to detect mild migration / orientation defects . to investigate possibility ( 1 )
, we performed co - electroporation of syni with syniii shrna and assessed the migration / orientation defect in p7 mouse slices .
we found that syni could not rescue syniii shrna effects , indicating that syni can not compensate for loss of syniii - specific functions during development ( figures 3a3d ) . to investigate possibility ( 2 ) , we performed iue  with a pcaggs - ires - tdtomato ( tom ) vector to visualize migrating pns in e15.5 syniii ko and wild - type ( wt ) mice . in agreement with the cortical phenotype of shrna - electroporated  animals
, we observed a misorientation of an 20% of pns at the level of the cp in all ko brains ( figures 3e3 g ) .
interestingly , in contrast to controls , in two out of seven ko animals analyzed , we observed pns in ectopic positions , with these cells occasionally exhibiting in orientation ( figures 3e and 3h ) .
while rescuing orientation defects in the cp ( figures 2h and 2i ) , the co - transfection of shrna rsyniii and shrna at a ratio 1:1 also led to ectopic cells in deep cortical layers ( figures s6a and s6b ) ; however , these cells did not exhibit orientation defects ( figure  s6c ) .
given the tight temporal regulation of syniii expression , we hypothesized that not only downregulation / deletion but also overexpression of syniii could interfere with proper radial migration of pns . to test this hypothesis
pns expressing either tom or syniii properly migrated through the cortex toward the cp ( figures 4a and 4b ) .
nonetheless , a significant percentage of syniii - overexpressing cells were misplaced at p7 ( figures 4c and 4f4h ) .
the effect of syniii on neuronal migration was long - lasting , as syniii overexpression also caused the appearance of ectopic cells at p14 ( figures s6 g and s6h ) .
thus , syniii overexpression , kd , and ko all affected migration and resulted in the ectopic placement of cells . however , syniii - overexpressing cells were all properly oriented ( figures 4c4h and s6d  s6h ) .
these effects could be due to subcellular mislocalization of syniii upon overexpression in  vivo , as we demonstrated in neuronal cultures ( figure  s6i ) .
similar to the phenotype found for syniii kd during development , cdk5 ko leads to a delay in radial migration and misoriented pns ( ohshima et  al . , 2007 ) .
by analyzing the amino acid sequence of syniii , we observed that it carries a conserved consensus sequence for cdk5 phosphorylation , with ser predicted to be the target ( songyang et  al . , 1996 ) .
moreover , we found that cdk5 was expressed in rat cortices during development as early as e15 ( figures 5a and 5b ) and that cdk5 and syniii expression are highly correlated at perinatal ages ( figure  5c ) .
interestingly , we found that syniii and cdk5 co - immunoprecipitated from lysates of rat primary cortical neurons grown for 4  days in  vitro ( div ) ( figure  5d ) , demonstrating that syniii and cdk5 physically interact .
syniii and the neuron - specific cdk5 activator p35 consistently colocalized in developing neurons in  vitro ( figure  5e ) . to test whether syniii is indeed phosphorylated by cdk5
we first generated a non - phosphorylatable syniii mutant in which ser was replaced with an ala .
when immunoprecipitated and dephosphorylated syniii or its s404a mutant was phosphorylated in  vitro by active p35/cdk5 complex ( figure  5 g ) , we observed that syniii was phosphorylated by cdk5 , whereas phosphorylation was significantly decreased by 31% in the s404a syniii mutant ( figures 5 g and 5h ) .
this result indicates that ser represents almost 1/3 of the total cdk5-mediated syniii phosphorylation . to test whether cdk5 phosphorylates syniii in  vivo , we performed liquid chromatography tandem mass spectrometry ( lc - ms / ms ) on endogenous syniii from rat brains at p1 , the peak of syniii expression . in figure  5i , the predicted m / z values of fragment ions are shown in blue or red , indicating identified b- and y - type ions , respectively .
finally , we generated a phosphorylation - state - specific antibody recognizing phospho - ser in syniii .
after testing its specificity for phospho - ser ( figure  s7b ) , we analyzed the phosphorylation of syniii on ser in  vivo .
we found that the absolute amount of phosphorylated syniii present in the cortex plateaued at p1p7 and then dropped to undetectable levels at p16p35 .
