SUPPLEMENTARY INFORMATION Supplementary Figure 1. The expression of ephrin-b2 H2BGFP persists in the post-hearingonset organ of Corti and is specifically restricted to supporting cells. Sox2 immunolabeling (b,c) in combination with DAPI staining (c) on transversal section of a three-week-old ephrin-b2 +/H2BGFP organ of Corti showing that ephrin-b2 H2BGFP expression is restricted to supporting cells (a,c). Scale bar = 5µm. BC = border cell, DCs = Deiters cells, HeCs = Hensen s cells, IHC = inner hair cell, IPhC = inner phalangeal cell, OHCs = outer hair cells, PCs = pillar cells.
Supplementary Figure 2. Expression patterns of EphB1, EphB2 and EphB3 in the developing organ of Corti. Representative images of in situ hybridization on transversal sections of E16 cochlea showing that EphB1 is expressed in supporting cells (a), EphB2 is absent from the organ of Corti (b) and EphB3 is expressed in pillar cells (PCs) and outer hair cells (OHCs) (c). Scale bar = 5µm. BC = border cell, DCs = Deiters cells, GER = greater epithelial ridge, IHC = inner hair cell, IPhC = inner phalangeal cell, MB = modiolar boundary, SB = strial boundary.
Supplementary Figure 3. GFAP is expressed in all supporting cells in the developing organ of Corti. GFAP immunolabeling alone (a) or in combination with DAPI (b) on a transversal section of E16 cochlea showing that GFAP is expressed in all supporting cells that are directly adjacent to inner (IHC) and outer hair cells (OHCs). Scale bar = 5µm. BC = border cell, DCs = Deiters cells, HeCs = Hensen s cells, IPhC = inner phalangeal cell, PCs = pillar cells.
Supplementary Figure 4. Extra hair cells develop stereocilia and are targets of innervation in GFAP +/cre ;ephrin-b2 lox/lox mice. (a) F-acting staining of whole-mount cochlea from newborn GFAP +/cre ;ephrin-b2 lox/lox mouse showing that extra inner (IHC) and outer hair cells (OHCs) develop stereocilia (arrowheads). (bd) Double parvalbumin NF-H immunolabeling on whole-mount cochlea from newborn GFAP +/cre ;ephrin-b2 lox/lox mouse showing that extra IHC (arrowhead) and OHCs (bracket) are targets of innervation. Scale bar = 5µm in (a) and 10µm in (b-d).
Supplementary Figure 5. Uncropped scans of the western-blots illustrated in Figure 3 of the manuscript. The panels illustrated in the paper are encircled. Left panel : ephrin-b2, right panel : actin. L = lipofectamine.
Supplementary Figure 6. Cell death does not change after transfection using ephrin-b2 compared to scrambled shrna. Active caspase-3 immunolabeling after transfection using scrambled or ephrin-b2 shrnas. Scale bar = 5µm.
Supplementary Figure 7. Ephrin-B2 expression silencing induces supporting cell transdifferentiation and integration in the inner hair cell layer. Representative example of an EGFP-labeled cell transfected with an ephrin-b2 shrna integrated in the inner hair cell (IHC) layer and co-expressing p27 kip1 (b,c) and myosin-vii (b,d). Scale bar = 5µm. MB = modiolar boundary, OHCs = outer hair cells.
Supplementary Figure 8. Quantification of EGFP + /p27 kip1+ and EGFP + /p27 kip1+ /myosin- VII + cells after electroporation using scrambled or ephrin-b2 shrnas.
Supplementary Figure 9. Stepwise supporting cell integration into hair cell layer by inhibition of ephrin-b2 signalling. Ex vivo electroporation of E16 cochleae using ephrin-b2 shrna. Double immunolabeling for p27 kip1 (a-c) and myosin-vii (a,b,d). Successive states of transfected EGFP-labeled p27 kip1+ supporting cells before transdifferentiation: quiescence (blue arrowhead) and translocation (yellow arrowhead). Note that supporting cells progressively lose p27 kip1 expression as they integrate the hair cell layer (b,c). Scale bar = 5µm. HeCs = Hensen s cells, OHCs = outer hair cells, SB = strial boundary.
Supplementary Figure 10. Blocking EphA4/EphB signalling results in supporting cell translocation into hair cell layer and switch in cell identity. In vitro organotypic assay using organs of Corti cultured in the presence of control (IgG-Fc, a- d) or soluble inhibitor ephrin-b2-fc (e-h) reagents. Triple immunolabeling for myosin-vii, p27 kip1 and sox2. (a-d) In control condition, the organ of Corti exhibits sharp boundaries (dotted line in a-c) with cells displaying a clear and unique phenotype (myosin-vii + or p27 kip1+ ). (e-h) When blocking EphA4/EphB signalling, a subset of cells become simultaneously positive for myosin-vii, p27 kip1 and sox2 (yellow arrowhead) or myosin-vii and sox2 (blue arrowhead) at the hair cell / supporting cell interface. As a consequence, myosin-vii + hair cell and p27 kip1+ supporting cell populations cannot be easily segregated along the hair cell / supporting cell boundary (dotted line in e-g). (i,j) The number of myosin-vii + /p27 kip1+ /sox2 + (i) and the number of myosin-vii + /p27 kip1- /sox2 + cells / 100µm of explant (j) is significantly increased in the presence of ephrin-b2-fc compared to control condition (n=3 independent experiments for each condition; *P=0.0142 for myosin-vii + /p27 kip1+ /sox2 + cells, *P=0.0469 for myosin- VII + /p27 kip1- /sox2 + cells). Statistical significance was determined using Student s t-test. Data are presented as mean s.e.m. Scale bar in (a) represents 10µm in (a-h). HeCs = Hensen s cells, OHCs = outer hair cells, SB = strial boundary.