Functional Genomics Tools for the Analysis of Zebrafish Pigment

Abstract

Genetic model organisms are increasingly valuable in the postgenomics era to provide a basis for comparative analysis of the human genome. For higher order processes of vertebrate pigment cell biology and development, the mouse has historically been the model of choice. A complementary organism, the zebrafish (Danio rerio), shares many of the signaling and biological processes of vertebrates, e.g. neural crest development. The zebrafish has a number of characteristics that make it an especially valuable model for the study of pigment cell biology and disease. Large-scale genetic screens have identified a collection of pigmentation mutants that have already made valuable contributions to pigment research. An increasing repertoire of genomic resources such as an expressed sequence tag-based Gene Index (The Institute for Genomic Research) and improving methods of mutagenesis, transgenesis, and gene targeting make zebrafish a particularly attractive model. Morpholino phosphorodiamidate oligonucleotide (MO)ÔknockdownÕ of pigment gene expression provides a nonconventional antisense tool for the analysis of genes involved in pigment cell biology and disease. In addition, an ongoing, reverse-genetic, MO-based screen for the rapid identification of gene function promises to be a valuable complement to other high-throughput microarray and proteomic approaches for understanding pigment cell biology. Novel reagents for zebrafish transgenesis, such as the Sleeping Beauty transposon system, continue to improve the capacity for genetic analysis in this system and ensure that the zebrafish will be a valuable genetic model for understanding a variety of biological processes and human diseases for years to come.

Autolysosome biogenesis and developmental senescence are regulated by both Spns1 and v-ATPase

“…S5A and B; MO2). 7,19,20 We found that further knockdown of atp6v0ca by the MOs was more effective in suppressing the opaque yolk phenotype in cis-spns1 hi891/hi891 ;atp6v0ca cm/cm animals compare with the single spns1 hi891/hi891 mutant ( Fig. 7A and B, and Fig.…”

“…7 The phenotype induced by the atp6v0ca knockdown during early development was particularly evident based on hypopigmentation observed at 48 hpf., 7,19,20 By further performing atp6v0ca MO1 injections into spns1-mutant embryos we demonstrated an increased number of viable larvae with temporally ameliorated yolk phenotype through 120 hpf (Fig. 1D).…”

“…To extend our studies, we initially relied on a morpholino approach. Because BafA specifically binds to the Atp6v0ca/subunit c of the v-ATPase and thereby inhibits its enzymatic and proton-pump activity, we examined a specific knockdown of the atp6v0ca gene by using an antisense morpholino oligonucleotide (MO; atp6v0ca MO1), 7,19,20 and found that the morphants recapitulated the phenotype of the BafA-treated animals resulting in reduced yolk opacity and SA-Glb1 activity compare with control spns1 mutants. 7 The v-ATPase is a lysosomal proton transporter, 21 and a proton-coupled symport permease function is proposed for Spns1.…”

“…The sequences of the atp6v0ca MO1 and MO2 are 5 0 -GTTCGGGAGACTAAAAC-TAAAGGCA-3 0 and 5 0 -CTTCTTTTGGCTGACAGGTCTTGC A-3 0 , respectively, which bound to the 5 0 -untranslated regions of zebrafish gene atp6v0ca mRNA, blocking translation. 7,19,20 The spns1 MO sequence 5 0 -ATCTGCTTGTGACATCACTGC TGGA-3 0 overlaps the 5 0 untranslated region-exon boundary at the 5 0 -splice junction of exon 1 in the zebrafish spns1 gene. The sequence of the standard control MO is 5 0 -CCTCTTACCT-CAGTTACAATTTATA-3 0 .…”

Genome Scanning by RNA Interference

“…There have been many prominent examples for this approach in model organisms such as bacteria, yeast, C. elegans, Drosophila, and zebrafish [21][22][23][24]. In contrast stands reverse genetics, in which one goes from a gene/sequence, often discovered via high-throughput Gene-phenotype correlation FIGURE 4.2 Schematic workflow of forward-and reverse genetic screens.…”