however , the fraction of phospho - syniii to total syniii was high at very early stages of development ( e15e18 ) and steadily decreased thereafter to become undetectable at p16 ( figures 5j and 5k ) . to test whether syniii is indeed a downstream effector of cdk5 during development in  vivo , we generated a pseudo - phosphorylated syniii mutant by replacing ser with an asp ( d ) , which mimics the negative charge of the phosphate group ( rsyniii s404d ) .
we next verified that both s404a and s404d rsyniii expression and subcellular distribution were similar to that of the wt protein ( figures s7c  s7e ) . when expressed alone
, rsyniii s404a caused a delay in radial migration at e21 similar to that observed in the syniii kd experiments ( figures 6a and 6b ) , indicating that this mutant may act in a dominant - negative manner on endogenous syniii .
interestingly , when overexpressed at p7 , rsyniii s404a resulted in the appearance of ectopic cells without orientation defects ( figures 6c , 6e , and 6f ) , suggesting that the presence of the endogenous syniii is sufficient to guarantee proper neuronal orientation .
consistent with the dominant - negative effect of the mutant on migration , when s404a rsyniii was co - expressed with shrna at a 0.5:1 or 1:1 ratio ( as was done for wt rsyniii ; figures 2h and 2i ) , it did not rescue the shrna - induced migration defects at either e21 ( figures 6a and 6b ) or p7 ( figures 6c and 6e ) .
moreover , the co - transfection of s404a rsyniii with shrna at either ratio did not rescue the orientation defects in pns located in the cp ( figure  6d ) or in ectopic pns ( figure  6f ) .
conversely , when co - electroporated with shrna at a 0.5:1 ratio , s404d rsyniii significantly rescued the delay in radial migration caused by syniii kd at e21 ( figures s7f and s7 g ) .
cdk5 is a downstream effector in the sema3a pathway , and alterations in this pathway at various levels all cause a phenotype similar to syniii kd in the cortex ( polleux et  al . , 2000 ; behar et  al . , 1996 ) . to test the possibility that syniii acts downstream of the sema3a - signaling cascade , we investigated syniii phosphorylation by cdk5 in 4-div cortical neurons after stimulation with 1  nm sema3a .
we observed that syniii was phosphorylated on ser only 5  min after the stimulus , and its phosphorylation significantly increased after 20 and 30  min in comparison to vehicle - treated cells ( figures 7a and 7b ) .
we next tested the causal involvement of syniii in the sema3a pathway in  vivo .
first , we investigated the expression of the sema3a receptor neuropilin 1 ( np1 ) ( chen et  al . , 2008 ) in rat brain cortices .
np1 expression was highly similar to that of syniii during development , with a maximum expression between e21 and p7 , followed by a sharp decrease immediately thereafter ( figures 7c7e ) .
moreover , as was seen for p35 , we observed a colocalization of syniii and np1 in developing neurons in  vitro ( figure  7f ) , suggesting that these three molecules may indeed be part of the same signaling cascade .
next , to investigate the sema3a / cdk5/syniii pathway in  vivo , we electroporated a shrna against np1 in rat embryos ( chen et  al . , 2008 ) . as expected ( chen et  al . ,
2008 ) , we observed a very strong effect on the radial migration of layer ii / iii pns at p7 , with only 35% of pns reaching the cp ( figures 7 g and 7h ) .
however , when we co - expressed s404d rsyniii and np1 shrna , we significantly rescued radial migration ( figures 7 g and 7h ) , with 50% more pns reaching the cp in comparison to np1 shrna - electroporated brains .
during cortical development , post - mitotic pns migrate away from the vz along rg fibers toward the surface of the cp .
on reaching the superficial layer , pns stop migrating , detach from rg fibers , and morphologically differentiate . in this study
, we showed that syniii , the syn isoform that is most precociously expressed , plays a key role in cortical development .
kd , ko , or upregulation of syniii caused defects in radial migration , with the appearance of ectopic cells at later stages .
moreover , syniii kd / ko caused significant orientation defects in ectopic pns as well as in pns that fully completed their radial migration .
interestingly , in misoriented pns , the centrosome was properly located on the side of the apical dendrite . if centrosome localization ahead of the nucleus in the direction of migration is necessary for neuronal migration ( solecki et  al . , 2009 )
, the question arises of how most of the syniii - kd - misoriented neurons completed their migration .
