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DEVELOPMENTALBIOLOGY 129,72-83(1988) Maternal-Effect Genes That Alter the Fate Map of the Drosophila Blastoderm Embryo GARY M. WINSLOW,SEAN B. CARROLL,' Department of Molecular, Cellular and Devebpmental ANDMATTHEW P. SCOTT~ Biology, Universi@ of Colorado at Boulder, Boulder, Colorado 8o$o$-osh7 Accepted April 26, 1988 The pattern of segmentation in the Drosophila embryo is controlled by at least 25 zygotically active genes and at least 20 maternally active genes. We have examined the pattern of expression of the protein product of the zygotically active segmentation gene&s& tarazu (ftz) at the cellular blastoderm stage in progeny of mutant females homozygous for each of six maternal-effect segmentation genes to observe the early effects of the maternal-effect genes on zygotic gene expression. The genes included ewperantia (a member of the anterior class of maternal-effect segmentation genes); staqen and vusa (members of the posterior class); and torso, trunk, and fs(l)N (members of the terminal class). Mutations in the genes caused a disruption of the normal pattern of ft.z stripes in regions of the embryo where gene activity is known to be required. The@ stripes provide a marker for segmental determination at the cellular blastoderm stage, making it possible to correlate aberrant patterns of& protein with defects in cuticle morphology at the end of embryogenesis. J%Zprotein expression in progeny of females mutant for combinations of the above genes was also examined. The changes in the& pattern in progeny of females doubly mutant for genes of the anterior and terminal classes or of the posterior and terminal classes can largely be understood as the result of the additive effects of the single mutations. In contrast, clearly nonadditive effects on thefiz pattern were seen when a mutation in a gene of the anterior class (ezuperantia) was combined with mutations in posterior class genes. 0 1988 Academic PEW, he. INTRODUCTION abnormalities that affect either contiguous blocks of segments, alternate segmental units, or a part of each segment (Niisslein-Volhard and Wieschaus, 1980). Among the zygotically active segmentation genes is fushi taraxu (J%z).Embryos homozygous for null alleles of ftx exhibit a pair-rule phenotype, i.e., alternate segment boundaries and adjacent pattern elements are missing so that embryos form only about one-half the normal number of segments (Wakimoto et al, 1984). Molecular and immunochemical analyses of the products of the ftx gene have demonstrated that at the cellular blastoderm stage& mRNA and protein molecules are localized in a series of seven transverse stripes (Hafen et al, 1984; Carroll and Scott, 1985). Each stripe of ftz protein initially corresponds to a parasegmental unit (Lawrence et al, 1987), sofx products are present in alternate parasegments. A parasegment (PS) consists of the posterior region of one segment and the anterior region of the next most posterior segment (Martinez-Arias and Lawrence, 1985). Thus,ftx expression occurs in the primordia that give rise to the segments that are missing infix- embryos. Cell fates are at least partially determined by the cellular blastoderm stage (Schubiger and Newman, 1982; Simcox and Sang, 1983), so the ftx protein stripes are convenient markers for cell determination. (For detailed fate maps of the blastoderm embryo see Lohs-Schardin et al. (1979) and Hartenstein et al. (1985).) Genetic analyses in Drosophila have shown that the development of the proper spatial organization of the embryo is dependent upon the activity of maternally encoded factors that are present in the egg cytoplasm (Anderson and Niisslein-Volhard, 1984a; Boswell and Mahowald, 1985; Mlodzik et al., 1985; Frohnhiifer and Niisslein-Volhard, 1986; Lehmann and Niisslein-Volhard, 1986; Macdonald and Struhl, 1986). Progeny of females that are homozygous for mutations at any of a number of maternally active genetic loci develop distinct pattern of abnormalities such as loss of normal anteroposterior or dorsal-ventral polarity or aberrant numbers of segments (Bull, 1966; Niisslein-Volhard, 19’77; Niisslein-Volhard et ah, 1980; Anderson and Niisslein-Volhard, 1984b; Mohler and Wieschaus, 1985; Frohnhiifer and Niisslein-Volhard, 1986; Sehiipbach and Wieschaus, 1986; reviewed in Ntisslein-Volhard et al., 1987). In addition to the maternally active genes, at least 20 zygotically active genes are required for proper segmentation (reviewed in Akam, 1987; Scott and Carroll, 1987). Homozygous embryos carrying mutations in zygotically active segmentation genes develop segment 1Present address: Laboratory of Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706. ’ To whom correspondence should be addressed. 0012-1606/88 Copyright All rights $3.00 0 1988 by Academic Press, Inc. of reproduction in any form reserved. 