Journal: Nature communications

Article Title: Ephrin-A5/EphA4 signalling controls specific afferent targeting to cochlear hair cells.

doi: 10.1038/ncomms2445

Figure Lengend Snippet: Figure 1 | Complementary expression patterns of ephrin-A5 and EphA4 in HCs and SGNs. (a,b) Confocal images of SG cells on transversal section of P0 cochlea processed for ISH with EphA4 probe (a) and for peripherin immunostaining (b). Note that peripherin þ type II SGNs (arrowheads in b) expressed lower levels of EphA4 as compared with peripherin cells. (c–f) Triple immunolabeling on transversal section of SG from P0 EphA4 þ /EGFP mouse showing that EphA4EGFP is expressed in SGNs (yellow arrowheads in c,d,f) but absent from peripherin þ type II SGNs (white arrowhead in d–f). (g,h) Immunolabeling of whole-mount P2 EphA4 þ /EGFP cochlea showing the presence of EGFP (arrowheads in g) in NF-H þ fibres (arrowheads in h) retained at IHCs. ISH on transversal sections of P2 (i) and P0 (j) cochleae showing the expression of EphA4 (i) and ephrin-A5 (j) along the basal-to-apical axis of the cochlea. (k–n) ISH (k) and immunolabeling (l–n) on transversal section of a newborn mouse OC showing a prominent expression of ephrin-A5 mRNA and protein in OHCs compared with IHC. (o–t) Confocal images of SG on transversal section of P0 cochlea processed for ISH with ephrin-A5 probe (o,q,s) and for bIII-tubulin (p,r) or peripherin immunostaining (t). Note that ephrin-A5 is expressed in a subset of SGNs (blue arrowhead in q, yellow arrowhead in r) but is absent from peripherin þ type II SGNs (blue arrowheads in s, yellow arrowheads in t). (u) Quantification of ephrin-A5 þ SGNs across developmental stages. This ephrin-A5 þ SGN subpopulation reaches a peak at P0. Data are presented as mean±s.e.m. (v) Schematic representation of ephrin-A5 (blue)/EphA4 (green) complementary expressions in HCs and SGNs in the perinatal cochlea. Note that ephrin-A5 expression likely overlaps EphA4 in a subset of type I SGNs. Scale bars in (a) represents 10 mm in (a,b), in (c), (g) and (k) 5 mm in (c–f,q–t), (g,h) and (k–n) respectively, in (i) and (o) 25 mm in (i,j) and (o,p) respectively.

Article Snippet: After incubation with pre-clustered ephrin-A5 for 15 min in a humidified atmosphere of 5% CO2/95% air, SGNs were then fixed in 4% paraformaldehyde and immunolabeled using peripherin (rabbit polyclonal antibody, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), EphA4 (tyr-602) phospho-specific (rabbit polyclonal antibody, 1:100; ECM Biosciences, Versailles, KY, USA), green fluorescent protein (rat monoclonal IgG2a, 1:100; Gentaur, London, UK), ephexin-1 (tyr-87) phosphospecific (rabbit polyclonal antibody, 1:100; ECM Biosciences, Versailles, KY, USA), phospho-myosin light chain 2 (ser-19) (rabbit polyclonal antibody, 1:100; Cell Signaling, Danvers, MA, USA) or phospho-cofilin (ser-3) (rabbit monoclonal antibody, 1:100; Cell Signaling, Danvers, MA, USA) antibodies and tetramethyl rhodamine isothiocyanate-phalloidin (1:500; Sigma-Aldrich, St Louis, MO, USA) as an F-actin marker.

Techniques: Expressing, Immunostaining, Immunolabeling

Journal: Nature communications

Article Title: Ephrin-A5/EphA4 signalling controls specific afferent targeting to cochlear hair cells.

doi: 10.1038/ncomms2445

Figure Lengend Snippet: Figure 2 | Radial shift in afferent HC innervation pattern in ephrin-A5 / cochlea. (a–f) NF-H immunolabeling alone (a,b) or in combination with myosin-VII (c–f) of whole-mount cochleae from post-hearing-onset WT (a,c,d) and ephrin-A5 / (b,e,f) mice. Loss of ephrin-A5 induces a radial shift in innervation pattern from the IHC to the OHC layers. The yellow dotted curve delimits the area of projections to OHCs. (g–j) Double immunolabeling for NF- H (g,i) and peripherin (h,j) on whole-mount cochleae from WTand ephrin-A5 / mice. (g,h) In WTmice, almost all projections to OHCs are identified as type II fibres (NF-H þ/peripherin þ) and exhibit extensive arborization (yellow arrowheads). (i,j) In ephrin-A5 / mice, the OHC innervation is more abundant and largely attributable to ectopic NF-H þ/peripherin projections. In addition, type II afferents (peripherin þ) show incomplete innervation (yellow arrowheads) compared with WT. Yellow brackets indicate the OHC area. (k–n) Double NF-H—CtBP2/RIBEYE immunolabeling focused on IHCs from WT (k,l) and ephrin-A5 / (m,n) mice. (o–r) Triple NF-H—parvalbumin—CtBP2/RIBEYE immunolabeling focused on OHCs from WT (o,p) and ephrin-A5 / (q,r) mice. (s) The number of synaptic ribbons per IHC is significantly reduced in ephrin-A5 / compared with WTmice (n ¼ 3 animals for both genotypes; **Po0.01). (t) The number of synaptic ribbons per row of OHCs is significantly increased in ephrin-A5 / compared with WT mice (n ¼ 3 animals for both genotypes; **Po0.01). (u) The number of NF-H þ/peripherin fibres targeting the OHC layer is significantly increased in ephrin- A5 / compared with WT mice (n ¼ 3 animals for both genotypes; ***Po0.001). (v) The percentage of type II fibres of which length isoor4to 200 mm after entering the OC is, respectively, significantly increased and decreased compared with WT (n ¼ 3 animals for both genotypes; **Po0.01). Statistical significance was determined using Student’s t-test. Data are presented as mean±s.e.m. Scale bars in (a) represent 50 mm in (a,b) and 10 mm in (c–f), in (g) 10 mm in (g–j), in (l) 5 mm in (k–r), respectively.