it is possible that syniii - kd neurons migrate along glial fibers when the leading process is directed to the pial surface and stop migrating when they change orientation .
conceivably , re - orientation of migrating pns occurs several times during migration , and  consistent with the higher levels of syniii shrna in ectopic pns  the more syniii is kd , the more migrating cells will re - orientate , resulting in a slower migration and ultimate ectopic positioning .
conversely , pns expressing lower levels of syniii shrna will reach the cp , detach from rg fibers , and , without the fiber s restriction , acquire all possible degrees of orientation before morphological maturation .
syniii kd causes the described phenotypes through a cell - autonomous mechanism , as confirmed by an intact rg scaffold in shrna - transfected brains .
the migration and orientation defects involved the sema3a - signaling cascade ; indeed , upon sema3a stimulation , syniii is phosphorylated by cdk5 . as shown by syniii phospho mutant experiments ,
/ cdk5 signaling to control neuronal migration and orientation ( figure  7i ) . given that syniii kd caused similar defects when performed in neuronal progenitors of both layer ii / iii and layer iv / v , and considering that cdk5 activity is essential for migration and dendrite development of layer v pns ( ohshima et  al . , 2007 ) , we hypothesize that the mechanism by which syniii regulates proper cortical organization is generalizable to the development of all cortical layers .
future studies should investigate whether syniii also regulates migration in other brain areas , as previously described for cdk5 ( jessberger et  al . , 2008 ) .
although many factors have been identified in recent years to be involved in cortical migration , we are still far from understanding the entire framework , and many actors await identification . among these , syniii was a good candidate .
first , the emergence of the syn3 gene correlates with the appearance of the telencephalic region during evolution ( kao et  al . , 1999 ) .
second , we demonstrated that syniii is highly expressed and phosphorylated on ser during development and has a well - defined spatial expression along cortical layers at distinct developmental stages .
finally , in  vitro data suggest a role for syniii in neuronal development ( ferreira et  al .
in particular , a tight regulation of syniii expression is necessary for proper cortical development . indeed , syniii expression peaks in a defined temporal window , beyond which it is detrimental for cortical pn development , as proven by the following facts : ( 1 ) both syniii kd and overexpression caused defects in radial migration and in the morphological maturation of pns ; ( 2 ) a shrna - insensitive rsyniii completely rescued the kd phenotypes only when co - expressed with shrna at optimal levels ; and ( 3 ) the cortical phenotype caused by syniii kd during early development was irreversible and persisted even when the expression of the protein was endogenously downregulated at later developmental stages , such as p14 ( also see the supplemental discussion ) .
data from the literature indicated no major brain abnormalities in syniii ko mice ( feng et  al . , 2002 ) , whereas our data demonstrated that acute kd of syniii by rnai profoundly affected cortical development .
interestingly , many other cases have been described where acute kd of a specific protein in a subset of neurons by iue leads to strong defects in brain development , whereas the constitutive ko of the same protein does not lead to an apparent phenotype ( e.g. , dcx , corbo et  al .
, 2003 ; p75 , lee et  al . , 1992 versus zuccaro et  al . , 2014 ; gabab receptor , schuler et  al . , 2001 versus bony et  al . , 2013 )
two main reasons could explain this finding : ( 1 ) the chronic and widespread depletion of the protein in ko mice could trigger redundancy or compensatory mechanisms by other proteins that overcome the deficits in brain development and/or ( 2 ) subtle developmental impairments in ko animals may be simply overlooked due to the complexity of the in  vivo system and/or the lack of adequate methods to study mild defects . here , we provided evidence for both possibilities . indeed , marking developing neurons with a fluorescent reporter via iue in syniii ko mice allowed us to examine their cortical development with an unprecedented focus on neuronal migration and orientation . using this technique allowed us to conclude that syniii ko animals display the same phenotype as observed in shrna - transfected brains , although to a milder extent .
because of the high homology between syniii and syni / ii , we hypothesized that they could compensate for the loss of syniii and that this compensation could result in a mild developmental phenotype in ko animals .
nevertheless , we showed that synii is not significantly expressed at perinatal ages in the cortex and that the overexpression of syni did not rescue syniii shrna - mediated effects .
