vibrissae provide input to specific thalamic nuclei and these nuclei have well - defined connections with the somatosensory cortical region where sensory information is processed ( woolsey and van der loos , 1970 ) .
these connections have a somatotopic organization , and sensory information from each vibrissa reaches a specific barreloid in the thalamic ventrobasal ( vb ) complex and is relayed to a specific cortical barrel field in the primary somatosensory cortex . during their developmental outgrowth ,
thalamocortical axons are guided by a variety of cues ( molnr et  al . , 2012 ) .
after crossing the subpallial - pallial border , thalamocortical axons advance within the intermediate zone ( iz ) and , approaching the cortex , they accumulate below the cortical plate ( cp ) at the subplate ( ghosh et  al . ,
the interaction of thalamocortical axons with the subplate is one of the most enigmatic processes in the development of thalamocortical fibers . even in reeler or p35 knockout ( ko ) mice ,
where the subplate is aberrantly located , thalamocortical axons cross the cp toward the misplaced subplate before connecting to their final targets ( hoerder - suabedissen and molnr , 2015 ) . however , the molecular mechanisms that control thalamocortical axon - subplate interaction and ultimately  the correct targeting of thalamic projections are largely unknown . in the developing brain , bioactive phospholipids like lysophosphatidic acid ( lpa ) play important roles in cortical migration ( fukushima et  al . , 2000 ) and neuronal apoptosis ( kingsbury et  al . , 2003 ) .
these effects are mediated by lpa receptors , namely lpa1-r and lpa2-r , which are expressed in the developing brain ( kingsbury et  al . , 2003 ) .
lpa is a well - described repellent factor for axons , eventually leading to growth cone ( gc ) collapse ( campbell and holt , 2003 ) .
however , while in  vitro experiments suggested an involvement of lpa1-r in  lpa - mediated axonal retraction , deletion of specific lpa receptors did not lead to significant alterations of fiber tracts in the brain ( contos et  al . ,
2002 ) and did not affect inhibitory  lpa effects on retinal gcs ( birgbauer and chun , 2010 ) .
lpa is enzymatically inactivated by dephosphorylation via lipid phosphate phosphatases ( lpps ) , which are cell - surface lipid enzymes and constitute a large family of phosphatidic acid phosphatases ( pap2 ) .
recently , it was shown that lpa signaling at the synapse is controlled by prg-1/lppr4 ( trimbuch et  al . , 2009 ) , which belongs to a new class of lpa - interacting molecules ( plasticity - related genes , prgs ) sharing transmembrane features of the lpps ( bruer et  al . , 2003 )
but which seem to have a different mode of action ( mcdermott et  al . , 2004 ) .
among these , prg-2/lppr3 is expressed in developing thalamocortical axons and has a high expression level in gcs . using both prg-2 animals and a thalamus - specific prg-2/gbx2/r26 mouse line
, we set out to analyze the role of this new lpa regulatory mechanism in outgrowing axons .
prg-2/lppr3 is prominently expressed in the thalamus ( as shown by in  situ hybridization by http://www.genepaint.org ; figure  s1b , available online ) and in developing thalamocortical fibers ( figures 1a and s1a ; for antibody specificity see figures s1g 
s1i ) , where prg-2 co - localized with the thalamic axonal marker l1 ( robichaux et  al . , 2014 ) . according to this expression pattern , we hypothesized that prg-2 plays a role in the developing thalamocortical projection .
we therefore performed biocytin tracing studies in thalamocortical brain slices of wild - type ( wt ) and prg-2 mice at embryonic day ( e)17 ( figures 1d , 1e , 1h , and 1i ) , a time point when thalamocortical axons accumulate below the subplate ( molnr et  al . , 1998 ) .
slices were injected with alexa 488-labeled biocytin into the vb complex of the thalamus and were incubated for 6  hr to allow for anterograde biocytin transport .
this procedure combines the advantages of maintaining slice integrity with precise tracer application in the vb . while wt thalamocortical fibers were restricted to the iz ( figures 1d and 1h ) , as described by others ( molnr et  al . , 1998 ) , prg-2 fibers aberrantly protruded into the cp ( figures 1e and 1i ) . to further substantiate this finding , we used a conditional prg-2 mouse line ( figures s1e and s1f ) , which was cross - bred with a gbx2 line , resulting in thalamus - specific creer expression ( chen et  al .
