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Fig. 6. Smad3 activity is both required and sufficient for the generation of
ventral interneurons. (A-F) HH stage 14 embryos electroporated with Smad3
or TßR-I were analyzed 24 hours later for the expression of selective IN
markers. Pax2+ (A-D) and Lhx1/5+ (E,F) were cell-autonomously induced in the
electroporated side. (G) Quantitative analysis shows an increase in
expression of Pax2+ and Lhx1/5+ cells after Smad3 or TßR-I transfection
compared with the non-electroporated control side. (H) Quantitative
analysis of p27kip1/Pax2 double labeled cells after electroporation
of Smad3 shows a increased proportion of Pax2+cells. Histograms show data
points as mean values ± s.d. (n=embryos, 6 sections).
(I,J) 48 hours co-electroporation of pSUPER with TßR-I
resulted in a dramatic increase in Pax2+ cells. (K,L)
Co-electroporation of pSUPER-Smad3 shRNA with TßR-I resulted in the
rescue of Pax2 ectopic expression. (M-R) HH stage 14-16 embryos
electroporated with pSUPER-Smad3 shRNA were analyzed 48 hours later for the
expression of selective LIM-HD factors. shRNA Smad3 and empty pSUPER vector
were co-electroporated with pCIG (5:1) to use GFP as a reporter protein. 48
hours after electroporation (M,N) the expression of interneuron markers such
as Pax2 (O,P) and Lhx1/5 (Q,R) is reduced. (S) Percentage of cells that
express Pax2, and Lhx1/5 in control vs. electroporated sides. Pax2+ cells are
reduced by 41.29%, Lhx1/5+ cells by 42.31% (n=5 embryos were assessed
in each experiment). Histograms show data points as mean values ±
s.e.m. Lhx1/5, P=0.00022; Pax2, P=0.000025; using the
two-tailed Student's t-test.
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