72 WINSLOW, CARROLL, AND SCOTT In order to understand the role of maternally encoded information during pattern formation and segment development, we have examined the spatial pattern of ftx protein expression in progeny of females homozygous for mutations in several maternal-effect genes that affect normal dorsal-ventral polarity or anterior-posterior segmentation (Carroll et aZ., 1986, 1987). Mutations in several of the genes that have been studied cause distinctive alterations in the normal spatial distribution offtz protein and it has been possible to correlate changes infix protein distribution with changes in cell fate. This information has been used to identify the parts of the embryonic primordia that require the function of specific maternal genes. In this study we have extended our earlier analyses of ftx protein expression to several other maternal-effect segmentation genes. In addition, the effects of combinations of mutant genes on the spatial expression of ftz have been studied. Each of the maternal-effect mutations alters both the normal pattern of ftx protein expression and proper segmental development. No single mutation or any combination of mutations causes a complete repression or derepression of ftx protein expression; we have not observed in any case an obvious change in the timing or level of ftz expression. In the progeny of females homozygous for combinations of mutant genes the alterations in the& pattern suggest that specific interactions occur among some maternaleffect gene products and that the interactions take place over long distances in the embryo. Related results have been reported by Mlodzik et al. (1987) and Frohnhijfer and Niisslein-Volhard (1987) using RNA and antibody probes to the ftx gene products. MATERIALS AND METHODS The patterns of ftx protein expression in embryos that were progeny of homozygous females of the following genotypes were examined: exuperantiaPJ, staufenHL, vasaPD, torsowK, trunpA, and fs(l)NasratY In addition, progeny of females that were doubly and triply mutant for some of the above genes were examined. All of the mutant genotypes, except fs(l)Nasrat, are from a collection of second chromosome maternal-effect mutants that has been described previously (Schiipbach and Wieschaus, 1986). fs(l)Nasrat (fs(l)N) is an X-linked EMS-induced maternal-effect mutation (Counce and Ede, 1957; Degelmann et al, 1987). A detailed analysis of the segmentation defects of fs(l)N has been presented (Degelmann et aZ., 1987). Although the strongest alleles of each of the genes available at the time were used in this study, there is at present no proof that the alleles examined in this study are nulls. Mutant embryos were obtained from females homo- jlz Expression in Maternal 73 Mutants zygous for the second chromosome maternal-effect mutations. The second chromosome carrying each maternal-effect mutation was marked with cn bw, which causes the eyes to be white, so white-eyed homozygous females were collected and placed in bottles with yeasted egg-lay plates at 25°C. Females homozygous for the X-linked mutation fs(l)Nasrat were isolated from a stock balanced with FM3. Two- to 6-hr-old embryos were collected, fixed, stained with anti-fix antibody, and photographed as described by Carroll and Scott (1985). At least 50 embryos were examined from each genotype. Cuticle preparations were made essentially as described by Van der Meer (1977). The wild-type ftx stripes are numbered l-7, from anterior to posterior. For terminology used to describe cuticular markers see Lohs-Schardin et al. (1979). RESULTS Closes of Maternal Phenotypes Niisslein-Volhard et al. (1987) have grouped the known maternal-effect genes that affect anteroposterior pattern into three phenotypic classes. The anterior class includes genes such as tic&d @cd), exuperantia (exu), and swallow (swa); mutations in this class of genes cause defects in anterior structures. Genes of the posterior class are required for proper development of abdominal structures and, in most cases, the pole plasm. This class includes oskar (osk), vasa (vm), staufen (stau), and tudor (bud). Finally, torso (tor) and trunk (trk) are characteristic of a class of genes that functions in development of pattern at both ends of the embryo. Mutations in these “terminal” genes eliminate the patterns in terminal regions of the embryo. The pattern of ftx protein expression and its correspondence to the cuticle morphology of a wild-type embryo are shown in Figs. la and lb and have been described in detail (Carroll and Scott, 1985). The wild-type and mutant patterns of ftx expression are summarized in a diagram (Fig. 4). ftz Protein Expression in Mutants of the Posterior Class Theftz pattern in progeny of females homozygous for mutations in several posterior class genes has been described (Carroll et al, 1986). Theftx pattern and cuticle phenotype of vasa progeny (Fig. lc) are characteristic of that class of genes. Alterations are seen in the regions that would normally give rise toftx stripes 3 to 6. There is a corresponding loss of all abdominal denticles (Fig. Id), indicating that the abdominal defects are heralded by changes in theftx pattern at the cellular blastoderm stage. FIG. 1. Changes in the pattern of ftz protein expression and larval cuticle caused by maternal-effect expression (a, c, e, g, i, k), in progeny from females homozygous mutant for maternal-effect segmentation form (b, d, f, h, j, 1). Polyclonal antibody specific to thefts portion of aftz+galactosidase fusion protein embryos. The antibody was visualized using a fluorescein-conjugated secondary antibody. Except where tion the anterior end of the embryo is at the left and the ventral side is at the bottom. Cuticular ventrolateral view, with anterior at the left. The identities and positions of some of the denticle belts are mutations. The pattern of ftz protein genes, and the cuticle structures that was affinity purified and used to stain noted, in photographs of ftz localizaphenotypes are shown in ventral or indicated. Scale bars are 100 pm. (a) A Wn~s~ow, CARROLL, AND SCOTT For the progeny of each mutant, defects in the ftx pattern have been correlated as precisely as possible with the cuticular defects visible in embryos at the end of embryogenesis. Because