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Autonomous requirements for the segment polarity gene armadillo during Drosophila embryogenesis
Cell, Vol. 49, 177-184,
April 24, 1987, Copyright
0 1987 by Cell Press
Autonomous Requirements for the Segment Polarity Gene armadillo during Drosophila Embryogenesis Eric Wieschaus and Robert Riggleman Department of Biology Princeton University Princeton, New Jersey 08544
Summary Embryos hemizygous for armadillo produce a %egment polarity” phenotype in which the naked posterior two-thirds of each segment is replaced by denticles with reversed polarity. Small patches of homotygous arm ceils induced by mitotic recombination also form such denticles, indicating that the changes in cellular fate observed in homozygous arm embryos are autonomous at the level of single cells. Clonally derived arm patches do not, however, show the characteristic arm polarity reversals, arguing that this feature of the phenotype depends on cell interactions in fully mutant embryos. Pew, if any, clones were found in the posterior-most regions of the naked cuticle, and none were found in the posterior compartments of the thorax. Introduction Although the final cuticle pattern of the Drosophila embryo is composed of discrete denticles, hairs, and sensilla, classical experiments suggest that the underlying anterior-posterior information is graded or continuous in nature (Locke, 1967; Bohn, 1970). When epidermis is wounded, cells are juxtaposed that normally would not have been adjacent to each other in the wild-type pattern. This juxtaposition induces an intercalary growth which eventually smoothes out the disparity in positional values. Intercalation can result in regeneration of the normal pattern, or in local polarity reversals and mirror images (French et al., 1976; Wright and Lawrence, 1981). The extent of regeneration in both cases, however, depends on the juxtaposition, each cell being only subtly different from its immediate neighbors, but very different from cells further away in the pattern. The graded nature of the positional field observed in these experiments can be modeled assuming a diffusible morphogen gradient (Lawrence et al., 1972) but the relative stability of positional fates following transplantation argue that these graded differences can somehow be maintained within cells, even following surgical manipulation. Among the mutants that affect segmental pattern in Drosophila (Niisslein-Volhard and Wieschaus, 1980) a number of loci have been identified that cause pattern alterations repeated in each segment. In the most common phenotype, the posterior two-thirds of the pattern is replaced by a mirror image of the anteriorly situated denticle rows. This ‘segment polarity” phenotype is produced by mutations in at least six different loci (armadillo, fused, wingless, gooseberry, hedgehog, and ciD) and is reminiscent of the polarity reversals produced in other insect
epithelia following surgical manipulation (Wright and Lawrence, 1981). These genes are required zygotically, at least during the first half of embryogenesis, and may play a role in the establishment or maintenance of positional information within each segment. In this paper we present a detailed analysis of one of the segment polarity loci, armadillo (Wieschaus et al., 1984). The major concern of our analysis was the extent to which the patterns observed in arm embryos are the result of local autonomous requirements for arm product in those cells whose fates are switched by the mutation. To test for such autonomy, we used mitotic recombination to produce very small patches of afm cells in the posterior regions of the embryonic segment primordia. Results The armadillo Phenotype In wild-type embryos, segmentation is most obvious in the repeated pattern of denticle band and intervening naked cuticle on the ventral side in the first instar larva. In embryos homozygous for armadillo, the naked cuticular part of the pattern is eliminated. In its place, an apparent mirror image of the denticle band is formed with reversed polarity. The size of this “mirror image” region is smaller than that which would normally be occupied by the missing naked cuticle, and although it is likely that some of the naked cuticle precursors contribute to the denticles (see below), the exact relationship is not known. The pattern in armadillo embryos is somewhat variable, even when the zygotic and maternal genotypes are kept constant. Comparison of the phenotypes produced by strong and weak alleles suggests that the naked regions of the cuticle are most sensitive to reductions in arm activity and the middle of the denticle band least sensitive (Figure lb-lc). In most of its basic features, the phenotype of armadillo embryos is similar to those produced in embryos homozygous for other segment polarity mutants. One of the peculiarities that distinguishes armadillo from the other mutants in this class is that afm+ transcription is absolutely required in female germ cells for them to complete oogenesis (Wieschaus and Noell, 1986). Some of this afm+ product remains in the egg and influences the phenotype of mutant embryos, making it less extreme. Since it is impossible to obtain eggs unless the mother had at least one active wild-type allele during oogenesis, the embryonic afm phenotype is always viewed in the background of this maternal afm+ component. Thus, although the arm alleles used in this study can be classified as amorphic in a genetic sense (see Experimental Procedures), the phenotypes that they produce clearly do not represent development in the total absence of arm product. Homozygosity for armadillo Causes Autonomous Changes in Cell Pate The absence of naked cuticle suggests that the afm pattern might arise because the cells that normally make
Figure 1. Phase-Contrast Micrographs of the Thoracic and Anterior Abdominal Regions in Wild-Type and armadillo Mutant Embryos (a) The wild-type pattern. The denticle bands are separated by large regions of naked cuticle, and most of the denticles point posteriorly. Each segment is about 13 cells long and the denticle band occupies the anterior 4tI% of that length. In the third larval instar (a stage when the epidermal cells can be most accurately counted), there are about 88 cells underlying the denticle region of the pattern and about 118 cells in the naked cuticular region that follows. Since each denticle band averages 243 denticles, each epidermal cell secretes about 2.8 denticles. No cell division occurs in the epidermis between embryogenesis, and the third larval instar and the denticle pattern changes only slightly. (b) A weak arm phenotype produced by a hypomorphic allele.(armXM1s) in an embryo derived from wild-type attached-X mother. The denticle band is expanded in size and patches of intervening naked cuticular region are observed in the thorax and occasionally in the abdomen (-). (c) A strong arm phenotype produced by an apparent amorphic allele (armyM) in an embryo derived from a heterozygous mother. The mirrorimage belts are now fused into a lawn of denticles with no intervening cuticle. The segmental organization is still apparent because of the repeated polarity reversals.
naked cuticle are reprogrammed to make denticles instead. Such change8 might occur by autonomous genetic switches in single ceils, or by a more global reprogramming associated with regeneration or shifts in diffusible gradients. No large scale nonautonomy was observed in large patches of arm cells produced by Ring-X loss (Gergen and Wieschaus, 1986) but a more rigorous test for autonomy would rely on the behavior of clones of one or two arm ceils in a naked cuticular region which is otherwise totally wild-type. One obvious way of producing such patches is by mitotic recombination in arm heterozygotes at the blastoderm stage. The blastodermal precursors for the epidermis divide only twice before they begin differentiation (Szabad et al., 1979), and clones produced by a blastoderm irradiation should therefore be only one to two cells in size when they make cuticle. Since each differentiated epidermal cell secretes about three denticles, it should be easy to detect any arm clones in the naked cuticle if they are shifted toward making denticles. In the first set of experiments, the irradiations were carried out using an unmarked armadillo chromosome, since the markers available for embryonic cuticle would either reduce the denticle size or make the arm clone difficult to detect. Three hundred and thirty-four heterozygous larvae were examined following irradiation at the blastoderm stage and a total of 95 denticle patches were detected in the naked cuticle (Figures 2a-2e). The frequency with which such apparent clones were produced depended on
the irradiation dosage: fewer were detected following the short two-minute irradiations than following four- or fiveminute irradiations, and no patches were identified in unirradiated heterozygotes (Table 1). Several feature8 of the denticle patches argue that they do in fact represent the armadillo homozygous clones. The average size of the clones was between six and seven denticles, only slightly larger than that predicted for homozygous arm clones two cells in size (see legend to Figure 1). The frequency of clones per animal (0.34) obsewed following four-minute irradiations is about that predicted from the known sensitivity of blastoderm cells to this dosage (1 mitotic recombination/300 irradiated cells; Wieschaus and Gehring, 1976) and the estimated number of blastoderm precursor cells that give rise to the storable naked cuticular regions of Al to A6 (133 cells, i.e., 6% of an epidermal primordium of 2200 cells; Lohs-Schardin et al., 1979; Szabad et al., 1979). As a final test for the clonal origin of the denticles, we carried out a second set of irradiations, using the shavenbaby mutation to mark the homozygous clones. The cuticular marker shavenbaby (Gergen and Wieschaus, 1986) reduces the size of the larger denticles and removes most of the smaller ones. arm shavenbaby homozygotes have only about a quarter the number of denticles as arm svb+ embryos, and all of these are very small. Only half as many clones were detected when arm svW+ blastoderms were irradiated, and the clones that were recovered averaged only 2.6 den-
Blastoderm 179
Figure
Clones
2. Homozygous
for armadillo
armadillo
Clones
Produced
by Irradiation
at the Blastoderm
Stage
(a-e) Unmarked clones produced in embryos heterozygous for arrrtXp~ or armyD35; (f-g) Marked shavenbaby armadillo clones produced zygous armxp33 svb embryos; (h) Extra denticle rows (+) at posterior margin of the naked cuticle in an irradiated svb arm heterozygous Such denticles are probably not of clonal origin, given that they do not show the svb phenotype.
