DEV02653 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. α- and β-Spectrin localization is disrupted in hypomorphic β-spectrin embryos. (A,B) Dorsal section of a wild-type embryo stained with (A) β-Spectrin and (B) α-Spectrin. (C) Merge of A and B showing almost complete colocalization. (D-F) When hypomorphic β-spectrin (G0108) hemizygous embryos (identified in our genetic screen) are stained with anti-β-Spectrin (D) and anti-α-Spectrin (E), we see reduced levels of each protein (yet levels are not as compromised as the null em6 allele); however, the α- and β-Spectrin proteins still present in the embryo are mislocalized and we observe punctate staining in many, but not all, cells of the CNS (arrowheads). Staining does not appear to favor particular cell types or cell surfaces. Intriguingly, we still observe colocalization of α- and β-Spectrin proteins (F) even though the overall staining pattern of both proteins is highly disrupted. This supports the hypothesis that α-Spectrin depends upon β-Spectrin for its localization and maintenance.

• Supplemental Figure 2 -

  Fig. S2. β-spectrin does not interact with the Netrin/Frazzled or Sema/Plexin signaling pathways. (A-D) Expressing low levels of Sema-1a from midline glia in a sema-1a mutant background (PUP background) results in a one-commissure phenotype. Instead of containing two commissures per segment as in wild-type embryos, Sema-1a gain-of-function embryos on average have only one commissure per segment, and removing genes involved in the Plex-Sema guidance pathway can dominantly modify this one-commissure phenotype (Ayoob et al., 2004). If β-spectrin participates in Sema-Plexin-regulated axon guidance, we might expect that β-spectrin mutations would alter the one-commissure phenotype. However, this is not what we see. Hypomorphic β-spectrin mutant embryos expressing low levels of Sema-1a have on average the same number of commissures per segment as embryos expressing low levels of Sema-1a that are wild type for β-spectrin. (A,B) Stage 15 embryos stained with BP102. Anterior is up. (A) β-spectrin heterozygous embryos overexpressing low levels of Sema-1a with P52-Gal4 in a sema-1a background (PUP) exhibit a one-commissure phenotype in many segments (arrowhead). (B) β-spectrin hemizygous mutant embryos in a PUP background still display the one-commissure phenotype. (C,D) Error bars indicate s.e.m. (C) Percentage of segments containing either zero, one or two commissures in a β-spectrin heterozygous plus PUP background (white bar, n=24 embryos) compared with a β-spectrin mutant plus PUP background (gray bar, n=17 embryos). (D) Average number of commissures per segment in a β-spectrin heterozygous plus PUP background (white bar, 1.21) compared with a β-spectrin hemizygous plus PUP background (gray bar, 1.12). (E,F) The attractive guidance receptor Frazzled is required for attraction of the posterior bundle of eagle neurons (EW) in response to Netrin. In frazzled mutants, these neurons fail to cross the midline (D.S.G and G.J.B., unpublished). Unlike the genetic interaction seen with the Slit-Robo pathway, β-spectrin null mutants do not modify Fra-dependent guidance trajectories of the eagle neurons in a frazzled heterozygous background. Although axons of the eagle neurons may be slightly more defasiculated, attraction toward the midline appears unaltered. (E) frazzled heterozygous embryos expressing UAS-TauMycGFP under the control of eagleGAL4 show both the EG and EW bundles crossing the midline. In a frazzled mutant, the EW bundle fails to cross the midline (D.S.G and G.J.B unpublished). (F) Removing β-spectrin in a frazzled heterozygous background does not alter attraction of the eagle neurons toward the midline. Together, these observations support a specific interaction between β-spectrin and the Slit-Robo pathway. Alternatively, β-spectrin still may have a more general effect on growth cone migration, and the specific interactions seen with slit and robo might reflect the fact that the Slit-Robo system is more susceptive to subtle perturbation.