Vesicle trafficking is essential for a wide range of cellular processes and defects in this process have been shown to underlie various human diseases. Intracellular protein transport and sorting by vesicles in the secretory and endocytic pathways requires the formation of a protein coat on the membrane. The heterotetrameric adaptor protein complex 1 (AP-1) sorts proteins into and formed clathrin-coated vesicles and they regulate maturation of early endosomes. The ubiqui- tously expressed AP-1A complex consists of four adaptins γ1, β1, μ1, and σ1A. In previous studies, we generated γ1 −/− mice; the knock-out mice cease development at the blastocyst stage and inner cell mass stem cells cannot be cultivated. Moreover, γ1 +/− mice grow slower than their wild type littermates. The data reveal that AP-1 is essential for viability and for the development of multicellular organism. Analysis of the zebrafish genome revealed the existence of two gamma subunits: γ1 and γ2. The expression of both subunits is widespread in the whole organism at 24 and 48 hpf. We knocked-down the synthesis of gamma1 and gamma2 transcript using a splicing-inhibiting morpholino specific for each transcript. We injected both morpholinos (γ1MO and γ2MO) and we analysed neuronal and vascular tissue as well notochord develop- ment in more detail. AP-1/γ1 and AP-1/γ2 are both essential for development of those tissues and γ1/γ2 double-deficiency induced even more severe phenotypes, and embryos dye at 48 hpf. In order to generate knock-out fish for gamma1 subunit, using the CRIPSR/Cas9 technique, we injected at one-cell stage the specific sgRNA for γ1 and the mRNA for Cas9. We obtained F0 mutants fish that showed the same phenotype of γ1 morphants. In F1 generation we found 7 heterozygous individuals out of 90 genotyped; the heterozygous fish carry a frame-shift muta- tion with insertion of two or four nucleotides and they showed hepatomegaly and splenomega- ly. Next, we generated the F2 generation showing the Mendelian distribution and we found that the homozygous individuals (21%) dye between 4 and 5hpf (blastula stage). The phenotype of −/− embryos is morphologically evident and the cells at the animal pole disaggregated in short time and they undergo the necrosis process. The preliminary results obtained by the injection of gamma1 mouse mRNA in embryos at one-two cell stages show that the −/− embryos may recov- er the phenotype and develop normal phenotype. We conclude that CRISPR/CAS can be used as a powerful reverse genetic screening strategy in vivo in a vertebrate system and our results confirm the central role of the AP-1/γ1 during the development of a vertebrate model, possibly completing the data obtained in the mouse model.

Early embryonic death of zebrafish knock-out for γ1 gene (AP1-adaptor complex)

FACCHINELLO, NICOLA;TISO, NATASCIA;ARGENTON, FRANCESCO;BORSANI, GIUSEPPE;ZIZIOLI, DANIELA
2017

Abstract

Vesicle trafficking is essential for a wide range of cellular processes and defects in this process have been shown to underlie various human diseases. Intracellular protein transport and sorting by vesicles in the secretory and endocytic pathways requires the formation of a protein coat on the membrane. The heterotetrameric adaptor protein complex 1 (AP-1) sorts proteins into and formed clathrin-coated vesicles and they regulate maturation of early endosomes. The ubiqui- tously expressed AP-1A complex consists of four adaptins γ1, β1, μ1, and σ1A. In previous studies, we generated γ1 −/− mice; the knock-out mice cease development at the blastocyst stage and inner cell mass stem cells cannot be cultivated. Moreover, γ1 +/− mice grow slower than their wild type littermates. The data reveal that AP-1 is essential for viability and for the development of multicellular organism. Analysis of the zebrafish genome revealed the existence of two gamma subunits: γ1 and γ2. The expression of both subunits is widespread in the whole organism at 24 and 48 hpf. We knocked-down the synthesis of gamma1 and gamma2 transcript using a splicing-inhibiting morpholino specific for each transcript. We injected both morpholinos (γ1MO and γ2MO) and we analysed neuronal and vascular tissue as well notochord develop- ment in more detail. AP-1/γ1 and AP-1/γ2 are both essential for development of those tissues and γ1/γ2 double-deficiency induced even more severe phenotypes, and embryos dye at 48 hpf. In order to generate knock-out fish for gamma1 subunit, using the CRIPSR/Cas9 technique, we injected at one-cell stage the specific sgRNA for γ1 and the mRNA for Cas9. We obtained F0 mutants fish that showed the same phenotype of γ1 morphants. In F1 generation we found 7 heterozygous individuals out of 90 genotyped; the heterozygous fish carry a frame-shift muta- tion with insertion of two or four nucleotides and they showed hepatomegaly and splenomega- ly. Next, we generated the F2 generation showing the Mendelian distribution and we found that the homozygous individuals (21%) dye between 4 and 5hpf (blastula stage). The phenotype of −/− embryos is morphologically evident and the cells at the animal pole disaggregated in short time and they undergo the necrosis process. The preliminary results obtained by the injection of gamma1 mouse mRNA in embryos at one-two cell stages show that the −/− embryos may recov- er the phenotype and develop normal phenotype. We conclude that CRISPR/CAS can be used as a powerful reverse genetic screening strategy in vivo in a vertebrate system and our results confirm the central role of the AP-1/γ1 during the development of a vertebrate model, possibly completing the data obtained in the mouse model.
10th European Zebrafish Meeting - Book of Abstracts
978-615-5270-37-6
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3238934
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