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Putting the model to the test: are APC proteins essential for neuronal polarity, axon outgrowth, and axon targeting?
Correspondence to M. Peifer: peifer{at}unc.edu
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.
Abbreviations used in this paper: APC, adenomatous polyposis coli; CB, central brain; GMC, ganglion mother cell; MARCM, mosaic analysis with a repressible cell marker; MB, mushroom body; MT, microtubule; NB, neuroblast.
© 2008 Rusan et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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One superb model of neuronal polarity is cultured hippocampal neurons (for review see Arimura and Kaibuchi, 2007). They undergo ordered changes from multipolar extension of many immature neurites to selection of one as the axon followed by rapid axon growth. Other neurites become dendrites. The Par3 complex localizes to nascent axon tips, and altering Par protein function disrupts polarity and thus axon outgrowth. To influence axon outgrowth, the axon selection machinery must influence the actin and microtubule (MT) cytoskeletons. The Par complex is proposed to regulate GSK3 kinase, which in turn regulates MT-associated proteins like Tau and adenomatous polyposis coli (APC), linking the Par complex, the cytoskeleton, and axon outgrowth (for reviews see Solecki et al., 2006; Arimura and Kaibuchi, 2007).
APC is an attractive candidate polarity regulator. APC and GSK3 negatively regulate the Wnt effector β-catenin (β-cat), and Wnt signaling can shape cell polarity (Näthke, 2006). APC plays a separate role in cytoskeletal regulation in part through effects on MT stability and cortical association (Näthke, 2006). One illustrative example involves polarized astrocyte migration in which APC is suggested to act at the end of a cdc42–GSK3–Par complex pathway (Etienne-Manneville and Hall, 2003). APCs may also regulate neural precursor polarity. In fly embryos, neural precursors (neuroblasts [NBs]) divide asymmetrically, whereas other ectodermal cells divide symmetrically. APC2 RNAi depletion led epidermal cells to divide asymmetrically like NBs (Lu et al., 2001). In other stem cell types, APCs help orient mitotic spindles (Yamashita et al., 2003) or regulate exit from the stem cell fate (van de Wetering et al., 2002), which are roles they might also play in neural precursors.
Several studies suggest that APC is essential for neuronal polarity and axon extension. The Snider and Jan laboratories provided the first connection (Shi et al., 2004; Zhou et al., 2004). NGF triggers GSK3 inactivation specifically at axon tips, allowing APC to localize there. When they misexpressed truncated APCs that they propose are dominant negative, axon outgrowth was significantly reduced. Zhou et al. (2004) proposed that local GSK3 inhibition in growth cones allows APC to regulate MTs. Shi et al. (2004) suggested that APC helps transport Par3 to the axon tip. Several papers extended these studies but suggested other mechanisms. One suggested that GSK3 and APC act, at least in part, through β-cat via direct cytoskeletal/adhesive effects and via Wnt signaling (Votin et al., 2005). The other suggested that GSK3 regulates membrane trafficking to neurites, perhaps via APC (Gartner et al., 2006; Collin et al., 2008). Finally, APC inactivation by CALI (chromophore-assisted laser inactivation) affects growth cone migration (Koester et al., 2007), suggesting an additional role in axon guidance. These studies suggest that APC is a GSK3 effector acting on the cytoskeleton to direct axon polarization, outgrowth, and guidance, a model now widely accepted in reviews (for reviews see Arimura and Kaibuchi, 2007; Goldstein and Macara, 2007) and textbooks (Squire et al., 2008).
These experiments have two important caveats. First, none used genetic loss of function to reduce or eliminate APC function; instead, they expressed truncated APC fragments, reasoning that these are dominant negative, or used CALI to inactivate APC and possibly its binding partners. Because APC has many cytoskeletal partners, truncated APCs may sequester these in inactive complexes and thus have indirect effects (McCartney et al., 2006). These issues are critical, as is shown by Par3. Although cell culture studies supported a Par3 role in neuronal polarity, (e.g., Shi et al., 2003) loss of function analysis in Drosophila melanogaster demonstrated that this is not an essential Par3 function (Rolls and Doe, 2004). Second, most studies did not consider the possibility that APC inactivation alters Wnt signaling. Because Wnts regulate asymmetric divisions, neuronal migration, and axon outgrowth in Caenorhabditis elegans (Silhankova and Korswagen, 2007) and inhibit NGF-induced neurite outgrowth in culture (Chou et al., 2000), APC might affect axon outgrowth by affecting Wnt signaling.