2013 ) , and an r26 line ( madisen et  al . , 2010 ) , which produces red fluorescent protein ( rfp ) upon cre - mediated recombination . 
using thalamus - specific prg-2-deficient mice ( prg-2/gbx2/r26 ) , we analyzed the effects of thalamus - specific prg-2 deletion induced by tamoxifen treatment at e12.5 ( prg-2 ; see also figures s1c and s1d ) , as described ( normand et  al . , 2013 ) . here , we found a similar aberrant phenotype as observed in constitutive prg-2 mice ( figures 1f and 1j ) . to prove that not only a fiber subpopulation aberrantly protruded into the cp , we analyzed the full thalamocortical projection , which was displayed by the rfp reporter , and confirmed the aberrant phenotype ( figure  1 g ) .
quantitative assessment of these slices revealed a significant disruption of the thalamocortical projection in constitutive prg-2 mice as well as in prg-2 mice ( figure  1k ) . to demonstrate that misrouting was also present in  vivo , we applied neurovue tracer in the thalamic vb at e17 in fixed whole brains and analyzed thalamocortical axons . while thalamocortical fibers formed well - defined projections restricted to the iz in wt animals ( figure  1l ) , in prg-2 mice , thalamocortical fibers invaded the cp , showing a similar misrouting as observed in biocytin - traced thalamocortical slices ( figure  1 m ) . to avoid remote effects , e.g. , by prg-2 deletion in the cortex , we analyzed the thalamocortical projection in  vivo in prg-2 mice , where the rfp reporter delineated the thalamocortical fibers with high accuracy , as described by others ( normand et  al . , 2013 ) . here , prg-2-deficient thalamocortical fibers aberrantly invaded the cp , while prg-2-expressing fibers were restricted to the iz ( figures 1b and 1c ) . interestingly , in prg-2 animals , the subplate had a normal appearance ( figures s1j and s1k ) and the cortical organization , as well as the synaptic connectivity , was not altered ( figures s1m 
s1 t ) , ruling out possible subplate or cortical defects that could induce mistargeting of thalamocortical fibers ( ghosh et  al . ,
since thalamocortical projection defects observed at prenatal stages might be a temporary feature that eventually disappears after formation of thalamocortical connections , we analyzed the thalamocortical fiber tract at postnatal day ( p)5 , a time point at which the connections of these fibers are established ( molnr et  al .
we applied lipophilic tracers in different brain areas ( motor cortex , somatosensory cortex , and visual cortex ) at p5 and found that retrogradely labeled thalamocortical fibers derived from their corresponding thalamic nuclei grossly maintained their termination pattern ( figures s2a  s2c ) . to analyze whether the thalamocortical projection to the somatosensory cortex follows the well - established somatotopic termination pattern to the appropriate barrels , we inserted lipophilic tracers in the vb and visualized the thalamic projection in the corresponding barrels in tangential cut cortical slices .
as shown in figure  1n , in the somatosensory cortex of wt animals lipophilic tracer - stained axons were restricted to their corresponding barrels . however , in prg-2 mice , thalamic axons readily crossed barrel borders aberrantly invading neighboring barrels ( figures 1o , 1p , and s1l ) . to further assess misrouting in prg-2 mice , we performed retrograde tracing of thalamocortical fibers by paired injection of retrogradely transported fluorescent latex microbeads into adjacent cortical barrels with 400  m separation between injection sites .
( 1995 ) , even injections placed in close proximity ( 75120  m apart ) resulted in segregated neuronal clusters of  retrogradely labeled cells in the thalamic vb ( agmon et  al . ,
1995 ) . while in wt animals , even nearby injections of differently labeled fluorescent beads ( red and green fluorescence
; see figure  s2d ) resulted in labeling of distinct subpopulations  in the corresponding vb thalamic nuclei ( figures 1q and 1s ) , in prg-2 mice , injections even at distant sites ( see figure  s2e ) resulted in double labeling of the corresponding subpopulations ( figures 1r and 1 t ; exemplarily retrograde stained neurons in the thalamus are shown in figures s2f and s2 g ) .
quantitative assessment of double - labeled neurons revealed a significantly higher number in prg-2 animals , while the total number of retrogradely traced neurons did not differ between genotypes ( figures 1u and 1v ) .
thus , prg2 depletion in mice leads to a misrouting in thalamocortical projections . to test whether the aberrant thalamic projection in prg-2 mice results from absence of prg-2 in thalamocortical axons
, we used thalamocortical slice cultures of e15 wt and prg-2 mice at the peak of thalamocortical outgrowth .
the thalamocortical projection was visualized by co - electroporation of a gfp - expressing construct into the thalamic vb at e15.5 . after 48  hr ,
slice cultures were fixed and the axonal projection was analyzed ( figure  2a ) . in wt slices ,
thalamocortical fibers were restricted to  the iz ( figure  2b ) , while prg-2 thalamocortical axons aberrantly invaded the cp ( figure  2c ) . however ,
when prg-2 was re - expressed ( together with gfp ) in thalamocortical axons , these fibers were again restricted to the iz and did not show  obvious differences to wt fibers ( figures 2d and s3a ) .
