Fig. 2

Analysis of efficiency of UM and SM morpholinos. (A–C) UM efficiently blocks EGFP expression encoded by six1a-gfp mRNA in the embryos sequentially injected with six1a-gfp mRNA and UM (60/60 injected with UM and 1/62 injected with CM). Bright field (A–C, left) and fluorescent (A–C, right) images. Embryos were injected with: (A) six1a-gfp mRNA and UM, (B) six1a-gfp mRNA and CM, (C) six1a-gfp mRNA alone and grown until 10 s. (D–F) SM morpholino effectively blocks the nascent splice donor site in six1a pre-mRNA. (D) Semi-quantitative RT-PCR (29 cycles) of six1a mRNA in SM morphants (sm) generated a shorter DNA fragment (asterisk) than in wild-type (wt) control indicative that an aberrant six1a transcript formed in morphants. Note that only the aberrant transcript was detected in SM morphants at 6 s and 10 s. Analysis of β-actin mRNA was done as a control. RT-negative control was carried out simultaneously and no fragment was detected. The primers used in PCR reactions are described (Materials and methods). (E) Sequencing of RT-PCR fragments revealed the “cryptic” splice donor site (arrowhead in panels E and F) located within six1a protein coding sequence and utilized in SM morphants. (F) Aberrant splicing resulted in the deletion of most of the homeodomain helix III and frameshift in the sequence of six1a mRNA. Region of six1a with intron (lower case) involved in splicing is shown on the top. Genomic sequence of six1a was identified by BLAST search in the clone DKEY-225H23 (accession no. BX649231). Sequence of SM morpholino is shown in bold. Deduced amino acid sequences of wild-type Six1a (regular style) and aberrant Six1a (bold style) proteins are shown below the nucleotide sequence. Amino acid residues that are absolutely conserved across homeodomain family of transcription factors are italicized and underlined and the residues that are conservatively substituted are italicized (Banerjee-Basu and Baxevanis, 2001). Asterisk indicates a stop codon in the aberrant six1a mRNA.