iln heteroembryo.
Cdl 160
Table
1. Clone
Frequency
in Irradiated
arm Heterozygotes Embryos with Clones
Embryos Examineda
Number of Clones
Frequency
Genotype
Age
Dose
arm/+
-
0
96
0
0
0
0
arm/.+ arm/+ arm/+
3 hr 3 hr 3 hr
2 4 5
70 106 156
6 31 56
6 37 73
6.6 34 47
5.7 6.4 7.4
arm/+ arm svb/ +
3 hr 3 hr
4 4
104
100
29 20
37 20
36 20
6.4 2.3
arm/+ arm/+ arm/+ arm/+ arm/ +
4 hr 5 hr 6’ hr 7 hr 6 hr
4 4 4 4 4
97 99 66 97 99
46 43 6 4 1
69 61 6 4
71 61 7 4 1
6.4 5.0 4.0 2.5
Sizeb
w-4
1
PI
a The number of embryos examined was based on the fraction of the total number of differentiated embryos from the cross that were expected to be of the appropriate genotype. This fraction is one half for all experiments listed above, except for that involving arm svb and the corresponding control, where only one quarter of the embryos are of the appropriate genotype. b Data excludes denticle patches in the posterior region of the naked cuticle (see text). Average clone size was calculated as the average number of denticles per clone; bracketed figures indicate values based on only one or two clones.
Table
2. Clone
Genotype
armadillo wingless QfJo=beny
T(l;2)BhVgoosebeny hedsehos +/+ control
Frequencies
Following
Irradiation
of Heterozygotes
Embryos Examineda
Embryos Clones
172 265 122
51 4 2
44 259 126
with
for Other Number Clones
Segment
Polarity
of
Frequency
Mutants
tw
SW
56 5 2
34 1.6 1.6
7.0 4.4 [12.5]
0
0
0
0
3 1
3 1
1.1 0.6
7.7 I51
Posterior ROWSC
Denticle
7 9 7 6 10 0
a The number of embryos examined was based on the fraction of the total number of differentiated embryos from the cross that were expected to be of the appropriate genotype. This fraction is one half for all experiments listed above, except for that involving T(1;2)E/d where only one-third of the differentiated embryos are of the appropriate genotype. b Size was calculated as the average number of denticles per clone; bracketed figures indicate values based on only one or two clones. c These are the questionable posterior clones described in the text. Only patches of five or more denticle have been included.
titles per clone. Moreover, excluding the exceptional posterior patches described below, all clonally derived denticles had the small size characteristic of svb (Figures a-29). The only area where clonal designation of denticles was ambiguous was at the posterior margin of the naked cuticle, immediately anterior to the adjacent belt. Among the irradiated arm/+ heterozygotes, we found several individuals with an additional, partial row of denticles in this area. Although extra denticles were occasionally observed in this area in the wild-type control, they were never arranged in large rows of more than five denticles (Table 2). In the arm/+ heterozygotes, the denticles were found much more frequently in the posterior abdominal segments and were often bunched together at the ventral midline in segment A7. Similar rows were occasionally found following irradiation of heterozyotes for other segment polarity mutations (Table 2). In the arm svb/+ experiment, the denticles in such rows were not shavenbaby (Figure 2h), and therefore we conclude that they are probably not the direct products of homozygous arm clones. Because of the uncertainty of their origin, they have been excluded from the data presented in Table 1 and given below.
Homozygous armadillo Clones Are Not Uniformly Distributed and Do Not Always Show the Polarity Reversals Characteristic of the Homozygous Embryo Although removal of the wild-type afm gene from the naked cuticle precursors at the blastoderm stage appears to shift the cell into a more anterior, denticle-secreting pathway, not all regions of the naked cuticle show the same sensitivity to the loss. Figure 3 shows the distribution and average polarity of 90 clones induced in abdominal segments Al through A6 following irradiation at the blastoderm stage. Almost all clones are restricted to the anterior two-thirds of the naked cuticle (Figure 3). Most of the exceptions were located along the posterior margin of the naked cuticle, in that region where the clonal nature of additional denticles is doubtful. The existence of a posterior boundary in clonal extent is also suggested by the drawnout morphology of those clones that extend into the posterior limit (Figure 26). To define this boundary more precisely, we scored a fraction of the preparations for clones in the thorax. Although the smaller size of the thoracic denticles makes the clones harder to detect, they can be localized rather precisely relative to the Keilin’s organs that mark the anterior-posterior compartment bor-
Blastoderm 181
Clones
for armadillo
”
0 1.’ m
the anterior-posterior extent of a single clone. The values to the right indicate relative positions within the naked cuticle. Each bar is shaded to indicate the fraction of the denticles in the clone that point anteriorly(i.e., potentially have reversed polarity). Although all clones are depicted as having identical lateral extents, the actual clone patterns varied greatly. The six bars marked with X’s mark the positions of dubious clones which were initially scored as being on the posterior margin. Data is based on a subset of the clones identified following the irradiations described in lines four and five of Table 1
0% <25% <50% <75%
der in each segment (Struhl, 1964). All 14 thoracic clones found were located anterior to, or exactly at the same level as, the Keilin’s organs. This suggests that arm clones induced in the posterior compartment either die or do not make denticles. Alternatively, it is possible that such clones migrate forward as a result of their anterior transformation in fate (see Discussion). In embryos homozygous for armadillo, the naked cuticle region of the pattern is replaced by denticles with reversed polarity, i.e., denticles that point anteriorly. In contrast, about 70% (359/526) of the denticles in the arm clones have normal polarity. Anteriorly pointing denticles are especially rare (
surprising, given that mitosis is needed to produce homozygous clones and little proliferation is observed in the ventral epidermis following 6 hr of development (Hartenstein and Campos-Ortega, 1965). Other Segment Polarity Mutants Do Not Produce Denticle Patches When Heterozygotes Are Irradiated at the Blastoderm Stage In addition to armadillo, we have also tested three autosomal segment polarity mutations for the ability of homozygous clones to cause patches of denticles in the naked cuticle. Blastoderms heterozygous for either wingless, gooseberry, or hedgehog (NDsslein-Volhard and Wieschaus, 1960) were irradiated following the same procedures as those used with armadillo and about 150 individuals scored for each locus. The yield of apparent clones was extremely low, only 5% of the frequencies obtained using armadillo (Table 2). Because we have no independent system for estimating the frequency of mitotic recombination on the autosomes, it is difficult to evaluate the negative nature of this result. In a detailed analysis of clone frequencies in abdominal histoblasts, GarciaBellido (1972) showed that autosomes undergo mitotic recombination at one-half to one-third the rate measured for the X chromosome. We believe it therefore unlikely that the extremely low yields of wg, hh, and goo clones simply reflect a lower induction rate for autosomal clones. This possibility has been eliminated for gooseberry by showing that even when a duplication (Dp(7;2)6/1$ was used to move the wild-type allele to the X chromosome, the clone frequency was not increased (Table 2). Instead, it seems that homozygous clones for these loci when produced at the blastoderm stage do not normally result in ectopic denticles in the naked cuticle. The occasional denticle patches we have observed using these mutants may represent rare autonomous exceptions to this rule, or cases
Cell 182
where a dying homozygous clone has altered the pattern of the adjacent wild-type cells. Given their low frequency, they might also represent unusual cases of pattern regulation following X-ray damage.
