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

• Autores: Carmen Alvarez Fernandez
• Directores de la Tesis: Emili Saló i Boix (dir. tes.), Enrique Martín Blanco (dir. tes.)
• Lectura: En la Universitat de Barcelona ( España ) en 2013
• Idioma: inglés
• Tribunal Calificador de la Tesis: Elisa Martí Gorostiza (presid.), Florenci Serras Rigalt (secret.), Marco Milán Kalbfleisch (voc.)
• Materias:
  • Ciencias de la vida
    • Biología animal (zoología)
      • Genética animal
    • Genética
    • Biología vegetal (botánica)
      • Genética vegetal
• Enlaces
  • Tesis en acceso abierto en:  TDX  Dipòsit Digital de la UB 
• Resumen

  • 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.