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Fig. 6. Hh signalling regulates two distinct steps of RGC differentiation. (A,B) In
situ hybridization of 24-hpf embryos with a ptc1 RNA probe. Embryos
were treated with DMSO (A) and forskolin (B) from 22 to 24 hpf. ptc1
expression is observed in the ventral CNS in DMSO-treated embryos (A), but is
drastically reduced in forskolin-treated embryos (B, asterisk), although weak
expression is still observed in the ventral forebrain. (C,D) ptc1
expression of embryos treated with forskolin between 22 and 26 hpf.
ptc1 expression is still low at 28 hpf (C, asterisk), but has
recovered at 32 hpf (D, arrowheads). (E) Schematic diagram of the time lag
between forskolin treatment and inhibition of the Hh signalling pathway.
ptc1 expression is inhibited within 2 hours of the start of forskolin
treatment. ptc1 expression is still low 2 hours after the removal of
forskolin, but has recovered within 6 hours of the removal of forskolin. (F)
ath5:GFP expression in retina treated with forskolin from 18 to 22 hpf;
ath5:GFP expression progresses throughout the neural retina. (G-G'')
ath5:GFP expression in retinas treated with forskolin from 18 to 24 hpf. The
inhibition of progression varies from severe (G), to mild (G') and
nearly normal (G''). In the severe case, ath5:GFP is observed only in the
ventronasal retina (white arrowhead). (H) ath5:GFP expression in retina
treated with forskolin from 22 to 26 hpf. The progression of ath5:GFP
expression is completely blocked (white arrowhead). (I) shh:GFP expression in
retina treated with forskolin from 22 to 26 hpf. shh:GFP expression also fails
to progress from the ventronasal retina (white arrowhead). (J) ath5:GFP
expression in retina treated with forskolin from 27 to 48 hpf. The propagation
of ath5:GFP expression occurs normally throughout the neural retina. (K)
shh:GFP expression in retina treated with forskolin from 27 to 48 hpf. The
progression of shh:GFP expression remains blocked (white arrowhead). Weak
background signals are observed in the skin surrounding the eye. (L)
ath5 RNA expression in the same embryo as shown in K. ath5
RNA expression spreads to whole region of the neural retina. (M) Pulse
treatment with forskolin in different time windows. Blue bars indicate the
period of treatment with forskolin. All embryos were examined at 48 hpf. +,
progression of ath5:GFP or shh:GFP expression occurs normally; –
indicates that progression is effectively blocked. (N) Time-window of Hh
signalling inhibition, estimated by considering the time lag between forskolin
treatment and the downregulation of ptc1 expression. Red bars
indicate the period when ptc1 expression is severely suppressed by
forskolin treatment. Pink bars indicate the time lag between the start of
forskolin treatment and the suppression of ptc1 expression, or
between the removal of forskolin and the recovery of ptc1 expression;
within these time periods Hh signalling may be reduced. Black and grey bars
indicate Hh-sensitive and possible Hh-sensitive periods for the progression of
ath5 expression and RGC maturation, respectively. FK, forskolin.
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