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Title: Mechanics and Cellular Mechanisms Driving Zebrafish Epiboly = Mecánica y mecanismos celulares responsables de la epibolia del pez cebra
Author: Hernández Vega, Amayra Noemi
Director: Martín Blanco, Enrique
Bueno i Torrens, David, 1965-
Keywords: Morfogènesi
Peix zebra
Zebra danio
Issue Date: 5-Oct-2015
Publisher: Universitat de Barcelona
Abstract: [eng] Epithelial layers constitute the skeleton of early embryos and most tissues and organs and their remodelling during development is essential for reshaping the embryo and for tissue architecture. Epithelial expansion in particular, has fundamental roles during early embryogenesis in both vertebrates and invertebrates. Some well-established invertebrate models have contributed greatly to our knowledge of this process. However, we are still far from having a global understanding of the cellular, molecular and mechanical changes involved in this process, the diversity, and relationship between them. With this in mind, we decided to explore a less well known model for epithelial expansion, epiboly in the vertebrate zebrafish. Epiboly is the expansion of the blastoderm around a big yolk cell to finally engulf it. Three different layers are involved in this process, an epithelial layer, a mesenquimal layer and a big yolk cell. Although teleost epiboly has been studied for many years a clear understanding of the process was still missing. We analysed the cellular, molecular and mechanical elements involved in this process and found that epithelial expansion is in this process a passive event driven by the pulling of the adjacent layer, the yolk syncytium. The increase in area of this epithelia is achieved by cell shape changes (flattening) and the RhoGTPase Rac1 activity seems to be necessary for these passive changes in shape. The contraction in the yolk syncytium is accompanied by the formation of membrane folds and endocytic vesicles in this area and the different mechanical properties of the elements at both sides of these contractile domains are, together with endocytosis, essential to understand the expansion. In relation to this, we found that Rab5ab activity in the yolk is essential for the expansion and for doming of the internal part of the yolk. In addition, we showed that the main constituent of the embryo at this stage, the yolk granules, behave as an hydrodynamic fluid at low Reynolds number that passively flow during epiboly by the activity at the surface. We learned that the spherical geometry of the embryo together with volume conservation and the transmission of forces between the different elements involved in the process are essential to understand the changes observed in the blastoderm during this process and its global coordination. We generated a non-intrusivemethodology to extract the mechanical changes involved in a given morphogenetic event from microscopy data based on the relation between the active elements (elastic, visco-elastic) and the passive ones (fluids). We applied this method to the process of epiboly and validated the results obtained by atomic force microscopy (AFM) indentation and laser microsurgery experiments. Finally, we generated an enhancer trap screen using the Gal4/UAS binary system with the aim of being able to spatially restrict gene expression during epiboly. However, and although we found several interesting lines that drove specific gene expression, we could not find any with sufficient early expression to be useful for our epiboly studies. Overall, we learnt that an isotropic actomyosin contraction generates an anisotropic stress pattern and movement by the properties of the surrounding elements. To get a precise understanding of epiboly we had to consider the transmission of forces between the different layer and volume conservation. For that, it was important to take into account both the contribution from the active elements (cortex) and from the passive ones (fluid). The role that unselective membrane removal has in morphogenesis has been barely explored. We anticipate that membrane tension and removal and its relationship to actomyosin contraction and shape changes will become an emerging and exciting field and that zebrafish epiboly will become a great model to study these relationships.
[spa] La expansión de epitelios es esencial durante la embriogénesis de muchos organismos tanto invertebrados como vertebrados y también juega un papel importante en el organismo adulto como, por ejemplo, durante el proceso de cicatrización de heridas. Para ampliar nuestros conocimiento sobre los mecanismo celulares, moleculares y mecánicos responsables de este cambio morfogenético estudiamos la epibolia del vertebrado pez cebra. La epibolia de pez cebra consiste en la expansión del blastodermo alrededor del vitelo para finalmente embolverlo. Elaboramos un análisis descriptivo, funcional y mecánico del procesos que nos llevó a concluir que la expansión de este epitelio (EVL) es pasiva y generada por la contracción y la endocitosis del córtex de la capa adyacente, el sincitio del vitelo. La actividad de la RhoGTPasa Rac en el epitelio parece importante para este cambio de forma celular pasivo, mientras que la endocitosis mediada por la RhoGTPasa Rab5ab en el sincitio adyacente es esencial para la expansión del epitelio. Encontramos que la contracción isotrópica del córtex del sincitio genera un movimiento unidireccional por las diferentes propiedades mecánicas de las dos estructuras adyacentes. Por otro lado, descubrimos que los gránulos del vitelo, el mayor componente del huevo en este estadio, se comportan como un fluido viscoso incompresible que se mueven pasivamente durante el proceso de expansión por la actividad generada en la superficie. Además, el movimiento de estos gránulos durante el proceso puede explicar el cambio de forma del blastodermo durante el proceso. Finalmente, generamos un método para extraer los cambios mecánicos durante la epibolia a partir de imágenes de microscopia, basado en la relación entre la actividad elástica del córtex y el movimiento del fluido. Los resultados obtenidos con este método fueron validados por microscopía de fuerza atómica y microcirugía del córtex. En resumen, hemos obtenido un conocimiento global de la epibolia y aprendido que para explicar este proceso es necesario considerar las diferentes propiedades mecánicas de los diferentes elementos involucrados y la transmisión de fuerzas entre estos teniendo en cuenta la conservación del volumen y la forma esférica del embrión. Creemos que estos conceptos resultarán aplicables a otros procesos morfogenéticos.
Appears in Collections:Tesis Doctorals - Departament - Genètica

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