quantitative analysis of thalamocortical cultures confirmed the significant amount of aberrant thalamocortical projections and the rescued targeting of these axons after prg-2 re - expression ( figure  2e ) . in sum , re - expression of prg-2 in thalamocortical axons was sufficient to completely abrogate aberrant targeting of the thalamocortical fiber projection in slices from prg-2 mice . since prg-2 is a member of the family of lpa - interacting molecules ( trimbuch et  al . ,
2009 ) , we analyzed the presence of autotaxin ( atx , enpp-2 , lysopld ) , the lpa - synthesizing enzyme , in the somatosensory cortex at e16 .
atx is expressed by subplate neurons , as has been shown on the mrna level ( hoerder - suabedissen et  al . , 2013 ) , and by a gfp reporter at corresponding age ( figures s4a and s4b ) .
due to its secreted and diffusible nature , we found strong atx expression at the lower border of the cp and at the upper border of the iz ( figure  3a ) . here
, atx immunoreactivity overlaps with that of lpa and prg-2 , which was expressed in fasciculated thalamocortical fibers in the iz ( figures 3b3d ; higher magnification in figures 3e3h ) .
these findings are in line with a putative role of atx / lpa in restricting prg-2-expressing thalamocortical fibers to the iz .
moreover , lpa - degrading enzymes like lpp1 and lpp3 are strongly expressed in the iz ( figures s6n and s6o ) , which suggests that proper regulation of phospholipids allows thalamocortical axons to occupy a corridor in the iz , possibly contributing to the fasciculated appearance of the thalamocortical projection .
we therefore perturbed phospholipid signaling and analyzed the effect of inhibited lpa synthesis on the correct targeting of thalamocortical axons .
we applied a recently characterized atx blocker ( pf8380 ) ( gierse et  al . , 2010 ) , which was shown to effectively decrease lpa concentrations ( vogt et  al . , 2015 ) ,
after atx inhibition ( 0.1  m ) , wt thalamocortical fibers aberrantly invaded the cp ( figure  3j ) , showing a similar phenotype as observed in slices from prg-2 mice ( figures 1e and 1i ) .
quantitative analysis revealed that this phenotype was observed in more than 92% of all pf8380-treated slices ( figure  3l ) . to further study whether this effect was due to cortical lpa production
inhibition of atx in the cp was sufficient to induce an aberrant targeting of thalamocortical fibers ( figure  3k ) .
in contrast , pf8380 injection into the thalamus did not significantly alter the thalamocortical targeting , supporting a role for cortical - derived lpa in thalamocortical guidance ( figure  3l ) .
taken together , our data provide strong evidence that correct guidance of thalamic fibers to the cortex depends on subplate - derived lpa , produced by atx , and on the presence of prg-2 on thalamic fibers .
the role of lpa in gc collapse is well described ( campbell and holt , 2003 ) ; however , the molecular mechanism transducing lpa action from the membrane to the cytoskeleton is not well understood .
while specific , g protein - coupled lpa receptors were reported not to affect lpa - induced collapse of retinal gcs ( birgbauer and chun , 2010 ) , authors of the same group showed that downstream inhibition of the g12/13-rho - rock pathway was capable of inhibiting lpa - induced retinal gc collapse ( fincher et  al . , 2014 ) .
this is in line with reports of downstream lpa signal transduction acting on the cytoskeleton ( campbell and holt , 2003 ) . to analyze the role of axonal prg-2 in lpa - mediated thalamic axon guidance
, we embedded thalamic explants in matrigel with a polarized concentration of lpa ( distance to tf - lpa - containing matrigel was 401.6   53.63  m for wt explants and 475   146.80  m for prg-2 explants ) and
on one side , the matrigel contained 10  m fluorescent - labeled lpa ( tf - lpa ) , while the adjacent side did not contain lpa ( see figures 4a
s5e , and 4d2 for the border of the tf - lpa - containing matrigel region at higher magnification ) .
the 3d matrigel structure , where fibers are able to radially expand , allows analysis of fiber growth into the lpa - rich zone and the adjacent control zones .
however , while wt axons were not able to invade this lpa - rich zone , prg-2 axons grew far inside it ( figures 4c and 4d1 ) .
quantitative analysis of fibers entering the lpa - rich zone at different distances revealed significantly higher invasion by fibers from prg-2 thalamic slices ( figure  4e ) , while no difference between wt and prg-2 axon fiber outgrowth was observed in the adjacent , non - lpa - containing control side ( figure  4f ) .
direct comparison of lpa - containing and control sides revealed that wt axons were significantly repelled by the lpa - rich zone , while prg-2 axons were not affected ( figure  4 g ) .
since prg-2 deficiency allowed axons to enter the lpa - rich zone , which is repulsive for wt axons , these data suggest that prg-2 is critically involved in mediating the repulsive lpa sensitivity in thalamic axons . during normal development
, thalamocortical axons do not collapse at the border to the cp but rather turn away from this border and continue to grow in the iz until reaching their final target .
this indicates that lpa provided by the subplate induces the turning of axon gcs when thalamocortical fibers approach lpa - rich areas .
