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Cell, Vol. 51, 689698, December 4, 1987, Copyright 0 1987 by Cell Press The Segmentation and Homeotic Gene Network in Early Drosophila Development Matthew P. Scott and Sean B. Carroll* Department of Molecular, Cellular and Developmental Biology University of Colorado Boulder, Colorado 80309 The Temporal In 1980, Nijsslein-Volhard and Wieschaus reported the first results of a systematic screen of an entire metazoan genome for genes that control a particular set of patternforming events, the segmentation of the fruit fly embryo (Nusslein-Volhard and Wieschaus, 1980). In total, they described about twenty zygotically active genes (including some that had previously been found in other ways), which they assigned to three different classes: those in which mutations cause multiple adjacent segments to be missing from the embryo (“gap” genes); those in which mutations cause alternate segment-size units to be missing (“pair-rule” genes); and those in which mutations delete part of every segment and replace the deleted part with a mirror image of some of the remaining structure (“segment polarity” genes). Niisslein-Volhard and Wieschaus proposed that the three classes of genes act to subdivide the embryo into progressively smaller developmental units. A fourth class of genes, the homeotic genes, controls segment identity; mutations in these genes cause parts of the fly to follow an incorrect developmental pathway and form structures normally found in another segment. Many of the segmentation loci have now been cloned and analyzed at the molecular level. These studies, and accompanying genetic studies, have revealed several important features about the segmentation and homeotic gene network. First, there is a temporal order to the expression and function of the various classes of genes. Second, the genes are expressed in precise spatial patterns that are achieved through the regulatory influence of previously acting genes and interactions with other genes of the same class. Third, these precise spatial patterns provide sufficient complexity to assign unique identities to cells at each position along the anterior-posterior axis. Fourth, many genes act at multiple times and/or in multiple germ layers to control pattern formation. Finally, many of the gene products share protein motifs that indicate functional similarities and perhaps evolutionary relationships among the loci. Our purpose here is to discuss some of the central issues and ideas in this field, not to provide a comprehensive review of all that is known. (For further information, readers should consult Scott and O’Farrell, 1988; Gehring and Hiromi, 1986; Duncan, 1987; Akam, 1987; Peifer et al., 1987; Niisslein-Volhard et al., 1987.) * Present address: Laboratory consin, Madison, Wisconsin of Molecular 53706. Biology, UniVerSity Of WiS- Order of Regulatory Review Gene Expression Embryonic pattern formation begins during oogenesis, when the oocyte is packed with maternally encoded mRNAs and proteins. Some of the maternally active genes are segmentation genes, as revealed by maternaleffect mutations that alter the segmentation pattern in the progeny of homozygous mutant female flies (e.g., Niisslein-Volhard, 1979; Boswell and Mahowald, 1985; Schiipbath and Wieschaus, 1986; Frohnhofer and NiissleinVolhard, 1986; Degelmann et al., 1986; Lehmann and Niisslein-Volhard, 1986; MacDonald and Struhl, 1986; reviewed in Niisslein-Volhard et al., 1987). Some of these genes encode products that are asymmetrically distributed in the oocyte, in two cases in graded amounts (Mlodzik et al., 1985; MacDonald and Struhl, 1986, Figure la; Frigerio et al., 1986; Mlodzik and Gehring, 1987). Presumably these genes participate in the initial events that trigger position-specific activation of zygotic gene expression. Experiments with ligated embryos suggest that position-specific activation of zygotically active segmentation genes does not depend upon precisely localized determinants that are placed in position during oogenesis (Schubiger and Newman, 1982). Rather, a dynamic process involving communication between cells (or perhaps nuclei) in different parts of the developing embryo is viewed as the more likely mechanism for establishing positional information. The first zygotically active genes to be transcribed include the segmentation genes. Transcripts of the gap genes are among the earliest detected; KfiipPe/ (Kr) mRNA, for example, can be observed shortly after the eleventh nuclear division, when the embryo is still a syncytium (Knipple et al., 1985). Transcripts of the pair-rule genes that have been examined, including fushi t8rezu (ftz, Hafen et al., 1984a), hairy (II, lngham et al., 1985) paired (prd, Kilchherr et al., 1986) and emen-skipped (eve, Harding et al., 1986; MacDonald et al., 1986), are all detected slightly later than the Kr transcripts. The mRNAs of the segment polarity genes, such as engrailed (en, Kornberg et al., 1985; Fjose et al., 1985), gooseberry (gsb, Bopp et al., 1986) and wingless (wg, Baker, 1987) appear later, during the thirteenth nuclear division. Finally, homeotic gene transcripts are initially detected at the cellular blastoderm stage of embryogenesis (e.g., Levine et al., 1983; Akam and Martinez-Arias, 1985). In general, the site of the highest level of RNA accumulation for each gene corresponds to the inferred site of gene function as determined from mutant