pattern during early Drosophila

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1 Development 104 Supplement, (1988) Printed in Great The Company of Biologists Limited The generation of embryogenesis periodic pattern during early Drosophila KEN HOWARD Howard Hughes Medical Institute, Department of Neurobiology and Behavior, Colombia University, 722 W 168th Street, New York, NY 10032, USA Summary The first indication of the formation of segment primordia in Drosophila is expression of the segmentpolarity genes in particular parts of each primordium. These patterns are controlled by another class of genes, the pair-rule genes, which show characteristic two-segment periodic expression. Each pair-rule gene has a unique domain of activity and in one view different combinations of pair-rule gene products directly control the expression of the segment-polarity genes. There is a hierarchy within the pair-rule class revealed by pair-rule gene interactions. It is unlikely that these interactions generate the periodicity de novo. Instead, pair-rule genes respond to positional information generated by a system involving zygotic gap and maternal coordinate genes. In this paper, I will concentrate on the problem of the mechanism that generates these pair-rule patterns, the first periodic ones seen during segmentation. I will review and discuss some of the relevant literature, illustrating certain points with data from my recent work. Key words: Drosphila, segmentation, gene interactions, periodic pattern, pair-rule gene. A summary of Drosophila segmentation The following brief synopsis of Drosophila segmentation is not intended to be exhaustive but merely to mention a few of the points which are more pertinent to this paper. There are a number of recent reviews (Akam, 1987; Niisslein-Volhard et al. I9B7; Scott & CarrolI, I9B7; Scott & O'Farrell, 1986) that cover the topic in greater depth. The early development of Drosophila has most recently been described by Zalokar & Erk (1976) and Foe & Alberts (1983). This summary is based on the later work (Foe & Alberts, 1983). Early development occurs in a syncitium, the first nine cleavage divisions in the central yolky part of the egg. By the early part of cell cycle L0 most of the nuclei have migrated to the periphery of the egg, where they undergo a further four syncitial divisions. A few nuclei enter the pole plasm in the posterior peripheral cytoplasm and cellularize at this stage to form the germ line. Others remain in the yolky core of the egg, becoming polyploid yolk nuclei. Only after the last of these divisions, in cell cycle 14, do the majority of the nuclei cellulartze: the nuclei elongate perpendicular to the surface of the embryo and cleavage furrows form between them. Gastrulation begins slightly in anticipation of the completion of cellularrzation, about 3 h after fefirhzation at 25'C. Experimental manipulation (Shubiger et al. 1977: Vogel, 1977; Simcox & Sang, 1983), temperaturesensitive mutants (Schi.ipbach & Wieschaus, I9B7), clonal analysis (Szabad et al. 1979) and fate-mapping studies (Lohs-Shardin et al. 1979) indicate that the segmental pattern is set up some time after the first hour of development and before cellularization of the blastoderm is complete. As the nuclei reach their fully elongated shape about 40 min into cycle 14 several segment-polarity genes are expressed in transverse stripes marking parts of each segmental primordium (Kornberg et al. 1985; Fjose et al. 1985; Baker, 1987). The signals that direct these segment-pol arity patterns, and therefore the formation of the segmental primordia, are provided by maternal coordinate and the zygotrc gap and pair-rule genes. In the next few parugraphs I will introduce these classes of genes. The maternally acting genes fall into three classes

2 36 K. Howard having effects on three distinct regions of the embryo (see French, this volume; Lehmaro, this volume). One of these, the torso group, causes defects in the terminal regions of the embryo. The other two classes of gen es (bicoid and oskar groups) affect the anterior and posterior parts, respectively, of the remaining central region. Transplantation experiments show that these two activities are localized at the anterior and posterior poles of the embryo and that their influence spreads into the more central regions. For both systems certain genes encode active factors (bicoid in the anterior, nanos in the posterior) and others function to localize these factors. (Frohnhofer & Ntisslein-Volhard, 1986; Lehmann & Ntisslein- Volhard, 1986; Schtipbach & Wieschaus, 1986: reviewed in Ntisslein-Volhard et al. 1987; Lehmann, this volume). The ways in which these three systems generate positional information are not yet understood. FIowever enough is known about bicoid to allow speculation: bicoid contains a homeobox sequence so the protein may act as a transcription factor. The mrna is localized at the anterior pole of the egg (Frigefio et al. 1986). This message is translated early in the development of the blastoderm and the protein diffuses forming a gradient from anterior to posterior (Driever & Ni.isslein-Volhard, 1988). A simple way for bicoid to function would be if the protein were a morphogen and activated different zygottc genes in different blastoderm cells and that these then determined the fates of those cells. In fact, the properties of the zygotically acting segmentation genes suggest that the mechanism is not this simple: zygotic genes are involved not only in responding to, but also in elaborating, the information set up by