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Precise Somatotopic Thalamocortical Axon Guidance Depends on LPA-Mediated PRG-2/Radixin Signaling

Summary Precise 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
The thalamocortical projection is essential for sensory processing. In rodents, vibrissae provide input to specific thalamic nuclei and these nuclei have well-defined connections with the so-matosensory 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 (Molná r 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., 1990). 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 Molná r, 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 LPA 1 -R and LPA 2 -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 LPA 1 -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 (Brä uer 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 À/À animals and a thalamus-specific PRG-2 DE12/DE12 /Gbx2 CreER /R26 tdTomato mouse line, we set out to analyze the role of this new LPA regulatory mechanism in outgrowing axons.

PRG-2 -/-Mice Have an Aberrant Thalamocortical
Projection 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 À/À mice at embryonic day (E)17 ( Figures 1D,  1E, 1H, and 1I), a time point when thalamocortical axons accumulate below the subplate (Molná r 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 antero-grade 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 (Molná r et al., 1998), PRG-2 À/À fibers aberrantly protruded into the CP ( Figures  1E and 1I). To further substantiate this finding, we used a conditional PRG-2 fl/fl mouse line (Figures S1E and S1F), which was cross-bred with a Gbx2 CreER line, resulting in thalamus-specific CreER expression (Chen et al., 2009;Normand et al., 2013), and an R26 tdTomato line (Madisen et al., 2010), which produces red fluorescent protein (RFP) upon cre-mediated recombination. Using thalamus-specific PRG-2-deficient mice (PRG-2 fl/fl / Gbx2 CreER /R26 tdTomato ), we analyzed the effects of thalamusspecific PRG-2 deletion induced by tamoxifen treatment at E12.5 (PRG-2 DE12/DE12 ; 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 À/À 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 1G). Quantitative assessment of these slices revealed a significant disruption of the thalamocortical projection in constitutive PRG-2 À/À mice as well as in PRG-2 DE12/DE12 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 À/À mice, thalamocortical fibers invaded the CP, showing a similar misrouting as observed in biocytin-traced thalamocortical slices ( Figure 1M). To avoid remote effects, e.g., by PRG-2 deletion in the cortex, we analyzed the thalamocortical projection in vivo in PRG-2 DE12/DE12 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 (A) PRG-2 is expressed in thalamocortical fibers and co-localizes with the axonal marker L1 at E16 (see also Figures S1A and S1B). (B) Thalamus-specific PRG-2 deletion by tamoxifen administration at E12.5 (PRG-2 DE12/DE12 ) resulted in aberrant thalamocortical fibers (displayed by the RFP reporter), which prematurely invaded the cortical plate (CP). (C) Higher magnification of the IZ/CP border shows aberrant thalamic fibers protruding into the CP while WT PRG-2-expressing fibers were restricted to the IZ (C 2 ). (D-F and H-J) Thalamocortical fiber tracing using in vivo tracer biocytin injected in the ventrobasal (VB) nuclei of acute thalamocortical slices. While in WT slices (D and H), thalamocortical fibers were restricted to the IZ, in PRG-2 À/À (E and I) and PRG-2 DE12/DE12 (F and J) slices, thalamic axons aberrantly invaded the CP. (G) Using the Cre-driven RFP reporter, the full extent of the thalamic projection in PRG-2 DE12/DE12 is shown together with biocytin-labeled fibers in the overview (G 1 ) and at higher magnification (G 2 ).
(K) Quantitative analysis shows significant amount of aberrant fibers in PRG-2 À/À and in PRG-2 DE12/DE12 slices (Fisher's exact test; n = 8 WT, 13 PRG-2 À/À , and 14 PRG-2 DE12/DE12 slices). (L and M) Lipophilic dye tracing in E17 fixed brains depicted normal thalamocortical tract in WT animals (L) and high amount of aberrant fibers in PRG-2 À/À mice (M). (N-P) Thalamocortical fibers at P5 are restricted to specific barrels in the WT brain (N), while in PRG-2 À/À brains numerous fibers (arrow heads) crossed barrel borders (O and P; see also Figure S1L). (Q and R) Following paired injection of fluorescent-labeled retrobeads in different barrels, retrograde-labeled neurons in the thalamic VPM nucleus of WT adult mice show clear segregation (Q; see also . In contrast, in PRG-2 À/À mice, numerous double-labeled neurons were found in the thalamic VPM (R). (S and T) Higher magnification of (Q) and (R). (U and V) Analysis of double-stained and total number of stained neurons in the thalamic VPM (t test [U] and Mann-Whitney U test [V]; n = 11 WT and 7 PRG-2 À/À mice). Bars represent mean ± SEM. ****p < 0.0001. Scale bars, 100 (A, B, D-J, and L-P), 50 (Q and R), and 10 mm (S and T). CP, while PRG-2-expressing fibers were restricted to the IZ (Figures 1B and 1C). Interestingly, in PRG-2 À/À 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-S1T), ruling out possible subplate or cortical defects that could induce mistargeting of thalamocortical fibers (Ghosh et al., 1990).
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 (Molná r et al., 1998). 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 À/À mice, thalamic axons readily crossed barrel borders aberrantly invading neighboring barrels ( Figures 1O, 1P, and S1L). To further assess misrouting in PRG-2 À/À mice, we performed retrograde tracing of thalamocortical fibers by paired injection of retrogradely transported fluorescent latex microbeads into adjacent cortical barrels with 400 mm separation between injection sites. As shown by Agmon et al. (1995), even injections placed in close proximity (75-120 mm 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 À/À mice, injections even at distant sites (see Figure S2E) resulted in double labeling of the corresponding subpopulations ( Figures 1R and 1T; exemplarily retrograde stained neurons in the thalamus are shown in Figures S2F and  S2G). Quantitative assessment of double-labeled neurons revealed a significantly higher number in PRG-2 À/À 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.

PRG-2 Function Is Required in Thalamocortical Afferents for Correct Targeting
To test whether the aberrant thalamic projection in PRG-2 À/À mice results from absence of PRG-2 in thalamocortical axons, we used thalamocortical slice cultures of E15 WT and PRG-2 À/À 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 À/À 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 À/À mice.
LPA Signaling Is Critical for Thalamocortical Axon Guidance in 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 3B-3D; higher magnification in . 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), to the medium of WT thalamocortical slice cultures and traced thalamocortical fibers using biocytin. After ATX inhibition (0.1 mM), WT thalamocortical fibers aberrantly invaded the CP ( Figure 3J), showing a similar phenotype as observed in slices from PRG-2 À/À 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, PF8380 was locally injected into the CP. Here, 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.

