Taken together, the above data support find FAQ the view that enzymatically active CD is present during zebrafish development, implying a potential role for this protease in this process. Assessment of Cathepsin D knock-down by two different morpholino oligonucleotides in Zebrafish Mature CD was not found in UFE and 30% epiboly embryos. Still, the mRNA analysis suggested the presence of maternal CD mRNA that could drive the synthesis of CD in post-fertilization stages. To achieve the extensive down-regulation of CD protein expression in fertilized eggs, we designed two different morpholino oligonucleotides targeting two different sites of the CD mRNA, so that both splicing and translation events could be disrupted (Fig. 4A).

The S-MPO (splicing morpholino) was aimed at impairing the exon 2-intron 2 splicing in newly synthesized CD RNA, while the T-MPO (translation morpholino) was designed to affect the translation process of both mature maternal (pre-existing) and immature neo-synthesized CD RNAs. T-MPO, in fact, targets a region containing the ATG starting codon. To verify the specificity of S-MPO, we cloned and sequenced from genomic DNA a region of 622 bp (Fig. 4B) that includes the complementary site of S-MPO. Based on sequencing data (Fig. 4C) we can exclude the presence of polymorphisms that could hamper the ability of S-MPO to specifically match with its target in this region. We micro-injected wild type zebrafish fertilized eggs at the one/two-cell stage with standard control morpholino oligonucleotide (control injection, CTRL), S-MPO, or T-MPO.

Micro-injections with either control MPO or the micro-injection solution alone generated larvae with identical wild type phenotype (not shown). Figure 4 Zebrafish cathepsin D down-regulation by two different morpholinos. To test the efficacy of this KD approach, we assayed by western blotting the expression of mature CD at 4 dpf stage of development, that is at the larva stage in which CD expression (apparently) reaches the highest level (see above). Data shown in Fig. 4D demonstrate that in 4 dpf larvae the S-MPO injection reduced by approximately 5-fold the expression of CD, while the T-MPO injection achieved a complete KD of CD expression. Thus, in S-MPO larvae a residual 20% of CD persisted, likely arising from translation of maternal mRNA.

Developmental effects of cathepsin D knock-down in Zebrafish Next, we investigated whether the expression of the 41 kDa single-chain Brefeldin_A CD was indeed functional for the development of zebrafish. We analyzed the effects of CD down-regulation by comparing the gross phenotypic alterations produced by the two morpholinos at 4 dpf larva stage. Zebrafish were grown in the absence or the presence of the melanin-synthesis inhibitor PTU, so that pigmented and completely translucent larvae could be studied. Control MPO-injected fish developed normally and met the predicted developmental milestones [48].