for help with keeping and selecting the mutant flies, Greg. Gibson, Leslie Pick and Alexander Schier for comments and suggestions on the manuscript, and Erika ...
Development 101. 421-435 (1987) Printed in Great Britain © The Company of Biologists Limited 1987
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Hierarchy of the genetic interactions that specify the anteroposterior segmentation pattern of the Drosophila embryo as monitored by caudal protein expression
MAREK MLODZIK and WALTER J. GEHRING Biozemrum, University of Basel, Klingelbergslr 70. CH-4056 Basel. Switzerland
Summary The establishment of the body pattern of Drosophila along the anteroposterior axis requires the coordinated functions of at least three classes of genes. First, the maternally active ccordinate genes define the polarity of the embryo and act as primary determinants; second, the segmentation genes divide the developing embryo into the correct number of segments and third, the segments become specified by the homeotic selector genes. We have examined the effects of mutations in the genes of the first two classes on the spatial distribution of the protein product(s) of the caudal (cad) gene, which in wild type shows a graded distribution along the anteroposterior axis during the syncytial blastoderm stage, whereas its persistent zygotic expression is confined to the telson region (the posterior terminal structures). Mutations in maternal genes that specify the spatial coordinates of the egg and the future embryo change the gradient distribution of cad according to the alterations of the fate
map which they produce. A second group of maternally expressed genes, the gap genes of the 'grandchildless-knirps' group, which are considered to represent posterior activities, do not have any effect on the cad gradient. The same is true for the zygotic segmentation genes that are active after fertilization. However, the same class of zygotic genes partly affects the zygotic cad expression in the telson. Therefore, the two phases of cad expression represent different levels within the genetic hierarchy. The cad protein gradient seems to form in response to the primary maternal determinants independent of the segmentation genes, whereas the latter influence zygotic cad expression in the telson region which corresponds to a homeotic selector gene function.
Introduction
of the anterior (die) or the posterior (bid) pattern (Bull, 1966; Lohs-Schardin & Sander, 1976; NussleinVolhard, 1977; Lohs-Schardin, 1982; Mohler & Wieschaus, 1986). More recently, other coordinate genes have been isolated (Frohnhofer & Niisslein-Volhard, 1986; Schupbach & Wieschaus, 1986). All of the anteroposterior coordinate genes, when mutated, alter the pattern along the entire axis. The maternal gap genes define the second group of maternally required genes. In contrast to the long-range effects of mutations of the coordinate genes, maternal gap mutants show strictly localized defects. For example, mutant embryos of the 'grandchildless-knirps' group
The anteroposterior body pattern of a developed Drosophila embryo consists of a defined number and sequence of segments. The formation of these metameric subdivisions depends on different classes of genes. The earliest acting class of genes are the maternal-effect genes, which can be subdivided into two groups: the coordinate genes and the maternal gap genes. The two classic examples of coordinate mutants are bicaudal (bic) and dicephalic (die), which in the most severe cases change the anteroposterior pattern completely, leading to bipolar embryos with duplications
Key words: maternal-effect genes, homeobox, protein gradient, immunofluorescence, segmentation genes, Drosophila.
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(Schiipbach & Wieschaus, 1986; Lehmann & Niisslein-Volhard, 1986; Boswell & Mahowald, 1985) show localized defects in the abdomen, while head, thorax and telson (i.e. the most posterior somatic structures) develop normally. An interplay among the various maternal-effect genes leads to a distinct specification of positional information along the anteroposterior axis, which is later interpreted and refined by the zygotically active segmentation genes (Niisslein-Volhard & Wieschaus, 1980) and by homeotic selector genes (Garcia-Bellido, 1975; Lewis, 1978; reviewed by Gehring & Hiromi, 1986). Thus, as early as the cellular blastoderm stage, when almost all cells look identical, specific groups of cells are already committed to specific developmental pathways (Chan & Gehring, 1971; Wieschaus & Gehring, 1976; Lawrence & Morata, 1977; Simcox & Sang, 1983). The effects of single maternal-effect loci on the blastoderm fate map can be visualized at this stage using cloned probes or antibodies directed against the segmentation genes, revealing local and long-range effects of the different genes (Carroll, Winslow, Schupbach & Scott, 1986; Mlodzik et al. 1987). The caudal (cad) gene was isolated by screening the Drosophila genome under reduced stringency conditions (McGinnis et al. 1984) using a homeobox clone as probe. It is expressed maternally as well as zygotically, and its maternal transcripts and protein distribution follow a concentration gradient along the anteroposterior axis during the syncytial blastoderm stage. Later in development cad is expressed in the most posterior region of the ectoderm (A 10 and hindgut), as well as in the endoderm (posterior midgut) (Mlodzik, Fjose & Gehring, 1985; Levine et al. 1985; Macdonald & Struhl, 1986; Mlodzik & Gehring, 1987). The presence of a molecular gradient suggested the possibility that cad is involved in the establishment and/or maintenance of positional information. The phenotypic analysis of maternal and zygotic mutants showed that cad~ embryos have graded defects along the anteroposterior axis which are most severe in the posterior part of the developed embryo. However, most of these defects can be rescued by either the maternal or the zygotic activity alone (Macdonald & Struhl, 1986). These findings suggest that cad is probably not a primary determinant of anteroposterior polarity but could be involved in the transfer of information between the primary maternal determinants and the zygotic genes. To investigate this and other possibilities we have monitored cad protein distribution in embryos derived from females carrying mutations in genes affecting the anteroposterior pattern. We find that mutants having a long-range effect on the blastoderm fate map alter the gradient pattern, and that mutants of the
'grandchildless-knirps' group, with the exception of staufen (stau) do not affect this pattern significantly compared to wild type. Another subset of segmentation mutants does not have any effect on the gradient, but eliminates the expression of cad in the region of the telson anlagen. Materials and methods Antibody preparation A 1-86kb Xhol-EcoRl fragment of the cDNA clone pSC33F (Mlodzik & Gehring, 1987) containing approx. 85 % of the cad open reading frame was inserted into the /J-galactosidase C-terminal expression vector pTRBO (Biirglin & De Robertis, 1987). The resulting recombinant plasmid pTRB.33 gives rise to a fusion protein of approx. ^SxKfiMr, and was induced in bacterial cultures grown in the presence of 2mM-IPTG for 2-4 h. The cells were pelleted, resuspended and lysed in a French pressure cell as described by Wirz, Fessler & Gehring (1986). The fusion protein was purified over a p-amino-phenyl-/S-D-thiogalactosidase affinity column, washed and eluted as described by Ullmann (1984). Mice were immunized with 50/zg of purified cad-fusion protein, boosted at 10-day intervals with 25 /ig of protein, and after the second boost serum showed specific labelling. The serum was preadsorbed to Sepharose coupled with E. coli extracts containing /S-galactosidase to remove nonspecific antibodies.
