A Second Drosophila APC
10 expressed sequence tags from the Berkeley Drosophila
Genome Project correspond to dAPC2
; we obtained sequence of several cDNAs and the corresponding genomic region (we reported partial sequence in van Es et al. 1999
; full sequence data available from EMBL/GenBank/DDBJ under accession no. AF091430). These predict a 1067 amino acid protein with striking similarity to other APC family members () (for review of hAPC features see Polakis 1999
). All share an NH2
-terminal conserved domain, 6 Arm repeats, and a series of βcat binding (15 and 20 amino acid repeats) and Axin binding (SAMP repeats; Behrens et al. 1998
) motifs. dAPC2 is shorter at its NH2
and COOH termini than other APCs. dAPC2 lacks the COOH-terminal basic region (the putative MT binding site) found in hAPC and dAPC (Hayashi et al. 1997
), as well as the hAPC region containing binding sites for Discs-large (DLG) and EB1. Substantial alternative splicing is unlikely, as there are only two small introns in coding sequences (63 and 197 nucleotides).
dAPC2 is most similar to other APC family members in the Arm repeats, where it most closely resembles dAPC; hAPC2 is more similar to hAPC ( B) (dAPC2 is 81% identical to dAPC and 57% identical to hAPC). Thus, there is no correspondence between individual human and fly proteins, even though both phyla show neural-enriched isoforms, dAPC and hAPC2, suggesting independent gene duplications. All APCs have six Arm repeats; a putative seventh Arm repeat is much more divergent and is not identifiable in dAPC2. The NH2
-terminal conserved region (61% identity to dAPC vs. 44% identity to hAPC) distantly resembles the Arm repeat consensus and may form one or two degenerate Arm repeats. APC family members also share similarity COOH-terminal to the Arm repeats. hAPC has two sets of repeated βcat binding sites, the 15 and 20 amino acid repeats (for review see Polakis 1999
; dAPC features are from Hayashi et al. 1997
; hAPC2 lacks 15 amino acid repeats). dAPC2 shares two of the three 15 amino acid repeats of dAPC. dAPC and dAPC2 have five 20 amino acid repeats, among which are interspersed SAMP repeats ( C). dAPC has four SAMP repeats, whereas dAPC2 has two. dAPC2 ends 40 amino acids after the last SAMP repeat.
We generated antisera to a dAPC2 fusion protein (amino acids 491–1061); antisera from two independent rats immunized with this antigen both recognize a single set of protein isoforms of ~155–170 kD in embryonic extracts ( A) (they occasionally weakly cross-react with proteins of ~120 and > 200 kD). In contrast, the preimmune sera do not recognize any proteins on immunoblots of embryo extract, supporting the specificity of the antisera. Further, as we show below, the migration on SDS-PAGE of the putative dAPC2 protein is altered in a dAPC2 mutant, consistent with these protein isoforms representing the genuine dAPC2 protein. The predicted molecular mass of dAPC2, 117 kD, is smaller than the observed molecular mass. However, an epitope-tagged version of the dAPC2 open reading frame expressed in human SW480 colon carcinoma cells also migrated at much higher apparent molecular mass than predicted from the sum of the predicted molecular mass of the dAPC2 coding sequence plus that of the epitope ( A). This suggests that the large apparent molecular mass of dAPC2 is a property of its migration on SDS-PAGE. We examined the developmental profile of dAPC2 expression during embryogenesis ( B). dAPC2 is present in the preblastoderm embryo (presumably maternally contributed), and levels remain relatively constant through the first half of embryogenesis, then drop sharply.
Figure 2 dAPC2 protein properties. (A) dAPC2 protein. Extracts from 5–23-h Drosophila embryos and from human SW480 colon carcinoma cells expressing epitope-tagged dAPC2 coding sequences (the fusion protein includes a six-myc tag and additional amino acids (more ...)