We tested the hypothesis that APCs play a fundamental cytoskeletal role in polarity of neuronal progenitors and neurons. In Drosophila, we have the unique ability to simultaneously eliminate function of both APCs using protein-null alleles, revealing how complete APC loss affects polarization of neural precursors and their neuronal progeny.
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20-fold. We confirmed APC2 depletion by APC2 antibody staining APC2d40 APC1Q8 clones; perdurant wild-type protein plus levels of truncated APC2d40 protein were reduced to background levels (Fig. S1, compare B with C). APC1 protein accumulates at low levels in NBs and high levels in neuronal axons; this staining is abolished in APC1Q8 mutants (Akong et al., 2002). To confirm that residual wild-type APC1 was depleted in APC2g10 APC1Q8 clones, we stained these with APC1 antibody (Hayashi et al., 1997). Staining was reduced to background levels (Fig. S2), which was especially apparent in CB neuron axons where staining is usually prominent. 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).
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APC mutant mammalian cells exhibit cell cycle alterations, although studies differ on whether anaphase onset is delayed (Draviam et al., 2006) or occurs prematurely (Dikovskaya et al., 2007). Thus, we explored cell cycle length in APC double mutant CB NBs. In APC2g10 APC1Q8 double null NBs, the cell cycle and mitosis duration were significantly prolonged (Table I), mimicking delays seen in some mammalian cells after APC loss (Draviam et al., 2006). APC2d40 APC1Q8 NBs had subtle changes in cell cycle length, whereas mitosis duration was slightly increased, consistent with this allele's hypomorphic nature (Table I). Thus, CB NBs completely lacking APC function have defects in cell cycle progression and spindle regulation, but these are not severe enough to disrupt asymmetric division.
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APCs are not essential for axon outgrowth or targeting in other larval neurons
APCs are also suggested to regulate growth cone guidance in culture (Koester et al., 2007). Thus, we tested whether APCs play general roles in this process. One disadvantage of CB NBs is that there are 100 CB NBs and neuronal progeny of each have distinct axon projection patterns (Pereanu and Hartenstein, 2006), making it very difficult to assess precision of axon targeting in clonally derived mutant neurons. Thus, we turned to two other larval neuronal populations with stereotyped axon projection patterns. We first examined lobular plug neurons, which have cell bodies at the lateral edge of the optic lobe and axons projecting medially to the laminar neuropil (Fig. 3 A, right). Clonal lobular neurons double null for both APCs had axonal projections indistinguishable from control wild-type clones; they polarized and sent out axons that projected medially to the neuropil (Fig. 3, K–M). Thus, APCs are not essential for axon outgrowth or guidance decisions of these neurons with relatively simple axon trajectories.
Our final test used some of the best characterized larval neurons, the mushroom body (MB; Heisenberg, 2003). They have highly reproducible axon and dendrite projections with fasciculated axons that first project ventrally and then turn and bifurcate in separate dorsal and medial projections (we visualized MBs using the MB-specific GAL4201Y driver; Fig. 4, A, B, E, and F; Yang et al., 1995). We generated control and APC2g10 APC1Q8 double null mutant clones and specifically visualized clones arising in MBs (see Materials and methods). In control clones, MB axon projections were normal (n = 12; Fig. 4, C and G). In APC2g10 APC1Q8 double null mutant clones, MB neurons differentiated, polarized, and sent out axons and dendrites normally. 25/26 clones had normal axon architecture (Fig. 4, D and H). One clone made an axon outgrowth error (Fig. 4, I and I'), which may reflect a modest role for APCs in this complex guidance decision or may be a random error unrelated to APC.