Immunohistochemical methods and embryo selections Embryos were fixed, permeabihzed and incubated with antibodies as described previously (Mlodzik & Gehring, 1987) and mounted in Gelvitol (Rodriguez & Deinhardt, I960). Embryos were collected from homozygous or transheterozygous mutant mothers in the case of maternal-effect mutants. The mutant mothers were selected by genetic markers from balanced populations. The loci and alleles are listed in Table 1. Homozygous zygotic mutants were obtained from balanced heterozygous parents. Embryos were collected on apple-juice agar plates with yeast paste at 22° or 25°C, except for pww680 which was collected at 18°C (temperature-sensitive allele; Lehmann, 1985). From each population of mutant embryos, cuticle preparations were analysed for expected phenotypes. We do not know if the alleles of the maternal loci are null alleles. However, all the alleles we used in this study represent typical phenotypes of each particular locus and are the strongest available. All of the alleles of the zygotic segmentation mutants are either amorphic or strong hypomorphic alleles. Results
cad protein expression during wild-type embryogenesis To visualize cad protein distribution throughout embryogenesis of wild-type and mutant embryos, we have raised polyclonal mouse antibodies directed
Effects on cad protein expression
against the cad portion of a ^S-galactosidase-cad fusion protein as described in Materials and methods. In wild-type embryos cad RNA and protein distribution follows a concentration gradient along the anteroposterior axis (Mlodzik etal. 1985; Levine etal. 1985; Macdonald & Struhl, 1986; Mlodzik & Gehring, 1987). To assist an interpretation of our findings in mutant embryos, the important stages of cad protein distribution are summarized below. The maternal RNA from the cad locus is synthesized in the nurse cells during oogenesis and becomes evenly deposited in the growing oocyte (Mlodzik etal. 1985; Mlodzik & Gehring, 1987). Following fertilization, the single nucleus of the zygote undergoes 13 largely synchronous divisions before formation of the cellular blastoderm (Zalokar & Erk, 1976; Foe & Alberts, 1983). These syncytial stages have been subdivided by Foe & Alberts (1983) into fourteen stages according to the nuclear divisions. During very early Drosophila development, stages 1-5, we do not detect any cad protein although minute amounts below the detection level cannot be excluded. Around stage 6 or 7 we begin to see faint specific staining in the cytoplasm. During nuclear migration (stages 7-9), most of the nuclei migrate towards the periphery of the egg to form the syncytial blastoderm and, during the same time, cad protein accumulates mainly in the posterior half of the embryo forming a concentration gradient along the anteroposterior axis. At the end of this period, peripheral nuclei at the posterior pole form the pole cells and cad protein can be detected in these cells from the time of their formation up to gastrulation stages (Figs 1,2). From stage 9 on, cad protein is mainly localized in the nuclei (Fig. 1A) although staining in the cytoplasm is still detectable up to stage 13. The differences in amounts of cad protein between the anterior and the posterior ends rise markedly during the later stages of the syncytium (stages 10-14) and the gradient becomes steeper (Fig. 1A-E). After stage 13, cad protein starts to disappear from the anterior end progressively towards the posterior half of the embryo (Fig. IE) and, in later stage-14 embryos, cad protein levels also decline in the most posterior 13 % of the embryo (except in the pole cells, Fig. IF). Moreover, the decline of protein from the anterior is faster on the ventral side, so that on the dorsal side cad protein extends more anteriorly in mid stage-14 embryos (Fig. IF; Macdonald & Struhl, 1986)". Following stage 13 up to cellularization, similar processes are detectable at the RNA level as well (Mlodzik & Gehring, 1987). However, it is interesting to note that the protein gradient precedes the RNA gradient: the RNA remains evenly distributed up to stage 12 of early embryogenesis.
