Figure 4

Sequence and functional analysis of the Β-catenin 3'UTR'. (A) Evolutionary conservation of the β-catenin 3'UTR in vertebrates. Individual plots show conservation between the indicated species and the mouse based on comparisons between genomic sequence starting at the β-catenin stop codon (asterisk) at the 5' limit of the 3'UTR and continuing 10 kb in the 5' direction encompassing the entire 3'UTR and downstream untranscribed sequence. The position of the polyadenylation signal in the mouse sequence, which approximates to the 3' limit of the 3'UTR, is marked poly A. Conservation score calculated as an averaged windowed identity (window size = 100 bp). Curves only show conservation score range 50-100%. Position indicates kilo bases from stop codon in mouse β-catenin. (Alignments calculated with M-LAGAN). (B-F) β-catenin 3'UTR directs GFP expression to the axon. (B) Diagram of GFP plasmid reporter constructs. CAGG promoter (unfilled box) myrd1EGFP cDNA (green box). pKR6: The myrd1EGFP stop codon (TGA) attached to β-catenin genomic sequence spanning from just 3'to the translation stop codon (TAA), through the 3'UTR (black) to approximately 300 bp 5'of the polyadenylation signal sequence (ATTAAA). pKR7 is identical to pKR6 except that most of the 3'UTR is deleted. (C, D) Examples of thalamic neurons electroporated with GFP reporter constructs and cultured for 2 days, GFP immunostaining gives black signal in these images (C) pKR6, (D) pKR7. Neurons are growing within a tissue explant so cell bodies (cb) and axons (a) are only visible if GFP is expressed in them. The red lines illustrate lines drawn to measure the distance GFP signal could be detected along an axon. Note that the red line in C extends to a growth cone. (E) Densitometric analysis of GFP signal intensity (in arbitrary units (AU)) in cell bodies following transfection with pKR6 and pKR7. (F) Distance GFP signal could be traced from the cell body along the axon. Transfecting thalamic explants with pTP6 to express a tau tagged GFP which completely fills axons gave a value for total axon length of 158 ± 14 μm. Mann-Whitney Rank sum test P-values for pairwise comparisons shown above bars, error bars are ± SEM. GFP values were taken from 84 and 30 (pKR6) and 116 and 30 (pKR7) cell bodies and axons respectively or 30 axons (pTP6) randomly selected from 3 independent cultures each including thalamic tissue from several embryos. Scale bars in C, D, 10 μm.