Fig. 5

Bmp4 morpholino knock down. (A) Genomic sequence of bmp4 showing exon1 (capitalized), intron1 (non-capitalized) and exon2 (capitalized). Location of the bmp4 splice MO is indicated and the primers used for RT-PCR are indicated by black arrows. (B–D) RT-PCR on mRNA from uninjected control embryos (lane 1) or embryos injected with 1 ng bmp4 splice MO (lane 2) and 0.5 ng bmp4 splice MO (lane 3) and control PCR (-RT) on control embryos (lane 4) or embryos injected with 1 ng bmp4 splice MO (lane 5) and 0.5 ng bmp4 splice MO (lane 6). PCR on the ef1a gene was used as a control for RNA isolation procedure and the RT reaction (B). PCR using the exon-intron primers 1 and 2 will amplify unspliced bmp4 RNA (C) which is induced by bmp4 splice MO injections. PCR using exon–exon primers 1 and 3 will results in the amplification of normal spliced bmp4 mRNA (lane 1) or alternatively spliced bmp4 mRNA after bmp4 splice MO injection (lanes 2 and 3). Band “A” in lane 1 indicates normal spliced bmp4 mRNA and band “B” indicates alternatively spliced bmp4 mRNA with 54 bp insertion (both determined by sequencing the fragments). The asterisk in lane 1 indicates non-specific amplification (determined by sequencing). Arrowheads indicate various alternatively spliced products induced by injection of 1 ng of bmp4 splice MO. (E, F) Injection of 1.6 pg of wt bmp4 mRNA results in a strong ventralization of the embryos (91%, n = 116) (E), while injection up to 40 pg of the bmp4 mRNA with the 54 bp insertion (bmp4-splice) has no effect (0% affected, n = 54) (F). (G–I) Injection of 0.8 ng of bmp4 splice MO results in the loss of ventral structures such as ventral tail fin (arrow) and blood islands (asterisks), both characteristics of mild C1 and C2 dorsalization (H) (in 49% of injected embryos, n = 138) which can be rescued by coinjection of bmp4 splice MO and 0.8 pg bmp4 RNA (I) (15% C1 and C2 dorsalization, n = 156).