Fig. 6

DSCAM impacts the topographic organization of ventral RGC axon fibers branching at the target. a Schematic illustrates the sequential electroporation of ventral RGCs with lissamine-tagged control or DSCAM MO, and dorsal RGCs with Alexa Fluor 488-dextran. b Coronal section of a stage 46 tadpole eye shows the distribution of the lissamine-tagged MO and Alexa Fluor 488-dextran after electroporation. Arrow points to fluorescent debris picked by glial cells. c Confocal projection of axon terminals of a tadpole transfected at stage 47 and imaged in vivo 48 h after transfection illustrates the organization of retinal fibers along the medial to lateral axis. Control MO lissamine-tagged axons (red) from ventral RGC terminate medially (M) within the tectal neuropil while Alexa Fluor 488 dextran-labeled axons (green) from dorsal RGCs terminate more laterally (L) with little to no overlap. d Confocal projections of axon terminals from three sample tadpoles with ventral RGCs transfected with lissamine-tagged Control MO or DSCAM MO (red) at stage 46 and imaged 48 h after transfection show some degree of overlap between RGC axons labeled with Alexa Fluor 488 dextran (green) and MO-transfected RGC axons (red). Single channel fluorescent signals for arbors of ventral RGC transfected with Control MO and DSCAM MO are shown in the insets. Transfection of DSCAM MO in ventral RGCs resulted in their axons projecting medially (red) but with less overlap than those in Control MO transfected tadpoles. e, f Quantitative analysis of the territory occupied by dorsal and ventral RGCs axons within the tectal neuropil in tadpoles transfected with either DSCAM MO or Control MO and imaged 48 h later. The area occupied by the ventral or dorsal RGC axon terminals was measured by separately creating a polygon surrounding the first branch point and the terminal tips of the lissamine-labeled arbors (red only; see white dashed line in d) or the Alexa 488-labeled arbors (green only; see magenta dashed lines in d). Isolated, unbranched axons that project medially (arrows in d) were not included in the analysis. e A significant difference in the area of arbor overlap is observed when comparing axon arbors from ventral RGCs that project medially in DSCAM MO vs Control MO treated tadpoles. Data was normalized across tadpole samples by calculating, in percent, the area of overlap with respect to the region occupied by the medial (red fluorescence), lateral (green fluorescence) and medial + lateral (red and green fluorescence) axons. f When comparing tadpoles treated with DSCAM MO vs Control MO (targeted to ventral RGCs), no difference in the area occupied by arbors from ventral RGCs that project medially or regions occupied by dorsal RGCs axons that project laterally. Statistical analysis was by unpaired, two-tailed t test with equal sample sizes (n = 8) for Control MO and DSCAM MO. Error bars indicate S.E.M. *** p ≤ 0.001, **** p ≤ 0.0001, ns = non-significant. Scale bars: 40 μm