Scl expression is knocked down by MO injection. (A) Genomic structure of the scl gene. Exons (ex) depicted as boxes, intron sizes not to scale. Atg and spliceMO binding sites and primers for RT-PCR analysis in panel C are shown (red lines and black arrows). The major splice variant obtained after spliceMO injection is indicated by the red line. (B) AtgMO injection blocked translation of a coinjected scl:GFP fusion mRNA that included the MO target sequence. GFP fluorescence was examined at germ ring stage (5.7 hpf) and was found to be strongly reduced in the presence of the atgMO (11 × magnification; views are animal [a] and lateral [l] as indicated). (C) RT-PCR analysis revealed formation of an alternative splice product following spliceMO injection (426 bp, upper red arrow), in addition to residual wt product (401 bp, lower red arrow). (D) DNA sequencing of the alternative splice product revealed the use of a cryptic splice donor 25 bp into the intron (A). These 25 bases (shown in red) caused a frameshift which, when translated, would give rise to a truncated Scl lacking the bHLH. (E) Live 24-hpf embryos (15 × original magnification); lateral views; anterior, left. While spliceMO-injected embryos exhibit normal overall morphology, atgMO-injected embryos displayed severe nonspecific defects by 24 hpf.