PRG-2 Mediates Axonal Sensitivity to LPA
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-LPAcontaining matrigel was 401.6 ± 53.63 mm for WT explants and 475 ± 146.80 mm for PRG-2 À/À explants) and analyzed axonal outgrowth. On one side, the matrigel contained 10 mM fluorescent-labeled LPA (TF-LPA), while the adjacent side did not contain LPA (see Figures 4A, 4B, S5A-S5E, and 4D 2 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 À/À axons grew far inside it ( Figures 4C and 4D 1 ). Quantitative analysis of fibers entering the LPA-rich zone at different distances revealed significantly higher invasion by fibers from PRG-2 À/À thalamic slices ( Figure 4E), while no difference between WT and PRG-2 À/À axon fiber outgrowth was observed in the adjacent, non-LPAcontaining 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 À/À axons were not affected ( Figure 4G). 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.

CP IZ
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 mM 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, we turned to live imaging of cultured thalamocortical axons. 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 mm 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 À/À axons, which even seemed to be attracted by LPA ( Figures 4L-4N; Movies S1 and S2). Quantitative analysis revealed a significant repulsion of WT axons by 500 nM LPA, which was not observed in PRG-2 À/À axons ( Figure 4O).  However, PRG-2 À/À axons were still repelled by other guidance cues like Sema3A ( Figures S5G and S5H), which are welldescribed 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.

IZ
Radixin Is a Downstream PRG-2-Interacting Molecule in the Axon GC 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, we confirmed that PRG-2 interacts with RDX (Figures 5A and S6E). 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 colocalization 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  S6M). 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., 2010). 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 À/À 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 mM LPA induced a significant and robust increase in pERM levels ( Figures 5G 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 mM 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 mM 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 5M and 5N). These data suggest that higher RDX phosphorylation upon LPA stimulation augmented PRG-2/RDX association. 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 m Array format (mSPOT) 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 T564A -GST, its non-phosphorylated form. We detected six putative interaction sites that showed significant difference in their binding to RDX-GST or to RDX T564A -GST, pointing to a lower binding affinity of the nonphosphorylated RDX T564A -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 WT ) or non-phosphorylated RDX-GFP (RDX T564A ) using the described PRG-2 antibody. In line with data from the peptide-microarraybased mapping, although binding of RDX T564A was present at lower levels when compared to RDX WT , PRG-2/RDX T564A 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 À/À neurons. Here, LPA stimulation of WT neurons resulted in a significant increase in pERM levels, while PRG-2 À/À 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.
PRG-2 Mediates pERM Increase at GC Membranes Induced by Extracellular LPA 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 À/À 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 À/À GCs ( Figures 6C, 6D, and 6G-6I). 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 À/À 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 À/À 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 À/À neurons did not show such a change ( Figures 6M 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 mM 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 7B-7E). 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 7G). 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.

RDX Deficiency Phenocopies PRG-2 Thalamocortical Axon Defect
To further substantiate the role of RDX in LPA-induced GC turning, we analyzed the thalamocortical projection in RDX À/À mice. Using biocytin tracing in living E17 thalamocortical slices, we observed misrouted thalamocortical axons in RDX À/À slices that aberrantly invaded the CP ( Figures 7I-7K),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 +/À /RDX +/À ) 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 7L-7O). 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 S6G and S6H), these data could explain how thalamocortical axons are finally able to enter into their target cortical region.

Functional and Behavioral Alterations in PRG-2 -/-Mice
To assess the functional outcome of altered thalamocortical projections in PRG-2 À/À 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 À/À mice. These data point to a more diffuse and broader cortical response extending beyond the border of the activated barrel ( Figures 8A-8C). Moreover, it reflects our findings using retrograde tracing of thalamocortical fibers in PRG-2 À/À mice showing a clear overlap in the termination pattern of neurons from neighboring thalamic barreloids in the VPM (Figures 1R,1T,and 1U). This is in agreement with enlargement of functional barrel columns in mice due to and PRG-2 À/À (B) mice exposed to 10 mM TF-LPA for 40 hr. TF-LPA-containing zone is located in the lower left part of the image. The opposite, LPA-free right side was regarded as control (C). Lines delineate regions located 100 or 200 mm within the LPA-containing or the control (C) region, respectively. Outgrowing axons stained for Tuj1 were color coded in green. (C and D) Higher magnification of WT axons at the TF-LPA interface shows a repulsive effect on outgrowing axons (C, arrows). In contrast, PRG-2 À/À axons were able to enter the LPA-containing region (D 1 ). Higher magnification shows the border of the TF-LPA-containing region color coded in red (D 2 ). (E) Quantitative analysis of fibers protruding 100 and 200 mm into the LPA-rich region (normalized to the number of fibers 100 mm before the TF-LPA-rich matrigel). (Mann-Whitney test; n = 8 WT and 8 PRG-2 À/À thalamic explants). (F) On the control side (C; lower right corner in A and B, containing no TF-LPA), no difference was observed when WT and PRG-2 À/À axons were analyzed at the same distances (as measured to the LPA front). (Mann-Whitney test for 100 mm and t test for 200 mm; n = 8 WT and 9 PRG-2 À/À thalamic explants). (G) Comparison of axon numbers at different depths on the LPA-rich side and on the control side. (Kruskal-Wallis test with Dunn's multiple comparisons test; n = 8 WT and 8 PRG-2 À/À thalamic explants). (H and I) When exposed to lower LPA concentrations (1 mM; H), thalamic WT axons displayed a turning behavior (I; red arrows pointing to turning axons) in front of the LPA-rich region. Border of the LPA-rich zone is marked by dotted line and visible by addition of red fluorescent beads. (J) Thalamic explants from PRG-2 À/À mice at the border of the LPA-rich area displayed no turning behavior and entered the LPA-rich zone in high numbers. (K) Quantitative analysis of the ratio of turning axons to the total number of axons revealed that WT axons displayed a turning behavior while PRG-2 À/À axons were not affected by 1 mM LPA (one-way ANOVA with Bonferroni correction for multiple comparisons; n = 8 WT thalamic explants exposed to 1 mm LPA and to control conditions, 11 PRG-2 À/À thalamic explants exposed to 1 mM LPA, and 8 to control conditions). (L and M) Live imaging of WT (L) and PRG-2 À/À GCs (M) exposed to low LPA concentrations (500 nM) at a distance of 40 mm for 60 min (see also Movies S1 and S2; Figure S5F). Line intersection represents GC starting point used for graphic display of axon growth shown in (N). (O) Analysis of GC behavior (unpaired t test; n = 9 WT and 9 PRG-2 À/À GCs). *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent mean ± SEM. Scale bars, 500 (A and B), 100 (C and D 1 ), 500 (H), 50 (I and J), and 10 mm (L-N).
(legend on next page) 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 À/À 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 À/À mice compared to WT controls ( Figures 8D-8F), which are the main target layers of the thalamocortical projection (Meyer et al., 2010). 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 À/À 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 mm] and 80 grade [grain size, 190 mm]; Figure 8G). 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 À/À 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 À/À mice in the Morris water maze (MWM). Escape latency for visible and hidden platforms was indistinguishable between PRG-2 À/À and WT littermates ( Figures S7E-S7G). Since learning per se was not impaired in PRG-2 À/À 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 in-formation, we also tested the above described thalamus-specific PRG-2-deficient mouse line (PRG-2 DE12/DE12 ). 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 À/À 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 DE12/ DE12 mice in the sandpaper radial maze revealed that these mice displayed the same phenotype as the constitutive PRG-2 À/À 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 À/À mice alter cortical information processing, resulting in a specific deficit in sensory discrimination at adult ages.