As hAPC is phosphorylated (e.g., Rubinfeld et al. 1996
), we suspected that the dAPC2 isoforms might be phosphorylation variants. To test this, we immunoprecipitated (IPed) dAPC2 from embryos and treated the IPs with protein phosphatase 2A (PP2A), a serine/threonine-specific phosphatase. PP2A treatment reduced the apparent molecular mass of dAPC2; this effect was abolished if the PP2A inhibitor okadaic acid was included during incubation ( C, left panel). Further, if embryonic cells were dissociated and incubated in tissue culture medium, the apparent molecular mass of dAPC2 decreased ( C, right panel); this effect was also abolished by okadaic acid, suggesting that it is mediated by endogenous phosphatases. Parallel alterations in Arm phosphorylation support this hypothesis ( C, right panel) (Peifer 1993
). Taken together, these data suggest that the dAPC2 isoforms reflect, at least in part, differential phosphorylation.
dAPC2 Interacts Directly with Arm
hAPC and dAPC (Hayashi et al. 1997
) bind to βcat and Arm, respectively. We tested whether dAPC2 also interacts with Arm in vivo. We immunoprecipitated Arm from embryonic extracts, and, in parallel, IPed proteins with a control mAb, anti-myc. dAPC2 specifically co-IPed with Arm from both early and older embryos ( D), but did not co-IP with the control anti-myc antibody. We were unable to detect Arm in anti-dAPC2 IPs (data not shown); because the antigen for the dAPC2 antisera includes the Arm binding region, these sera might not recognize a dAPC2–Arm complex. We also found that a dAPC2 fragment containing the putative βcat binding sites co-IPed with βcat when expressed in the human colorectal cancer cell line SW480 (data not shown).
The hAPC–βcat interaction is direct, and is mediated by the 15 and 20 amino acid repeats of hAPC and the Arm repeats of βcat (for review see Polakis 1999
); the analogous region of dAPC binds Arm (Hayashi et al. 1997
). To test whether dAPC2 directly interacts with Arm, we used the yeast two-hybrid system ( E), examining whether dAPC2's 15 and 20 amino acid repeats interact with the full set of Arm repeats of Arm (R1–13), or with the centralmost Arm repeats (R3–8; the binding site for Drosophila
E-cadherin and dTCF). For comparison, we tested the 15 and 20 amino acid repeats of dAPC (Hayashi et al. 1997
). The full 15 and 20 amino acid repeat regions of both dAPC and dAPC2 strongly interact with the entire Arm repeat region and with R3–8. We also tested 31–34 amino acid fragments carrying individual 15 or 20 amino acid repeats of dAPC and dAPC2 (selected as good matches to the consensus). Individual 15 amino acid repeats of either dAPC or dAPC2 interacted with both the entire Arm repeat region of Arm and with R3–8. An individual 20 amino acid repeat of dAPC also interacted with both Arm fragments. A single 20 amino acid repeat of dAPC2 interacted strongly with Arm repeats 1–13; its interaction with R3–8 was much weaker.
dAPC2 Localization and the Actin and MT Cytoskeletons
To demonstrate that our anti-dAPC2 antisera are specific in situ, we determined that preimmune sera do not specifically stain any structures in Drosophila embryos, even at concentrations 10-fold higher than those we used below (data not shown). Our anti-dAPC2 sera also specifically stain mammalian cells engineered to express dAPC2 but not nontransfected cells (data not shown). The specificity of staining in situ is further supported by the change in intracellular localization seen in a dAPC2 mutant (see below), and by the fact that antisera from a second rat immunized with this antigen recognize a similar set of cellular structures (at least during midembryogenesis, the stage we examined).
Thus, we used our anti-dAPC2 antisera to characterize its expression and subcellular localization. During nuclear division cycles 10–13, which take place without cytokinesis in the peripheral cytoplasm of the embryo, dAPC2 shows dynamic changes in subcellular localization, coincident with those of actin (). Sequential changes in MT organization as nuclei proceed through mitosis direct reorganization of the cortical actin cytoskeleton (for review see Foe et al. 1993
). Before nuclei migrate to the periphery, actin is found at the cortex in a random reticulum. When nuclei reach the periphery, actin condensations appear in interphase and prophase above each nucleus, forming an actin bud which overlays a cytoplasmic bud. This separates the mitotic apparatus of one nucleus from that of its neighbor. As division proceeds to metaphase, actin redistributes from the crown of the bud to its lateral cortex, forming an oblong ring around each spindle. During anaphase, actin redistributes into discs above each newly formed nucleus. Centrosomes and their associated MTs direct the changes in actin distribution, although the mechanism responsible for this interaction is not known.
