The expression pattern during Drosophila development of a new adenomatous polyposis coli (APC) homolog called E-APC was examined. E-APC protein is expressed in all embryonic and larval cells. In the early blastoderm embryo, a striking concentration of E-APC is seen in the cortical actin caps. Microtubules are closely associated with these caps. Since human APC has been reported to bind to microtubules, an investigation was carried out to see whether the cortical E-APC co-localizes with tubulin. However, this is not the case, implying that the putative tubulin-binding property of human APC is not well conserved (Yu, 1999).

The most striking subcellular localization of E-APC is seen during the syncytial blastoderm. At this early embryonic stage, E-APC is associated with the cortical actin caps that lie above each nucleus. Staining with phalloidin visualizes the filamentous actin in these caps, which also contain membrane-associated antigens such as phosphotyrosine proteins, spectrin and E-cadherin. E-APC staining in the caps is remarkably punctate. It does not correspond precisely to phalloidin staining: the latter is seen at the periphery of the embryonic cortex, whereas the former is associated with the inward-facing cytoplasmic side of the actin meshwork. Some E-APC staining is also seen in the cytoplasm and around the nuclear envelopes. Cortical actin caps lie above the dividing mitotic spindles, and their formation and positioning is thought to depend on the centrosomes and microtubules that lie underneath them (Yu, 1999).

The ability of Drosophila Apc to down-regulate beta-catenin levels in a mammalian cell line raised the possibility that Apc might regulate its Drosophila homolog Armadillo in vivo. The best-characterized modulation in Arm levels occurs during embryonic development, when Arm protein accumulates in a striped pattern in response to expression of the segment polarity gene wingless. To address whether Apc might be a component of that system, Apc expression was examined during stages when Arm localizes in a striped pattern. When embryos collected from parents heterozygous for a deficiency covering Apc are hybridized with probes for APC mRNA, all preblastoderm stages show large amounts of RNA, likely derived from the wild-type allele present during oogenesis in the heterozygous mother and stored in the egg. Transcripts begin to decline at the syncytial blastoderm stage, and little RNA is detected in embryos by the end of cellularization. This condition persists through gastrulation and the extended germ-band stages when wg is first transcribed and Arm proteins are expressed in the striped pattern. APC transcripts are observed only after the completion of germ-band shortening, but not seen in Apc null embryos. This late zygotic transcription is concentrated in the CNS, although some RNA is detected in epidermis after stage 13. If Apc protein plays a role in Wg mediated Arm regulation, the absence of detectable APC mRNA during extended germ-band stages would require that any Apc protein be derived from the maternal RNA, which is degraded in late cleavage embryos. However, little antiserum detected protein is observed in wild-type embryos until germ-band shortening. At this point, the protein distribution parallels the distribution of zygotic APC transcripts and is highly concentrated in the nervous system. Because little Apc protein is detected in stages prior to shortening, the maternally supplied RNAs observed during cleavage do not appear to contribute a significant amount of protein to the embryo. Failure to detect Apc protein at stages when Wg and Arm protein localize in a striped pattern suggests that Apc may not play a role in Arm regulation at those stages (Hayashi, 1997).

During the late stages of embryogenesis, Apc is expressed in the CNS. The RNA expression is first seen slightly after the onset of germ-band shortening and its level increases as germ-band shortening proceeds. High levels of RNA persist at least until cuticle formation. No significant expression is observed in neuroblasts; instead, the expression is restricted to the postmitotic population in the CNS. This result is similar to that observed in the rat, where APC is expressed in postmitotic neurons in the CNS. The Apc protein derived from Drosophila zygotic expression localizes in axon fiber tracts and motor neurons. When staining is first observed, the levels are similar in both commissural and longitudinal tracts, but after completion of germ-band shortening, staining is significantly more intense in the longitudinal tracts. Within the tracts the pattern is not regular but appears distributed in patches or streaks within specific fibers. This fibrous pattern contrasts with the more uniform distributions of other CNS markers, such as Arm, tubulin, and horseradish peroxidase. In double-immunofluorescence staining for Apc and Arm, the two proteins show a general colocalization. Highest levels of Apc, however, are not associated with high levels of Arm. A close association is observed in the fiber tracts between Apc and tubulin staining, but again colocalization is not complete. A possible interaction between Apc and Arm is supported by the late expression of both proteins in the CNS. If Apc down-regulates Arm protein, one might expect to see relatively high levels of Arm protein where the Apc protein level is low and vice versa. The generally uniform Arm expression argues that such regulation would have to be very transient, if it exists at all. Very high levels of the Arm protein were not observed in the axon fiber tracts of Apc null embryos. The high level of Apc RNA expression in the Drosophila CNS parallels similar observations in the rat. In both organisms, expression of APC appears to be restricted to postmitotic neurons. In Drosophila the pattern of APC expression in fiber tracts changes with time. APC protein can be observed clearly in the commissural fibers of stage 13 embryos, but it does not increase as the CNS develops. This contrasts with the increases in commissural staining levels observed with antibodies to Arm, tubulin, and horseradish peroxidase, and with the increases in Apc observed in the longitudinal fiber tracts during the same period (Hayashi, 1997).

Given the late timing of its expression, a role for Apc in the CNS would be temporally distinct from any well characterized process involving wingless signaling. In Drosophila CNS development, wg functions largely in the specification of neuroblasts, at stages much earlier than the postmitotic stages when Apc is expressed. One other Wnt family gene (D-wnt3) is expressed in the CNS at late stages. However, it does not appear to regulate Arm, at least not in an in vitro tissue culture system (Fradkin, 1995) and thus, would be a poor candidate for countering an Apc-based affect on the Arm protein. Thus, Wg and Apc might have distinct functions in CNS development as well (Hayashi, 1997).


In the larval eye disc, Arm is present at the apical junctions of all epithelial cells but concentrated at the cell surface at points of photoreceptor contact, thus appearing in a star pattern, reflecting lack of cytoplasmic staining and predominance of junctional staining. Apc is found apically in the cytoplasm of photoreceptor cells, but not at the junctional perifery. In the pupal disc, while the contacts between photoreceptors extend nearly to the neighboring ommatidia, both Arm and Apc relocate to those points of neuronal contact at the very center of the ommatidium. While Apc and Arm are both found apically within photoreceptor cells, there is no detectable overlap in their distribution. Arm appears to surround regions of Apc localization closely. Although biochemical experiments reveal that Apc directly interacts with Arm (Hayashi, 1997), there is no detectable overlap in their distribution within photoreceptors. To explain this, it is hypothesized that Apc might function to regulate Arm levels negatively, such that at those places in the cell where Apc levels are high, Arm levels would be low (Ahmed, 1998).

APC2 and APC1 have overlapping roles in the larval brain despite their distinct intracellular localizations

The tumor suppressor APC and its homologs, first identified for a role in colon cancer, negatively regulate Wnt signaling in both oncogenesis and normal development, and play Wnt-independent roles in cytoskeletal regulation. Both Drosophila and mammals have two APC family members. The functions of the Drosophila APCs is further explored using the larval brain as a model. Both proteins are expressed in the brain. APC2 has a highly dynamic, asymmetric localization through the larval neuroblast cell cycle relative to known mediators of embryonic neuroblast asymmetric divisions. Adherens junction proteins also are asymmetrically localized in neuroblasts. In addition they accumulate with APC2 and APC1 in nerves formed by axons of the progeny of each neuroblast-ganglion mother cell cluster. APC2 and APC1 localize to very different places when expressed in the larval brain: APC2 localizes to the cell cortex and APC1 to centrosomes and microtubules. Despite this, they play redundant roles in the brain; while each single mutant is normal, the zygotic double mutant has severely reduced numbers of larval neuroblasts. These experiments suggest that this does not result from misregulation of Wg signaling, and thus may involve the cytoskeletal or adhesive roles of APC proteins (Akong, 2002).

APC2 localizes asymmetrically in larval neuroblasts. This study focused on central brain neuroblasts, which are located medially to the proliferation centers of the optic lobes. Neuroblast divisions are asymmetric in size and fate, with the larger daughter remaining a neuroblast and the smaller daughter becoming a ganglion mother cell (GMC). Each central brain neuroblast divides a series of times to produce a 'cap' of GMCs that remain joined to the mother, and divide mitotically themselves (Akong, 2002).

To characterize APC2 dynamic localization during the cell cycle, two parallel approaches were taken. Immunofluorescence and confocal microscopy were used to colocalize APC2, microtubules, and mitotic DNA (via the phosphohistone H3 epitope) in fixed tissue, and an APC2-GFP fusion under the control of the GAL4-UAS system was used; APC2 was driven in a subset of neuroblasts by prospero-GAL4 (pros-GAL4). The localization of APC2 revealed by these approaches is quite similar, though not identical. APC2-GFP accumulated somewhat more uniformly around the cortical circumference; accumulation was present at higher levels in GMCs; it is suspected that these differences reflect the elevated levels of APC2-GFP, although they could be due to localization differences between the GFP fusion and endogenous APC2. Attempts were made to acquire images from neuroblasts that were dividing in the apical/basal plane (Akong, 2002).

During interphase, APC2 forms a strong asymmetric crescent, with APC2 accumulating at the border between the neuroblast and its previous daughters; this region is where the next daughter will be born. The APC2 crescent remains strong through prophase, as the centrosomes separate and begin to set up the spindle. The crescent becomes less pronounced at metaphase. The orientation of the neuroblast spindle at metaphase determines where the GMC is born. Two different relationships were observed between the cortical APC2 crescent and the mitotic spindle, which were present in approximately equal numbers. In about half of the neuroblasts, the forming mitotic spindle was directed toward the center of the APC2 crescent. In the other neuroblasts, the forming mitotic spindle was directed toward one edge of the crescent. The difference was first apparent during late prophase, and continued through metaphase and anaphase. The reason for this difference in APC2 localization in different neuroblasts remains to be determined. One speculative possibility is that the relationship of GMC birth position to the APC2 crescent differs depending on how many GMCs have already been born. The new GMC daughter is usually born adjacent to one of the earlier daughters. At early stages, when there are relatively few GMC daughters, the spindle may be directed toward the middle of the APC2 crescent. In later divisions, when there are more GMC daughters, the new GMC may be born at the edge of the APC2 crescent, which is found at the interface between the neuroblast and the cap of previous GMC daughters (Akong, 2002).

As neuroblasts enter anaphase, APC2 remains cortical. In neuroblasts where the spindle is pointed toward the center of the APC2 crescent, APC2 surrounds the budding daughter. In cells in which the spindle is directed toward the edge of the crescent, APC2 localizes along one side of the GMC. As cytokinesis begins, APC2 localizes prominently to the cleavage furrow in all cases. This enrichment is even more prominent at the end of cytokinesis; at times, the cleavage furrow localization resolved into an apparent double ring. APC2 remains enriched at the division site after cytokinesis, marking the spot where the last daughter was born, and it only gradually reexpands to the interface between the neuroblast and all of its GMC daughters (Akong, 2002).

The live imaging of APC2-GFP also provided a glimpse of the timing of neuroblast cell cycles. Mitosis lasts ~15-20 min. In two cases, a single neuroblast that divided twice was observed, allowing an assessment of the minimum cell cycle length. In these cases, the two mitoses were completed in 106-120 min. This is comparable to the cell cycle length of embryonic neuroblasts (about 50 min), and fits previous estimates of neuroblast cell cycle length from BrdU-labeling experiments. Other neuroblasts within the same brain, however, divided only once, while still others did not divide at all during the ~2.5 h of the movie (Akong, 2002).

One striking feature of the asymmetric localization of APC2 is that it is present throughout the cell cycle and is particularly strong during interphase. During embryonic neuroblast divisions, most asymmetric markers are localized only during mitosis. However, less is known about their localization in larval neuroblasts. Several asymmetric markers in larval neuroblasts were examined, and their localization was compared with that of APC2. In embryonic neuroblasts, the transcription factor Prospero (Pros) and its mRNA are GMC determinants that are asymmetrically localized to the GMC daughter. Pros protein then becomes nuclear and helps direct cell fate. In larval neuroblasts, a similar localization is observed. Pros is not detectable in interphase neuroblasts, when the cortical APC2 crescent is strongest. A small amount of Pros transiently localizes to an asymmetric crescent during mitosis. Pros is present at low levels in GMC nuclei and at higher levels in the nuclei of ganglion cells (Akong, 2002).

Mira is basally localized in embryonic neuroblasts, and required there for localization of Pros protein and mRNA. In central brain neuroblasts, Mira is diffusely cytoplasmic during interphase, when the APC2 crescent is the strongest. As cells enter mitosis, Mira first becomes cortical and then begins to accumulate asymmetrically on the side of the neuroblast where the daughter will be born. By metaphase, Mira asymmetry is very pronounced. The center of the Mira crescent is always precisely aligned with one spindle pole. As a result, in cells with the spindle pointing toward the center of the APC2 crescent, the Mira and APC2 crescents substantially overlap, while in cells in which the spindle points to the edge of the APC2 crescent, the two crescents are offset. Mira is partitioned into the GMC during anaphase, while APC2 relocalizes to the cleavage furrow. Mira could still be detected in some GMCs, which are thought to be those that were recently born (Akong, 2002).