in some mutants the phenotype varies somewhat, we cannot always correlate the defects in theftx pattern to the mutant cuticle morphology with absolute precision. ftz Expression in Maternal ftx Expression Class 75 Mutants in Mutants of the Terminal Embryos that are progeny of females homozygous mutant for trk, tor, or fs(l)N exhibit phenotypes in which pattern elements at the middle of the embryo appear “expanded” anteriorly and posteriorly with concomitant loss of pattern elements at the posterior- and anterior-most regions of the embryo. The eighth dentiexuperantia, A Member of the Anterior Class, Causes cle band is entirely absent or greatly reduced in embryos from trk or fs(l)N mutant females (Figs. lh and an Anterior Shift in the ftx Pattern lj). All telson structures are absent, and the cephaloIn embryos that are the progeny of homozygous mu- pharyngeal skeleton is reduced (Schiipbach and Wieschaus, 1986). Progeny of tar females often exhibit more tant exu females the normal number of sevenftx stripes is seen, but the spacing and width of the stripes are extensive pattern deletions, leaving only abdominal aberrant. The most anterior fin stripe (hereafter re- denticle bands Al-A6 (Fig. 11). ferred to as stripe 1) in exu progeny is found at about The pattern of ftx staining in progeny of trk females 80% egg length (EL) (Fig. le, arrowhead). (The anterior is altered such that only six stripes of protein are visible (Fig. lg). The first five of these stripes have approxiedge of stripe 1 in wild-type embryos is normally found at 65% EL, where the posterior end of the embryo is mately normal positioning, width, and spacing, but are defined as OS..) The first stripe is followed by an ab- followed by an enlarged interstripe and a sixth stripe normally wide interstripe of about 20 nuclei, a second that is abnormally wide (five nuclei). The pattern of stripe offtx protein of 12-13 nuclei, an interstripe of 10 staining in fs(l)N embryos is identical to that seen in nuclei, and a series of five posterior stripes that are the progeny of females homozygous for trk (Fig. li). slightly compressed (Fig. le, bracket). The seventh Mutations in a zygotically active gene, tailless (tZZ), stripe is often only three nuclei in width (not shown), cause a similar alteration in theftx pattern (Mahoney instead of the usual five nuclei. The first stripe some- and Lengyel, 1987). times lacks a well-defined posterior boundary of ftx exIn embryos from homozygous tar females, one sees a pression. At the end of embryogenesis, when recognizftx pattern displacement similar to that of trk and able cuticular markers are visible, defects are seen at fs(l)N progeny, but with the difference that the sixth the anterior end of the embryo. The head skeleton is stripe is often positioned still more posteriorly (Fig. lk, reduced (Fig. If, arrow) and the median tooth is absent. arrowhead) or in some cases is absent (not shown). In The pattern of thoracic and abdominal denticles is in addition, the first five ftx stripes and interstripes are most cases normal, with only occasional defects seen in often slightly wider than those seen in wild-type or trk, posterior structures. This result was unexpected, be- and are shifted posteriorly. In some cases, there is a cause the region of the embryo in which the j’tx pattern slight anterior shifting of stripe 1 as well. Thus, the tar is most affected by loss of exu function gives rise to mutation appears to cause a shift of all the ftx stripes, thoracic structures, which nonetheless have a normal or while trk and fs(l)N mutations affect primarily the nearly normal cuticle. Frohnhiifer and Ntisslein-Volmost posterior two stripes. It is possible that the difhard (1987) have reported that the thorax is slightly ferences seen between the genes of the terminal class expanded in progeny of females carrying the same exu are due to characteristics of the particular alleles used allele. A more dramatic change in the cuticle might be in this study and that tw, trk, fs(l)N, and tll are involved expected from the changes in ftx expression. in the same pattern-regulating processes. wild-type embryo at the cellular blastoderm stage (3 hr) exhibits seven stripes offtz protein. (b) A wild-type embryo at the end of embryogenesis (22 hr). Some segments are indicated. T, thoracic; A, abdominal; tl, telson. (c) vdpD/vc&? The broad region offtz staining that occurs in place of stripes 3 to 6 does not form a continuous stripe around the embryo. vf, ventral furrow. There are often isolated nuclei expressing theftz protein at normal levels. (d) wosrD/waP! All abdominal denticles are absent. sp, spiracles. (e) ezuPJ/ezuP< Note the anterior shift of the first ftz stripe (arrowhead), the abnormally wide second stripe, and the compression of the posterior stripes (bracket). (f) exupJ/exup< The pattern of thoracic and abdominal denticles is essentially normal, but note the defects in the head skeleton (arrow). (g) trkRA/trkRA.This embryo exhibits fiveflz stripes of normal width and spacing followed by an enlarged interstripe and an abnormally wide sixth stripe. In some embryos, the posterior stripe extends to the very end of the embryo, forming a “cap” of staining at the end of the embryo (not shown). (h) trkR4/trkRA. Abdominal segment eight and the telson are absent. (i) fs(l)~l’/fs(l)N”‘l (dorsal view). The pattern of&z expression is identical to that seen in trk. Only six stripes of protein are visible. (j) fs(l)A@“/fs(l)N? Only seven complete abdominal denticle bands are visible. An incomplete eighth denticle band is found at the terminus of the embryo. (k) tar WK/torWKThe ftz stripes are slightly wider than in trk and shifted posteriorly. The posterior stripe covers the entire end of embryo (arrowhead). (1) torWK/torWK.Only six abdominal segments are visible. FIG. Z.ftz protein expression and cuticular phenotype of progeny from females doubly or triply homozygous for mutations in maternal-effect segmentation genes. (a, c, e, g, i) fez localization. (b, d, f, h, j) Cuticle phenotypes. (a) stm.