to mimic this situation in our in  vitro outgrowth assay , we exposed thalamocortical explants to lower lpa concentrations ( 1  m lpa ; figure  4h ) . here
, wt axons displayed a typical turning behavior in front of the lpa - rich zone ( figures 4i and 4k ) . to confirm this observation
axon gcs were exposed to a low , but continuous , lpa concentration ( 500  nm lpa ) . to avoid bias by perfusion flow , which may repel axons , gcs were exposed to an lpa - covered pipette tip at 40  m distance ( see figure  s5f for experimental overview ) .
while wt axons stopped growing and turned away from the lpa source , this behavior was not observed for prg-2 axons , which even seemed to be attracted by lpa ( figures 4l4n ; movies s1 and s2 ) .
quantitative analysis revealed a significant repulsion of wt axons by 500  nm lpa , which was not observed in prg-2 axons ( figure  4o ) .
however , prg-2 axons were still repelled by other guidance cues like sema3a ( figures s5 g and s5h ) , which are well - described repulsive cues ( mintz et  al . , 2008 ) . in sum , these data indicate that prg-2 at the gc mediates the repellent effect of lpa for axonal guidance .
prg-2/lppr3 has an intracellular 400 aa - long c - terminal tail , which is putatively involved in signal transduction . to determine interaction partners of this intracellular domain
, we first performed a y2h screen using this prg-2 c - terminal tail as a bait and found a positive interaction with the ferm domain of radixin ( rdx ) ( fehon et  al . , 2010 ) . using immunoprecipitation ( ip ) on e17 brain lysate
this interaction is specific for prg-2 since only prg-1 and prg-2 possess an intracellular c terminus , and prg-1 is not expressed at embryonic ages ( figure  s6j ) . to further elucidate the role of this interaction in proper guidance of thalamocortical fibers to the developing cortex ,
we performed co - localization studies at e16 and found co - expression of prg-2 and rdx in the thalamocortical tract ( figures 5b , 5c , s6k , and s6i ) . expression analysis of prg-2 and rdx showed a similar expression pattern , where expression for both molecules increased from e11.5 , a time point where thalamic neurons are already present and start to differentiate ( figure  s6f ) . to prove that the rdx / prg-2 interaction in fact occurs in the axon gc , we performed subcellular immunofluorescent co - localization studies of rdx and prg-2 and found a strong signal in gcs ( figure  5d ) . here
, prg-2 is expressed at the gc membrane and co - localizes with rdx , which shows an asymmetric distribution , as described by others ( mintz et  al . , 2008 ) .
rdx belongs to the ezrin , radixin , and moesin ( erm ) protein family .
however , due to their embryonal expression pattern at e14.5 , ezrin and moesin do not seem to be involved in the outgrowth of the thalamocortical projection ( figures s6l and s6 m ) .
the c terminus of rdx can directly bind to filamentous actin ( f - actin ) , thereby acting as a crosslinker between the cortical f - actin cytoskeleton and the plasma membrane ( fehon et  al . ,
the activation of rdx involves the phosphorylation of a critical c - terminal threonine residue ( t564 ) , which is highly conserved in the erm protein family .
therefore , we analyzed the role of prg-2 in rdx activation using a specific antibody that recognizes the phosphorylation of t564 in rdx , t567 in ezrin , and t558 in moesin ( hausrat et  al . , 2015 ) .
interestingly , we found significantly lower phosphorylated erm ( perm ) levels in prg-2 neurons ( figures 5e and 5f ) , indicating diminished activation of erm proteins . to prove the role of lpa in erm activation , we assessed the effect of different lpa concentrations .
while 100  nm lpa already revealed an increase in erm phosphorylation , 1  m lpa induced a significant and robust increase in perm levels ( figures 5 g and 5h ) .
we further analyzed perm levels over time and found a fast and constant increase in erm phosphorylation in the course of lpa stimulation ( figures 5i , 5j , and s6a  s6c ) .
to understand the functional significance of  the lpa - dependent perm expression , we analyzed the rdx / prg-2 interaction under lpa - free conditions and after 1  m lpa stimulation for 15  min .
here , we used rdx - gfp transfected , stable prg-2-expressing hek cells , which have comparable prg-2 expression , and performed coips of rdx and prg-2 using an anti prg-2 antibody ( figure  5k ) . quantitative analysis ( rdx levels were adjusted according to the prg-2 ip signal ) revealed a significantly increased rdx / prg-2 interaction upon 1  m lpa stimulation ( figure  5l ) .
interestingly , analysis of the ip product revealed that phosphorylation of the prg-2-interacting rdx was significantly higher upon lpa stimulation when compared to serum - starved cells ( figures 5 m and 5n ) .
