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, 92 (1), 126-142

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

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

Jin Cheng et al. Neuron.

Abstract

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.

Figures

Figure 1
Figure 1
PRG-2 Deficiency Alters Thalamocortical Projection (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-2ΔE12/ΔE12) 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 (C2). (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−/− (E and I) and PRG-2ΔE12/ΔE12 (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-2ΔE12/ΔE12 is shown together with biocytin-labeled fibers in the overview (G1) and at higher magnification (G2). (K) Quantitative analysis shows significant amount of aberrant fibers in PRG-2−/− and in PRG-2ΔE12/ΔE12 slices (Fisher’s exact test; n = 8 WT, 13 PRG-2−/−, and 14 PRG-2ΔE12/ΔE12 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−/− mice (M). (N–P) Thalamocortical fibers at P5 are restricted to specific barrels in the WT brain (N), while in PRG-2−/− 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 Figures S2D–S2G). In contrast, in PRG-2−/− 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−/− mice). Bars represent mean ± SEM. ∗∗∗∗p < 0.0001. Scale bars, 100 (A, B, D–J, and L–P), 50 (Q and R), and 10 μm (S and T).
Figure 2
Figure 2
PRG-2 Reconstitution Rescues PRG-2−/− Phenotype (A) Schematic drawing of the thalamocortical slice preparation, GFP/PRG-2 electroporation, and incubation time. Using a cutting angle of 45°, thalamic neurons and their cortical projection region are maintained in a single slice. (B) WT thalamocortical slice cultures transfected with GFP at E15.5 display a typical thalamocortical projection. (C) In contrast, in PRG-2−/− thalamocortical slice cultures, aberrant fibers prematurely entered the CP. (D) Re-expression of PRG-2 in PRG-2−/− slices from the same litter rescued the aberrant phenotype, resulting in a thalamocortical projection confined to the IZ (see also Figure S3A). (E) Statistical analysis revealed a significant aberrant thalamocortical projection in slices from PRG-2−/− mice that was restored to WT levels by PRG-2 re-expression (Fisher’s exact test; number of analyzed slices = 11 WT, 18 PRG-2−/−, and 10 PRG-2−/− with PRG-2 re-expression). ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Scale bars, 200 (B) and 100 μm (C and D).
Figure 3
Figure 3
Thalamocortical Fiber Tract Defect in PRG-2−/− Mice Is LPA Dependent (A–D) Autotaxin (ATX; A), the LPA-synthetizing enzyme, and LPA (B and D) are highly expressed in the upper part of the IZ and subplate (see also Figures S4A and S4B). PRG-2-positive fibers are restrained in the IZ (C and D). (E–H) Higher magnification of the boxed region confirms ATX (E) and LPA (F and H) expression in close proximity to PRG-2-positive thalamocortical fibers (G and H). (I and J) Biocytine-traced thalamocortical axons develop a normal fiber projection (I), while slices from the same litter incubated with the ATX inhibitor PF8380 (applied to the medium) displayed a severe phenotype with aberrant thalamocortical axons (J). (K) Cortical injection of PF8380 led to an aberrant thalamocortical fiber projection. (L) Analysis of aberrant termination of thalamocortical fibers under different conditions revealed significantly disturbed axon guidance after ATX inhibition (Fisher’s exact test; number of slices = 8 WT, 14 WT + PF8380 applied to the culture media, 13 WT + cortical PF8380 injection, and 11 WT + thalamic PF8380 injection). p < 0.05, ∗∗∗∗p < 0.0001. Scale bars, 100 (A–D), 10 (E–H), and 200 μm (I–K).
Figure 4
Figure 4
PRG-2 Deficiency Abolishes Thalamic Axon Sensitivity to LPA (A and B) Thalamic explants from WT (A) and PRG-2−/− (B) mice exposed to 10 μM 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 μm 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−/− axons were able to enter the LPA-containing region (D1). Higher magnification shows the border of the TF-LPA-containing region color coded in red (D2). (E) Quantitative analysis of fibers protruding 100 and 200 μm into the LPA-rich region (normalized to the number of fibers 100 μm before the TF-LPA-rich matrigel). (Mann-Whitney test; n = 8 WT and 8 PRG-2−/− 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−/− axons were analyzed at the same distances (as measured to the LPA front). (Mann-Whitney test for 100 μm and t test for 200 μm; n = 8 WT and 9 PRG-2−/− 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−/− thalamic explants). (H and I) When exposed to lower LPA concentrations (1 μM; 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−/− 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−/− axons were not affected by 1 μM LPA (one-way ANOVA with Bonferroni correction for multiple comparisons; n = 8 WT thalamic explants exposed to 1 μm LPA and to control conditions, 11 PRG-2−/− thalamic explants exposed to 1 μM LPA, and 8 to control conditions). (L and M) Live imaging of WT (L) and PRG-2−/− GCs (M) exposed to low LPA concentrations (500 nM) at a distance of 40 μm 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). (N) Schematic diagram showing starting point and end point of GCs during live imaging. Axons extending during live imaging (60 min) into the quadrant facing the LPA source were assigned positive values; axons growing in other quadrants were assigned negative values. (O) Analysis of GC behavior (unpaired t test; n = 9 WT and 9 PRG-2−/− GCs). p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Bars represent mean ± SEM. Scale bars, 500 (A and B), 100 (C and D1), 500 (H), 50 (I and J), and 10 μm (L–N).
