TheDrosophilaARF6-GEF Schizo controls commissure formation by regulating Slit

Abstract

The CNS of bilateral symmetric organisms is characterized by intensive contralateral axonal connections. Genetic screens in Drosophila have identified only a few genes required for guiding commissural growth cones toward and across the midline. Two evolutionarily conserved signaling molecules, Netrin and Slit, are expressed in the CNS midline cells. Netrin acts primarily as an attractive signaling cue, whereas Slit mediates repulsive functions. Here, we describe a detailed analysis of the Drosophilagene schizo, which is required for commissure formation. schizo leads to a commissural phenotype reminiscent of netrin mutant embryos. Double-mutant analyses indicate that Netrin and Schizo act independently. The schizo mutant phenotype can be suppressed by either expressing netrin in the CNS midline cells or by a reduction of the slit gene dose, indicating that the balance of attractive and repulsive signaling is impaired in schizo mutants. Overexpression of the schizo RNA in the CNS midline using the GAL4/UAS system leads to a slit phenocopy, suggesting that schizo primarily antagonizes Slit signaling. This is further supported by cell type-specific rescue experiments. The schizo gene generates at least two proteins containing a conserved Sec7 and a pleckstrin homology domain (PH) characteristic for guanine nucleotide exchange factors(GEF) acting on ARF GTPases, which are known to regulate endocytosis.In support of the notion that schizo regulates Slit expression via endocytosis, we found that block of endocytosis leads to a schizo-like phenotype. We thus propose that the balance of the two signaling cues Netrin and Slit can be regulated, controlling membrane dynamics.

Spermatocyte cytokinesis requires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes

“…Homozygous arf6 1 progeny from homozygous mutant mothers (maternal/zygotic mutants) were viable until adulthood, presenting no overt external morphological phenotype. Recent reports show that expression of a GDP-bound dominant negative ARF6 protein (ARF6TN) impairs myoblast fusion and axon path finding during embryogenesis (Chen et al, 2003;Onel et al, 2004). Both developmental events occurred normally in arf6 null mutant embryos (see Fig.…”

“…Via these mechanisms, ARF6 affects processes such as adherens junction disassembly and cell migration, including the formation of cord-like structures by hepatocytes in the mouse liver in response to hepatocyte growth factor (D'Souza-Schorey and Chavrier, 2006;Suzuki et al, 2006). In Drosophila, the ARF6-GEF Loner/Schizo is necessary for myoblast fusion (Chen et al, 2003) and midline crossing of axons (Onel et al, 2004).…”

Myoblast fusion: lessons from flies and mice

“…Crk, in turn, can recruit actinpolymerizing machinery to points of cell-cell contact [ (Kim et al, 2007) see below]. The cytoplasmic domain of Kirre recruits both Rolling pebbles/Antisocial (Rols/Ants) (Chen and Olson, 2001;Menon and Chia, 2001;Rau et al, 2001), which contains numerous motifs for protein-protein interaction, and Schizo/Loner, a guanine nucleotide exchange factor (GEF) for the Arf6 GTPase (Arf51F -FlyBase) (Chen et al, 2003;Onel et al, 2004) to points of cell contact (Chen and Olson, 2001;Chen et al, 2003;Bulchand et al, 2010). Rols functions in a positive-feedback loop to stabilize Kirre at the myotube membrane, thereby ensuring its continued availability for interaction with FCMs (Menon et al, 2005).…”

The BRAG/IQSec family of Arf GEFs

“…This latter observation suggests that shizo functions to reduce the surface level of Slit on midline glia, by promoting its endocytosis. 28 It should be noted however that slit is a secreted protein and lacks a transmembrane domain, but remains closely associated with membranes, 29 and it is not yet clear how it is endocytosed.…”