The Autonomy of the armadillo Phenotype and the Role of the armadillo Gene Product in lntrasegmental Patterning The best developed models for intrasegmental patterning in insects involve either a diffusible gradient which establishes concentration differences along the anterior-posterior axis (Locke, 1987; Lawrence et al., 1972; Meinhardt, 1982) or local cell-cell interactions between cells of slightly different positional identity (French et al., 1978; Wright and Lawrence, 1981). In both models, cell fate depends heavily on the communication between cells or long-range properties of the field. Indeed, the pattern of denticles and sensilla in each segment is so complex that it is hard to imagine its being constructed by single cells without cell interactions and other inherently nonautonomous phenomena. In the experiments described in this paper, we used genetic mosaics to test whether the transformations observed in embryos mutant for different segment polarity genes are autonomous at the level of single cells. Of the four segment polarity loci we tested, only armadillo produced small denticle patches with the morphology predicted of autonomous clones. We were able to produce such clones by irradiations made any time up until midextended germ band stages. The negative results obtained with later irradiations can be explained by the difficulty of inducing homozygous clones at stages with little or no proliferation. The shifts in fate observed following irradiations at 8 hr imply that either the perdurance of zygotic arm product is very small, or that the gene may not be active prior to that point. In contrast to the results obtained with armadillo, irradiated wingless, gooseberry, and hedgehog heterozygotes produced no more denticle patches than did the wild-type controls, except that they, like irradiated armadillo heterozygotes, occasionally showed an additional large row of denticles immediately anterior to the normal denticle band. We have not interpreted these rows as clones, even though they were only found after irradiation of segment polarity heterozygotes, as they were not always marked with shavenbaby when arm svb/+ heterozygotes were irradiated. Although the otherwise negative results obtained with wingless, gooseberry, and hedgehog might be explained by perdurance of wild-type products made prior to clonal induction, it seems more likely to us that at least some of these mutants produce their phenotypes by disrupting the system in nonautonomous ways. The products of the wild-type genes may actually diffuse from one cell to the next. On the other hand, their segment polarity phenotype may be a secondary consequence of autonomous, local disturbances which have long range effects on the patterning (i.e., cell death followed by regeneration).
The autonomy observed in the armaaulo clones also contrasts with the more regulative behavior of cells observed following surgical manipulation of the insect epidermis. The results of the latter experiments are usually interpreted in models where the fates of epidermal cells depend either on local cell interactions or on diffusible morphogen whose concentrations are graded across the segment. Regardless of the nature of the determinative system, however, some components required for proper specification might still be restricted to single cells. Given its autonomy, the wild-type arm+ product might function as a cellular interpreter of external morphogen levels, transducing or internalizing the positional information within each cell. If, for example, the posterior-most fates are determined by high morphogen levels, then mutants that reduce theperceivedlevel of morphogen would cause anterior shifts in cell fates. Cells that would normally make naked cuticle would under such conditions make denticles. The transformation would be cell-autonomous, because it is a local cellular property rather than the morphogen distribution itself that is affected. The only exception to the general autonomy observed with armadillo is that the clones do not show the same polarity reversals found in the transformed regions of nonmosaic mutant animals. In the gradient model, the polarity of hair and denticles reflects the slope of the morphogen distribution (Stumpf, 1985; Lawrence, 1988). In the clock model, the polarity of a cell depends on interactions with neighbors of slightly different positional identity (French et al., 1978). In both cases, the polarity of the pattern is a secondary consequence of a more basic mechanism which defines positional differences between the cells within each segment. The behavior of arm clones provides an interesting test of this model in that the denticle patches found in the naked cuticle are out of their normal positional context. In contrast to the mirror images and polarity reversals observed in arm embryos that are entirely mutant, most of the denticles in the clone show the same polarity as the wild-type cells that surround them. This suggests that polarity and alignment may be imposed on a cell by its environment. Moreover, the result argues that the primary role of the armadillo gene product is to control cell fate, rather than cell polarity. The polarity reversal in armadillo embryos appears to arise secondarily, smoothing out discontinuities that may arise when the entire posterior two-thirds of each segment is shifted toward a more anterior fate. Are Different Regions of the Field Differentially Sensitive to Loss of armadillo? Our failure to obtain embryonic arm clones in the posterior regions of each segment was unexpected. Arm+ product is clearly required for posterior pattern formation, since posterior naked cuticle is eliminated in homozygous embryos. Given our other results with armadillo, we assume that this requirement is cell-autonomous. One possibility is that homozygous clones produced in the posterior compartment die, possibly because in that region the maternal component alone is insufficient for viability. The same cell death might be invoked to explain the loss of posterior
Blastoderm 183
Clones
for armadillo
compartment cells in homozygous embryos. Given the reduced size of arm embryos, only the anterior fraction of the naked cuticle precursor cells may survive or be shifted to an anterior denticle-secreting fate (see, also, MartinezArias and Ingham, 1985). Extensive cell death has been observed in the posterior compartment of embryos homozygous for other segment polarity mutants (fused; Martinez-Arias, 1985). Although subsequent regeneration might account for the mirrorimage belts observed in those mutants, it is very hard to invoke regeneration to account for the autonomous production of small denticle patches found in the naked cuticle of arm heterozygotes. Instead, our results indicate that reduced arm+ levels in the anterior cells autonomously alter cell fate. The failure to detect clones induced in the posterior compartment might also be explained if such clones survive and are transformed to anterior fates, but, because of a changed surface affinity, they no longer remain in the posterior compartment. Similar shifts in cell position have been demonstrated in the Dysdercus epidermis when large grafts are rotated (Nubler-Jung, 1979). Such grafts do not migrate as a whole, but the cells at the interface between graft and host (i.e., those cells which suffer the greatest juxtapositional abnormalities) migrate around the graft until they achieve a more appropriate position. This is thought to produce the whirling pattern characteristic of rotated insect transplants. The small clones produced in our experiments might be capable of even more substantial movement, especially if the anteriorly directed changes in positional identity occur only gradually, such that the juxtapositional abnormalities suffered by the clone are never very great. Preferential adhesion and cell migration allows the position dependent recovery of arm clones to be explained using the same mechanism postulated for the transformation of fate. Cell death in the posterior compartment, on the other hand, is not so easily compatible with a simple role for armadillo in transducing positional information; lowered levels of arm should result in anterior shifts of cell fate, regardless of where in the segment the clones are produced. Preferential adhesion is an intriguing possibility in that it would couple both the armadillo gene product and positional information to cell surface properties that might be investigated by complementary technologies.
