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Fig. 2. Effects of RA signaling on the expression of PITX2c,
Ad4BP/SF-1 and cell proliferation in chick. (A-F) RA and
RAA beads were implanted into the left (A,B) and right (C,D) presumptive gonad
regions of both sexes, respectively, at stage 18-19. As controls, DMSO beads
were implanted (E,F). 48 hours after implantation (stage 27), the embryos were
subjected to whole-mount in situ hybridization with Ad4BP/SF-1
(A,C,E) and PITX2c (B,D,F) probes, then the embryos were sectioned.
Sections of female (ZW) samples are shown. Arrowheads in A and C indicate
affected expression of Ad4BP/SF-1. (G-N) The effects of
implantation of RA (G) and RAA (I) beads on cell proliferation were examined
at stage 27 in female embryos. DMSO beads were used as a control (H,J). BrdU
was injected into the operated embryos 1 hour before fixation, then gonads
were stained with antibodies for BrdU (green) and cytokeratin (red). Effects
of RA (K) and RAA (M) beads on cortical thickness were examined by staining
with anti-cytokeratin antibody 72 hours after the implantation (stage 29).
DMSO beads were used as controls (L,N). (O-R) Cell proliferation was
analyzed quantitatively in gonads of embryos implanted with RA (O,P), RAA
(Q,R) and DMSO beads at stage 27. Total DAPI-stained cells (data not shown)
and BrdU-positive cells in the gonadal cortex (cytokeratin-positive) (O,Q) and
medulla (cytokeratin-negative) (P,R) were counted. Relative fold changes in
the number of BrdU-positive cells in the cortex and medulla are plotted, with
the number of BrdU-positive cells in the right cortex of DMSO-bead-implanted
embryos set at 1. Scale bars: 100 µm in A-J; 50 µm in K-N.
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