In contrast to Mira and Pros, Inscuteable (Insc) and Bazooka (Baz) localize to the apical sides of embryonic neuroblasts, where they play essential roles in asymmetric divisions. Insc is asymmetrically localized in larval neuroblasts. Insc localizes to the side of the neuroblast opposite that of APC2 through much, if not all, of the cell cycle. Interestingly, there is a weak Insc crescent during interphase, that becomes stronger through prophase and metaphase. During anaphase, Insc localizes to the neuroblast cortex but not the GMC daughter. Baz localization was similar to that of Insc, though no cortical localization during interphase was detected. During prophase and metaphase, Baz localizes to a crescent opposite APC2, and as the chromosomes begin to separate, Baz localizes to a tight cap opposite the future GMC. Together, these data confirm that larval and embryonic neuroblasts asymmetrically localize many of the same proteins, and that APC2 localizes on the GMC side (basal) of the neuroblast, overlapping Mira and opposite Baz and Insc, which localize apically (Akong, 2002).

Arm also localizes asymmetrically in neuroblasts. Extending this, an examination was made of the localization of Arm's adherens junction partners DE-cadherin and ß-catenin. When central brain neuroblasts undergo a sequential series of asymmetric divisions, the GMCs remain associated with their neuroblast mother, resulting in a cap of GMCs in association with each neuroblast. APC2 localizes strongly to the boundary between the neuroblast and each GMC, and more weakly to the borders between the GMCs. APC2 is present at lower levels in ganglion cells and differentiating neurons (Akong, 2002).

The adherens junction proteins DE-cadherin, Arm, and ß-catenin all show a striking and asymmetric localization pattern in central brain neuroblasts. All precisely colocalize both at the boundary between neuroblasts and GMCs and at the boundaries between GMCs. DE-cadherin, Arm, and ß-catenin are also all expressed in epithelial cells of the outer proliferation center. The localization of DE-cadherin and the catenins is consistent with the idea that cadherin-catenin-based adhesion could help ensure that GMCs remain associated with each other, via association with their neuroblast mother (Akong, 2002).

To further explore this, how successive GMCs are positioned relative to their older GMC sisters was examined using two different approaches. First Mira was used to mark the newborn GMCs and DE-cadherin was used to mark the neuroblast and all of her GMC daughters. Mira localizes to a crescent on the side of the neuroblast where the daughter will be born (basal side), and then is segregated into the daughter. Mira persists for some time in newborn GMCs, and it remains detectable in the other GMCs as well, thus allowing the position of newborn GMCs to be examined relative to their older sisters. In many cases, new GMCs are clearly born at the edge of the cluster of older GMCs. This is particularly striking in neuroblasts with many progeny. It is worth noting that the cluster of daughters is three-dimensional, comprising a 'cap' of daughters in three dimensions rather than a two-dimensional line of daughters. It is thus suspected that new daughters are born near the edge of this cap (Akong, 2002).

These data suggest that neuroblasts and their GMC progeny remain closely associated. The GMCs then divide to form ganglion cells and ultimately neurons. The data further suggest that these latter cells may also remain associated and send their axons together toward targets in the central brain. When sections were made more deeply into the brain, below each cluster of neuroblasts and GMCs, structures that appear to be axons were detected projecting from these groups of cells. These axons label with Arm, DE-cadherin, and APC1. Arm also localizes to the axons of the neuropil, while DE-cadherin and APC2 are present at low levels or are absent from this structure (Akong, 2002).

Despite its striking localization, APC2 is not essential for brain development. One possible explanation is that APC1 is also expressed in larval neuroblasts and plays a redundant role there. The localization and function of APC1 in brain development was examined. In embryos, APC1 is expressed in primordial germ cells and in CNS axons. Anti-APC1 antibody was used to examine its expression in the larval brain, using the null allele APC1Q8 as a negative control. APC1 accumulates at apparently low levels throughout the brain. It is largely diffuse throughout the cell, with slight cortical enrichment. There was a small amount of residual staining in the APC1 mutant, that might represent slight crossreactivity with APC2 (cross-reactivity is detectable in immunoblotting). There is a strong accumulation of APC1 in the axons emerging from clusters of neuroblast, GMCs, and their progeny. Together, these data indicate that low levels of APC1 accumulate in neuroblasts, GMCs, and their progeny, with higher levels found in axons projecting from these cells. Thus, APC1 could potentially compensate for loss of APC2 in the larval brain (Akong, 2002).

The cell biological properties of APC1 and APC2 were compared. The four known APC family members, two in flies and two in mammals, share certain structural features but differ in others. All share a block of N-terminal Arm repeats, followed by a set of short repeated sequences that serve as binding sites for Arm/ßcat and Axin. These regions comprise most of fly APC2, which is the shortest family member. Fly APC1 is significantly longer at its N and C termini. Its extended C-terminal region contains a sequence similar to the microtubule-binding domain of human APC (Akong, 2002).

Given these structural differences between fly APC1 and APC2, it was asked whether they would exhibit similar or distinct cell biological properties when both were expressed at equivalent levels. The GAL4-UAS system to overexpress each protein in the same cellular environments, and where each localized under these conditions was examined. When APC2-GFP was overexpressed, it localized asymmetrically to the cortex of the larval neuroblasts, as well as to the junctions between the GMCs and thus paralleled the localization of endogenous APC2. In contrast, when APC1 was overexpressed, it had a strikingly different localization. APC1 localized very strongly to the centrosome and the microtubules emanating from it. It also localized to interphase microtubule arrays in cells of the inner proliferation center. However, it did not strongly colocalize with all microtubule-based structures; for example, it was only marginally enriched in the spindle at metaphase. Similar localization experiments were carried out after mis-expression in the embryonic epidermis, with similar results; APC2 localizes to the cell cortex, while APC1 localizes to centrosomes and microtubules. Together, the data from brains and embryos suggest that sequence differences between APC1 and APC2 direct them to quite different intracellular locations, despite their strong similarity in the core region of the protein (Akong, 2002).

Mammalian APC protein can oligomerize, and thus whether fly APC1 and APC2 proteins might interact was examined. To begin to examine this, the effect of APC1 overexpression on the localization of endogenous and overexpressed APC2 was examined. This study suggested that APC1 and APC2 may interact, either directly or indirectly, allowing APC1 to recruit APC2 to a new location. APC2 may recruit APC1 to the cell cortex; however, this is subject to the caveat that the APC1 antibody may weakly cross-react with APC2 (Akong, 2002).

Given the striking localization of APC2 in the brain, it was surprising to find that APC2 mutants have no apparent brain defects. The brain is only one of several tissues where neither single mutant had any apparent phenotype; this was surprising as Wg signaling plays an important role in several of these tissues. The realization that APC1 is also expressed in the brain raised the possibility that it might play a partially redundant role in this tissue. To test this, the phenotype of animals mutant for both APC1 and APC2 was examined (Akong, 2002).

APC1 and APC2 single mutants are both zygotically viable to adulthood. The only known problem in APC1 mutants are morphological defects and inappropriate apoptosis in the photoreceptors of the eye, while APC2 zygotic mutants are morphologically wild type, with a phenotype only emerging in embryos maternally and zygotically mutant. In contrast, APC2;APC1 double mutants are zygotically lethal. Two allelic combinations, APC2deltaS;APC1Q8 and APC2g10;APC1Q8, die as second instar larvae, while the third, APC2d40;APC1Q8, dies primarily during the first larval instar. None of the double mutants had any apparent defects in segment polarity as first instar larvae. Mitotic activity during larval stages is restricted to the imaginal tissues and the brain, and imaginal discs are dispensable for larval development. Given this and the observed expression pattern of APC2 and APC1 in the CNS, brain development was examined in double mutant larvae (Akong, 2002).

During normal brain development, most embryonic neuroblasts exit the cell cycle during late embryogenesis. Immediately after hatching, the only mitotically active neuroblasts are the so-called mushroom-body neuroblasts, which are at a ventrolateral position. Eight hours after hatching, other neuroblasts become mitotically active, such that by 20 h after hatching, 20-30 central brain neuroblasts per hemisphere are dividing. During the early first instar, the cells of the optic anlage become epithelially arranged and also begin dividing. The number of proliferating neuroblasts continues to increase, plateauing 20-50 h after egg laying (Akong, 2002).

APC2deltaS;APC1Q8 zygotic double mutants die during the second larval instar. Wild-type and double mutant animals were compared immediately after they completed the second instar larval molt. Double mutant brains are essentially normal in size, and the optic anlage becomes epithelial. However, double mutants have a strikingly different pattern of neuroblast proliferation than wild type. While presumptive mushroom body neuroblasts continued to proliferate, the number of other mitotic neuroblasts is drastically reduced relative to wild type, as assessed both by phosphohistone H3-labeling and by Mira and Pros staining. A similar, though less drastic, block in mitotic activity was seen in APC2g10;APC1Q8. To determine whether DNA synthesis was initiated but mitosis blocked, larvae were labelled with BrdU throughout the first instar, identifying cells that replicated their DNA during this period. The results were similar to those seen with mitotic markers. APC2deltaS;APC1Q8 double mutants have drastically reduced numbers of BrdU-labeled cells; most labeled cells remaining appeared to be mushroom-body neuroblasts. It was next examined whether embryonic CNS development was altered, by examining the axonal scaffold produced by progeny of the embryonic neuroblasts, using the marker BP102 that labels all axons. Zygotic double mutants were distinguished from wild-type siblings by the presence or absence of APC1 in the CNS. The axonal scaffold was unaltered in APC2deltaS;APC1Q8 double mutants, suggesting that embryonic neuroblast proliferation is at least roughly normal. Finally, whether defects could be detected in asymmetric divisions in double mutants was examined, as assessed by examining asymmetric localization of Mira. In the wild-type first and second instar brain, the localization of Miranda is less strikingly asymmetric, with reasonably high levels in the cytoplasm. However, crescents of Mira could be detected and they localize to smaller daughters. In double mutants, many fewer Mira-positive neuroblasts were seen, but neuroblasts with Mira crescents or Mira segregated to smaller daughters were found. In addition, Prospero-positive daughters are found in the double mutants, though in reduced numbers (Akong, 2002).

These data thus suggest that asymmetric divisions still can occur, though it remains possible that they are not entirely normal or occasionally fail. Together, these data suggest that most likely explanation for the mutant phenotype is that double mutant embryonic neuroblasts do not reenter the cell cycle during the first larval instar (Akong, 2002).

APC1 and APC2 regulate Wg signaling, and thus one mechanism that could underlie the zygotic double mutant phenotype is the failure to properly regulate Arm levels. To test this, Arm accumulation was examined in zygotic double mutant embryos. Stage 16 double mutants were identified by the absence of APC1 accumulation in the CNS, and the level of Arm accumulation was examined relative to wild-type embryos. Arm accumulation appeared completely normal in the epidermis of APC2g10;APC1Q8 double mutant embryos, suggesting that maternally contributed protein from the two loci is sufficient for normal Arm regulation in the embryo (Akong, 2002).

Arm accumulation was examined in the brains of midfirst instar larvae. In the wild-type brain, Arm accumulates heavily in axons of the ventral nerve cord and neuropil. Arm accumulation in the cellular portion of the first instar brain is much lower. In the brain lobes, weak cortical staining of most cells was found. The only significant Arm accumulation outside the neuropil is in cells that are believed to be epithelial cells of the developing optic lobe. The accumulation of Arm in zygotic double mutant brains is not substantially elevated from that of wild-type. Arm levels in the neuroblasts and cell bodies are unchanged, while Arm levels in axons are similar or only slightly elevated. These data thus do not support the hypothesis that elevated Arm levels cause the phenotype (Akong, 2002).

These data demonstrate that APC1 and APC2 play redundant roles in larval brain development. Further, the data suggest that this role is Wg-independent, since it is not mimicked by elevating Arm levels. This contrasts with the situation in embryos and imaginal discs, where the two proteins also have overlapping functions but where these clearly involve Wg signaling. APC1 and APC2 have also been found to play redundant roles in cell adhesion during oogenesis. Maternal contribution of the two proteins appears sufficient for many if not all aspects of embryogenesis, since double mutant embryos hatch with a normal cuticle pattern and an apparently normal brain. However, striking differences between the larval brains of wild-type and double mutant animals were seen. During wild-type development, most neuroblasts become quiescent during late embryogenesis, with only the mushroom body neuroblasts mitotically active upon hatching. In the middle of the first instar, however, other embryonic neuroblasts reenter the cell cycle and proliferate. In the APC2;APC1 double mutant, this appears not to occur, as far fewer total neuroblasts are seen, and the number of brain cells in mitosis and the number that have gone through S-phase is substantially reduced (Akong, 2002).

EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells

Tissue-specific adult stem cells are commonly associated with local niche for their maintenance and function. In the adult Drosophila midgut, the surrounding visceral muscle maintains intestinal stem cells (ISCs) by stimulating Wingless (Wg) and JAK/STAT pathway activities, whereas cytokine production in mature enterocytes also induces ISC division and epithelial regeneration, especially in response to stress. This study shows that EGFR/Ras/ERK signaling is another important participant in promoting ISC maintenance and division in healthy intestine. The EGFR ligand Vein is specifically expressed in muscle cells and is important for ISC maintenance and proliferation. Two additional EGFR ligands, Spitz and Keren, function redundantly as possible autocrine signals to promote ISC maintenance and proliferation. Notably, over-activated EGFR signaling could partially replace Wg or JAK/STAT signaling for ISC maintenance and division, and vice versa. Moreover, although disrupting any single one of the three signaling pathways shows mild and progressive ISC loss over time, simultaneous disruption of them all leads to rapid and complete ISC elimination. Taken together, these data suggest that Drosophila midgut ISCs are maintained cooperatively by multiple signaling pathway activities and reinforce the notion that visceral muscle is a critical component of the ISC niche (Xu, 2011).