Irmdistlons
and Cuticls
Pqwstions
To produce mitotic recombination during embryonic stages, embryos were collected at hourly intervals from y w f females that had been mated to armyD36/yZY67g males. Eggs were maintained on the apple juice-agar collecting dish at 25% until they had reached the appropriate age for the irradiation. After the irradiation, the embryos were kept an additional 10 hr at 18oc. until they had at least reached late germ band elongation stages. They were then dechorionated in commercial bleach (~5% sodium hypochlorite), allowed to complete embryonic development underwater, and mounted in Hoyet’s solution following previously described procedures (Wieschaus and NiissleinWhard, 1986). Irradiations involving other segment polarity mutants were carried out in similar manner, except that the y w f females were mated to males heterozygous for the given mutation. Control irradiations utilized eggs collected from y w f females mated to w males. In one experiment, heterozygous goo females were mated to T(7;2)Bld,goo+/
goo males to allow production of gooseberry clones by recombination on the X chromosome.Marked arm clones also homozygousfor shavanbaby were produced by irradiating embryos collected from amxp33 svh/fm7females mated to wild-type males. Controls in that experiment were derived from similarly mated armxpj3/-Fm7 females. Embryos heterozygous for arm or other segment polarity mutants were irradiated using a Cs-137 source (Gammator Model B) with an ap proximate dosage of 270-290 Rlmin. Since the animals were irradiated on the petri dishes, the dose delivered to the cells are unknown but probably less. Following a 4 min (-1150 R) irradiation at 3 hr + 0.5, the embryonic hatch rate was 70% (160/227), similar to that obtained in earlier studies with a dosage of 1.1. kR (Wieschaus and Gehring, 1976). Embryonic cuticles were examined for clones using a compound microscope with phase-contrast optics at 400x. Between IO and 20 individuals were mounted on a single slide, and although the identity of the slides was known, experimental and control slides from a given experiment were examined alternately to maximize the probability that they were scored with equal rigor. In the final analysis, only data from the naked cuticular regions of the abdominal segments Al to A8 were used in the tables because of the predominance of questionable clones in segment A7 (see text).
Acknowledgments We thank our colleagues at Princeton for many helpful comments on the manuscript and Nick Baker, Norbert Perrimon, Alfonso MartinezArias, and Bob Holmgren for sharing unpublished data with us. The experiments were supported by a National Institutes of Health Research Grant (PHS HD15567) to E. W. The costs of publication of this article were defrayed in part by the payment of page charges. This article musl therefore be hereby marked “adveftisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received
December
9, 1986; revised
February
6, 1987.
References Bohn, H. (1970). lnterkalare regeneration und segmentale gradienten bei den extremititen von Leucophaea-larven (Blattaria), I. Femur und tibia. Roux’s Arch. Dev. Biol. 765, 303-341.
Expsrlmental Gsnotypss
Procedures and Stocks
The two alleles used in the clonal analysis (armxP3 and armyD35, are both EMS-induced and are viable in males with duplications of the locus (y2Y67g; Dp(7;4)MG). They are apparently close to amorphic in that their phenotype in hemizygous embryos is strong and not enhanced in trans heterorygotes for synthetic deficiencies (the XP3D segregant of T(7;3)oR34). afmXMls produces a weaker phenotype which is slightjy temperature-sensitive. The alleles of gooseberry (goollx@$ wingless (wg1’G22). and hedgehog (hhlJ32) are all strong, apparently amorphic alleles obtained in our original mutagenesis screen (Niisslein-Volhard and Wieschaus, 1980). The shave&a& marker mutation is described in Wieschaus 81 al. (1964) and Gergen and Wieschaus (1986).
French, V., Bryant, P J., and Bryant, S. V. (1976). Pattern epimorphic fields. Science 793, 969-981.
regulation
Garcia-Bellido, A. (1972). Some parameters of mitotic recombination Drosophila melanogastar. Mol. Gen. Genetics 775, 54-72. Gergen, J. P, and Wieschaus, E. F. (1985). The localized for a gene affecting segmentation in Drosophile: analysis saic for runt. Dev. Biol. 709, 321-335.
in in
requirements of larvae mo-
Gergen, J. P, and Wieschaus. E. F. (1986). Localized requirements for gene activity in segmentation of Drosophile embryos: analysis of armadillo, fused, giant and unpaired mutations in genetic mosaics. Roux’s Arch. Dev. Biol. 795, 49-62. Hartenstein, V., and Campos-Ortega, J. (1965). Fate mapping in wild type Drosophila melanogaste~ I. The pattern of the embryonic cell divisions. Roux’s Arch. Dev. Biol. 794, 181-195.
Cell 184
Lawrence, P A. (1966). Gradients in the insect segment: the orientation of hairs in the milkweed bug, Oncopeltus fasciatus. J. Exp. Viol. 44, 807-820. Lawrence, positional
P A, Crick, F. H. C.. and Munro, M. (1972). A gradient of information in an insect Rhodius. J. Cell Sci. 77, 815-853.
Locke, M. (1967). The development of patterns sects. Adv. Morphogenesis 6, 33-88.
in the integument
of in-
Lohs-Schardin, M., Cremer, C., and Nirsslein-Volhard, C. (1979). A fate map for the larval epidermis of Drosop/ri/a melanogaster localized cuticle defects following irradiation of the blastoderm with an ultraviolet laser microbeam. Dev. Biol. 73, 239-255. Martinezdrias, A. (1985). The development of fused- embryos sophila melanogaster. J. Embryol. Exp. Morph. 87, 99-114. Martinez-Arias, A., and Ingham, duplications in segment polarity Exp. Morph. 87, 129-135. Meinhardt, H. (1982). Models York: Academic Press). Nubler-Jung, intersegmental
of Dro-
P W. (1985). The origin of pattern mutants of Drosophila. J. Embryol. of Biological
Pattern
Formation
K. (1979). Pattern stability in the insect segment. region. Roux’s Arch. Dev. Biol. 786, 211-233.
(New II. The
Niisslein-Volhard, segment number
C., and Wieschaus, E. (1980). Mutations affecting and polarity in Drosophila. Nature 287, 795-801.
Struhl, G. (1984). 308,454-457.
Splitting
the bithorax
complex
of Drosophila.
Nature
Stumpf, H. (1968). Deutung der Richtungsmuster der Schuppen von Galleria mellonella auf Grund eines Konzentrationsgef8lles. Naturwissenschaften 52, 522. Szabad, J., ScMpbach, T, and Wieschaus, E. (1979). Cell lineage and development in the larval epidermis of Drosophila melanogaster Dev. Biol. 73, 258-27f. Wieschaus, E., and Gehring, disc cells in the early embryo 50, 249-283.
