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First published online 11 June 2008
doi: 10.1242/dev.009019

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The neural adhesion molecule TAG-1 modulates responses of sensory axons to diffusible guidance signals

Chris O. Law^*, Rebecca J. Kirby^*, Soheil Aghamohammadzadeh and Andrew J. W. Furley

Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.

View larger version (98K): [in this window] [in a new window]   Fig. 1. Expression of TAG-1 and L1 on spinal sensory afferents. (A-H) Transverse sections of E12.5 spinal cord immunolabelled for TrkA and L1 (A-D) or L1 and TAG-1 (E-H). Magnified images of the dorsal funiculus (DF) from C and G shown in D and H, respectively. Asterisks indicate L1+, TAG-1/TrkA- region. (I-P) Transverse sections of E15.5 spinal cord with TrkA and L1 (I-L) or TAG-1 and L1 (M-P). Higher magnification of the DF from K and O is shown in L and P. Asterisks indicate L1+, TAG-1/TrkA- region. Arrows indicate position of TrkA- proprioceptive fibres emanating from the L1+, TAG-1/TrkA- region. Arrowheads indicate TrkA+ fibres. Scale bar: 100 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 2. Aberrant projections of sensory afferents. (A-L) Spinal cord sections immunolabelled for TrkA at E12.5 (A-D), E13.5 (E-H) and E14.5 (I-L) in wild-type (WT; A,B,E,F,J), TAG-1-null (C,G,I,K) and L1 null (D,H,L) mice. Boxes in A and E indicate typical regions shown in B-D and F-H, respectively, at higher power; boxes in I are magnified in K; broken lines indicate the dorsal midline. Arrowheads highlight aberrant projections. Scale bars: 100 µm. (M) Mean number of aberrant projections calculated at E12.5, E13.5 and E14.5 in wild-type (n=8, 4 and 6 embryos, respectively), L1-null (n=6, 3 and 5) and TAG-1-null mice (n=7, 5 and 6). Horizontal bars indicate significant differences (unpaired t-test, ***P<0.001, **P<0.01, *P<0.05). Error bars throughout are s.e.m. (N) Proportion of axons projecting into medial (green) or lateral (blue) regions. Red lines indicate significant differences (Chi-squared test; significance as above). (O) Proportion of axons projecting from medial (orange) or lateral (cyan) regions of the DF. Red lines indicate significant differences (Chi-squared test; significance as above).

View larger version (36K): [in this window] [in a new window]   Fig. 3. Mutant sensory axons fail to respond to ventral spinal cord. (A-C) Co-cultures of wild-type E13.5 ventral spinal cord (VSC) with DRG from E13.5 wild-type (A), L1-null (B) or TAG-1-null (C) embryos in collagen gels. (D) Quantitation of axon growth: DRG were divided into quadrants and axons in each counted to give proximal:distal (P/D) ratio. (E) Graph showing the P/D ratios in wild type (WT; mean=0.269, s.e.m.=0.06, n=27), L1 null (mean=0.713, s.e.m.=0.043, n=15), TAG null (mean=1.03, s.e.m.=0.08, n=15), wild type plus polyclonal anti-TAG-1 (mean=0.67, s.e.m.=0.02, n=7) and wild type plus PI-PLC (mean=0.72, s.e.m.=0.06, n=7). ***P<0.001, **P<0.01, *P<0.05; unpaired t-test. Similar results were obtained counting axon length (not shown). Scale bars: 100 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 4. Mutant sensory axons fail to respond to Sema3A chemorepulsion. (A-C) Co-cultures of Sema3A-transfected Cos7 cell aggregates (S3A) with E13.5 DRG from wild-type (A), L1-null (B) or TAG-1-null (C) embryos. (D) Wild-type DRG co-cultured with Sema3A-transfected Cos7 cells in the presence of soluble NRP1-AP. (E) P/D ratios of axons numbers in wild type (WT; mean=0.096, s.e.m.=0.03, n=25), L1 null (L1-; mean=0.88, s.e.m.=0.047, n=20), TAG-1 null (TAG-; mean=0.71, s.e.m.=0.05, n=15) and wild type plus soluble NRP1-AP (WT+NRP1; mean=1.0, s.e.m.=0.04, n=10). (F,G) Wild-type DRG and VSC cultured together with soluble NRP1-AP (F) and plotted with data from Fig. 3E for comparison (G). There was no significant difference (P>0.05) between L1- and WT+NRP1 in either E or G. Scale bars: 100 µm. (H) NGF-dependent growth cones from wild type (WT) or TAG-1 null (TAG-) with (+Sema3A) or without Sema3A. Growth cones are immunolabelled with anti-L1. Scale bar: 20 µm. (I) Percentage of collapsed growth cones from wild-type or TAG-1-null DRG with (S3A) or without (cont.; supernatant from mock-transfected Cos7 cells) Sema3A. Over 600 growth cones were assessed for each DRG; each datapoint is the mean of data from n=18 (WT+S3A), n=17 (WT cont.), n=7 (TAG- +S3A) and n=5 (TAG- cont.) DRG. ***P<0.001, **P<0.01; unpaired t-test.