&~~/~td~~~tor? The embryo has just begun gastrulation. Note the anterior shift and abnormal width of the first stripe and the wide band offtz staining that covers the entire posterior of the embryo. The cephalic furrow (cf) is also shifted anteriorly. (b) .+taz&or wK/sta~HLt~“? There are patches of abdominal denticles at the m/vd%rwK. The position of the anterior border of the first stripe is posterior of the embryo. No telson structures are visible. (c) ~u8~tar The thorax is expanded and a patch of abdominal denticles is located at the posterior end of the embryo knal, (d) vn~Dtw~/wa$lDtwWK. where the telson should be. (e} t&%rrWK/t~kRKtorwK. Only five stripes are visible, The two anterior-most stripes are wider than the corresponding stripe in trk or tar and are shifted anteriorly. The first stripe is found at about 76% EL, as compared to 66% in wild-type and trk WINSLOW, CARROLL, AND SCOTT The Pattern of ftx Expression in Progeny of Females Doubly Mutant for tar and stau or vas In order to learn whether interactions occur among maternal-effect genes from the different phenotypic classes, we have examined the ftx pattern in some double-mutant combinations. Strictly additive effects would not suggest interactions, while nonadditive effects on theftx pattern would suggest that the different classes of maternal-effect loci affect each other. In general, combining mutations of the posterior and terminal classes produces additive effects. Embryos that are progeny of mutant females homozygous for both stau (a posterior class gene) and tar (a terminal class gene) exhibit two anterior stripes that are followed by one large region of ftx staining that extends from what would normally be the anterior border of stripe 3 to the posterior end of the embryo (Fig. 2a). Stripe 1 is shifted anteriorly and lies just posterior to the cephalic furrow, which has also been shifted anteriorly (as in stau progeny). Thus, the tur mutation does not alter the effect of the stau mutation on the two most anterior stripes, and the changes caused by the tar mutation are not further altered (or diminished) by the loss of stau function. The cuticular phenotype of the double-mutant stau tar progeny reveals defects in pattern elements that correspond to the pattern alterations seen with the ftz antibody. Posterior structures are absent apparently due to the loss of tar function, and the abdominal segments are fused, as is characteristically seen in stau progeny (Fig. 2b). Therefore, the changes in the.@ pattern seen in stau tar progeny appear to be due to the independent, additive effects of the two mutations. Subtle deviations from a strictly additive effect occur in progeny of homozygous VCLStar females (vas, like stau, is a posterior class gene). A single broad region of posterior ftx staining again replaces stripes 3 to 6, but the apparent effect of tcw is stronger in the vas tor case than in the stau tar case (Fig. 2~). The tor mutation causes a slight posterior shift of the.@ stripes (Fig. lk), but the degree of posterior shifting is often more pronounced in vas tar double-mutant progeny. The anterior border of the third stripe is now found at only 20% EL, as compared to 50% in wild-type or 44% in stau tar progeny (Fig. 2~). As in stau tar progeny, the third stripe in vas to-r progeny now extends as a broad band to cover the entire posterior of the embryo. The cuticular phenotype ftz Expression in Maternal Mutants 77 of vas tar progeny shows a posterior shift of pattern elements relative to either of the single mutant progeny (Fig. 2d). The thoracic segments are wider than in wild-type and encompass a large portion of the embryo, while the residual abdominal denticles are found in only a small patch at the very posterior. Combinations of tar, trk, and exu Progeny of doubly mutant females were examined in order to determine whether interactions occur between genes of the terminal class (trk, tw) and the anterior class (exu). When progeny of females mutant for both trk and tar are examined, the defects in the@ pattern are similar to either single mutant, but stronger (Fig. 2e), even though both genes are in the same class. There is a clear difference in the positioning and width of the first two anterior stripes and the posterior stripe when the progeny of the double-mutant females are compared to those of the single mutants (Fig. 2e, arrowheads). In trk tar progeny, the fifth.