since we could show that rdx binds to the intracellular c terminus of prg-2 ( figure  s6d ) , to further analyze whether rdx phosphorylation is a prerequisite for prg-2 binding and to determine the prg-2 binding sites , we performed a systematic peptide scan using a celluspot  array format ( spot ) and mapped the rdx / prg-2 interaction ( figure  5o ) . here
, prg-2 intracellular c - terminal overlapping peptides ( 69 overlapping peptide fragments , 14 aa in length ) were analyzed regarding their binding affinity to rdx - gst or to rdx - gst , its non - phosphorylated form .
we detected six putative interaction sites that showed significant difference in their binding to rdx - gst or to rdx - gst , pointing to a lower binding affinity of the non - phosphorylated rdx - gst to prg-2 ( see also figure  s6p ) .
we have therefore performed ips from stable prg-2-expressing hek293 cells transfected either with wt rdx - gfp ( rdx ) or non - phosphorylated rdx - gfp ( rdx ) using the described prg-2 antibody . in line with data from the peptide - microarray - based mapping , although binding of rdx was present at lower levels when compared to rdx , prg-2/rdx binding could be clearly detected ( figures 5p and 5q ) .
this points to the fact that rdx phosphorylation , although not a prerequisite for rdx / prg-2 interaction , significantly increases rdx / prg-2 interaction . to answer the question of whether rdx / prg-2 association at the membrane is important for lpa - dependent rdx phosphorylation in neurons
, we analyzed the effect of lpa stimulation on perm expression in wt and prg-2 neurons . here , lpa stimulation of wt neurons resulted in a significant increase in perm levels , while prg-2 neurons failed to show any significant change ( figures 5r and 5s ) .
these results point to a yet unknown , but critical role of prg-2 in lpa - induced rdx activation .
rdx is concentrated below the plasma membrane , a critical position linking transmembrane signaling to the actin cytoskeleton . since phosphorylation influences rdx activity in connecting the actin - cytoskeleton with the membrane ( fehon et  al . ,
2010 ) , transmembrane signaling events leading to rdx activation are optimally suited to act in extracellular cue mediated axon guidance .
we therefore analyzed the effect of extracellular lpa in inducing perm changes at the gc membrane in wt and prg-2 neurons . in wt gcs
, perm was concentrated in a compartment directly adjacent to the gc membrane ( figures 6a and 6b ) and strongly increased upon lpa stimulation ( figures 6e , 6f , and 6i ) . in contrast , no change of perm was observed in  prg-2 gcs ( figures 6c , 6d , and 6g6i ) . moreover ,
direct comparison of perm levels in the center of the gc and in the periphery revealed a shift of the perm signal toward the membrane of wt gcs , but not of prg-2 gcs ( figure  6j ) . to prove whether rdx is dynamically recruited to the membrane via prg-2 binding induced by a local extracellular lpa source , we transfected wt or  prg-2 neurons with gfp - tagged rdx and performed live imaging studies .
while in wt neurons , local lpa stimulation rapidly induced a translocation of rdx - gfp to the membrane next to the lpa source ( figures 6k and 6l ) , prg-2 neurons did not show such a change ( figures 6 m and 6n ) . in sum
, these data show that prg-2 is the mediator of lpa - induced localization  of rdx to the gc membrane , where it is present in its phosphorylated form , in turn increasing binding to prg-2 .
to prove that erm phosphorylation and activation play a role in lpa - induced gc turning , we analyzed the subcellular localization of perm in gcs of thalamic explants exposed to an lpa - rich environment ( 1  m lpa ) , as shown in figure  4i .
detailed analysis of turning gcs , exemplarily encircled in figure  7a , revealed strong perm signal co - localized with f - actin at the tip of gcs turning away from the lpa - rich zone ( figures 7b7e ) . to confirm that prg-2 interacts with rdx at the gc , we performed a proximity ligation assay ( pla ) , a method that allows the study of in  situ protein - protein interactions at resolutions < 40  nm ( rhett et  al . , 2011 ) . here , we found a strong signal at the gc tip , confirming the prg-2/rdx interaction at the site of lpa action ( figures 7f and 7 g ) . taken together , these results confirm the critical role of prg-2/rdx interaction for the induction of rdx activation in gcs by extracellular lpa , which eventually leads to axon turning . to further substantiate the role of rdx in lpa - induced gc turning
, we analyzed the thalamocortical projection in rdx mice . using biocytin tracing in living e17 thalamocortical slices
, we observed misrouted thalamocortical axons in rdx slices that aberrantly invaded the cp ( figures 7i7k),while in slices from control littermates , the thalamocortical projection was confined to the iz ( figures 7h and 7k ) . in order to better understand the functional role of the prg-2/rdx interaction in thalamocortical axon guidance
, we analyzed the effect of partial  reduction of these proteins in transheterozygous mice ( prg-2/rdx ) in thalamocortical slices at e17 . here , the thalamocortical projection aberrantly invaded the cp , suggesting that it is indeed the interaction of prg-2 and rdx that is important for correct guidance of these fibers by an lpa gradient ( figures 7l7o ) .
