Elul TM , Kimes NE , Kohwi M , Reichardt LF .

F32 MH 12613 NIMH NIH HHS, P01 NS016033-24 NINDS NIH HHS , F32 MH012613 NIMH NIH HHS, P01 NS016033 NINDS NIH HHS , R01 CA094143 NCI NIH HHS

	Figure 1. β-catenin is expressed in RGC axon arbors in the tectum. Confocal image (single optic section) of a tectal section taken from a stage 45/46 tadpole stained with anti β-catenin antibody (red) shows expression in the neuropil area, the target region for RGC axons (A). Confocal image of a GFP-expressing RGC axon arbor (green) in the same optic section shows that the axon arbor is in the neuropil region of the tectal section (B). Higher magnification zoom of boxed regions in A and B shows that β-catenin staining is punctate in the neuropil (C) and that the GFP-expressing RGC axon arbor contains β-catenin puncta (E, arrows, double arrow, arrowhead). β-catenin puncta are located at branch points (D, arrows), in growth cones (D, double arrow), and along branch shafts (D, arrowhead). np, Neuropil region of the tectum; cb, cell body region of the tectum. Scale bars: A, B,10 μm; C-E, 5 μm.
	Figure 2. Schematic diagram of mutant β-catenin constructs. Wild-type β-catenin consists of three distinct functional and structural domains: an N-terminal domain, a central core consisting of 12 armadillo repeats, and a C-terminal domain. Within these three domains, β-catenin contains binding sites for α-catenin (N-terminal domain and armadillo repeats R1-R2; amino acid residues 120-150), cadherins (binding spans throughout armadillo repeats R3-R12), and PDZ-domain-containing proteins (C-terminal domain; amino acid residues 777-781). The deletion mutant ΔARM lacks almost the entire armadillo repeat region found in wild-type β-catenin; it consists of the N-terminal residues 1-151 of wild-type β-catenin fused to the C-terminal residues 648-781. Thus, ΔARM retains binding to α-catenin and PDZ-binding proteins but not to cadherin. The deletion mutants NTERM and CTERM are simply the N- and C-terminal domains of β-catenin that together constitute ΔARM. Each protein had GFP fused to its N terminus and was expressed using the Xenopus expression vector pCS2. ΔARM and CTERM also contained a myc-epitope tag at their C termini. We also confirmed the effects of ΔARM without an attached GFP tag and of CTERM without attached GFP or myc tags (see Results).
	Figure 3. Axonal arborization phenotypes of RGCs expressing β-catenin deletion mutants. Projected confocal z-series images taken of axon arbors in live tadpoles show arbor morphologies for RGCs expressing GFP (A) or GFP-tagged-β-catenin deletion mutants (B-E). RGC axons that express the β-catenin deletion mutant ΔARM have two distinct phenotypes. They either do not arborize (B) or they arborize but contain longer, tangled branches (C). RGC axons expressing the β-catenin deletion mutant NTERM form severely reduced arbors (D) that are similar to the subpopulation of ΔARM arbors with no or few branches (B). RGC axons expressing CTERM form arbors with long, tangled branches (E) that are similar to the subpopulation of ΔARM arbors with effusive branching (C). RGC axons that express ΔARM and NTERM and form severely reduced arbors (B, D) also have bulbous endings at their axon/branch tips (B, D, arrows) and appear thicker than RGC arbors that express GFP, ΔARM, and CTERM (compare axon/branch thicknesses in B, D with those in A, C, E). Note that at the level of magnification of these projected confocal z-series (40×), all of the GFP-tagged-β-catenin deletion mutant constructs appear to be uniformly distributed within the axon terminals and arbors. Scale bars: A-E, 20 μm.
	Figure 4. Tracings of projected confocal z-series of RGC axon arbors expressing β-catenin deletion mutants confirm morphological phenotypes. Compared with controls, RGC axons that express ΔARM have two distinct phenotypes. They either do not arborize (B) or they arborize but have abnormally long and tangled branches (C). NTERM-expressing arbors (D) are similar to the subpopulation of ΔARM arbors with no or few branches (B). CTERM-expressing arbors (E) are similar to the subpopulation of ΔARM arbors with abnormally long, tangled branches (C). For each condition (A-E), two examples of arbors are shown. In each example of an arbor, two (or three) (B, left) tracings are shown that correspond to confocal images taken ∼ 24 hr apart. The first tracing of each example corresponds to a confocal image taken at stage 45/46. The left most arbor tracing in A-E corresponds, respectively, to the actual confocal z-series projection image shown in Figure 3A-E. Stippling over a single arbor branch in C and E illustrates a particularly tangled branch. Rostral is to the top and lateral is to the right in all images. Scale bars, 20 μm (A-E).
	Figure 5. Quantification of morphological parameters for arbors from RGCs expressing β-catenin deletion mutants confirm phenotypes shown in Figures 3 and 4. The distribution of branch numbers in arbors at stage 45 shows that GFP axon arbors are distributed about a mean of ∼11 branches (A). In contrast, ΔARM axons are distributed about two distinct means: ΔARM arbors either have very few (1-2) branches or they have approximately the same number of branches as control GFP arbors (A). In addition, NTERM arbors have no or very few (1-6) branches, whereas CTERM arbors have numbers of branches comparable with GFP arbors (A). Plot of mean numbers of branches in arbors over 2 d shows that GFP arbors acquire more branches over time (B). ΔARM arbors with branches and CTERM arbors behave similarly to GFP arbors (B). In contrast, ΔARM axons without arbors and NTERM axons form few or no additional branches over time (B). Branched ΔARM arbors (with 9 or more branches) and CTERM arbors also grow faster than GFP arbors (C) (p < 0.05 for both, Student's t test). We did not measure the growth rate for unbranched ΔARM and NTERM arbors because many RGCs in these two classes did not form arbors (C). The number of axons analyzed is as follows: A, GFP (21), ΔARM (24), NTERM (7), CTERM (10); B, GFP (10), ΔARM-branched (6), ΔARM-unbranched (6), NTERM (7), CTERM (10), (C) GFP (9), ΔARM-branched (6), CTERM (10).

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