Adult stem cells commonly interact with special microenvironment for their maintenance and function. Many adult stem cells, best represented by germline stem cells in Drosophila and C. elegans, require one primary maintenance signal from the niche while additional signals may contribute to niche integrity. ISCs in the Drosophila midgut do not seem to fit into this model. Instead, they require cooperative interactions of three major signaling pathways, including EGFR, Wg and JAK/STAT signaling, for long-term maintenance. Importantly, Wg or JAK/STAT signaling over-activation is able to compensate for ISC maintenance and proliferation defects caused by EGFR signaling disruption, and vice versa. Therefore, ISCs could be governed by a robust mechanism, signaling pathways could compensate with each other to safeguard ISC maintenance. The mechanisms of the molecular interactions among these pathways in ISC maintenance remains to be investigated. In mammals, ISCs in the small intestine are primarily controlled by Wnt signaling pathways, and there are other ISC specific markers not controlled by Wnt signaling. In addition, mammalian ISCs in vitro strictly depend on both EGFR and Wnt signals, indicating that EGFR and Wnt signaling may also cooperatively control mammalian ISC fate. It is suggested that combinatory signaling control of stem cell maintenance could be a general mechanism for ISCs throughout evolution (Xu, 2011).

The involvement of EGFR signaling in Drosophila ISC regulation may bring out important implications to understanding of intestinal diseases, in which multiple signaling events could be involved. For example, in addition to Wnt signaling mutation, gain-of-function K-Ras mutations are frequently associated with colorectal cancers in humans. Moreover, activation of Wnt signaling caused by the loss of adenomatous polyposis coli (APC) in humans initiates intestinal adenoma, but its progression to carcinoma may require additional mutations. Interestingly, albeit controversial, Ras signaling activation is suggested to be essential for nuclear β-catenin localization, and for promoting adenoma to carcinoma transition. In the Drosophila midgut, loss of APC1/2 genes also leads to intestinal hyperplasia because of ISC overproliferation. Given that EGFR signaling is generally activated in ISCs, it would be interesting to determine the requirements of EGFR signaling activation in APC-loss-induced intestinal hyperplasia in Drosophila, which might provide insights into disease mechanisms in mammals and humans (Xu, 2011).

Previous studies suggest that intestinal VM structures the microenvironment for ISCs by producing Wg and Upd maintenance signals. This study identified Vn, an EGFR ligand, as another important ISC maintenance signal produced from the muscular niche. Therefore, ISCs are maintained by multiple signals produced from the muscular niche. In addition, Spi and Krn, two additional EGFR ligands, were identified that function redundantly as possible autocrine signals to regulate ISCs. These observations are consistent with a previous observation that paracrine and autocrine EGFR signaling regulates the proliferation of AMPs during larval stages, suggesting that this mechanism is continuously utilized to regulate adult ISCs for their maintenance and proliferation. The only difference is that the proliferation of AMP cells is unaffected when without autocrine Spi and Krn, due to redundant Vn signal from the VM, whereas autocrine Spi/Krn and paracrine Vn signals are all essential in adult intestine for normal ISC maintenance and proliferation. It was found that Vn and secreted form of Spi have similar roles in promoting ISC maintenance and activation, but additional regulatory or functional relationships among these ligands require further investigation, as the necessity of multiple EGFR ligands is still not completely understood. It is known that secreted/activated Spi and Krn are diffusible signals, but clonal analysis data show that Spi and Krn can display autonomous phenotypes. This observation indicates that these two ligands could behave as very short range signals in the intestinal epithelium, or they could diffuse over long distance but the effective levels of EGFR activation could only be achieved in cells where the ligands are produced. Interestingly, palmitoylation of Spi is shown to be important for restricting Spi diffusion in order to increase its local concentration required for its biological function. Whether such modification occurs in intestine is unknown, but it is speculated that Vn, Spi and Krn, along with the possibly modified forms, may have different EGFR activation levels or kinetics, and only with them together effective activation threshold could be reached and sustained in ISCs to control ISC behavior. Therefore, a working model is proposed that ISCs may require both paracrine and autocrine mechanisms in order to achieve appropriate EGFR signaling activation for ISC maintenance and proliferation.

Mechanisms of JAK/STAT signaling activation is rather complex. In addition to Upd expression from the VM, its expression could also be detected in epithelial cells with great variability in different reports, possibly due to variable culture conditions. Upon injury or pathogenic bacterial infection, damaged ECs and pre-ECs are able to produce extra cytokine signals, including Upd, Upd2 and Upd3, to activate JAK/STAT pathway in ISCs to promote ISC division and tissue regeneration. Several very recent studies suggest that EGFR signaling also mediates intestinal regeneration under those stress conditions in addition to its requirement for normal ISC proliferation. Therefore, in addition to basal paracrine and autocrine signaling mechanisms that maintain intestinal homeostasis under normal conditions, feedback regulations could be employed or enhanced under stress conditions to accelerate ISC division and epithelial regeneration (Xu, 2011).

Evidence so far has indicated a central role of N signaling in controlling ISC self-renewal. N is necessary and sufficient for ISC differentiation. In addition, the downstream transcriptional repressor Hairless is also necessary and sufficient for ISC self-renewal by preventing transcription of N targeting genes in ISCs. Therefore, N inhibition could be a central mechanism for ISC fate maintenance in Drosophila. High Dl expression in ISCs may lead to N inhibition, though how Dl expression is maintained in ISCs at the transcriptional level is not clear yet. Hyperactivation of EGFR, Wg or JAK/STAT signaling is able to induce extra Dl+ cells, suggesting that these three pathways might cooperatively promote Dl expression in ISCs. It is also possible that these pathways regulate Dl expression indirectly. As Dl-N could have an intrinsically regulatory loop for maintaining Dl expression and suppressing N activation, these pathways could indirectly regulate Dl expression by targeting any component within the regulatory loop. Identifying their respective target genes by these signaling pathways in ISCs would be an important starting point to address this question (Xu, 2011).

Effects of Mutation or Deletion

Arm regulation plays a direct role in the establishment of a segmental pattern for the embryonic cuticle. Failure to down-regulate Arm protein results in an uniform "naked cuticular" fate, whereas uniform low levels result in a homogenous lawn of denticles. To further investigate the function of Apc, the cuticle pattern and Arm protein expression in Apc homozygous null embryos was examined. Df(3R)3450 embryos lacking zygotic Apc show a normal Arm stripe pattern. When such embryos contain the duplication DpB152, they differentiate a normal cuticle pattern, even though they lack all zygotic APC. These observations are consistent with the view that zygotic Apc does not play a role in Arm regulation in ventral cuticle pattern formation (Hayashi, 1997).

The changing pattern of Apc protein expression during the CNS development suggests a possible role in the patterning of the fiber tracts. To test this possibility, the CNS development in Df(3R)3450 and DpB152 embryos was examined. Such embryos lack Apc and an undefined number of adjacent genes but form normal cuticles. Df(3R)3450 and DpB152 embryos show no dramatic morphological disorganization of the CNS. The only defect observed was in the longitudinal fiber tracts, which appear thinner and less developed than those of wild-type embryos at a similar stage. The specificity of the defects is consistent with the generally higher expression of Apc in longitudinal fibers at these stages. Therefore, loss of Apc may result in abnormalities or retardation of their development, although a role for other unidentified gene(s) normally present in the deleted region cannot be excluded (Hayashi, 1997).

Since Arm is a multifunctional protein, specific mutants of arm that allow a dissection of its distinct functions were examined in a Apc mutant background to delineate regions that are required in the induction of cell death. The armH8.6 mutant allele creates a truncation of Arm's carboxyl terminus that reduces Arm's ability to mediate Wingless signaling in vivo. Flies heterozygous for the armH8.6 allele were examined to determine whether deletion of the carboxyl terminus reduces Arm's ability to induce apoptosis in a Apc mutant background. Reduction of the wild-type gene dosage of arm by one-half due to the introduction of the null allele armYD35 rescues many photoreceptors from death and thereby dominantly suppresses the Apc mutant phenotype. In contrast, the mutant allele armH8.6 acts like a wild-type copy of the gene; all photoreceptors in all ommatidia degenerate completely. This unexpected finding suggests that the carboxyl terminus of Arm is not required for Arm's ability to induce photoreceptor death in the Apc mutant. A similar assay was utilized to analyze the requirement of other regions of Arm for the induction of cell death. A series of transgenes containing deletions within Arm have differential effects on Arm's roles in cell adhesion and Wingless signal transduction. These transgenes were expressed under control of the Arm promoter, and protein levels of these mutated forms of Arm were shown to be equivalent to wild-type Arm levels (Orsulic, 1996). Flies heterozygous for these mutated arm genes were examined to determine whether they retain the ability to induce apoptosis in a Apc mutant background. A series of deletions in Arm, termed S5, S15, and S12, eliminate Armadillo repeats 5, 8, and portions of 10 and 11, respectively. Each deletion severely disrupts Arm's ability to induce cell death in the Apc mutant. In contrast, a deletion in which Arm's alpha-catenin binding site is eliminated retains the ability to induce cell death. This finding is consistent with the results obtained by overexpression of stabilized Arm. Although this stabilized mutant Arm protein lacks amino acids required for binding to alpha-catenin, it retains the ability to induce photoreceptor death. Together, these findings suggest that while the carboxyl terminus and alpha-catenin binding site are dispensable for Arm-induced cell death, Arm's central repeats are required for this activity (Ahmed, 1998).

Inactivation of the Adenomatous Polyposis Coli tumor suppressor triggers the development of most colorectal carcinomas. APC is required for targeted degradation of ß-catenin, the central transcriptional activator in the Wnt/Wingless (Wg) signal transduction pathway; however, the precise biochemical functions of APC remain uncertain. The two Drosophila homologs of APC (Apc1 and Apc2) appear to have predominantly different tissue distributions, different subcellular localizations and mutually exclusive phenotypes upon inactivation. Unexpectedly, despite these differences, simultaneous reduction in both Drosophila Apc proteins results in the global nuclear accumulation of ß-catenin and the constitutive activation of Wg transduction throughout development. This redundancy extends even to functions previously thought to be specific to the individual Apc homologs. Together, these results reveal that the combined activity of Apc1 and Apc2 allows a tight regulation of transcriptional activation by ß-catenin and suggest that APC proteins are required for the regulation of Wnt transduction in all cells (Ahmed, 2002).

The in vivo analyses of loss-of-function mutations in the two Drosophila homologs of Apc have been crucial in providing conclusive evidence that transcriptional transactivation by ß-catenin can in fact be negatively regulated by APC. However, previous studies using loss-of-function mutations in either of the two Drosophila Apc genes have failed to establish an absolute requirement for Apc in regulating Wg signaling throughout development, since many Wg transduction events proceed normally, particularly during post-embryonic stages. These findings raised questions as to whether Apc is required to prevent the constitutive activation of Wg transduction in only a subset of cells, and whether Apc function could be compensated for by other mechanisms elsewhere. Simultaneously reducing the activities of both Drosophila Apc proteins is reported in this study. An absolute requirement is found for Apc proteins in preventing the constitutive activation of Wg signaling in many epithelial cells throughout development. In those limited situations for which the inactivation of one of the two Drosophila Apc proteins does lead to hyperactivation of transcriptional activation by Arm, the other Apc protein can functionally substitute if provided in sufficient quantity. This result argues against a specific function for either Apc protein in regulating Wg transduction (Ahmed, 2002).

Apc1 is highly (though not exclusively) expressed in neurons, while Apc2 is highly (though not exclusively) expressed in most epithelial cells, leading to the proposal that the two Apc proteins function in a tissue-specific manner. The data presented here argue against a tissue-specific division in Apc expression or function. The dramatic and global constitutive activation of Wg transduction that is revealed only by simultaneous reduction in both Drosophila Apc proteins demonstrates that both Apc proteins are found and function in many tissues that are not restricted by cell type or developmental stage (Ahmed, 2002).

These results reveal that the combined activity of Apc1 and Apc2 within the same cell enables these two proteins to tightly regulate Arm levels. Thus, specific phenotypes that are found upon inactivation of either Apc1 or Apc2 singly (leading to cell death in pupal retinal neurons and cell fate transformation in the embryonic epidermis, respectively) denote the relatively rare situations in which the activity of one of the two Apc proteins is not sufficient to compensate for reduction in the other. The data reveal that even in the embryonic epidermis, Apc1 and Apc2 function to prevent the ectopic activation of Wg transduction. When Apc2 activity is reduced, ectopic Wg transduction is very sensitive to the dose of Apc1, since cutting the wild-type dose of Apc1 in half either maternally or zygotically has dramatic effects. In this tissue, Apc1 has a subsidiary role though, and the normal levels of Apc1 are not sufficient to compensate for Apc2 loss. These data, coupled with the rescue of Apc2 reduction by Apc1 overexpression, suggest that the absolute levels of Apc1 and Apc2 are important in enabling the two Apc proteins to compensate for each other (Ahmed, 2002).

It was not possible to determine whether the converse situation is also true -- whether reducing levels of endogenous Apc2 would exacerbate defects resulting from mutations in Apc1, because a hypomorphic allele of Apc1 to use as a sensitized background for genetic interaction tests is unavailable. Retinal neuronal apoptosis is exquisitely sensitive to total Apc2 activity, since increasing the dose of Apc2 by only one copy is sufficient to prevent apoptosis in the Apc1 mutant. Together, these data suggest that the absolute levels, or total 'dose' of intracellular Apc1 and Apc2 is important in preventing the hyperactivation of Arm. Whether the dose sensitivity that is revealed in these situations reflects differences not only in total levels, but also in the relative binding affinities of the two Apc proteins for Arm, Axin or Zw3 remains to be investigated (Ahmed, 2002).

