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Genetic studies of the targets of the Hox genes have revealed only the tip of the iceberg. Recent microarray studies that have identified hundreds more transcriptional responses to Hox genes in Drosophilawill help elucidate the role of Hox genes in development and evolution.

Although the molecular pathways that pattern the early embryo of Drosophila melanogaster are well understood, how these pathways differ in other types of insect embryo remains largely unknown. We have examined the expression of three markers of early patterning in the embryo of the African plague locust Schistocerca gregaria, an orthopteran insect that displays a mode of embryogenesis very different from that of Drosophila. Transcripts of the caudal gene are expressed maternally and are present in all cells that aggregate to form the early embryonic rudiment. First signs of a posterior-to-anterior gradient in the levels of caudal transcript appear in the early heart-stage embryo, shortly before gastrulation. This gradient rapidly resolves to a defined expression domain marking segment A11. The decapentaplegic (dpp) gene, which encodes a transforming growth factor β family ligand, is first expressed in a circle of cells that delimit the margins of the embryonic primordium, where embryonic and extra-embryonic tissues abut. Patterned transcription of wingless reveals that the first segments are delineated in the Schistocerca embryo substantially earlier than previously thought, at least 14-16 hours before the onset of engrailed expression. By the late heart-stage, gnathal and thoracic segments are all defined. Thus, with respect to the molecular patterning of segments, the short germ Schistocerca embryo differs little from intermediate germ embryos. The expression of these marker genes suggests that embryonic pattern formation in the grasshopper occurs as cells move together to form the blastodisc.

The arthropods are the most speciose, and among the most morphologically diverse, of the animal phyla. Their evolution has been the subject of intense research for well over a century, yet the relationships among the four extant arthropod subphyla — chelicerates, crustaceans, hexapods, and myriapods — are still not fully resolved. Morphological taxonomies have often placed hexapods and myriapods together (the Atelocerata) [1, 2], but recent molecular studies have generally supported a hexapod/crustacean clade [2–9]. A cluster of regulatory genes, the Hoxgenes, control segment identity in arthropods, and comparisons of the sequences and functions of Hoxgenes can reveal evolutionary relationships [10]. We used Hoxgene sequences from a range of arthropod taxa, including new data from a basal hexapod and a myriapod, to estimate a phylogeny of the arthropods. Our data support the hypothesis that insects and crustaceans form a single clade within the arthropods to the exclusion of myriapods. They also suggest that myriapods are more closely allied to the chelicerates than to this insect/crustacean clade.

The Drosophila haltere is a much reduced and specialised hind wing, which functions as a balance organ. Ultrabithorax (Ubx) is the sole Hox gene responsible for the differential development of the fore-wing and haltere in Drosophila. Previous work on the downstream effects of Ubx has focused on the control of pattern formation. Here we provide the first detailed description of cell differentiation in the haltere epidermis, and of the developmental processes that distinguish wing and haltere cells. By the end of pupal development, haltere cells are 8-fold smaller in apical surface area than wing cells; they differ in cell outline, and in the size and number of cuticular hairs secreted by each cell. Wing cells secrete only a thin cuticle, and undergo apoptosis within 2 hours of eclosion. Haltere cells continue to secrete cuticle after eclosion. Differences in the shape of wing and haltere cells reflect differences in the architecture of the actin cytoskeleton that become apparent between 24 and 48 hours after puparium formation. We show that Ubx protein is not needed later than 6 hours after puparium formation to specify these differences, though it is required at later stages for the correct development of campaniform sensilla on the haltere. We conclude that, during normal development, Ubx protein expressed before pupation controls a cascade of downstream effects that control changes in cell morphology 24-48 hours later. Ectopic expression of Ubx in the pupal wing, up to 30 hours after puparium formation, can still elicit many aspects of haltere cell morphology. The response of wing cells to Ubx at this time is sensitive to both the duration and level of Ubx exposure.

Clones homologous to the 5′ region of the Ultrabithoraxgene of Drosophila melanogasterhave been isolated from D. pseudoobscura, D. funebrisand Musca domestica. Regions that encode most of the Ubxprotein have been sequenced in all three of these species, and the 5′ upstream region has been sequenced in D. funebristo a point ˜1000 bases upstream of the probable mRNA start site. Here we compare these sequences with those described elsewhere for D. melanogaster. Deduced amino acid sequences of the Ubxprotein show 8% (D. pseudoobscura), 15% (D. funebris) and 22% (M. domestica) divergence from D. melanogaster. However, these figures mask very different rates of evolution in different regions of the protein. A glycine‐rich (‘hinge’) region is conserved in each of these species, although its length is variable. Comparison of D. funebrisand D. melanogastersequences in the long 5′ untranslated leader region of the mRNA, and in the region immediately upstream of the start point of transcription, reveals tightly conserved elements embedded in an otherwise non‐homologous sequence. These conserved elements include a 118‐bp region that spans the mRNA start site, an internally repetitive (TAA)nregion in the untranslated leader and a short repeated motif immediately upstream of the ATG codon that initiates the major open reading frame of the Ubxprotein. Two other conserved elements were identified upstream of the transcription start site; both elements have structural features consistent with a role as recognition sites for regulatory proteins.