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Together, our data provide the first direct test of the hypothesis that APC is a universal mediator of symmetric versus asymmetric divisions or axon polarization and outgrowth in vivo. APCs play key roles in asymmetric divisions in male germline stem cells (Yamashita et al., 2003). RNAi in the embryonic ectoderm also suggested a role in division symmetry (Lu et al., 2001), although a subsequent loss of function study called this into question (McCartney et al., 2006). CB NBs do not require APCs for division asymmetry or persistent spindle orientation despite APC2's striking asymmetric localization. APC-null cells also undergo asymmetric divisions in the larval medulla (Hayden et al., 2007), whose architecture is epithelial and thus more like the mammalian cortex. Thus, APCs do not play a key instructive role in matching cortical polarity to spindle cues in CB NBs. We cannot rule out a supporting role in CB NBs, which helps to ensure extremely high fidelity asymmetric division, perhaps acting in parallel with other pathways like those mediated by Mud or the apical polarity cues. Perhaps in different tissues, different pathways work additively but the contributions of each vary. Clearly, in the male germline, loss of APCs disrupts the fidelity of asymmetric division, but even in that tissue APC mutant spindles are mispositioned in only a subset of cells (Yamashita et al., 2003). This could be tested by double mutant analysis.
APC loss did prolong the cell cycle, as occurs in mammalian cells (Draviam et al., 2006), but this delay did not prevent successful completion of mitosis. Spindle structure was also affected with an increased frequency of bent spindles, but these defects did not disrupt asymmetric division.
We next tested the hypothesis that APCs are key effectors of neuronal polarity, axon outgrowth, and growth cone guidance (Shi et al., 2004; Zhou et al., 2004; Votin et al., 2005; Koester et al., 2007). However, in three different neuronal populations, axon outgrowth and fasciculation were normal in APC's absence, and in lobular plug and MB neurons, axon targeting was also unaffected. These data compellingly demonstrate that APCs do not play conserved roles in axon polarization and outgrowth in all neurons.
How do we explain the discrepancies between our and earlier data? First, APCs may not play a universal role in neuronal polarity in either flies or mammals, with effects in hippocampal neurons the result of methods used to disrupt APC function. Because GSK3 has many substrates, including MAPs like Tau, GSK3 manipulation may affect substrates other than APC. Misexpression of APC fragments may sequester binding partners (e.g., GSK3, ASEF, and KIF3) with important roles in cytoskeletal regulation, blocking polarization even if APCs themselves play no essential role. Consistent with this, truncated APC2 has dominant-negative effects on cytoskeletal events in early embryos in which APC2 plays only a modest role (McCartney et al., 2006). Second, effects in hippocampal neurons could reflect APC's role in Wnt signaling rather than a direct role in polarization. Wnts regulate axon outgrowth in C. elegans, flies, and mammals (Yoshikawa et al., 2003; Lu et al., 2004; Hayden et al., 2007; Silhankova and Korswagen, 2007). Perhaps Wnts regulate hippocampal axon outgrowth. Interestingly, while our manuscript was under review, a study appeared suggesting that Wnt signals regulate growth cone structure and migration in cultured dorsal root ganglion neurons, perhaps via changes in APC localization (Purro et al., 2008). Of course, it is possible that machinery regulating neuronal polarity differs between flies and mammals, although this is not the case for epithelial polarity (for review see Goldstein and Macara, 2007).
These possibilities can now be tested in mice. Conditional APC knockout in the developing brain would directly test whether APC is critical for axon outgrowth. Furthermore, if effects are seen, one can test whether these are mediated by Wnt signaling using activated β-cat and dominant-negative TCF (T cell factor) to activate or block Wnt signaling. Similar work revealed that APC's role in chromosome instability is mediated, at least in part, via Wnt signaling (Aoki et al., 2007). Our results suggest that these experiments should be a high priority for future research.