& stripe is shifted posteriorly, and a sixth stripe is never visible although it is always present in trk progeny and often visible in tar progeny. The overall effect is that the extent of posterior and anterior shifting of theftx stripes is greater than that seen in either of the single-mutant progeny. One possible explanation is that the’ tw and trk gene products perform a similar function or participate in the same process and each mutation only partially eliminates this function. The cuticle phenotype (Fig. 2f) of progeny of the doubly mutant females is not significantly different from that seen in progeny of females homozygous for tar or trk alone, although theftx pattern in the trk tar progeny suggests that additional morphological defects might be observed. Thus, the ftx pattern is a more sensitive marker for changes in the blastoclerm fate map than is the pattern of cuticle markers seen at the end of embryogenesis, perhaps because later in embryogenesis the pattern is partially corrected. A clearly nonadditive effect on thefiz pattern is seen when tor (terminal class) is combined with exu (anterior class). Only five stripes of staining are visible and the two anterior stripes are wider and are shifted anteriorly compared to those of wild-type (Fig. 2g). However, the extent of the shifts and the altered size of the anterior stripe in exu tar progeny a0 not appear as pro- and about 67% in tar progeny. (f) trkR%wWK/trkRKtw ‘? The pattern of adominal denticles is similar to that of tar. (g) exu’%r m/exupJtor wK. Thefiz pattern shows similarity to trk and tm-.The fluorescence at the dorsal anterior surface is due to the contact with another embryo and is notjk protein. (h) exupJtorwK/exup%r ? (i) exupJtormtrkRA/exupJtormtr~A. The pattern is essentially identical to that in (e) and (g). (j) exupJtorw~t~kRA/e3cupJtorwxt~~ The defects in the head structures are more extensive than in single- and double-mutant progeny. In some cases, abdominal denticle bands 5 and 6 are fused or band 6 is entirely absent (not shown). 78 DEVELOPMENTAL BIOLOGY nounced as in exu alone. The anterior-most stripe in exu to 80% in exu (Fig. Zg, arrowhead), suggesting that normal tcw function is involved in the anterior shift seen in the absence of only exu. The extent of posterior shifting of theft2 stripes in exu tar is greater than that in tar alone, and is similar to that seen in the trk tar double-mutant progeny (Fig. 2e). This result suggests that exu function is required in the posterior of the embryo, at least in progeny of tcrr females. The pattern of abdominal dentitles in the exu tar progeny is similar to the tw or trk tor progeny. Denticle bands A7 and A8 and telson structures are entirely absent. In contrast to tar, where head structures are partially deleted, most head and gnathal structures are deleted in exu tor progeny (Fig. 3h), indicating additive effects of the two mutations on head structures. When progeny of females mutant for trk, exu, and tar are examined, the pattern of ftx expression is similar to that seen in exu tar progeny. The anterior-most stripe in the triple-mutant progeny appears wider than that in exu progeny (compare Figs. lc and Zi) and is often curved anteriorly along the dorsal-ventral axis. At the end of embryogenesis the pattern of abdominal denticles is similar to that of the double-mutant trk tm- or exu tw progeny (Fig. Zj), although more extensive pattern deletions sometimes occur. In addition, most, if not all, head and gnathal structures are absent. Thus, in all combinations of tar, trk, and exu, some nonadditive effects are seen on theftx pattern (summarized in Fig. 4), suggesting that some interactions are occurring among the gene products during early development. tar progeny is found at 70-75% EL, as compared ftx Expression in Progeny of Females Homozygous for exu and stau or vas A simple model would predict that additive effects on theftx pattern would be observed when a mutation that affects anterior development (exu) is combined with ones that affect primarily posterior development (stau or vas). However, dramatic nonadditive pattern alterations in the pattern offtx protein expression were seen in embryonic progeny of females homozygous for exu and either stau or vas. In stau exu progeny the sevenftx stripes are replaced by a large solid band of protein that extends from about 20 to 75% EL (Fig. 3a). In some embryos a partial stripe is observed at the anterior of the embryo, at about 90% EL (not shown). Considerable variation in the stripe pattern is seen in the stau exu double-mutant progeny, including embryos that have three to sixftx stripes of aberrant width and spacing, or mirror-image pattern duplications of the fcx pattern. The variability in theftz pattern is likely to be due to the residual function of the stau allele used in this VOLUME 129.1988 study. Figure 3a shows the most extreme effect on the ftx pattern of the loss of stau and exu function. The cuticle phenotype of stau exu double-mutant progeny exhibits a similar variability. In what appears to be the strongest phenotype, the embryonic cuticle consists of thoracic segments that form mirror-image duplications (Fig. 3b, arrows), followed immediately by a very reduced telson (Schiipbach and Wieschaus, 1986). No head structures or abdominal denticles are present, and an inverted telson is often found at the anterior end of the embryo. In progeny of homozygous vas exu females, a similar broad band of ftx protein is visible (Fig. 3~). When ftx protein first appears, at the cellular blastoderm stage, it is of uniform intensity along the embryo, but at the onset of gastrulation the nuclei at both the anterior and posterior borders of the broad band have high levels of fcx protein, while nuclei at the center of the embryo have lower levels of ftx protein. The intensity of the ftx staining appears to decrease toward the middle of the embryo in a graded fashion (Fig. 3e, arrow). No posterior stripe of the sort seen in stau exu progeny has been seen in vas exu progeny. In contrast to stau exu, in vas exu progeny, the fez pattern and the cuticle phenotype show little variability. No thoracic or abdominal denticles are visible (Fig. 3d), and the only discernible cuticle structures are