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Fig. 6. RA treatment does not increase the number of
osterix:nuGFP-positive osteoblasts in the centrum. (A-D)
osx:mCherry expressing osteoblasts in a vertebra of a 20-day-old
zebrafish. (A) osx-expressing cells are positioned around arches and
at the anterior and posterior edges of each centrum, which is counterstained
with calcein in B. Part of the overlay in C is shown in D. Osteoblasts are
positioned distal to the produced bone matrix (examples are indicated with
arrows). (E-H) Alizarin Red bone staining of 20-day-old zebrafish
embryos treated with DMSO (E, enlargement in F) or 0.1 µM RA (G,
enlargement in H) showing over-ossification of the vertebral column in
RA-treated specimen. (I-N) osx:nuGFP expressing osteoblasts in
20-day-old zebrafish embryos treated with DMSO (I, enlargement in J) or 0.1
µM RA (L, enlargement in M). Single scans of one focal plane (K) were used
to count cells. Cells indicated with arrows in projections (J,M) are not seen
in single scans (K,N) as they belong to the arch at the opposite site. In
single scans, cells can be distinguished from each other whereas in
projections this is not always clear (encircled spot in J was resolved to
represent two different cells in K). (M,N) High magnifications of a haemal
arch that were actually not used for counting. (O) Schematic
representation of vertebrae shows defined area in which cells were counted per
segment. (P) Cell counts show no difference between DMSO controls and
treated embryos in amount of osx:nuGFP positive osteoblasts. Scale
bars: 25 µm in A-C; 500 µm in E,G; 50 µm in I,L.
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