The functional redundancy in the Apc proteins suggests that the C-terminal half of Apc1 might not be required for targeted degradation of Arm, since this region of the protein is completely lacking in Apc2. However, this region of Apc1 might be important in previously proposed roles for APC that might be independent from ß-catenin degradation. These include the alteration of cell migration through regulation of the actin cytoskeleton, the planar positioning of mitotic spindles with respect to the polarized epithelial cell membrane, and in kinetochore-microtubule attachment. While the data demonstrate that both Drosophila Apc proteins function in the regulation of Wg transduction, further analysis employing the Apc1;Apc2 double mutant will be required to address their possible redundancy in functions that are independent of ß-catenin degradation (Ahmed, 2002).

Is there an absolute requirement for APC in the targeting of ß-catenin to a degradation pathway? In cell culture experiments, overexpressed Axin is able to downregulate ß-catenin levels even in cells that lack wild-type APC. Furthermore, even after deletion of its RGS domain, which is required for the interaction of Axin with APC, overexpressed Axin is still able to induce the degradation of ß-catenin. These data have led to the hypothesis that APC may facilitate, but not be absolutely necessary for, the Axin-mediated degradation of ß-catenin. If APC were to merely facilitate Axin mediated degradation of ß-catenin, it would be expected that phenotypes found upon reduction in APC would not be as severe as those found upon inactivation of Axin, since residual Axin-mediated degradation of ß-catenin would persist in the absence of APC. Instead, it is found that inactivation of APC results in phenotypes that completely mimic inactivation of Axin, with respect to both their scope and their severity. These data argue against a secondary role for APC in the degradation of ß-catenin, and provide in vivo evidence for an absolute requirement for APC in preventing the constitutive activation of Wg transduction in virtually all epithelial cells (Ahmed, 2002).

Human APC has been found to shuttle between the nucleus and cytoplasm. Nuclear export of human APC is dependent on both nuclear export sequences within APC and on the CRM1 export receptor. Treatment of cells in culture with the CRM1-specific export inhibitor leptomycin B results in the nuclear accumulation of APC, as well as the nuclear accumulation of ß-catenin. These findings have led to the proposal that APC is required for the nuclear export of ß-catenin. However this hypothesis must be reconciled with studies employing oocytes and semipermeabilized cultured cells to investigate ß-catenin export, which reveal that ß-catenin can be exported from the nucleus in a manner that is independent of the CRM1 pathway and independent of APC (Ahmed, 2002).

In epithelial cells that lack both wild-type Drosophila Apc1 and Apc2, Arm accumulates within the nucleus. Nuclear accumulation of Arm is found only in the Apc1Q8;Apc2d40 maternal/zygotic double mutant, and occurs during gastrulation. The nuclear localization of Arm, and the temporal pattern of the nuclear accumulation of Arm in the absence of wild-type Apc1 and Apc2, is similar to that seen upon inactivation of Axin, and in contrast to that seen upon inactivation of Zw3, in which the increased levels of Arm appear uniformly dispersed between nucleus and cytoplasm. These data are therefore completely consistent with the model that there is a second role for APC in the nuclear export of ß-catenin, in addition to the role APC serves in the targeting of ß-catenin to degradation (Ahmed, 2002).

However, an alternate model for APC function in Arm localization incorporates three observations: (1) a similar temporal pattern of nuclear Arm accumulation is seen in Axin mutants and in Apc1;Apc2 double mutants; (2) the interaction of Axin with ß-catenin is critically dependent on APC, and (3) ß-catenin is freely diffusible from nucleus to cytosol. In this model, an Axin/APC complex would serve as a cytoplasmic anchor for ß-catenin and would dictate, in part, the steady-state subcellular localization of ß-catenin. Axin would serve as the primary cytoplasmic anchor for ß-catenin, but its physical interaction with ß-catenin would be greatly enhanced by APC. The elimination of either Axin or APC, or their functional inactivation in the presence of Wg transduction, would not only increase the total levels of ß-catenin, but would also shift the steady state localization of ß-catenin to the nucleus. While further experiments will be necessary to distinguish between roles for APC in the nuclear export and/or cytoplasmic anchoring of ß-catenin, these data suggest that together, APC and Axin exercise two levels of control of ß-catenin activity: APC and Axin not only initiate the destruction of ß-catenin, but also modulate the ability of ß-catenin to accumulate in the nucleus where it can serve as a transcriptional activator (Ahmed, 2002).

The results of this study reveal an absolute requirement for APC in the targeting of ß-catenin for destruction and may have implications for the function of the human APC proteins in the regulation of Wnt transduction. In mouse and humans, as in Drosophila, there are two known APC homologs: APC and APC2/APCL. The mammalian APC homologs are expressed at high levels in the nervous system, with lower levels in many other tissues analyzed. Although human APC is widely expressed, germline mutations in APC result in a relatively narrow spectrum of disease. This includes the development of adenoma in the gastric and small and large bowel epithelia, as well as osteomas, desmoid fibromatosis, and lesions in retinal neurons and pigment epithelium. While hyperactivating mutations in ß-catenin are also associated with colonic carcinoma and desmoid fibromatosis, these hyperactivating mutations have been found in several carcinomas that are not detected in people with germline mutations in APC. Several scenarios could account for this discrepancy in sites of disease induced by APC loss versus ß-catenin hyperactivation. Perhaps human APC has a key role in controlling the degradation of ß-catenin in only a subset of epithelial tissues. Alternatively, in a manner directly analogous to that found for the two Drosophila Apc proteins, inactivation of one human APC homolog might be compensated for by the activity of the other in most tissues. Homozygous inactivation of human APC would induce disease states in only those tissues in which APC, rather than APC2, is the predominantly expressed gene, and would be dependent on the absolute levels of the two APC proteins in any given cell (Ahmed, 2002).

Testing hypotheses for the functions of APC family proteins using null and truncation alleles in Drosophila

Adenomatous polyposis coli (APC) is mutated in colon cancers. During normal development, APC proteins are essential negative regulators of Wnt signaling and have cytoskeletal functions. Many functions have been proposed for APC proteins, but these have often rested on dominant-negative or partial loss-of-function approaches. Thus, despite intense interest in APC, significant questions remain about its full range of cellular functions and about how mutations in the gene affect these. Six new alleles of Drosophila APC2 were isolated. Two resemble the truncation alleles found in human tumors and one is a protein null. Ovaries and embryos null for both APC2 and APC1 were generated, and the consequences of total loss of APC function was assessed, allowing several previous hypotheses to be tested. Surprisingly, although complete loss of APC1 and APC2 resulted in strong activation of Wingless signaling, it did not substantially alter cell viability, cadherin-based adhesion, spindle morphology, orientation or selection of division plane, as predicted from previous studies. The hypothesis that truncated APC proteins found in tumors are dominant negative was tested. Two mutant proteins have dominant effects on cytoskeletal regulation, affecting Wnt-independent nuclear retention in syncytial embryos. However, they do not have dominant-negative effects on Wnt signaling (McCartney, 2006).

Despite substantial interest in Wnt signaling and its regulation in development and disease, important questions remain about the nature of the null phenotype and thus the full range of processes in which APC family proteins play a crucial role. Experiments in vitro, in cultured cells and in Drosophila suggested novel roles for APCs in cadherin-based adhesion, spindle structure and chromosome segregation. Although some of these effects are subtle, APC family function was not completely eliminated, suggesting that APCs may play essential roles in one or more of these processes. Alternatively, because these phenotypes were assessed in cells expressing truncated or otherwise mutant proteins, or expressing transfected APC fragments, it is possible that these effects result from dominant interference with binding partners of APC that work in a process in which APC proteins themselves are not essential (McCartney, 2006).

To distinguish between these possibilities, null mutations removing the function of both APCs must be characterized. In mammals, all work has been done in single mutants and most was done with cells or animals expressing one truncated APC allele. Recently, Cre-lox technology was used to generate mouse APC alleles that may be null; these delete exon 14, and are predicted to truncate APC before the Arm repeats. Although the phenotype of homozygous animals has not been reported, Cre induction was used to create homozygous mutant clones of colon cells. This triggers polyp formation, with mutant cells assuming stem cell properties consistent with Wnt activation. Other phenotypes were not assessed, however, and tests to confirm that this allele is protein null were not reported, so splicing variations might produce residual mutant protein (McCartney, 2006).

Ovaries and embryos for APC2, or double null for both APC2 and APC1, were examined null for essential roles in cadherin-based adhesion. No phenotypes were observed consistent with substantial disruption of cadherin-catenin function, which disrupts both oogenesis and embryonic epithelial integrity. In ovaries, loss of APC2 and APC1 had no apparent effect on adhesion, and in embryos no significant alterations were observe in DE-cadherin or alpha-catenin localization at adherens junctions. Thus APC family proteins do not play an essential role in cell adhesion. However, subtle modulatory effects cannot be rule doult (McCartney, 2006).

Proposed roles for APC proteins in spindle assembly and orientation were tested. Embryos null for both APC proteins had no defects in spindle structure in syncytial embryos, apart from those in regions of spindle detachment or defective metaphase furrows, and no defects in spindle orientation or cell division symmetry in the ectoderm during gastrulation. Thus APC family proteins are not essential for spindle function in these tissues. A subtle but significant lengthening of syncytial spindles was seeen during cycle 13. Subtle defects were not assessed in chromosome segregation, which might lead to a slow accumulation of aneuploid cells in tumors - this will require other assays. How can the earlier data suggesting that APC family proteins have roles in adhesion and cytoskeletal regulation be reconciled when full loss-of-function experiments indicate that they do not? One possibility is that truncated fragments of APC may have dominant effects on processes in which APC does not play an essential role - the data on the phenotype of APC2DeltaS in spindle tethering, which is discussed in more detail below, provide an example of this (McCartney, 2006).

Unlike most other tumor suppressors, APC homozygous null colon tumors are either rare or non-existent. Instead, one allele encodes a protein truncated in the 'mutation-cluster region' (MCR), lacking the SAMP repeats and all sequences located between that region and the C-terminal end, suggesting strong selection for this event during tumor development. Several models propose that the truncated APC proteins found in tumors are dominant negative. One suggests that this affects Wnt signaling, with truncated APC proteins promoting stem cell proliferation. Most models suggest that truncated proteins affect cytoskeletal functions. Different studies come to different conclusions, however. For example, some suggest that truncated APC interferes with microtubule-kinetochore attachments, leading to genomic instability, but others suggest these effects are subtle. A dominant-negative role of truncated APC is not essential for disease, as some FAP patients inherit germline-null APC mutations. Their adenomas carry truncating mutations in the other allele; in this case, there was no wild-type APC to be affected by a dominant-negative truncation. Furthermore, the putative dominant-negative effect is not sufficient for oncogenesis - mice engineered to express truncated APC in a wild-type background do not develop polyps or tumors (McCartney, 2006 and references therein).

Genetic data provide new insight into this question. Little evidence for dominant-negative effects on Wg signaling. Heterozygotes are viable and adults are wild type in phenotype, and wild-type paternal APC2 effectively rescues eight of the nine mutants, suggesting that mutant proteins cannot be strongly dominant negative. The exception is APC2c9, where there appears to be some interference with paternal rescue. Likewise, the data suggest that truncated APC2 does not substantially affect spindle structure (whether APC1 truncations behave dominantly was not assessed) (McCartney, 2006).

Compelling evidence was found for dominant-negative effects on nuclear retention in syncytial embryos. APC2DeltaS and APC2d40 heterozygotes exhibit elevated levels of nuclear loss, and the frequency of abnormal embryos is higher in APC2DeltaS homozygotes than in APC2 null mutants. Thus, the cytoskeletal functions of APCs may be more sensitive to dominant-negative effects of truncated proteins, and this may affect chromosome segregation and contribute to tumor progression (McCartney, 2006).

These data also illuminate the mechanisms of dominant-negative activity. Loss of APC1 did not enhance the nuclear-loss phenotype of APC2 null embryos, suggesting that APC1 does not play a significant role in this process. This suggests that the dominant-negative effect is not on maternally contributed APC1. As nuclear retention is not completely disrupted in double null mutants, alternative mechanisms of nuclear retention partially compensate for the lack of APC1 and APC2. Because APC2DeltaS has a nuclear retention defect more severe than that of embryos M/Z null for both APCs, this mutant protein may not only block residual APC2 function, but may also interfere with a parallel, APC2-independent means of nuclear retention (McCartney, 2006).

The data also provide insights into the domains of APC2 required for nuclear retention, suggesting roles for the Arm repeats and the C terminus. Because APC2DeltaS mutants exhibit much more nuclear loss than do APC2N175K and APC2c9, Arm repeat 5 may have a special importance, perhaps by interfering with binding of a particular partner; APC2DeltaS may also more profoundly affect the overall structure of the Arm repeats (McCartney, 2006).

The experiments provide an in vivo test of the function of proteins truncated in the MCR. APC2d40 and APC2g41 strongly reduce the ability to regulate Wg signaling, but are not as strong as the null APC2g10, or APC2f90, truncating APC2 at the end of the Arm repeats. A similar severely truncated allele of mammalian APC led to higher levels of Wnt reporter activity in cultured cells than did a truncation in the MCR. The data support the 'just right' hypothesis (Albuquerque, 2002), which posits selection in tumors for mutations in which Wnt signaling is elevated, but not too much. In this model, proteins truncated in the MCR retain some ability to regulate ß-catenin, resulting in levels of Wnt signaling that are above the threshold for polyp formation but not 'too high', which might be cell lethal. This study is the first direct test of this hypothesis using null alleles (McCartney, 2006).

