Analysis and coordination of different mechanisms controlling tube elongation during the development of the embryonic system in Drosophila melanogaster

Abstract

The tracheal (respiratory) system of the Drosophila embryo is a network of interconnected epithelial tubes that supplies and exchanges the gas to maintain the homeostasis of the entire organism. Maintaining a controlled tube diameter and fitting tube length are two major requirements for proper tube function. The Epidermal growth factor receptor (EGFR), a Tyrosine Kinase Receptor (RTK), triggers one of the principal conserved signalling pathways operating in development and homeostasis. EGFR is known to be use reiteratively for organ and tissue formation in the fruitfly. One of the aims of this work was to gain new insights into the activity of EGFR during Drosophila development. We focused on the requirements of EGFR during the formation of the tracheal system. EGFR was previously known to be required for different aspects of tracheal development, such as for invagination, branching and epithelial integrity. In this work, we identify a new requirement for EGFR during tracheal formation in controlling the length of the tracheal tubes. We found that EGFR regulates the size of the Dorsal trunk (DT), the main branch of the Drosophila tracheal network. The constitutively activation of EGFR (EGFRCA) displays shorter tubes whereas a downregulation of EGFR (EGFRDN) activity leads to an overelongated and convoluted DT. This phenotype correlates with cell shape regulation, resulting in more cuboidal cells in the case of EGFRCA and more elongated cells in EGFRDN. This work shows that EGFR act as a hub to coordinate cell intrinsic and extrinsic tube elongation mechanisms. This role is performed through the regulation of the luminal deposition of the extracellular matrix regulator Serpentine (Serp) (extrinsic factor) and of the apical determinant Crumbs (Crb) in the DT (intrinsic factor). In EGFR downregulation conditions the accumulation of both proteins is altered, and this may lead to the defective control of tube size observed. We show that Crb and Serp are loaded in common endosomes, which require EGFR activity for the proper organisation, ensuring delivery of both cargoes to their final destination. The regulation of endosomal sorting of cargoes could be one of the molecular mechanisms downstream of EGFR, and therefore could regulate different morphogenetic and pathological EGFR-mediated events. We also report that during tracheal development Crb undergoes a complex pattern of recycling, which involves internalisation and different sorting pathways that ensures its apical subcellular accumulation. We propose that Crb recycles using the Rab11-Recycling Endosome route to accumulate preferentially to the Subapical Region (SAR) and a Rab4/Retromer short-loop from the endosome to the plasma membrane to enrich in the Apical free region (AFR). Concerning the accumulation of Crb in the SAR, our results indicate that Crb is not equally distributed in all cellular junctions, but instead it is more clearly accumulated in longitudinal cell junctions (LCJ) than in circumferential cell junctions (CCJ). Crb has been proposed to control tube length through apical membrane expansion, so we hypothesize that Crb promotes a polarised longitudinal/axial tube growth. Because Src42A was

previously proposed to mediate polarised cell shape changes in DT cells by regulating membrane growth in the longitudinal/axial axis, we evaluated a possible Crb/Src42A interaction. We find that in loss of function conditions for Src42A, in which the DT remains shorter, Crb is more evenly distributed in all cellular junctions. The results suggest that Src42A may contribute to Crb preferential accumulation in the LCJ. FRAP experiments point to a role of Src42A in controlling the dynamics of Crb protein accumulation in junctions. Conversely, in conditions of constitutive activation of Src42A, where there is an overelongation of the DT, we find that Crb is lost from all junctions. In addition, Serp accumulation in the lumen is also severely affected. Altogether, these results suggest a possible role for Src42A in regulating trafficking.

El sistema traqueal embrionario de Drosophila está compuesto por una red de tubos epiteliales interconectados, los cuales necesitan una regulación constante tanto del diámetro como de la longitud para su correcto funcionamiento. Durante el desarrollo traqueal, el receptor de EGF (Epidermal Growth Factor Receptor) está implicado en la regulación de la invaginación e integridad epitelial de las diferentes ramas traqueales. Uno de los principales objetivos de este trabajo es obtener nuevos conocimientos sobre la implicación de EGFR en el desarrollo traqueal. Nuestros datos indican que EGFR regula la longitud del tronco dorsal (DT), ya que en embriones con una disminución de su actividad (EGFRDN), la longitud del DT se ve afectada produciendo una elongación de éste. Asimismo, cuando alteramos la actividad de EGFR, observamos que la localización apical de Crumbs (Crb) y la acumulación luminal de Serpentine (Serp), dos proteínas que regulan el crecimiento del DT, se ven afectadas. La alteración en la acumulación de ambas proteínas desencadena un defecto en el control de la elongación del tubo, tal y como se observa en embriones EGFRDN. Observamos que Crb y Serp localizan en endosomas comunes que necesitan de la actividad de EGFR para organizarse correctamente y transportar las proteínas a su destino final. Además, nuestros resultados demuestran que Crb sufre un complejo proceso de transporte y reciclaje durante el desarrollo traqueal. En estadios tardíos de la embriogénesis, Crb se localiza en la región Subapical (SAR) pero lo hace de manera anisotrópica: Crb presenta mayor acumulación en las uniones longitudinales (LCJ) que en las circunferenciales (CCJ). Cuando disminuimos la actividad de Src42A, un regulador axial del crecimiento del tubo, Crb se distribuye homogéneamente, pero en condiciones de sobreactividad, Crb desaparece de esas uniones. Nuestros resultados indican que Src42A contribuye a una mayor acumulación de Crb en las LCJ regulando su dinámica, y sugieren una posible implicación de Src42A en el reciclaje de proteínas.