Spatial expression of the Drosophila segment polarity gene armadillo is posttranscriptionally regulated by wingless

Cell, Vol. 63, 549-560,

November

2, 1990, Copyright

0 1990 by Cell Press

Spatial Expression of the Drosophila Segment Polarity Gene armadillo Is Posttranscriptionally Regulated by wingless Bob Riggleman,’ Paul Schedl, and Eric Wieschaus Department of Molecular Biology Princeton University Princeton, New Jersey 08544

Summary armadillo (arm) is one of a group of Drosophila segment polarity genes that are required for normal patterning within the embryonic segment. Although arm RNA is uniformly distributed in embryos, arm protein accumulates at higher levels in regions that contain wingless, another segment polarity gene which encodes a secreted protein that regulates patterning via cell-cell communication. These local increases in arm protein require wingless activity, and mutations that alter wingless dfstribution produce corresponding changes in the arm protein pattern. These results sug gest that wingless regulates accumulation of arm protein by a posttranscriptional mechanism. Two other segment polarity genes, porcupine and dishevelled, are required for this effect. We also show that arm protein is closely associated with the plasma membrane in virtually ail cell types and often coiocalizes with P-actin. Introduction During Drosophila embryogenesis, the embryonic body is divided into a series of homologous segments along the anterior-posterior axis. Within each segment, ceils also have specific anterior-posterior positional identities. A group of genes required for the formation of different intrasegmental ceil types has been identified; these are referred to as segment polarity genes (Nusslein-Voihard and Wieschaus, 1980). Nine of the known segment polarity genes are required for proper patterning in the posterior region of the segment. Mutations in any of these genes cause posterior ceils to form anterior structures, often producing a mirror image of the anterior region (Figures la and lb). Because these genes share a common phenotype, it seems likely that they affect the same developmental process and may function together as a system. Recently, one of these genes, wingless (wg), was found to be homologous to the mammalian proto-oncogene Ml (Rijsewijk et al., 1987) a secretory factor whose normal function and cellular effects are largely unknown (Papkoff et al., 1987). The molecular and genetic properties of the wg gene have suggested that it is involved in a cellular communication process that determines positional fate. Although the wg phenotype affects a broad region within each segment, wg transcripts are found only in a narrow band of ceils along the parasegmental border (Baker, Present address: Department of Human Genetics, School of Medicine, Salt Lake City, Utah 64112. l

University

of Utah

1987, 1988a). The wg protein, however, is seaeted by these ceils and travels to neighboring regions, affecting their positional fates (van den Heuvei et al., 1989). These findings have led to a model in which wg protein acts as a morphogenetic signal that determines positional fate beyond its site of production. The other segment polarity genes with phenotypes similar to wg would be required to produce or transmit the wg signal, or would be required for the target ceils to detect anchor respond to this signal. Mutations in the armadillo (am?) gan8 produce an embryonic phenotype that is neariy identical to that of wg (Wieschaus et al., 1984; Klingensmith et ai., 1989) and these genes also share a very similar adult phenotype (E. W. et al., unpublished data). Because arm and eg affect two different developmental processes in the same way, it seems likely that these genes have closely associated functions. There is, however, one striking difference between the two loci: the pattern transformations in arm embryos are ceil autonomous (Gergen and Wieschaus, 1988; Wieschaus and Riggleman, 1987) suggesting that arm acts downstream of the extracellular wg signal. In this context, arm may be required for individual target cells to perceive, interpret, or respond to wg signal. To investigate the role of arm in this process further, we cloned the arm gene and examined its transcript distribution (Riggieman et al., 1989). In contrast to wg, we found that arm RNAs are uniformly distributed in the segment. Furthermore, arm transcripts are present in virtually ail ceil types throughout development, including cells that are not known to respond to wg. This observation, as well as the requirement for arm during oogenesis (Wieschaus and Noell, 1986) suggested that arm may be a general factor needed for a variety of cellular processes in addition to those involving wg. in this paper, we use antibodies to arm protein to examine its cellular and subcellular distribution. Results Production and Specificity of Antisera to arm Protein Using fusion proteins, we produced affinity-purified polyclonal antibodies to two different N-terminal regions of the arm protein (see Experimental Procedures). Both antibodies gave similar, strong staining patterns in wild-type embryos, but only weak staining in embryos hemizygous for an arm deficiency (Figure lc), indicating that they both probably recognize epitopes specific for the arm protein. Because the weak staining seen in deficiency embryos had the same distribution as in wild type (Figure lc; see also Figure 5C) and became even weaker in older stages, we suspected that it was due to maternal arm product contributed to the embryo during oogenesis (Wieschaus and Noell, 1986; Riggleman et al., 1989). Support for this view was obtained from Western blots of protein extracts from wild-type and deficiency embryos. As illustrated in Figure 28, the antibody detects proteins of the same size (be-

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df

tact in DF

Figure

W-r

Figure 1. The eon Phenotype tein in Wild-Type and Mutant

and the Relative Embryos

Abundance

of arm Pro-

In wild-type embryos (a), each segment produces denticles in the anterior region and naked cuticle in the posterior region. In embryos hemizygous for arm point mutations (b), cells in the posterior region also produce denticles. This phenotype suggests that arm is required for cells in the posterior region of the segment to assume their proper positional fates. (c) Wild-type (Wr, bottom) and arm deficiency (DF, top) embryos stained with antibody to arm fusion protein. The embryos were fixed at 12 hr of development (stage 13; Campos-Ortega and Hartenstein, 1965). The arm deficiency embryo can be identified by its broad segmental morphology and short body. Note that while arm protein levels are greatly reduced in the 81171deficiency embryo, significant amounts of maternally derived arm protein are still detected, especially in the hindgut (hg) and proventriculus (pv), where arm protein is normally very abundant.

