Figure 3

The boundary cell marker FGF3 shows persistent segmental expression in embryos expressing truncated EphA4. (A-E): Flat-mounted hindbrains from different-staged embryos in situ hybridized with FGF3 probe. (A): 8 ss embryo showing FGF3 expression in r4-r6 and low levels in r2. (B): 16 ss embryo showing expression in r2/r4/r6. (C): 20 ss embryos showing expression in r4/r6. FGF3 expression is also becoming apparent at rhombomere borders. (D): 25 ss embryo showing FGF3 transcripts in r6 and in boundaries. (E): 30 ss embryo showing FGF3 localization to boundary cells. (F-I): Flat-mounted hindbrains from embryos electroporated unilaterally with dnEphA4 (G, I) or control (F, H) constructs at 8 ss, and left to develop for further 30 hrs. FGF3 transcripts are restricted to boundaries in control electroporations (F, H), while dnEphA4-embryos show FGF3 within even-numbered rhombomeres (G, I white arrowheads). (J, K): Views of hindbrains electroporated at 22 ss with dnEphA4 (K) or control (J) constructs and left to develop for further 18 hrs. For both, expression of FGF3 is restricted to boundary cells. Embryos in (H-K) were immunostained with anti-GFP antibody followed by Alexa-488 (H, I) or HRP (J, K) secondary antibodies to label electroporated cells. Asterisks mark the electroporated side, anterior is at the top and rhombomere numbers are indicated.