analyses. The developmental profiles of the protein products of Kr (Gaul et al., 1987, Figure lb), ffz (Carroll and Scott, 1985, Figure lc), eve (Frasch et al., 1987) en (DiNardo et al., 1985, Figure le), and the three homeotic genes Ultrabithorax(Ubx, White and Wilcox, 1984; Beachyet al., 1985, Figure If), Antennapedia (An@, Carroll et al., 1986a; Wirz et al., 1986) and Sex combs reduced (Scr, Mahaffey and Cdl 690 Kaufman, 1967; Riley et al., 1987; Figure lg) have been examined using antibodies. The protein patterns generally correspond to the RNA patterns. The most notable exception is Kr (Gaul et al., 1987); Kr transcripts in certain parts of the embryo are not translated due to an unknown control mechanism that may involve alternative forms Of the Kr RNA. The timing of homeotic protein synthesis also appears to be controlled; the proteins are first detected hours after the transcripts are completed. This lag may be due to inefficient translation or to posttranscriptional regulation of homeotic RNAs. Drosophilaembryogenesis is complete in less than one day, and the blastoderm stage, when the segmentation and homeotic genes become active, is reached in just the first few hours (see Campos-Ortega and Hartenstein, 1985). During this early period, the patterns of expression of the segmentation genes change rapidly. The ftz and h transcripts, for example, are initially found in most or all cells spanning the length of the embryo, but these “solid” patterns evolve, in about 30 min, into patterns of seven transverse stripes. The change occurs before cell membranes are completely formed (Hafen et al., 1984a; Weir and Kornberg, 1985). It has been suggested that the pattern refinement is due to rapid turnover of the RNA in all cells, combined with a shut-off of transcription in the nuclei that will come to be outside the stripes (Edgar et al., 1988). A different type of pattern change is observed for eve RNA (MacDonald et al., 1988) and protein (Frasch et al., 1987), and for prd RNA (Kilchherr et al., 1988). Both of these pair-rule genes are expressed at the blastoderm stage in the transverse seven-stripe pattern, but shortly after gastrulation acquire new fourteen-stripe (one per segment) patterns. In the case of eve, seven new stripes are initiated; in the case of prd, the seven initially broad stripes are split by the disappearance of transcripts from cells central to each early stripe. The observed changes in the pattern of expression of segmentation genes presumably arise in response to input from other genes in the network. Figure 1 shows the patternsof expression of representative examples from the different classes of segmentation and homeotic genes. The simpler patterns (Figure la-c) seen at the blastoderm stage, when the embryo is made up of a monolayer of about 8000 cells (Figure Id), are characteristic of the segmentation genes. At the later germ band elongation stage (Figure lh), as the metameric divisions of the embryo appear, the later-acting segmentation genes (Figure le) and the homeotic genes (Figures lf,g) are expressed in more refined patterns. The Emergence of Precise through Gene Interactions Gene Expression Patterns In order to study the interactions between genes involved in segmentation, molecular probes have been used to detect the product(s) of one gene in embryos mutant for other segmentation or homeotic gene functions. Altered spatial patterns of gene expression indicate that the missing gene function controls the product being assayed. In this way, it has been shown that gap, pair-rule, and seg- ment polarity genes interact. Generally, each gene class is influenced by the action of earlier-acting genes that control larger units of pattern and by some members of the same class. Thus, gap genes influence the pattern of pairrule (Carroll and Scott, 1986; lngham et al., 1988), segment polarity (Ingham et al., 1986) and homeotic genes (White and Lehmann, 1986; Riley et al., 1987), while segment-polarity genes do not (so far as has been tested) affect gap or pair-rule genes. Some gap genes are mutually negative regulators of each other (J&ckle et al., 1988). Maternally active genes have also been shown to regulate the ftz pair-rule gene (Carroll et al., 1988b; Mlodzik et al., 1987). It is also useful to note the lack of any change in the product, which indicates, within the limits of detection, that the mutation tested does not interfere with the expression of the gene whose product is being assayed. All four zygotically active gap genes and some of the pair-rule genes are required for the ftz pair-rule gene to be activated in its normal striped pattern (Carroll and Scott, 1986). Mutations in each of the gap genes alter some of the ffz stripes while leaving the other stripes apparently unaffected. In contrast, the pair-rule genes that regulate ftz affect all the stripes. Of the seven pair-rule genes other than ftz, only the eve, h, and runt genes influence the ftz striped pattern. The pair-rule gene interactions are hierarchical in at least some cases. For example, h regulates ftz, but ftz does not regulate h (Howard and Ingham, 1986). Furthermore, the three genes that regulate ftz do not simply shut off or turn on ftz: the response of the ftz gene in individual cells to abnormal eve, h, or runt function depends upon the position of the cell in the embryo, and therefore upon the activities of other genes that may simultaneously regulate ftz. This sort of multipleinput