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Additional Supporting Information may be found in the online version of this article.

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DVDY_22515_sm_SuppInfoFigS1.tif3671KSupp. Fig. S1. Gogocyto-myc localizes to R axons in larvae. A: Rs are detected by myc antibody in the brains of late 3rd instar larvae expressing Gogocyto-myc. B: Control. Scale bars = 10 μm.
DVDY_22515_sm_SuppInfoFigS2.tif2591KSupp. Fig. S2. Hts physically interacts with all Gogo deletion constructs. A: Schematic of the deletion constructs used to confine the Hts binding site in the cytoplasmic part of Gogo. B: The indicated myc-tagged Gogo deletion constructs and his-tagged ShAdd were coexpressed in Schneider cells. Gogo deletion constructs were immunoprecipitated using anti-myc antibody. ShAdd-his coimmunoprecipitated with all deletion constructs, but binding to GogoδC12 and GogoδC1 was weaker compared to the other deletion constructs.
DVDY_22515_sm_SuppInfoFigS3.tif2106KSupp. Fig. S3. Defects caused by hts01103 are rescued by Add1 and HtsPD but not ShAdd. The defects in the medulla of (A) hts01103 hemizygous flies are rescued by the expression of (B) Add1 and (C) HtsPD but not (D) ShAdd under direct control of the GMR promoter.
DVDY_22515_sm_SuppInfoFigS4.tif4308KSupp. Fig. S4. hts antagonizes gogo overexpression. A: Overexpression of Gogo causes abnormally thick swellings of R8 axons marked by GFP at the M1 layer and at the R8 termini in M3 (9 of n=11). B: Co-overexpression of Add1 under direct control of the GMR promoter reduces the swellings both at M1 and M3 (8 of n=10).
DVDY_22515_sm_SuppInfoFigS5.tif4069KSupp. Fig. S5. hts does not affect Gogo immunostaining. A: Gogo immunostaining of a 3rd instar larval optic lobe containing htsnull homozygous clones marked by the absence of (A') KO fluorescence (dashed lines). Gogo immunostaining does not differ from the mutant clones to the surrounding heterozygous tissue. B,C: Pupal medullae containing htsnull homozygous mutant clones marked by (C') the absence of KO or (D') the presence of GFP fluorescence (brackets). Gogo immunostaining does not differ from the surrounding heterozygous tissue at (C) 24 APF or (D) 40 APF. Scale bars = 10 μm.
DVDY_22515_sm_SuppInfoFigS6.tif2265KSupp. Fig. S6. Hts level is not increased in gogo mutant clones. A,B: Homozygous gogo mutant clones in the 24APF pupal (A–A') or adult (B–B') medulla can be detected by the absence of (A',B') KO fluorescence (brackets). The intensity of (A,B) Hts immunostaining is not noticeably increased in these clones. C: Hts expressed under direct control of the GMR promoter can be detected in the adult medulla. Intensity of Hts immunostaining is not increased in gogo mutant clones identified by the absence of (C') KO immunofluorescence (brackets). Scale bars = 10 μm.
DVDY_22515_sm_SuppInfoFigS7.tif6187KSupp. Fig. S7. F-Actin and excessive Add1 or Gogo. Medullae of adult flies with the indicated genotypes have been stained with phalloidin to visualize F-Actin. Compared to the (A) wild type control, F-actin can be detected more clearly in the axons of flies overexpressing (B) Add1 or (C) Gogo. Scale bars = 10 μm.

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