View larger version (28K): [in this window] [in a new window]   Fig. 5. TAG-1 does not bind neuropilin 1, plexin A4 or Sema3A. Cos7 cells transfected with full-length L1 (A,D), TAG-1 (B,E,I,J), NRP1 (C,F) or plexin A4 (K,L) and probed with L1-Fc (A-C), TAG-1-Fc protein (D-F,K,L) or Sema3A-Flag (I,J). Quantification of the percentage of cells binding the probe is presented in G (L1-Fc), H (TAG-1-Fc) and M (Sema3A-Flag). ***P<0.001, **P<0.01; unpaired t-test. The transfection efficiency (as assessed by immunolabelling; shown for anti-TAG-1 (I) and anti-Myc (detects plexin A4-myc; K) was similar in each case.

View larger version (39K): [in this window] [in a new window]   Fig. 6. Construction and analysis of a TAG-1 mutant allele that encodes N-terminally truncated TAG-1 protein. (A) Recombination strategy showing part of TAG-1 gene locus, including exon 2 (ATG and leader) and exons 3-13 (Ig domains 1-5), targeting construct and targeted TAG-1a locus. Red lines indicate diagnostic BamHI fragments; black box, probe PP; arrowheads, polymerase chain reaction (PCR) primers. (B) Southern analysis of targeted ES cells with BamHI digest and probe PP. (C) PCR detection of wild-type (+/+) allele (orange primers in A) 450 bp product; TAG-1a allele (green primers in A) 260 bp product. +/a, heterozygote; a/a, homozygote. (D) A major 135 kDa product detected by anti-TAG-1 in western analysis of +/+ and +/a mice, but not in a/a. Novel bands at 100 kDa and 110 kDa present only in +/a and a/a (see J). (E) Aberrant splicing of mRNA from TAG-1a allele and primers (arrows) used for reverse-transcription (RT) PCR in F and G. (F) RT-PCR detects aberrant exon 2-7 splice using a and b primers (see E); 391 bp in a/a animals instead of normal 1017 bp product (+/+). (G) RT-PCR using a and c primers detects exon 2-9 splice (230 bp) in a/a instead of 1133 bp product (+/+). Faint band at 507 bp=exon 2-7 splice. (H) Cloning and sequencing of exon 2 to 9 product (a and d primers in E) confirms splice is in-frame. (I) Surface immunolabelling of wild-type and TAG-1a/a sensory neurons with anti-TAG-1 (see Materials and methods). (J) Predicted proteins. Full-length TAG-1 mRNA encodes a 1041 aa protein, calculated MW 113 kDa (compare with observed MW of 135 kDa) (D). Exon 2-7 splice: 831 aa; calculated MW 89.9kDa; observed 110 kDa. Exon 2-9 splice: 740 aa; calculated MW 79.7 kDa; observed 100 kDa.

View larger version (44K): [in this window] [in a new window]   Fig. 7. Deletion of L1-binding domains from TAG-1 disrupts responses to Sema3A and ventral spinal cord. (A) E13.5 TAG-1a/a DRG co-cultured with Sema3A-expressing Cos7 cells. (B) P/D ratio of number of axons from TAG-1a/a DRG cultured with Sema3A (mean=0.677, s.e.m.=0.068, n=8) compared with wild type, TAG-1-null and L1-null results (from Fig. 4E). (C) E13.5 TAG-1a/a DRG co-cultured with VSC. (D) P/D ratio of number of axons from TAG-1a/a DRG cultured with ventral spinal cord (mean=0.68, s.e.m.=0.042, n=15) compared as in B. ***P<0.001, **P<0.01; unpaired t-test. Scale bar: 100 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 8. Internalisation of L1 and NRP1 is affected in TAG-1 mutants. Immunolabelling of growth cones from wild-type (A,C,E,G; WT) and TAG-1-null (B,D,F,H; null) sensory neurons shows that L1 (A,B) and NRP1 (C,D) reach the surface of TAG-1-null growth cones at normal levels, as quantitated in I (L1) and J (NRP1); n=16 for wild type, n=21 for null (see Materials and methods). Quantitation of surface immunolabelling after Sema3A treatment of wild-type and TAG-1-null growth cones shows that surface levels of L1 (E,F,I) and NRP1 (G,H,J) are significantly reduced in wild type (E,G; WT + Sema) but not TAG-1-null (F,H; Null + Sema) growth cones (**P<0.01, *P<0.05; unpaired t-test). Results from both collapsed and extended growth cones are pooled for each genotype in both conditions (and are present in each population in the proportions indicated in Fig. 4I) but results are expressed as average intensity per unit area to normalise for differences in growth cone size [similar results were obtained when comparing growth cones from the different genotypes according to morphology (i.e. extended versus collapsed; not shown)]. (K) FITC-labelled dextran is taken up to a lesser extent by TAG-1-null growth cones (null + Sema) than by wild-type growth cones (WT + Sema) after Sema3A treatment. (L) Cell-surface TAG-1 levels (red) in wild-type growth cones (counterlabelled with phalloidin; green) fall significantly (P<0.0001) after Sema3A treatment (+Sema; n=13) compared with no treatment (NT; n=12). Panels on the right in each pair show TAG-1 only. Quantitation as for L1 and NRP1 above. (M-Q) Colocalisation of TAG-1 and clathrin heavy chain in wild-type growth cones after Sema3A treatment. Area outlined by white box in P shown in Q. Scale bars: 10 µm in A-H,Q; 20 µm in L.

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