Figure 2

Disruption of boundary cells in embryos expressing truncated EphA4. Flat-mounted views of hindbrains electroporated unilaterally with dnEphA4- (B, D, F, H, J, L, N, O, P) or control (A, C, E, G, I, K, M) constructs at 6–8 ss, then left to develop for further 40 hrs. (A-H): Confocal imaging of hindbrains labeled with anti-CSPG or anti EphA4 antibodies, showing loss or disruption of boundary cells (B, F) and altered rhombomere shape (D, white arrows) upon dnEphA4-electroporation, compared to controls (A, C, E). (G, H): Overlay images of embryos shown in C-F, including GFP expression in the electroporated cells (green). (I-N): In situ hybridized embryos transfected with control (I, K, M) or dnEphA4 (J, L, N) constructs, using probes against pax6 (I, J), NSCL1 (K, L), hoxb1 (M, N). Unilateral loss or decrease in pax6 and NSCL1 boundary staining is observed in the dnEphA4-expressing hindbrains (J, L), in contrast to controls (I, K, white arrows). (M, N): Hoxb1 expression in r4 is altered or absent in some cells in the dnEphA4-trasfected side of the hindbrain (N, white arrows), in contrast to control (M). (O, P): Bright-field and fluorescent images of a frontal paraffin sectioned-embryo electroporated with dnEphA4. Typical morphology of a segmented hindbrain is evident in the control hemi-neural tube (boundaries are marked by lines), in contrast to the transfected side where constrictions and bulges are lost. Asterisks mark the electroporated side, labeling is stated at the bottom, anterior is at the top and rhombomeres are numbered.