scenario is likely to be a general phenomenon. Although the formal regulatory relationships among the genes are becoming clear, the mechanisms involved are still mysterious. It is not known how the gene interactions result in patterns such as stripes and, in particular, whether the interactions are direct or mediated through other genes. The mechanisms must be both precise, in that adjacent cells must in some cases behave very differently, and flexible, in that the number and exact arrangement of cells is different in different embryos. The mechanisms cannot work through counting cells, since embryos with drastically different numbers of nuclei can still form stripes of the normal size (Sullivan, 1987). Furthermore, the simplicity of the striped patterns conceals regulatory subtlety. Different stripes depend on different sets of upstream functions; not all ftz stripes, for example, require the same gap gene function. The en segment polarity gene is expressed in the posterior part of each segment (Kornberg, 1981; Kornberg et al., 1985; DiNardo et al., 1985). Because the en expression pattern of fourteen stripes (Figure le) emerges after the pair-rule patterns, it seems likely that the en stripes are derived from the preexisting wider stripes; it is not necessary to invoke a system for forming stripes de novo as it is for the pair-rule genes. (The broad gap gene stripes, as in Figure lb, could provide only some of the pair-rule stripe boundaries.) Indeed, the normal striped en pattern Review: 691 Segmentation and Homeotic Gene Network Figure 1. Expression Patterns of Segmentation and Homeotic Gene Products a-c, e-g. Whole mount embryos were permeabilized and stained with antibodies against segmentation and homeotic gene products. Anterior is to the left and ventral is down in each panel. The embryo is about 0.6mm long. a-d show embryos at the blastoderm stage (25-35 hr of development) when the approximately 6C06 cells form a monolayer covertng the yolk. e-h show embryos at the elongated germ band stage (5-6 hr of development), at the time when the homeotic protein products become detectable. The wrapping of the segments around the posterior brings the most posterior segments to their location behind the head. The plane of focus in each case shows only the epidermal expression of each gene; the internal tissues have related but different patterns. PS: parasegment (see Figure 2). (a) Expression of the caudel gene product (MacDonald and Struhl, 1966). a nuclear protein that is distributed in a gradient in the early blastoderm embryo. Staining is with peroxidase, so the protein signal appears black. Photograph courtesy of P MacDonald and G. Struhl. (b) Expression of the Kriippel gap gene nuclear protein (Gaul et al., 1967) detected with peroxidase stain. The broad band corresponds approximately to PS4-6. Photograph courtesy of U. Gaul and H. Jackie. (c) Expression of the lushi farazu pair-rule gene product in the blastoderm stage embryo, detected by immunofluorescence so the nuclear protein signal appears white. The anterior edges of the stripes mark the anterior edges of the parasegments (Lawrence et al., 1967); the extent of PS2 is indicated. (d) Diagram of an early blastoderm stage embryo. bc, blastoderm cells; y, yolk; pc, pole cells (the primordial germ cells). (e) Expression of the nuclear protein encoded by the engreiled segment polarity gene (DiNardo et al., 1966) in an embryo at the elongated germ band stage. The stripes mark the posterior part of each segment primordium, and therefore the anterior part of each pamsegme nt. The tracheal pits (tp) are visibte. PS2 includes the posterior maxillary and anterior labial compartments. (f) Expression of the U/tmbIthorax homeotic gene as a nuclear protein (White and Wilcox, 1964; Beachy et al., 1965) at the extended germ band stage. PS6 and PSI3 are indicated. (g) Expression of the Sex oontba reduced homeotic gene product, also a nuclear protein (Riley et al., 1967; Mahaffey and Kaufman, 1967) at the extended germ band stage. This embryo is turned relative to the embryos in (e) and (f); the arrowhead marks the ventral midline. The major site Of Sex combs reduced expression at this stage is in PS2. Later, as the germ band retracts, Sex combs reduced becomes active in PS3 as well. (h) Diagram of an extended germ band stage embryo. The parasegments are numbered. tp, tracheal pits. Cell 692 forms only if the gap and pair-rule genes function properly. Different en stripes require input from different upstream genes, just as different ftz stripes require different gap genes for their formation. Alternate en stripes do not appear in the absence of ftz function (DiNardo and O’Farrell, 1987); the other set of alternate en stripes require the pair-rule gene hairy (Howard and Ingham, 1988); and all of the en stripes require ewe function (Harding et al., 1988; MacDonald et al., 1986). The products of all three of the segment polarity genes that have been studied, in contrast to pair-rule gene products, persist throughout embryogenesis, therefore overlapping with the next class of regulatory gene products, encoded by the homeotic genes. Homeotic gene transcripts are first detectable at the blastoderm stage as the cell membranes form (e.g., Akam and Martinez-Arias, 1985; Levine et al., 1983); this is when the segmentation gene products are at their peak abundance. In three cases, it has been demonstrated that homeotic gene expression is initially