these data corroborate the idea that the lpa - prg-2-rdx / perm signal transduction axis is critical for correct guidance and restriction of thalamocortical axons to the iz .
moreover , in line with decreased rdx expression after e16 ( figures s6 g and s6h ) , these data could explain how thalamocortical axons are finally able to enter into their target cortical region . to assess the functional outcome of altered thalamocortical projections in prg-2 mice
, we first measured intrinsic optical responses in the corresponding barrel upon single - whisker back - and - forth deflection . this approach measures blood - flow changes in the representation of single whiskers in the barrel cortex upon activation . using this method , we found that the response area upon single - whisker stimulation , which corresponds to the stimulated barrel ( for morphological barrel assessment , see figure  s7a ) , was larger in prg-2 mice .
these data point to a more diffuse and broader cortical response extending beyond the border of the activated barrel ( figures 8a8c ) .
moreover , it reflects our findings using retrograde tracing of thalamocortical fibers in prg-2 mice showing a clear overlap in the termination pattern of neurons from neighboring thalamic barreloids in the vpm ( figures 1r , 1 t , and 1u ) .
this is in agreement with enlargement of functional barrel columns in mice due to impaired precision of the thalamocortical projection , as reported in other mouse models ( lokmane et  al . , 2013 ) .
to get a layer - specific resolution of neuronal activity , we performed in  vivo electrophysiological recordings using multi - channel electrodes .
we observed in prg-2 mice a slight decrease in multi - unit activity ( mua ) upon single - whisker deflection in layer iv and a significant decrease in layers ii / iii for prg-2 mice compared to wt controls ( figures 8d8f ) , which are the main target layers of the thalamocortical projection ( meyer et  al .
recent data suggest that layers ii / iii contain neurons specifically responsive for texture coarseness ( garion et  al . , 2014 ) .
we therefore assessed the ability of prg-2 mice to distinguish texture coarseness using a modified radial arm maze , where arms were covered with sandpaper of two slightly different grades ( 60 grade [ grain size , 265  m ] and 80 grade [ grain size , 190  m ] ; figure  8 g ) . only arms covered with the coarser ( 60 grade ) sandpaper
were rewarded by accessible food pellets and counted as correct entrance . since experiments were performed in darkness and no other cues were presented , mice had to orient by sensing sandpaper roughness . while wt mice showed a significant improvement in their ability to choose the correct arm , prg-2 mice did not improve beyond chance levels even after 9  days of continuous training ( figure  8h ) .
to  exclude bias by an impaired learning ability , we tested prg-2 mice in the morris water maze ( mwm ) .
escape latency for visible and hidden platforms was indistinguishable between prg-2 and wt littermates ( figures s7e 
was not impaired in prg-2 mice , we conclude that their performance at chance level in the texture discrimination task is a direct result of impaired thalamocortical axon projection precision . in line with unaltered learning ability , motor performance was also unaltered , as shown by analysis in the mwm or supporting limb responses ( figures s7h  s7k ) . to exclude an effect of prg-2 deletion outside the thalamus , e.g. , in the cortex , which could affect cortical processing of somatosensory information
, we also tested the above described thalamus - specific prg-2-deficient mouse line ( prg-2 ) . as shown in figures s8l
 s8o using retrograde tracing of thalamocortical fibers projecting to the cortical barrels , these animals display a similar phenotype as described for the constitutive prg-2 animals , where we could show that different barrels were aberrantly innervated by the same neuron in the thalamic vpm .
furthermore , staining for prg-2 revealed that fibers originating from gbx-2-expressing thalamic neurons ( identified by the rfp reporter ) did not express prg-2 ( figures s7p  s7s ) .
behavioral analysis of the prg-2 mice in the sandpaper radial maze revealed that these mice displayed the same phenotype as the constitutive prg-2 mice and were not able to distinguish between the two sandpapers of different coarseness ( figure  8h ) .
together with our in  vivo imaging and electrophysiological data , these findings strongly indicate that misguided thalamocortical axons in prg-2 mice alter cortical information processing , resulting in a specific deficit in sensory discrimination at adult ages .
an intriguing phenomenon in cortical development is the specific outgrowth of thalamocortical fibers that reach the appropriate cortical area before their target cells migrate to their final position in the cp ( kostovic and rakic , 1990 ) .
as shown in other systems , thalamocortical axons accumulate in the iz below the cp ( ghosh et  al . ,
, they interact with a transient neuronal population , the subplate neurons , which keep them in place and are thereby critically important for correct patterning of the thalamocortical connections . although the role of subplate neurons has long been recognized and gene expression patterns of subplate neurons have been described ( for review , see hoerder - suabedissen and molnr , 2015 ) , molecular mechanisms regulating the subplate neurons / thalamocortical fiber interaction are far from being understood . here
, we analyzed the role of prg-2/lppr3 , a phospholipid - interacting molecule strongly expressed in thalamocortical axons during development ( figure  1a ) . using  a constitutive prg-2 mouse line as well as a thalamus - specific prg-2 ko line ( prg-2/gbx2/r26 ) , where tamoxifen application at e12.5 leads to specific deletion of prg-2 in the thalamus ( prg-2 ; normand et  al .