Figure 3 dAPC2 associates with actin in preblastoderm embryos. (A–C and E–P) Wild-type embryos labeled for dAPC2 (A, E, I, and M), actin (B, F, J, and N), and β-tubulin (G, K, and O). In the merged images of the triple-labeled wild-type (more ...)
In cycle 10–13 embryos, dAPC2 colocalizes with actin at all stages of mitosis (we could not test for colocalization with Arm, as its levels at these stages are too low to detect its localization). The dAPC2/actin colocalization is most prominent in the microvillar projections at the surface of the bud in interphase and prophase (, A–C). At metaphase and anaphase, dAPC2 and actin condensations are observed at the lateral cortex of the bud (, I–L); dAPC2 staining is somewhat less intense here relative to actin. Toward the base of the bud, condensations of actin and dAPC2 are also found in the region of the centrosome and asters (, E–H, arrows). These dAPC2 condensations occur within 0.3–0.5 μm of the surface of the embryo (data not shown), and thus are most prominent above the spindle apparatus; kinetochore MTs are not in uniform focus until ~1.25 μm from the surface of the embryo. The location of these dAPC2/actin condensations above the plane of the spindle places them in a position to interact with the astral MTs as they reach toward the cortex. During later nuclear cycles when pseudocleavage furrows are present, more defined dots of actin and dAPC2 staining are sometimes observed (, I–P, arrows) in the region of the centrosomes. In one of our wild-type stocks, which was infected with the bacterial endosymbiont Wolbachia
(visible as small propidium iodide–positive bodies), we saw an additional APC2 localization. Wolbachia
associate with astral MTs in Drosophila
and thereby disperse into newly formed cells (Callaini et al. 1994
; Kose and Karr 1995
). In infected embryos, dAPC2 localizes with the actin cytoskeleton as in uninfected stocks, and also associates with bacteria at the asters ( D). Another astral MT-associated protein, the kinesin-like protein KLP67A, is also reported to associate with bacteria (Pereira et al. 1997
). EM studies have shown that the bacteria are encapsulated within a cytoplasmic vacuole attached to astral MTs via an electron-dense bridge, possibly composed of cellular MT-associated proteins (Callaini et al. 1994
). dAPC2's localization to the aster region of noninfected embryos and its association with bacteria suggest that dAPC2 may contribute to the binding of the vacuole to the asters.
After cellularization, dAPC2 is still enriched in the region of MTs. Increased levels of cytoplasmic dAPC2 are observed in mitotic domains (groups of cells undergoing synchronous mitosis) ( D). Here, cytoplasmic condensations of dAPC2 are observed in the region of the spindle in metaphase and anaphase ( and , arrows), but are absent in prophase or telophase (the other cells in the mitotic domain in and , are in prophase); serial sections revealed that these cytoplasmic condensations are most prominent within 2–4 μm of the cell apex. In mitotic domains of a Wolbachia-infected strain, we observed punctate condensations of dAPC2 near the spindle poles, presumably astrally associated bacteria ( G), consistent with dAPC2 localization to bacteria associated with preblastoderm asters.
Figure 4 dAPC2 is associated with the cortex in cellularized embryos and with cytoplasmic condensations in dividing cells. (A–C) Wild-type embryos triple-labeled for dAPC2 (A), actin (B), and β-tubulin (C). dAPC2 is associated with the cortex in (more ...)
dAPC2 is also expressed in dividing cells of the larval brain (). The optic lobes contain two proliferative regions, the inner and outer proliferative zones. dAPC2 is highly expressed in dividing cells of the proliferative zones and in their immediate progeny, but not in differentiated neurons ( and ). In contrast, Arm is not enriched in the proliferative zones ( and ) but is enriched in axons. In the ventral nerve cord, Arm is found in axons, whereas dAPC2 is found in midline glial cells ( and ). In contrast, dAPC localizes to axons, at least in embryos (Hayashi et al. 1997
Figure 5 dAPC2 localization in dividing cells of the larval brain. (A–D) Third instar larval brain and ventral nerve cord double-labeled for dAPC2 (A and C) and Arm (B and D). OPZ, outer proliferative zone; IPZ, inner proliferative zone; (more ...)
