CD has been shown to play important roles in eukaryotic cell functioning and tissue turnover and remodeling, being involved in protein maturation and degradation nothing and in cell death as well as in cell proliferation [61]�C[63]. On this ground, it is expected that the lack of CD altered the autophagy flux and the rate of cell death vs cell proliferation in several organs in T-MPO zebrafish. Investigations in this direction are underway in our laboratory. Defective ontogenesis of the swim bladder, altered development of the eye and of the retina, defective absorption of the yolk and shortened lifespan have been reported in zebrafish carrying inactivating mutations in genes involved in the biogenesis, trafficking and fusion of endosomes, lysosomes and lysosome related organelles [35], [36], [49], [64].

The similarities of these phenotypes underscore the role of lysosomal proteolysis in development. In conclusion, the present findings show that CD plays critical roles in tissue homeostasis and organ development in zebrafish. Given the similarity of organ development, organization and function between zebrafish and mammals [30], these data suggest that defective CD-mediated lysosomal proteolysis may contribute to several pathologies in humans also, including the blinding diseases, such as the Leber’s congenital amaurosis, the Best macular dystrophy and the age-related macular degeneration, in which the RPE layer is primarily affected [65], [66]. In this respect, it is worth noting that defective CD activity in human RPE cells has been linked to age-related macular degeneration [67], [68].

Materials and Methods Zebrafish husbandry Zebrafish (Danio rerio) were maintained as previously described [34] and staged based on developmental time and morphological criteria according to published guidelines [39], [48]. Fish were kept under a 14 hours-light and 10 hours-dark photoperiod at approximately 28��C. Following fertilization, eggs were collected and embryos (wild type or micro-injected) were raised at 28��C under standard laboratory conditions. Unless otherwise specified, embryos were grown in the presence of 0.003% 1-phenyl-2-thiourea (PTU) to prevent formation of melanin pigment. Experimental procedures related to fish manipulation followed the recommendation reported in [69] and were conform to the Italian regulations protecting animals used for research purposes, including those of the DL 116/92.

Reverse-Transcription Polymerase Chain Reaction Total RNA was extracted according to the TRIzol LS reagent protocol (Invitrogen Co, Carlsbad, CA, US) from UFE, embryos at 30% epiboly (corresponding to approximately 4.66 hpf), or at 1 and Cilengitide 2 dpf and larvae at 3 and 4 dpf (n=50 of each sample). Aliquots (3.5 ��g) of DNAse I-treated total RNA were retro-transcribed using the RevertAiD H Minus First Strand cDNA Synthesis Kit (Fermentas, Burlington, CA, US).