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Putting the model to the test: are APC proteins essential for neuronal polarity, axon outgrowth, and axon targeting?

Rusan NM, Akong K, Peifer M - J. Cell Biol. (2008)

Bottom Line: Experimental data based on dominant-negative approaches suggest that the tumor suppressor adenomatous polyposis coli (APC), a regulator of Wnt signaling and the cytoskeleton, regulates polarity of neuroectodermal precursors and neurons, helping specify one neurite as the axon, promoting its outgrowth, and guiding axon pathfinding.However, such dominant-negative approaches might affect processes in which APC is not essential.Likewise, CB, lobular plug, and mushroom body neurons do not require APCs for polarization, axon outgrowth, or, in the latter two cases, axon targeting.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
The highly polarized architecture of neurons is important for their function. Experimental data based on dominant-negative approaches suggest that the tumor suppressor adenomatous polyposis coli (APC), a regulator of Wnt signaling and the cytoskeleton, regulates polarity of neuroectodermal precursors and neurons, helping specify one neurite as the axon, promoting its outgrowth, and guiding axon pathfinding. However, such dominant-negative approaches might affect processes in which APC is not essential. We completely removed both APCs from Drosophila melanogaster larval neural precursors and neurons, testing whether APCs play universal roles in neuronal polarity. Surprisingly, APCs are not essential for asymmetric cell division or the stereotyped division axis of central brain (CB) neuroblasts, although they do affect cell cycle progression and spindle architecture. Likewise, CB, lobular plug, and mushroom body neurons do not require APCs for polarization, axon outgrowth, or, in the latter two cases, axon targeting. These data suggest that proposed cytoskeletal roles for APCs in mammals should be reassessed using loss of function tools.

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CB NBs do not require APCs for persistent spindle positioning. (A–C) Time-lapse images; NB clones followed for two rounds of division. Tau∷GFP (A and B) or CD8∷GFP (C). Arrowheads, GMCs from first round of division; arrows, GMCs from second round. (A) Control clone. (B) APC2d40APC1Q8 clone. (C) APC2g10 APC1Q8 clone. The GMC from the first round does not remain in focus during the second round. (D–H) Bent spindle phenotype. (D and E) Fixed samples. (D) Wild type (WT). (E) APC2g10APC1Q8. (F–H) Time lapse of NB clones, prophase, metaphase, and one frame before anaphase onset (AO). (F) Control. (G) APC2d40APC1Q8 transitioning through bent spindle intermediate. Dashed circles outline NB. (H) APC2d40APC1Q8 without bent spindle. Time is given in hours:minutes:seconds. Bars,10 μm.
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fig2: CB NBs do not require APCs for persistent spindle positioning. (A–C) Time-lapse images; NB clones followed for two rounds of division. Tau∷GFP (A and B) or CD8∷GFP (C). Arrowheads, GMCs from first round of division; arrows, GMCs from second round. (A) Control clone. (B) APC2d40APC1Q8 clone. (C) APC2g10 APC1Q8 clone. The GMC from the first round does not remain in focus during the second round. (D–H) Bent spindle phenotype. (D and E) Fixed samples. (D) Wild type (WT). (E) APC2g10APC1Q8. (F–H) Time lapse of NB clones, prophase, metaphase, and one frame before anaphase onset (AO). (F) Control. (G) APC2d40APC1Q8 transitioning through bent spindle intermediate. Dashed circles outline NB. (H) APC2d40APC1Q8 without bent spindle. Time is given in hours:minutes:seconds. Bars,10 μm.

Mentions: We hypothesized that loss of both APCs would abrogate asymmetric division or eliminate persistent spindle alignment. Surprisingly, neither property was disrupted. We assessed asymmetric divisions using CD8-GFP, revealing membranes, nuclei, and spindles (Fig. 1, D and E), or Tau-GFP, marking spindles (Fig. 1, F and G). Double mutant NBs (both APC2g10 APC1Q8 and APC2d40 APC1Q8) exhibited apparently normal asymmetric divisions with high fidelity (Fig. 1, D–G; Table S1, and Videos 1–3, available at http://www.jcb.org/cgi/content/full/jcb.200807079/DC1). Furthermore, mutant NBs maintained a persistent division axis as in wild type, with the new daughter always born next to the previous daughter (Fig. 2, A–C; Table S1, and Videos 4–6). Because cortical polarity proteins regulate spindle alignment in many cell types, we examined the relationship between cortical polarity and spindle orientation. This was normal for both apical (aPKC; Fig. S3, A and B) and basal (Miranda; Fig. S3, C and D) markers. Finally, adherens junction proteins remained asymmetrically localized (Fig. S3, E–J). Thus, APCs are not essential for the decision between symmetric and asymmetric divisions here, as was suggested in embryos (Lu et al., 2001). Furthermore, APC loss must not eliminate MT–cortical interactions critical for CB NB spindle orientation (Siller et al., 2005, 2006), as loss of APC does in some cultured mammalian cells (Green and Kaplan, 2003; Caldwell et al., 2007).