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After cellularization, at the onset of gastrulation, zygotic expression of cad is detected in a stripe of about four cells (Fig. 2A) extending approximately from 13 to 19 % egg length (0 % represents the posterior pole) and this band lies just at the posterior edge of the ventral furrow (Fig. 2B). As gastrulation proceeds and the germband starts to elongate, a ring of cells (3-4 cells wide) at the posterior end of the germ band shows staining for the cad protein. Note that the cad protein in the pole cells (arrowheads in Fig. 2) results from maternal expression since no cad RNA is detectable over the pole cells (Mlodzik et al. 1985; Mlodzik & Gehring, 1987), and in embryos that do not express cad protein zygotically, the pole cells still show antibody staining due to the maternal contribution (Macdonald & Struhl, 1986). In subsequent development, i.e. throughout further embryogenesis and larval development, cad is not only expressed in ectodermal tissue, for example in the anal pads, but also in endodermal tissue, in the posterior midgut (Fig. 2D, Mlodzik et al. 1985; Macdonald & Struhl, 1986; Mlodzik & Gehring, 1987). cad protein distribution in embryos derived from maternal coordinate mutant females
The presence of a molecular gradient along the anteroposterior axis suggests that cad could be involved in specification or interpretation of positional information along this body axis. Head plays a role in specifying the body pattern or in transmitting instructive information for its formation, altered cad protein distribution should be correlated with global changes along the anteroposterior axis that are caused by mutations in the maternal-effect genes of the coordinate class. It has been shown previously that this is the case for the BicD locus, where embryos derived from mutant mothers develop double abdomens in mirror-image symmetry (Mohler & Wieschaus, 1986); this abnormal polarity is already reflected by cad protein distribution during syncytial blastoderm stages. The protein is first evenly distributed over the embryo and then forms two mirror-symmetric posterior rings (Macdonald & Struhl, 1986; Mlodzik & Gehring, 1987). Mutations in the bicoid gene (bed) also lead to the absence of head and thoracic structures, but instead of duplication of the abdomen as in BicD embryos, only the telson (anal pads, tuft and posterior spiracles with filzkorper) is duplicated at the anterior pole (Frohnhofer & Niisslein-Volhard, 1986). In addition, in embryos derived from bed" females all abdominal structures that are present seem to be shifted more anteriorly (Frohnhofer & Niisslein-Volhard, 1986), and this is clearly reflected by the expression patterns
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of segmentation genes (Mlodzik et al. 1987). The distribution of cad protein in bcd~ embryos is essentially even along the anteroposterior axis during the syncytial blastoderm until early stage 14. Fig. 3A,B
show cad protein distribution in embryos derived from homozygous bcdBl females at stages 10 and 13 respectively: the staining resembles very much that observed in BicD embryos (Macdonald & Struhl,
Table 1. cad protein expression in mutants affecting the anteroposterior pattern Gene class locus and alleles studied
Larval phenotypes
MATERNAL COORDINATE bicaudal D double abdomen in mirror-image 7 w symmetry; head, thoracic and B i c D niE48 BicD ' anterior abdominal structures missing bicoid head, thorax and anterior bcd EI abdomen missing, telson duplication at anterior end anterior head structures missing exuperantia
2 posterior rings in mirror-image symmetry
evenly distributed at high levels, asymmetric disappearance (Fig 3) weak expression towards the anterior tip, normal in posterior half weak expression towards the anterior tip, normal in posterior half gradient 'normal'
2 posterior rings, slightly asymmetric
gradient 'normal'
no cad expression in ectoderm
no effect
no effect
most of the abdomen missing
no effect
no effect
most of the abdomen and anterior head structures missing
no effect
parts of the abdomen missing
weak expression towards the anterior tip. weak evenly distributed in posterior half (Fig. 7) no effect
no effect
most of the abdomen missing
no effect
no effect
all of the abdomen missing
no effect
no effect
thoracic and anterior abdominal structures missing, A6 duplicated most of abdominal structures missing gnathal and thoracic segments missing, defects in A8 A8 and telson absent: shortened pharyngeal ridges
no effect
no effect
no effect
no effect
no effect
cad stripe 1-2 cells broader (Fig. 9) no cad expression in ectoderm (Fig. 9)
„,,,,, 1497
anterior head structures and structures posterior to A7 missing anterior head structures and structures posterior to A7 missing
torso t o r ™ , tor p v trunk trk R K , trk PI
MATERNAL oskar osk 166 pumilio pum 68 " staufen stau" L , stau D 3
GRANDCHILDLESS-KNIRPS* all of the abdomen missing
tudor tudwc valois vls R B , vls P E vasa vas P D ZYGOTIC GAP Kruppel K r , Kr 581 knirps kni I I D 4 8 , kni 7 M 4 S hunchback hb 1 4 F 2 1 tailless til 1 -'"- 22 ZYGOTIC hairv h 5Hb7
Effect on zygotic cad expression
evenly distributed at high levels, symmetric disappearance from middle of the embryo
anterior head structures missing
swallow
Effect on cad protein gradient
no effect
abnormal cad expression in anterior endoderm after germband extension some embryos show expression in anterior endoderm after germband extension no cad expression in ectoderm (Fig. 8)
PAIR-RULE
hlL79K
fusht larazu ftz 9H34 ; Df(3R)9A99
deletion of even denticle bands and odd naked cuticle deletion of odd denticle bands, even naked cuticle and parts of the telson
no effect
no effect
no effect
no initial cad expression during blastoderm and early gastrulae (Fig. 9)
* In addition to the abdominal phenotype. all mutants lack pole cells except pumilio.