tween 99 and 103 kd) in extracts from both genotypes; however, consistent with the specificity observed in whole mounts, the levels are substantially reduced in the deficiency embryos. In comparison, the amount of actin in the deficiency and wild-type extracts is essentially equivalent (bottom). The similar size of the protein detected in embryos of both genotypes suggests that the weak staining observed in whole-mount deficiency embryos is not a cross-reaction with other arm-like proteins, but instead represents true arm product present in deficiency embryos, presumably of maternal origin.

2. The am, Protein

Is Posttranslationally

Modified

(A) In vitro translation products representing the E9 and El6 forms of arm RNA both migrate at 92 kd on SDS polyacrylamide gels, which coincides well with the 91.1 kd size predicted by the amino acid sequence. (B) Western blots containing approximately equal amounts of protein from wild-type (wt) or arm deficiency (df) embryos were stained with arm antibody (top) or an actin antibody (bottom). Although both extracts contain about the same amounts of actin, the major 99 and 103 kd bands detected by the arm antibody are greatly reduced in the arm deficiency extract. This demonstrates that these proteins are produced by the arm gene, and their large size relative to the in vitro translation products suggests that the arm protein exists in at least two modified forms in vivo.

arm Protein Accumulates at High Levels in Specific Regions of the Embryo Although mutations in arm cause pattern deletions in specific regions of every segment, our previous work had shown that arm RNAs are uniformly distributed within the embryonic segment and are present at similar levels in all embryonic tissues (Riggleman et al., 1969). A quite different result is obtained with arm antibody; instead of uniform staining, we found that the antigen appears to accumulate in a segmental pattern during mid-stages of embryogenesis. Initially, at the blastoderm stage, arm protein is uniformly distributed in all cells of the embryo (Figure 3A). Most of staining is closely associated with the plasma membrane. Because the predicted arm amino acid sequence does not contain a membrane-spanning domain or secretory sequence, we assume that the protein is located in the surface cortex, below the plasma membrane. The first indication of nonuniform arm distribution is observed early in gastrulation (stage 6; Campos-Ortega and Hartenstein,

wingless 551

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armadillo

Protein

Accumulation

1985) when slightly higher levels can be detected in the procephalic lobe, prospective foregut, and the invaginating posterior midgut (Figure 38). A more dramatic alteration in the staining pattern is evident at mid-stage 8. Segmental stripes of arm protein begin to appear, with the ventral regions of the anteriormost stripes arising first (Figure 3C). The striped pattern is even more pronounced at stage 9, and the position and general appearance of the stripes evolve from this stage to stage 13. At stages 9-10, the final number of 15 stripes is completely formed in parasegments O-14, and all of the stripes extend fully to the dorsal edge of the germband (Figures 3E and 3F). Prior to the first postblastoderm cell division cycle, the segments are about six cells wide, and arm expression is elevated in an average of three cells across the segment (Figure 3D). The cells in the interstripe region also stain, but at a lower level. By the time that cell division is completed, the stripes become narrower and they have a more graded appearance (compare Figure 3D with Figure 3H). In addition, they partially separate into dorsal and ventral domains (Figure 3G; see also Figure 4F). The epidermal stripes become less obvious at stage 12 as a result of increased arm expression in the neural and mesodermal cells, but can still be detected until the middle of stage 13, when the ventral epidermal cells flatten out. Modulation of arm protein staining is also evident in nonepidermal derivatives. For example, at stage 9, staining in the head resolves into distinct domains in the stomodeum, labral, and antenna1 regions, while staining in the posterior region is seen in the proctodeum (Figure 3F). In stage 13 embryos, high levels of arm are seen in the hindgut, proventriculus, Malpighian tubules, anus, and posterior spiracles (see Figure 4H). In addition, arm protein begins to accumulate at increased levels in the brain, ventral nerve cord, and certain sensory organs and persists at these locations through stage 18. Alignment of arm Protein Stripes Relative to the Domains of wg Expression We determined the position of the arm protein stripes within the segment using several morphological features. In whole mounts of stage 11 embryos (Figure 3G), the arm stripes appear to lie between the tracheal pits and the parasegmental grooves. This positioning was refined using plastic sections of whole-mount stained embryos and double labeling experiments. The photographs in Figures 3J and 3K show that the heavy accumulation of arm protein stops just anterior to the parasegmental groove and that it extends from the epidermis through the mesoderm. The double label experiment in Figure 31 shows that the engrailed-expressing cells of the posterior compartment (red) lie immediately posterior to the arm stripe (brown). These findings indicate that the cells with elevated levels of arm staining are located in the anterior compartment. The width of the stripes and the fact that they extend only up to the tracheal pits (Figure 3G) suggest that they are restricted to the posterior half of the anterior compartment. While this positioning is consistent with all of our observations, the graded margin of the arm stripe makes it difficult to determine the boundary on a

