Figure 3. Dynamic properties of the Reissner fiber revealed in scospondin-GFP^ut24 knock-in zebrafish.

(A) Inverted greyscale maximal Z-projection of confocal stack of scospondin-GFP^ut24/+ embryo at 3 dpf. Expression in the subcommissural organ (SCO) and flexural organ (FO) in the head with Reissner fiber (red arrowhead) (A). Merge of DIC image and pseudocolored SCO-spondin-GFP expression (Green) (A’). Scale bar: 50 μm

(B) Inverted greyscale frames from a time-lapse confocal dataset from a scospondin-GFP^ut24/+ embryo head at 3 dpf. At time (t)=0, a region was photobleached using a short, high energy pulse from a 488nm solid state laser which allowed for manual tracking of the movement of the bleached region from rostral to caudal. Scale bar 10μm.

(C) Inverted greyscale maximal Z-projection of confocal stack of scospondin-GFP^ut24/+ embryo tail at 3 dpf. The Reissner fiber (red arrowhead) and floor plate (red bracket) are labeled with GFP (C). Merge of DIC image and pseudocolored SCO-spondin-GFP expression (Green) (C’). Scale bar 20μm.

(D) Inverted greyscale frames from a time-lapse confocal dataset from a scospondin-GFP^ut24/+ embryo head at 3 dpf. At time (t)=0, two regions were photobleached as in B which allowed for manual tracking of the movement of the bleached region from rostral to caudal. Scale bar 10μm.

(E) Average velocity as nanometers (nm) per second (sec) were calculated manually for multiple embryos at 3, 5, and 7 dpf in experiments depicted in B and D (n=13, 9, and 8 respectively). The average velocity for each individual embryo was plotted as box plots (mean ± SD). (**** denotes p-value <10⁻⁴)

(F-K) Frames from a time-lapse confocal dataset taken during tail bud development (20-30 hours post fertilization, see Video S2) presented as inverted greyscale maximal Z-projections. Red brackets highlight faint expression, extended fibers of SCO-spondin-GFP, while red arrows point out a bolus SCO-spondin-GFP material which is observed to travel rapidly in a rostral to caudal direction. Inset in the lower left-hand side of each panel are digitally enlarged portions of the region containing SCO-spondin-GFP-labeled material. Scale bars: 100 μm in main, 50 μm in inset. Time stamp is hr:min post-fertilization.

(L) Frames from a time-lapse confocal dataset taken during tail bud development (20-22 hours post fertilization, see Video S2) presented as inverted greyscale maximal Z-projections. Red arrows highlight SCO-spondin-GFP-labeled bolus material, while red arrowheads indicate bolus material leading the Reissner fiber. Scale bars: 10 μm

See also Figure S3 and Video S1–5.