Effects on cad protein expression 1986; Mlodzik & Gehring, 1987). Although the protein distribution looks even, it cannot be excluded that small differences below the detection level are present. During the formation of the cellular blastoderm the staining declines from a region of the embryo at approx. 60-70 % egg length ( 0 % represents the posterior pole), as well as in narrow zones at both poles. At the end of cellularization two stable rings of cad protein expression can be detected close to each pole (Fig. 3C). The ring at the normal posterior end resolves slightly faster than the one at the anterior end, and the distances of the two rings from the respective poles of the embryo are also different, the anterior one being closer to the pole
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than the posterior one (Fig. 3C). The pole cells (arrowhead in Fig. 3C) indicate the normal posterior end. However, the location of both rings relative to the corresponding poles is different from wild type, in both cases (compare Figs2A, 3C). The embryo in Fig. 3C is viewed from its dorsal side, note that between the two rings a longitudinal stripe (5-7 cells) at the most dorsal position still contains detectable levels of cad protein; in wild-type embryos the dorsal expression usually extends to 4 5 - 5 0 % (Fig. IF). Throughout later embryogenesis of bcd~ embryos cad protein is detectable in the ectoderm and the endoderm at both poles (data not shown).
Fig. 1. cad protein distribution during the syncytial blastoderm in wild-type embryos. The anterior points always to the left and dorsal up, except in (F) which shows a dorsal view of an embryo. (A) Embryo during stage 10 (after Foe & Alberts, 1983). (B) Embryo at stage 11. (C) Embryo at stage 12. (D) Embryo at stage 13. (E) Embryo during early stage 14. (F) Embryo during mid stage 14 just prior to cellularization, dorsal view. Note that in (F) cad protein is still detectable in the most dorsal region between 13% and 60% egg length. Bar, 0 1 mm.
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Another type of coordinate mutant is represented by exuperantia {exu, Schiipbach & Wieschaus, 1986), and swallow (swa) (Stephenson & Mahowald, 1987), formerly called fs(l)1502 (Gans, Audit & Masson, 1975; Niisslein-Volhard, Wieschaus & Jurgens, 1982). Mutations in the exu locus lead to partial deletions of the head region and duplication of the posterior midgut invagination at the anterior pole (Schiipbach
& Wieschaus, 1986). Similar deletions of head structures are observed in mutants of the swa locus (Niisslein-Volhard etal. 1982). However, the fate map at the blastoderm stage in both these mutants shows
Fig. 3. Expression of the cad protein in embryos derived from females homozygous for a strong bed mutation. Anterior points to the left. (A,B) Embryos during the syncytial blastoderm at stage 12 and 13, respectively. (C) Embryo at the cellular blastoderm stage, dorsal view. Note the asymmetric localization of the two cad stripes. Pole cells are indicated by an arrowhead. Note also that cells in the most dorsal region still contain detectable levels of cad protein. Bar, 0 1 mm. Fig. 2. cad protein distribution at cellular blastoderm, gastrulation and germband extension stages in wild-type embryos. The anterior points always to the left. (A) Embryo at the cellular blastoderm stage. Pole cells are indicated by an arrowhead. (B) Ventral view of a gastrulating embryo. The ventral furrow is indicated by little arrows. (C) Lateral view of an embryo at the beginning of germband extension. Pole cells are indicated by an arrowhead, dorsal points up. (D) Lateral view of an embryo at the extended germband stage, dorsal up. Note the weaker staining in the posterior midgut rudiment (pmg). Bar, 0 1 mm.
Effects on cad protein expression
more severe alterations than those that are obvious from the larval cuticle. Using molecular probes for segmentation and homeotic genes, it can be shown that posterior head and thoracic anlagen are shifted anteriorly almost to the anterior tip of the mutant embryos, and that the abdominal anlagen are pushed slightly in the posterior direction (Mlodzik et al. 1987). In situ hybridization experiments with cad probes to embryos from exu mothers did not reveal any changes compared to wild-type embryos prior to the germband-extension stage (Mlodzik et al. 1987). However, using antibodies directed against the cad protein, differences from wild-type distribution are detectable in embryos from exu~ and swa~ mothers. From stage 6 throughout stage 13 of early embryogenesis cad protein extends to the very anterior tip in exu~ embryos (Fig. 4A). The posterior part still shows a cad distribution which is hardly different from the one observed in wild-type embryos (Fig. 4). A comparison of scanning curves that reflect the cad staining intensity in exu~ and wild-type embryos is shown in Fig. 5. During early stage 14 and cellularization the cad protein gradient also sharpens to the posterior end as in wild-type (Fig. 4B), and focuses into the one stripe close to the posterior end. However, the gradient observed in exu~ embryos is always
less steep than the one found in wild-type embryos (see Fig. 5). Very similar changes of cad protein distribution are detected in embryos derived from swa females, although the alterations are less extreme than in exu~ embryos. At the extended germband stage and later in development of exiC embryos, the duplication of posterior midgut tissue at the anterior end is reflected through expression of cad RNA and protein in endodermal tissues at both embryonic ends as visualized by in situ hybridization and antibody staining (Mlodzik et al. 1987, and our unpublished results). Analysis of cad protein distribution in mutants of coordinate genes affecting the dorsoventral axis (the three mutants tested were: the dorsalizing loci dorsal (dll) and windbeutel (wblRP), and the partially ventralizing locus cactus (cac1"0) for review see Anderson, 1987), did not reveal any differences from wild-type pattern(s), except that the small difference between dorsal and ventral resolution of the posterior
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Fig. 4. Expression of the cad protein in embryos derived from exu females. Very similar distribution is observed in embryos derived from swa females. Anterior points to the left. (A) Embryo during syncytial blastoderm stage 12. (B) Embryo during early stage 14. Bar, 0 1 mm.