cell-by-cell basis. For example, elevated staining is sometimes evident in single cells immediately posterior to the parasegmental grooves. These would correspond to the anteriormost cells of the posterior compartment. The positions of the arm stripes correspond rather closely with the regions of wg RNA accumulation described by Baker (1988b). Using whole-mount in situ hybridization (Tautz and Pfeifle, 1989) we compared wg RNA distribution with that of arm protein at different stages of embryogenesis. wg stripes first appear in stage 8-7 embryos. Initially, they are detected only in the ventral regions of the anteriormost segments, but this pattern rapidly evolves into the full 1Cstripe complement (Figure 5A). Elevated levels of arm protein appear at stage 8 in the same locations that contain wg RNA (Figures 4A and 48) and like wg, the arm stripes are first seen in the ventral cells of the most anterior segments. While the evolution of the arm stripes and their position in the embryo mimic that of wingless, wg RNAs could be detected at a given position slightly before the appearance of arm protein. This raised the possibility that wg product may be required for increased arm staining. By mid-stage 9, patterns of wg RNA and arm protein are virtually identical, although the segmental stripes of arm protein are slightly wider than the wg RNA stripes (Figures 4C and 4D). Thus, the arm protein stripes probably correspond more closely with the broader domains of wg protein accumulation that have been reported by van den Heuvel et al. (1989). On the other hand, it is difficult to define borders of the arm stripes precisely, given that the stripes are graded and arise in a background of uniform arm staining. The correspondence between wg and arm patterns, however, becomes even more striking at stage 10, when both sets of segmental stripes separate into distinct dorsal and ventral domains (Figures 4E and 4F). In regions of the embryo outside the segmental stripes, the relationship between wg RNA and arm staining is complicated. For example, at stage 13, both arm protein and wg RNA accumulate at high levels in the proventricuIus, hindgut, anus, and posterior spiracles (Figures 4G and 4H). In contrast, other regions of the embryo (the ventral midline, the margins of the tracheal pits, the mesoderm, and certain structures in the central and peripheral nervous system) show elevated levels of arm in the absence of wg transcript. arm Protein Stripes Do Not Appear to Depend on Transcription The striped pattern of arm protein staining contrasts sharply with the uniform distribution of arm RNA observed by in situ hybridization (Riggleman et al., 1989). This difference is illustrated in the whole mounts shown in Figures 5A and 58 and would argue that elevated levels of transcription are not responsible for formation of the protein stripes. To confirm this suggestion, we analyzed arm protein distribution in deficiency embryos that do not contain the arm gene and can only produce arm protein from uniformly distributed maternal arm RNAs. Although the overall level of arm protein in such embryos is greatly reduced, we observe a pattern of arm stripes essentially identical

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Figure

3. arm Protein

Accumulates

at Specific

Regions

during

Embryogenesis

The distribution of arm protein at different stages of embryogenesis was detected using affinity-purified polyclonal antibodies. Whole embryos are shown with their dorsal sides to the top of the page and their anterior end to the left. (I) and (K) show details of the dorsal region of stage IO and 11 embryos, respectively. Because of germband extension, anterior is to the right in these photos. (A) Cellular blastoderm (stage 5). afm protein is uniformly distributed in the metameric region. Some embryos show slight increases in arm protein levels near the anterior and posterior ends at this stage. (6) Early gastrula (stage 6). arm protein accumulates at high levels in the procephalic lobe (pl) and posterior midgut (pm). (C) Early germband extension (stage 6). The first afm protein stripes (arrowheads) become visible along the ventral side of the most anterior segments. (D) At higher magnification along the ventral midline of a stage 6 embryo, the arm protein stripes include an average of three cells across the segment (about one-half segment width). (E) and (F) Mid-extension (stage 9). On the surface (E), the full number of 15 stripes is visible in parasegments O-14. In an optical section (F), high levels of arm protein are also seen in the prospective labral (lb) and antenna1 (at) regions and the stomodeum (St) and proctodeum (pr).

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Figure

Regulates

4. Comparison

armadillo

Protein

Accumulation

of wg RNA and arm Protein

Distribution

Whole Drosophila embryos were stained for wg RNA (A, C, E, and G) or arm protein (6, D, F, and H) as described. All embryos are oriented with their anterior end to the left; (A)-(F) are shown with their dorsal sides to the top, while (G) and (H) are dorsal views, (A) and (B) In stage 7 embryos, both wg RNA (A) and arm protein (6) are found in the posterior midgut and in a series of segmental stripes that first appear on the ventral side in the anteriormost segments. Because the appearance of arm protein follows that of wg RNA, the wg stripes are complete at this stage while the arm stripes are still forming. (C) and (0) At stage 9, wg RNA (C) and arm (D) protein are found at the same positions in the head, segment, and gut. Note that the arm protein stripes are several cells wider than those of wg RNA. (E) and (F) By stage 11, both the wg RNA stripes (E) and the arm protein stripes (F) have separated into dorsal and ventral domains. As with the initial stripe formation, the change in arm protein location occurs after wg RNA changes position. (G) and (H) In stage 13 embryos, both wg (G) and arm (H) products are found at elevated levels in the stomodeum (St), proventriculus (pv), hindgut (hg), posterior spiracles (ps), and anus (an). The segmental stripes of arm protein cannot be seen easily in this view.