Article Snippet: After incubation with pre-clustered ephrin-A5 for 15 min in a humidified atmosphere of 5% CO2/95% air, SGNs were then fixed in 4% paraformaldehyde and immunolabeled using peripherin (rabbit polyclonal antibody, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), EphA4 (tyr-602) phospho-specific (rabbit polyclonal antibody, 1:100; ECM Biosciences, Versailles, KY, USA), green fluorescent protein (rat monoclonal IgG2a, 1:100; Gentaur, London, UK), ephexin-1 (tyr-87) phosphospecific (rabbit polyclonal antibody, 1:100; ECM Biosciences, Versailles, KY, USA), phospho-myosin light chain 2 (ser-19) (rabbit polyclonal antibody, 1:100; Cell Signaling, Danvers, MA, USA) or phospho-cofilin (ser-3) (rabbit monoclonal antibody, 1:100; Cell Signaling, Danvers, MA, USA) antibodies and tetramethyl rhodamine isothiocyanate-phalloidin (1:500; Sigma-Aldrich, St Louis, MO, USA) as an F-actin marker.

Techniques: Immunolabeling

Journal: Nature communications

Article Title: Ephrin-A5/EphA4 signalling controls specific afferent targeting to cochlear hair cells.

doi: 10.1038/ncomms2445

Figure Lengend Snippet: Figure 4 | Radial shift in HC afferent innervation pattern in post-hearing-onset EphA4 forward-signalling-deficient mice. (a–f) NF-H immunolabeling alone (a,b) or in combination with myosin-VII (c–f) of whole-mount cochleae from post-hearing-onset WT (a,c,d) and EphA4EGFP/EGFP (b,e,f) animals. Loss of EphA4 forward signalling induces a radial shift in innervation pattern from IHC to OHC layers. The yellow dotted curve delimits the area of projections to OHCs. (g–j) Double immunolabeling for NF-H (g,i) and peripherin (h,j) on whole-mount cochleae from WT and EphA4EGFP/EGFP mice. (g,h) In WT mice, almost all projections to OHCs are identified as type II fibres (NF-H þ/peripherin þ) and exhibit extensive arborization (yellow arrowheads). (i,j) In EphA4-knocking mice, the OHC innervation is more abundant and largely attributable to ectopic NF-H þ/peripherin projections. In addition, type II afferents (peripherin þ) show incomplete innervation (yellow arrowheads) compared with WT. Yellow brackets indicate the OHC area. (k,l) Double parvalbumin—CtBP2/RIBEYE immunolabeling focused on OHCs from WT (k) and EphA4EGFP/EGFP (l) mice. (m,n) CtBP2/RIBEYE immunolabeling focused on IHCs from WT (m) and EphA4EGFP/EGFP (n) mice. (o) The number of synaptic ribbons per IHC is significantly reduced in ephrin- A5 / compared with WT mice (n ¼ 3 animals for both genotypes; ***Po0.001). (p) The number of synaptic ribbons per row of OHCs is significantly increased in EphA4EGFP/EGFP compared with WT mice (n ¼ 3 animals for both genotypes; *Po0.05). (q) The number of NF-H þ/peripherin fibres targeting the OHC layer is significantly increased in EphA4EGFP/EGFP compared with WT mice (n ¼ 3 animals for both genotypes; **Po0.01). (r) The percentage of type II fibres whose length isoor4to 200 mm after entering the OC is, respectively, significantly increased and decreased compared with WT (n ¼ 4 animals for both genotypes; *Po0.05). Statistical significance was determined using Student’s t-test. Data are presented as mean±s.e.m. Scale bar in (a) represents 50 mm in (a,b), in (c) and (g) 10 mm in (c–f) and (g–j), respectively, in (k) 5 mm in (k–n).