The nine alleles also allow the roles of different domains in Wg regulation to be assessed. The role of the Arm repeats of APC has been unclear. In APC mutant tumor cells, transfection of constructs encoding the 15- and 20-amino acid repeats and the SAMP repeats alone restores ß-catenin turnover. However, because these cells retain the Arm repeat-containing truncated protein found in the tumor, these two APC fragments might exhibit intra-allelic complementation. The data suggest that the Arm repeats are crucial for full function of APC2 in the destruction complex. Since missense mutations are not null for Wg regulation, APC proteins may retain residual function in Wg signaling without the Arm repeats. Alternatively, the mutations that were isolated may not fully disrupt the repeats. Each Arm repeat comprises three alpha-helices. Together, the Arm repeats form a superhelical structure with a large groove where partner proteins bind. Structure-based sequence alignments indicate that four out of the five missense mutations (APC2e90, APC2N175K APC2c9 and APC2b5) are in helix 3 of Arm repeats 3, 6 and 7. These mutations affect residues predicted to be in the protein core rather than those predicted to form the binding surface. They may thus destabilize individual Arm repeats, or the Arm repeat domain, without totally eliminating its function. This is consistent with the temperature sensitivity of four out of the five Arm repeat mutations (McCartney, 2006).

APC2 and APC1 function redundantly in Wg signaling throughout Drosophila development, despite differences in domain structure and subcellular localization. This redundancy suggests that the shared domains - the Arm repeats, the 15- and 20-amino acid repeats, the SAMP repeats and the conserved sequences A and B - are sufficient for Wg regulation. It is hypothesized that the Arm repeats are the docking site for a binding partner important for destruction-complex function. Using the temperature-sensitive allele APC2DeltaS, it was found that the phenotype and the membrane association of the mutant protein vary in parallel. Two of the weakest new alleles also exhibit residual membrane association, thus the Arm repeats may bind a partner mediating cortical localization of the destruction complex. However, this does not explain how APC1 and APC2 can have different predominant localizations and yet be redundant. Perhaps low-level cortical accumulation of APC1, especially in the absence of APC2, is sufficient for function. Future tests of this model and identification of the relevant binding partner are needed (McCartney, 2006).

Adenomatous polyposis coli regulates Drosophila intestinal stem cell proliferation

Adult stem cells define a cellular reserve with the unique capacity to replenish differentiated cells of a tissue throughout an organism's lifetime. Previous analysis has demonstrated that the adult Drosophila midgut is maintained by a population of multipotent intestinal stem cells (ISCs) that resides in epithelial niches. Adenomatous polyposis coli (Apc), a tumor suppressor gene conserved in both invertebrates and vertebrates, is known to play a role in multiple developmental processes in Drosophila. This study examined the consequences of eliminating Apc function on adult midgut homeostasis. This analysis shows that loss of Apc results in the disruption of midgut homeostasis and is associated with hyperplasia and multilayering of the midgut epithelium. A mosaic analysis of marked ISC cell lineages demonstrates that Apc is required specifically in ISCs to regulate proliferation, but is not required for ISC self-renewal or the specification of cell fate within the lineage. Cell autonomous activation of Wnt signaling in the ISC lineage phenocopied Apc loss and Apc mutants were suppressed in an allele-specific manner by abrogating Wnt signaling, suggesting that the effects of Apc are mediated in part by the Wnt pathway. Together, these data underscore the essential requirement of Apc in exerting regulatory control over stem cell activity, as well as the consequences that disrupting this regulation can have on tissue homeostasis (Lee, 2009).

This study reports that loss of Apc results in a disruption of midgut homeostasis and is associated with hyperplasia and multilayering of the midgut epithelium. Mosaic analyses show that Apc is required specifically in ISCs to regulate stem cell proliferation. By contrast, loss of Apc did not detectably affect self-renewal or cell fate specification in the ISC lineage. Activation of Wnt signaling in the ISC lineage phenocopied Apc loss and Apc mutants were suppressed in an allele-specific manner by abrogating Wnt signaling, suggesting that the effects of Apc are mediated in part by the Wnt pathway. The finding that Apc differentially affects ISC proliferation without obviously altering self-renewal or multipotency highlights the ability of the stem cell to fine-tune lineage output to meet homeostatic need; loss of Apc appears to short-circuit this regulation, providing increased cellular output in the absence of true physiological demand for new cells (Lee, 2009).

Previous analysis of Wnt signaling in the midgut has led to the assertion that Wnt functions as the primary maintenance signal for ISCs; cell-autonomous loss of Wnt transduction components results in a failure of ISC maintenance, while ectopic expression of Wnt ligand leads to an increase in the number of Dl-expressing cells. These observations led to the following model: transduction of the Wnt signaling pathway in ISCs adjacent to a Wnt source maintains the stem cell population by preventing lineage differentiation. A central prediction of the model is that cell-autonomous activation of the Wnt signaling pathway in ISCs should lead to the production of daughter cells, which constitutively transduce the Wnt signal and, as such, remain undifferentiated. The predicted consequence of this manipulation is an expansion in the number of ISCs at the expense of differentiated cells within the lineage, as has been observed in the case of N loss. This study directly tested this prediction by analyzing marked ISC lineages. The experiments clearly demonstrate that in contrast to N loss, ISCs lacking Apc generate both differentiated cell types of the adult midgut: enteroendocrine (ee) cells and enterocytes (ECs). Importantly, these findings suggest that ISCs and their daughters are not distinguished solely on the basis of Wnt signal transduction (Lee, 2009).

Several explanations could account for these apparent disparities. First, it is worth noting that although Dl might be a reliable marker for certain stem cells in wild-type midguts, this might not be the case in every mutant background examined. For example, a previous analysis in Drosophila has demonstrated that Wnt activation is sufficient to stimulate high levels of Dl expression in a cell-autonomous manner. Thus, it is possible that Wnt pathway activation can uncouple Dl expression from stem cell identity, thereby diminishing the utility of Dl as a reliable stem cell marker. A second possible explanation is methodological; the use of genetic mosaic analyses to analyze individual ISC lineages, as in this study, might provide a different view of the Wnt pathway activation phenotype to that seen following the use of Gal4 driver lines. Third, it is possible that Wnt can affect stem cell maintenance upstream of Apc via a non-canonical pathway. For example, studies have demonstrated that Wnt signaling can act directly via dishevelled (dsh) to inhibit the N signaling pathway. Such effects might not be detected in the Apc, Axin or activated armS10 ISC lineages analyzed in this study (Lee, 2009).

This study demonstrated an increase in the number of dividing cells following Apc loss, consistent with what has previously been reported for mutations that activate the Wnt pathway. And yet, the presence of differentiated cells within marked Apc mutant lineages strongly suggested that the increase in dividing cells could not be explained solely by a N-dependent change in cell fate within the ISC lineage as was proposed by Lin (2008). Based on the current analysis of Apc, it is hypothesized that the increased proliferation following Apc loss reflects a cell-autonomous requirement for Apc specifically in ISCs. This view is further supported by the observation that Apc loss can lead to an increase in mitotic index when N and Apc are simultaneously removed from the ISC lineage. Thus, there is a requirement for Apc specifically in ISCs to regulate proliferation that is separable from N-dependent cell fate specification. Taken together, it is concluded that a primary requirement for Apc in the ISC lineage is to autonomously restrict the proliferation of ISCs, and not to regulate the choice of cell fate (Lee, 2009).

On the basis of the current findings it is proposed that ISC activity is regulated by the level of Wnt signal transduction. In this model, Wnt functions as a permissive signal for ISC self-renewal, since it was shown that constitutive Wnt activation is not a sufficient criterion to convert all ISC progeny to stem cells, nor is activation sufficient to alter the fidelity of ISC self-renewal. Intermediate levels of Wnt define an adaptive homeostatic range, which permits the midgut to respond to environmental changes that the organism encounters. ISCs transducing Wnt at levels outside this range appear refractory to homeostatic input, as low levels of Wnt are associated with ISC loss, whereas Wnt activation leads to hyperplasia (Lee, 2009).

Nevertheless, the data do not rule out additional roles for Apc in the ISC lineage. For example, it is possible that the hyperplasia observed in Apc mutants reflects the combined requirement of Apc to regulate proliferation both in the ISCs and in nascent EB daughters. Similarly, Apc might also play a role in regulating cell turnover in the midgut. Indeed, several of observations support the view that Apc might, in fact, be required for EC differentiation. First, mosaic analysis of Apc shows that cells of the lineage contribute to the multilayering phenotype, which suggests that mutant cells might have failed to properly establish appropriate adhesive contacts with the monolayer and/or the surrounding extracellular matrix. Second, although ECs lacking Apc appear to have large, polyploid nuclei and express molecular markers such as Pdm1, their nuclei are often detectably smaller than those of wild-type ECs. Furthermore, ECs lacking Apc often exhibit a restricted basal profile, failing to develop the tiled morphology that is characteristic of wild-type ECs. Finally, in the absence of Apc, ECs often display reduced cytoplasm, suggesting a requirement for cellular growth, a requirement that is not observed in either Axin mutant or armS10- expressing ICS lineages (Lee, 2009).

The discovery that Apc is somatically mutated in cells of the smallest human adenomas and that the frequency of Apc mutations detected among early adenomas is roughly the same as the frequency of Apc mutations in more advanced carcinomas were among the seminal observations that established Apc as the rate-limiting step for gastrointestinal tumor initiation. Subsequently, a number of Apc models have been established in both mouse and zebrafish to study gastrointestinal tumorigenesis. Yet, the precise cell(s) in which Apc is required remained unknown. Recently, through the use of refined genetic cell lineage tracing methodologies in the mouse, specific subpopulations of cells in the intestinal mucosa have been identified (e.g. Lgr5+ and Bmi1+), which display the ability to self-renew and undergo multilineage differentiation. Subsequent studies have demonstrated that deletion of Apc specifically within the Lgr5+ cell population leads to the formation of rapidly proliferating cells in both the large and small intestine. Thus, in the case of both Drosophila ISCs and mouse Lgr5+ cells, loss of Apc leads to a disruption of homeostasis in the intestinal stem cell lineage. The remarkable parallels that exist between the dipteran and mammalian gastrointestinal tract suggest that the Drosophila midgut will continue to be a powerful genetic model system with which to dissect the molecular mechanisms underlying tumor initiation (Lee, 2009 and references therein).

Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites

In many differentiated cells, microtubules are organized into polarized noncentrosomal arrays, yet few mechanisms that control these arrays have been identified. For example, mechanisms that maintain microtubule polarity in the face of constant remodeling by dynamic instability are not known. Drosophila neurons contain uniform-polarity minus-end-out microtubules in dendrites, which are often highly branched. Because undirected microtubule growth through dendrite branch points jeopardizes uniform microtubule polarity, this system was used to understand how cells can maintain dynamic arrays of polarized microtubules. It was found that growing microtubules navigate dendrite branch points by turning the same way, toward the cell body, 98% of the time and that growing microtubules track along stable microtubules toward their plus ends. Using RNAi and genetic approaches, it was shown that kinesin-2, and the +TIPS EB1 and APC, are required for uniform dendrite microtubule polarity. Moreover, the protein-protein interactions and localization of Apc2-GFP and Apc-RFP to branch points suggests that these proteins work together at dendrite branches. The functional importance of this polarity mechanism is demonstrated by the failure of neurons with reduced kinesin-2 to regenerate an axon from a dendrite. It is concluded that microtubule growth is directed at dendrite branch points and that kinesin-2, APC, and EB1 are likely to play a role in this process. It is proposed that kinesin-2 is recruited to growing microtubules by +TIPS and that the motor protein steers growing microtubules at branch points. This represents a newly discovered mechanism for maintaining polarized arrays of microtubules (Mattie, 2010).

Using Drosophila dendrites as a model system, this study demonstrates that growing microtubule plus ends almost always turn toward the cell body at branch points and that they track stable microtubules through branches. Kinesin-2, EB1, and APC are all required for maintaining microtubule polarity and are linked in an interaction network (Mattie, 2010).

On the basis of these results, a model for directed growth of microtubules in dendrites is proposed. Apc2 most likely contains a branch localization signal because Apc2-GFP localizes well to dendrite branches even when overexpressed. Localization of Apc2 to dendrite branch points can recruit Apc to the branch. Apc can interact with the Kap3 subunit of kinesin-2 and so could increase concentration of the motor near the branch. EB1-GFP does not concentrate at branches, therefore it is proposed that a growing microtubule plus end coated with EB1 is transiently linked, through the interaction between Apc and the EB1 tail, to kinesin-2 as it passes through the branch. SxIP motifs in Apc and Klp68D could also contribute to this interaction. Because both Kap3 and the SxIP motif in Klp68D are in the kinesin-2 tail, the motor domain would be free to walk along a nearby stable microtubule toward the plus end and cell body (Mattie, 2010).

Even a very brief application of force pulling the growing microtubule toward the cell body should be sufficient to steer growth toward the cell body. Once the tip of the microtubule turns, growth should be constrained by the dendrite walls. The association of the growing plus end with a stable microtubule would probably only need to be maintained over a distance of a micron. This model is consistent with the observations that kinesin-2 has shorter run lengths than kinesin-1 and that individual EB1 interactions with the microtubule plus end persist for less than a second (Mattie, 2010).

Observations of plus-end behavior in vivo also favor a model in which only transient interactions of the motor and microtubule plus end are involved; frequently plus ends are seen turning sharply. Stable microtubules do not accommodate such sharp turns, so the sharp turns of plus ends are not likely to occur while the plus end is tracking along a stable microtubule. Instead, they probably represent a switch from a freely growing plus end to one that is following a track (Mattie, 2010).

