5A). T-MPO 4 dpf larvae presented the following phenotype: 1. reduction of the whole body length (standard length, SL) and of the oro-anal segment (snout-vent length, SVL) (Fig. 5A); 2. skin hyper-pigmentation selleckbio with melanophores dispersed over the yolk (Fig. 5D); 3. absence of inflated swim-bladder (Fig. 5A,D); 4. impairment of yolk absorption (Fig. 5A,D); 5. microphtalmia (Fig. 5B,E). We also observed a reduced dimension of the posterior otolith of inner ear and of the semicircular canals (Fig. 5A,D). However, this phenotype was not evident in a portion of T-MPO larvae and therefore was not further investigated in the present work. SL and SVL in T-MPO larvae were 10% and 18%, respectively, shorter than in paired controls (Fig. 5C). In addition, the whole area of T-MPO larvae eyes was some 20% smaller than that of controls (Fig.

5C). It is to be noted that (apparently) the iridophore was not affected by the lack of CD during development (Fig. 5E). The lack of CD activity has been shown to be causally associated with neuronal lipofuscinosis [24], [26]�C[29]. However, preliminary observations did not reveal such a phenotype in zebrafish larvae at 4 dpf, probably because of the relative short time. Figure 5 Phenotype of zebrafish cathepsin D following down-regulation by two different morpholinos at 4 dpf. The absence of a phenotype in S-MPO zebrafish could be explained by the presence of maternally supplied mature mRNA which guaranteed a sufficient amount of CD protein in the initial stages of embryo development.

Rescue of T-MPO phenotype by mutated zebrafish CD mRNA To definitively demonstrate that the lack of CD underlies all the phenotypic alterations observed in T-MPO zebrafish, a rescue experiment was performed. For this purpose we used an in vitro synthesized CD mRNA carrying eight nucleotide mutations in the matching sequence targeted by the morpholino (Fig. 6A). We micro-injected wild type zebrafish fertilized eggs at the one/two-cell stage with standard control morpholino oligonucleotide (control, Cilengitide CTRL), T-MPO, or T-MPO along with the mutant CD mRNA (RESCUED). We have assessed the optimal amount of exogenous mRNA needed to restore CD expression in the rescue experiments. In total we analyzed n=40 embryos at 2 dpf (from two independent experiments) and n=60 larvae at 4 dpf (from three independent experiments) for each condition. As shown by western blotting analysis (Fig. 6B), the mutant CD mRNA escaped the targeting by T-MPO and could be efficiently translated. The accumulation level of the CD protein in RESCUED zebrafish decreased with time according to the physiological decay of mRNA, which indirectly prove that its synthesis was indeed driven by the exogenous mRNA (Fig. 6B).