However, in larval neuroblasts (neural stem cells) dAPC2 and Arm share a striking asymmetric distribution. Neuroblasts divide asymmetrically to produce a large neuroblast and a smaller ganglion mother cell, which will divide symmetrically to produce two neurons (for review see Fuerstenberg et al. 1998
). The asymmetric division requires specific orientation of the mitotic spindle. Inscuteable (Insc), localized in a crescent opposite the future daughter cell during prophase and metaphase, is required for both spindle orientation and localization of the neural determinants Prospero and Numb (Kraut et al. 1996
). In larval neuroblasts, both dAPC2 ( E, arrow) and Arm ( F, arrow) colocalize to a cortical crescent next to the future daughter cell; this crescent also includes the neural determinant Prospero ( and , arrow). In contrast to other asymmetric neuroblast components (for review see Fuerstenberg et al. 1998
), the dAPC2 and Arm crescents are present even at interphase ( E, lower neuroblast). In some neuroblasts, cortical actin also accumulates in a crescent with dAPC2 ( and , arrows), whereas in others this association is less apparent ( and , arrows). To examine the relationship between dAPC2 and the spindle, we triple-labeled neuroblasts with antibodies against phosphohistone, β-tubulin, and dAPC2 (, N–P). One pole of the spindle apparatus colocalizes with the dAPC2 crescent; dAPC2 is enriched at this point relative to the rest of the crescent (, arrows). We also observed low levels of dAPC2 at the opposite cortex at this stage of the cell cycle, the position of which often coincided with the other spindle pole (, arrows). Whereas cortical dAPC2 associated with spindle poles, neuroblasts did not have cytoplasmic condensations of dAPC2 around the central spindle as were observed in epidermal cells. dAPC2 is also asymmetrically localized in embryonic neuroblasts ( G, arrows).
In nondividing cells, dAPC2 also associates with the cell cortex, and colocalizes with actin. In the embryo, dAPC2 is most strongly expressed in the epidermis and other epithelial cells. In the epidermis, dAPC2 is enriched at the cell cortex and is also found throughout the cytoplasm in a punctate distribution ( A). At the cortex, dAPC2 appears as numerous punctate condensations of protein ( A) which are most prevalent at the apical end of the lateral cell surface but are also found more basally. The most intense staining of dAPC2 appears at points of contact between multiple epidermal cells ( A, arrows). dAPC2 condensations often colocalize with condensations of actin ( and , arrows) and phosphotyrosine (data not shown), although actin and phosphotyrosine associate with the cortex more continuously. In fully polarized epithelial cells like the embryonic hindgut ( and ) or the larval imaginal discs ( and ), dAPC2 is enriched in adherens junctions, where it colocalizes with Arm; dAPC2 also accumulates on the apical plasma membrane ( and ). The intracellular distribution of dAPC2 ( E), in contrast to that of Arm ( F), is not modulated in a segmental fashion. A strikingly different localization of dAPC2 occurs in the epidermis after stage 15. dAPC2 becomes organized into very large apical structures in segmentally repeated subsets of ventral epidermal cells ( and ), just before the stage at which these cells initiate denticle formation. The dAPC2 structures occur specifically in anterior epidermal cells of each segment and colocalize with similar actin structures (, I–K), which likely represent larval denticle precursors.
Figure 6 Localization of dAPC2 in epidermal and epithelial cells. The embryonic hindgut (A and B), wing imaginal disc (C and D), and early epidermis (E and F), double-labeled for dAPC2 (A, C, and E) and Arm (B, D, and F). (G) Stage 15 embryo labeled for dAPC2. (more ...)
Although dAPC2 colocalizes with actin in many tissues, it does not colocalize with actin in all contexts. For example, during cellularization, actin is prominent at the cellularization front, whereas dAPC2 is enriched at the apical cortex (data not shown). In addition, as we noted previously, at the cortex of epidermal cells actin is present at the membrane in a continuous fashion, whereas dAPC2 is restricted to regions of most intense actin staining. Finally, dAPC2 is not found with actin in cytokinesis furrows ( and ). Thus, although dAPC2 associates with the actin cytoskeleton, the context-dependent nature of this association suggests that it is regulated.
