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During formation of neuronal circuits, axons navigate long distances to reach their target locations in the brain. When axons arrive at their target tissues, in many cases, they extend collateral branches and/or terminal arbors that serve to increase the number of synaptic connections they make with target neurons. Here, we investigated how Adenomatous Polyposis Coli (APC) regulates terminal arborization of optic axons in living Xenopus laevis tadpoles. The N-terminal and central domains of APC that regulate the microtubule cytoskeleton and stability of β-catenin in the Wnt pathway, were co-expressed with GFP in individual optic axons, and their terminal arbors were then imaged in tectal midbrains of intact tadpoles. Our data show that the APCNTERM and APCβ-cat domains both decreased the mean number, and increased the mean length, of branches in optic axonal arbors relative to control arbors in vivo. Additional analysis demonstrated that expression of the APCNTERM domain increased the average bifurcation angle of branching in optic axonal arbors. However, the APCβ-cat domain did not significantly affect the mean branch angle of arbors in tecta of living tadpoles. These data suggest that APC N-terminal and central domains both modulate number and mean length of branches optic axonal arbors in a compensatory manner, but also define a specific function for the N-terminal domain of APC in regulating branch angle in optic axonal arbors in vivo. Our findings establish novel mechanisms for the multifunctional protein APC in shaping terminal arbors in the visual circuit of the developing vertebrate brain.
Fig. 1. APC N-terminal and central domains alter branching in optic axonal arbors in vivo. We constructed truncated mutants consisting of the N-terminal and central domains of Xenopus laevis APC (A). The APCNTERM mutant consisted of the N-terminal region of APC (amino acids 1-1034) containing the oligomerization domain and armadillo repeats of APC (A). The APCβ-cat mutant contained the middle third of APC (amino acids 1034-1984) containing the β-catenin binding site of full length APC (A). Example images (B) and reconstructions (C) of optic axonal arbors expressing GFP (controls) or GFP together with an APC mutant (experimentals) in tectal midbrains of intact, living tadpoles (stages 46/47) show alterations in optic axon branching induced by expression of the APC domains. The left most tracing of each group of arbors (C) is based on the arbor image shown in (B). Scale Bar – 30 μm (B); 40 μm (C).
Fig. 2. Quantification of effects of APC domains on optic axonal arbors in vivo. Plots of number of branches (A), total arbor branch length (B), and mean branch length (C) confirm observed differences between optic axonal arbors expressing GFP or GFP together with an APC mutant. Data in A-C is shown as percent of control mean with SEM. * above data bar indicates pâ¯<â¯0.05 for control versus APC mutant. * above horizontal line indicates pâ¯<â¯0.05 for APCNTERM versus APCÃ-cat condition. Additional scatter plots of number of branches versus mean branch length with regression lines show inverse correlation between these parameters in optic axonal arbors expressing APC domains (D, E). Sample numbers: A) GFP-12, APCNTERMâ18 APCβ-cat-25; B) GFP-12, APCNTERM-16, APCβ-cat-25; C) GFP-11, APCNTERM-16, APCβ-cat-25.
Fig. 3. APC domains alter target region morphologies and branch angles of optic axonal arbors. Representative convex bounding polygons outlining control and APC-mutant expressing optic axonal arbors in vivo depict differences in overall morphologies of the control and APC domain expressing arbors (A). Illustration of how made measurements of bifurcation angles on a schematic optic axonal arbor (B). Zoomed in regions of images of optic axonal arbors expressing GFP, or GFP with APC domains (left) and tracings of these images (right) show how APC domains alter bifurcation angle (C). Scale Bar- A) 40â¯Î¼m; C) 10â¯Î¼m.
Fig. 4. Quantification of morphology and bifurcation angle of optic axonal arbors in vivo. Quantification of size (A, B) and shape (C) of convex hull polygons confirm additional differences between morphologies of control and APC mutant expressing optic axonal arbors in vivo. Plot and histogram of measurements of mean branch angle in control and APC mutant expressing optic axonal arbors also show alterations in bifurcation angles (D, E). Data in AâD are presented as percent of control mean with SEM. * above data bar indicates pâ¯<â¯0.05 in comparison between control and APC mutant arbors. * above horizontal line indicates pâ¯<â¯0.05 in comparison between APCNTERM and APCÃ-cat arbors. Sample Numbers: AâC) GFP-13 arbors, APCNTERM-17 arbors, APCβ-cat-29 arbors; D, E) GFP - 92 angles in 10 arbors, APCNTERM - 93 angles in 16 arbors, APCβcat â 104 angles in 18 arbors.