Directed growth of microtubules at dendrite branch points allows dendrites to maintain uniform minus-end-out polarity despite continued microtubule remodeling. This mechanism is also probably necessary to establish uniform microtubule polarity in branched dendrites, but it probably cannot account for minus-end-out polarity on its own. It is hypothesized that directed microtubule growth is used in concert with an unidentified mechanism to control microtubule polarity in dendrites. Transport of oriented microtubule pieces has been proposed to contribute to axon microtubule polarity, and a similar mechanism could play a role in dendrites. For example, a kinesin anchored to the cell cortex by its C terminus could shuttle minus-end-out microtubule seeds into dendrites. Alternatively, polarized microtubule nucleation sites could be localized within dendrites. Identifying directed growth as one mechanism that contributes to dendrite microtubule polarity should facilitate identification of the missing pieces of this puzzle (Mattie, 2010).

Because kinesin-2, APC, EB1, and polarized microtubules are found in many cell types, directed growth of microtubules along stable microtubule tracks could be very broadly used for maintaining microtubule polarity. This is a newly identified function for both the +TIP proteins and kinesin-2. APC has previously been localized to junctions between a microtubule tip and the side of another microtubule at the cortex of epithelial cells, and sliding of microtubule tips along the side of microtubules was also seen in this system, but there was no association with a particular direction of movement or overall microtubule polarity (Mattie, 2010).

Kinesin-2 has previously been shown to be enriched in the tips of growing axons in cultured mammalian neurons, and it is possible that they could mediate tracking of growing microtubules along existing microtubule tracks in the growth cone. In fact, this type of microtubule tracking behavior has been observed in axonal growth cones under low-actin conditions. Thus, directed growth of microtubules could also be important during axon outgrowth. Indeed, the same principle of directed growth by a motor protein connected to a growing microtubule plus end could be involved in aligning microtubules in many circumstances (Mattie, 2010).

Drosophila apc regulates delamination of invasive epithelial clusters

Border Cells in the Drosophila ovaries are a useful genetic model for understanding the molecular events underlying epithelial cell motility. During stage 9 of egg chamber development they detach from neighboring stretched cells and migrate between the nurse cells to reach the oocyte. RNAi screening led to the identification of the dapc1 gene as being critical in this process. Clonal and live analysis showed a requirement of dapc1 in both outer border (oBC) cells and contacting stretched cells (SCs) for delamination. This mutant phenotype was rescued by dapc1 or dapc2 expression. Loss of dapc1 function was associated with an abnormal lasting accumulation of β-catenin/Armadillo and E-cadherin at the boundary between migrating border and stretched cells. Moreover, β-catenin/armadillo or E-cadherin downregulation rescued the dapc1 loss of function phenotype. Altogether these results indicate that Drosophila Apc1 is required for dynamic remodeling of β-catenin/Armadillo and E-cadherin adhesive complexes between outer border cells and stretched cells regulating proper delamination and invasion of migrating epithelial clusters (De Graeve, 2012).

Cell migration is a dynamic process involving multiple cell-cell and cell-substrate interactions. It is therefore important to better characterize the molecular mechanisms underlying cell adhesion during all stages of cell invasion. BC migration represents a powerful in vivo model, as cells become motile through a multi-step process involving cluster assembly and cohesion, delamination from the follicular epithelium, and labile interactions with nurse cells throughout migration. All these processes require the dynamic remodeling of DE-cadherin and β-catenin/Arm during adhesion. Indeed, an artificial DE-cadherin-β-catenin/Arm fusion protein can act as a strong dominant negative preventing BC migration (De Graeve, 2012).

Several mechanisms can regulate DE-cadherin - β-catenin/Arm interactions. In cell culture systems, Apc proteins have been shown to be able to compete with E-cadherin for β-catenin/Arm binding. As a result, β-catenin/Arm is continuously incorporated into and released from adherens junctions. Hence β-catenin/Arm exchange is strongly affected in cells containing mutations in the apc gene. Nevertheless, little is known about the molecular events involved in cell delamination and about the role of Apc in this process. In order to better understand how cell-cell and cell-substrate interactions control BC migration, an RNAi-based genetic screen was performed and dapc1 was identified as a key regulator of BC delamination (De Graeve, 2012).

The results show that dApc1 regulates BC delamination through DE-cadherin - β-catenin/Arm remodeling at the interface between oBC and SC. Indeed, loss of dapc1 function in oBC and adjacent SC led to abnormal persistence of DE-cadherin and β-catenin/Arm proteins at their boundary preventing them to detach from neighboring cells. Down regulation of β-catenin/arm or DE-cadherin rescued the dapc1 mutant phenotype indicating that loss of dapc1 function indeed increases adhesion strength between oBC and adjacent SC. The results fit with a model in which dApc1 regulates BC-SC adhesion acting at two levels. First, dApc1 competes with DE-cadherin for β-catenin/Arm binding, hence regulating the interaction between DE-cadherin and β-catenin/Arm and thereby adhesion remodeling. Second, dApc1 also favors β-catenin/Arm degradation, thereby controlling the level of proteins involved in adhesion. Altogether, this allows dApc1 to regulate negatively global adhesion strength in between oBC and SC and control BC delamination (De Graeve, 2012).

In the absence of dApc1, the half-life of β-catenin/Arm is sustained, its interaction with DE-cadherin is favored and as a consequence cells display at their surface a higher number of stable adhesions. The dapc1 over-adhesive phenotype can be rescued by lowering β-catenin/Arm levels, rendering delamination again possible. Surprisingly, overexpression of β-catenin/Arm in dapc1 mutant cells also generated a partial rescue of the dapc1 mutant phenotype. Although most of the BC clusters did not reach the oocyte in time, some mutant clusters were able to delaminate. Overexpression of β-catenin/Arm probably bypasses the need of dApc. Indeed excess of β-catenin/Arm molecules generates inter-molecular competition for DE-cadherin binding, rescuing partially BC delamination. In contrast to wild type β-catenin/Arm, the overexpression of ArmS10 did not rescue the dapc mutant phenotype. ArmS10 lacks a sequence (aa 34-87) that contains a consensus GSK-3β phosphorylation site leading to the degradation of wild type β-catenin/Arm protein after phosphorylation. This suggests that β-catenin/Arm phosphorylation by GSK3-β kinase is required for BC delamination. The results are consistent with previous data in mammals showing the presence of Apc and β-catenin/Arm containing complexes that are phosphorylated by GSK3β/CKI, favoring their degradation (De Graeve, 2012).

The dapc mutant phenotype requires loss of dapc both in oBC and adjacent SC. Indeed, when mutant BCs interact with wild type SCs, or vice-versa, abnormal accumulation of β-catenin/Arm and DE-cadherin is no longer detectable in between the mutant and wild type cells and BC delaminate, migrate and reach the oocyte normally. This suggests that in the absence of dapc, DE-cadherin from the mutant cell can still establish a functional interaction with DE-cadherin from the wild type cell. The remodeling of β-catenin/Arm-DE-cadherin in the wild type cell is probably sufficient to allow the release of the mutant cell (De Graeve, 2012).

Live imaging of migrating BC clusters revealed that oBC change their position within the cluster throughout migration. Loss of dapc function led to persistent β-catenin/Arm and DE-cadherin at cell boundaries (oBC-oBC and oBC-SC interfaces), therefore potentially increasing cluster stiffness. However, no defect was observed in mutant oBC tumbling or cluster velocity, suggesting that dApc1 is not essential for regulating inter-oBCs interactions during migration (De Graeve, 2012).

Several models from cell culture to mouse have been used to study Apc function. Wild type Apc acts as a scaffold for many proteins including F-actin, microtubules, β-catenin/Arm, and regulates multiple biological processes independent of Wg signaling, such as chromosomal segregation, cell adhesion, cell migration and apical cell extrusion. The current results show that BC migration provides a new powerful model, out of Wg influence, unraveling mechanisms regulating collective cell migration in vivo with important implications for wound healing and tumor metastasis (De Graeve, 2012).

Non-autonomous crosstalk between the Jak/Stat and Egfr pathways mediates Apc1-driven intestinal stem cell hyperplasia in the Drosophila adult midgut

Inactivating mutations within adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, are responsible for most sporadic and hereditary forms of colorectal cancer (CRC). This study used the adult Drosophila midgut as a model system to investigate the molecular events that mediate intestinal hyperplasia following loss of Apc in the intestine. The results indicate that the conserved Wnt target Myc and its binding partner Max are required for the initiation and maintenance of intestinal stem cell (ISC) hyperproliferation following Apc1 loss. Importantly, it was found that loss of Apc1 leads to the production of the interleukin-like ligands Upd2/3 and the EGF-like Spitz in a Myc-dependent manner. Loss of Apc1 or high Wg in ISCs results in non-cell-autonomous upregulation of upd3 in enterocytes and subsequent activation of Jak/Stat signaling in ISCs. Crucially, knocking down Jak/Stat or Spitz/Egfr signaling suppresses Apc1-dependent ISC hyperproliferation. In summary, these results uncover a novel non-cell-autonomous interplay between Wnt/Myc, Egfr and Jak/Stat signaling in the regulation of intestinal hyperproliferation. Furthermore, evidence is presented suggesting potential conservation in mouse models and human CRC. Therefore, the Drosophila adult midgut proves to be a powerful genetic system to identify novel mediators of APC phenotypes in the intestine (Cordero, 2012).

Using the Drosophila adult midgut as a model system this study has uncovered a key set of molecular events that mediate Apc-dependent intestinal hyperproliferation. The results suggest that paracrine crosstalk between Egfr and Jak/Stat signaling is essential for Apc1-dependent ISC hyperproliferation in the Drosophila midgut (Cordero, 2012).

Previous studies have demonstrated that Myc depletion prevents Apc-driven intestinal hyperplasia in the mammalian intestine. This study provides evidence that such a dependency on Myc is conserved between mammals and Drosophila. It was further demonstrated that endogenous Myc or Max depletion causes regression of an established Apc1 phenotype in the intestine. Taken together, these data highlight the importance of developing Myc-targeted therapies to inhibit Apc1-deficient cells. Since not all roles of Myc are Max dependent, present efforts are focused on developing inhibitors that interfere with Myc binding to Max and would therefore be less toxic. These data provide the first in vivo evidence in support of the Myc/Max interface as a valid therapeutic target for CRC (Cordero, 2012).

Recent work showed that loss of the tuberous sclerosis complex (TSC) in the Drosophila midgut leads to an increase in cell size and inhibition of ISC proliferation. Reduction of endogenous Myc in TSC-deficient midguts restored normal ISC growth and division. These results might appear contradictory to the current work, where Myc is a positive regulator of ISC proliferation. However, in both scenarios, modulation of Myc levels restores the normal proliferative rate of ISCs (Cordero, 2012).

Previous work in mouse showed that Myc upregulation is essential for Wnt-driven ISC hyperproliferation in the intestine. However, Myc overexpression alone only recapitulates some of the phenotypes of hyperactivated Wnt signaling. This study shows that overexpression of Myc is capable of mimicking some aspects of high Wnt signaling in the Drosophila midgut, such as the activation of Jak/Stat, but is not sufficient to drive ISC hyperproliferation. Multiple lines of evidence have shown that forced overexpression of Myc in Drosophila and vertebrate models results in apoptosis partly through activation of p53. Therefore, driving ectopic myc alone is unlikely to parallel Apc deletion in the intestine, where the activation of multiple pathways downstream of Wnt signaling is likely to contribute cooperatively to hyperproliferation (Cordero, 2012).

Understanding the contribution of Jak/Stat signaling to the Apc phenotype in the mammalian intestine has been complicated by genetic redundancy between Stat transcription factors. Constitutive deletion of Stat3 within the intestinal epithelium slowed tumor formation in the ApcMin/+ mouse, but the tumors that arose were more aggressive and ectopically expressed Stat1. Using the Drosophila midgut, direct in vivo evidence is provided that activation of Jak/Stat signaling downstream of Apc1/Myc mediates Apc1-dependent hyperproliferation (Cordero, 2012).

The data on the Drosophila midgut and in mouse and human tissue samples suggest that blocking Jak/Stat activation could represent an efficacious therapeutic strategy to treat CRC. Currently, there are a number of Jak2 inhibitors under development and it would be of great interest to examine whether any of these could modify the phenotypes associated with Apc loss (Cordero, 2012).

Previous studies have demonstrated that enterocytes (ECs) are the main source of Upds/interleukins in the midgut epithelium. The results show that activation of Wnt/Myc signaling in ISCs leads to non-autonomous upregulation of upd3 within ECs. Furthermore, Spitz/Egfr signaling appears to mediate the paracrine crosstalk between Wnt/Myc and Jak/Stat in the midgut. Overexpression of a dominant-negative Egfr in ECs blocks upd3 upregulation and ISC hyperproliferation in response to high Wnt signaling. A previous EC-specific role for Egfr has been demonstrated during midgut remodeling upon bacterial damage. Nevertheless, the downstream signaling that mediates such a role of Egfr remains unclear given that the activation of downstream MAPK/ERK occurs exclusively within ISCs. Therefore, the current evidence would suggest that Egfr activity in ECs does not involve cell-autonomous ERK activation. Consistent with these observations, p-ERK (Rolled -- FlyBase) localization was not detected outside ISCs in response to either Apc loss or overexpression of wg in the Drosophila midgut. Reports on the Apc murine intestine have also failed to detect robust ERK activation. Since MAPK/ERK is only one of the pathways activated downstream of Egfr, it is possible that ERK-independent mechanisms are involved. It is important to explore this further because ERK-independent roles of Egfr signaling have not yet been reported in Drosophila. Thus, what mediates Upd3 upregulation in ECs in response to Egfr signaling activation and whether Spitz-dependent upregulation of Upd3 involves a direct role of Egfr in ECs remain unclear. A potential alternative explanation is that intermediate factors induced in response to Spitz/Egfr activation in ISCs might drive Upd3 expression (Cordero, 2012).