W. (1976). Clonal analysis of primordial of DrosophL me/anogasteL Dev. Biol.
Wieschaus, E., and Noell, E. F. (1988). Specificity of embryonic lethal mutations in Drosophila analysed in germ line clones. Roux’s Arch. Dev. Biol. 195, 83-73. Wieschaus, E., and Niisslein-Volhard, In Drosophila: A Practical Approach
C. (1986). Looking at embryos. (Oxford: IRL Press), pp. 199-227.
Wieschaus, E., Niisslein-Volhard, C., and Jiirgens. G. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogastee III. Zygotic loci on the X-chromosome and fourth chromosome. Rouxs Arch. Dev. Biol. 193, 296-387. Wright, D. A., and Lawrence, P A. (1981). Regeneration boundary in Oncopeltus. Dev. Biol. 85, 317-327.
of the segment
April 24, 1987, Copyright
0 1987 by Cell Press
Autonomous Requirements for the Segment Polarity Gene armadillo during Drosophila Embryogenesis Eric Wieschaus and Robert Riggleman Department of Biology Princeton University Princeton, New Jersey 08544
Summary Embryos hemizygous for armadillo produce a %egment polarity” phenotype in which the naked posterior two-thirds of each segment is replaced by denticles with reversed polarity. Small patches of homotygous arm ceils induced by mitotic recombination also form such denticles, indicating that the changes in cellular fate observed in homozygous arm embryos are autonomous at the level of single cells. Clonally derived arm patches do not, however, show the characteristic arm polarity reversals, arguing that this feature of the phenotype depends on cell interactions in fully mutant embryos. Pew, if any, clones were found in the posterior-most regions of the naked cuticle, and none were found in the posterior compartments of the thorax. Introduction Although the final cuticle pattern of the Drosophila embryo is composed of discrete denticles, hairs, and sensilla, classical experiments suggest that the underlying anterior-posterior information is graded or continuous in nature (Locke, 1967; Bohn, 1970). When epidermis is wounded, cells are juxtaposed that normally would not have been adjacent to each other in the wild-type pattern. This juxtaposition induces an intercalary growth which eventually smoothes out the disparity in positional values. Intercalation can result in regeneration of the normal pattern, or in local polarity reversals and mirror images (French et al., 1976; Wright and Lawrence, 1981). The extent of regeneration in both cases, however, depends on the juxtaposition, each cell being only subtly different from its immediate neighbors, but very different from cells further away in the pattern. The graded nature of the positional field observed in these experiments can be modeled assuming a diffusible morphogen gradient (Lawrence et al., 1972) but the relative stability of positional fates following transplantation argue that these graded differences can somehow be maintained within cells, even following surgical manipulation. Among the mutants that affect segmental pattern in Drosophila (Niisslein-Volhard and Wieschaus, 1980) a number of loci have been identified that cause pattern alterations repeated in each segment. In the most common phenotype, the posterior two-thirds of the pattern is replaced by a mirror image of the anteriorly situated denticle rows. This ‘segment polarity” phenotype is produced by mutations in at least six different loci (armadillo, fused, wingless, gooseberry, hedgehog, and ciD) and is reminiscent of the polarity reversals produced in other insect
epithelia following surgical manipulation (Wright and Lawrence, 1981). These genes are required zygotically, at least during the first half of embryogenesis, and may play a role in the establishment or maintenance of positional information within each segment. In this paper we present a detailed analysis of one of the segment polarity loci, armadillo (Wieschaus et al., 1984). The major concern of our analysis was the extent to which the patterns observed in arm embryos are the result of local autonomous requirements for arm product in those cells whose fates are switched by the mutation. To test for such autonomy, we used mitotic recombination to produce very small patches of afm cells in the posterior regions of the embryonic segment primordia. Results The armadillo Phenotype In wild-type embryos, segmentation is most obvious in the repeated pattern of denticle band and intervening naked cuticle on the ventral side in the first instar larva. In embryos homozygous for armadillo, the naked cuticular part of the pattern is eliminated. In its place, an apparent mirror image of the denticle band is formed with reversed polarity. The size of this “mirror image” region is smaller than that which would normally be occupied by the missing naked cuticle, and although it is likely that some of the naked cuticle precursors contribute to the denticles (see below), the exact relationship is not known. The pattern in armadillo embryos is somewhat variable, even when the zygotic and maternal genotypes are kept constant. Comparison of the phenotypes produced by strong and weak alleles suggests that the naked regions of the cuticle are most sensitive to reductions in arm activity and the middle of the denticle band least sensitive (Figure lb-lc). In most of its basic features, the phenotype of armadillo embryos is similar to those produced in embryos homozygous for other segment polarity mutants. One of the peculiarities that distinguishes armadillo from the other mutants in this class is that afm+ transcription is absolutely required in female germ cells for them to complete oogenesis (Wieschaus and Noell, 1986). Some of this afm+ product remains in the egg and influences the phenotype of mutant embryos, making it less extreme. Since it is impossible to obtain eggs unless the mother had at least one active wild-type allele during oogenesis, the embryonic afm phenotype is always viewed in the background of this maternal afm+ component. Thus, although the arm alleles used in this study can be classified as amorphic in a genetic sense (see Experimental Procedures), the phenotypes that they produce clearly do not represent development in the total absence of arm product. Homozygosity for armadillo Causes Autonomous Changes in Cell Pate The absence of naked cuticle suggests that the afm pattern might arise because the cells that normally make
Figure 1. Phase-Contrast Micrographs of the Thoracic and Anterior Abdominal Regions in Wild-Type and armadillo Mutant Embryos (a) The wild-type pattern. The denticle bands are separated by large regions of naked cuticle, and most of the denticles point posteriorly. Each segment is about 13 cells long and the denticle band occupies the anterior 4tI% of that length. In the third larval instar (a stage when the epidermal cells can be most accurately counted), there are about 88 cells underlying the denticle region of the pattern and about 118 cells in the naked cuticular region that follows. Since each denticle band averages 243 denticles, each epidermal cell secretes about 2.8 denticles. No cell division occurs in the epidermis between embryogenesis, and the third larval instar and the denticle pattern changes only slightly. (b) A weak arm phenotype produced by a hypomorphic allele.(armXM1s) in an embryo derived from wild-type attached-X mother. The denticle band is expanded in size and patches of intervening naked cuticular region are observed in the thorax and occasionally in the abdomen (-). (c) A strong arm phenotype produced by an apparent amorphic allele (armyM) in an embryo derived from a heterozygous mother. The mirrorimage belts are now fused into a lawn of denticles with no intervening cuticle. The segmental organization is still apparent because of the repeated polarity reversals.
naked cuticle are reprogrammed to make denticles instead. Such change8 might occur by autonomous genetic switches in single ceils, or by a more global reprogramming associated with regeneration or shifts in diffusible gradients. No large scale nonautonomy was observed in large patches of arm cells produced by Ring-X loss (Gergen and Wieschaus, 1986) but a more rigorous test for autonomy would rely on the behavior of clones of one or two arm ceils in a naked cuticular region which is otherwise totally wild-type. One obvious way of producing such patches is by mitotic recombination in arm heterozygotes at the blastoderm stage. The blastodermal precursors for the epidermis divide only twice before they begin differentiation (Szabad et al., 1979), and clones produced by a blastoderm irradiation should therefore be only one to two cells in size when they make cuticle. Since each differentiated epidermal cell secretes about three denticles, it should be easy to detect any arm clones in the naked cuticle if they are shifted toward making denticles. In the first set of experiments, the irradiations were carried out using an unmarked armadillo chromosome, since the markers available for embryonic cuticle would either reduce the denticle size or make the arm clone difficult to detect. Three hundred and thirty-four heterozygous larvae were examined following irradiation at the blastoderm stage and a total of 95 denticle patches were detected in the naked cuticle (Figures 2a-2e). The frequency with which such apparent clones were produced depended on
the irradiation dosage: fewer were detected following the short two-minute irradiations than following four- or fiveminute irradiations, and no patches were identified in unirradiated heterozygotes (Table 1). Several feature8 of the denticle patches argue that they do in fact represent the armadillo homozygous clones. The average size of the clones was between six and seven denticles, only slightly larger than that predicted for homozygous arm clones two cells in size (see legend to Figure 1). The frequency of clones per animal (0.34) obsewed following four-minute irradiations is about that predicted from the known sensitivity of blastoderm cells to this dosage (1 mitotic recombination/300 irradiated cells; Wieschaus and Gehring, 1976) and the estimated number of blastoderm precursor cells that give rise to the storable naked cuticular regions of Al to A6 (133 cells, i.e., 6% of an epidermal primordium of 2200 cells; Lohs-Schardin et al., 1979; Szabad et al., 1979). As a final test for the clonal origin of the denticles, we carried out a second set of irradiations, using the shavenbaby mutation to mark the homozygous clones. The cuticular marker shavenbaby (Gergen and Wieschaus, 1986) reduces the size of the larger denticles and removes most of the smaller ones. arm shavenbaby homozygotes have only about a quarter the number of denticles as arm svb+ embryos, and all of these are very small. Only half as many clones were detected when arm svW+ blastoderms were irradiated, and the clones that were recovered averaged only 2.6 den-
Blastoderm 179
Figure
Clones
2. Homozygous
for armadillo
armadillo
Clones
Produced
by Irradiation
at the Blastoderm
Stage
(a-e) Unmarked clones produced in embryos heterozygous for arrrtXp~ or armyD35; (f-g) Marked shavenbaby armadillo clones produced zygous armxp33 svb embryos; (h) Extra denticle rows (+) at posterior margin of the naked cuticle in an irradiated svb arm heterozygous Such denticles are probably not of clonal origin, given that they do not show the svb phenotype.