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01 Too
Fig. 5. Scanning of aid intensity in exu and wild-type embryos. (A) exu' embryos at stage 13 (two upper panels) and early stage 14 (lower panel). (B) Wild-type embryos of same stages as the respective exu embryos shown in (A). Horizontal axis: egg length along the anteroposterior axis, given in % from 0 to 100 (0 % represents the posterior pole). Vertical axis: cad staining intensity, 0, background level; 1, maximal intensity. Immunofluorescence pictures of the embryos were scanned along the anteroposterior axis using standard scanning equipment. Note that in exu~ embryos (A) the intensity raises above background levels immediately at the anterior end, and that the gradient is less steep.
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stripe in stage 14 was absent in dl and wbl embryos. In the cac allele that was tested no difference to the wildtype pattern was observed (data not shown). cad protein expression in mutants of the maternal 'grandchildless-knirps' group A common phenotypic feature of the larval cuticle of all the different loci of the maternal-effect 'grandchildless-knirps' (grd-knirps) group is the total or partial lack of the development of the abdomen - a feature also seen in the embryonic phenotype produced by the zygotically acting lethal mutation knirps (Nusslein-Volhard & Wieschaus, 1980). All these genes, tudor (tud, Boswell & Mahowald, 1985), valois (vis), vasa (yas), staufen (stau) (Schupbach & Wieschaus, 1986), oskar (osk, Lehmann & Nusslein-Volhard, 1986), and pumilio (pum, Lehmann, 1985), cause deletions or fusions of abdominal segments; in the most severe cases all abdominal segments are missing, (e.g. oskar, vasa). Detailed cuticular descriptions of each of these mutant embryos have been presented (see References cited). The second feature seen in mutant embryos of all these complementation groups is the complete absence of pole cells (the exception is pumilio, which forms normal pole cells). The cad protein distribution in almost all these mutant embryos (for alleles and allelic strength, see Table 1 and Materials and methods) looks like that
of wild-type embryos. From the earliest detectable stages throughout syncytial and cellular blastoderm, a gradient as observed in wild-type embryogenesis forms. The gradient sharpens up to the posterior half and focuses into the stripe close to the posterior end just prior to the onset of gastrulation. The zygotic expression of cad during embryogenesis is also unaffected. Representative embryos of this group of mutants (derived from oskar~ females) are shown in Fig. 6. The only observed difference from wild type during these stages of embryogenesis is the lack of pole cells and therefore the lack of pole cell staining. The only exception within this group is stau, which shows different cad protein distribution than in wild type. However, stau differs also from the other genes of this group with respect to its cuticular phenotype. The developed embryos not only show defects in the abdomen, but also have similar head defects to embryos from torso-like mothers: structures corresponding to the embryonic labral segment are reduced or absent (Schupbach & Wieschaus, 1986). The distribution of cad protein in embryos derived from stau females has two different features compared to wild type. First, protein is present at detectable levels (almost) to the anterior end of the embryos, similar to the change observed in exu~ embryos; second, there is no gradient-like increase towards the posterior end, but rather a uniform weak
Fig. 6. Expression of the cad protein in embryos derived from osk females. Anterior always points to the left. (A) Embryo during syncytial blastoderm stage 10. (B) Embryo during stage 13. (C) Embryo during early stage 14. (D) Embryo at the cellular blastoderm stage. Note the absence of pole cells. Bar, 01 mm.
Effects on cad protein expression staining all over the embryo (Fig. 7). The staining observed in stau~ embryos is actually weaker compared to wild type than is apparent in Fig. 7 (panel a and b: stauHL; panel c: stauDi), and is generally of similar intensity as the staining seen in the anterior part of exu~ embryos (Fig. 4A). The s/a« HL -allele shows variable phenotypes, ranging from almost normal abdomens up to complete fusion of abdominal segments A 1 - A 7. This variability is also reflected by antibody staining of ftz protein (Carroll etal. 1986, and our unpublished observations), and cad protein, where some embryos (10-20 %) show a more or less normal cad gradient (data not shown) and others show cad protein distribution as shown in Fig. 7. The
Fig. 7. cad protein expression in embryos derived from stau females. Anterior always points to the left. (A-C) Embryos during syncytial blastoderm stages 11, 12 and 13, respectively. Note that cad protein expression extends more anteriorly than in wild type, and is present only at low levels in the posterior part of the embryos. A and B are derived from stauHL and (C) from stau03 females. Bar, 0 1 mm.
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other tested 5faw-allele (stauDi) does not show this variability either in phenotype or in antibody staining and behaves like the strong phenotype of the stauHL allele. The zygotic expression of cad is not affected in stau~ embryos. cad expression in the maternal-effect mutants torso and trunk Fully developed embryos from torso (tor) and trunk (trk) mothers show similar although less severe head defects to those formed in exu, swa and stau females: the median tooth (labrum) is always absent and, in addition, the pharyngeal arms of the chitinous mouth skeleton are reduced. Furthermore, at the posterior end the structures corresponding to the telson are always absent, and the 8th abdominal segment (A8) and parts of A7 are also deleted, so that torso~ or trunk' embryos end with abdominal denticles corresponding to the anterior part of A7 (Schiipbach & Wieschaus, 1986). cad protein distribution in these mutant embryos shows no clear difference from wild type, from its appearance at stage 6-7 throughout syncytial blastoderm (early stage 14) forming a 'normal' gradient (Fig. 8A). However, it cannot be excluded that the gradient is less steep than in wildtype embryos. During late stage 14, when in wild-type
Fig. 8. Expression of cad protein in embryos derived from tor females. Embryos derived from trk mothers show the same distribution pattern(s). Anterior points to the left. (A) Embryo during syncytial blastoderm stage 12. (B) Embryo at the cellular blastoderm stage. Note that cad protein can be detected only in the pole cells (indicated by an arrowhead). Bar, 0-1 mm.