to that in wild type (Figure 92). Thus, uniform maternal transcripts can give rise to arm protein stripes in the absence of zygotic arm transcription. Formation of arm Stripes Is Mediated by wg The close correspondence between wg and arm stripes suggests that wg may be responsible for the increased

arm protein levels. To test this hypothesis directly, we examined the distribution of arm protein in wg mutant embryos. In wg embryos, no localized increases in arm protein levels were observed in the epidermis at the time when the stripes would be apparent in wild-type embryos (Figure 5D). Moreover, elevated levels were no longer detected in those other regions that normally express high

(G) In late germband extension (stage 11) the stripes have split into distinct dorsal and ventral lobes and are located posterior to the tracheal pits (arrow). (H) At higher magnification, the stripes have narrowed significantly at the central and the lateral regions and have a distinctly graded appearance. (I) In embryos doubly stained with antibodies to arm (brown) and engrai/ed (red), the stripe of engrailed expression is observed to lie immediately posterior to the stripe of highest erm staining. (J) and (K) In a plastic section of a stage 11 embryo, the arm stripes are seen to extend about 40% of one segment width anterior from parasegmental grooves. The arm stripes include cells in the epidermal, neural, and mesodermal layers. Note that arm protein has begun to accumulate at high, uniform levels in the mesoderm. In the posterior region, arm protein accumulates in the cells that will give rise to the hindgut and Malpighian tubules (hg) and at the end of the posterior midgut (pm). In (I) and(K), arrows indicate the position of the parasegmental groove and point toward the anterior of the segment.

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Figure

5. The wg Gene

Is Responsible

for Localized

Accumulation

of arm Protein

The distributionsof arm RNA and protein were assessed in wild-type and different pattern formation mutant embryos. All embryos are oriented dorsal to the top and anterior to the left. (A), (B), (C), (D), (G), and (H) are surface views; (E) and (F) are optical sections. (A) and(B) arm RNA in wild-type embryos (A) is uniformly distributed and shows no regional increases that could account for the localized accumulation of arm protein (B). (C) In arm deficiency embryos (=Df(l)OR34/Y), arm protein continues to appear in stripes at the proper location, although these stripes are much weaker than normal. These stripes are formed from low levels of uniformly distributed maternal arm RNA, demonstrating that zygotic arm transcription is not required for their formation. (D) In wg mutant embryos, no localized increases in arm protein are seen in the wg expression domains in the head, gut, and segments. Note that these embryos still have low, uniform levels of arm protein. (E) and (F) In fushi tarazu embryos, arm protein is found in a reduced number of broad stripes at stage 9 (E). By stage 11 (F), the arm protein stripes in the epidermis have become restricted to a narrow region anterior to the parasegmental grooves (arrows). fushi tarazu affects wg RNA distribution in a similar fashion. (G) and (H) In porcupine mutants(G), the arm stripes are much thinner and weaker than normal. As with wg itself, dishevelled embryos (H) do not produce any segmental stripes of arm protein.

levels of arm and wg product (e.g., the head and gut). Thus, wg product is required for increased accumulation of arm protein at these locations as well. Although local increases in arm protein do not occur in the wg expression domains of wg mutant embryos, arm protein is still found in several contexts in these mutants. First, there is still a moderate amount of uniformly distributed arm protein in wg embryos that is approximately equal to the inter-stripe level of arm in wild-type embryos. Thus, the presence of wg product in the stripe region appears to increase the accumulation of arm protein above the low level that would be produced in its absence. Second, increased levels of arm protein continue to occur in wg mutant embryos at positions, such as the ventral mid-

line, that do not correspond to detectable wg expression domains. This suggests that wg is not absolutely required for arm protein accumulation and that other factors might regulate arm levels at these locations. To investigate the relationship between arm and wg further, we examined arm protein distribution in a pair-rule mutant that rearranges the pattern of wg expression. In early stage 9 fushi tarazu mutant embryos, wg RNA accumulates in a reduced number of broad stripes (Ingham et al., 1988; Martinez-Arias et al., 1988). We observed similar changes in the distribution of arm protein (Figure 5E), supporting our hypothesis that the presence of wg product leads directly to increased accumulation of arm protein. In the ectoderm of stage 11 fushi tarazu embryos,