iln heteroembryo.
Cdl 160
Table
1. Clone
Frequency
in Irradiated
arm Heterozygotes Embryos with Clones
Embryos Examineda
Number of Clones
Frequency
Genotype
Age
Dose
arm/+
-
0
96
0
0
0
0
arm/.+ arm/+ arm/+
3 hr 3 hr 3 hr
2 4 5
70 106 156
6 31 56
6 37 73
6.6 34 47
5.7 6.4 7.4
arm/+ arm svb/ +
3 hr 3 hr
4 4
104
100
29 20
37 20
36 20
6.4 2.3
arm/+ arm/+ arm/+ arm/+ arm/ +
4 hr 5 hr 6’ hr 7 hr 6 hr
4 4 4 4 4
97 99 66 97 99
46 43 6 4 1
69 61 6 4
71 61 7 4 1
6.4 5.0 4.0 2.5
Sizeb
w-4
1
PI
a The number of embryos examined was based on the fraction of the total number of differentiated embryos from the cross that were expected to be of the appropriate genotype. This fraction is one half for all experiments listed above, except for that involving arm svb and the corresponding control, where only one quarter of the embryos are of the appropriate genotype. b Data excludes denticle patches in the posterior region of the naked cuticle (see text). Average clone size was calculated as the average number of denticles per clone; bracketed figures indicate values based on only one or two clones.
Table
2. Clone
Genotype
armadillo wingless QfJo=beny
T(l;2)BhVgoosebeny hedsehos +/+ control
Frequencies
Following
Irradiation
of Heterozygotes
Embryos Examineda
Embryos Clones
172 265 122
51 4 2
44 259 126
with
for Other Number Clones
Segment
Polarity
of
Frequency
Mutants
tw
SW
56 5 2
34 1.6 1.6
7.0 4.4 [12.5]
0
0
0
0
3 1
3 1
1.1 0.6
7.7 I51
Posterior ROWSC
Denticle
7 9 7 6 10 0
a The number of embryos examined was based on the fraction of the total number of differentiated embryos from the cross that were expected to be of the appropriate genotype. This fraction is one half for all experiments listed above, except for that involving T(1;2)E/d where only one-third of the differentiated embryos are of the appropriate genotype. b Size was calculated as the average number of denticles per clone; bracketed figures indicate values based on only one or two clones. c These are the questionable posterior clones described in the text. Only patches of five or more denticle have been included.
titles per clone. Moreover, excluding the exceptional posterior patches described below, all clonally derived denticles had the small size characteristic of svb (Figures a-29). The only area where clonal designation of denticles was ambiguous was at the posterior margin of the naked cuticle, immediately anterior to the adjacent belt. Among the irradiated arm/+ heterozygotes, we found several individuals with an additional, partial row of denticles in this area. Although extra denticles were occasionally observed in this area in the wild-type control, they were never arranged in large rows of more than five denticles (Table 2). In the arm/+ heterozygotes, the denticles were found much more frequently in the posterior abdominal segments and were often bunched together at the ventral midline in segment A7. Similar rows were occasionally found following irradiation of heterozyotes for other segment polarity mutations (Table 2). In the arm svb/+ experiment, the denticles in such rows were not shavenbaby (Figure 2h), and therefore we conclude that they are probably not the direct products of homozygous arm clones. Because of the uncertainty of their origin, they have been excluded from the data presented in Table 1 and given below.
Homozygous armadillo Clones Are Not Uniformly Distributed and Do Not Always Show the Polarity Reversals Characteristic of the Homozygous Embryo Although removal of the wild-type afm gene from the naked cuticle precursors at the blastoderm stage appears to shift the cell into a more anterior, denticle-secreting pathway, not all regions of the naked cuticle show the same sensitivity to the loss. Figure 3 shows the distribution and average polarity of 90 clones induced in abdominal segments Al through A6 following irradiation at the blastoderm stage. Almost all clones are restricted to the anterior two-thirds of the naked cuticle (Figure 3). Most of the exceptions were located along the posterior margin of the naked cuticle, in that region where the clonal nature of additional denticles is doubtful. The existence of a posterior boundary in clonal extent is also suggested by the drawnout morphology of those clones that extend into the posterior limit (Figure 26). To define this boundary more precisely, we scored a fraction of the preparations for clones in the thorax. Although the smaller size of the thoracic denticles makes the clones harder to detect, they can be localized rather precisely relative to the Keilin’s organs that mark the anterior-posterior compartment bor-
Blastoderm 181
Clones
for armadillo
”
0 1.’ m
the anterior-posterior extent of a single clone. The values to the right indicate relative positions within the naked cuticle. Each bar is shaded to indicate the fraction of the denticles in the clone that point anteriorly(i.e., potentially have reversed polarity). Although all clones are depicted as having identical lateral extents, the actual clone patterns varied greatly. The six bars marked with X’s mark the positions of dubious clones which were initially scored as being on the posterior margin. Data is based on a subset of the clones identified following the irradiations described in lines four and five of Table 1
0% <25% <50% <75%
der in each segment (Struhl, 1964). All 14 thoracic clones found were located anterior to, or exactly at the same level as, the Keilin’s organs. This suggests that arm clones induced in the posterior compartment either die or do not make denticles. Alternatively, it is possible that such clones migrate forward as a result of their anterior transformation in fate (see Discussion). In embryos homozygous for armadillo, the naked cuticle region of the pattern is replaced by denticles with reversed polarity, i.e., denticles that point anteriorly. In contrast, about 70% (359/526) of the denticles in the arm clones have normal polarity. Anteriorly pointing denticles are especially rare (
surprising, given that mitosis is needed to produce homozygous clones and little proliferation is observed in the ventral epidermis following 6 hr of development (Hartenstein and Campos-Ortega, 1965). Other Segment Polarity Mutants Do Not Produce Denticle Patches When Heterozygotes Are Irradiated at the Blastoderm Stage In addition to armadillo, we have also tested three autosomal segment polarity mutations for the ability of homozygous clones to cause patches of denticles in the naked cuticle. Blastoderms heterozygous for either wingless, gooseberry, or hedgehog (NDsslein-Volhard and Wieschaus, 1960) were irradiated following the same procedures as those used with armadillo and about 150 individuals scored for each locus. The yield of apparent clones was extremely low, only 5% of the frequencies obtained using armadillo (Table 2). Because we have no independent system for estimating the frequency of mitotic recombination on the autosomes, it is difficult to evaluate the negative nature of this result. In a detailed analysis of clone frequencies in abdominal histoblasts, GarciaBellido (1972) showed that autosomes undergo mitotic recombination at one-half to one-third the rate measured for the X chromosome. We believe it therefore unlikely that the extremely low yields of wg, hh, and goo clones simply reflect a lower induction rate for autosomal clones. This possibility has been eliminated for gooseberry by showing that even when a duplication (Dp(7;2)6/1$ was used to move the wild-type allele to the X chromosome, the clone frequency was not increased (Table 2). Instead, it seems that homozygous clones for these loci when produced at the blastoderm stage do not normally result in ectopic denticles in the naked cuticle. The occasional denticle patches we have observed using these mutants may represent rare autonomous exceptions to this rule, or cases
Cell 182
where a dying homozygous clone has altered the pattern of the adjacent wild-type cells. Given their low frequency, they might also represent unusual cases of pattern regulation following X-ray damage.
