Resumen de Functional analysis of the pdz protein canoe/af-6 during drosophila neural differentiation
In the developing nervous system, a precise neural network is formed in a step-wise fashion through a series of recognition processes, such as axon guidance and neuron-glia interactions, both highly relevant for the correct wiring of the nervous system. The Drosophila central nervous system (CNS) midline at the ventral cord, which is homologous to the vertebrate floorplate, is the first choice point for the newly born neurons extending their axons. A key regulator of the axon decision of whether or not to cross the midline is the well-conserved Slit/Robo signalling pathway. However, the intracellular mechanisms associated with Robo function remain poorly defined. In this work, in which we focus on the role of the PDZ domain-containing protein Canoe (Cno), the Drosophila orthologue of AF-6/Afadin, during the processes of neural differentiation, we show that Cno is essential for Slit/Robo signalling. Cno, is expressed at the midline along axonal pioneers tracks and in cno mutant embryos, longitudinal Robo/Fasciclin2 positive axons are detected crossing the midline. In addition, cno strongly interacts genetically with Slit, Robo and other known upstream and downstream modulators of the Robo pathway, such as Neurexin-IV (Nrx-IV) and Rac genes. Moreover, the commissureless (comm) phenotype (complete lack of axon commissures due to increased levels of the Robo receptor on the surface in all neurons) was suppressed in comm, cno double mutants both in embryos and in neuron cell cultures. Finally, co-IPs experiments show that Cno is in a complex in vivo with Robo and culture experiments prove that Cno is necessary to stabilize Robo at the membrane. Hence, our results show a novel function of Cno during axon pathfinding at the CNS midline regulating Slit/Robo signalling at multiple levels.
Additionally, we found that Cno is expressed in the midline glia (MG) where it is highly restricted to the interface between MG and commissural axons. Drosophila MG ensheath and separate both axonal commissures, though the proteins involved in these events are scarcely known. Given the glial expression pattern of Cno, we investigated the role of Cno in MG-axonal interactions during commissural axons separation and ensheatment by the MG. Intriguingly, in cno mutants, we found failures in these processes. We observed that the DE-cadherin Shotgun (Shg) shows a similar phenotype and localizes, as Cno, at the MG, at the close contacts between MG and commissural axons. Moreover, cno genetic interactions and co-IPs assays support a Cno function modulating neuron-glia interactions in a complex with Shg, the glial protein Wrapper and its interacting protein Nrx-IV, present at commissural axons and homologue of vertebrate Caspr/paranodin.
Taking all data together, our results unveil a novel function of Cno as a regulator of cell-cell interactions and signalling events during neural differentiation.
