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Fig. 4. Hh signalling inhibits RGC development in vitro. (A-C) Brn3b staining in
E12 retinal explants cultured for 48 hours under control conditions (A) or in
the presence of recombinant Shh-N (B) or anti-Hh (C). The most dramatic change
in Brn3b staining is observed in the peripheral region (closer to the lens) of
the retinal explants. (D) Co-localization of silver grains marking
[3H]thymidine labelled cells and Brn3b staining. RPC were labelled
with [3H]thymidine for 4 hours, washed and cultured for an
additional 48 hours. Arrows indicate double-labelled cells, arrowheads
indicate Brn3b+ [3H]thymidine– cells.
(E) Dissociated cell scoring to quantify the proportion of RGCs among the
[3H]thymidine+ cohort in E12 retinal explants cultured
for 48 hours under the indicated treatment conditions. Treatment with
isotype-matched anti-LFA antibodies (1E6) was no different than control and is
not shown. n
3 for each condition. (F) Proportion of S-phase,
Brn3b+ and the non-RGC component of the Tuj1+ population
(determined by subtracting the number of Brn3b+ from the number of
Tuj1+ cells) in E12 explants cultured for 48 hours under the
indicated conditions and analyzed by dissociated cell scoring.
*P<0.005, ** P<0.005. Scale bars: 100 µm in A-C; 25
µm in D.
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