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Figure 5


Fig. 5. Wnt signalling controls Gli3 expression in the dorsal spinal cord. (A-C) Embryos were electroporated with TcfDN and analysed for the expression of Gli3 and Gli2 by in situ hybridization. (A) Eight hours PE, TcfDN caused a reduction of Gli3. (B) Twenty-four hours PE, co-electroporation of TcfDN resulted in the loss of Gli3 expression. (C) Twenty-four hours PE, co-electroporation of TcfDN caused the reported growth arrest of the electroporated side, without changing expression of Gli2. (D-F) Analysis of mouse Gli3 expression in wild-type (Wnt1+/+; Wnt3a+/+), double heterozygous (Wnt1-/+; Wnt3a-/+) and double homozygous (Wnt1-/-; Wnt3a-/-) 10.5 dpc mouse embryos, by in situ hybridization. (G) Schematic representation of the human GLI3 locus. Conserved coding sequences are depicted in blue and conserved non-coding sequences in pink. Grey arrow indicates the length of the GLI3 gene and the direction of transcription. Tcf-binding sites are depicted in green. (H-R) Activity of HCNR1-R4 as putative enhancers tested by in ovo electroporation. (H) Embryos were co-electroporated with pCMV-DsRed as electroporation control, and analysed 24 hours later for GFP and RFP expression. (I,J) Embryos electroporated with the control empty vector showed only red expression. (K,L) Embryos electroporated with R1 showed only weak dorsal GFP expression. (M,N) HCNR2 electroporation resulted in strong dorsal GFP expression. (O,P) HCNR3 electroporation resulted in dorsal GFP expression, although weaker. (Q,R) HCNR4 electroporation resulted in only weak GFP expression. (S) In vivo quantitative analysis of the transcriptional activities of HCNR1-4. Embryos were electroporated with each of the amplified HCNRs (R1-R4) alone, or together with β-cateninCA or TcfDN. Embryos were all co-electroporated with a renilla-luciferase reporter construct for normalization, harvested after 24 hours of incubation and luciferase activity quantitated. Graph shows normalized luciferase units.





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