of gnathal origin (Schiipbach and Wieschaus, 1986). DISCUSSION In order to learn more about the relationship between the maternally controlled processes of anteroposterior pattern specification and the expression of the wellcharacterized zygotic segmentation genes, we have examined the spatial pattern of ftx protein expression at the cellular blastoderm stage in embryos obtained from mutant females. A summary of the patterns of ftx protein expression is presented in Fig. 4. The effects on ftx expression were compared to the pattern of embryonic cuticle observed at the end of embryogenesis. Each of the maternal-effect mutations examined causes reproducible alterations of both the ftx protein expression pattern and the cuticular morphology. In general, the ffx stripes are very good markers for cell fate at the blastoderm stage. Because proper segmental development requires the combined activities of multiple segmentation genes, it is likely that the expression patterns of other segmentation genes are altered as well. The analysis of double-mutant progeny suggests that the maternal genes of the anterior and terminal classes or the posterior and terminal classes act independently or show only subtle interactions. In contrast, genes of the anterior and posterior classes interact very WINSLOW, CARROLL, AND SCOTT jIz Expressionin Maternal Mutants 79 FIG. 3. ftz protein expression and cuticular phenotypes of progeny from females doubly homozygous for emL and stau or vas. (a) stauHL exuPJ/stHLexuPJ. The broad band of&z staining is followed by a weak posterior stripe (arrowhead). (b) stauHLexuPJ/stHLexupJ. Arrows indicate polarity of segments. vm, vitelline membrane (removed from the embryos shown in Figs. l-2, but not from this preparation). (c) vasPDexupJ/ va8DextiP( The&z protein is distributed in a broad band from about 15-75% EL. (d) vasPDexuPJ/va?DexuPJ. Note the absence of all thoracic expression within the and abdominal denticles. (e) A higher magnification view of va.8Dex~pJ/v&Dex~ pJ at the beginning of gastrulation.fiz large stripe occurs as a gradient (arrows), from high levels anteriorly and posteriorly to low levels in the middle of the embryo. (f) exUPJ/exUPJ. A gradient of ftz expression is visible at the posterior edge of the most anterior stripe (arrow). strongly. Simultaneous loss of both anterior and posterior genes may cause the entire central region of the embryo to assume a similar fate. Changes in the ftx Pattern Can in Most Cases Be Correlated with Changes in the Cuticle Morphology Theftx protein is expressed at the early cellular blastoderm stage in even-numbered parasegments (PS) (Lawrence et al., 1987), which correspond to the primordia of the posterior maxillary (pMx) and anterior labial (aLb) segment (PS2), posterior prothorax (pTl), and anterior mesothorax (aT2) segment (PS4), and so on (Fig. 4). The stripes (and interstripes) of ftz protein thus serve as molecular markers for cell fates in the blastoderm embryo. For the most part, in embryos that are progeny of mutant females, the segmental primordia of the embryo where the ftx pattern is altered and the regions of the cuticle that are defective are closely correlated. This indicates that the maternal genes have most of their effects on embryonic patterning prior to the cellular blastoderm stage. 80 DEVELOPMENTAL BIOLOGY VOLUME 129.1988 In stau progeny, the widening and shifting of the stripes that mark the thoracic and gnathal primordia Wt (PS2-PS4) also correlate with the cuticle phenotype. 2 4 6 8 10 12 14 Cuticular structures derived from PS2-4 occupy a larger part of the embryo than those in wild-type. The stau anterior shift offtx stripes 1 and 2 in stau may occur as a result of the loss of a function that would under norvas \\J mal circumstances prevent the anterior stripes and interstripes from expanding to the anterior. Precedent for such a mechanism is seen in the interactions among gap genes. The domain of one gap gene expands in the absence of an adjacent gap gene function (Jackie et al, 1986). trk 71 In trk and fs(l)N progeny there is a very good correlation between the segmental primordia that appear shifted or absent as shown by theftx pattern and the structures that are defective in the larval cuticle. The sixth ftz stripe marks PS12 in the blastoderm anlage (the primordia for pA6 and aA7) (Fig. 4). The presence of the sixthftz stripe in trk and fs(l)N progeny suggests that the A7 denticle band will be present in the cuticle, as is the case. (The denticle bands originate from the anterior of each segment primordium and form in the anterior region of each segment.) Although the eighth exu tar b denticle band is derived from the region of the embryo marked by the sixth interstripe (PS13, i.e., pA7 and aA8) in wild-type embryos, no eighth denticle band is trk tar 11 seen in trk progeny. This may be because the sixth interstripe seen at the posterior end of trk embryos contains only the primordia for pA7 structures and lacks exutrktar(~J aA primordia. There are no morphological markers for pA7; correspondingly, only naked cuticle is seen at the posterior end of trk progeny (Fig. lg). In fs(l)N progeny, however, the sixth interstripe seen in the embryo probably includes primordia for at least part of aA8, as a small patch of A8 denticles are present in the cuticle of fs(l)N progeny (Fig. lj). FIG. 4. A summary of the patterns of & staining in progeny of In tor progeny, the A7 denticle band is often missing, females homozygous for maternal-effect mutations. In the first panel, segmental (S) and parasegmental (PS) boundaries are indicated, as and A6 is positioned at the posterior of the embryo. In well as the approximate positions of & protein stripes in wild-type addition, all abdominal denticle bands appear to be embryos. In the following panels, the positions of ftz stripes in the shifted posteriorly to a greater degree than those in trk mutant progeny are shown. In each case, the average positions of the or fs(l)N. This phenotype correlates with the ftx patftz stripes were determined by measurements of at least four emtern. Stripes 1 to 5 are shifted posteriorly, and the sixth bryos. Measurements were made using the ends of the embryo as j%z stripe (PS12, i.e., pA6 and aA7) is either entirely reference points. M, maxillary segment; L, labial segment. missing or is positioned at the very posterior of the embryo (Fig. 4). In exu tar, trk tar and exu trk tor progeny, only five ftz In the stau and vas mutant phenotypes, theft2 pattern is altered primarily in the region of fix stripes 3 to stripes are seen, and what appears to be the fifth ftz interstripe (PSll, i.e., pA5 and aA6) is found at the 6 (PS6-PS12), which comprise the segmental primordia posterior of the embryos. The& pattern predicts corfor Al through A7 (Fig. 4). It is these pattern elements that are largely absent in embryos from stau or vas rectly that A6 is the most posterior segment observed in homozygous females. Thus, both stau and vas are re- the cuticle of progeny of females of these three genoquired to direct cells to form the primordia for the ab- types. The anterior shifting of stripes 1 and 2 that is observed is consistent with the defects occurring in dominal segments at the cellular blastoderm stage. ‘thorax’ S tOrI vasexu I abdomen’ (M~Lt112131112l3~4151617181 WINSLOW, CARROLL, AND SCOTT head structures, although the thoracic segments appear normal in these progeny. Paradoxically, the head defects in the double- and triple-mutant progeny appear more severe than those in exu alone, yet the j?ftx stripes appear to be shifted anteriorly to a lesser extent. As molecular markers for anterior cell fates become available, it maybe possible to resolve this paradox. Additive and Nonadditive Eflects Are Observed in Double-Mutant Progeny ftz Expression in Maternal Mutants 81 ening of the anterior-most ftx stripes in a manner not observed in either single mutant. Considering the similarity in the cuticle phenotypes of tor and trk, one might have expected that the double-mutant progeny would show a pattern offtx stripes similar to that of the single mutants. This result would have suggested that tar and trk act in a common pathway of cell determination. The observed results suggests either that the two genes act in parallel pathways, both contributing to anteroposterior patterning, or that the alleles examined only partially inactivate two genes that act in the same pathway. The tar mutation causes a posterior shifting of theftx stripes, and the stau mutation causes a widening of the Interactions between exu and Genes of two most-anterior stripes and interstripes and the forthe Posterior Class mation of a large band offtx staining in place of stripes An exact correlation between the extent of pattern 3 to 6 (Fig. 4). In the double-mutant stau tar progeny, the broad band of ftx protein in stau progeny appears to disruption of theftx stripes and the cuticle defects is not seen in the progeny of homozygous exu females. The have spread to the posterior end of the embryo, presumably due to the absence of tor function. The ftx pattern two anterior ftx stripes and interstripes, which normally correspond to the primordia of PS2 through PS5 can therefore be understood as the result of additive stau and tor effects, suggesting that tor (a terminal (i.e., pMx through aA3), are abnormally positioned and of incorrect width. In the larval cuticle, however, the gene) and stau (a posterior gene) act largely independently of one another in the oocyte or embryo. thoracic segments, which include PS3 through PS5, apThe effect of combining vas with tar is very similar to pear normal or are slightly expanded (Frohnhofer and that seen in stau tor progeny. In this instance, however, Niisslein-Volhard, 1987). The most obvious defects in some deviation from strict additivity of the ftz pattern exu progeny appear in the head skeleton. The head dechanges is seen. The posterior shift caused by the tar fects are probably due to respecification of the fate of mutation is often more pronounced in the double-muthe cells that are normally found at positions anterior tant progeny. The two anterior stripes and interstripes, to 65% EL and are therefore outside the region where which mark PS2 through PS5, are wider than in either ftx is expressed. The anterior expansion of theftz patsingle mutant and are shifted posteriorly. The embrytern suggests that the cells that normally give rise to onic cuticle phenotype reflects this posterior shift, in the most anterior structures have adopted more postethat structures derived from PS2 through PS5 (pMx rior fates. through aT3) occupy nearly the entire embryo. Thus, Frohnhijfer and Niisslein-Volhard (1987) have postuwhile the phenotype of vas tor progeny is to a first lated that the exu phenotype is associated with the alapproximation the sum of the vu.s and tar phenotypes, tered activity of bed, a gene which has been shown to be there are some changes in theftx pattern that would not required for organizing anterior development in the have been predicted from an examination of either sin- embryo. Cytoplasmic transplantation experiments