The Autonomy of the armadillo Phenotype and the Role of the armadillo Gene Product in lntrasegmental Patterning The best developed models for intrasegmental patterning in insects involve either a diffusible gradient which establishes concentration differences along the anterior-posterior axis (Locke, 1987; Lawrence et al., 1972; Meinhardt, 1982) or local cell-cell interactions between cells of slightly different positional identity (French et al., 1978; Wright and Lawrence, 1981). In both models, cell fate depends heavily on the communication between cells or long-range properties of the field. Indeed, the pattern of denticles and sensilla in each segment is so complex that it is hard to imagine its being constructed by single cells without cell interactions and other inherently nonautonomous phenomena. In the experiments described in this paper, we used genetic mosaics to test whether the transformations observed in embryos mutant for different segment polarity genes are autonomous at the level of single cells. Of the four segment polarity loci we tested, only armadillo produced small denticle patches with the morphology predicted of autonomous clones. We were able to produce such clones by irradiations made any time up until midextended germ band stages. The negative results obtained with later irradiations can be explained by the difficulty of inducing homozygous clones at stages with little or no proliferation. The shifts in fate observed following irradiations at 8 hr imply that either the perdurance of zygotic arm product is very small, or that the gene may not be active prior to that point. In contrast to the results obtained with armadillo, irradiated wingless, gooseberry, and hedgehog heterozygotes produced no more denticle patches than did the wild-type controls, except that they, like irradiated armadillo heterozygotes, occasionally showed an additional large row of denticles immediately anterior to the normal denticle band. We have not interpreted these rows as clones, even though they were only found after irradiation of segment polarity heterozygotes, as they were not always marked with shavenbaby when arm svb/+ heterozygotes were irradiated. Although the otherwise negative results obtained with wingless, gooseberry, and hedgehog might be explained by perdurance of wild-type products made prior to clonal induction, it seems more likely to us that at least some of these mutants produce their phenotypes by disrupting the system in nonautonomous ways. The products of the wild-type genes may actually diffuse from one cell to the next. On the other hand, their segment polarity phenotype may be a secondary consequence of autonomous, local disturbances which have long range effects on the patterning (i.e., cell death followed by regeneration).
The autonomy observed in the armaaulo clones also contrasts with the more regulative behavior of cells observed following surgical manipulation of the insect epidermis. The results of the latter experiments are usually interpreted in models where the fates of epidermal cells depend either on local cell interactions or on diffusible morphogen whose concentrations are graded across the segment. Regardless of the nature of the determinative system, however, some components required for proper specification might still be restricted to single cells. Given its autonomy, the wild-type arm+ product might function as a cellular interpreter of external morphogen levels, transducing or internalizing the positional information within each cell. If, for example, the posterior-most fates are determined by high morphogen levels, then mutants that reduce theperceivedlevel of morphogen would cause anterior shifts in cell fates. Cells that would normally make naked cuticle would under such conditions make denticles. The transformation would be cell-autonomous, because it is a local cellular property rather than the morphogen distribution itself that is affected. The only exception to the general autonomy observed with armadillo is that the clones do not show the same polarity reversals found in the transformed regions of nonmosaic mutant animals. In the gradient model, the polarity of hair and denticles reflects the slope of the morphogen distribution (Stumpf, 1985; Lawrence, 1988). In the clock model, the polarity of a cell depends on interactions with neighbors of slightly different positional identity (French et al., 1978). In both cases, the polarity of the pattern is a secondary consequence of a more basic mechanism which defines positional differences between the cells within each segment. The behavior of arm clones provides an interesting test of this model in that the denticle patches found in the naked cuticle are out of their normal positional context. In contrast to the mirror images and polarity reversals observed in arm embryos that are entirely mutant, most of the denticles in the clone show the same polarity as the wild-type cells that surround them. This suggests that polarity and alignment may be imposed on a cell by its environment. Moreover, the result argues that the primary role of the armadillo gene product is to control cell fate, rather than cell polarity. The polarity reversal in armadillo embryos appears to arise secondarily, smoothing out discontinuities that may arise when the entire posterior two-thirds of each segment is shifted toward a more anterior fate. Are Different Regions of the Field Differentially Sensitive to Loss of armadillo? Our failure to obtain embryonic arm clones in the posterior regions of each segment was unexpected. Arm+ product is clearly required for posterior pattern formation, since posterior naked cuticle is eliminated in homozygous embryos. Given our other results with armadillo, we assume that this requirement is cell-autonomous. One possibility is that homozygous clones produced in the posterior compartment die, possibly because in that region the maternal component alone is insufficient for viability. The same cell death might be invoked to explain the loss of posterior
Blastoderm 183
Clones
for armadillo
compartment cells in homozygous embryos. Given the reduced size of arm embryos, only the anterior fraction of the naked cuticle precursor cells may survive or be shifted to an anterior denticle-secreting fate (see, also, MartinezArias and Ingham, 1985). Extensive cell death has been observed in the posterior compartment of embryos homozygous for other segment polarity mutants (fused; Martinez-Arias, 1985). Although subsequent regeneration might account for the mirrorimage belts observed in those mutants, it is very hard to invoke regeneration to account for the autonomous production of small denticle patches found in the naked cuticle of arm heterozygotes. Instead, our results indicate that reduced arm+ levels in the anterior cells autonomously alter cell fate. The failure to detect clones induced in the posterior compartment might also be explained if such clones survive and are transformed to anterior fates, but, because of a changed surface affinity, they no longer remain in the posterior compartment. Similar shifts in cell position have been demonstrated in the Dysdercus epidermis when large grafts are rotated (Nubler-Jung, 1979). Such grafts do not migrate as a whole, but the cells at the interface between graft and host (i.e., those cells which suffer the greatest juxtapositional abnormalities) migrate around the graft until they achieve a more appropriate position. This is thought to produce the whirling pattern characteristic of rotated insect transplants. The small clones produced in our experiments might be capable of even more substantial movement, especially if the anteriorly directed changes in positional identity occur only gradually, such that the juxtapositional abnormalities suffered by the clone are never very great. Preferential adhesion and cell migration allows the position dependent recovery of arm clones to be explained using the same mechanism postulated for the transformation of fate. Cell death in the posterior compartment, on the other hand, is not so easily compatible with a simple role for armadillo in transducing positional information; lowered levels of arm should result in anterior shifts of cell fate, regardless of where in the segment the clones are produced. Preferential adhesion is an intriguing possibility in that it would couple both the armadillo gene product and positional information to cell surface properties that might be investigated by complementary technologies.