In summary, this study has elucidated a novel molecular signaling network leading to Wnt-dependent intestinal hyperproliferation. Given the preponderance of APC mutations in CRC, the integration of Egfr and Jak/Stat activation might be a conserved initiating event in the disease (Cordero, 2012).

Integrin signaling is required for maintenance and proliferation of intestinal stem cells in Drosophila

Tissue-specific stem cells are maintained by both local secreted signals and cell adhesion molecules that position the stem cells in the niche microenvironment. In the Drosophila midgut, multipotent intestinal stem cells (ISCs) are located basally along a thin layer of basement membrane that composed of extracellular matrix (ECM), which separates ISCs from the surrounding visceral musculature: the muscle cells constitute a regulatory niche for ISCs by producing multiple secreted signals that directly regulate ISC maintenance and proliferation. This study shows that integrin-mediated cell adhesion, which connects the ECM and intracellular cytoskeleton, is required for ISC anchorage to the basement membrane. Specifically, the alpha-integrin subunits including alphaPS1 encoded by mew and alphaPS3 encoded by scb, and the beta-integrin subunit encoded by mys are richly expressed in ISCs and are required for the maintenance, rather than their survival or multiple lineage differentiation. Furthermore, ISC maintenance also requires the intercellular and intracellular integrin signaling components including Talin, Integrin-linked kinase (Ilk), and the ligand, Laminin A. Notably, integrin mutant ISCs are also less proliferative, and genetic interaction studies suggest that proper integrin signaling is a prerequisite for ISC proliferation in response to various proliferative signals and for the initiation of intestinal hyperplasia after loss of adenomatous polyposis coli (Apc). These studies suggest that integrin not only functions to anchor ISCs to the basement membrane, but also serves as an essential element for ISC proliferation during normal homeostasis and in response to oncogenic mutations (Lin, 2013).

Conserved mechanisms of tumorigenesis in the Drosophila adult midgut

Whereas the series of genetic events leading to colorectal cancer (CRC) have been well established, the precise functions that these alterations play in tumor progression and how they disrupt intestinal homeostasis remain poorly characterized. Activation of the Wnt/Wg signaling pathway by a mutation in the gene APC is the most common trigger for CRC, inducing benign lesions that progress to carcinomas due to the accumulation of other genetic alterations. Among those, Ras mutations drive tumour progression in CRC, as well as in most epithelial cancers. As mammalian and Drosophila's intestines share many similarities, this study explored the alterations induced in the Drosophila midgut by the combined activation of the Wnt signaling pathway with gain of function of Ras signaling in the intestinal stem cells. Compound Apc-Ras clones, but not clones bearing the individual mutations, were shown to expand as aggressive intestinal tumor-like outgrowths. These lesions reproduce many of the human CRC hallmarks such as increased proliferation, blockade of cell differentiation and cell polarity and disrupted organ architecture. This process is followed by expression of tumoral markers present in human lesions. Finally, a metabolic behavioral assay shows that these flies suffer a progressive deterioration in intestinal homeostasis, providing a simple readout that could be used in screens for tumor modifiers or therapeutic compounds. Taken together, these results illustrate the conservation of the mechanisms of CRC tumorigenesis in Drosophila, providing an excellent model system to unravel the events that, upon mutation in Apc and Ras, lead to CRC initiation and progression (Martorell, 2014).


Ahmed, Y., et al. (1998). Regulation of armadillo by a Drosophila APC inhibits neuronal apoptosis during retinal development. Cell 93(7): 1171-1182. PubMed Citation: 9657150

Ahmed, Y., Nouri, A. and Wieschaus, E. (2002). Drosophila Apc1 and Apc2 regulate Wingless transduction throughout development. Development 129: 1751-1762. 11923210

Akong, K., McCartney, B. M. and Peifer, M. (2002). Drosophila APC2 and APC1 have overlapping roles in the larval brain despite their distinct intracellular localizations. Dev. Bio. 250: 71-90. 12297097

Albuquerque, C., Breukel, C., van der Luijt, R., Fidalgo, P., Lage, P., Slors, F. J., Leitao, C. N., Fodde, R. and Smits, R. (2002). The 'just-right' signaling model: APC somatic mutations are selected based on a specific level of activation of the ß-catenin signaling cascade. Hum. Mol. Genet. 11: 1549-1560. 12045208

Andreu, P., et al. (2005). Crypt-restricted proliferation and commitment to the Paneth cell lineage following Apc loss in the mouse intestine. Development 132(6): 1443-51. 15716339

Ashton, G. H., et al. (2010). Focal adhesion kinase is required for intestinal regeneration and tumorigenesis downstream of Wnt/c-Myc signaling. Dev. Cell 19(2): 259-69. PubMed Citation: 20708588

Behrens, J., et al. (1998). Functional interaction of an axin homolog, Conductin, with beta-Catenin, APC, and GSK3beta. Science 280(5363): 596-599. PubMed Citation: 9554852

Benchabane, H., et al. (2008). Adenomatous polyposis coli is present near the minimal level required for accurate graded responses to the Wingless morphogen. Development 135: 963-971. PubMed Citation: 18234723

Benhamouche, S., et al. (2006). Apc tumor suppressor gene is the 'zonation-keeper' of mouse liver. Dev. Cell 10(6): 759-70. 16740478

Berrueta, L., et al. (1998). The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules. Proc. Natl. Acad. Sci. 95(18): 10596-601. PubMed Citation: 9724749

Berrueta, L., et al. (1999). The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain. Curr. Biol. 9(8): 425-8. PubMed Citation: 10226031

Brancolini, C., et al. (1997). Dismantling cell-cell contacts during apoptosis is coupled to a caspase-dependent proteolytic cleavage of beta-catenin. J. Cell Biol. 139: 759-771. PubMed Citation: 9348292

Browne, S. J., et al. (1994). Loss of APC protein expressed by human colonic epithelial cells and the appearance of a specific low-molecular-weight form is associated with apoptosis in vitro. Int. J. Cancer 59(1): 56-64. PubMed Citation: 7927905

Chazaud, C. and Rossant, J. (2006). Disruption of early proximodistal patterning and AVE formation in Apc mutants. Development 133(17): 3379-87. 16887818

Choi, S. H., Estaras, C., Moresco, J. J., Yates, J. R. and Jones, K. A. (2013). alpha-Catenin interacts with APC to regulate beta-catenin proteolysis and transcriptional repression of Wnt target genes. Genes Dev 27: 2473-2488. PubMed ID: 24240237

Cliffe, A., Hamada, F. and Bienz, M. (2003). A role of Dishevelled in relocating Axin to the plasma membrane during Wingless signaling. Curr. Biol. 13: 960-966. 12781135

Colnot, S., Decaens, T., Niwa-Kawakita, M., Godard, C., Hamard, G., Kahn, A., Giovannini, M. and Perret, C. (2004). Liver-targeted disruption of Apc in mice activates ß-catenin signaling and leads to hepatocellular carcinomas. Proc. Natl. Acad. Sci. 101: 17216-17221. 15563600

Cordero, J. B., Stefanatos, R. K., Myant, K., Vidal, M. and Sansom, O. J. (2012). Non-autonomous crosstalk between the Jak/Stat and Egfr pathways mediates Apc1-driven intestinal stem cell hyperplasia in the Drosophila adult midgut. Development 139: 4524-4535. PubMed ID: 23172913

Cox, R. T., et al. (1999). Membrane-tethered Drosophila Armadillo cannot transduce Wingless signal on its own. Development 126: 1327-1335. PubMed Citation: 10021350

De Graeve, F. M., et al. (2012). Drosophila apc regulates delamination of invasive epithelial clusters. Dev. Biol. 368(1): 76-85. PubMed Citation: 22627290

Deka, J., Kuhlmann, J. and Muller, O. (1998). A domain within the tumor suppressor protein APC shows very similar biochemical properties as the microtubule-associated protein tau. Eur. J. Biochem. 253(3): 591-597. PubMed Citation: 9654054

Draviam, V. M., Shapiro, I., Aldridge, B. and Sorger, P. K. (2006). Misorientation and reduced stretching of aligned sister kinetochores promote chromosome missegregation in EB1- or APC-depleted cells. EMBO J. 25(12): 2814-27. 16763565

Efstathiou, J. A., et al. (1998). Intestinal trefoil factor controls the expression of the adenomatous polyposis coli-catenin and the E-cadherin-catenin complexes in human colon carcinoma cells. Proc. Natl. Acad. Sci. 95(6): 3122-3127. PubMed Citation: 9501226

Fagotto, F., et al. (1999). Domains of axin involved in protein-protein interactions, wnt pathway inhibition, and intracellular localization. J. Cell Biol. 145(4): 741-756. PubMed Citation: 10330403

Fancy, S. P., et al. (2009). Dysregulation of the Wnt pathway inhibits timely myelination and remyelination in the mammalian CNS. Genes Dev. 23(13): 1571-85. PubMed Citation: 19515974

Farr, G. H., et al. (2000). Interaction among GSK-3, GBP, Axin, and APC in Xenopus axis specification. J. Cell Biol. 148: 691-701

Faux, M. C., et al. (2004). Restoration of full-length adenomatous polyposis coli (APC) protein in a colon cancer cell line enhances cell adhesion. J. Cell Sci. 117(Pt 3): 427-39. 14679305

Feng, Y., Li, X., Ray, L., Song, H., Qu, J., Lin, S. and Lin, X. (2014). The Drosophila tankyrase regulates Wg signaling depending on the concentration of Daxin. Cell Signal 26: 1717-1724. PubMed ID: 24768997

Fradkin, L. G., Noordermeer, J. N. and Nusse, R. (1995). The Drosophila Wnt protein DWnt-3 is a secreted glycoprotein localized on the axon tracts of the embryonic CNS. Dev. Biol.168: 202-213.

Green, R. A., Wollman, R. and Kaplan, K. B. (2005). APC and EB1 function together in mitosis to regulate spindle dynamics and chromosome alignment. Mol. Biol. Cell 16(10): 4609-22. 16030254

Hamada, F., et al. (1999). Negative regulation of Wingless signaling by D-Axin, a Drosophila homolog of Axin. Science 283(5408): 1739-42

Hamada, F. and Bienz, M. (2004). The APC tumor suppressor binds to C-terminal binding protein to divert nuclear ß-Catenin from TCF. Dev. Cell 7: 677-685. 15525529

Hart, M. J., et al. (1998). Downregulation of beta-catenin by human Axin and its association with the APC tumor suppressor, beta-catenin and GSK3 beta. Curr. Biol. 8(10): 573-581

Hayashi, S., et al. (1997). A Drosophila homolog of the tumor suppressor gene adenomatous polyposis coli down-regulates beta-catenin but its zygotic expression is not essential for the regulation of Armadillo. Proc. Natl. Acad. Sci. 94(1): 242-247

He T.-C., et al. (1998). Identification of c-MYC as a target of the APC pathway. Science 281(5382): 1509-1512

Honnappa, W., et al. (2005). Structural insights into the EB1-APC interaction. EMBO J. 24: 261-269. Medline abstract: 15616574

Ikeda, S., et al. (1998). Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin. EMBO J. 17(5): 1371-1384

Jimbo, T., et al. (2002). Identification of a link between the tumour suppressor APC and the kinesin superfamily. Nat. Cell Biol. 4(4): 323-7. 11912492

Kim, S. E., Huang, H., Zhao, M., Zhang, X., Zhang, A., Semonov, M. V., MacDonald, B. T., Zhang, X., Garcia Abreu, J., Peng, L. and He, X. (2013). Wnt stabilization of beta-catenin reveals principles for morphogen receptor-scaffold assemblies. Science 340: 867-870. PubMed ID: 23579495

Kita, K., Wittmann, T., Nathke, I. S., Waterman-Storer, C. M. (2006). Adenomatous polyposis coli on microtubule plus ends in cell extensions can promote microtubule net growth with or without EB1. Mol. Biol. Cell 17: 2331-2345. Medline abstract: 16525027

Kishida, S., et al. (1998). Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin. J. Biol. Chem. 273(18): 10823-10826

Korinek V., et al. (1997). Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma. Science 275(5307): 1784-1787

Kuraguchi, M., et al. (2006). Adenomatous Polyposis Coli (APC) Is Required for Normal Development of Skin and Thymus. PLoS Genet. 2(9). 17002498

Lee, E., et al. (2003). The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway. Plos Biol. 2(3): E89. 14551908

Lee, W. C., Beebe, K., Sudmeier, L. and Micchelli, C. A. (2009). Adenomatous polyposis coli regulates Drosophila intestinal stem cell proliferation. Development 136(13):2255-64. PubMed Citation: 19502486

Lebensohn, A.M., Dubey, R., Neitzel, L.R., Tacchelly-Benites, O., Yang, E., Marceau, C.D., Davis, E.M., Patel, B.B., Bahrami-Nejad, Z., Travaglini, K.J., Ahmed, Y., Lee, E., Carette, J.E. and Rohatgi, R. (2016). Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling. Elife [Epub ahead of print]. PubMed ID: 27996937

