Fig. S3

Morpholino controls (A-C) Isl2^aTG or ^e4i4 injected embryos do not show enhanced cell death. TUNEL labeling was used to detect apoptotic cells in wild type, and isl2^ATG morphants at two different doses. Although some neural death was observed, cell death in vascular regions was not elevated over that observed in wild type controls. (D-E) Isl2^ATG MO injected embryos show motoneuron stalling defects as previously demonstrated: Isl2^ATG MO was injected into Tg(mnx1:GFP)^ml2 embryos expressing GFP in primary motoneurons. At 24 hpf wild type embryos show normal growth of the caudal primary motoneurons with axons that extend from the spinal cord to the ventral aspect of the embryo (arrow), Embryos injected with isl2^ATG MO show stalled axons with increased branching (asterisk). Scale bar represents 50 µm. (F) Knockdown efficiency of isl2 morpholino cDNA from uninjected controls or Isl2 morphants (injected with either 1 ng or 8 ng morpholino) underwent PCR with primers for the housekeeping control gene (EF1α), or for isl2. In morphants injected with isl2e4i4, EF1α levels are unchanged while the amount of wild-type isl2 product is diminished (arrow) and a new higher molecular weight band appears (*), representing mis-splicing caused by the morpholino. Injection of 1 ng of morpholino causes partial mis-splicing, while mis-splicing is almost complete with 8 ng of morpholino. The predicted product size for wild type Isl2 is 547bp. We found a 1663bp mis-spliced band in morphants corresponding to the inclusion of an intron in the mRNA, as detected by sequencing.