Fig. 3.

Morpholino induced knock-down of invadolysin affects fish viability and results in a decreased number of neuromast clusters. (A) Graph comparing mortality at 24 hpf (black bars) and hatching by 96 hpf (grey bars) in non-injected, Con-MO, ATG-MO and Ex6-MO injected fish. The data were obtained from 3 experiments, with a minimum of 100 fish per sample. (B) RT-PCR analysis of cDNA obtained from non-injected, Ex6-MO, and ATG-MO injected 72 hpf embryos. Primers were designed to detect β-actin (250 bp), invadolysin exons 3–6 (250 bp), and invadolysin exons 3–8 (510 bp). An aberrant, larger splice product was observed with the primers flanking exons 3 and 8 in the larvae injected with the Ex6-MO. (C) 4-Di-2-Asp staining in 76 hpf ATG-MO (left) and non-injected (right) embryos. Arrows indicate aberrant melanophore distribution and pericardial oedema. (D) 4-Di-2-Asp staining in 96 hpf non-injected (top) and ATG-MO (bottom) injected embryos. Fewer neuromast clusters were observed in the ATG-MO injected fish (arrows). Scale bar=250 μm. (E) 4-Di-2-Asp staining in 96 hpf non-injected fish (longer exposure) showing specific clusters that were scored. (F) Comparison of neuromast clusters scored in non-injected, ATG-MO, Ex6-MO, invadolysin transcript injected, and rescued 76 hpf embryos. Non-injected samples (black), ATG-MO (green), ATG-MO+transcript (dark green) Ex6-MO (red) Ex6-MO+transcript (dark red) invadolysin transcript only (striped). A minimum of 24 fish were scored for each sample.