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the broad wg RNA stripes evolve into narrow stripes at the posterior margin of each parasegment (Martinez-Arias et al., 1988); we found that the arm protein redistributes in a similar fashion (Figure 5F). The narrowing of the arm stripes argues that the continued presence of wg product is necessary to maintain increased accumulation of arm. A similar conclusion is suggested by the concomitant dorsal-ventral separation of both wg and arm stripes described above for stage 10 wild-type embryos. To examine the role of other segment polarity genes in arm protein accumulation, we stained mutant embryos with arm antibodies. Three of these genes, naked, patched, and engrailed, affect the expression of wg RNA at midembryonic stages (stage 11, Martinez-Arias et al., 1988) although the initial patterns of wg expression are normal. Mutations in naked, patched, and engrailed do not effect the initial establishment of arm stripes at stage 9, but, as the case with fushi tamzu, all of these genes produced effects on the accumulation of arm protein at later stages, which correspond to their effects on wg expression (data not shown). Of the segment polarity mutants that produce wg-like phenotypes (Cell, cubitus interuptus dominant, disheveled, fused, gooseberry, fused, hedgehog, and porcupine), only two mutations noticeably affected arm protein expression. In disheveled embryos, arm stripes could not be detected and the staining was quite similar to that observed in wg mutants (Figure 5H). The stripe pattern was also disrupted in porcupine embryos. In those embryos, the stripes were often quite difficult to detect, and where they could be observed, they were much narrower and weaker than normal (Figure 5G). Subcellular Distribution and Biochemical Properties of the arm Protein To examine the distribution of the arm protein at later stages of development, we used the N2 antibodies to stain tissues from larvae, pupae, and adults. As the case with arm RNA (Riggleman et al., 1989), we detected arm protein in all cell types that we examined, predominantly at the cell surface (Figures 8a, 8b, and 8~). In larvae, arm protein is at highest levels in imaginal discs and brain, at intermediate levels in gut and salivary gland, and barely detectable in muscle. In some cell types, afm protein is not uniformly distributed around the periphery of the cell, but is concentrated along one side. In some of these situations, we found that the subcellular distribution of arm protein closely resembles that of F-actin. In embryos, for example, both arm protein and actin preferentially localize to the base of the cleavage furrow in cellularizing blastoderms (Figure 8d). Similarly, both are found at the highest levels along the apical surface of the posterior midgut cells in stage 7 embryos (Figure 8e) and appear to have a graded distribution along the lateral surfaces of these cells. Figures 8f and 8g show that both arm and actin are strongly concentrated along the basal and lateral surfaces of ovarian follicle cells. This colocalization may reflect an association between arm and the actin cytoskeleton: following standard cytoskeletal extraction procedures (BenZe’ev et al., 1979) both the 103 and 99 kd arm proteins partition with the cytoskeletal pellet as well as the soluble

supernatant (Riggleman, 1989; B. R., unpublished data). Not all actin-rich structures, however, contain high levels of arm protein. For example, arm protein is not associated with F-actin in muscle fibers or ovarian ring canals (Figure 89). As described above, the major forms of arm protein present in embryos have apparent molecular masses between 99 and 103 kd. This is also true of the arm protein identified in Western blots of extracts from later stages (data not shown). Apparent molecular masses of 99 to 103 kd are significantly larger than those predicted from the amino acid sequence (i.e., 91 kd; Riggleman et al., 1989), suggesting that the protein may be modified in vivo. Consistent with this suggestion, in vitro translation of RNAs representing the two major arm transcripts (E9 and E18) produced proteins with an apparent molecular mass of 92 kd (Figure 2A), very close to that which was predicted from the sequence. Discussion In this study, we found that the arm protein accumulates at high levels in specific regions of the segment. Because the arm protein pattern can be produced both by uniform zygotic RNA (Figure 5A versus 58) and by uniformly distributed maternal transcripts in arm deficiency embryos (Figure 5C), zygotic transcription is probably not responsible for this effect. Instead, some localized factor (or factors) must interact with a uniformly distributed arm product to produce regional increases in arm protein. This interaction might occur at the level of translation, such that uniform arm RNA would be translated at a higher rate in certain cells. Alternatively, it is possible that some localized factor stabilizes uniformly distributed arm protein at certain sites, or alters the accessibility of the N terminal region of the arm protein to our antibody. Several lines of evidence suggest that the wg gene is responsible for the segmental pattern of arm protein accumulation. First, we found that all regions that contain wg RNA in wild-type embryos also show increased accumulation of arm protein. Because the pattern of arm accumulation corresponds more closely with the distribution of the secreted wg protein (van den Heuvel et al., 1989) rather than that of wg RNA (Baker, 1987), it seems likely that it is the protein product of the wg gene that affects arm accumulation. Second, mutations that rearrange the pattern of wg expression produce similar changes in the pattern of arm protein accumulation. Third, in the absence of wg function, arm protein does not accumulate at increased levels within the wg expression domains. Considered together, these results predict that the presence of wg product at a given position in the embryo leads to increased accumulation of arm protein. This interaction represents the only known effect of wg on target cells in the anterior compartment and confirms the close association between arm and wg predicted by their similar phenotypes. These experiments also revealed several general features about the relationship between the arm and wg proteins For example, because the segmental stripes of arm

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Figure

6. The arm Protein

Is Associated

with the Plasma

Membrane

and Colocalizes

with F-actin

In all of the tissues we examined, the majority of arm staining is closely localized with the plasma membrane. (a) Stage 6 embryo (early gastrulation; Campos-Ortega and Hartenstein, 1965) viewed along the ventral furrow. (b) Third-instar larval salivary gland cells. (c) Follicle cells covering a stage 6 (King, 1970) ovarian egg chamber. (d) and (e) Cryostat sections of embryos stained with arm antibodies. In the cellularizing blastoderm, high levels of arm staining are observed at the base of the cleavage furrows (arrow). In the cross-sectioned gastrula, arm staining occurs at highest levels along the apical surface of the invaginating ventral furrow (VF) and posterior midgut cells (PMG). Much lower levels are observed along the basal surface of the posterior midgut cells and in the surrounding epithelial cells. On the lateral surfaces of some posterior midgut cells arm protein has a graded distribution, with the highest levels of arm being present at the apical end. (f) and (g) Optical section of a stage 6 ovarian egg chamber stained with bodipy-phallacidin to detect F-actin (f) and with arm antibody(g), arm protein and F-actin have very similar patterns of distribution along the plasma membranes of the nurse cells (NC) and follicle cells (FC). Both proteins are especially concentrated along the basal surface of the follicle cell epithelium and at junctions between follicle cells. Note that arm protein is not found with actin in the ring canals (RC) or in the muscle fibers of the ovarian muscle cells. The weak arm staining seen around the nuclei of the nurse cells is probably artifactual, since it is not detected by the horseradish peroxidase staining method.

