Fig. 6

Fig. 6 Adult rapunzel mutants have overgrowth and morphological changes in the axial skeleton. A, B. In two week-old larvae stained with calcein, the vertebrae of rapunzel heterozygotes (A) are less scalloped (arrows) compared to their wild type siblings (B). C, D. Similarly, alizarin red and Alcian blue staining show a loss of scalloping (arrows) in the vertebral bodies in 3 week-old heterozygous rapunzel larvae (C) in comparison to their wild type siblings (D). No skeletal hyperossification is seen at these early developmental stages (A–D). E, F. The loss of scalloping persists into adulthood (18 months old) and can be seen (asterisk) in Sirius red-stained sections from rapunzel heterozygotes (E) compared to their wild type siblings (F). Sirius red-stained sections through the vertebral column also demonstrate that rapunzel heterozygotes have increased bone deposition compared to wild type fish (arrows). G–J. MicroCT′s of 18-month-old fish demonstrate marked hyperossification in rapunzel heterozygotes compared to wild type siblings, most easily seen in the vertebral (G, I) and ventral craniofacial (H, J) skeletons. K–L. In higher power views of the mid vertebrae, hyperossification is also readily apparent in 18-month-old rapunzel heterozygotes (K) compared to wild type (L). M, N. Hyperossification is not seen in the vertebrae of 2-month-old rapunzel mutants (M) when compared to age-matched siblings (N). O. This finding is corroborated by measurements of total bone mineral density, where significant differences between rapunzel mutants and their age-matched siblings are seen at 8 and 18 months, but not at 2 months (data represent mean ± SD).