We describe the structure and expression of an exceptional bithorax complex mutation, C1. This deletion mutation removes major portions of both the Ultrabithorax(Ubx) and abdominal‐A(abd‐A) protein coding regions, yet retains many of the functions normally executed by these genes. We show that the ends of the C1deletion map to analogous positions within the Ubxand abd‐Atranscription units, and that the deletion chromosome gives rise to a set of novel fusion transcripts that may encode hybrid abd‐A/Ubxproteins. These fusion products are transcribed from the abd‐Apromoter, but exhibit a novel spatial pattern of expression that combines aspects of the normal Ubxand abd‐Adistributions. This pattern provides evidence for the existence of regulatory elements located in the 3′ region of the Ubxtranscription unit that can act on the abd‐Apromoter at a distance of at least 20 kb.

We have used in situhybridization to monitor the distribution of Ultrabithorax (Ubx)transcripts during the early stages of Drosophilaembryogenesis. When first detectable, in the late syncytial blastoderm, Ubxtranscripts are broadly distributed from 20 to 50% egg length (measured from the posterior pole). By the completion of cellular blastoderm formation, a precisely bounded zone one segment wide is defined by the accumulation of high levels of Ubxtranscripts. This zone is probably the primordium for parasegment 6, that is, the posterior compartment of the third thoracic segment (T3) and the anterior compartment of the first abdominal segment (A1). Following gastrulation, seven metameric units of the germ band express Ubxtranscripts in most or all cells of both the ectoderm and the mesoderm. This principal domain of Ubxexpression is sharply bounded at superficial grooves in the embryo which probably mark the A/P compartment boundaries in T3 and A7, and so consists of parasegments 6‐12. Outside this region, Ubxtranscripts can be detected in only a few cells of the ectoderm of parasegments 5 and 13, and in the amnioserosa. This distribution suggests that the major role of Ubxin the early embryo lies in the morphological domain generally ascribed to the control of the bithoraxoidgene. Later in embryogenesis, the pattern of Ubxtranscript accumulation is different in each of the major germ layers, suggesting that different controls regulate Ubxin each. In the ectoderm, but not in the mesoderm, Ubxtranscripts accumulate differentially in anterior and posterior compartments. Probes prepared from different regions of the Ubxtranscription unit show related but different patterns of hybridization. These patterns suggest that the 3.2‐kb Ubxtranscript is distributed throughout the wider domain of Ubxexpression, but that the 4.7‐kb transcript accumulates preferentially in parasegment 6.

Using sequence homology to Drosophila homeobox-containing genes, we have cloned a homologue of abdominal-A from the locust Schistocerca gregaria. The Schistocerca clone encodes a stretch of 78 amino acids including the homeodomain and its flanking regions identical to the corresponding region of abdominal-A. We have shown by in situ hybridization that this gene is transcribed and have used an antibody raised against its protein product to examine the expression of abdominal-A during early Schistocerca embryogenesis. Schistocerca is a short germ insect. Although the segmented body plan is very similar to that of Drosophila, the segments are generated sequentially by a process of growth, not simultaneously by subdivision of a syncytial blastoderm. In both organisms, abdominal-A is expressed throughout the abdomen from a sharp anterior boundary located within the first abdominal segment (Al). The initial activation of the genes in the two species differs. Schistocerca initiates expression in a small group of cells in the anterior of A2, shortly after this segment is defined by the appearance of engrailed protein. This contrasts with the appearance of abdominal-A expression in Drosophila, which appears simultaneously throughout the entire abdomen.

In Drosophila, the Hox gene Ultrabithorax (Ubx) specifies the development of two different metameres - parasegment 5, which is entirely thoracic, and parasegment 6, which includes most of the first abdominal segment. Here we investigate how a single Hox gene can specify two such different morphologies. We show that, in the early embryo, cells respond similarly to UBX protein in both parasegments. The differences between parasegments 5 and 6 can be explained by the different spatial and temporal pattern of UBX protein expression in these two metameres. We find no evidence for multiple threshold responses to different levels of UBX protein. We examine in particular the role of Ubx in limb development. We show that UBX protein will repress limb primordia before 7 hours, when Ubx is expressed in the abdomen, but not later, when UBX is first expressed in the T3 limb primordium. The regulation of one downstream target of UBX, the Distalless gene, provides a model for this transition at the molecular level.