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Materials and methods |
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Live cell imaging of NBs
Crawling third instar larvae were dissected in Schneider's Drosophila medium (Invitrogen). The entire brain was explanted and placed in a 40-µl drop of Schneider's Drosophila medium in the middle of a gas-permeable membrane (petriPERM; Sigma-Aldrich). This drop was surrounded by four drops of Halocarbon oil 700 (Halocarbon Products Corp.) to form the shape of the five on dice. A #1.5 glass coverslip was placed on top of the five drops. A kimwipe was used to wick away oil and media from the edge of the coverslip until the coverslip came in full contact with the brain. The brain was left intact and was not allowed to explode under the weight of the coverslip.
Samples were imaged using a spinning-disk confocal (Yokogawa; PerkinElmer) mounted on a microscope (Eclipse TE300; Nikon), which is equipped with an interline transfer-cooled charge-coupled device camera (ORCA-ER; Hamamatsu Photonics), a z-focus motor (Prior Scientific), an excitation and emission wheel controlled by the Lambda 10–2 controller (Sutter Instrument Co.), and emission filters (Semrock). 100x 1.4 NA, 60x 1.4 NA, and 40x oil 1.3 NA objectives were used. All microscope hardware was controlled by MetaMorph (MDS Analytical Technologies). All images were processed for brightness and contrast and prepared for publication using Photoshop (CS3; Adobe Systems, Inc.).
Immunostaining
Brains were fixed in 3.7% formaldehyde in PBS for 15 min, blocked in 1% normal goat serum for 3 h, and stained in a microcentrifuge tube in primary antibody and 1% normal goat serum in PTA (PBS + 0.1% Tween 20 + 0.2 g/liter sodium azide) overnight at 4°C. Brains were washed and incubated in secondary antibodies for 2 h at RT. The following antibodies were used: E7 mouse anti–
-tubulin (1:250; Developmental Studies Hybridoma Bank [DSHB]), DCAD2 rat monoclonal anti–DE-cadherin (1:200; DSHB), N271A mouse anti-Arm monoclonal (1:200; DSHB), rat polyclonal anti-APC2 (1:1,000; McCartney et al., 2006), rabbit anti-APC1 (1:500; Y. Ahmed, Dartmouth Medical School, Hanover, NH; Hayashi et al., 1997), rabbit anti-Miranda (1:2,000; F. Matsuzaki, RIKEN, Kobe, Japan; Ikeshima-Kataoka et al., 1997), rabbit anti-aPKC (1:2,000; Santa Cruz Biotechnology, Inc.), and Alexa-phalloidin (1:500; Invitrogen). Secondary antibodies were Alexa 488 or 546 (Invitrogen) and were used at a final concentration of 1:500. Images were acquired on the same system as mentioned in the Live cell imaging of NBs section.
Statistical methods
A Fisher's exact test was used to analyze the mutant bent spindle phenotype. An unpaired two-sample Student's t test was used to compare cell cycle and mitosis durations.
Online supplemental material
Fig. S1 and Fig. S2 present data that APC double mutant clones show only background levels of APC2 and APC1 staining, respectively. Fig. S3 shows that APC mutant NBs exhibit normal polarity. Table S1 shows NB asymmetric and persistent division. Video 1 shows a wild-type NB clone undergoing asymmetric division. Video 2 shows a APC2g10 APC1Q8 mutant NB undergoing asymmetric division. Video 3 shows an APC2d40 APC1Q8 mutant NB undergoing asymmetric division. Video 4 shows a control NB clone through two rounds of mitosis showing a persistent division axis. Video 5 shows an APC2d40 APC1Q8 mutant NB through two rounds of mitosis showing a persistent division axis. Video 6 shows an APC2g10 APC1Q8 mutant NB through two rounds of mitosis showing a persistent division axis. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200807079/DC1.
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Acknowledgments |
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N.M. Rusan is supported by American Cancer Society grant PF-06-108-CCG. This work was supported by National Institutes of Health grant GM67236.
Submitted: 14 July 2008
Accepted: 12 September 2008
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