Irmdistlons
and Cuticls
Pqwstions
To produce mitotic recombination during embryonic stages, embryos were collected at hourly intervals from y w f females that had been mated to armyD36/yZY67g males. Eggs were maintained on the apple juice-agar collecting dish at 25% until they had reached the appropriate age for the irradiation. After the irradiation, the embryos were kept an additional 10 hr at 18oc. until they had at least reached late germ band elongation stages. They were then dechorionated in commercial bleach (~5% sodium hypochlorite), allowed to complete embryonic development underwater, and mounted in Hoyet’s solution following previously described procedures (Wieschaus and NiissleinWhard, 1986). Irradiations involving other segment polarity mutants were carried out in similar manner, except that the y w f females were mated to males heterozygous for the given mutation. Control irradiations utilized eggs collected from y w f females mated to w males. In one experiment, heterozygous goo females were mated to T(7;2)Bld,goo+/
goo males to allow production of gooseberry clones by recombination on the X chromosome.Marked arm clones also homozygousfor shavanbaby were produced by irradiating embryos collected from amxp33 svh/fm7females mated to wild-type males. Controls in that experiment were derived from similarly mated armxpj3/-Fm7 females. Embryos heterozygous for arm or other segment polarity mutants were irradiated using a Cs-137 source (Gammator Model B) with an ap proximate dosage of 270-290 Rlmin. Since the animals were irradiated on the petri dishes, the dose delivered to the cells are unknown but probably less. Following a 4 min (-1150 R) irradiation at 3 hr + 0.5, the embryonic hatch rate was 70% (160/227), similar to that obtained in earlier studies with a dosage of 1.1. kR (Wieschaus and Gehring, 1976). Embryonic cuticles were examined for clones using a compound microscope with phase-contrast optics at 400x. Between IO and 20 individuals were mounted on a single slide, and although the identity of the slides was known, experimental and control slides from a given experiment were examined alternately to maximize the probability that they were scored with equal rigor. In the final analysis, only data from the naked cuticular regions of the abdominal segments Al to A8 were used in the tables because of the predominance of questionable clones in segment A7 (see text).
Acknowledgments We thank our colleagues at Princeton for many helpful comments on the manuscript and Nick Baker, Norbert Perrimon, Alfonso MartinezArias, and Bob Holmgren for sharing unpublished data with us. The experiments were supported by a National Institutes of Health Research Grant (PHS HD15567) to E. W. The costs of publication of this article were defrayed in part by the payment of page charges. This article musl therefore be hereby marked “adveftisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received
December
9, 1986; revised
February
6, 1987.
References Bohn, H. (1970). lnterkalare regeneration und segmentale gradienten bei den extremititen von Leucophaea-larven (Blattaria), I. Femur und tibia. Roux’s Arch. Dev. Biol. 765, 303-341.
Expsrlmental Gsnotypss
Procedures and Stocks
The two alleles used in the clonal analysis (armxP3 and armyD35, are both EMS-induced and are viable in males with duplications of the locus (y2Y67g; Dp(7;4)MG). They are apparently close to amorphic in that their phenotype in hemizygous embryos is strong and not enhanced in trans heterorygotes for synthetic deficiencies (the XP3D segregant of T(7;3)oR34). afmXMls produces a weaker phenotype which is slightjy temperature-sensitive. The alleles of gooseberry (goollx@$ wingless (wg1’G22). and hedgehog (hhlJ32) are all strong, apparently amorphic alleles obtained in our original mutagenesis screen (Niisslein-Volhard and Wieschaus, 1980). The shave&a& marker mutation is described in Wieschaus 81 al. (1964) and Gergen and Wieschaus (1986).
French, V., Bryant, P J., and Bryant, S. V. (1976). Pattern epimorphic fields. Science 793, 969-981.
regulation
Garcia-Bellido, A. (1972). Some parameters of mitotic recombination Drosophila melanogastar. Mol. Gen. Genetics 775, 54-72. Gergen, J. P, and Wieschaus, E. F. (1985). The localized for a gene affecting segmentation in Drosophile: analysis saic for runt. Dev. Biol. 709, 321-335.
in in
requirements of larvae mo-
Gergen, J. P, and Wieschaus. E. F. (1986). Localized requirements for gene activity in segmentation of Drosophile embryos: analysis of armadillo, fused, giant and unpaired mutations in genetic mosaics. Roux’s Arch. Dev. Biol. 795, 49-62. Hartenstein, V., and Campos-Ortega, J. (1965). Fate mapping in wild type Drosophila melanogaste~ I. The pattern of the embryonic cell divisions. Roux’s Arch. Dev. Biol. 794, 181-195.
Cell 184
Lawrence, P A. (1966). Gradients in the insect segment: the orientation of hairs in the milkweed bug, Oncopeltus fasciatus. J. Exp. Viol. 44, 807-820. Lawrence, positional
P A, Crick, F. H. C.. and Munro, M. (1972). A gradient of information in an insect Rhodius. J. Cell Sci. 77, 815-853.
Locke, M. (1967). The development of patterns sects. Adv. Morphogenesis 6, 33-88.
in the integument
of in-
Lohs-Schardin, M., Cremer, C., and Nirsslein-Volhard, C. (1979). A fate map for the larval epidermis of Drosop/ri/a melanogaster localized cuticle defects following irradiation of the blastoderm with an ultraviolet laser microbeam. Dev. Biol. 73, 239-255. Martinezdrias, A. (1985). The development of fused- embryos sophila melanogaster. J. Embryol. Exp. Morph. 87, 99-114. Martinez-Arias, A., and Ingham, duplications in segment polarity Exp. Morph. 87, 129-135. Meinhardt, H. (1982). Models York: Academic Press). Nubler-Jung, intersegmental
of Dro-
P W. (1985). The origin of pattern mutants of Drosophila. J. Embryol. of Biological
Pattern
Formation
K. (1979). Pattern stability in the insect segment. region. Roux’s Arch. Dev. Biol. 786, 211-233.
(New II. The
Niisslein-Volhard, segment number
C., and Wieschaus, E. (1980). Mutations affecting and polarity in Drosophila. Nature 287, 795-801.
Struhl, G. (1984). 308,454-457.
Splitting
the bithorax
complex
of Drosophila.
Nature
Stumpf, H. (1968). Deutung der Richtungsmuster der Schuppen von Galleria mellonella auf Grund eines Konzentrationsgef8lles. Naturwissenschaften 52, 522. Szabad, J., ScMpbach, T, and Wieschaus, E. (1979). Cell lineage and development in the larval epidermis of Drosophila melanogaster Dev. Biol. 73, 258-27f. Wieschaus, E., and Gehring, disc cells in the early embryo 50, 249-283.
W. (1976). Clonal analysis of primordial of DrosophL me/anogasteL Dev. Biol.
Wieschaus, E., and Noell, E. F. (1988). Specificity of embryonic lethal mutations in Drosophila analysed in germ line clones. Roux’s Arch. Dev. Biol. 195, 83-73. Wieschaus, E., and Niisslein-Volhard, In Drosophila: A Practical Approach
C. (1986). Looking at embryos. (Oxford: IRL Press), pp. 199-227.
Wieschaus, E., Niisslein-Volhard, C., and Jiirgens. G. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogastee III. Zygotic loci on the X-chromosome and fourth chromosome. Rouxs Arch. Dev. Biol. 193, 296-387. Wright, D. A., and Lawrence, P A. (1981). Regeneration boundary in Oncopeltus. Dev. Biol. 85, 317-327.
of the segment