β-Spectrin functions independently of Ankyrin to regulate the establishment and maintenance of axon connections in theDrosophilaembryonic CNS

Abstract

DEVELOPMENT 273 RESEARCH ARTICLE INTRODUCTIONDuring central nervous system (CNS) development, growth cones navigate a series of choice points to find their appropriate targets. These guidance decisions are shaped by a balance of attractive and repulsive cues found in the extracellular environment that can act locally or at a distance (Tessier-Lavigne and Goodman, 1996). Slit ligands and Robo receptors play conserved roles in regulating midline axon crossing in both invertebrate and vertebrate nervous systems. In the Drosophila embryonic CNS, ipsilateral neurons (neurons that do not cross the midline) express the Robo receptor on their surface, which senses Slit to prevent midline crossing (Kidd et al., 1999;Kidd et al., 1998). By contrast, commissural neurons can overcome Slit repulsion because expression of Robo on their surface is prevented prior to midline crossing by the Commissureless (Comm) protein (Keleman et al., 2002). Thus, the specific receptors that a growth cone expresses as it encounters the midline greatly influences its guidance decision. The question of how guidance receptors and their downstream effectors are targeted to and distributed within functional domains of the growth cone plasma membrane remains key to the understanding of the mechanisms of axon path finding.The Spectrin molecule, a long rod-shaped heterotetramer consisting of two ␣ and two ␤ subunits, is the defining element of a ubiquitous sub-membrane cytoskeletal network in nearly all metazoan cells (Bennett and Baines, 2001). Most ␤-Spectrin isoforms contain an actin-binding domain at their N termini, an Ankyrin-binding domain, and a pleckstrin homology (PH) domain near their C termini. The PH domain of ␤-Spectrin interacts with the plasma membrane in transfected COS cells (Wang et al., 1996). ␣-Spectrin contains an SH3 domain and two EF-hand motifs (Bennett and Baines, 2001).In red blood cells, the Spectrin network was originally shown to have a role in supporting cell shape (Bennett and Chen, 2001); however, more recently it has been shown to participate in the formation of specialized membrane sub-domains (Bennett and Chen, 2001). Through its binding partner Ankyrin, the Spectrin network links many integral membrane proteins to the actin cytoskeleton. Genetic studies have revealed that many membrane proteins are mislocalized in the absence of Spectrin or Ankyrin (Dubreuil et al., 2000;Jenkins and Bennett, 2001;Komada and Soriano, 2002;Zhou et al., 1998). In addition, mutations in the vertebrate ␤-Spectrin and Ankyrin genes have been linked to various diseases, such as hereditary hemolytic anemias and spinocerebellar ataxia in humans, and auditory and motor neuropathies in mice (Ikeda et al., 2006;Mohler and Bennett, 2005;Parkinson et al., 2001). It has been proposed that Ankyrin and ␤-Spectrin are interdependent, and mutually stabilize the formation of polarized domains at axon initial segments and nodes of Ranvier (Komada and Soriano, 2002). Yet the relationship between Ankyrin and ␤-Spectrin, and the role that each plays in c...

Distinct functions of α-Spectrin and β-Spectrin during axonal pathfinding

“…Because wild-type Slit levels are required to position the longitudinal fascicles, a reduction of slit gene dosage results, as expected, in a further medial positioning of the longitudinal connectives, explaining the increase in the number of ectopic midline crosses in these animals. Similar phenotypes were also observed by Garbe et al (Garbe et al, 2007).…”

Cell organization, growth, and neural and cardiac development require αII-spectrin

“…We also note that in Drosophila, the genetic loss of a-spectrin leads to a redistribution and mislocalization of b-spectrin (Garbe et al, 2007).…”

Independent Functions of Slit–Robo Repulsion and Netrin–Frazzled Attraction Regulate Axon Crossing at the Midline inDrosophila

“…4C). This defect can be rescued by providing robo function specifically in the ap neurons in an otherwise mutant background, indicating that Robo guides later projecting neurons independently of its earlier function in the longitudinal pioneers (Garbe et al, 2006). Interestingly, a time course analysis has shown that midline crossing defects in the ap neurons can arise at two different times.…”

“…Previous studies have established that the axon guidance phenotypes of both robo and fra can be significantly rescued by expressing these genes in postmitotic neurons using the Gal4-UAS system (Kolodziej et al, 1996;Kidd et al, 1998a;Garbe et al, 2006). Therefore, we tested whether transgenic expression of either Fra or Robo in the double mutants would "rescue" the phenotype and restore it to one that more closely resembled the single mutants (Fig.…”

“…Interestingly, a time course analysis has shown that midline crossing defects in the ap neurons can arise at two different times. In the complete absence of Robo (or Slit), almost all of the ap axons extend medially, directly across the midline at stage 15, never making their correct anterior turn (Table 1), whereas in embryos mutant for ␤-spectrin or carrying genetic combinations that partially limit Slit-Robo signaling, the ap axons initially make the correct ipsilateral anterior turn, but several hours later, at stage 17, ectopic midline crossing is observed (Garbe et al, 2006). We have interpreted these findings to suggest that Slit-Robo repulsion is required continuously to establish and maintain appropriate pathway selection (Garbe et al, 2006).…”