controlled by some of the boundaries set up by the segmentation genes. The hunchback (hb) and Kr gap genes (White and Lehman, 1986; lngham et al., 1986), and the ffz pair-rule gene (Duncan, 1986; lngham and Martinez-Arias, 1988) all regulate the spatial patterns of homeotic gene expression in embryos. Wild-type hb function prevents Ubx expression in the anterior of the embryo; Kr mutations appear to alter Ubx expression by altering pair-rule gene expression; and ffz acts as a positive regulator of certain aspects of homeotic gene transcription. Whether all segmentation genes regulate homeotic genes is an open question. Although the initial patterns of expression of the homeotic genes appear to be set up by the segmentation genes, the maintenance of those patterns requires the actions of at least two other classes of genes: the homeotic genes themselves, and the Polycomb-like class of genes (Hafen et al., 1984b; Struhl and White, 1985; Wedeen et al., 1988). For example, extra sex combs (esc) gene function is required to keep homeotic genes off where they should be off after the initial pattern is set (Struhl and Akam, 1985). Homeotic genes are expressed at high levels in the parts of the embryo where their functions are most easily detected genetically and, in at least some cases, at lower levels in more posterior regions of the embryo. For example, An@ RNA (and protein) is concentrated in the posterior Tl and anterior T2 compartments (also known as parasegment 4; parasegments are described in Figure lh and Figure 2) and is present at lower levels in more posterior regions, while Ubx products are expressed at their highest levels in posterior T3 and anterior Al, (i.e., parasegment 6; Figure If) and in lower amounts in the rest of the abdomen. The regulated levels of expression in the posterior regions appear to be maintained by the negative influences of other homeotic genes that are expressed more posteriorly. For example, if the Ubx gene is inactivated, the high level of An@ expression extends back through the region where Ubx is normally at a high level (Hafen et al., 1984b; Harding et al., 1985; Carroll et al., 1986a). The homeotic genes that act even further posteriorly, abdominal A and Abdominal 6, are negative regulators of Ubx (and abdominel A also negatively regulates An@) in the posterior regions (Struhl and White, 1985; Hafen et al., 1984b). All of these interactions may serve to maintain and elaborate patterns that are initially set up by the actions of segmentation genes upon homeotic genes. In addition to regulatory interactions among the segmentation and homeotic genes, some of these genes presumably control other types of genes that carry out differentiation. Potential downstream target genes include growth-controlling genes, genes encoding cell surface components, and genes encoding specialized products such as cuticle components or neurotransmitters. Other apparently independent regulatory networks, such as the sex determination gene network (Maine et al., 1985; Belote et al., 1985) presumably also converge on some of the same target genes, since segmental differentiation in the fly is also sexually dimorphic. Control of Tleeueepeciflc Gene Expression The actions of segmentation and homeotic genes are not limited to the control of epidermal precursor cell fates. High levels of segmentation and homeotic gene products have been observed in the central nervous system and, for some genes, in the mesoderm and peripheral nervous system (reviewed in Doe and Scott, 1988). The expression of homeotic genes is differentially controlled in neural ectoderm, epidermal ectoderm, and mesoderm (Akam and Martinez-Arias, 1985; Martinez-Arias, 1986; MartinezArias et al., 1987). Similarly, segmentation genes can be differentially activated in different tissues. The proteins encoded by ftz (Carroll and Scott, 1985) and eve (Frasch et al., 1987) are found in subsets of the cells in the central nervous system after their blastoderm patterns of stripes have disappeared. Furthermore, these two pair-rule genes are expressed in each segment of the developing nervous system, as opposed to the alternate parasegmental pattern in the blastoderm stage embryo. These observations suggest that the control of ftz and eve may be affected by multiple tissue-specific cis-acting control elements and corresponding Pans-acting factors. The cis-acting control elements involved in ftz expression have been dissected using what have become standard Drosophila molecular genetic techniques: transposon-mediated transformation and promoter-reporter gene fusions (Hiromi et al., 1985; Hiromi and Gehring, 1987). By joining different parts of the ftz gene to the E. coli 8galactosidase (lacz) gene, at least four regions have been identified that control different facets of ftz gene expression. The “zebra” element is responsible for the striped distribution of halactosidase and is within 0.62 kb of the translation initiation site. Most or all of the segmentation genes that affect ftz act upon the zebra element. A second region further upstream of the “zebra” element is responsible for activation of ftz in the ventral nervous system. A third region 3’ of the gene has a positive influence upon the quantitative expression of the gene. An enhancer found at -6.1 to -3.4 kb upstream of the ATG is necessary to elevate ftz expression to the normal level in the ectoderm, and this sequence appears to respond to ftz protein