The data here reported demonstrate that zebrafish expresses only one isoform of CD mRNA (of the expected size 1380 bp) that drives the synthesis of a pro-CD of 43 kDa which is then converted into a mature, sellckchem enzymatically active, single-chain protein of 41 kDa. The role of CD in zebrafish development was determined by morpholino-mediated KD and was definitively confirmed by rescue experiments. We observed a phenotype of zebrafish CD KD, showing multi-systemic anomalies, only when CD mRNA was targeted by a morpholino (T-MPO) interfering with the translation process. S-MPO, which targeted a splicing sequence in newly synthesized CD pre-mRNA in fertilized eggs, greatly, yet not completely, down-regulated CD expression. The phenotype of S-MPO larvae was indistinguishable from wild type.

These observations indicate that: 1. maternal mature CD mRNA pre-exists in UFE and effectively drives the synthesis of CD upon fertilization, and 2. the translation of this mRNA provides a sufficient amount of CD to guarantee the (apparent) normal development of the embryo. At 4 dpf T-MPO-mediated CD KD zebrafish larvae presented with several phenotypic alterations, including failure of yolk absorption, reduced growth of the whole body and of the digestive tract, the lack of the swim bladder, microphtalmia and the disorganization of the RPE. All these alterations could be attributed uniquely to the lack of CD, based on the fact that rescuing CD protein synthesis by co-injecting a non-T-MPO sensitive mutant CD mRNA along with T-MPO led to the complete rescue of the normal phenotype, including the correct organization of the RPE layer.

��Rescued�� larvae at 4 dpf expressed approximately 20% of the CD protein present in controls, an amount comparable with that found in S-MPO 4 dpf larvae. Thus, a relatively small amount of active CD in the very early stage of development is sufficient to allow the normal growth of zebrafish embryo. This strengthens the importance of CD-mediated proteolysis in embryo-morphogenesis of zebrafish. It is to be noted that T-MPO larvae eventually die at around 10 dpf (data not shown). It is conceivable that the absence of the swim bladder, which compromises the navigation, in conjunction with the defective development of the digestive tract negatively impact on feeding and nutrients absorption, which may concur to premature death.

S-MPO larvae also showed a reduced survival rate (10% less compared Drug_discovery to controls), further indicating that the amount of available CD in the early stage of development may be a limiting factor for zebrafish survival. Zebrafish embryo development fully depends on yolk in the first 3�C4 days [35], [39]. Vitellogenin is the main precursor of yolk proteins in eggs of oviparous animals [50]. Accumulating evidence indicate that the ovarian hydrolase responsible for the conversion of vitellogenin into yolk proteins is actually the lysosomal protease CD.