Cell elongation and cell intercalation in dorsal branch extension during the development of the emrbyonic tracheal system of drosophila melanogaster
- Autores: Sandra Casani Miravalls
- Directores de la Tesis: Marta Llimargas Casanova (dir. tes.), Jordi Casanova Roca (dir. tes.), Pedro Martínez Serra (tut. tes.)
- Lectura: En la Universitat de Barcelona ( España ) en 2020
- Idioma: inglés
- Tribunal Calificador de la Tesis: Jérôme Solon (presid.), Sofía J. de Moura Minguez Araujo (secret.), Véronique Brodu (voc.)
- Programa de doctorado: Programa de Doctorado en Genética por la Universidad de Barcelona
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
Morphogenesis shapes organs and organisms. The Drosophila melanogaster embryonic tracheal system was used in this thesis to understand the contribution of distinct cell behaviours in organ formation. This study focused on the behaviour of the stalk cells (SCs) that form the dorsal branches (DBs), which respond to pulling forces from the tip generated by the Bnl/Btl signalling pathway. SCs undergo two types of cell behaviours during DB extension: cell elongation and cell intercalation. Cell elongation is a change in cell shape, and cell intercalation is an exchange of cells’ positions. In DBs, cell intercalation involves an exchange of intercellular adherens junctions (AJs) by autocellular ones (referred to as junctional cell intercalation) and is modulated by the intracellular trafficking of AJ components.
The aims were to unveil the relationship between cell elongation and junctional cell intercalation, to determine their respective contribution to DB extension, and to explore the role of an actin polymerising protein in the SC elongation. With these objectives, I searched for mutant conditions that could potentially modify the actin cytoskeleton, which is known to have a huge impact on cell shape, and, on the other hand, I used a mutant condition already known to affect the intracellular trafficking.
A constitutively active (CA) Diaphanous (Dia) mutant protein was used to affect the actin cytoskeleton. DiaCA tracheaeexhibited rounded-shape cells that were shorter than the control ones and had lesser DB extension than the controls. However, SCs were still able to intercalate. These short DBs were not due to a reduced number of SCs forming the DBs or to failure of the tip cells to respond to the Bnl/Btl signalling. In vivo experiments showed that once DiaCA cells adopt a rounded shape, this change endures. Moreover, I saw that DiaCA has a cell-autonomous effect on the shape of tracheal cells. Using MoeGFP to visualise actin in vivo, DiaCA cells showed ubiquitous and more stable distribution of MoeGFP than the control trachea, where the actin cytoskeleton is very dynamic.
To impair the intracellular trafficking of AJ components, I used a dominant negative (DN) form of the Rab5 small GTPase (Rab5), which is involved in early endocytosis. The DBs expressing Rab5DN displayed junctional cell intercalation defects (from more severe to weak), although some SCs intercalated. The cells that did not intercalate were able to elongate even more than control SCs. The slight increase in the number of SCs in Rab5DN condition was not the cause of the severe junctional cell intercalation defects. In vivo experiments confirmed that Rab5DN SCs can elongate and that this condition induces a high accumulation of E-cadherin, an AJ component. Moreover, when DiaCA and Rab5DN where co-expressed, the DB cells exhibited both defective phenotypes, indicating an additive effect of these mutant conditions.
The role of Dia was explored as a possible regulator of cell elongation. Its downregulation using a DiaDN form produced cytoplasmic bridges in the DBs, usually in the most distal SCs. These cytoplasmic bridges correlated with over elongated cells, suggesting a role for Dia in the regulation of the SCs length. Conversely, DiaDN did not disrupt epithelial integrity and the SCs intercalated normally.
In summary, this study has unveiled a major role for cell elongation in DB extension, which in turn is not affected by the SCs number. It has also shown that cell elongation and junctional cell intercalation are two separable processes. Moreover, the actin cytoskeleton dynamics is important for cell elongation and Dia might regulate the cell shape during DB extension. How cell elongation and cell intercalation are coordinated to achieve DB extension would need to be addressed in future research.