itself; the enhancer has no effect in an embryo that lacks Review: Segmentation and Homeotic Gene Network 693 Figure 2. The Relationships between Segments, Parasegments, and Compartments A segment is composed of an anterior and a posterior compartment. Parasegments are also composed of compartments, but are offset from seg merits by one compartment (Martinez-Arias and Lawrence, 1985). Compartments are defined by boundaries respected by cell lineages (GarciaBellido ei al., 1973; Crick and Lawrence, 1975). At the blastoderm stage. posterior compartments are approximately one cell wide in the anterior-posterior axis, while anterior cumpartments are about three cells wide. The first visible metameric divisions to appear in the embryo, while the germ band is elongated, are the paraeegments. The divisions of the body seen later in development are segmental. Md, mandibular segment; Mx, maxillary segment; Lb, labial segment; Ti-T3, thoracic segments; Al-A9, abdominal segments. ftz function. Thus, ftz expression in the ectoderm involves a positive autoregulatory loop that is required for maintenance, not initiation, of the striped pattern. In contrast, neural expression of ftz does not appear to depend on ftz function. A fourth region has been identified using certain constructs lacking upstream sequences; these transformants express figaiactosidase stripes in the head, where ftz is not normally expressed. Structural Themes among the Homeotic and Segmentation Gene Products Clues about the molecular mechanisms used by the segmentation and homeotic gene products have come from sequence analysis. Certain amino acid sequences appear as recurrent motifs in many of the proteins. The first such theme to be recognized was the homeodomain (McGinnis et al., 1984b; Scott and Weiner, 1984), a 61amino-acid sequence common to the protein products of at least six homeotic genes (Harding et al., 1985), at least five segmentation genes (ffz, eve, prd, en, and gsb [Sopp et al., 1986; C&e et al., 19871), and at least two maternally active genes (bicoid, Frigerio et al., 1986; and caodal, Mtodzik et al., 1985; MacDonald and Struhl, 1986). The similarity between part of the homeodomain and sequences in bacterial DNA-binding proteins (Laughon and Scott, 1984) and in transcriptional regulators encoded by the MAT locus in yeast (Shephard et al., 1984; Laughon and Scott, 1984) suggests that homeodomain-containing proteins may be transcriptional regulators. Sequence-specific DNA binding by a homeodomain has been observed in vitro (Desplan et al., 1985), but the importance of DNA binding to the functions of homeodomains in vivo has yet to be proven. A second structural motif is the “zinc finger” structure that was first proposed for the Xenopus transcription factor IIIA (Miller et al., 1985) and has now been found as an apparently related sequence in the Drosophila gap genes Kr (Rosenberg et al., 1986) and hb (Tautz et al., 1987), and agroup of genesof unknown function (Schuh et al., 1986). Again, a transcriptional regulatory function is suggested, and the possibility of a role in posttranscriptional ContrOl is also raised. Translational control in Drosophila embryos has been observed for the RNA products of at least two genes, the segmentation gene caudel (MacDonald and Struhl, 1986) and the gap gene Kr (Gaul et al., 1987). A third type of repeat is the paired box, a region of homology of 128-135 amino acids found in paited and at least two other loci (Bopp et al., 1986). The paired box appears to be unrelated to any previously determined protein structure. A fourth recurrent theme is the abundance of poiyamino sequences in many of the regulatory gene products. The most striking are the “opa” or “M” or CAG repeat (McGinnis et al., 1984a; Wharton et al., 1985; Laughon et al., 1985) which encodes poiyglutamine in several homeotic genes; the paired (PRD) repeat, which encodes a sequence of alternating histidines and prolines and is found in paired and other genes (including bicoid, an important regulator of embryonic polarity; Frigerio et al., 1986; Frohnhilfer and Nilsslein-Volhard, 1986); and the pen repeat (GGX triplets), which has been found in Ubx (Beachy et al., 1985), in a maternal-effect homeotic gene (fsfl)h), and in many genes of unknown function (Haynes et al., 1987). The discovery of these protein motifs has prompted speculation on their origins and functions. Several possibilities seem reasonable. The genes containing similar sequences could ail be derived from a common ancestral gene. This would imply that genes with apparently very different roles in the control of development-for example, the maternal-effect polarity control genes, segmentation genes, and the homeotic genes-ail evolved from the same ancestral gene, since some genes in each of these classes contain homeoboxes. Another possibility is that different types of genes were assembled from repetitive (and nonrepetitive) components, and then evolved to perform different functions depending in part on how the components were combined. This type of evolutionary pathway could have involved transposition of repetitive components (e.g., homeoboxes) and their subsequent assembly into functional genes through deletions or other chromosomai rearrangements. Homeoboxes are often found as exon units (although additional 3’ coding sequence is usually also present in the same exon), which is consistent with a piecemeal gene assembly model. This sort of model is also supported by the finding that different combinations of protein themes occur in different proteins. For example, the f&red segmen- Cell 694 tation gene contains a homeobox and a PRD repeat (Frigerio et al., 1988), the bicoid polarity control gene contains a homeobox and a PRD repeat (Bopp et al., 1988) the Antp gene contains a homeobox and a CAG repeat (but no PRD repeat) (Schneuwly et al., 1988; Stroeher et al., 1988; Laughon et al., 1986) and some of the genes of unknown function that contain a PRD repeat also contain CAG repeats (but not known homeoboxes). Whatever the evolutionary history of these elements, it is clear that while most segmentation genes are dispersed throughout the genome, the clustered homeoboxcontaining loci (Regulski et al., 1985) of the Antennapedia complex (ANT-C, Kaufman et al., 1980) and bithorax complex (6X-C Lewis, 1978) probably are a classic example of gene duplication and divergence. Not only the homeodomain sequences have been conserved during evolution, however. The protein encoded by the Deformed (Dfd) homeotic gene, a gene in the ANT-C, has been found to be strikingly related in sequence to proteins encoded by a frog and a human homeobox gene (Regulski et al., 1987). Therefore certain aspects of homeotic protein structures other than the homeodomain appear to have predated the divergence of vertebrates and invertebrates, and to have been conserved since then, The cross-hybridization of the DNA sequences encoding the related protein domains has proven to be extremely useful in allowing the isolation of genes that control early development or that are good candidates for doing so (e.g., McGinnis et al.,1984a; Fjose et al., 1985; Harding et al., 1986; Schuh et al., 1986; Bopp et al., 1986; Frigerio et al., 1986; MacDonald et al., 1986; MacDonald and Struhl, 1986). This success implies that the protein themes, and presumably the biochemical functions they encode, are specific to genes that serve as central regulators during development. The data so far tend to support this idea, although there is still no direct proof. The Determination of Cell Identities Segmentation Genes by Pair-Rule Three of the pair-rule segmentation genes, ffz (Scott and Weiner, 1984; McGinnis et al., 1984a), eve (Harding et al., 1986; MacDonald et al., 1986) and prd (Frigerio et al., 1986) contain homeoboxes. Sequences for the five other genes in this class have not yet been reported. The ffz, eve, and prd genes are each expressed in stripes and no two of them appear to have stripes in phase. If homeodomain-containing proteins are transcriptional regulators, then different cells along the anterior-posterior axis of the blastoderm would contain different arrays of them. The striped patterns of the pair-rule gene products therefore lead to a simple model for how each blastoderm cell is instructed to be different from its immediate neighbors just anterior or posterior (Gergen et al., 1986). At the blastoderm stage (Figure Id), a segment primordium is about four cells wide in the anterior-posterior axis, so stripes in alternate segment-size (four cell) units define a repeat unit of eight cells (four “on,” four “off”) (in some cases, the stripes become narrower after they first appear, Figure lc). Because the stripes of one gene product are offset from stripes of others, each cell in the repeat unit will express a particular combination of gene products. In theory, only four different gene products expressed in outof-frame stripes (with three to four cells per stripe) would suffice to distinguish each cell in the repeat unit from each of the other cells. There are at least eight pair-rule genes, so ample genetic complexity for such a system exists. Some of the pair-rule genes control the expression of others, so a simple combinatorial model cannot be the whole story. The pair-rule genes are not all equivalent. Some may serve only to control other segmentation genes, helping to direct the formation of their expression patterns, and may play no role in directly controlling the downstream target genes (whatever they may be) that actually carry out differentiation processes. It is also possible that some of the pair-rule genes control other segmentation genes and control downstream target genes. Among the target genes are the segment polarity genes, at least three of which, en, gsb, and wg, are expressed in about fourteen stripes, one per segment. If most of the other segment polarity genes are expressed in offset stripes, then the eight cells in the repeat unit could be distinguished both by the transient action of the pair-rule gene products and by the longer-lived segment polarity gene products. The determination of individual embryonic cells may thus be viewed as the activation (or repression) of certain sets of regulatory genes in precise spatial patterns that together direct the cell into a particular developmental pathway. The embryonic fate map may now be drawn based upon knowledge of the spatial domains of regulatory genes. After the initial expression of genes in stripes, cell interactions are likely to occur that result in further pattern refinements (discussed in Scott and C’Farrell, 1988). The nonautonomous action in the embryo of the segment polarity gene wingless (Baker, 1987) suggests that some of the segment polarity genes may be involved in communication between cells. Cell-cell communication may lead to the formation of smoothly graded and complete cuticular patterns. Some of the segment polarity genes may therefore be involved in the sensing by cells of the character of neighboring cells. All of the genes discussed here are regulators of anterior-posterior patterning. If a set of genes with similar behaviors exists for the dorsal-ventral axis, then each cell or small group of cells could be determined by position along the two axes, that is by the array of anteriorposterior and dorsal-ventral regulators activated within it. A substantial number of genes controlling dorsal-ventral differentiation have been found (e.g., Simpson, 1983; Anderson and Nijsslein-Volhard, 1984) but whether they act by the same set of molecular mechanisms as the anterior-posterior genes is not yet known. How Do Homeotic Genes Make Cells Different? Homeotic genes are cell-autonomous in their actions (e.g., Garcia-Bellido and Lewis, 1978); a cell will behave according to its homeotic genotype, regardless of the genotype of the surrounding cells. In the extreme case, a Review: Segmentation and Homeotic Gene Network 695 single cell that has an active Ubxgene would develop into, for example, a T3 bristle, even if the surrounding cells lacked Ubx function and made a l2 pattern of bristles. However, a cell does need positional information to respond appropriately to the homeotic signal. A cell in one part of a segment might respond to the presence of, for example, Ubx protein by making a bristle, while a cell that also contains Ubx protein but is in a different position would respond to the Ubx signal by elongating in a T3-like way. A cell must integrate information about its position, presumably information derived from segmentation gene functions, with the information about its segmental identity that comes from homeotic genes. Cells expressing the same homeotic gene(s) in different parts of the same segment primordium could behave differently if: 1) the homeotic proteins are made at different levels in different cells; 2) different combinations of homeotic proteins are made in different cells within a segment (or parasegment); 3) there are variant forms of homeotic proteins made in cells in different positions (if these variants are transcription factors, they could control different target genes or differentially affect the same target gene); and 4) the segmentation gene products that acted earlier, or that continue to be active, modify the effects of homeotic gene products. It seems likely that all four mechanisms are used. Substantial cell-to-cell variations in the levels of homeotic proteins have been observed. Notably, minor differences in Ubx (Lewis, 1978) or Scr (Kaufman et al., 1980) gene dosage are sufficient to change the cell phenotype. It is also clear that many cells contain more than one homeotic protein (e.g., Carroll et al., 1988a) and, at least in the case of Ubx, that variant forms of the protein exist (Beachy, 1988). The persistence of some segmentation gene products through the time when homeotic gene products are made is consistent with the idea that some type of interaction between them is important for determining cell fate. In some compartments there appears to be a relatively uniform amount of, for example, Ubx protein in all of the epidermal primordial cells (White and Lehmann, 1988; Figure If). If the cells do, in fact, all contain the same amount of the same Ubx protein, or the same mix of Ubx isoforms, cells in the primordium must behave differently according to their position, and they “know” their positions as a result of the earlier actions of segmentation genes. Because the pair-rule segmentation genes are only transiently active, one possibility is that the early action of pair-rule (or other) genes could leave a lasting imprint on the cell, possibly through a transcription priming mechanism that renders certain target genes responsive to the homeotic proteins that appear later. Alternatively, the cell “memories” could be due to the continued activities of segmentation genes. eng&ed(Kornberg et al., 1986; DiNardo et al., 1985) and wingless (Baker, 1987) segment polarity gene products, for example, persist throughout embryogenesis. These and other proteins could modify the effects of the homeotic proteins. Thus in deciding cell fates within a segment-in deciding which cells should make bristles and which should not in forming the stereotypical segmental pattern-cells appear to respond both to the homeotic genes active within them and to the positional information that has been provided by the segmentation genes. Speculations of Homeotic on the Evolution Genes and Function A possible series of evolutionary events that may have led to the formation of homeotic genes is suggested by the location of the ftz gene in the ANT-C (Wakimoto and Kaufman, 1981), the strong similarity between the homeodomains in the ffz protein and the homeotic proteins (Scott and Weiner, 1984; McGinnis et al., 1984b), the autoregulation of ffz (Hiromi and Gehring, 1987), and the activation of homeotic genes by ftz (Ingham and Martinez-Arias, 1988). The development of the largely identical segments of an annelid-like insect ancestor may have depended on the function of a gene similar to fiz (or eve) that was expressed in stripes and directed the formation of repeating pattern elements. The homeotic genes could have arisen by duplication and divergence of the ftz-like gene-an extension of the proposal by E. Lewis (1951) that homeotic genes arose by duplication and divergence. This would be consistent with the clustering of the Drosophila homeotic genes in two groups, the ANT-C and BX-C, with one group including ftz. The existence of the two groups may have resulted from a splitting apart of one ancestral cluster. Indeed, in a beetle species, the ANT-C- and BX-C-like homeotic genes appear to be clustered together (Beeman, 1987). If the ancestral ftz-like protein had positively regulated its own gene, like the present day ftz (Hiromi and Gehring, 1987), it would also have positively regulated the duplicated gene(s). The ffz gene has been shown to activate three homeotic genes, Scr Antp and Ubx, in their positions of highest expression and major function, i.e., parasegments 2, 4, and 8, respectively (Ingham and Martinez-Arias, 1988). These parasegments are regions where ftz is normally expressed; thus the homeotic genes are active at their highest levels in places that correspond to part of the ftz pattern. But what prevents the homeotic genes from being expressed in all of the places where ftz is active? The newly reduplicated genes could come to be expressed differently in different segments (thereby taking a step toward becoming homeotic genes) by acquiring c&-acting elements that turn off some of the stripes. These elements could receive signals from regionally expressed earlier-acting genes such as the gap genes. A current example might be the repression of the Ubx homeotic gene in the anterior of the embryo by the hunchback gap gene (White and Lehmann, 1986). While frz is also regulated by gap genes (Carroll and Scott, 1986), the result is a reiterated striped pattern, not expression in only certain segments. How difficult would it be for the ftz striped expression pattern to change into a more homeotic sort of pattern? A clue is provided by recent experiments showing that the ftz upstream sequences, specifically the enhancer element mentioned earlier, can direct a homeotic pattern rather than an evenly striped pattern if the enhancer is merely inverted (Hiromi Cell 696 and Gehring, 1987). The new pattern seen is similar to that of transcripts from one of the Antp promoters (Ingham and Martinez-Arias, 1988)-that is, most of the expression is detected in a single stripe in parasegment 4. These experiments with ftz demonstrate that it does not necessarily require elaborate changes in c&acting elements to go from a striped pattern to a modified striped pattern in which one of the stripes is much stronger than the others. The change observed, moreover, was shown to depend upon the function of the gap gene Kr (Hiromi and Gehring, 1987), again implicating the gap genes in region-specific expression. Thus in the evolution of homeotic genes, the responses to the gap genes would merely have to be modified from the existing responses of a ftz-like gene; it would not be necessary for an entirely new kind of gene interaction to evolve. Segmental organization is a relatively simple repeating pattern, but homeotic genes need to direct the formation of quite complex patterns. The DNA sequences that regulate homeotic genes in cis appear to be very complex (see Peifer et al., 1987), which perhaps is another reason for the large size of these genes. One homeotic gene, Anfp, appears to have evolved by acquiring a second promoter that controls the same protein-coding sequence (Schneuwly et al., 1988; Stroeher et al., 1988; Laughon et al., 1988). In this way the same protein can be regulated by two sets of cis-acting sequences, allowing divergence of functions similar to that generated by gene duplication. The Antp promoters are differentially controlled by tmns-acting regulators (Ingham and Martinez-Arias, 1988), and are differentially expressed in imaginal discs (Jorgensen et al., 1987); thus, in this case, two different sets of cis-acting sequences are in fact used. Gene duplication, multiple promoters, the acquisition of large arrays of cis-acting elements, and changes in protein sequences all appear to have played a role in the evolution of the homeotic genes. Conclusions The advanced understanding of systems as diverse as phage assembly and yeast mating type has relied upon the systematic isolation of mutations that affect most or all components of the system. The same approach to Drosophila development, as first applied by a few pioneers such as E. Lewis, C. Niisslein-Volhard, and E. Wieschaus, has also been successful. We anticipate that the continued detailed study of genes that control embryonic pattern formation will yield many additional ideas about the biochemical nature of positional information and the molecular basis of cell determination. In particular, we expect that the most provocative information will come from the analysis of the expression and function of maternal gene products, the identification of the molecular mechanisms controlling interactions between the different tiers of the zygotic gene hierarchy, and the characterization of cisacting elements of pattern-regulating genes that respond to specific spatial, temporal, and tissue-specific regulatory proteins. Acknowledgments We thank the other members of the laboratory for stimulating discussions. Thanks to Drs. Robert Boswell, Margaret Fuller, David Gubb, and William Wood for comments on the manuscript. We are grateful to Drs. Paul MacDonald, Gary Struhl, Ulrike Gaul, and Herbert Jbkle for donating photographs, to Dn. 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