protein were not visible at even the earliest stage in wg mutant embryos, wg appears to be required to initiate accumulation of arm protein. This contrasts with the effects of wg on the engrailed gene, where it is only required to maintain late engrailed expression (DiNardo et al., 1988; Martinez-Arias et al., 1988). However, because the arm pattern evolves along with that of wg, the continued presence of wg product also seems to be necessary to maintain increased accumulation of arm protein. We also found that arm protein accumulates in several regions that are not wg expression domains, and continues to occur at these positions in wg mutant embryos. Thus, wg is not always required for the accumulation of arm protein, and there may be other localized factors at these sites that influence arm accumulation in the same way that wg does. Because the wg protein is thought to act primarily as an extracellular signal (van den Heuvel et al., 1989) it seems unlikely that it interacts directly with either arm RNA or arm

protein. Instead, it is probable that at least several other gene products intervene in the process, establishing arm stripes. The most obvious candidates would be those segment polarity genes that produce embryonic phenotypes similar to arm or wg. We examined the distribution of arm protein in seven other segment polarity mutants in this class and found that only two, porcupine and dishevelled, affect the early striping pattern of arm protein. Interestingly, these loci are thought to be required for the distribution of wg signal, or the ability of neighboring cells to respond to it (Klingensmith, personal communication). Their effects on the arm protein pattern may therefore be mediated through their effects on wg. Our results suggest that these four loci (arm, wg, dishevebd, and porcupine) may have a unique, early role in the patterning that occurs within each segment. Mutations in all four loci produce very similar embryonic phenotypes (Perrimon et al., 1989; Klingensmith et al., 1989) and their

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Figure 7. Spatial Relation of arm Protein Distribution and arm Pattern Defects In the schematic segment shown, trapezoids represent the denticle belts and horizontal lines indicate the segmental and parasegmental boundaries. The regions that are affected by loss of arm function are marked by cross-hatching and the regions containing elevated levels of arm protein during mid-embryogenesis are marked by dark stippling. Anterior is to the top.

products appear to regulate one another during the course of embryogenesis (Klingensmith, personal communication). This interaction is not limited to the effects of wg, dishevelled, and porcupine on arm protein levels reported here. arm expression, for example, is also required to maintain the normal wg pattern and, although stripes of wg RNA are established normally in an arm gastrula, they begin to decay early during germband extension and are completely abnormal by stage 11 (Fiauskolb and E. W., unpublished data). The other segment polarity genes (hedgehog, gooseberry etc), which do not affect the initial arm stripes, do not appear to be a part of this early system and may be required primarily for the later maintenance of the intersegmental pattern and thus function downstream of these four loci. Alternately, they may identify early pathways (e.g., maintenance of engrailed; Mohler, 1988) that operate initially in parallel to, and thus independent of, the armlwg process. What Are the Cellular and Developmental Effects of Increased am Levels? Each embryonic segment consists of two developmentally distinct regions, an anterior compartment and a smaller posterior compartment, separated by a parasegmental boundary (Martinez-Arias and Lawrence, 1985). The arm protein stripes occupy the posterior half of the anterior compartment and correspond with the region that is affected by loss of arm function in the anterior compartment (Figure 7). It is this region of the segment where clones homozygous for arm induced at the blastoderm stage produce cell-autonomous anterior transformations in fate (Gergen and Wieschaus, 1986; Wieschaus and Fliggleman, 1987; Klingensmith et al., 1989). This autonomy argues for a direct effect of arm in this region. Normal development of the posterior compartment also requires that the embryo possess a wild-type arm gene (Figure 7) although arm protein is not found at high levels there. arm

activity may only be required at low levels in the posterior compartment. The integrity of the posterior compartment, however, requires the continued presence of a normal anterior compartment (DiNardo et al., 1988; Martinez-Arias et al., 1988). The arm patterning defects in that region might be an indirect result of a requirement for arm in the anterior compartment. Although all cells express arm protein, the regions within the segment with the highest levels correspond to the cells where arm ‘s effects have been shown to be autonomous and therefore direct. This suggests that the striped accumulation of arm, which occurs at gastrulation, is a significant aspect of its segmental phenotype. In one extreme view, the absolute level of arm might specify different positional fates in the segment (Wolpert, 1969; Lawrence, 1981; Driever and Nijsslein-Volhard, 1988). Given that the level of arm is established in response to wg signal, this level may be an intracellular reflection of the extracellular wg cues. In an alternate view, absolute levels of arm may not be important, as long as the levels are above a certain threshold. In this model, wg might induce high arm levels to ensure adequate levels of arm product, but the actual level would not carry positional information. These two possibilities might be distinguished when transgenic lines are obtained that express the arm protein at elevated levels throughout the segment. Until the cellular function of arm protein is better understood, its role in establishing anterior fates is speculation. Our preliminary characterization suggests that arm is extremely abundant in the Drosophila embryo (Riggleman et al., 1989) and that a substantial fraction is membrane associated and colocalizes with actin. Interestingly, a number of proteins involved in signal transduction (e.g., the nonreceptor tyrosine kinase v-sfc; Hamaguchi and Hanafusa, 1987) are localized to the cell surface and may be associated with the actin cytoskeleton. Genetic arguments suggest a role for the arm protein in signal transduction. Its putative actin association may simply provide a passive anchor, ensuring a location of the transduction mechanism to the cell cortex. On the other hand, it is possible (by analogy to Drubin et al., 1990) that a cytoskeletalbased system is one of the major targets for the wg signal and that arm may be a major component of that system. Many of the subcellular regions where arm protein is found at the highest levels, such as the furrow canals during cellularization and the apical surface of the posterior midgut, are regions subject to increased mechanical stress. Although the cells in the stripe that show high levels of arm are morphologically indistinguishable from the neighboring cells with low arm levels, it is possible that because they are involved in wg signal transduction, they have special adhesive or structural requirements, which are filled by the arm-associated cytoskeleta/ system. These cellular properties would have to be an essential part of the wg signaling process, since embryos homozygous for arm or wg are phenotypically indistinguishable in their morphology at intermediate stages, in their effects on engraiki expression, and in their final differentiation pattern (Klingensmith et al., 1989; Rauskolb and E. W., unpublished data). This raises the possibility that all of the