the maternal genes. Ntisslein-Volhard & Wieschaus (1980) defined three phenotypic classes of zygotic segmentation gene: segment-polarity, pair-rule, and gap. Segmentpolarity genes are required in every segment; the generic mutant phenotype is removal of a particular region of each segment. Pair-rule genes are required with a two-segment periodicity, and pair-rule mutants lack structures in alternate segments. Gap genes are required aperiodically in contiguous groups of segments which are missing in mutant animals. Several of these genes have been isolated by molecular techniques and reagents that detect the transcripts (labelled nucleic acid probes) and the protein products (antibodies) have been used to describe the patterns of gene activity. These patterns correlate roughly with the phenotypes of the mutations so that the gap genes are expressed in broad aperiodic regions of the blastoderm t plrr-rule genes are expressed in patterns with two-segment periodicity and segment-polarity genes are expressed in parts of each segment. The phenotypes of these mutants suggest a sequential subdivision of the embryo (Ntisslein-Volhard & Wieschaus, 1980) and therefore a hierarchy of gene function. This has been confirmed by a number of observations of the dependence of the expression pattern of particular segmentation genes on the activity of others. The gap genes are regulated by influences from two classes of genes: the maternal genes and the gap genes themselves (Jiickle et al. 1986; Gaul & Jiickle, 1987; Tautz, 19BB; Jtickle, this volume). Pair-rule expression patterns are changed in maternal and gap mutant embryos (Mohler & Wieschaus, 1985; Ingham et al. 1985; Carroll & Scott, 1986; Carroll et al. 1986; Ingham et al. 1986; Macdonald & Struhl, 1986; Frohnhofer & Ntisslein- Volhard, 1987; Mlodzic et al. 1987; Frasch & Levine, 1987; Ingham & Gergen, this volume). There is also hierarchy within the pair-rule genes so that some pairrule gene patterns are dependent on the activity of other pair-rule genes (Howard & Ingham, 1986; Carroll & Scott, 1986; Harding et al. 1986; Martinez- Arias & White, 1988; Ingham & Gergen, this volume). There is also good evidence that one of the pair-rule genes, fushi-tarazu ffiz), has an autocatalytic activity (Hiromi &. Gehring, 1987). The pair-rule genes then direct the single-segment periodic expression of segment-polarity genes (Howard & Ingham, 1986; DiNardo & O'Farrell, 1987; Ingham et al. L988). No part of the segmentation is understood in mechanistic detail and, zt the moment, some of the basic organizational principles are still in doubt. For instance, there are two current views of the ways in which pair-rule genes instruct segment-polarity expression. In one view, it is by combinatorial action of some of the pair-rule genes (see, for example Ingham et al. 1988). In the other, the pair-rule genes serve to define boundaries within which positional information is defined (Lawrence et al. 1987, see Lawrence, 1987). Another controversial issue is that of compartments. These are lineage units consisting of groups of cells, polyclones, which are destined to give rise to a particular part of the pattern (see, for example, Crick & Lawrence, 1975). The existence of compartments is well established for the development of the imaginal structures, where the segment-polarity gene engrailed serves to define posterior compartments. However, it is not clear, that compartments exipt in the larval epidermis (Szabad et al. 1979; Gergen et al. 1986). Another unsolved issue, the one that I will address here, is the question of how the gap and maternal-effect genes direct the periodic expression of the pair-rule genes.

3 Generation of periodicity in Drosophila 37 The expression patterns of the zygotic segmentation genes hairy and Kruppel In this section, I will introduce the patterns of expression of transcripts from one gene of the gap class, Kruppel (Kr), and one of the pair-rule genes, hairy. The Kr pattern was first described by Knipple et al. (1985), the hairy pattern by Ingham et al. (1985) and Ish-Horowicz et al. (1985). Certain features of the development of the hairy pattern I report here have not been described previously. Fig. 1 shows a series of in situ hybridizations to alternate sections of wild-type embryos: Kr signal on the left and in the centre (in dark- and bright-field illumination); hairy signal on the right (in dark-field illumination). The levels of Kr RNA present just after the nuclei have migrated to the periphery and the pole cells have formed at stage 10 afe very low (Fig. 1A). Differential expression of Kr RNA can first be detected at stag e 12 when a relatively low level of transcript is present in a broad band in the middle of the embryo, the 'central domain' (Knipple et al. 1985). By the end of the next cell cycle, Kr RNA is present at high levels in the central domain (Fig. 1 panels bl and 2, ci and 2, d1, and 2). The expression domain is more extensive ventrally than on the dorsal surface. By the early part of cycle 14, this domain shrinks appreciably, especially on its ventral surface, so that by the time the periodic pattern of hairy expression has formed (see next section), it lies between approximately 40 and 55 % egg length (measured from the posterior pole), about the same extent reported for the central domain ( %) by Knipple et al. (1985) and for the protein ( to 54+ I%) by Gaul et al. (1987). Note that the early transcript domain is significantly more extensive. Subsequently, zn anterior (Fig. LJ,K) and a posterior domain (Fig. 1L) of. Kr expression