it is possible that the differences between syni and syniii in terms of their amino acid sequence , location of phosphorylation sites , and spatio - temporal expression profile could account for their lack of involvement in neuronal migration and orientation . in conclusion
, we hypothesize that proteins other than syni and ii may compensate for syniii loss in ko animals , and we propose the use of iue for expressing fluorescent reporters as a means to uncover subtle developmental phenotypes in ko animals .
our data consistently show that syniii kd leads to a peculiar effect in the orientation of neurons located in the cp and also in the ectopic cells .
interestingly , the orientation defects in cortical neurons described in the literature are primarily associated with the faulty expression of genes involved in the sema3a - signaling pathway ( behar et  al . , 1996 ;
, 1998 , 2000 ; sasaki et  al . , 2002 ; shelly et  al . ,
sema3a is a secreted protein well known for its role as a guidance signal upon binding to its receptor np1 .
then , fyn activation promotes cdk5 binding to plexin a2 and eventually activates cdk5 by phosphorylating it on tyr ( figure  7i ) .
we demonstrated that syniii physically interacts with cdk5 and is a substrate for the kinase by showing that ( 1 ) syniii and cdk5 are both expressed at perinatal ages in the cortex and colocalize in developing neurons in culture ; ( 2 ) syniii and cdk5 co - immunoprecipitate ; and ( 3 ) syniii is phosphorylated by cdk5 both in  vitro and in  vivo .
the rescue experiments performed with phospho mutants in  vivo proved that ser phosphorylation has a biological significance and that the phenotype we observed in syniii - null pns is at least partially due to a missing step in cdk5 signaling .
moreover , we showed that ser phosphorylation increased upon sema3a stimulation in  vitro and that the pseudo - phosphorylated mutant s404d rsyniii partially compensated for the loss of sema3a signaling caused by np1 kd in  vivo .
thus , our data demonstrate that syniii is part of the sema3a pathway ( figure  7i ; also see the supplemental discussion ) .
little is known regarding syniii interactors . in general , syn family members can bind to synaptic vesicles ( svs ) , actin ( benfenati et  al . , 1989a , b , 1992 ) , and tubulin ( petrucci and morrow , 1991 ) through
the highly conserved a , c , and e domains ( candiani et  al .
indeed , syniii also binds svs and regulates their trafficking within nerve terminals ( kao et  al . ,
are requisites for membrane rearrangements during cell migration ( itofusa and kamiguchi , 2011 ) , syniii may participate in radial migration by the latter processes ( figure  7i ) .
the phenotypic features of syniii - kd or -overexpressing pns support this hypothesis : delayed migration , ectopic positioning , and morphologically impaired leading processes are typical of neurons that lose their contact with rg fibers ( elias et  al . , 2007 ; gupta et  al . , 2003 ; sanada et  al . , 2004 ; valiente et  al . , 2011 ) .
the dysregulation of syniii activity could indeed interfere with the cytoskeletal dynamics and membrane rearrangements involved in the formation and disassembly of cell adhesions and , consequently , interactions between migrating neurons and rg fibers ( rivas and hatten , 1995 ; also see the supplemental discussion ) . in this study
, we showed that syniii acts downstream sema3a and regulates the radial migration and orientation of pns .
neuronal migration is fundamental for the development of a fully functional brain , as demonstrated by the implication of defective neuronal migration in a number of neurodevelopmental disorders ( ayala et  al . , 2007 ) .
indeed , sema3a - signaling dysregulation in brains from individuals with schizophrenia ( eastwood et  al . , 2003 ) and a genetic linkage between syniii mutations and schizophrenia have been reported ( hall et  al .
, 2007 ; lachman et  al . , 2005 ; porton et  al . ,
moreover , as we observed in syniii kd / ko animal model brains , reduced syniii expression ( porton and wetsel , 2007 ) , abnormal cytoarchitecture , and dendritic aberrations ( arnold et  al . , 1991 ; benes et  al . , 1991
; glantz et  al . , 2000 ; mirnics et  al . , 2000 ; rajkowska et  al . , 1998 ; rosoklija et  al . , 2000 )
interestingly , the altered directionality of axonal elongation in syniii - kd neurons that we describe in the present study is consistent with the disruption of axonal fiber connectivity in schizophrenic patients ( zalesky et  al . , 2011 ) .