DISCUSSION
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., 1990). Here, 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 Molná r, 2015), molecular mechanisms regulating the subplate neurons/thalamocortical fiber interaction are far (K and L) CoIP (using an antibody against PRG-2) of RDX-GFP and PRG-2 after serum starvation of stable PRG-2-expressing HEK cells (K) revealed significantly increased RDX-PRG-2 interaction after 15 min of 1 mM LPA stimulation (L; one-sample t test; n = 6 per group). (M and N) Western blot of immunoprecipitated RDX-GFP (M; contained in the IP product shown in K) displayed significantly higher pERM levels after LPA stimulation (N; one-sample t test; n = 5 per group).
(O) Peptide-microarray-based mapping of the RDX/PRG-2 interaction in the C-terminal PRG-2 region was assessed for RDX WT and RDX T564A . RDX WT displayed a different binding profile when compared to RDX T564A , as shown for the marked PRG-2 C-terminal cytoplasmic fragments quantitatively analyzed in Figure S6P (see also Table S1). (P and Q) Western blot (P) and quantitative analysis of IP product from PRG-2-expressing HEK293 cells transfected with RDX WT or RDX T564A (Q; one-sample t test; n = 5 per group). (R and S) Western blot (R) of PRG-2 À/À neurons stimulated with LPA (1 mM for 15 min) did not display altered pERM levels (S; one-sample t test; n = 18 WT and n = 7 PRG-2 À/À ). *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent mean ± SEM. Scale bars, 100 (A and B) and 1 mm (D). 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 À/À mouse line as well as a thalamus-specific PRG-2 KO line (PRG-2 fl/fl /Gbx2 CreER /R26 tdTomato ), where tamoxifen application at E12.5 leads to specific deletion of PRG-2 in the thalamus (PRG-2 DE12/ DE12 ; 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.

Role of Phospholipids in Subplate/Thalamocortical Axon Interaction
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. Here, we found that PRG-2 À/À thalamocortical axons lose their guidance by the subplate. This specific phenotype relies mechanistically on (E-H) LPA stimulation increased pERM expression at the GC membrane in WT axons (E and F), but not in PRG-2 À/À axons (G and H). (I) Signal intensity analyses of pERM at the GC membrane upon LPA stimulation (Mann-Whitney test; number of analyzed GCs = 21 non-stimulated and 23 stimulated WT GCs, and 22 non-stimulated and 24 stimulated PRG-2 À/À GCs). (J) Ratio of pERM expressed in the GC center and in the periphery significantly increased upon LPA stimulation in WT GCs, while no change was observed in PRG-2 À/À GCs (one-way ANOVA with Bonferroni correction; number of analyzed GCs = 21 non-stimulated and 20 stimulated WT GCs, and 22 non-stimulated and 24 stimulated PRG-2 À/À GCs). (K and L) RDX-GFP-transfected GC of a WT neuron at DIV2 before (K) and 20 min after 1 mM LPA stimulation (L). White arrow indicates site of LPA stimulation (500 mM LPA at a distance of 40 mm); yellow arrow indicates RDX-GFP translocation from the GC center to the GC periphery at the site of the LPA application. (M and N) RDX-GFP distribution in a PRG-2 À/À neuron at DIV2 before (M) and after LPA stimulation (N) did not markedly change. **p < 0.01, ***p < 0.0001. Bars represent mean ± SEM. Scale bars, 5 mm.
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 DE12/DE12 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.
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 À/À neurons, RDX phosphorylation did not increase, even upon 15 min of 1 mM 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 T564A -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 À/À 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.
In Vivo Effects of Altered Thalamocortical Guidance in Adult PRG-2 -/-Mice Using in vivo imaging and high-density multi-electrode recordings in the barrel cortex of PRG-2 À/À mice and performing behavioral tests both in PRG-2 À/À mice and PRG-2 DE12/ DE12 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). Interestingly, Cossell et al. (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 À/À 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.

Mouse Lines
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. 177-07/G 13-1-073). Details of the generation and breeding of PRG-2 À/À , PRG-2 DE12/ DE12 /Gbx2 CreER /R26 tdTomato , and RDX À/À mice are provided in the Supplemental Experimental Procedures.

Immunofluorescent Stainings and Tracing Experiments
Immunofluorescent (IF) stainings, PLA for detection of in situ protein interaction, and anterograde and retrograde tracing experiments were performed following standard protocols.

Slice Culture Preparation, Biocytin Labeling, and Slice Electroporation
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 4 C 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).

Assessment of pERM Levels in Cortical Neurons
Cortical neurons were prepared as described (Vogt et al., 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
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 and In Vivo Electrophysiological Recordings
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.

Animal Behavior
Sandpaper discrimination test and MWM were performed according to standard procedures.

Statistical Analysis
For animal experiments, mice from the same litter or of similar age were chosen. The investigator was blinded to the genotype of the animals. 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. p value was adjusted for multiple comparisons. 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, a two-way ANOVA was used. Statistical analyses of data were performed using GraphPad Prism 5/6 or SPSS.  LGLPDEASPVHLRA . PRG-2 and L1 expression in the brain at E16 show prominent co-localization in the thalamo-cortical projection (arrowheads; T = thalamus). Scale bar: 500µm. B). In situ hybridization for PRG-2 at E14.5 (using mRNA probe 1193 characterized in the genepaint open source data base, as described by (Diez-Roux et al., 2011)). Note the strong cellular PRG-2 mRNA expression in the thalamus reflecting the PRG-2 expression in thalamic fibers shown in Figure S1A. Scale bar: 300 µm.
E). Schematic design of the construct for coPRG-2 fl/fl mice generation (upper row) and after cre-mediated gene deletion (here, using a Del-Cre expressing mouse line, lower row). PCR primers for genotyping are indicated.
I). Western Blot results using a custom made antibody against PRG-2 (used for immunofluorence in G and H) directed against the amino acid sequence 701-716 located in the C-terminal part of PRG-2. Note the decreased expression in PRG-2 +/and the missing signal in PRG-2 -/tissue.
J,K). Subplate neurons as depicted by Ctgf staining were not altered in PRG-2 -/mice. Scale bar: 100µm. L). Overview of a tangential cut brain slice of a NeuroVue traced (NeuroVue insertion in thalamic VB) PRG-2 -/brain. Note the many aberrant thalamo-cortical fibers (arrow heads) crossing the barrel borders visible by the additional DAPI staining.
M,N). Cortical layering as displayed by Ctip2 and DAPI staining in WT and in PRG-2 -/mice. Scale bar: 100µm.
O). Nissl staining of WT and PRG-2 -/brain slices showing cortical layering of the somatosensory barrel cortex (S1BF), Hippocampus and parts of the thalamus (T) including the PO and VPM do not display visible differences. Scale bar: 500µm. P). Higher magnification of the cortical layers of the S1BF from WT and PRG-2 -/mice shows well preserved cortical layering in both conditions. Scale bar: 100µm.

Figure S3 related to Fig. 2 Re-expression of PRG-2 in PRG-2 -/thalamo-cortial slice cultures rescues aberrant phenotype
A). Thalamo-cortical slice cultures from PRG-2 -/mice prepared at E15 and electroporated with a PRG-2 expressing construct containing an IRES-GFP insert, re-express PRG-2 at E17 and were located in the IZ. Note the strong colocalization of the GFP-reporter and PRG-2 in the cell bodies located in the thalamus (lower left insert). Single fiber resolution shows PRG-2 expression in GFP-expressing axons (upper right insert). Scale bar: 75µm.  F). Exemplary overview of the live imaging experiments. A pipette tip covered with 500 nM LPA was positioned at a distance of 40 µm away from the growth cone. The effect of LPA on axon outgrowth was observed for 60 minutes in a live imaging mode. Scale bar: 10 µm G,H). Axon growth of thalamic axon growth cones exposed for 60 min to a Sema3A matrigel-coated pipette tip located at a distance of 40µm away from the growth cone. Center of the cross-hair represents growth cone position at the beginning of the experiment. Dot represents axon growth cone position after 60 min (n = 9 WT and 7 PRG-2 -/growth cones, one sample t-test). *P<0,05, **P<0,01. Bars represent mean ± s.e.m.  (18), 5 min (9), 10 min (9), 15 min (18), 60 min (9); one way ANOVA with Bonferroni correction).
B). Representative Western Blot showing pERM expression 60 min after LPA stimulation.
C). Densitometric analysis of the pERM expression 60 min after LPA stimulation revealed a significant increase in ERM phosphorylation as compared to the control (n = 18 (control); 9 (60 min); one-sample-t-test). *P<0.05, **P<0.01. Bars represent mean ± s.e.m. D). WB of lysate and IP-product from a stable transfected HEK293 cell line expressing only the transmembrane part of PRG-2 (PRG-2ΔCT) but not its intracellular C-term. PRG-2ΔCT was tagged with HA. Prior to IP, HEK293 cells stably expressing PRG-2 ΔCT were transfected with RDX-GFP. IP and WB were performed using an antibody against HA (6E2 mouse AB, Cell Signaling). WB using the HA antibody (left) and a GFP antibody (right) shows that PRG-2ΔCT was not able to interact with RDX-GFP. E). In parallel to the experiments performed in D, IP was performed in HEK293 cells stably expressing full length PRG-2. IP was performed either with the described PRG-2 antibody or with IgG. F). RNA Expression analysis (NCBI GEO Profile GDS 3442) for plppr4/lppr4 (PRG-1), plppr3/lppr3 (PRG-2) and RDX in mouse brain at embryonic stages E9.5 (n = 6), E11.5 (n = 4) and E13.5 (n = 6) revealed a strong increase in gene expression levels for PRG-2 and RDX concurrently with the start thalamo-cortical axon outgrowth but not for PRG-1 gene expression (two-way ANOVA). G). WB shows developmental expression pattern of RDX in the cortex, the hippocampus and the thalamus. Note the strong decrease of RDX expression during postnatal development.
I 1,2 ). Full-embryo in situ hybridization (ISH) for PRG-2 (from gene paint, mRNA probe 1193 characterized in the genepaint open source data base, as described by (Diez-Roux et al., 2011)). PRG-2 mRNA expression is specifically found in the CNS reaching from the forebrain up to the spinal cord. Higher magnification containing the thalamus is shown in SI2 and S1B and PRG-2 protein expression in thalamic fibers shown in S1A. Scale bar: 900 µm (I 1 ) and 600µm (I 2 ). J). ISH for PRG-1 (from genepaint, mRNA probe 3074). Note the absent PRG-1 expression in telencephalic and diencephalic structures. P). Mean binding intensities and s.e.m. of 6 partially overlapping fragments of the C-terminal tail of PRG-2 (amino acid sequence is shown on the x-axis) that directly interacted with RDX WT or with RDX T564A , respectively. Binding to each fragment was calculated from n = 3 experiments. To avoid background signal, threshold for binding was set at 0.4. Full binding data is shown in Supplemental Table S1. Binding of RDX WT to PRG-2 fragments displayed an altered binding profile when compared to RDX T564A binding to the corresponding PRG-2 fragments (two-way ANOVA, ****P<0.001 for differences in PRG-2 binding and for differences of binding between RDX WT and RDX T564A , respectively). O). Higher magnification of a retrogradely labeled neuron (labeled by the RFP-expression) shows accumulation of red and green tracers. Scale bar: 5 µm. P,Q). PRG-2 deficient and PRG-2 expressing fibers close to the internal capsule. Note the missing PRG-2 expression in the RFP-expressing thalamic axons. Scale bar: 25µm.
R,S). Higher magnification of a RFP-expressing axon running in parallel to PRG-2 expressing axons. Note the clear negative profile of the RFP-expressing axon in the PRG-2 staining. Scale bar: 5µm.

Figure S8 related to Figures 1-7 Thalamo-cortical axon guidance by the ATX-LPA/PRG-2/RDX signal transduction pathway
As shown on the mRNA level (Hoerder-Suabedissen et al., 2013) and on the protein level ( Figure 3A-H), subplate cells located at the lower border of the cortical plate express ATX, a diffusible LPA synthesizing enzyme. PRG-2, an LPA-interacting molecule located at the growth cone membrane of thalamcortical axons, interacts with RDX. Our data show that: 1. LPA leads to a PRG-2 dependent RDX phosphorylation, 2. RDX phosphorylation enhances RDX/PRG-2 binding, 3. PRG-2/RDX interaction is significantly increased by LPA, 4. asymmetric RDXaccumulation at the membrane at the site of the LPA-source is PRG-2 dependent, and 5. thalamo-cortical axon growth cone turning and growth cone collapse induced by LPA is PRG-2 dependent, pointing to a critical role for LPA/PRG-2/RDX signaling axis in thalamo-cortical axon guidance.
Supplemental Table S1 related to Fig. 5 Averaged SPOT responses (from 3 experimental runs) for 69 different 14AA peptide fragments as well as their respective root mean square (r.m.s.) derivations when tested against RDX WT and RDX T564A under identical conditions.
Supplemental Video S1 of a WT growth cone related to Fig. 4 Live-imaging of a WT growth cone exposed to an extracellular LPA-source in the upper right corner for 60 min. Note the initial retraction and the subsequent growth cone turning away from the extracellular LPA-source.
Supplemental Video S2 of a PRG-2 -/growth cone related to Fig. 4 Live-imaging of a PRG-2 -/growth cone exposed to an extracellular LPA-source in the upper right corner for 60 min. Note the unaffected filopodial extensions and the continuous axon outgrowth in spite of the extracellular LPAsource.

Immunofluorescent stainings (IF) and tracing experiments
For IF, mice or time-mated animals were anesthetized (0.5% ketamine i.p.) and transcardially perfused with 4% PFA for IHC. Brains were postfixed overnight in 4% and further processed. Brains were sectioned by vibrating microtomes (Thermo Microm HM650V) and brain sections were blocked for 3h (10% normal goat serum and 0,2% Triton-X) before overnight incubation with primary antibodies at 4°C. Sections were finally immunostained with Alexa-labeled secondary antibodies (Alexa 488, Alexa 568 or Alexa 647, all Invitrogen, Germany). Colocalization studies were performed using antibodies against PRG-2 (custom-made antibody against directed against the AA sequence 701-716 located in the C-terminal part of PRG-2; dilution: 1:6000; for characterization see Figure  Confocal images were taken on a Leica SP8 equipped with standard lasers and a white light laser (WLL). Maximum intensity projection images used for display were produced using Leica confocal software. Stiched images were produced using Leica confocal software or Adobe Photoshop CS6. Linear alterations to original images were performed to enhance contrast and brightness.

Retrograde in-vivo tracings
For retrograde in-vivo tracings at adult ages (mean age at perfusion of WT and PRG-2 -/animals was 10,24 ± 1,79 weeks, mean age at perfusion for PRG-2 ΔE12/ ΔE12 mice was 15,10 ± 0,70 weeks), animals were anesthetized using isofluorane and fixed in a stereotactic apparatus (David Kopf Instruments, California, USA). After exposing the barrel cortex using a dental drill, 50-70nl of red and green retrograde beads (diluted 1:4 in PBS; Lumafluor Inc., Naples, FL, USA) were injected 400 µm apart at a depth of 530µm (corresponding to layer 4) according to standard protocols using micromanipulator controlled pipettes. Stereotactic coordinates were selected according to "The Mouse Brain in Stereotaxic Coordinates" by Paxinos and Franklin, 4th edition, Elsevier Academic Press. Injections were placed at the following coordinates (anterior-posterior [AP] from Bregma, medio-lateral [ML] measured from midline of central sinus, and dorso-ventral [DV] from the dura surface): AP: 1.3mm, ML: 3.1mm, DV:530µm. This depth corresponds around 400µm from the pial surface and targets the cortical barrels containing layer IV. To avoid cortical damage and tracer overlap, retrobeads were slowly injected over 45 minutes. To allow for retrograde tracer transport, animals were perfused 11 days after injection with 4% paraformaldehyde. Subsequently, sections perpendicular to the pial surface (tangential) were obtained on a vibratome, and stained with DAPI (Thermo Fisher, Germany) and mounted for further imaging. Thalamic sections showing the neurons of origin of retrogradely traced thalamo-cortical fibers were cut coronally.

Preparation, quantification and immunofluorescent staining of thalamic explants
Embryonic brains (E14) were sliced using a McIlwain tissue chopper or a vibrating microtome. The 400*400*250μm thalamic tissue was positioned around 400 μm away from the 0,5*0,2cm matrigel rectangle containing 10 µM TF-LPA (which delineates a clear visible border by the fluorescent TF-LPA concentration as shown in Figure 4D 2 ) or containing 1 µM LPA mixed with fluorescence coated beads (which are clearly detectable as shown in Figure 4I,J)). Subsequently, the thalamic tissue and the LPA-containing matrigel were immediately embedded in matrigel (Corning, UK). After matrigel polymerization at 37°C for 10 min, the explant was cultured for 40h in 300μl DMEM/F12 medium (Invitrogen, 21041) supplemented with 17.5 mM glucose, 2.5 mM L-glutamine, 5% FBS, 2% B27 and 15 units penicillin/15 μg streptavidin. Subsequently slices were fixed with 4% PFA and were blocked in 5% NGS and 0,2% Triton. For staining of thalamic axons, slices were incubated with an antibody against Tuj1 (1:1000, MMS-435P, Covance, USA), and were visualized with a secondary Alexa 488 or Alexa 568 goat anti mouse antibody (1:000). Thalamic explants were imaged using a confocal laser microscope (Leica SP8 equipped with a white light laser). In order to avoid bias e.g. by a different positioning to the LPA-containing matrigel, axon numbers were counted 100µm in front of the 10 µM TF-LPA-containing matrigel and 100µm and 200µm beyond the border of the LPA-containing matrigel. The border of the 10µM TF-LPA-rich matrigel was delineated by its TFfluorescent label (see Figure 4D2). This procedure allows normalization of axon numbers in the LPA-containing matrigel for each thalamic explant avoiding bias which might arise from putative differences in manual positioning of the thalamic explants (see also Supplemental Figure S5E). The ability of WT and PRG-2 -/fibers to enter a TF-LPA rich zone was than calculated as a percentage of the normalized WT fibers and is shown in Fig 4E. Furthermore, to assess for the endogenous outgrowth capacity of the thalamic fibers, we have assessed the number of fibers at the same distances from the explant border in the neighboring quadrant (which did not contain LPA) and normalized these values in the same way as described above and displayed them as a percentage of the WT fibers as shown in Figure 4F. In order to compare the ability of the WT and PRG-2 -/fibers to enter a TF-LPA rich zone (in comparison to their endogenous controls), we have normalized the number of fibers entering the TF-LPA-rich zone to the number of fibers at the same distances from the explant border in the neighboring quadrant (not containing LPA and therefore used as endogenous controls). Data is shown in Fig 4G.

Y2H screen for interaction partners of the intracellular C-terminal part of PRG-2
Mouse PRG-2 cDNA of the C terminal cytoplasmic domain was amplified by OneStep RT-PCR and cloned into pGBT9 plasmid to get pGBT9-mPRG-2CD as bait expressing vector in yeast. For IPs, E17 brain lysate or lysate from stable PRG-2 expressing HEK293 cells, which were transiently transfected with a construct expressing a RDX-GFP fusion protein, was used. RDX was cloned into pEGFP-N2 vector (BD Bioscience, Germany) as described earlier (Hausrat et al., 2015). After transfection, cells were incubated for another 48h and were than lysed in 2 x RIPA lysis buffer for 1 h, cleared by centrifugation, and protein concentrations were assessed using an infinite M200 PRO (Tecan, Switzerland) using a Pierce BCA Protein Assay Kit (Thermo Scientific, USA). IPs were performed according to standard protocols. Briefly, after pre-clearing an appropriate amount of lysate with 50 µl 50% slurry Protein G agarose beads and incubation of 5 µg anti GFP (rb, Abcam) or rb anti-PRG-2 antibody (described above) with another 50 µl of 50% slurry Protein G agarose beads, precleared lysate was added to the antibody-bound beads and incubated at 4°C under rotation for 2.5 h. After supernatant removal, beads were washed three times with 2 x RIPA lysis buffer. Finally, each IP sample was resuspended in 40 µl lysis buffer and 10 µl 5 x SDS-PAGE loading buffer, heat shocked in a thermomixer (Eppendorf, Germany) at 95°C for 10 min and analyzed by Western blotting according to standard procedures. Briefly, samples were separated by SDS-PAGE using 10% polyacrylamide gels, and blotted on a nitrocellulose membrane (0.45 µm; BIO-RAD, USA) by semi-dry transfer cell (BIO-RAD, USA) at 100 mA for 40 min. Blots were blocked overnight at 4°C in 10% non-fat dry milk, washed twice, and incubated at room temperature (RT) for 3 h with a rabbit anti-GFP antibody (1:4000 dilution; Abcam, UK), a rabbit anti PRG-2 antibody (1:3000; described above) or a HA antibody (6E2 mouse AB, Cell Signaling) in 1% non-fat BSA. After three washes, membranes were incubated with a donkey anti-rabbit IgG horseradish peroxidase-conjugated antibody (1:5000 dilution; dianova, Germany) in 1% non-fat BSA at RT for 1 h. Immunoreactive bands were detected using SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermo Scientific, USA) according to standard protocols.

Analysis of pERM distribution in growth cone
After 2 days in vitro (DIV2), 1 μM LPA was added in culture medium for 20min. Neurons were subsequently fixed and stained as described (Brandt et al., 2007). After immunofluorescent staining and confocal imaging using a Leica SP8, pERM staining intensity in growth cones was quantified using ImageJ. To differentiate between the signal adjacent to the membrane and the signal in the center of the growth cone, we traced the growth cone contour and applied the enlarge function of ImageJ using negative values to draw concentric contours towards the center of the growth cone at a distance of 1 µm. The ROI containing the area below the growth cone membrane was assigned as periphery, the ROI containing the area in the center of the growth cone was assigned as center. The signal intensity was measured by ImageJ.

Analysis of pERM expression in dissociated neurons
For Western Blot assessment of pERM levels (see below for WB methods) cultured cortical neurons (DIV7) from WT and PRG-2 -/animals were treated as described in the respective experiments. Afterwards cells were lyzed and pERM expression was performed according to standard methods as described elsewhere (Hausrat et al., 2015).
Live imaging of LPA/Sema3A effect on axon outgrowth Dissociated thalamic neurons prepared at E14 (plated on matrigel coated glass slides) and kept for 40h in vitro were used for the collapse assay. To assay for LPA/Sema3A-dependent growth cone affection, a coated pipette tip (500nM LPA or 8µg/ml Sema3A prepared in matrigel) was placed 40μm (LPA) or 20µm (Sema3A) away from the center of the growth cone at an angle of 45° with respect to the initial direction of axon extension. This procedure provided continuous substance release but prevented growth cone affection by fluid flow due to continuous perfusion which might result in growth cone collapse. Live-imaging was performed 20 min before and 60 minutes after LPA/Sema3A-application at a rate of 1frame/second. Only non-collapsed growth cones were used for live imaging. For quantification, axon growth in the quadrant containing the substance coated pipette was accounted with positive values, axon growth in the opposing quadrant or in the quadrants below the starting point were accounted with negative values.
Live imaging of RDX distribution in the growth cone 24 h after preparation, cortical neurons (either WT or PRG-2 -/-) were transfected by Effectene Transfection Reagent (Qiagen) using 0.5µg pEGFP-N2-RDX plasmid per well. At DIV3, live imaging experiments were performed using an inverted TCS SP5 confocal microscope (Leica) equipped with a hybrid detector (HyD, Leica) and a 63x oil objective in a 35°C heated incubation chamber. The LPA-coated pipette tip (500nM LPA in matrigel) was placed 40μm away from the center of the growth cone at an angle of 45° with respect to the initial direction of axon extension. In order to avoid bleaching of RDX-GFP, scanning using a 488 nm Ar-laser was performed every 7 min for the following 60 min.

Analysis of pERM levels:
To assess the effect of LPA on ERM phosphorylation, DIV7 primary cortical (hippocampus) neurons derived from PRG-2 -/mice or littermate controls were prepared according to standard protocols , incubated over time at 37 °C, 5 % CO 2 and saturated humidity with different concentrations of LPA (Avanti Polar Lipids, Alabaster, AL) as indicated. The solvent (aqua dest.) was used as a control. The following steps were performed at 4 °C: Cells were washed once in PBS and harvested in PERM-lysis buffer, containing 1% (v/v) TX-100, 2 mM EDTA, 1 mM PMSF (Applichem, Germany), PhosSTOP phosphatases inhibitors and Complete proteases inhibitors (Roche, Germany) in PBS. After incubation for 30 min lysates were centrifuged at 1,000 x g for 10 min. The resulting supernatants were boiled in SDS sample buffer after adjustment of protein concentrations using a BCA assay (Pierce Biotechnology, USA). Samples were analyzed by western blotting. pERM levels were assessed using ImageJ. ERM levels were used as loading controls.