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embryos the protein distribution focuses in to a 3- to 4-cell-wide stripe at the posterior end (Figs IF, 2A), a continuous decrease of cad protein towards the posterior pole can be observed in tor~ or trk~ embryos and no new zygotic expression is detectable, so that at the onset of gastrulation cad protein is detectable only in the pole cells (Fig. 8B). No zygotic cad expression in the ectoderm was detected throughout further embryogenesis of tor~ or trk~ embryos (data not shown) and the most posterior structures (A8 and more posterior) are missing later in the mutant embryos. Zygotic mutants affecting cad expression in early embryogenesis Following the maternal genes, the zygotic gap genes are the first genes to be activated after fertilization to refine positional information along the anteroposterior axis, followed by the pair-rule segmentation genes, which specify double segment periodicity (Nusslein-Volhard & Wieschaus, 1980). None of the loci that were tested (see Table 1) affect the cad protein gradient distribution throughout syncytial blastoderm.
The zygotic cad expression, the posterior ring in the region of the telson anlagen in wild-type embryos is, however, affected by some of the zygotic segmentation genes analysed. Mutations in the gap gene tailless (til) lead to deletions of A8 and more posterior ectodermal structures (Strecker, Kongsuwan, Lengyel & Merriam, 1986). til eliminates cad expression in the posterior ring at the onset of gastrulation and during germband extension (Fig. 9B,C). The resulting staining pattern looks similar to that of tor' and trk~ embryos, in which only the pole cells show detectable amounts of cad protein (compare Fig. 9A- C to Fig. 8B). Similar findings at the cellular blastoderm stage were made in mutants of the pairrule gene fushi tarazu (ftz) (Wakimoto & Kaufman, 1981), which also affects the formation of the telson. The stripe of aid labelling is absent \nftz~~ embryos, very similar to tll~ (Fig. 9B). However, during later embryonic development (germband retraction, approx. 8h after fertilization) of ftz' embryos a small set of cells at the end of the abnormal germband contains detectable levels of cad protein (data not shown). Another gap gene, hunchback (hb, Lehmann & Nusslein-Volhard, 1987), also has an effect on the cad stripe. In homozygous hb embryos the posterior
Fig. 9. Expression of cad protein in the zygotic segmentation mutants hunchback (hb), tailless (til) and fushi tarazu (ftz). Anterior points always to the left. (A) Homozygous ftz embryo at the cellular blastoderm stage. (B) Homozygous til embryo at the beginning of gastrulation. Note that in A and B no cad protein is detectable in the stripe of the telson anlagen. Pole cells are indicated by an arrowhead. (C) Homozygous til embryo during germband extension, no zygotic cad expression is detected. Pole cells are indicated by an arrowhead. (D) Ventral view of the posterior part of a homozygous hb embryo. Note that the cad stripe is approx. 2 cells broader than in a wild-type embryo of the same stage (E). Ventral furrow is indicated by little arrows. Bar, 0 1 mm in A-C, and 005mm in D and E.
Effects on cad protein expression stripe, after it has sharpened to its final width at the beginning of gastrulation, is 5-6 cells broad instead of 3-4 cells as in wild-type embryos (Fig. 9D; compare to wild type of same stage, Fig. 9E). The other segmentation genes that were tested (see Table 1) did not reveal notable changes of zygotic cad expression during early embryogenesis. Discussion In this report we have examined cad protein distribution in maternal and zygotic mutants. All the mutations analysed visibly affect embryonic pattern formation along the anteroposterior body axis, with the exception of dorsal, windbeutel and cactus (cac) which have either a dorsalizing or ventralizing (cac) phenotype. Table 1 summarizes all the mutants that were analysed in this respect and their effects on cad protein expression.
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1982; Frohnhofer, Lehmann & Niisslein-Volhard, 1986; Stephenson & Mahowald, 1987), and also the cad protein distribution corresponds to that found in exu~ embryos. Embryos derived from tor' or trk" mothers have similar but less severe head deletions as compared to exu~ embryos, and in addition they lack all structures distal to the anterior part of A7 (Schiipbach & Wieschaus, 1986). However, the cad protein gradient in tor~ and trk~ embryos is very similar to that found in wild-type embryos (Fig. 8A), although small relative changes cannot be excluded. These different effects on the blastoderm fate map are also reflected by the striped pattern of the segmentation gene fushi tarazu (ftz) for which long-range effects on the entire embryo are detectable in exu~, but not in tor~ and trk~ embryos (Mlodzik et al. 1987).