Biochemical Properties of dAPC2
Biochemical analyses also suggest that dAPC2 associates with the cell cortex. When we fractionated 0–6-h-old embryos into soluble (S100) and membrane-associated (P100) fractions, dAPC2 partitioned almost equally into these two fractions ( A). In contrast, Arm was almost exclusively in the membrane fraction at this stage. The isoforms of dAPC2 in the membrane fraction migrated more rapidly on SDS-PAGE than those in either the soluble fraction or the total cell lysate ( A); because these isoforms are not detectable in total lysate, we suspect that they may arise during fractionation by dephosphorylation. To examine whether dAPC2 might associate with the membrane via a glycoprotein, we used Con A–Sepharose, which can be used to isolate membrane glycoproteins as well as proteins associated with them (e.g., Arm) (Peifer 1993
). A subset of dAPC2 specifically bound to Con A in extracts from 0–6-h embryos ( A; BicD was a negative control). Thus, dAPC2 may be anchored to the cortex via a transmembrane glycoprotein.
Figure 7 Biochemical properties of dAPC2. (A) dAPC2 is found in the membrane fraction. 0–6-h embryonic extract was fractionated into membrane (P100) and soluble (S100) fractions, and sequentially immunoblotted with anti-dAPC2, anti-Arm, and anti–β-tubulin. (more ...)
Identification of a dAPC2 Mutation
We mapped dAPC2
to polytene region 95F1–2 on the third chromosome by in situ hybridization to wild-type and deficiency chromosomes. dAPC2
is removed by Df(3R)crb89-4
but not by Df(3R) crb87-5
(data not shown). All three deficiencies remove crumbs
and thus have a null crumbs
phenotype (Tepass and Knust 1990
); thus, the severe epidermal fragmentation made examination of cuticular pattern impossible. During a genetic screen for suppressors of wg
, we isolated a temperature-sensitive mutation which mapped to this genomic interval by complementation with the same deficiencies, and had a phenotype consistent with that of a negative regulator of Wg signaling (see below). Thus, we evaluated it as a candidate dAPC2
mutation, sequencing dAPC2
from the mutant and comparing its sequence to that of dAPC2
in the parental stock from which the mutant was derived, and in several other wild-type stocks. The mutant and parental chromosomes share 33 polymorphisms relative to the wild-type Canton S; only 8 altered the protein, and most changes are conservative ( D). There is only a single difference between the parental chromosome and the mutant: deletion of three nucleotides, leading to deletion of serine 241. This serine residue falls within an alpha-helix in the third Arm repeat (by analogy to the Arm repeats of β-catenin) ( D). The length of this alpha-helix is invariant among APC family members, and this residue is either serine or alanine (a conservative change) in all APCs. Thus, we refer to this allele as dAPC2ΔS
Whereas homozygous mutant embryos accumulate normal levels of dAPC2, mutant dAPC2 migrates more rapidly on SDS-PAGE than wild-type protein ( M). A portion of dAPC2 in heterozygous mutants, which are wild-type in phenotype, also migrates abnormally (data not shown), suggesting that this is an intrinsic property of mutant dAPC2 rather than a consequence of the mutant phenotype. The subcellular localization of dAPC2 in dAPC2ΔS mutants was dramatically altered at both the permissive (18°C) and restrictive (25°C) temperatures. At the restrictive temperature, dAPC2 association with the cell cortex is essentially abolished, rendering the protein almost completely cytoplasmic ( A vs. D). At the permissive temperature, some cortical dAPC2 remains ( C). In heterozygotes, dAPC2 protein localization is intermediate between mutant and wild-type, as if mutant protein localizes incorrectly despite the presence of wild-type protein ( B). The loss of phosphorylated dAPC2 isoforms observed above ( M) may be a consequence of the loss of cortical association.
Figure 8 dAPC2ΔS mutant protein is mislocalized. Embryos in A–D, K, and L are stage 9 of development. (A) Wild-type embryo labeled for dAPC2. dAPC2 is localized to the cell cortex and the cytoplasm. (B) dAPC2ΔS/+ embryo displays less cortical (more ...)
