Apc-like

DEVELOPMENTAL BIOLOGY

Embryonic

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).

Larval

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).

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 arm^H8.6 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 arm^H8.6 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 arm^YD35 rescues many photoreceptors from death and thereby dominantly suppresses the Apc mutant phenotype. In contrast, the mutant allele arm^H8.6 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 Apc1^Q8;Apc2^d40 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 APC2^DeltaS 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 APC2^c9, 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. APC2^DeltaS and APC2^d40 heterozygotes exhibit elevated levels of nuclear loss, and the frequency of abnormal embryos is higher in APC2^DeltaS 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 APC2^DeltaS 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 APC2^DeltaS mutants exhibit much more nuclear loss than do APC2^N175K and APC2^c9, Arm repeat 5 may have a special importance, perhaps by interfering with binding of a particular partner; APC2^DeltaS 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. APC2^d40 and APC2^g41 strongly reduce the ability to regulate Wg signaling, but are not as strong as the null APC2^g10, or APC2^f90, 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 (APC2^e90, APC2^N175K APC2^c9 and APC2^b5) 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 APC2^DeltaS, 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).

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