, 2013 ) , we discovered that prg-2-deficient thalamocortical axons prematurely and aberrantly entered the cp .
this altered the precise somatotopic termination of thalamocortical fibers in the somatosensory barrel field cortex and led to a deficit in sensory discrimination .
molecular analysis revealed a signal transduction axis in the gc leading from the lpa - synthesizing molecule atx to lpa , prg-2 , and finally to phosphorylated rdx , a well - described linker between the gc membrane and the actin cytoskeleton .
we demonstrate this atx / lpa / prg-2/prdx signaling axis on thalamocortical axon gc turning and  suggest that this mechanism is responsible for keeping thalamocortical axons in the iz before entering the cp .
the role of subplate cells was concluded from ablation experiments , where thalamocortical fibers , no longer able to contact subplate cells , escaped the subcortical waiting period and extended beyond their target cortical regions .
this specific phenotype relies mechanistically on the lost sensitivity of thalamocortical axons toward extracellular lpa gradients synthesized by atx , a diffusible molecule expressed in subplate neurons ( hoerder - suabedissen et  al . , 2013 ) .
eventually , this leads to loss of proper axon guidance and to an invasion of inappropriate cortical compartments .
since we also analyzed thalamus - specific prg-2 ko animals ( normand et  al .
, 2013 ) , we could rule out that this misrouting is due to defects , e.g. , derived from cortical prg-2 deficiency .
interestingly , the same phenotype was detected when the catalytic function of atx was blocked , thereby impeding lpa production .
our data suggest a thalamocortical guidance mechanism with a mutual dependence on lpa / prg-2 interaction .
the critical importance of prg-2-mediated extracellular lpa effects on thalamic axon guidance was further clarified by single - axon analysis .
while downstream mechanisms mediating lpa gc collapse have been described ( campbell and holt , 2003 ) , direct and specific lpa - interacting molecules involved in this mechanism remain unclear . in line with live imaging data ,
wt axons were repelled / collapsed when in contact with 10  m lpa , while lower lpa concentrations ( 1  m ) induced a turning behavior in gcs .
however , prg-2 axons were not affected even by high lpa concentrations ( 10  m ) . searching for downstream interacting partners , we found rdx to directly interact and be co - expressed with prg-2 in thalamocortical axons . moreover , subcellular analyses revealed rdx to interact with prg-2 in gcs as shown by pla .
importantly , ip experiments revealed that the prg-2/rdx binding was highly increased upon lpa stimulation .
the role of this prg-2/rdx interaction was functionally confirmed , showing that prg-2 is critically important for rdx activation by extracellular lpa .
while in wt neurons , concentrations as low as 100  nm lpa induced clear rdx phosphorylation , in prg-2 neurons , rdx phosphorylation did not increase , even upon 15  min of 1  m lpa stimulation .
our data showing rdx phosphorylation upon lpa stimulation in full - length prg-2-expressing hek293 cells suggest that prg-2/rdx association increased rdx phosphorylation upon lpa stimulation . however , when analyzing binding to prg-2 to either wt rdx - gfp or its non - phosphorylated form ( rdx - gfp ) , we detected binding under both conditions , albeit with a significant reduction of binding to prg-2 of the non - phosphorylated form .
these data show that rdx phosphorylation is not a prerequisite for prg-2/rdx binding ; however , it influences this binding .
our finding that transheterozygous loss of prg-2 and rdx already results in altered thalamocortical fiber projection indicates an important role of the lpa / prg-2/rdx signal axis . since the active form of erms ( perm ) is critical for gc guidance ( mintz et  al . ,
2008 ) , we investigated the role of this signal axis at the gc , the critical location for axon guidance . here
, lpa stimulation induced strong erm phosphorylation at the gc membrane of wt axons , while prg-2 gcs failed to show these changes . in line
, rdx was rapidly translocated to the membrane directly next to a local lpa source , indicating an active role of lpa / prg-2/rdx signaling in gc guidance . together with the detection of perm adjacent to f - actin at the tip of turning gcs , this signaling axis appears to directly control lpa - induced axon turning . using in  vivo imaging and high - density multi - electrode recordings in the barrel cortex of prg-2 mice and performing behavioral tests both in prg-2 mice and prg-2 animals
, we elucidated the functional consequences of prg-2 deficiency and subsequent altered thalamocortical connectivity in cortical layers iv and ii / iii as well as in the characteristic discrimination abilities associated with these layers ( garion et  al . , 2014 ) .
it appears that rather specific deficits can be observed even upon drastic developmental defects resulting in grossly impaired cortical lamination and subsequent changes in the thalamocortical projection ( wagener et  al . , 2016 ) , as shown by preserved functional sensory maps in the somatosensory barrel cortex observed in reelin - deficient mice ( guy et  al . , 2015 ) .