Homeotic genes confer identity to the different segments of Drosophila. These genes are expressed in many cell types over long periods of time. To determine when the homeotic genes are required for specific developmental events we have expressed the Ultrabithorax, abdominal-A and Abdominal-Bm proteins at different times during development using the GAL4 targeting technique. We find that early transient homeotic gene expression has no lasting effects on the differentiation of the larval epidermis, but it switches the fate of other cell types irreversibly (e.g. the spiracle primordia). We describe one cell type in the peripheral nervous system that makes sequential, independent responses to homeotic gene expression. We also provide evidence that supports the hypothesis of in vivo competition between the bithorax complex proteins for the regulation of their down-stream targets.

The identities of the second through ninth abdominal segments of Drosophila are specified by two genes of the bithorax complex (BX-C), abdominal-A (abd-A) and Abdominal B (Abd-B). The correct deployment of these two genes requires an extensive region (the iab region) located between the two protein-coding transcription units. We show here that one iab mutation affects the pattern of expression of Abd-B. We also show that most or all of the DNA in this regulatory iab region is transcribed. In blastoderm stage embryos we can define three distinct domains within the iab DNA, each transcribed in a region that extends from a characteristic anterior limit to the posterior end of the segmented part of the embryo. The anterior limits of expression for the three regions are colinear with the sequence of the domains on the chromosome, and lie at about two-segment intervals. We suggest that these early transcription patterns reflect the initial activation of the BX-C.

Homeotic genes control the diversity of segment development, but the domains of action of homeotic genes do not obviously correspond with the major morphological subdivisions of the insect body. We suggest that this lack of correspondence is misleading, because the spatial domains defined by genetics mask fundamental differences in the roles played by individual genes in different regions. In one or more parasegments, each homeotic gene is expressed `metamerically'; that is, it is expressed from blastoderm stages onwards in all or virtually all cells of the parasegment primordium. Elsewhere, the same homeotic gene may be deployed adventitiously, only in subsets of cells and at later stages of development. We argue that the early `metameric' domains of gene expression do correlate with the major morphological subdivisions of the fly. This suggests a relatively direct relationship between the expression of particular homeotic genes and the establishment of the `ground plan' that characterizes segments within each major tagma of the body. This relationship allows us to suggest a scenario for the evolution of homeotic genes in relation to the evolving morphological organization of the arthropod body plan in the insect-myriapod lineage.

The metameric organization of the Drosophila embryo is generated in the first 5 h after fertilization. An initially rather simple pattern provides the foundation for subsequent development and diversification of the segmented part of the body. Many of the genes that control the formation of this pattern have been identified and at least twenty have been cloned. By combining the techniques of genetics, molecular biology and experimental embryology, it is becoming possible to unravel the role played by each of these genes. The repeating segment pattern is defined by the persistent expression of engrailed and of other genes of the ‘segment polarity’ class. The establishment of this pattern is directed by a transient molecular prepattern that is generated in the blastoderm by the activity of the ‘pair-rule’ genes. Maternal determinants at the poles of the egg coordinate this prepattern and define the anteroposterior sequence of pattern elements. The primary effect of these determinants is not known, but genes required for their production have been identified and the product of one of these, bicoid is known to be localized at the anterior of the egg. One early consequence of their activity is to define domains along the A–P axis within which a series of ‘cardinal’ genes are transcribed. The activity of the cardinal genes is required both to coordinate the process of segmentation and to define the early domains of horneotic gene expression. Further interactions between the homeotic genes and other classes of segmentation genes refine the initial establishment of segment identities.

In Drosophila intracellular gradients establish the pattern of segmentation by controlling gene expression during a critical syncytial stage, prior to cellularization. To investigate whether a similar mechanism may be exploited by other insects, we examined the timing of cellularization with respect to blastoderm formation in an insect with extreme short-germ development, the African desert locust, Schistocerca gregaria. Using light and electron microscopic techniques, we show that the islands of cytoplasm surrounding cleavage nuclei are largely isolated from their neighbours, allowing cleavage to proceed asynchronously. Within a short time of their arrival at the surface and prior to blastoderm formation, nuclei become surrounded by complete cell membranes that block the free uptake of dye (10,000 kDa) from the yolk. Our results imply that the formation of the blastoderm disc involves the aggregation of cells at the posterior pole of the egg and not the migration of nuclei within a syncytial cytoplasm. These findings suggest that the primary cleavage syncytium does not play the same role in patterning the locust embryo as it does in Drosophila. However, we do identify a syncytial nuclear layer that underlies the forming blastoderm and remains in continuity with the yolk.