cellular roles of wg in segmentation are mediated via arm. If an arm-associated cytoskeletal system plays the role outlined above, then identifying that system and elucidating its role in the signal transduction process may be crucial to understanding how positional information is established and maintained across the segment. Exparlmental

Procedures

Fly Stocks and Methods All wild-type embryos were from the Oregon R strain (Lindsley and Grell, 1966). Mutant stocks, their respective alleles, and the source of the mutations used in this study are as follows: gooseberry (2X62), engrailed (lK57), patched (lN106), and wg (lG22) from Niisslein-Volhard et al. (1964); wg(CX4) from Baker (1987); fushi tarazu (7817), hedgehog (13C), and neked (7E89) from Jurgens et al. (1984); a deficiency uncovering both Cell and cubitus interuptusD (My from Orenic et al. (1987); fused (94) from Bob Holmgren (unpublished data); dishevelled o/26) from Perrimon and Mahowald (1967); and porcupine from Perrimon et al. (1989). porcupine and dishevelled germline clones were produced as described in Perrimon and Mahowald (1987). The erm deficiency embryos were produced as XP30 segregants of the stock T(1,3) OR34/FM6 (Lindsley and Grell, 1966). All flies were raised under standard conditions as described by Roberts (1966). Production of Antibodles to the arm Protein Our general strategy for antibody production was to attach small regions of the arm protein to two different bacterial proteins and to use one of these proteins to immunize rabbits and the other to affinitypurify the arm-specific antibodies. We cloned Sau3A restriction fragments from the arm ES cDNA into the SamHI site of pTRB-0 (Burghlin and deRobertis, 1987) and pATH2 (Dieckmann and Tzagoloff, 1985) to produce 3-galactosidase and TrpE fusions, respectively. The regions of the arm E9 cDNA used were: Nl, 1880 bp to 2110 bp (47 amino acids); and N2, 2011 bp to 2175 bp (56 amino acids). Base pair positions are as given in Riggleman et al. (1989). The fusion proteins were prepared in one of several ways. The NP-La&! fusion was purified in its native tetrameric form by column chromatography as described by Carroll and Laughon (1987). The Ni-LacZ fusion protein was prepared by boiling the cell pellets from a 500 ml culture in 5 ml of 2x SDS sample buffer for 5 min, sonicating the mixture briefly, and separating 2 ml of the resulting protein solution on a 7% preparative polyacrylamide SDS gel (Laemmli, 1970). The fusion protein was excised from the gel and collected by electroelution; residual SDS was removed by dialysis against PBS. All trpE fusion proteins were prepared from insoluble aggregates. Cell pellets of 1 liter cultures of JMlOl cells (Messing, 1963) producing these fusion proteins were resuspended in 10 ml of buffer A (50 mM MOPS [pH 7.41, 10 mM EDTA, 10 mM j3-mercaptoethanol, 200 pglml PMSF) containing 4 mg of lysozyme and held on ice for 30 min. The cell pellet was frozen at -7oOC, thawed rapidly, and sonicated until the viscosity was reduced. The extract was centrifuged at 13,000 x g for IO min and resuspended in 30 ml of buffer A containing 6 M urea. The suspension was centrifuged at 13,000 x g for 10 min, and the supernatant was dialyzed against 50 mM MOPS (pH 7.4) until protein began to precipitate. Fusion proteins prepared in this fashion were about 90% pure, while those prepared by the other two methods were greater than 95% pure. Antisera were produced by injecting (subcutaneously and intradermally) young New Zealand rabbits with *l mg of LacZ fusion protein in 1 ml of a I:1 emulsion of PBS:Freund’s complete adjuvant. At 21,31, and 41 days after the initial injection, rabbits were boosted with 208 wg of fusion protein in Freund’s incomplete adjuvant. Rabbits were bled every 2 weeks beginning on day 41. Serum was prepared by centrifugation and stored at -20°C until needed for affinity purification. Affinity matrices were prepared by coupling 50-100 mg of trpE fusion protein to 4 ml of a 1:l mixture of Affigel-10 and Affigel-15 (Biorad; Richmond, CA) according to the manufacturer’s directions. The matrices were washed with TENT buffer (100 mM NaCI, 1 mfvl EDTA, 50 mM Tris [pH 7.51, and 0.1% Triton X-100), mixed for 5 min with TENT buffer containing 7 M urea, and washed 3 times with normal TENT buffer. Postimmune serum was bound to the matrix by shaking overnight at 4OC, and the matrix was rinsed 3 times with TENT buffer; it