develop. All three of these domains are present as gastrulation begins (Fig. L panels m1 and 2). The first differential expression of. hairy transcripts can be clearly seen at stage 13 when RNA accumulates at low levels. These patterns are quite variable from embryo to embryo. Often one SeeS expression between about 20 % egg length to the anterior pole (Fig. 1C) which is sometimes broken into two domains, an anterior one covering the anteriot 15 % of the egg and a posterior one from about 20 to S0 % egg length. By the middle of stage 14 (the final blastoderm cell cycle), hairy is expressed in a pattern consisting of seven stripes, L -7, and an antero-dorsal region, 0 (Ingham et al. 1985; Ish-Horowicz et al. 1985; Fig. 1 panel 13). There are intermediate stages in the development of this pattern that have not been noted before. A11 of these occur in the early part of cycle 74 before the nuclei begin to elongate when it is not possible to stage the embryos showing the different patterns by morphological criteria. However, there are a series of patterns and, in the following description, I assume that the more developed patterns represent later stages. At first, stripes 3 and 4 are not resolved from one another and stripes 2, 5 and 6 canbarely be detected (Fig. 1 panel f3). Next, stripe 2 is expressed at high levels before stripes 5 and 6 (Fig. 1 panel g3). Subsequently, stripes 5 and 6 are diffusely expressed so that stripes 5,, 6 and 7 can barely be distinguished from each other (Fig. 1 panel h3). These then resolve from one another before stripes 3 and 4 so that there is a stage in which stripes 3 and 4 are fused but all the others are distinct (Fig. 1 panels i3 and j3). After this, os the nuclei begin to elongate, the pattern of seven stripes can be clearly seen (Fig. 1 panels k3 and 13) and persists through early gastrulation (Fig. L panel m3). At this time,?r additional eighth stripe of hairy expression can be seen in the posterior of the embryo (Fig. L panel n2 and 3; Ingham personal communication). One striking feature of hairy is that it seems to be expressed in alternate primordia throughout the entire segmental region of the animal: the posteriormost hairy stripe lies over the primordia for parasegment 15, the posterior part of the tenth abdominal segment and the anterior part of the eleventh. Jtirgens (1987) has demonstrated that this is the most posterior segment in the Drosophilq embryo. The anteriormost element of the hairy pattern, region 0, lies over the primordium for the labrum whilst region I lies over the anterior part of the mandibular segment (Ingham et al. 1985; Ish-Horowicz et al. 1985). In the simplest case, one would expect only one segment to arise between these two regions of expression. In fact, two segmental structures, the antennal segment and the intercalary segment, derive from this region (Jtirgens et al. 1986). These two segments seem to arise at the same anteroposterior level and it is possible that they form from one segmental primordium, defined by pair-rule gene expression, which is split in two dorsoventrally. In between these terminal regions, hairy is expressed with a two-segment periodicity, approximately in the primordia for the odd-numbered parasegments (Martinez-Arias & Lawrence, 1985). A two-segment periodic expression pattern is characteristic of all pair-rule genes for which these type of data are available. In the case of ftz, there are seven stripes that form from a relatively homogeneous region of expression, apparently by repression of ftz expression in the interbands (Hafen et al. I9B4; Edgar et al. 1986). The most anterior of these stripes lies approximately in between the first and second hairy stripes, the last of them posterior to the last strong hairy stripe seen at blastoderm. In fact, ftz

4

5 Generation ol' pcriodic'itt' irr Drosophila 3 39 Fig. l. Kr ancl lruirt,cxpression in wild-type blastoderms. Each row shows two alternate scctit-rtrs of a wilcl-typc blastgclcrm cmbryo. Thc lcft-hancl panel (number l) shows a dark-ficld view of thc Kr hybriclizatiott sigrtal: thc miclcllc pancl (lumbcr 2) shows a bright-fielcl view of the same field; the right-hand panel (numbcr 3) shows a clark-ficlcl vicw tlf the huir'l'sigpal. Thc cliffcrcnt embryos arc at progressively latcr stagcs clf dcveltlpmcrtt: (A) stagc 12, (B-E) stagc l3: (F-L) micl-stagc- l-l (not e longating nuclei), (M,N) early gastrulae. Tlrc Kl pattern is gelteratecl well in advance of the hui,'y one and can be clearly seelt in stase l3 (B-E). The Kr' clomairr lalrows tts ltuirt'bcgins to bc expressed and goes through its characteristic developmetttitl patterll in stage l't (F-L). Towalcl t[c cncl of this clcvcklpmcnt Kr becomes expresscd at thc ends of the cmbrvt) (J-M). Art cighth stri;rc of huirt' cxpt'cssiott ctttr bc scctl in carly gastrulac (N ). is not entirely complementary to hairy. The polarity of the pattern is revealed by the fact that the ftz, stripes slightly overlap the ltuirv stripes lying posterior to them so that there are narrow regions, the first of which lies behind the first ltairv stripe., where neither sene is expressed at high levels toward the end of stage 11 (lngham et ul. 1985). The other pairrule genes, even skippcd (Harding ct al. 1986; Macdonald et al. 1986), paired (Kilchher et al. 1986) and runr (Gergen & Butler, 1988: Ingham & Gergen., this volume), go through stages at which they are expressed in every segmental primordium, but at differ-

