DEV02842 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Deletion of the EF-hand or GAS2 motif in Shot impairs its interaction with Kra. S2 cells transiently expressing both HA-Kra and Shot L(A)-GFP, Shot L(A)-ΔGAS2-GFP or Shot L(A)-ΔEF-GFP were lysed. Soluble extracts were immunoprecipitated with an anti-GFP antibody. The precipitates were analyzed by western blot analysis using anti-HA and anti-α-tubulin antibodies. HA-Kra and endogenous α-tubulin were also detected in 5% of the total extracts to control for protein levels.

• Supplemental Figure 2 -

  Fig. S2. kra and shot genetically interact with slit at the CNS midline. (A-D) The CNS of stage 16-17 embryos stained with mAb 1D4 (anti-Fasciclin II). Wild-type embryos show three parallel fascicles of longitudinal axons on each side of the ventral midline; these fascicles never approach or cross the midline (Fig. 4D). (A) In slit²/+ heterozygous embryos, the longitudinal axons ectopically cross the midline. (B,C) The slit²/+ phenotype is significantly enhanced when one copy of kra or shot is also removed in slit²/+; kra²/+ (B) or slit²,+/+,shot³ (C) transheterozygous embryos, respectively. In kra²/+ or shot³/+ control embryos, the longitudinal axon tracts appear the same as in the wild-type (Fig. 5A,B). (D) A further enhancement in ectopic midline crossing is observed in slit²,+/+,shot³; kra²/+ transheterozygous embryos. Anterior is to the top. (E) Quantification of the midline crossing frequency per segments in each of the indicated genotypes (slit²/+, n=220; slit²/+; kra²/+, n=486; slit²,+/+,shot³, n=445; slit²,+/+,shot³; kra²/+, n=350). Statistically significant differences are denoted on the top of bars by an asterisk (control, slit/+; P<0.001). Scale bar: 10 μm.

• Supplemental Figure 3 -

  Fig. S3. Expression of Repo and Wrapper is normal in kra mutant embryos. Stage 16 wild-type (A,B) and kra mutant (C,D) embryos carrying the apC-tau-lacZ marker were doubly stained with anti-β-galactosidase (red) and either anti-Repo (green) or anti-Wrapper (green). (A) Repo is expressed by all CNS glial cells except for the midline glia in wild-type embryos. (B) Wrapper is specifically expressed by the midline glia in wild-type embryos. (C,D) In kra¹/kra² embryos, apparently normal levels and patterns are observed for Repo (C) and Wrapper (D) even in the segments where Ap axons ectopically cross the midline (arrowheads). Scale bar: 10 μm.

• Supplemental Figure 4 -

  Fig. S4. eIF2β is required for midline axon guidance. (A) The genomic structure of the eIF2β locus. The eIF2β gene is located at 69C4 on the third chromosome. The insertion sites of P-element are indicated as inverted triangles. The transposons of G6433 and G6414 (purchased from Genexel, Korea) are inserted at -81 and -69, respectively, of the eIF2β open reading frame (ORF). Molecular lesions of the deletion lines eIF2β¹ and eIF2β² that were generated by the imprecise excision of G6414 and G6433, respectively, are indicated. Homozygotes and transheterozygotes for eIF2β¹, eIF2β² and Df(3L)ED4486 are embryonic lethal. For the predicted transcripts of eIF2β and its neighboring gene CG10627, black boxes represent coding regions and white boxes represent untranslated regions; predicted translation initiation and stop sites are indicated. (B-E) Mutations in eIF2β lead to defects in midline axon repulsion. (B) A stage 16 eIF2β mutant embryo carrying the apC-tau-lacZ marker was stained with anti-β-galactosidase. The Ap axons abnormally cross the midline in this embryo (white arrows). (C) A stage 13 eIF2β mutant embryo was stained with mAb 1D4. The pCC axons also show ectopic midline crossing (black arrows). (D) A stage 16 eIF2β mutant embryo was stained with mAb 1D4. Axons from the innermost longitudinal tract frequently cross the midline (black arrow). Scale bar: 10 μm. Anterior is to the top. (E) Quantification of midline crossing defects per segments in each of the indicated genotypes (eIF2β¹/+, n=280 segments; eIF2β²/+, n=224; eIF2β¹/eIF2β², n=650; eIF2β¹/eIF2β¹, n=496; eIF2β²/Df(3L)ED4486, n=344). Scale bar: 10 μm.