The cad protein gradient in mutants of the 'grdknirps '-group A second set of maternally expressed genes comprises Effects on the gradient distribution of cad in maternalthose that belong to the grandchildless-knirps (grdcoordinate mutants knirps) group. All the different mutants of this group Almost all of the genes of the maternal-coordinate have similar or identical phenotypes; they lack abclass have an effect on the cad gradient, suggesting dominal structures either totally or partially, and that cad is expressed after or in parallel to these form no pole cells (see Table 1). However, almost all genes. The observed changes of cad protein distriof these mutants (staufen is the only exception) have bution in the coordinate mutants reflect the cuticular no effect on the cad gradient, which is somewhat phenotypes that are visible at the end of embryogenunexpected since the highest levels of cad protein are esis. The changes in the fate map correlate largely found in the abdominal anlagen in wild type, which with the altered levels of cad protein. Genes that do not develop in mutant embryos of this class, stau affect head development change cad protein exthe only exception of this group concerning cad pression in the anterior part(s) of the mutant embryos expression (see Results, Fig. 7) - is also exceptional so that cad protein is detected at higher levels than in with respect to its phenotype and its effects on the wild-type embryos. However, we cannot say whether blastoderm fate map. None of the other genes has an cad itself inhibits head structures. effect on the development and the fate map of head The even distribution of cad protein found in BicD and thoracic structures. However, in embryos derived (MacDonald & Struhl, 1986; Mlodzik & Gehring, from stau mothers, not only anterior head structures 1987) and bcd~ embryos during the syncitial blastoare deleted (Schiipbach & Wieschaus, 1986), but also derm stages can be correlated with the observed the fate map in the anterior part of the embryo is embryonic phenotype: only posterior abdominal changed in a way similar to that observed in exu' and structures are formed in these mutant embryos. In swa~ embryos, namely that posterior head and thoexu embryos, anterior head structures are deleted racic anlagen are shifted anteriorly (our unpublished (Schiipbach & Wieschaus, 1986), and posterior head results; Carroll et al. 1986). The effects of stau on cad and thoracic anlagen, which are already visible at the protein distribution are twofold: first, cad protein blastoderm stage, expand almost to the very anterior extends further anteriorly than in wild type (similar end (Mlodzik et al. 1987). These changes in the fate but not as extreme as in exu~ embryos) and, second, map are reflected even earlier by cad protein distribution during the syncytial blastoderm (Figs 4,5). cad cad protein expression is reduced to about 50% of wild-type levels in the posterior part of stau~ emprotein can be detected at abnormally far anterior bryos. This suggests that similar to the maternalpositions in these mutant embryos, albeit only at low coordinate genes (BicD, bed, exu, swa), stau could be levels. The posterior half of these mutant embryos shows a more or less normal cad protein distribution, involved directly or indirectly in the translational control or stabilization of the gene products of cad. as would be expected, since the development of the These findings support the notion that staufen is not a abdomen is virtually normal in exu" embryos. Similar typical member of the 'grandchildless-knirps' group, phenotypic observations were made in embryos debut a combination of a coordinate and a gap gene. rived from swa~ females (Niisslein-Volhard et al.
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The cad protein distribution in members of the 'grd-knirps' group suggests that cad acts independently of most of these genes or earlier in the specification of abdominal structures. Nevertheless, it is required for normal formation of the abdomen, since by far the most severe defects in cad~ embryos are found in the abdomen (Macdonald & Struhl, 1986). However, it seems unlikely that cad acts earlier than the products of the genes of the 'grd-knirps' group, and for one, oskar, it has been shown that it is required before the cad gradient. Lehmann & Niisslein-Volhard (1986) have shown that the temperaturesensitive period for the oskar product is during oogenesis, when no cad protein is detectable yet. In addition, the phenotypic defects in the abdomen observed in embryos derived from 'grd-knirps' females (Lehmann & Niisslein-Volhard, 1986; Schiipbach & Wieschaus, 1986) are generally more severe than those observed in embryos that lack both maternal and zygotic cad contributions (Macdonald & Struhl, 1986). A possibility of how cad might interact with the gene products of the 'grd-knirps' group could be that first the genes of the 'grd-knirps' group deposit the necessary information for the generation of abdominal anlagen in the posterior region of the egg and later in embryogenesis the independently formed cad gradient (and possibly other genes) refine and stabilize this information to give the abdomen the correct number and sequence of segments. Moreover, the 'grd-knirps' genes and cad alter the pattern of expression of the pair-rule segmentation gene fushi tarazu in the same region of the embryo (Carroll etal. 1986; Macdonald & Struhl, 1986). Genes affecting zygotic cad expression in the telson anlagen All the maternal-effect genes that affect telson formation result in altered cad expression pattern concerning the posterior stripe. First, in BicD and bcd~ embryos which have duplicated telsons at both embryonic ends, the cad protein is present in two stripes, each close to one of the embryonic poles. In BicD embryos, the two stripes reflect the mirror-image symmetry of the developing embryo and are expressed symmetrically (MacDonald & Struhl, 1986; Mlodzik & Gehring, 1987). In bed" embryos, the development is not symmetrical (only one abdomen forms; Frohnhofer & Nusslein-Volhard, 1986) and the formation and position of the two posterior stripes is asymmetrical as well (see Results, Fig. 3). Second, embryos of torso and trunk do not develop a telson, and accordingly no cad protein is detectable in the posterior region of the mutant embryos (except for the pole cells, Fig. 8B). All these mutants lead to changes in the blastoderm fate map and disrupt normal expression of all zygotically expressed genes
