Fig 7. Endocytosis motifs are required for ectopic repulsion in vivo.

The projection pattern of all axons of the ventral nerve cord of late stage 14 Drosophila embryos are imaged with HRP (A-D) and the fascicles of the Eg commissural subset are imaged with a Tau-myc-GFP transgene (E-H). (A) In wild-type embryos, all segments (3 shown here) have two horizontal commissures, which are quantified as 0% of segments with error in the histogram (L n = 88). (B) Overexpressing wild-type Robo transgene in all neurons causes gain of repulsion from midline Slit, resulting in a loss of commissures in 76% of embryonic segments (L, n = 99). In contrast, overexpressing similar levels of Robo transgene that is missing its AP-2 binding motifs (C, D) can not signal ectopic repulsion from the midline, with all segments projecting in a commissural pattern indistinguishable from embryos without transgene (L ΔYQAGL n = 152, ΔYLQY n = 136, ΔYLQYΔYQAGL n = 88). (E) The Ew commissural subset of axons, schematized on the right, cross the midline in each embryonic segment, quantified as 0% error in (M, n = 88). (F) Expressing wild-type Robo transgene (I) specifically in the Ew commissural subset of axons is sufficient to cause ectopic repulsion, with loss of projection across the midline (schematized in dotted gray) in 96% of embryonic segments (M, n = 99). In contrast, expressing either Robo∆YQAGL (G, J) or Robo∆YLQY (H, K) does not cause ectopic repulsion of the Ew projection pattern, with a 0% error in (M, ΔYQAGL n = 152, ΔYLQY n = 136, ΔYLQYΔYQAGL n = 88). Error bars indicate standard error of the mean. See also S5 Fig.