We also examined the localization of dAPCΔS mutant protein at the restrictive temperature in other tissues. Although dAPCΔS is found in apical buds in the preblastoderm embryo ( and ), it no longer associates with actin structures as does the wild-type protein (, A–C). Furthermore, dAPC2ΔS ( G) does not associate with the apical plasma membrane in the wing imaginal epithelia, marked by the presence of cortical actin ( H). In the larval neuroblasts, dAPC2ΔS is largely cytoplasmic ( and ), although an association with the cortex is sometimes observed ( I, arrow).
dAPC2 Is a Negative Regulator of Wg Signaling in the Embryonic Epidermis
dAPC2ΔS is viable and fertile at the permissive temperature (18°C). At the restrictive temperature (25°C), dAPC2ΔS homozygous mutants derived from heterozygous mothers are viable, indicating that maternal contribution of dAPC2 is sufficient for embryonic development. Heterozygous embryos derived from homozygous mutant mothers are wild-type and survive to adulthood, suggesting that zygotic function is also sufficient. Mutant embryos derived from mutant mothers (referred to below as dAPC2ΔS maternal/zygotic mutants) have severe abnormalities in their embryonic body plan. On the ventral surface, wild-type embryos show segmentally repeated denticle belts interspersed with naked cuticle ( A). In dAPC2ΔS maternal/zygotic mutants, denticle belts are replaced with an almost uniform expanse of naked cuticle ( B), as is observed when wg is ubiquitously expressed ( D). The dorsal surface also has an array of pattern elements marking specific cell fates ( E); cells receiving Wg signal secrete fine hairs. On the dorsal surface of dAPC2ΔS maternal/zygotic mutants, many more cells secrete fine hairs ( F), as they do when wg is ubiquitously expressed (data not shown). Thus, maternal/zygotic loss of dAPC2 function activates Wg signal transduction both dorsally and ventrally, suggesting that wild-type dAPC2 helps negatively regulate this pathway.
Figure 9 dAPC2 is a negative regulator of Wg signaling. (A) Wild-type cuticle pattern with alternating denticle belts and naked cuticle. (B) dAPC2ΔS maternal/zygotic mutants show excess naked cuticle. (C) dAPC2ΔS/Df(3)crb87-4 embryos derived from (more ...)
function at defined developmental time points supports this hypothesis. At the permissive temperature, dAPC2
mutant embryos develop normally into adults and a homozygous mutant stock can be maintained. When we shifted homozygous mutant embryos up to the restrictive temperature at 4 h after egg laying (AEL), they secreted uniform naked cuticle, like animals at the restrictive temperature throughout development. Progressively later upshifts result in intermediate cuticle defects, with increasing numbers of denticles secreted, until by 10 h AEL the pattern is essentially wild-type (data not shown). Conversely, shifts from the restrictive temperature down to the permissive temperature at 4 h AEL fully rescue the pattern, whereas progressively later downshifts result in more and more naked cuticle replacing the ventral denticle belts. Thus, dAPC2
activity is required between 4–10 h AEL, the same time window during which wg
acts (Bejsovec and Martinez-Arias 1991
; Heemskerk et al. 1991
). Somewhat surprisingly, dAPC2
function may be dispensable for adult patterning; mutant embryos shifted up to the restrictive temperature after 10 h and cultured continuously at this temperature develop into apparently normal adults. This could be the result of partial activity of the dAPC2ΔS
allele. However, we suspect that dAPC2ΔS
is at least a strong hypomorph, as placing this allele over a deficiency for the region both in the mother and the zygote, does not increase the severity of the embryonic mutant phenotype at restrictive temperature ( C).
