Utilize este identificador para referenciar este registo: http://hdl.handle.net/10362/13155
Título: Using cilia mutants to study left-right asymmetry in zebrafish
Autor: Sampaio, Pedro Rafael Martins de Almeida
Orientador: Lopes, Susana
Ludovice, Ana
Palavras-chave: Kupffer’s vesicle
Motile cilia
KV flow
Left-right asymmetry
Dnah7
DeltaD mutant
Data de Defesa: 2014
Editora: Faculdade de Ciências e Tecnologia
Resumo: In vertebrates, internal organs are positioned asymmetrically across the left-right (L-R) body axis. Events determining L-R asymmetry occur during embryogenesis, and are regulated by the coordinated action of genetic mechanisms. Embryonic motile cilia are essential in this process by generating a directional fluid flow inside the zebrafish organ of asymmetry, called Kupffer’s vesicle ﴾KV). A correct L-R formation is highly dependent on signaling pathways downstream of such flow, however detailed characterization of how its dynamics modulates these mechanisms is still lacking. In this project, fluid flow measurements were achieved by a non-invasive method, in four genetic backgrounds: Wild-type (WT), deltaD-/- mutants, Dnah7 morphants (MO) and control-MO embryos. Knockdown of Dnah7, a heavy chain inner axonemal dynein, renders cilia completely immotile and depletes the KV directional fluid flow, which we characterize here for the first time. By following the development of each embryo, we show that flow dynamics in the KV is already asymmetric and provides a very good prediction of organ laterality. Through novel experiments, we characterized a new population of motile cilia, an immotile population, a range of cilia beat frequencies and lengths, KV volumes and cilia numbers in live embryos. These data were crucial to perform fluid dynamics simulations, which suggested that the flow in embryos with 30 or more cilia reliably produces left situs; with fewer cilia, left situs is sometimes compromised through disruption of the dorsal anterior clustering of motile cilia. A rough estimate based upon the 30 cilium threshold and statistics of cilium number predicts 90% and 60% left situs in WT and deltaD-/- respectively, as observed experimentally. Cilia number and clustering are therefore critical to normal situs via robust asymmetric flow. Thus, our results support a model in which asymmetric flow forces registered in the KV pattern organ laterality in each embryo.
Descrição: A thesis submitted in fulfillment of the requirements for the degree of the Masters in Molecular Genetics and Biomedicine
URI: http://hdl.handle.net/10362/13155
Aparece nas colecções:FCT: DF - Dissertações de Mestrado

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