have gle mutant. demonstrated that wild-type bed activity can cause the Other deviations from a strictly additive effect on the development of ectopic anterior structures (FrohnhMer ftz pattern can be seen when the anterior class mutation and Niisslein-Volhard, 1986). In addition, bed RNA is exu is combined with mutations of the terminal class. In observed to form a gradient in the embryo, from high progeny of exu tar females, the ftx pattern exhibits tar- levels anteriorly to low levels posteriorly (Frigerio et ah, like posterior shifts and exu-like anterior shifts. How- 1986). It was proposed that exu functions to localize bed ever, the extent of posterior shifting caused by tar muactivity to the anterior pole and that the level of bed tations is exaggerated in the absence of exu function as gene activity determines the degree of anterior developit is in the absence of vas, and the extent of anterior ment. The loss of exu causes a more even distribution of shifting is not as pronounced as it is in exu progeny bed gene product, and consequently the loss of anterior alone. The tor mutation in the double-mutant exu tar structures and the expansion of thoracic positional progeny in some manner reduces the anteriorly directed values (Frohnhofer and Niisslein-Volhard, 1987; Bershifting of the ftz pattern that is caused by the exu leth et ah, 1988). mutation. When exu, which primarily affects anterior patternProgeny of mutant females homozygous for trk and ing, is combined with vas, which has its greatest effect tar exhibit an anterior and posterior shifting and widin the abdominal primordia, the embryonic progeny ex- 82 DEVELOPMENTAL BIOLOGY hibit one very broad band offtx protein (Fig. 4). During gastrulation, this band of ftx expression forms a gradient of ftx protein that decreases toward the middle of the embryo. A similar gradient of fez staining is sometimes visible at the posterior border of stripe one (PS2) in exu progeny (Fig. 3f). An interpretation of the ftx pattern in exu vas progeny is that the single large ftz stripe is in fact an expansion and mirror-image of the first ftz stripe. This interpretation is supported by the cuticular phenotype in which the vestigial cuticle is entirely of gnathal origin, which is derived in part from PS2 (pMx and aLb). In contrast to the vas exu case, the large stripe of ftx staining visible in progeny of homozygous stau exu females may correspond to an enlarged PS4ftx stripe (i.e., pT1 and aT2). This interpretation is supported by the cuticle phenotype, in which T2 is duplicated and flanked anteriorly by Tl and, in some cases, telson structures, and posteriorly by a reduced telson (Schiipbach and Wieschaus, 1986). The large stripe of fix staining possibly marks the primordia for the thoracic structures, and the ftz stripes seen at the anterior and posterior ends of the embryo are probably a normal and duplicated/inverted seventh ftx stripe, which marks the primordia for the terminal segmented structures. Theftx patterns and the cuticle phenotypes described above for stau exu and vas exu may be a result of the altered activity of bed, as proposed for exu (Frohnhofer and Ntisslein-Volhard, 1987; Berleth et ah, 1988). It has been inferred that the activity of a gene of the posterior class, oskur, can inhibit bicoid activity. Injection of cytoplasm from oskur+ embryos into the anterior of wildtype embryos leads to a reduction of anterior structures; cytoplasm from oskar- embryos has no effect (Lehmann and Niisslein-Volhard, 1986). The findings suggest that oskur, and possibly other genes of the posterior class such as vas and stau, inhibit bed activity at the posterior pole. In the absence of exu function, which would normally localize bed function in the anterior, and without vas or stau, which may act negatively upon bed function in the posterior, bed may act uniformly in the embryo. This altered bed activity would then cause all cells to assume anterior developmental fates. In the case of vas exu the anterior cells have a gnathal identity, while in stuu exu they have a thoracic identity. This suggests that bed activity is roughly uniform, but at different levels in the two double-mutant progeny. In vas exu we propose that bed gene activity is at a level that would specify gnathal development (PS2), while in stau exu, bed gene activity is at a level that specifies thoracic development (PS4). The appearance of terminal structures and the absence of ftx expression at the poles in the double mutants may be due to the normal activity of the terminal genes. The differences in the VOLUME 129,1988 phenotypes seen in vas exu and stau exu may be because other genes of the posterior class) functions in the anterior of the embryo as well and thus interacts differently with bed and/or exu. stuu (unlike We thank Drs. Trudi Schiipbach, Adelheid Degelmann, and Anthony Mahowald for sending stocks and Vern Twombly for care and maintenance of the stocks. We also thank Trudi Schiipbach for her insightful comments, encouragement, and suggestions; Joan Hooper and David Gubb for critical comments on the manuscript; and Wolfgang Driever for a stimulating discussion. We thank Cathy Inouye for skilled preparation of the manuscript. This work was supported by an NIH Postdoctoral Fellowship GM09756 to S.B.C., NIH Grant HD18163, and a Searle Scholar Award to M.P.S. REFERENCES AKAM, M. (1987). The molecular basis for metameric pattern in the Drosophila embryo. Develomnt lOl, l-22. ANDERSON, K. V., and N~SSLEIN-VOLHARD, C. (1984a). 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