Fig. S5

Validation of an Antisense Morpholino for Zebrafish meis2b Knockdown Shown in Figure 5 (A) Diagram of the first seven exons of zebrafish meis2b (NM_130910.1), shown approximately to scale, except for introns 2 and 6. Shown are the locations of binding sites for two antisense meis2b morpholinos, one targeting the start codon (ATG MO) to block translation, and one targeting the splice donor site for intron 3 (E3I3 MO) to block mRNA processing. Also shown are the locations of the primers (P1, P2) used in (B). (B) Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of meis2b mRNA structure in wild-type, Standard control MO (control MO), and meis2bE3I3MO embryos. Sequencing of these RT-PCR products revealed wild-type and control MO embryos express wild-type meis2b transcript, while meis2bE3I3MO embryos express two transcript variants (diagrammed on the right). Variant 1 (var 1) retains intron 3, which encodes a premature stop codon (*) that occurs upstream of the Meis2b homeodomain. Variant 2 (var 2) results from aberrant splicing to an upstream cryptic splice donor, resulting in the loss of 51 base pairs of exon 3 but maintenance of the correct open reading frame in exon 4. (C and D) Live 72 hpf control MO and meis2bE3I3MO embryos. meis2bE3I3MO embryos show mild pericardial edema (arrow), as well as variable smaller heads and body curvature phenotypes. Anterior is to the right.