Putting the model to the test: are APC proteins essential for neuronal polarity, axon outgrowth, and axon targeting?

Rusan NM, Akong K, Peifer M - J. Cell Biol. (2008)

CB NBs do not require APCs for persistent spindle positioning. (A–C) Time-lapse images; NB clones followed for two rounds of division. Tau∷GFP (A and B) or CD8∷GFP (C). Arrowheads, GMCs from first round of division; arrows, GMCs from second round. (A) Control clone. (B) APC2d40APC1Q8 clone. (C) APC2g10 APC1Q8 clone. The GMC from the first round does not remain in focus during the second round. (D–H) Bent spindle phenotype. (D and E) Fixed samples. (D) Wild type (WT). (E) APC2g10APC1Q8. (F–H) Time lapse of NB clones, prophase, metaphase, and one frame before anaphase onset (AO). (F) Control. (G) APC2d40APC1Q8 transitioning through bent spindle intermediate. Dashed circles outline NB. (H) APC2d40APC1Q8 without bent spindle. Time is given in hours:minutes:seconds. Bars,10 μm.
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fig2: CB NBs do not require APCs for persistent spindle positioning. (A–C) Time-lapse images; NB clones followed for two rounds of division. Tau∷GFP (A and B) or CD8∷GFP (C). Arrowheads, GMCs from first round of division; arrows, GMCs from second round. (A) Control clone. (B) APC2d40APC1Q8 clone. (C) APC2g10 APC1Q8 clone. The GMC from the first round does not remain in focus during the second round. (D–H) Bent spindle phenotype. (D and E) Fixed samples. (D) Wild type (WT). (E) APC2g10APC1Q8. (F–H) Time lapse of NB clones, prophase, metaphase, and one frame before anaphase onset (AO). (F) Control. (G) APC2d40APC1Q8 transitioning through bent spindle intermediate. Dashed circles outline NB. (H) APC2d40APC1Q8 without bent spindle. Time is given in hours:minutes:seconds. Bars,10 μm.
Mentions: We hypothesized that loss of both APCs would abrogate asymmetric division or eliminate persistent spindle alignment. Surprisingly, neither property was disrupted. We assessed asymmetric divisions using CD8-GFP, revealing membranes, nuclei, and spindles (Fig. 1, D and E), or Tau-GFP, marking spindles (Fig. 1, F and G). Double mutant NBs (both APC2g10 APC1Q8 and APC2d40 APC1Q8) exhibited apparently normal asymmetric divisions with high fidelity (Fig. 1, D–G; Table S1, and Videos 1–3, available at http://www.jcb.org/cgi/content/full/jcb.200807079/DC1). Furthermore, mutant NBs maintained a persistent division axis as in wild type, with the new daughter always born next to the previous daughter (Fig. 2, A–C; Table S1, and Videos 4–6). Because cortical polarity proteins regulate spindle alignment in many cell types, we examined the relationship between cortical polarity and spindle orientation. This was normal for both apical (aPKC; Fig. S3, A and B) and basal (Miranda; Fig. S3, C and D) markers. Finally, adherens junction proteins remained asymmetrically localized (Fig. S3, E–J). Thus, APCs are not essential for the decision between symmetric and asymmetric divisions here, as was suggested in embryos (Lu et al., 2001). Furthermore, APC loss must not eliminate MT–cortical interactions critical for CB NB spindle orientation (Siller et al., 2005, 2006), as loss of APC does in some cultured mammalian cells (Green and Kaplan, 2003; Caldwell et al., 2007).

Bottom Line: Experimental data based on dominant-negative approaches suggest that the tumor suppressor adenomatous polyposis coli (APC), a regulator of Wnt signaling and the cytoskeleton, regulates polarity of neuroectodermal precursors and neurons, helping specify one neurite as the axon, promoting its outgrowth, and guiding axon pathfinding.However, such dominant-negative approaches might affect processes in which APC is not essential.Likewise, CB, lobular plug, and mushroom body neurons do not require APCs for polarization, axon outgrowth, or, in the latter two cases, axon targeting.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
The highly polarized architecture of neurons is important for their function. Experimental data based on dominant-negative approaches suggest that the tumor suppressor adenomatous polyposis coli (APC), a regulator of Wnt signaling and the cytoskeleton, regulates polarity of neuroectodermal precursors and neurons, helping specify one neurite as the axon, promoting its outgrowth, and guiding axon pathfinding. However, such dominant-negative approaches might affect processes in which APC is not essential. We completely removed both APCs from Drosophila melanogaster larval neural precursors and neurons, testing whether APCs play universal roles in neuronal polarity. Surprisingly, APCs are not essential for asymmetric cell division or the stereotyped division axis of central brain (CB) neuroblasts, although they do affect cell cycle progression and spindle architecture. Likewise, CB, lobular plug, and mushroom body neurons do not require APCs for polarization, axon outgrowth, or, in the latter two cases, axon targeting. These data suggest that proposed cytoskeletal roles for APCs in mammals should be reassessed using loss of function tools.

Show MeSH
Related in: MedlinePlus