6 40 K. Howard Fig. 2, Regulatory mutations at hairy. Pairs of dark-field photographs showing the pattern of hairy expression at the blastoderm stage and the final cuticular phenotype of three of the region-specific hairy alleles: (A,B) wild-type; (C,D) h^3; (E,F) h-t; (G,H) hkr. Note that the alleles express partial versions of the wild-type pattern and have corresponding defects in the cuticle. For example, h-3 fails to express stripes 3 and 4 and does not produce the denticle belts of the third thoracic (73) and second abdominal (A2) segments. ent levels in each one so that there is still a twosegment periodicity to the pattern. Mutations al hairy indicate that it is decoding positional information Some time ago, Ingham et al. (1985) reported an allele of hairy, h^3' which showed defects predominantly in the posterior thorax and anterior abdomen. This allele fails to express hairy transcript in the third and fourth stripes of the normal hairy pattern. Three other hairy alleles, hut, h^7 and h^8, also fail to express particular hairy stripes (Fig. 2 and Howar d et al. 1988). The alleles fall into a series: hkt expresses only the anterior region 0, h^7 expresses regions 0, 1 and 5, ft-8 is similar to h^7 although it shows some expression in region 2, finally, h^3 expresses in regions 0, I,2, 5, 6 and 7. These alleles appear to mutate cis-acting regulatory regions of hairy, a view confirmed by the molecular analysis of the lesions: the cause of these mutations is deletion of progressively more DNA 5' to the coding sequence of hairy so that the strongest of the alleles, hut, has the least 5' DNA remaining intact (Howard et al. 1988). It has yet to be established if there are discrete elements that respond to different signals that initiate the expression of the individual stripes of the hairy pattern. Preliminary evidence (Howard, unpublished observations) suggests that this is so. Whatever the precise mechanism, it is clear that hairy is responding to different signals in different places in the blastoderm. The gap-gene Kruppel attects pair-rule gene expression In this section, I will describe the effect of lack of Kr function on hairy and ftz patterns and the relationship between them. I will then present the phenotypic

7 Generation of periodicity in Drosophila 4I Fig. 3. hairy and ftz expression in Kr mutants. The panels show two examples of embryos at the late blastoderm stage homozygous for the amorphic Kr allele Krt. The left-hand panels show serial section of one embryo, the right-hand panels another one. (A,,8) hairy in situ signal; (C,D) ftz signal; (E,F) mixed hairy and ftz signal. The drawings in panels G and H interpret these data: ftz expression in clear enclosed regions: hairy in dotted regions and overlaps in the black regions. Note the reversal of the polarity of the pattern around and posterior to the weak second ftz stripe (see Fig. 5C,E,G and I for data on the wild type). series of ftz patterns in Kr mutants. These observations extend previous work on the pair-rule phenotype of Kr (Carroll & Scott, 1986; Ingham et al. 1986; Frasch & Levine, 1987). The following description of the terminal Kr phenotype is based on the work of Wieschaus et al. (1984): in the complete absence of Kr function (Kr acts entirely zygotically) all of the head structures and structures posterior to the sixth abdominal segment form. These is a deletion of the entire thorax and the anterior abdomen back the naked cuticle of the fifth, or the denticle band of the sixth, abdominal segment on the ventral surface of the larval cuticle (the deletion is somewhat less extensive dorsally). A mirror-image sixth abdominal segment is formed with reversed polarity in front of the correctly oriented one. This polarity reversal is not seen ir, any of the weaker partial loss-of-function Kr alleles. Fig. 3 shows two examples of in situ hybridizations to three serial sections of late blastoderm embryos homozygous for the amorphic allele Krr. The top panels show the sections hybridized with hairy probe, the second panels the next section hybridized with ftz probe, the third panels the third section which was hybridized with mixed hairy and ftz probe. The last panels shows schematic representations of the hybridization patterns. In this Kr mutant, hairy is expressed in four stripes: the first and third of these are not very different from those seen in the wild type (although the posterior one is slightly larger and lies more anteriorly than it would in wild type). The second stripe is very broad and not well separated from the third, which is about the same size as the last stripe. The novel pattern of ftz expression consists of four strong regions of expression with a weak region lying in between the first two strong ones. Again the anterior and posterior stripes are in approximately normal positions whilst the intervening ones are

8 42 K. Howard displaced. By comparing these patterns with one another and with the double-label hybridization, it is possible to determine the relationship between the two patterns and this gives some information about polarity at this stage in the Kr mutant. Recall that in the normal situati on hairy strip es 2-7 overlap the posterior parts of ftz stripes I-6, respectively, hairy stripe 1' and ftz stripes 7 do not overlap and lie anterior and posterior to the other stripes, respectively. Consequently in favourable specimens of a late-st age-i4 embryos hybridrzed with mixed hairy and ftz probes it is possible to distinguish eight regions of strong hybridrzation separated by very small interband regions (Fig. 5, p?nel G2; Ingham et al. 1985; Ish-Horowicz et al. 1985). The mixed-probe experiment performed on amorphic Kr mutants shows four distinct regions (Fig.3). As in the wild type, the anterior and posterior stripes represent isolate d hairy and ftz signals, respectively. The third mixed stripe in the mutant seems to be like the seventh one in wild type and consists of the fourth ftz stripe overlapping the fourth hairy one. The large second mixed stripe represents a region where the first, second and third ftz stripes overlap the second and third hairy stripes. These relationships are summ arized in the schematics in Fig. 3 which are based on superposition of the hairy and ftz patterns and the pattern generated in the mixed hybridizatron The relationships between ftz and hairy show a small polarity reversal in the mutant around and posterior to the weak second ftz stripe. It is tempting to propose that this is the direct causal precursor of the reversed pol anty seen in the cuticular phenotype of Kr amorphs. If this were so, one would expect that this weak ftz stripe would not be present in any of the weaker Kr phenotypes. This is indeed what is observed. Fig. 4 shows a series of. Kr mutant embryos stained with anti-ftz antibody. Two alleles were employed in this study, Krr, vfl amorph and IirrrrAroz ) a strong hypomorph which never shows the polarity reversal in the ventral cuticle typical of the amorph (Wieschaus et al. 1984). The weakest phenotype, seen in Krt l* embryos involves a thinning of the third ftz stripe and slight compensatory movement of the surrounding stripes to fill the gap (Fig. a panel A). After this (in grrrraro2 homozygotes) stripe 4 also weakens (Fig. a panel B) and then moves closer to stripe 5 (Fig. 4 panel C) before disappearing (Fig. a panel D). By this time, stripe 2has become very weak so that the pattern consists of strong stripes I, 5, 6 and 7 with weak stripes 2 and 3 lying in between L and 5. Also note that stripes 5 and 6 have moved appreciably toward the anterior from their wild-type positions (Fig. 4 panel D). After this, stripe 2 disappears and the weak stripe 3 fuses with stripe 5 (Fig. 4 panel E). A11 of these patterns are observed in the strong hypomorph yrrrraro2. Presumably the weak and compressed stripes of ftz, cannot direct the formation of proper segment primordia in the limited space (see Carroll & Scott, 1986, Gaul & Jiickle, I9B7 and Frohnhofer & Ntisslein-Volhard, 1987 for discussion of this point). The next stage in the development of the pattern is seen tn Krr homozygotes, embryos with no Kr gene activity. These show the four major ftz stripes which correspond to the wild-type stripes 1, 5, 6 and 7. Stripes I and 7 are in approximately their normal positions whilst stripes 5 and 6 have moved forward appreciably. It is as if the gap created by removal of stripes 2, 3 and 4 had been filled by stretching the posterior pattern over the gap. In addition, these embryos show a weaker and variable stripe in between the first two strong stripes. This corresponds to the polarity reversal seen in the amorphs and probably has the quality of the wild-type stripe 5 (Fig. 4 panels F and G). The gap-gene hunchback has different effects in the anterior and posterior Here I examine the changes in hairy pattern caused by lack of zygotic hunchback (hb) function and relate these to the changes seen in ftz expression (Carroll & Scott, 1986). The following description of the terminal hb phenotype is based on the work of Lehmann & Niisslein-Volhard (1987): complete lack of hb activity causes deletion of all the gnathal segments of the head, the thorax and abdomen back to the fourth abdominal segment and the formation of two or three abdominal segments of inverted polarity. The naked cuticle of the seventh and the denticle band of the eighth abdominal segment are also deleted. Normally some hb activity is supplied by the mother and the phenotype caused by lack of hb in the zygote alone is not so severe: the head is present with the exception of the labial segment. Only the thorax is deleted. The first abdominal segment is enlarged and the defect in the seventh and eighth abdominal segment remains. However, there is no polarity reversal in the abdomen. The maternal effect of hb is rescuable by zygotic expression so that perfectly normal animals can develop from mutant eggs provided that one copy of the gene is contributed paternally. The expression pattern of hb has been reported for both the RNA (Tautz et al. I9B7) and protein (Tautz, 1988) products of the locus. As expected, there is a maternal contribution of hb product and the RNA is present throughout the newly laid egg. This RNA is translated early in development. At cell cycle B, the protein is expressed uniformly in the anterior third of the embryo and drops off in the posterior two thirds

9 Generation of periodicity in Drosophila 43 q il e E G Fig. 4. The phenotypic series of ftz in Kr mutants. The panels show a series of late-blastoderm-stage Kr mutant embryos stained with anti-ftz antibody. The drawings on the right are schematic interpretations of these patterns. (A) The dominant phenotype of the amorphic allele Krr. Note the weakening of stripe :; (B-E) different phenotypes seen in embryos homozygous for the strong hypomorph Kr Irr^to'; (B) stripes 2 and 3 are weak and compressed; (C) stripe 3 begins to disappear, merging with stripe a; (D) stripe 2 becomes weak; (E) stripe 2 disappears and stripe 3 is very weak, merging with stripe 4; (F,G) Krl homozygotes showing the remaining stripes 1,5,,6 and 7. Note the weak stripe in between 1 and 5. This probably marks the polarity reversal and has the quality of stripe 5.

10 44 K. Howard Fig. 5. hairv and ftz expression in zygotic hb mutants. Wild-type (on the left) and zygotic hb mutant (on the right) embryos at late stage 14. The panels show serial section of one embryo showing in situ hybridization signals: (A,B) hb: (C,D) hairy; (E,F) ftz; (G,H) mixed huirv and ftz. The drawings in panels I and J interpret these data: ftz expression in clear enclosed regions; /lairy in dotted regions and overlaps in the black regions. in a linear gradient. As zygotic expression of the gene begins at around cell cycle 12, the protein-staining pattern evolves into a simple bipartite one in which the anterior half of the embryo expresses the gene and the posterior half does not. Fig. 4 shows two sets of four serial sections processed to reveal the patterns of distribution of transcripts from hb (top panels); hairy (second panels); ftz (third panels) and hairy and frz (fourth panels). The final set of panels show a schematic representation of the ftz and hairy transcript patterns. The embryo on the left is wild type, the one of the right shows the zygotic phenotype of hb. The low level of hb signal present in this embryo is attributable to hb transcript remaining from the maternal contribution. In the anterior part of the pattern, the effects are consistent with the cuticular phenotype: hairy stripes 2, 3 and 4 are missing as are ftz stripes 1, 2 and 3. These regions of expression are replaced by one broad region of ftz expression (which sometimes resolves into an anterior stripe 2 and a broad region consisting of stripes 3 and 4) which lies anterior to, and extensively overlaps, a broad region of hairy expression. Thus the primordia for parasegments 2 to 7 seem to be replaced with one large segmental primordium with the correct polarity (ftz anterior to but overlapping hairy). According to its position with regard to the posterior pattern, this /re stripe should

11 Generation of periodicity in Drosophila 45 overly an enlarged parasegment 6. It seems reasonable to propose that part of this region of the blastoderm gives rise to the enlarged first abdominal segment seen in the terminal phenotype of these animals, a conclusion that is consistent with the laser defect fatemap of zygotic hunchback mutants (Lehmann & Ntisslein-Volhard, 1986), and also with the work of White & Lehmann (1986). The situation is quite different in the posterior region. The posterior two ftz stripes ate barely resolved in zygotic hb mutants. This correlates with the defect seen in the eighth abdominal segment of these mutants. However, the hairy RNA pattern is essentially normal in this region (Fig. 5 panel D arrow). Thus it seems that the posterior region of hb expression is acting after hairy and is not necess ary for the earliest stages of segmentation in this region. Discussion The pair-rule genes must be responding to signals that carry information about position in the embryo. The output generated by their response is periodic and has a polarity (revealed in the relationships between the different pair-rule patterns). The genetic data show that these signals are dependent upon gap and maternal segmentation gene activities. Here I will discuss how these genes might function in order to generate these signals. A simple way to make the pair-rule pattern would be if pair-rule genes responded to information which was itself periodic and polar. Reaction-diffusion mechanisms (Turing, 1952) can generate such patterns de novo (Meinhardt, this volume, also See Harrisoil, 1982). This mechanism could function either by generating a pattern based on chemical reactions in the cytoplasm to which pair-rule genes responded or on a network of pair-rule gene interactions. In the latter case, one would expect the generation of pair-rule periodicity to be dependent on pair-rule gene activity. This does not seem to be so for the initial peridic expression of the pair-rule genes at the top of the hierarchy (Ingham & Gergen, this volume). In either case, the naive expectation would be that the pair-rule genes would be responding to the same signals in each region of expression. The regulation of hairy indicates that this is not true: the regulatory mutations reveal distinct responding elements for distinct signals that activate the different stripes. The developmental sequence is consistent with this view: the different stripes form at different times. These results indicate that there is information that changes aperiodically across the egg and is sensed by the hairy promoter. At least one other pair-rule gene must respond to this information in order to 'see' polarity. But it is not necess ary for all of the pair-rule genes to respond to aperiodic information since there is a regulatory heirarchy within the class. (The other pairrule genes runt and even-skipped may well respond independently to the information; see Ingham & Gergen, this volume). There are two extreme types of aperiodic information that can be envisioned a graded morphogen and a complex set of localtzed cues (see Meinhardt, this volume, for a discussion of the relationships between these two alternatives). Pair-rule genes could respond directly to a graded distribution of a morphogen by becoming active over several different concentration ranges. In this case, the problem of generating the pattern would have two parts: how to make the graded distribution and how to interpret it directly into a periodic pattern. One way to make a graded distribution is by simple diffusion (see Crick, 1970) and I have already mentioned a very simple scheme for the action of. bicoid involving diffusion of the protein to form a gradient. An alternative to simple diffusion is reaction-diffusion, the Same mechanism that can generate a periodic pattern. The essential feature of this mechanism is that the reactions have a characteristic spatial frequency. So if the reaction vessel (the embryo) is small compared with the characteristic frequency it can also form monotonic gradients. A detailed model of such a mechanism in insect segmentation has been put forward by Meinhardt (1977). In either case, the response mechanism has to take a monotonic gradient and produce a periodic output. This is simply achieved if a pair-rule gene is activated between different appropriately spaced morphogen concentrations. The polarity of the gradient could be preserved if another pair-rule gene is activated by approximately the same morphogen concentrations, but with each threshold a little higher (or lower) so that the two output patterns are out of phase with one another. The alternative to a gradient of morphogen is a complex set of cues consisting of combinations of different factors that serve to activate each pair-rule stripe. In this case, the different pair-rule genes respond to qualitatively different cues in different places. Of course these cues themselves must be generated in response to some positional information, most likely the concentration of a morphogen. In the extreme version of this model, the gap genes, which are expressed in broad regions of the embryo, would respond to a graded morphogen generating a number of overlapping regions which would direct the expression of the pair-rule genes. More sophisticated models involving gap gene ac' tivity modulating a reaction-diffusion, pair-rule mechanism with intrinsic periodicity have been proposed (Meinhardt, this volume).

12 46 K. Howard hairy ft2 hairy ftz Fig. 6. The Kr phenotype. A shows the relationship between prospective fate in the wild-type (hortzontal axis) and in Kr mutants (vertical axis). The weak alleles produce only a slight distortion of the pattern, this is reflected in a small deviation of the line depicting the relationship from the straight slanting line representing wild type. In more extreme mutants the line deviates further until in the most extreme case it changes slope reflecting the polarity reversal in the amorphic mutant. B shows a similar diagram using two lines to depict the relationship. These represent the anterior (bicoid) and posterior (oskar grovp) organtzing gradients in the egg and suggest how they might be changed in the Kr mutants. See text for discussion. The respective roles of the gap and maternal genes A distinction can be made between genes involved in the generation of the information and in the response. Mutations in the first class of gene should affect the positional information. They should distort the fate map, &S if it were drawn on a sheet of rubber and stretched. Mutations in genes of the other type, response genes, would not do this. They might abolish or enhance particular responses, but they would not affect the overall shape of the fate map: they should erase particular regions. For the present discussion, I will consider the patterns of pair-rule gene expression to represent the fatemap. The maternal mutations do indeed distort the pairrule fatemap. Frohnhof er & Ntisslein-Volhard (1987) have reported the changes in ftz pattern in the anterior group of maternal-effect mutants. In bicoid mutants, the anterior two ftz stripes disappear and the remainitrg posterior ones move forward to occupy the deleted region. Similarly mutants of the different maternal-effect posterior group genes all distort the fatemap as revealed by their effects on ftz expression (Carroll et al. 1986; Lehmann, this volume). This contrasts with the zygotrc gap-gene knirps (Carroll & Scott, 1986). Although this has a similar terminal phenotype, the changes in ftz expression are limited to the region of the primordia for the deleted structures where ftz becomes expressed uniformly. Mutations in the terminal group of genes are associated with deletions of the terminal regions of the fatemap and compensatory movement of the adjacent subterminal regions. This effect has been seen for the two pair-rule genes ftz and hairy in embryos mutant for the terminal group genes torso and trunk (Mlodzik et al. 1987). The general conclusion is that, in maternal segmentation mutants, the pair-rule patterns look as if certain parts of the fate map have been removed and the remainitrg parts stretched over the gaps. Interestingly some of the zygotic gap genes also do this. In this paper, I have shown that mutations at the zygotically acting gap gene Kr distorts the pair-rule fatemap. Mutations at other gap genes, tailless (Mahoney & Lengyel, 1987) and hunchback (Carroll & Scott, 1986), also seem to have this effect. How could gap genes act so that the fatemap is distorted in mutant animals? Meinhardt (1986, also see Meinhardt, this volume) presented a detailed model in which gap genes interacted with one another under the influence of maternal information. The consequent gap patterns then acted on pair-rule genes. This model is capable of explaining the movement of pairrule patterns in gap mutants: in the absence of one gap gene the others move in to its normal domain and activate pair-rule genes in ectopic positions. In this model, the interactions between the gap genes produce regulative properties, the stretchitrg of the fatemap. An alternative is that the pair-rule genes respond directly to graded information and that this information is changed in gap-gene mutant embryos. In this case, gap genes would respond to and alter the information generated by the maternal functions, the pair-rule genes responding directly to this infor-

13 Generqtion of periodicity in Drosophila 47 mation and not to the gap genes themselves. On the basis of the cuticular phenotype of Kr, Ni.isslein- Volhard & Wieschaus (1980) and Wieschaus et al. (1984) suggested that Kr mutations disturb the final shape of the maternal gradient rather like the maternally acting bicaudal loci. It has been argued (Knipple et al. 1985; Gaul et al. 1987; Gaul & Jiickle, L987) that since the Kr expression domain is smaller than the region of the embryo deleted rn Kr mutants the effect of. Kr on the deleted region must be mediated by some Kr-dependent activity. Another piece of evidence that suggests that Kr is having an indirect effect on the fatemap is the observation that the changes in Kr expression domain seen in the maternal and gap mutants that affect it do not correlate precisely with the phenotypes produced by these loci (Gaul & Jiickle, 1986). The blastoderm Kr phenotypic series I report here indicates that the mutant phenotype involves deletion of the middle part of the pattern associated with an expansion of the posterior pattern into the gap. Associated with this expansion is a polarity reversal. These changes can be formally described by the diagram of the relationship between the fatemap of the wild type and the mutant shown in'fig. 6A. The weak phenotypes cause a small deviation in the relationship. As the phenotype becomes stronger the deviation is greater until an apparent discontinuity arises associated with the polarity reversal. This description may actually represent the mechanism underlying the Kr phenotype, so that the changes in fate are directly related to changes in graded positional information. Fig. 68 shows a similar diagram representing the Kr phenotypic series, but this time in terms of a two-gradient model: the anterior bicoiddependent gradient and the posterior oskar-groupdependent gradient which shows selforg antzing properties and generates the polarity reversal. Interestingly, Redmann et al. (1988) have shown that the Kr protein expression pattern at the blastoderm stage is normal in an amorphic Kr allele that produces a mutant protein. This is a surprising result. It is not consistent with Meinhardts model: in this case one would expect that a nonfunctional Kr mutant would fail to repress the adjacent gap genes hb and knirps and that these would 'invade' the Kr territory and repress the Kr gene. Nor is it consistent with the idea that Kr acts via the graded information generated by maternal gene products: in this case, the Kr expression domain would depend on information that would itself be modified by Kr function. In this case, too, the Kr pattern would be Kr dependent. It is not. It remains possible that there is some temporal feature, such as a short half-life for an active form of Kr protein coupled with a long half-life for the still antigenic inactive form, which could explain these results. The zygotic hb phenotype may also arise as a consequence of deletion and reaffangement of the pair-rule patterns. In this case, the deletions do not extend over the entire blastoderm region of hb expression. This is probably a consequence of the small maternal contribution of hb which can, to a large extent, rescue the hb phenotype. The amorphic phenotype suggests that the deletions extend beyond the posterior border of hb expression and may be associated with the same kind of pol arity reversal in the blastoderm seen with Kr. It is possible that this is also an effect mediated through modification of graded information. In the posterior region (where hb is also expressed in a rather broad stripe) it is not required to form periodic hairy expression, but is required for ftz striping. This suggests that, in this region, hb is acting in parallel with or after the primary segmentation mechanism in order to repress ftz. Tautz (1987) has shown that this posterior region of expression is under different regulation. The gap-gene tailless has been shown to delete the terminal regions of the embryo (Strecker et al. 1986) this deletion is associated with a compensatory movement of the surrounding regions of the fate map (Mahoney & Lengyel, I9B7). Data on the pattern of ftz expression in amorphic knirps (Carroll & Scott, 1986; CarroII et al. 1986), and giant (Carroll & Scott, 1986) mutants suggest that the effects of these mutants are local, in one case involvittg failure to repress ftz and in another failure to maintain ftz expression. In the case of giant, genetic data suggest that it is acting after Kr (Petschek et al. I9B7). The situation with caudal is more complex. Like hb it is a paternally rescuable maternal-effect gene (Macdonald & Struhl, 1986). Complete lack of function has a variable phenotype: ftz stripe 4 is expanded and ftz stripes 5 and 6 generally disappear. There is a weak stripe 7 in approximately the correct position. This phenotype does not easily fit into either the stretch or the erase category. No data are published on the pair-rule phenotype of the maternal effect of the zygotic lethal 1(1) hopscotch (Perrimon & Mahowald, L986). We do not yet have a complete picture of the mechanism lying behind the generation of pair-rule periodicity. Meinhardt (1986 and this volume) proposed that the gap genes are the proximate cause of the patterns of pair-rule gene expression. In this model, the gap genes interact with one another under the influence of maternally generated cues so that they become active in contiguous regions which are the size of 3'5 segmental primordia. The entire embryo is thus divided into five 'cardinal regions', which organize two pair-rule patterns out of frame with oneanother by a distance of 0'5 of a segment primordium. In this way, the two pair-rule frames are

14 48 K. Howard set out of phase and polarity is preserved. The available data do not fit very well with the details of this model, but do not necessarily rule out its basic features (See Meinhardt, this volume). An alternative possibility is that pair-rule genes directly respond to graded morphogen distributions with different and slightly overlapping thresholds. If the pair-rule pattern were to be produced in direct response to a gradient then one function of some gap genes, in particular Kruppel, hunchback and tailless, ffi?y be to alter the graded information. This viewpoint may prove incorrect; moreover, other gap genes may have functions that are altogether different only new experiments can decide. I would like to thank Gary Struhl and the Howard Hughes Medical Institute for support. The Lucille P. Markey Charitable Trust for a Visiting Fellowship. Phil Ingham, Paul Macdonald, Alfonso Martinez-Arias, Chris Rushlow and Gary Struhl for advice. 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