Please use this identifier to cite or link to this item: http://hdl.handle.net/2445/43585
Title: Genomic analysis of epithelial fusion and wound healing morphogenetic processes in Drosophila / Análisis genómico de los procesos morfogenéticos de fusión de epitelios y cicatrización en Drosophila
Author: Alvarez Fernández, Carmen
Director: Martín Blanco, Enrique
Saló i Boix, Emili
Keywords: Epiteli
Genòmica
Genètica animal
Drosòfila melanogaster
Epithelium
Genomics
Animal genetics
Drosophila melanogaster
Issue Date: 26-Apr-2013
Publisher: Universitat de Barcelona
Abstract: [spa] Esta tesis se basa en el estudio de los procesos morfogenéticos de fusión de epitelios y cicatrización utilizando Drosophila melanogaster como organismo modelo. Ya que esta tesis se basa en un análisis por microarray de las células involucradas activamente en el proceso de cicatrización en comparación con aquellas células vecinas no implicadas activamente en dicho proceso, se estandarizó un nuevo sistema para cultivar discos imaginales in vitro. Gracias a este sistema, que recapitula todos los procesos que ocurren durante la cicatrización, se pudieron aislar una gran cantidad de tanto de células activamente involucradas en la cicatrización (puc-GFP positivas) como de sus vecinas no involucradas (puc-GFP negativas). Una vez obtenidas las distintas poblaciones celulares se realizaron las extracciones de RNA y posteriores hibridaciones en chip de Affymetrix. Los datos obtenidos se analizaron mediante diferentes estudios estadísticos, resaltando 302 genes regulados positivamente y 209 genes regulados negativamente con un fold change por encima o por debajo de 2 y -2 respectivamente. También se realizaron análisis de la ontología génica (GO) de los genes que aparecían modificados, lo que nos permitió ver que categorías génicas aparecían mas representadas (como por ejemplo genes implicados en la regulación de la actina, la respuesta inmune, el movimiento celular o factores de activación de la transducción). Una vez realizados los análisis estadísticos se procedió al análisis funcional de aquellos genes que aparecían modificados. Ya que realizar un cribado en cicatrización era un proceso largo y tedioso se decidió hacer un cribado previo utilizando el modelo de fusión del tórax. Para ello se sobreexpresaron RNA de interferencia mediante un driver que digiere su expresión a la zona dorsal del tórax. Se testaron 206 líneas UAS-RNAi para disminuir la expresión de 123 genes y 21 líneas UAS para sobreexpresar aquellos genes que aparecían regulados negativamente. De estos 89 líneas UAS-RNAi (43 % de los testados) correspondiente a 54 genes (44 %) y 18 líneas UAS (42 %) correspondientes a 11 genes (44%) presentaban problemas de fusión de tórax. Aquellos genes que producían fenotipos mas fuertes estaban relacionados con factores de transcripción, con la regulación de la actina o las adhesiones celulares (esperable ya que ya se ha descrito previamente la importancia de estos factores durante la cicatrización), y de forma preliminar, fueron estudiados durante el proceso de cicatrización, identificando nuevos genes implicados en el mismo.
[eng] This thesis is based on a genomic study of the epithelial fusion and wound healing processes using Drosophila melanogaster as a model system. The aimed of the work is to study the genomic differences between cells actively involved in wound healing and neighboring cells not actively involved in wound healing. To achieve this objective we standardized a new system to culture imaginal discs in vitro. This new system that is healing permissive recapitulate the entire wound healing processes. Using the line pucE69-A-Gal4/UAS-GFP, which express puc-GFP in the leading edge cells of the wound, a large amount of cells actively involved in healing (puc-GFP positives) and their neighboring cells (puc-GFP negatives) were sorted. RNA from the different cells populations were extracted and hybridized to affymetrix chips. The obtained data were further analyzed using different statistical analyses. Finally, 302 genes appeared upregulated and 209 genes were downregulated with a fold chage higher and lower of 2 and -2 respectively. However, during the normal development of imaginal discs, puc-GFP is not only express in cells involved in healing, it is also present in cells of the stalk and the peripodial sheet of the disc. This cells that also express puc-GFP are not involved in the wound healing process and could lead to contamination. To solve this problem a second analysis including wounded and not wounded discs were performed. Dual statistical analyses allow us to identify three different subpopulation: dw1_dnw1_dd1 subpopulation, in which genes appear modified not only in wounded discs but also in unwounded discs, but differentially; dw1_dnw0_dd1 subpopulation, in which genes appear modified only in wounded dicsc; dw0_dnw1_dd1 subpopulation, in which genes appear modified only in unwounded discs. Gene ontology (GO) analyses were done for those genes that were modified during wound healing showing overrepresented categories during wound healing (for example genes related to actin regulation, immune response, cells movements, of transduction factors). To investigate the potential role of selected genes in wound healing, we aimed to employ an in vivo healing model using the Gal4-UAS system. Different Gal4 drivers were used to interfere with the expression of those genes upregulated in “healing” cells (UAS-RNAi lines) and to reload back by overexpression those genes downregulated (UAS lines). To test all these lines in the wound healing model is an enormous task that would take long time, for that reason we decided to perform a previous analysis of their functionality in a heterologous morphogenetic epithelial model (similar to wound healing), the thorax fusion. In brief, 89 UAS-RNAi lines (43 % of those tested) corresponding to 54 genes (44 %) displayed thorax closure phenotypes ranging from weak cleft to embryonic lethality. For the UAS construct, 18 of them (42 %) corresponding to 11 genes (44%) also lead to thorax fusion defects. Once identified a set of genes whose overexpression or interference elicit defects in thorax closure and may have a role in wound healing, a secondary screen employing an imaginal disc healing assay was performed for those lines that produces stronger phenotypes during thorax closure. In summary, downregulation of different genes that appear modified during healing by RNAi overexpression has leaded us to identify new genes necessaries for a proper closure completion. Fundamental processes for wound healing are affected, like actin accumulation in leading edge cells, peripodial epithilia movement, or tissue relaxation. The aimed of this project was not only to generate a comprehensive database of those genes whose expression is altered during wound repair in Drosophila, but also identify genes and highly conserved pathways which are likely to be of crucial importance for wound healing by comparing our data to those obtained in vertebrates healing model systems. We set up a collaborative project with a series of groups performing transcriptional analysis in humans and mice in order to identify those genes conserved through the phylogeny. After a comparative study to identify Drosophila homologues for those genes that appear modified in vertebrates functional analysis were done in vertebrates using a scratch assay and in Drosophila using the Gal4-UAS system. These genes, functionally relevant in healing models across phylogeny constitute potential candidates for deep translational studies.
URI: http://hdl.handle.net/2445/43585
Appears in Collections:Tesis Doctorals - Departament - Genètica

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