so far studied. The altered zygotic cad expression reflects the observed changes in the respective fate maps at the beginning of gastrulation just after the onset of expression of the zygotic segmentation genes. Several of the zygotic segmentation genes (see Table 1) were analysed for their effects on zygotic cad expression. In two mutants, /// and ftz, cad is not expressed in the posterior stripe near the telson anlagen. Both genes also affect the formation of the embryonic telson as observed on the cuticular phenotypes. In addition, ftz expression clearly overlaps in its most posterior stripe with the cad band (our unpublished results) and ftz is required for correct initiation of expression of some homeotic genes of the Bithorax- and Antennapedia-complex at the blastoderm stage (Ingham & Martinez-Arias, 1986). Although in til and ftz mutants the final staining pattern at the onset of gastrulation looks very similar to that observed in tor" and trk" embryos (only the pole cells contain cad protein), in late stage-14 embryos the transient staining patterns between these two groups differ. In ftz mutants the graded disappearance of cad protein appears to be similar as observed in wild type (see Fig. IF), but later, at stage 14 when the band of cad expression becomes prominent in wild type, no new protein is detectable in the mutant embryos. In contrast to this, in ///, tor" and trk" embryos, cad protein disappears continuously towards the posterior pole during stage 14, no transient patterns as shown in Fig. IF are observed. Another segmentation mutant, hunchback (hb), affects zygotic cad expression in a different way. The band of expression is approximately two cells broader in homozygous hb embryos. Phenotypic analysis of hb embryos shows defects in abdominal segment A8 (Lehmann & Nusslein-Volhard, 1987) and most likely cad is expressed more anteriorly at the expense of other genes being normally expressed in A8 anlagen. hb could act directly or indirectly to repress cad anterior to its wild-type domain of zygotic expression. Only the zygotic genes with notable effects on cad expression during cellular blastoderm and early gastrulation stages are mentioned here, and it is likely that other genes, including the ones listed in Table 1, have other more subtle effects on cad expression at the blastoderm stage and during later embryogenesis. Conclusions
The presence of a molecular gradient and the phenotypic analysis of cad mutants suggested the possibility that cad might be involved in specifying the body pattern along the anteroposterior axis. This notion correlates with our observations in maternal-coordinate mutants: changes in the fate map and later in the
Effects on cad protein expression
larval cuticle are reflected as early as the syncitial blastoderm stage by an altered cad protein distribution. However, it is unlikely that absolute levels of cad protein during early stages of syncitial blastoderm are required to specify structures along the anteroposterior axis. This is supported by the finding that abnormal zygotic cad expression alone can rescue most of the phenotypic defects. In these embryos that lack the maternal contribution, cad protein levels during the syncitial blastoderm are atypically low and only rise significantly by stage 14 (Macdonald & Struhl, 1986). These observations might indicate that threshold levels of cad protein are not required before stage 14. Interestingly, the control of cad protein distribution is mediated through anterior determinants (e.g. bed, exu and swa), whereas the posterior activities of the grd-knirps group have no effect on the graded distribution. Moreover, in exu and swa, the RNA distribution of bed itself is altered (T. Berleth & C. Niisslein-Volhard, personal communication). This suggests the possibility that bed is involved directly or indirectly in establishing the cad gradient through repression of cad mRNA translation in anterior regions of the embryo. cad itself affects the expression of at least one segmentation gene,^fz (Macdonald & Struhl, 1986); most likely it also affects the expression of most other zygotic segmentation genes, since they seem to interact in a hierarchical system (Howard & Ingham, 1986; Harding et al. 1986; Carroll & Scott, 1986). The localized zygotic expression of cad in the anlagen of the telson seems to be similar to the function of a homeotic selector gene and is affected by some of the zygotic segmentation genes that are expressed and required in these posterior regions of the embryo. This suggestion is supported by the observation that cad is expressed in parts of the genital imaginal disc (Mlodzik & Gehring, 1987). All homeotic selector genes are required during metamorphosis in those segments which they specify and, therefore, are expressed in the respective imaginal disc. The positional information is established by the primary maternal determinants, e.g. bed, whose transcripts are localized already during oogenesis at the anterior end of the egg (Frigerio et al. 1986). Later, during early embryogenesis, the cad gradient could be involved in maintaining or transferring this positional information to the zygotically active segmentation genes. Some of these zygotic segmentation genes affect the persistent zygotic cad expression in the telson region, and the zygotic cad expression itself can be placed at the level of the homeotic selector genes, specifying (parts of) the telson. In the hierarchy model proposed by Ingham & Martinez-Arias (1986) the gradient expression of cad is placed after the
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primary maternal determinants (e.g. bed), and before the zygotic gap genes. The second phase of cad expression, the persistent zygotic expression in the telson would be placed in the model at the level of segment specification after the expression of the zygotic segmentation genes, when the segmental subdivision of the developing embryo is established. This dual function of cad is also clearly divided by the presence of two-stage specific promoters (Mlodzik & Gehring, 1987). How the gradient is established and how it is involved in specifying the body pattern remains to be investigated. The molecular mechanisms of interaction between cad and the maternal and zygotic genes affecting its expression remain to be elucidated and the cloning and molecular analysis of other maternal-effect genes should provide insights into the problem of how these genes interact. We are very indebted to Trudy Schiipbach, Erie Wieschaus, Ruth Lehmann, Christiane Nusslein-Volhard and John Merriam for generously providing us with the mutant stocks. We thank Ursula Weber and Stefan Baumgartner for help with keeping and selecting the mutant flies, Greg Gibson, Leslie Pick and Alexander Schier for comments and suggestions on the manuscript, and Erika WengerMarquardt for efficient typing of it. This work was supported by the Swiss National Science Foundation and the Kan tons of Basel.
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{Accepted 12 August 1987)