Article Snippet: After incubation with pre-clustered ephrin-A5 for 15 min in a humidified atmosphere of 5% CO2/95% air, SGNs were then fixed in 4% paraformaldehyde and immunolabeled using peripherin (rabbit polyclonal antibody, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), EphA4 (tyr-602) phospho-specific (rabbit polyclonal antibody, 1:100; ECM Biosciences, Versailles, KY, USA), green fluorescent protein (rat monoclonal IgG2a, 1:100; Gentaur, London, UK), ephexin-1 (tyr-87) phosphospecific (rabbit polyclonal antibody, 1:100; ECM Biosciences, Versailles, KY, USA), phospho-myosin light chain 2 (ser-19) (rabbit polyclonal antibody, 1:100; Cell Signaling, Danvers, MA, USA) or phospho-cofilin (ser-3) (rabbit monoclonal antibody, 1:100; Cell Signaling, Danvers, MA, USA) antibodies and tetramethyl rhodamine isothiocyanate-phalloidin (1:500; Sigma-Aldrich, St Louis, MO, USA) as an F-actin marker.

Techniques: Immunolabeling

Journal: Nature communications

Article Title: Ephrin-A5/EphA4 signalling controls specific afferent targeting to cochlear hair cells.

doi: 10.1038/ncomms2445

Figure Lengend Snippet: Figure 5 | Ephrin-A5 induces growth cone collapse on type I SGNs through activation of EphA4. (a,b,f,g) Representative examples of in vitro growth cone collapse assay on dissociated SGNs from newborn mice using control IgG-Fc or pre-clustered ephrin-A5-Fc. (a) In the control condition, SGNs show a large growth cone with no sign of EphA4 phosphorylation. (b) Upon incubation with pre-clustered ephrin-A5-Fc, F-actin marker tetramethyl rhodamine isothiocyanate-phalloidin (red) reveals severe growth cone collapse simultaneously co-localized with EphA4 phospho-specific (tyr-602) immunolabeling. (f) Growth cones of type II SGNs (peripherin þ, green) are not sensitive to clustered ephrin-A5, and their structure remains similar to those observed in the control condition. (g) Type I SGN growth cones from EphA4EGFP/EGFP mice, in which EGFP is expressed, do not collapse in the presence of ephrin-A5-Fc. (c–e,h–j) Signalling proteins downstream of EphA4 activation. Ephrin-A5-induced growth cone collapse coincides with phosphorylation of ephexin-1 (h), cofilin (i) and myosin light chain-2 (MLC-2) (j) compared with control condition (c,d,e). (k–p) In vitro organotypic model using SG and OC cultured in the presence of dimethylsulphoxide (k,n), Y-27632 (l,o) or ML-7 (m,p). (k,n) Under control conditions, almost all afferent fibres are retained to IHCs and some stopping fibres (arrowheads) can be observed at the limit between IHC and OHC layers (yellow dotted line). When blocking endogenous ROCK (l,o) or MLCK (m,p) signalling pathways, a majority of fibres project to the OHC layer. (q) The proportion of type I growth cone collapse is significantly increased upon stimulation with ephrin-A5-Fc compared with the control condition, but not when type I SGNs lack EphA4 forward signalling. This effect is significantly rescued by pre-treatment with either Y-27632 or ML-7. By contrast, the proportion of type II growth cone collapse is not increased upon stimulation with ephrin-A5-Fc compared with the control condition. n ¼ 3 independent experiments for each condition. **Po0.01, ***Po0.001. (r) The percentage of afferent fibres targeting the OHC layer is significantly increased in the presence of either Y-27632 or ML-7 inhibitors. n ¼ 3 independent experiments for each condition. **Po0.01 Statistical significance was determined using one-way ANOVA test followed by a Dunnett’s post-test. Data are presented as mean±s.e.m. Scale bars in (a) and (k) represent 5 mm in (a-j) and (k–p), respectively.

Article Snippet: After incubation with pre-clustered ephrin-A5 for 15 min in a humidified atmosphere of 5% CO2/95% air, SGNs were then fixed in 4% paraformaldehyde and immunolabeled using peripherin (rabbit polyclonal antibody, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), EphA4 (tyr-602) phospho-specific (rabbit polyclonal antibody, 1:100; ECM Biosciences, Versailles, KY, USA), green fluorescent protein (rat monoclonal IgG2a, 1:100; Gentaur, London, UK), ephexin-1 (tyr-87) phosphospecific (rabbit polyclonal antibody, 1:100; ECM Biosciences, Versailles, KY, USA), phospho-myosin light chain 2 (ser-19) (rabbit polyclonal antibody, 1:100; Cell Signaling, Danvers, MA, USA) or phospho-cofilin (ser-3) (rabbit monoclonal antibody, 1:100; Cell Signaling, Danvers, MA, USA) antibodies and tetramethyl rhodamine isothiocyanate-phalloidin (1:500; Sigma-Aldrich, St Louis, MO, USA) as an F-actin marker.

Techniques: Activation Assay, In Vitro, Control, Phospho-proteomics, Incubation, Marker, Immunolabeling, Cell Culture, Blocking Assay