finally , syniii ko mice exhibit abnormal sensory motor gating in response to prepulse inhibition ( geyer et  al . , 2001 ) , similar to what is observed in patients with schizophrenia and in mouse models of schizophrenia .
thus , the clarification of the role of syniii in the sema3a pathway could be important in shedding light on the pathogenesis of neurodevelopmental diseases and further increase our knowledge of the complex mechanisms that regulate cortical development in health and disease .
for detailed cloning procedures and list of used primers , see the supplemental experimental procedures .
cos7 cells were cultured in dulbecco s mem ( dmem ) ( gibco ) supplemented with 10% fetal calf serum ( gibco ) , 1% l - glutamine , 100  u / ml penicillin , and 100  g / ml streptomycin ( biowhittaker ) and maintained at 37c in a 5% co2 humidified atmosphere .
primary cultures of dissociated cortical neurons were prepared from e18 rats and maintained in neurobasal medium supplemented with 2% b-27 , 0.5  mm glutamine , 50  u / ml penicillin , and 50  g / ml streptomycin ( invitrogen ) .
the neurons were maintained at 37c in a 5% co2 humidified atmosphere . for stimulation experiments ,
the neurons were washed with neurobasal medium and incubated for 30  min at 37c .
msema3a - fc ( 1  nm ; r&d systems ) was added to the medium and , after incubation , the cells were lysed and processed for western blot .
total cell lysates were obtained from cos7 cells 48  hr after transfection or from cortical neuron cultures at 4 div . for detailed procedures , reagents , and a list of antibodies ,
following transient transfection , cos7 cells were lysed and syniii and s404 syniii were immunoprecipitated and dephosphorylated .
the samples were then subjected to in  vitro phosphorylation . for detailed procedures and reagents ,
syniii ko mice were generated by homologous recombination ( feng et  al . , 2002 ) and backcrossed onto the c57bl/6j background for over ten generations .
all of the experiments were performed in accordance with the guidelines established by the european communities council ( directive 2010/63/eu of september 22nd , 2010 ) and were approved by the italian ministry of health .
slice histology and immunostaining were performed as previously described ( bony et  al . ,
the images were acquired using a confocal laser - scanning microscope ( tcs sp5 ; leica microsystems ) or with an epifluorescence microscope equipped with neurolucida ( microbrightfield ) software . for detailed procedures on acquisition and analysis , see the supplemental experimental procedures .
tryptic digests were performed as previously described ( ballif et  al . , 2006 ) , and extracted peptides were subjected to lc - ms / ms analysis in a linear ion trap - orbitrap mass spectrometer , as previously described ( ballif et  al . , 2008 ; buel et  al . ,
if not indicated otherwise in the legend , the numbers in parentheses on the  histograms indicate the number of processed animals .
statistical analysis between control and experimental groups  was performed with student s t test or , in the case of more than two experimental groups , with one - way anova followed by post hoc multiple comparisons . for
non - normally distributed data , anova on ranks test and non - parametric post hoc comparisons were used .
l.e.p . participated in the design of the experiments and performed the molecular biology and biochemistry experiments , iue , immunocytochemistry , immunohistochemistry , image acquisition , and data analysis . l.e.p . also wrote the manuscript and made the figures .
performed the in  vitro phosphorylation , co - ip experiment , and neuronal cell culture experiments .
 summarysynapsin iii ( syniii ) is a phosphoprotein that is highly expressed at early stages of neuronal development . whereas in  vitro evidence 
 suggests a role for syniii in neuronal differentiation , in  vivo evidence is lacking . here , we demonstrate that in  vivo downregulation of  syniii expression affects neuronal migration and  orientation . 
 by contrast , syniii overexpression affects neuronal migration , but not orientation . 
 we identify a cyclin - dependent kinase-5 ( cdk5 ) phosphorylation site on syniii and use phosphomutant rescue experiments to demonstrate its role in syniii function . 
 finally , we show that syniii phosphorylation at the cdk5 site is induced by activation of the semaphorin-3a ( sema3a ) pathway , which is implicated in  migration and orientation of cortical pyramidal neurons ( pns ) and is known to activate cdk5 . 
 thus , fine - tuning of  syniii expression and phosphorylation by cdk5 activation through sema3a activity is essential for proper neuronal migration and orientation . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions