Figure 4. Loss of the Reissner fiber is associated with scoliosis in additional independent scoliosis mutant zebrafish strains.

(A-A’) Inverted greyscale maximal Z-projections of confocal images of Et(druk-GFP^dut26/+; scospondin-GFP^ut24/+ demonstrating typical assembly of the Reissner fiber, floor plate and terminal ampulla expression in these mutant knock-in embryos at 5 dpf. Note that GFP expression is contributed both by the Et(druk-GFP^dut26/+) transgene insertion and from the scospondin-GFP^ut24/+ allele. Scale bars are 25μm in (A) and 5μm in (A’).

(B, C, D) Bright field images of a wild-type juvenile scospondin-GFP^ut24/+ (B), Et(druk-GFP^dut26/+) (C), and Et(druk-GFP^dut26/+); scospondin-GFP^ut24/+ (D) displaying the onset of mild scoliosis. Scale bar is 1mm, standard length in upper right corner.

(B’, C’, D’) Inverted greyscale maximal Z-projections of confocal images of the same fish in B, C and D to highlight the spinal cord. At 19 dpf, in wild-type scospondin-GFP^ut24/+ we observe high expression of the Reissner fiber (100%, n=14 size range 6.3-6.8mm)(B’), in scoliosis mutant Et(druk-GFP^dut26/+); +/+ the Reissner fiber is not labeled but some spinal cord cells express GFP (n=6 size range 6.0-6.5mm), while in double mutant Et(druk-GFP^dut26/+); scospondin-GFP^ut24/+ we observed curvature of the spinal canal and a complete loss of a Reissner fiber (94%, n=16 size range 6.1–7.0mm). Scale bars are 10μm.

(E, F) Inverted greyscale maximal Z-projections of confocal imaging of AFRU-stained zebrafish embryos at 1 dpf (30hpf). The Reissner fiber is observed in heterozygous kinesin family member 6 (kif6^sko/+) and homozygous kif6^sko mutants (8/8, E-E”). Scale bars are 10μm.

(G-H”) Inverted greyscale maximal Z-projections of confocal imaging of AFRU and ZO-1 stained zebrafish at 3 dpf. ZO-1 localizes to tight junctions and shows the location of the central canal epithelium. Heterozygous kif6^sko/+ animals have an intact Reissner fiber in the central canal (13/13) (G-G”), whereas in homozygous kif6^sko the Reissner fiber is disassembled and Reissner material fills the space of the central canal (12/13) (H-H”). Scale bars are 25μm

(I-J) Merge of maximal Z-projections of confocal imaging of AFRU and ZO-1 stained zebrafish at 5 dpf. Heterozygous kif6^sko/+ animals have an intact Reissner fiber in the central canal (I), but in homozygous kif6^sko animals diffuse Reissner material fills the central canal lumen (J). Scale bars are 10μm.