Analysis of developmental protein expression patterns
To analyze protein expression levels at different developmental stages in different brain regions, the respective tissue was dissected in ice cold PBS and homogenized in lysis buffer (PBS, 1% Triton-X-100) supplemented with Complete Mini Protease inhibitor cocktail (Roche, Mannheim, Germany). The lysates were incubated for 30 min on ice and subsequently centrifuged at 1,000 × g for 10 min at 4°C. Protein concentration was determined using a BCA assay kit (Pierce, Bonn, Germany). Subsequently, protein samples were subjected to SDS PAGE and Western blotting.

Analysis of RNA expression levels
To analyze the gene expression levels in the developing mouse brain for lppr3 (PRG-2) and lppr4 (PRG-1), the GEO profile data set GDS 3442 was downloaded from NCBI. The data set is based on whole genome Affymetrix GeneChip microarray (Hartl et al., 2008). The data points for the respective genes at embryonic stage E9.5 (n=6), E11.5 (n=4) and E13.5 (n=6) were extracted and statistically analyzed using a two-way ANOVA.
SPOT assay (Peptide array analysis). 20 peptide arrays including 69 PRG-2 peptide fragments of the C-terminal cytoplasmic region were bought in microarray CelluSpot TM format from Intavis (Intavis Bioanalytical Instruments AG, Cologne, Germany). After rinsing and blocking of the peptide microarrays with SPOT-Buffer (137mM NaCl, 2.7 mM KCl, 12 mM H 2 PO 4 , pH 7.4, 0.05% Tween-20, 0.1% BSA) the arrays were incubated with GST-tagged RDX WT , RDX T564A and GST alone in parallel for 30 minutes at a concentration of 1 µM in SPOT Buffer. After extensive washing, the arrays were incubated for 30 minutes with an anti-GST Antibody HRP-conjugate (Thermo Scientific) at a concentration of 100 ng/ml. Bound GST, GST-RDX WT or GST-RDX T564A was detected by chemiluminescence using the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) based on the chemiluminescent bio-imaging system MicroChemi (DNR Bio-imaging Systems). The resulting dot-blots were analyzed using the array analyze software (Active Motif) which defines the error range of each data set by comparing the intensities of each peptide duplicate on the analyzed array. For calculations peptide array data sets were included if > 90% of the peptide SPOTs were within an < 5% error margin. The SPOT densities of the accepted data sets were normalized and based on the evaluation of three peptide arrays, average Spot densities and r.m.s. deviations were calculated (full binding data is shown in Supplemenary Table S1).

Proximity ligation assay (PLA)
PLA was performed using Duolink reagents (Sigma, Germany) according to standard protocols. Briefly, primary neurons (derived from E17 WT pups) were cultured on poly-L-lysine coated glass coverslips and transfected with a construct expressing RDX-GFP (construct details are described in the IP section). Afterwards, neurons were cultured for 2 days in vitro (DIV) and fixed with ice cold 4% PFA for 10 min. Subsequently, cells were permeabilized with Triton X-100 and incubated with blocking solution at 37°C for 30 min. Primary antibodies (rb anti PRG-2 1:3000, and mouse anti GFP 1:1000, Abcam, UK) diluted in antibody diluent were incubated at 37°C for 90 min. After washing, slides were incubated with PLA-probes (anti mouse and anti rabbit IgG antibodies) diluted 1:5 at 37°C for 60 min. Following washing, slides were incubated in 1:40 diluted ligation solution at 37°C for 30 min and washed afterwards. Amplification of the signal was performed using an 1:80 diluted polymerase solution incubated at 37°C for 10 min. After final washing, slides were dried, mounted and confocal imaged using a Leica SP8 confocal microscope equipped with a WLL.

Cytochrome oxidase staining
After mice perfusion, brains were incubated in 30% sucrose overnight and were subsequently cut into 50-100μm thick tangential sections. Sections were mounted on glass slides, air-dried and stained for cytochrome oxidase using standard protocols (Wong-Riley and Welt, 1980). Briefly, slices were processed in 0.06% cytochrome C, 4.44% sucrose, 0.032% DAB, 0.0008% Cobalt Chloride, 0.0007% Nickel Ammonium Sulfate and 0.46% catalase for 45min at 37°C. Analysis of barrel surface was performed using ImageJ.

Intrinsic Signal Optical Imaging (IOI) Surgery preparation
For IOI experiments 23 to 28 postnatal days aged mice were used (n = 7 WT and 7 PRG-2 KO animals). Mice were anesthetized with intraperitoneal injection of mixture of Urethane (1g/kg, company) and Chlorprothixene (7mg/kg, company), mixed in the ratio of 3:1 respectively. Heart and breathing rates were continuously monitored during the experiment. Body temperature was maintained at 37°C using a heating pad (ATC 2000, World Precision Instruments, Florida). Xylocaine gel (2%, AstraZeneca, Germany) was applied on the skin before surgery. After scalp dissection, a custom built head post was attached to the right side of the skull with cyanoacrylate adhesive and dental cement to facilitate head fixation during imaging. Since vasculature was clearly visible, there was no need of skull thinning. All whiskers on the contralateral side of the snout were cut except C2. To stimulate the C2 whisker, a custom built magnetic driven whisker stimulator driven by TTL voltage pulses was used. Whisker stimulation was synchronized with imaging using an A/D converter (Micro3 1401, Cambridge Electronic Design Limited, UK) controlled with the spike2 software.

Image Acquisition
For image acquisition, an Andor Zyla 5.5 sCMOS camera (5.5 megapixels) was used, which was mounted on an upright Olympus Microscope. The camera was controlled by Micro-Manager (Vale's Laboratory, UCSF) software. In order to obtain pictures of superficial blood vessels, we used 120 W Fluorescence Microscope Excitation Light Source (X-cite 120Q, Excelitas Technologies), filtered through 488 nm filter. A Nikon 10X objective with a numerical aperture of 0.45 and a field of view of 1.8 x 1.6 mm was used for imaging while no excitation or emission filter was applied. For functional imaging, the focal plane was moved 300-400 µm deeper from the pial surface and red LED (625nm, Thor labs, USA) was used to emphasize rapid changes in blood oxygenation levels. The light intensity was adjusted every time, just below the camera saturation.
To calculate the area of the intrinsic signal evoked by single whisker (C2) stimulation, full resolution data frames were acquired at a frame rate of 15 fps and with spatial binning of 3 X 3. Individual trials were 15 sec long, with an initial 5 sec baseline, a subsequent 5 sec stimulation (5 Hz) and 5 sec post stimulus recording. 30 trials were averaged in order to improve signal-to-noise ratio. Additionally 10 trials were taken without stimulation (as baseline recording) for normalization.