We carried out epistasis analysis to position dAPC2 with respect to other components of the signal transduction pathway. wg; dAPC2ΔS double mutant embryos (with dAPC2ΔS mutant mothers) show a partial rescue of the wg phenotype, with restoration of the normal diversity of cuticular pattern elements and small expanses of naked cuticle ( and ), suggesting that dAPC2 is downstream of wg. There are two possible explanations for the fact that the double mutant does not show the same phenotype as the dAPC2 single mutant: either dAPC2ΔS is not null, or the negative regulatory machinery remains partially active in the absence of dAPC2. If dAPC2ΔS is not null, we reasoned that repeating the epistasis test with dAPC2ΔS in trans to a deficiency removing dAPC2 (Df(3R)crb87-4) might further reduce dAPC2 function, producing a double mutant phenotype more similar to that of dAPC2ΔS alone. However, when we did this, there was no change in the double mutant phenotype ( I), suggesting that dAPC2ΔS may be genetically null for this function. Other components of the Wg signal transduction pathway act downstream of dAPC2. Embryos maternally and zygotically mutant for both dishevelled (dsh) and dAPC2 ( K) show a phenotype indistinguishable from the dsh single mutant ( J), as do embryos maternally mutant for both dsh and dAPC2 that are zygotically dsh/Y; dAPC2ΔS/Df(3R)crb87-4 ( L). Likewise, arm; dAPC2 and dAPC2; dTCF double mutants (derived from dAPC2 homozygous mothers) () are indistinguishable from arm or dTCF single mutants ( and ). Thus, dsh, arm, and dTCF all act genetically downstream of dAPC2; this was expected for arm and dTCF, but was surprising for dsh.
Loss of dAPC2
also leads to ectopic activation of Wg-responsive genes. One target is wg
itself. If the Wg pathway is constitutively activated by removing zw3
function (Siegfried et al. 1992
) or by expressing constitutively active Arm (Pai et al. 1997
), an ectopic stripe of wg
RNA is induced in each segment. A similar ectopic stripe of wg
RNA is seen in dAPC2ΔS
maternal/zygotic mutants ( and ). Similarly, the domain of expression of a second Wg target gene, engrailed
), is expanded relative to wild-type ( and ), as it is in zw3
mutants or in the presence of activated Arm. In addition, a novel phenotype was observed. In dAPC2ΔS
maternal/zygotic mutants ( and ), the levels of Wg protein are higher and Wg extends more cell diameters away from wg
-expressing cells than in wild-type ( C). These effects on Wg protein do not appear to be accounted for solely by ectopic activation of wg
RNA, as they are detected beginning at stage 9 before induction of ectopic wg
, and they are not observed in embryos expressing activated Arm ( F). Thus, the efficiency of Wg protein transport (Dierick and Bejsovec 1998
) appears to be enhanced in dAPC2
Figure 10 Molecular responses in dAPC2ΔS mutants mimic Wg hyperactivity. (A) Wild-type wg RNA is expressed in one row of cells per segment. (B) In dAPC2ΔS maternal/zygotic mutants, an ectopic stripe of wg is induced. (C) In wild-type embryos, Wg (more ...) dAPC2
mutant embryos still respond to Wg signaling, as segmental stripes of stabilized Arm remain ( and ). In dAPC2ΔS
maternal/zygotic mutants, levels of cytoplasmic Arm in all cells are elevated, but cells receiving Wg signal continue to accumulate more Arm than their neighbors ( J). In contrast, zw3
loss of function results in uniform accumulation of cytoplasmic Arm in all cells, eliminating the Arm stripes (Peifer et al. 1994
). Immunoblot analysis of Arm protein from dAPC2ΔS
maternal/zygotic mutants revealed an accumulation of hypophosphorylated Arm ( M). This effect was not as dramatic as that seen in a zw3
mutant ( M), but was similar to that seen upon ubiquitous expression of Wg using the e22c-GAL4
driver (data not shown). Thus, the effect of dAPC2ΔS
on Arm levels is intermediate between that of wild-type and that of zw3
loss of function, suggesting that negative regulation of Arm is reduced but not completely abolished in dAPC2ΔS
activates Wg signaling, we examined whether the change in its localization was simply a consequence of pathway activation. When we activated Wg signaling by ubiquitous Wg expression (via the e22c-GAL4
driver) or by removing zw3
function, the localization of dAPC2 was essentially unchanged, suggesting that pathway activation is not sufficient to eliminate cortical dAPC2 ( and ; data not shown). There was also no apparent change in dAPC2 protein levels or isoforms in zw3
mutants relative to wild-type ( M); this was somewhat surprising as GSK phosphorylates hAPC (Rubinfeld et al. 1996
), and suggests that dAPC2 can be phosphorylated by another kinase.