( 2015 ) suggest microcircuits in layers ii / iii are important for specific stimulus orientation ( cossell et  al . , 2015 ) , supporting the idea that layer ii / iii neurons mediate rather specific functions .
this implies that functional changes in layers ii / iii seen in prg-2 mice can best explain the sensory discrimination deficits of the animal .
taken together , we describe a novel molecular guidance mechanism involving phospholipid signaling influencing thalamocortial axon guidance at a critical time point , at which axons exhibit a waiting period at the cortical subplate .
the alteration of the lpa / prg-2/prdx signal transduction pathway resulted in distinct electrophysiological and behavioral deficits in adult animals and highlighted the importance of this pathway for proper thalamocortical connectivity .
all experiments were conducted in accordance with the national laws for the use of animals in research and with the european communities council directive 86/609/eec , and approved by the local ethical committee ( landesuntersuchungsamt rheinland - pfalz 23 .
details of the generation and breeding of prg-2 , prg-2/gbx2/r26 , and rdx mice are provided in the supplemental experimental procedures .
immunofluorescent ( if ) stainings , pla for detection of in  situ protein interaction , and anterograde and retrograde tracing experiments were performed following standard protocols .
organotypic slices were prepared as described in the supplemental experimental procedures . for biocytin tracing experiments ,
pressure injections were performed into e17 embryonic slices using glass capillaries attached to a toohey spritzer ( toohey company ) to induce biocytin alexa fluor 488 ( thermo fisher scientific ) .
slices were then incubated for 6  hr and fixed with 4c pfa ( 4% ) . for electroporation experiments ,
corresponding plasmids were injected into organotypic slices from e15.5 brains that were electroporated by needle tungsten electrodes and an nepa21 super electroporator ( nepa gene ) .
. , 2012 ) , cultivated for 2  days in  vitro ( div ) and used for assessment of perm levels in the gc . if staining and perm assessment were performed as described in the supplemental experimental procedures .
ip studies were performed on e17 brain material or on cell lysate from stably prg-2-expressing hek293 cells according to standard protocols .
intrinsic signal optical imaging ( ioi ) and in  vivo electrophysiological recordings in the barrel cortex of adult mice were performed according to standard protocols as described in the supplemental experimental procedures .
sandpaper discrimination test and mwm were performed according to standard procedures . for animal experiments , mice from the same litter or of similar age
following experiments , results were analyzed , animals were re - genotyped , and corresponding final statistical analyses were performed .
briefly , after assessing for normal distribution ( using the kolmogorov - smirnov test ) , comparison between two groups , if not otherwise stated , was performed using a two - tailed unpaired t test for normal distributed data or a mann - whitney test for nonparametric data . when data were normalized to controls ( set as 100% ) , a one - sample t test was used . for quantitative assessment of aberrant fibers in traced or electroporated slices , a fisher s exact test was applied .
comparison between more than two groups was performed using a one - way anova for parametric data or a kruskal - wallis test for nonparamatric data .
variance was similar between groups and was estimated using either the f - test or the brown - forsythe test and bartlett s test .
post hoc analysis for more than two groups for parametric data was performed using the bonferroni adjustment for multiple comparisons and dunn s test for nonparametric data . for behavioral analyses assessing genotype effects and time effects , as well as for comparisons of the multiple binding sites of prg-2 ,
h.j . , n.s . , h.m.m . , a.h .- s . , p .- h.p . , w.f . , and t.t .
provided reagents ; writing  original draft , j.v . ; review and editing , j.v . , r.n . , j.c .
 summaryprecise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information . here , we show that prg-2 , a phospholipid - interacting molecule , is important for thalamocortical axon guidance . developing thalamocortical fibers both in prg-2 full knockout ( ko ) and in thalamus - specific ko mice prematurely entered the cortical plate , eventually innervating non - corresponding barrels . 
 this misrouting relied on lost axonal sensitivity toward lysophosphatidic acid ( lpa ) , which failed to repel prg-2-deficient thalamocortical fibers . 
 prg-2 electroporation in the prg-2/ thalamus restored the aberrant cortical innervation . 
 we identified radixin as a prg-2 interaction partner and showed that radixin accumulation in growth cones and its lpa - dependent phosphorylation depend on its binding to specific regions within the c - terminal region of prg-2 . in  vivo recordings and whisker - specific behavioral tests demonstrated sensory discrimination deficits in prg-2/ animals . 
 our data show that bioactive phospholipids and prg-2 are critical for guiding thalamic axons to their proper cortical targets . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions