DEV034405 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Eph receptor expression in the developing cortex. (A-N) E14.5 cerebral walls stained for Nissl (A,I) and hybridized with cRNA probes for EphA1 (B), EphA2 (C), EphA3 (D), EphA4 (E), EphA5 (F), EphA6 (G), EphA7 (H), EphB1 (J), EphB2 (K), EphB3 (L), EphB4 (M) and EphB6 (N). (O) Quantification of the expression of EphA receptors (green) and EphB receptors (blue) in the embryonic zones of the E14.5 cerebral wall. CP, cortical plate; IZ, intermediate zone; PZ, proliferative zone.

• Supplemental Figure S2 -

  Fig. S2. EphA4 and EphA7 expression in developing brain. Images of in situ hybridizations of EphA4 (A) and EphA7 (B) in sagittal sections of an E12.5 brain. Similar levels of expression but in opposing gradients are visible within the tectum (T), but levels of EphA4 are much higher than EphA7 in the cortex. A, anterior; D, dorsal; LV, lateral ventricle.

• Supplemental Figure S3 -

  Fig. S3. EphA4 is expressed by cortical progenitors. (A-I) E14.5 cerebral wall stained for EphA4 (A,D,G; green) and RC2 (B), Tuj1 (E) or nestin (H) (red) with merged images (C,F,I). EphA4 overlaps with RC2 and nestin (C,I) but not Tuj1 (F).

• Supplemental Figure S4 -

  Fig. S4. EphA receptor expression and activation. (A-C) RT-PCR of EphA receptors in E14.5 cortex (A), neurosphere (Nsph) cultures (B) and HEK293T cells (C). Patterns in Nsph (B) mirror the E14.5 cortex profile (A), whereas there is little detectable EphA expression in HEK293T cells (C). (D) Western analysis of EphA activation in HEK293T cells transfected with YFP (C, left panel), YFP and EphA4 (EphA4, middle panel), or YFP, EphA4 and DN (EphA4 & DN, right panel) before stimulation (0′) or upon incubation with clustered ephrin A5 for 2 or 10 minutes (2′ and 10′, respectively). There is no detectable EphA activation in control cells (left). Transfection with EphA4 results in some autoactivation (0′, middle), with elevated levels in the presence of ephrin A5 (2′ and 10′, middle). Receptor activation is eliminated when DN is coexpressed (right), demonstrating the ability of DN to silence ephrin A5-mediated EphA4 stimulation. Actin (bottom) reveals equal loading.

• Supplemental Figure S5 -

  Fig. S5. Initial transfection rates of neurospheres are similar. Quantification of YFP⁺ (gray), DN⁺ (black) and ephrin B1⁺ (white) cells 9 hours after transfection. Comparable levels of transfected cells per neurosphere were initially observed.

• Supplemental Figure S6 -

  Fig. S6. Quantification of EphA4^−/− cortex. (A) Anteroposterior length is similar between WT and EphA4^−/− mice. (B). BrdU incorporation is decreased in the PZ and IZ of EphA4^−/− cortex compared with WT. (C,D) TUNEL⁺ cells are rare in WT (C) and EphA4^−/− (D) cortex. (E-H) In situ hybridization of EphA5 in coronal sections at E14.5 (E,G) and sagittal sections at P0 (F,H) in WT (E,F) and EphA4^−/− (G,H) cortex revealed persistent expression of EphA5 after birth in EphA4^−/− (H) but not in WT (F).

• Supplemental Figure S7 -

  Fig. S7. Characterization of transfected cerebral walls. (A,B) Representative images of YFP-transfected cortex 1 (A) and 3 (B) days after IUE. (C) Proportions of YFP⁺ cells in cortical embryonic zones 1 (left) and 3 (right) days after IUE reveals stereotyped developmental maturation. (D) Quantification of transfected cells 1 day after IUE with YFP (dark gray), sA4 (light gray), DN (black) or FL (white). Similar numbers of cells are initially transfected.

• Supplemental Figure S8 -

  Fig. S8. Cell death in transfected cortex. Levels of TUNEL⁺ cells in YFP-, sA4- and DN-transfected cortex, illustrated according to the proportion of transfected (T) and untransfected (U) cells. Cell death increased only with the introduction of DN (right bar) and only in untransfected cells in that condition.

• Supplemental Figure S9 -

  Fig. S9. Rescue of knockdown phenotype. Proportion of transfected cells present in the PZ 3 days after IUE with YFP (C, gray bar), DN or sA4 (black bars), or DN or sA4 plus FL (white bars). The addition of FL increased the proportion of cells remaining in the proliferative zone.

• Supplemental Figure S10 -

  Fig. S10. Ephrin ligand expression in the developing cortex. (A-I) E14.5 cerebral walls stained for Nissl (A,G) and hybridized with cRNA probes for ephrin A1 (B), ephrin A2 (C), ephrin A3 (D), ephrin A4 (E), ephrin A5 (F), ephrin B1 (H), ephrin B2 (I) and ephrin B3 (J). (K) Quantification of the expression of ephrin A (purple) and ephrin B (red) ligands in the embryonic zones of the E14.5 cerebral wall. CP, cortical plate; IZ, intermediate zone; PZ, proliferative zone.

• Supplemental Figure S11 -

  Fig. S11. Ephrin B1 is expressed by cortical progenitors. (A-I) E14.5 cerebral wall stained for ephrin B1 (A,D,G; green) and RC2 (B), Tuj1 (E) or nestin (H) (red) with merged images in C, F and I. Ephrin B1 overlaps with RC2 and nestin (C,I), but not Tuj1 (F).