Figure 5
Figure 5
PRG-2 Interacts with RDX and Mediates LPA-Dependent pERM Expression in Cortical Neurons (A) CoIP of RDX and PRG-2 from E17 brain lysates. Antibodies against RDX co-precipitate PRG-2. Signal below PRG-2 band represents heavy chains of the antibody used for IP. (B) RDX and PRG-2 show clear co-localization along the thalamocortical tract at E16. (C) Higher magnification of (B). (D) PRG-2 and RDX are mainly localized at the GC tips. (E and F) Western blot of PRG-2−/− neurons cultivated for 7 days (DIV7; E) shows a lower expression of phosphorylated ERM (pERM) when compared to WT controls (F; one-sample t test; n = 5 cultures per genotype). (G and H) Western blot (G) and quantification of pERM levels in DIV7 WT neurons stimulated for 15 min with 1 μM LPA (H; one-sample t test; n = 9 cultures per group). (I and J) Western blot of DIV7 WT neurons stimulated with 1 μM LPA (I) shows significantly increased pERM levels starting 10 min after LPA stimulation (J; one-way ANOVA with Bonferroni correction; n = 18 cultures for 0 and 15 min, n = 9 cultures for 5 and 10 min; see also Figures S6A–S6C). (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 μM 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 RDXWT and RDXT564A. RDXWT displayed a different binding profile when compared to RDXT564A, 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 RDXWT or RDXT564A (Q; one-sample t test; n = 5 per group). (R and S) Western blot (R) of PRG-2−/− neurons stimulated with LPA (1 μM for 15 min) did not display altered pERM levels (S; one-sample t test; n = 18 WT and n = 7 PRG-2−/−). p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Bars represent mean ± SEM. Scale bars, 100 (A and B) and 1 μm (D).
Figure 6
Figure 6
PRG-2 Induces LPA-Dependent pERM Expression at the GC Membrane (A–D) RDX, pERM, and NCAM expression in a WT (A and B) and in a PRG-2−/− GC (C and D) of a DIV2 neuronal culture. Note the prominent pERM expression located at the WT GC membrane (B). (E–H) LPA stimulation increased pERM expression at the GC membrane in WT axons (E and F), but not in PRG-2−/− 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−/− 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−/− 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−/− GCs). (K and L) RDX-GFP-transfected GC of a WT neuron at DIV2 before (K) and 20 min after 1 μM LPA stimulation (L). White arrow indicates site of LPA stimulation (500 μM LPA at a distance of 40 μm); 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−/− 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 μm.
Figure 7
Figure 7
RDX−/− Phenocopies PRG-2−/− Thalamocortical Defect (A) Thalamic WT axons growing toward an LPA-containing (1 μM) environment as shown in Figures 4H and 4I. (B–D) Higher magnification of a Tuj1-positive turning GC (D) highlighted in (A) shows clear pERM expression at the GC tip (B; arrow heads), which contains high amounts of F-actin (C; depicted by Phalloidin in red). (E) 3D reconstruction of the turning GC shown in (B)–(D). Arrowheads point to pERM expression. (F and G) Proximity ligation assay (PLA) for PRG-2 and RDX-GFP (F) shows co-localization of PRG-2 and RDX at the GC tip of a DIV2 neuron (G). (H–K) Biocytin tracing revealed normal distributed thalamocortical fibers in WT slices (H) but an aberrant projection in RDX−/− slices (I). Higher magnification of aberrant fibers is shown in (J). Quantitative analysis is shown in (K) (Fisher’s exact test; n = 11 WT and 11 RDX−/− slices). (L–O) Biocytine tracing in thalamocortical slices from WT (L) and PRG-2+/−/RDX+/− animals (M and N) at E17 shows an altered thalamocortical projection in PRG-2+/−/RDX+/− slices. Quantitative analysis is shown in (O). (Fisher’s exact test; n = 14 WT and 12 PRG-2+/−/RDX+/− slices). ∗∗p < 0.01. Scale bars, 10 (A), 2.5 (B–D), 1.5 (D), 1 (F and G), 200 (H, I, L, and M), and 100 μm (N and J).
Figure 8
Figure 8
PRG-2−/− Mice Have Altered Somatosensory Cortical Processing and a Deficit in Somatosensory Discrimination (A and B) Color-coded images represent changes in reflection of hemodynamic response (ΔR/R0) after single-whisker stimulation using intrinsic optical imaging (see also Figures S7C and S7D) in WT (A) and PRG-2−/− animals (B). Threshold set at 5% ΔR/R0 reflecting steepest drop of hemodynamic response is delineated. High hemodynamic response to single-whisker stimulation in WT animals (A) typically corresponds to one barrel with a sharp delineation toward the neighboring barrels. (C) PRG-2−/− mice revealed a significantly broader hemodynamic response (Mann-Whitney test; n = 7 mice per group). p < 0.05 at 5% threshold. (D) Averaged traces of evoked multi-unit activity (MUA) responses recorded in six WT (black trace) and six PRG-2−/− (red trace) mice. Blue dashed line indicates time point of whisker deflection and blue braces indicate period from 0 to 100 ms after whisker deflection used for calculation of MUA shown in (E) and (F). (E and F) MUA in layers II/III (E) and layer IV (F), both marked with green star in (D). Unpaired t test; n = 17 barrel columns from 6 animals per genotype). (G) Image of the eight-arm maze designed for somatosensory perception where arms are covered with sandpaper of different grain sizes. (H) Constitutive PRG-2−/− mice and thalamus-specific PRG-2-deficient mice (PRG-2ΔE12/ΔE12) show significant differences in correct performance when compared to WT mice (two-way ANOVA [genotype, time]; n = 8 WT, 20 PRG-2−/−, and 11 PRG-2ΔE12/ΔE12 animals). p < 0.05, ∗∗∗∗p < 0.0001. Bars represent mean ± SEM. Scale bar, 200 μm (A).

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