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Fig. 3. Ubiquitous Sema3d overexpression slows midline growth by increasing growth cone pausing and decreasing growth cone advancing. (A) Overall growth rates in regions before, at and after the midline, reported as mean+s.e.m. Sema3d overexpression significantly decreased RGC growth rate in the midline region. Sema3d-overexpressing embryos, n=14 growth cones in 12 embryos; control embryos, n=2 growth cones in two wild-type embryos, 15 growth cones in 13 heat-shocked wild-type embryos, and two growth cones in two non-heat-shocked Tg(hsp70:sema3d^myc) embryos. (B) Percentage of time growth cones spent advancing, pausing and retracting. Sema3d overexpression significantly increased time spent pausing and significantly decreased time spent advancing in the midline region. P, pausing; R, retracting; A, advancing. Sema3d-overexpressing embryos, n=15 growth cones in 12 embryos; control embryos, n=3 growth cones in two wild-type embryos, 15 growth cones in 12 heat-shocked wild-type embryos, and one growth cone in one non-heat-shocked Tg(hsp70:sema3d^myc) embryo. ^*P<0.05; ^**P<0.02. (C,D) Frames taken from timelapse movies of growth cones in representative control (C) and Sema3d-overexpressing (D) embryos. Over a similar time period, the control growth cone extended from the midline into the optic tract, but the growth cone in the Sema3d-overexpressing embryo showed very limited forward growth. Times are indicated in hours:minutes; arrows below frame sequences indicate the midline. Anterior is up in all frames. Scale bar: 10 µm.

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