Establishing a System to Test Genetic Interactions that Regulates Layer Specificity in the Drosophila Visual System
Authors:Yi Chen, Matthew Y. Pecot, C.Y. Kimberly Tsui
Mentor:S. Lawrence Zipursky, Distinguished Professor, Biological Chemistry; Investigator, Howard Hughes Medical Institute, University of California, Los Angeles
In the Drosophila visual system, synaptic connections are organized into discrete layers for precise neuronal network formation. Lamina neuron L3 targets its target medulla layer M3 in two steps: First, L3 target to a common domain in the outer medulla by N-Cadherin (CadN) adhesion and Semaphorin-1a (Sema-1a)/Plexin A repulsion. Next, L3 segregates into M3 partially by Sema-1a signaling. Using mosaic analysis with a repressible cell marker (MARCM), we demonstrated Cdc42 acts cell-autonomously to regulate the second step of L3 targeting. In a RNA interference screen, Cdc42’s reported activator dPix was identified to also be involved in L3 targeting. We hypothesize that CadN and Sema-1a regulate specific steps in L3 targeting by precisely modulating cytoskeletal dynamics through interaction with different downstream effectors, Cdc42 and dPix. We developed a genetic system to cell-autonomously study these interactions at different stages of L3 targeting. Utilizing MARCM, individual mutant L3s, rendered homozygous for null-mutation in our genes-of-interest by mitotic recombination in multiple chromosomes, are specifically labeled. Labeling of heterozygous cells is prevented by a repression system. We first examined L3s with null-mutations in Cdc42 and Sema-1a. Mutant L3 were successfully generated and appropriate mis-targeting phenotypes were observed. However, wild-type cells were labeled due to ineffectiveness of the repression system on one chromosome, giving false-positive results. We are currently repeating the study with a stronger repressor that has been tested to be effective.