was then poured into a column and washed with TENT buffer extensively. Antibodies were eluted by adding 15 ml of elution buffer (100 mM glycine adjusted to pH 2.7 with acetic acid, 1 mg/ml BSA, 0.15 mM sodium azide) and collecting the elutate into a tube on ice containing 0.75 ml of 1 M Tris (pH 6.3) after the pH of the eluate had dropped below pH 5. Remaining antibodies to 6-galactosidase and other bacterial proteins were removed by adsorbing the eluate to a matrix containing 200 mg of total protein extract from JMIOI cells containing the pTRB-0 plasmid with no arm insert. Antibodies were stored at -2OOC after the addition of glycerol to 40% final concentration. Antlbody Stalnlng of Embryos and Tissues Embryos were fixed and stained with primary antibody according to the method of Mitchison and Sedat (1963). Primary antibody was detected using biotinylated secondary antibody, horseradish peroxidase-conjugated avidin, and diaminobenzidine from the Vectastain ABC Elite kit (Vector Systems, Burlingame, CA). To double label embryos for both arm and engraifed protein distributions, fixed embryos were first incubated with a mouse monoclonal antibody to engrai/ed/i’cted(Patel et al., 1969) and stained with Promega goat antimouse alkaline phosphatase-conjugated secondary antibody, using Vector Red (Vector Labs) as the color substrate. The embryos were then processed with arm antibody and stained with HRP as usual. Ovaries, salivary glands, and imaginal discs were prepared by dissecting these organs from well-fed animals in PBS and fixing them in 4% formaldehyde for 30 min on ice. Cryostat sections (7 urn thick) were prepared as described by Pesacreta et al. (1969). In the immunofluorescence experiments, arm antibody was detected with a Lissamine rhodamine-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA). F-actin was detected using biodipy-conjugated phallacidin (Molecular Probes, Eugene, OR). For plastic sectioning, embryos were stained darker than usual, dehydrated, embedded in Epcn 812, and cut into 4 nm sections using a glass knife according to Wieschaus and Sweeton (1988). Biochemical Analysis of arm Pmteln arm RNAs were prepared in vitro as described by Melton et al. (1984) with T7 RNA polymerase (Boehringer Mannheim, Indianapolis, IN) using the E9 and El6 arm cDNA clones (Riggleman et al., 1989) as a template. These RNAs were translated in vitro using a rabbit reticulocyte system (Promega, Madison, WI) according to the manufacturers directions. The proteins were electrophoresed on a 7% polyacrylamide SDS gel (Laemmli, 1970) and transferred to a nitrocellulose filter with a semi-dry blotting apparatus, and the filter was exposed to film. This method was used to allow the sizes of the radiolabeled proteins to be compared directly (i.e., on the same gel) with unlabeled Drosophila proteins. To compare levels of arm protein in wild-type and deficiency embryos, ~150 arm deficiency embryos were collected by selecting embryos that exhibited the deficiency phenotype at about 6 hr of develop ment under a dissecting microscope. After 6 more hr, these embryos were ground in 200 ul of 2x SDS sample buffer in a mortar. The extracts were boiled for 2 min and 40 ul was loaded on a 7% SDS polyacrylamide gel alongside 40 PI of protein extract prepared by the same method from an equal number of wild-type embryos. The proteins were transferred to nitrocellulose and incubated with a I:100 dilution of arm N2 antibody (upper section of the filter) or a I:1000 dilution of a monoclonal anti-actin antibody (Amenham, Arlington Heights, IL) (lower section of the filter) as described by Driever and N(isslein-Volhard (1988). The primary antibodies were detected using alkaline phosphatase-conjugated secondary antibody and NBT/BCIP substrate (Promega, Madison, WI). RNA Detection Methods wg RNA was detected using the whole-mount in situ hybridization procedure described by Tautz and Pfeifle (1989). The probe was was the 0.9 kb Hindlll-EcoRI fragment of the full-length wg cDNA2 clone (Rijsewijk et al., 1987).

We wish to thank Bob Holmgren and John Klingensmith for providing stocks, Roe1 Nusse for providing the wg cDNA, and John Klingen-

wingless 559

Regulates

armadillo

Protein

Accumulation

smith, Norbert Perrimon, Marcel van den Heuvel, Peter Lawrence, and Roe1 Nusse for communicating results prior to publication. We would also like to thank Edward Kennedy, Alan Frey, and Ann Fling for advice on antibody production; Dari Sweeton for preparing plastic sections of arm antibody-stained embryos; Don Doering, Shige Sakonju, and especially Mark Peifer for helpful comments on the manuscript; and our Drosophila colleagues for useful discussions during the course of this work. This research was supported by NIH Grant 5ROlHD22780 to E. W. and grants from the NIH and March of Dimes Foundation to I? S. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact.

Lawrence, I? A. (1981). The cellular Cell 26, 3-10.

Received

Messing, 20-77.

May 15, 1990; revised

July 9, 1990.

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