Lin, G., Xu, N. and Xi, R. (2008). Paracrine Wingless signaling controls self-renewal of Drosophila intestinal stem cells. Nature 455: 1119-23. PubMed Citation: 18806781

Lin, G., Zhang, X., Ren, J., Pang, Z., Wang, C., Xu, N. and Xi, R. (2013). Integrin signaling is required for maintenance and proliferation of intestinal stem cells in Drosophila. Dev Biol 377: 177-187. PubMed ID: 23410794

Lin, R., Hill, R. J. and Priess, J. R. (1998). POP-1 and anterior-posterior fate decisions in C. elegans embryos. Cell 92(2): 229-239

Liu, J., et al. (2001). Siah-1 mediates a novel ß-Catenin degradation pathway linking p53 to the Adenomatous polyposis coli protein. Mol. Cell 7: 927-936. 11389840

Liu, J., et al. (2006). The third 20 amino acid repeat is the tightest binding site of APC for ß-catenin. J. Mol. Biol. 360(1): 133-44. 16753179

Louie, R. K., Bahmanyar, S., Siemers, K. A., Votin, V., Chang, P., Stearns, T., Nelson, W. J., Barth, A. I. M. (2004). Adenomatous polyposis coli and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes. J. Cell Sci. 117: 1117-1128. Medline abstract: 14970257

Lu, B., et al. (2001). Adherens junctions inhibit asymmetric division in the Drosophila epithelium. Nature 409: 522-525. 11206549

Marikawa, Y. and Elinson, R. P. (1999). Relationship of vegetal cortical dorsal factors in the Xenopus egg with the Wnt/beta-catenin signaling pathway. Mech. Dev. 89: 93-102

Martorell, O., Merlos-Suarez, A., Campbell, K., Barriga, F. M., Christov, C. P., Miguel-Aliaga, I., Batlle, E., Casanova, J. and Casali, A. (2014). Conserved mechanisms of tumorigenesis in the Drosophila adult midgut. PLoS One 9: e88413. PubMed ID: 24516653

Matsumine, A., et al. (1996). Binding of APC to the human homolog of the Drosophila Discs Large tumor suppressor protein. Science 272: 1020-1024

Mattie, F. J., et al. (2010). Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites. Curr. Biol. 20(24): 2169-77. PubMed Citation: 21145742

McCartney, B. M., et al. (1999). Drosophila APC2 is a cytoskeletally-associated protein that regulates wingless signaling in the embryonic epidermis. J. Cell Biol. 146: 1303-1318. PubMed Citation: 10491393

McCartney, B. M., et al. (2006). Testing hypotheses for the functions of APC family proteins using null and truncation alleles in Drosophila. Development 133(12): 2407-18. 16720878

Mendoza-Topaz, C., Mieszczanek, J. and Bienz, M. (2011). The Adenomatous polyposis coli tumour suppressor is essential for Axin complex assembly and function and opposes Axin's interaction with Dishevelled. Open Biol. 1(3): 110013. PubMed Citation: 22645652

Mizumoto, K. and Sawa, H. (2007). Cortical beta-catenin and APC regulate asymmetric nuclear beta-catenin localization during asymmetric cell division in C. elegans. Dev. Cell 12(2): 287-99. PubMed Citation: 17276345

Molenaar, M., et al. (1996). XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86: 391-399

Morin, P. J., et al. (1997). Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275(5307): 1787-1790

Morrison, E. E., et al. (1998). EB1, a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle. Oncogene 17(26): 3471-7

Moseley, J. B., et al. (2007). Regulated binding of adenomatous polyposis coli protein to actin. J. Biol. Chem. 282(17): 12661-8. Medline abstract: 17293347

Munemitsu, S., et al. (1994). The APC gene product associates with microtubules in vivo and promotes their assembly in vitro. Cancer Res. 54(14): 3676-81

Munemitsu, S., et al. (1995). Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. Proc. Natl. Acad. Sci. 92(7): 3046-3050

Munemitsu S., et al. (1996). Deletion of an amino-terminal sequence beta-catenin in vivo and promotes hyperphosporylation of the adenomatous polyposis coli tumor suppressor protein. Mol. Cell Biol. 16(8): 4088-4094

Muñoz-Descalzo, S., et al. (2011). Modulation of the ligand-independent traffic of Notch by Axin and Apc contributes to the activation of Armadillo in Drosophila. Development 138: 1501-1506. PubMed Citation: 21389052

Nathke, I. S., et al. (1996). The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration. J. Cell Biol. 134(1): 165-79

Nusse, R. (1997). A versatile transcriptional effector of wingless signaling. Cell. 89: 321-323

Orsulic, S. and Peifer, M. (1996). An in vivo structure-function study of Armadillo, the beta-catenin homologue, reveals both separate and overlapping regions of the protein required for cell adhesion and for wingless signaling. J. Cell Biol. 134, 1283-1300

Orsulic, S., et al. (1999). E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. J. Cell Sci. 112(Pt 8): 1237-1245

Peifer, M., et al. (1994). Wingless signal and Zeste-white 3 kinase trigger opposing changes in the intracellular distribution of Armadillo. Development 120: 369-380

Perrimon, N. (1996). Serpentine proteins slither into the Wingless and Hedgehog Fields. Cell 86: 513-516

Phelps, R. A., et al. (2009). A two-step model for colon adenoma initiation and progression caused by APC loss. Cell 137(4): 623-34. PubMed Citation: 19450512

Pollack, A. L., et al. (1997). Dynamics of beta-catenin interactions with APC protein regulate epithelial tubulogenesis. J. Cell Biol. 137(7): 1651-1662

Reilein, A. and Nelson, W. J. (2005). APC is a component of an organizing template for cortical microtubule networks. Nat. Cell Biol. 7(5): 463-73. 15892196

Robbins, P. F., et al. (1996). A mutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes. J Exp Med. 183(3): 1185-1192

Rogers, S.L., Rogers, G.C., Sharp, D.J., and Vale, R.D. (2002). Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J. Cell Biol. 158: 873-884. 12213835

Rosin-Arbesfeld, R., Townsley, F. and Bienz, M. (2000). The APC tumor suppressor has a nuclear export function. Nature 406(6799): 1009-12

Rosin-Arbesfeld, R., Ihrke, G. and Bienz, M. (2001). Actin-dependent membrane association of the APC tumour suppressor in polarized mammalian epithelial cells. EMBO J. 20: 5929-5939. 11689433

Rosin-Arbesfeld, R., Cliffe, A., Brabletz, T. and Bienz, M. (2003). Nuclear export of the APC tumour suppressor controls ß-catenin function in transcription. EMBO J. 22: 1101-1113. 12606575

Rubinfeld, B., et al. (1996). Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science 272(5264): 1023-1026

Rubinfeld, B., et al. (1997). Loss of beta-catenin regulation by the APC tumor suppressor protein correlates with loss of structure due to common somatic mutations of the gene. Cancer Res. 57(20): 4624-4630

Sakanaka, C., Weiss, J. B. and Williams, L. T. (1998). Bridging of beta-catenin and glycogen synthase kinase-3beta by axin and inhibition of beta-catenin-mediated transcription. Proc. Natl. Acad. Sci. 95(6): 3020-3023

Salic, A., et al. (2000). Control of beta-Catenin stability: reconstitution of the cytoplasmic steps of the Wnt pathway in Xenopus egg extracts. Molec. Cell 5: 523-532

Sansom, O. J., et al. (2004). Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev. 18: 1385-1390. 15198980

Seeling, J. M., et al. (1999). Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A. Science 283(5410): 2089-91

Senda, T., et al. (1998). Localization of the adenomatous polyposis coli tumour suppressor protein in the mouse central nervous system. Neuroscience 83(3): 857-866

Sierra, J., Yoshida, T., Joazeiro, C. A. and Jones, K. A. (2006). The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes. Genes Dev. 20(5): 586-600. 16510874

Smith, K. J., et al. (1994). Wild-type but not mutant APC associates with the microtubule cytoskeleton. Cancer Res. 54(14): 3672-5

Smits, R., et al. (1999). Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli protein involved in tumorigenesis and development. Genes Dev. 13(10): 1309-21

Spink, K. E., Polakis, P. and Weis, W. I. (2000). Structural basis of the Axin-adenomatous polyposis coli interaction. EMBO J. 19: 2270-2279

Spink, K. E., Fridman, S. G. and Weis, W. I. (2001). Molecular mechanisms of ß-catenin recognition by adenomatous polyposis coli revealed by the structure of an APC-ß-catenin complex. EMBO J. 20: 6203-6212. 11707392

Strater, J., et al. (1995). In situ detection of enterocytic apoptosis in normal colonic mucosa and in familial adenomatous polyposis. Gut 37(6): 819-825

Su, L. K., et al. (1995). APC binds to the novel protein EB1. Cancer Res. 55(14): 2972-7

Subramanian, A., et al. (2003). Shortstop recruits EB1/APC1 and promotes microtubule assembly at the muscle-tendon junction. Curr. Biol. 13: 1086-1095. 12842007

Takizawa, S., et al. (2006). Human scribble, a novel tumor suppressor identified as a target of high-risk HPV E6 for ubiquitin-mediated degradation, interacts with adenomatous polyposis coli. Genes Cells 11(4): 453-64. Medline abstract: 16611247

Tanaka, S., et al. (1998). A novel frizzled gene identified in human esophageal carcinoma mediates APC/beta-catenin signals. Proc. Natl. Acad. Sci. 95(17): 10164-9

Trzepacz, C., et al. (1997). Phosphorylation of the tumor suppressor adenomatous polyposis coli (APC) by the cyclin-dependent kinase p34. J. Biol. Chem. 272(35): 21681-21684

van Es, J. H., et al. (1999). Identification of APC2, a homologue of the adenomatous polyposis coli tumour suppressor. Curr. Biol. 9(2): 105-8

Vleminckx, K., et al. (1997). Adenomatous polyposis coli tumor suppressor protein has signaling activity in Xenopus laevis embryos resulting in the induction of an ectopic dorsoanterior axis. J. Cell Biol. 136(2): 411-420

Wang, Z., Tacchelly-Benites, O., Yang, E., Thorne, C. A., Nojima, H., Lee, E. and Ahmed, Y. (2016a). Wnt/Wingless pathway activation is promoted by a critical threshold of axin maintained by the tumor suppressor APC and the ADP-ribose polymerase Tankyrase. Genetics 203: 269-281. PubMed ID: 26975665

Wang, Z., Tian, A., Benchabane, H., Tacchelly-Benites, O., Yang, E., Nojima, H. and Ahmed, Y. (2016b). The ADP-ribose polymerase Tankyrase regulates adult intestinal stem cell proliferation during homeostasis in Drosophila. Development 143: 1710-1720. PubMed ID: 27190037

Wang, X. P., et al. (2009). Apc inhibition of Wnt signaling regulates supernumerary tooth formation during embryogenesis and throughout adulthood. Development 136(11): 1939-49. PubMed Citation: 19429790

Wen, Y., et al. (2004). EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration. Nat. Cell Biol. 6(9): 820-30. 15311282

Wong, M. H., et al. (1996). Forced expression of the tumor suppressor adenomatosis polyposis coli protein induces disordered cell migration in the intestinal epithelium. Proc. Natl. Acad. Sci. 93(18): 9588-9593

Xing, Y., Clements, W. K., Kimelman, D. and Xu, W. (2003). Crystal structure of a ß-catenin/Axin complex suggests a mechanism for the ß-catenin destruction complex. Genes Dev. 17: 2753-2764. 14600025

Xu, N., et al. (2011). EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells. Dev. Biol 354(1): 31-43. PubMed Citation: 21440535

Yamamoto H., et al. (1998). Axil, a member of the Axin family, interacts with both glycogen synthase kinase 3beta and beta-catenin and inhibits axis formation of Xenopus embryos. Mol. Cell. Biol. 18(5): 2867-2875. PubMed Citation: 9566905

Yamashita, M., Jones. D. L. and Fuller, M. T. (2003). Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome. Science 301: 1547-1550. PubMed Citation: 12970569

Yamulla, R. J., Kane, E. G., Moody, A. E., Politi, K. A., Lock, N. E., Foley, A. V. and Roberts, D. M. (2014). Testing models of the APC tumor suppressor/β-catenin interaction reshapes our view of the destruction complex in Wnt signaling. Genetics [Epub ahead of print] PubMed ID: 24931405

Yang, E., Tacchelly-Benites, O., Wang, Z., Randall, M. P., Tian, A., Benchabane, H., Freemantle, S., Pikielny, C., Tolwinski, N. S., Lee, E. and Ahmed, Y. (2016). Wnt pathway activation by ADP-ribosylation. Nat Commun 7: 11430. PubMed ID: 26975665

Yu, X. and Bienz, M. (1999). Ubiquitous expression of a Drosophila adenomatous polyposis coli homolog and its localization in cortical actin caps. Mech. Dev. 84: 69-73

Zeng, L., et al. (1997). The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation. Cell 90(1): 181-192

Zhang, J., Ahmad, S. and Mao, Y. (2007). BubR1 and APC/EB1 cooperate to maintain metaphase chromosome alignment. J. Cell Biol. 178(5): 773-84. Medline abstract: 17709426

Zhou, F.-Q., et al. (2004). NGF-induced axon growth is mediated by localized inactivation of GSK-3ß and functions of the microtubule plus end binding protein APC. Neuron 42: 897-912. 15207235

Zumbrunn, J., et al. (2001). Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3ß phosphorylation. Curr. Biol. 11: 44-49. 11166179

Apc-like: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 21 November 2016

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