Data Analysis
The images recorded were analyzed using a plugin (Intrinsic Signal Processor VSD1) for ImageJ (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij). Multi-frame image stacks were averaged across 30 trials. Next, the images of baseline periods and stimulation periods were each averaged. After Gaussian filtering of 10 in the X and Y dimensions, we compared baseline and response either by calculating their ratio or by dividing their difference by the baseline (ΔR/R0) (Harrison et al., 2009).

In-vivo electrophysiological recordings
In-vivo electrophysiological recordings in the barrel cortex of adult mice (6 WT and 6 PRG2 KO mice) was performed as previously described (Yang et al., 2013). Briefly, initial intraperitoneal urethane anesthesia (1.6-2 g/kg, Sigma-Aldrich), the head of the mouse was fixed into the stereotaxic apparatus using a custom-made aluminum holder fixed with dental cement on the occipital bones. The mice were kept at a constant temperature of 37°C by placing them on a heating blanket. The positions of target barrel columns (C1, C2 and C3) were identified using intrinsic optical imaging. One silver wire was inserted into the cerebellum and served as a ground electrode. A cranial window of 2x2 mm2 was opened above the center of the mapped barrel columns and the dura was kept intact. A four-shank 80-channel electrode (150 µm horizontal shank distance and 50 µm vertical inter-electrode distance, 1-2 MW, NeuroNexus Technologies, Ann Arbor, MI, USA) was labeled with DiI (Molecular Probes, Eugene, OR, USA) and inserted perpendicularly into the mapped barrel columns. 60 min later after electrode insertion, single whiskers were stimulated approximately 1 mm from the snout using a miniature solenoid actuator (modified from (Krupa et al., 2001) that generated for 16 ms a deflection in the rostral-to-caudal direction. LFP data was recorded at a sampling rate of 20 kHz using a multi-channel extracellular amplifier and the MC_RACK software (Multi Channel Systems, Reutlingen, Germany). At the end of each experiment, animals were deeply anesthetized with an overdose of ketamine (240 mg/kg) and xylazine (31 mg/kg) Then mice were perfused with sodium phosphate buffer and fixed with 4% paraformaldehyde. The mouse brain was tangentially sectioned at 200 µm thickness and processed for cytochrome oxidase histochemistry to identify positions of four-shank probe in recorded barrel columns.

Multi unit activity (MUA)
MUA was detected after filtering the LFP traces at 800-5000 Hz and setting the detection threshold at 7.5-fold baseline SD.

Behavioral assessment Sandpaper discrimination test
Sandpaper discrimination test was specifically designed to assess for behavioral deficits in processing of somatosensory whisker-related information (Morita et al., 2011) with minimal involvement of visual and/or olfactory cues. Briefly, we used a reward based strategy in the 8-Arm Maze, where mice had to discriminate surfaces with different roughness in order to get reward -chocolate flavored sweet food pellet. To provide required level of motivation, mice were first food deprived reducing their body weight to 80% of initial body weight (Rowland, 2007), which was maintained during the whole experiment. Water was freely available during food deprivation period. After habituating mice (mixed gender) with chocolate flavored food pellets for 1h daily for 7 days, animals were habituated for 7 days in the 8-Arm maze (10 min daily) where pellets were available at the end of each arm. At the end of maze habituation all mice showed no signs of anxiety in the maze, moved freely and consumed not less than 40 pellets within 10 min. For the discrimination task we used two different grades of sandpaper (P60 and P80, Bosch): P60 (coarse) being with bigger sand particle size than P80 (fine). The inner side walls of the arms were covered with sand paper: 4 arms with P60, and other 4 with P80.To prevent animals from learning the alternatevisiting strategy, arms of the 8-arm radial maze were arranged in the following fashion: P60-P60-P80-P60-P80-P60-P80-P80. Position of arms was daily shifted while keeping the arrangement. The arms with P60 sandpaper were all rewarded with pellets in the end of the arms. The bulk of non-accessible food pellets was placed behind a wire mesh at the end of each arm in order to prevent animals to use a pellet scent as a cue. To avoid bias by visual cues, light intensity during experiments was dimmed to 0.7 lux which was barely enough for the experimenter to score. Each animal was tested for 10 min. The number of correct (if animal entered P60 arm) and incorrect visits was manually scored. The criterion to score for the visit was when the animal passed at least 3/4th of the arm length.

Morris-Water-Maze
MWM was performed in a large circular tank (diameter 1.2 m, depth 0.4 m) filled with water (25± 1˚C, depth 0.3 m) and an escape platform (10x10 cm), which was submerged 1 cm below the water surface. Mouse performance was recorded using a PC-based video-tracking system "Ethovision" (Noldus Inc., Netherland). Escape latency, swim speed, path length, and trajectory of swimming were recorded for each mouse. During the first two days mice were trained to swim to a clearly visible platform ("visible platform" task marked with a 15 cm high black flag). After two days of "visible platform" training, "hidden platform" training (spatial training) was performed. Mice were trained for 8 days to find a hidden platform (the flag was removed) located at the center of one of the four quadrants of the pool. Mice had to navigate using visible cues. Mic received four daily trials with a random start and with an inter trial interval of 5 min. The test ended when mice found the platform, or after 90 s. After completion of the "hidden platform" training, a "probe trial" was conducted where the mouse was allowed to swim freely for 90 s. The time spent in the each quadrant of the pool was recorded.

Supporting Responses
For supporting forelimb responses, the animal was hold by the nape of its neck and allowed to touch a 2mm wide stick, which was located horizontally, 20cm above the table top. After the animal grasped the stick with its forelimb, time grabbing the stick was measured. For supporting hindlimb responses, the test was done in a similar fashion as the previous test, however, the animal was only scored when it hold the stick by its forelimbs but also using synergistic support of the body with the hindlimbs.

Statistical analysis
After assessing for normal distribution (using the K.-S. 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. Comparison between more than two groups was performed using an one-way ANOVA. Post hoc analysis for more than two groups was performed using the Bonferroni adjustment for multiple comparisons. For behavioral analyses assessing genotype effects and time effects, a 2-way ANOVA was used. Statistical analyses of data (including testing for normality using the K.-S. test) were performed using GraphPad Prism 5/6 (La Jolla, CA, USA) or SPSS (Chicago, IL, USA).