Fig. 5. Effect of FGF8a overexpression on neural patterning. (A) FGF8a induces posterior neural genes in ectodermal explants. Embryos were injected into the animal hemisphere at the one-cell stage. Explants were excised at stage 9 and cultured until stage 20. Whole embryo (WE) stage-control embryo sample; whole embryo lysate processed without reverse transcriptase (RT-); explants from uninjected embryo (UI). Embryos were injected as indicated along the top. EF1, loading control; muscle actin (MA) is an indicator of dorsal mesoderm. Endogenous neural gene expression domains are as follows: sox2, general neural tissue; otx2, forebrain and midbrain; en2, MHB; krox20, hindbrain r3 and r5; hoxD1, posterior hindbrain and spinal cord; hoxB9 and cad3, spinal cord. (B) Uninjected X. laevis tadpole; (C) FGF8a-injected tadpole. (D-V) Embryos displayed dorsoanteriorly; red staining indicates the lineage tracer. Embryos injected into a dorsal blastomere at the four-cell stage with 50 pg of FGF8a mRNA and cultured until neural tube stage 19/20. (D-I) FGF8a does not expand expression of the mesodermal genes myoD (32/32 embryos) or coll II (6/6), but sox2 expression domains are mispatterned and expanded (25/25). (M) Fluorescein-conjugated control MO was injected with the FGF8a mRNA as a lineage tracer (pink). (J-V) FGF8a expands posterior neural domains and reduces anterior neural domains. The displayed phenotypes are representative of the effects of FGF8a mRNA quantitated as follows: (K) otx2, 13/14; (M) rx1, 17/17; (O,P) ephA4, 33/35; (R,S) en2, 25/32; (U,V) krox20, 45/52; hoxB9, 43/44.