Abd-B expression in Porcellio scaber Latreille, 1804 (Isopoda: Crustacea): conserved pattern versus novel roles in development and evolution.

The Hox genes are intimately involved in patterning the animal body during development and are considered to have had a pivotal role in the evolution of different body plans among the metazoans. From this perspective, crustaceans, a group that has evolved an extreme diversity of body structures, represent a choice group in which to study the evolution of these genes and their expression. The expression of one of these genes, Abdominal-B (Abd-B), has only been studied in two distantly related crustaceans, Artemia and Sacculina, where it shows dissimilar patterns, highly differentiated from the one described in other arthropods. Moreover, we have no information for the Malacostraca. Thus, we cloned the gene Abd-B and followed its expression through development by in situ hybridization in the isopod Porcellio scaber. We found a highly dynamic expression pattern of PsAbd-B during embryonic development. In early stages, it is expressed in the posterior-most part of the germ band, in a domain common to several arthropods studied to date, and later it is expressed in the developing limb buds of the pleon and still later in the endopodites of the third to fifth pleopodites. This raises the interesting possibility of the involvement of this gene in the later respiratory specialization of these appendages. In association with the above expression domain, Abd-B appears to be expressed in later stages also in the ventral ectoderm, raising the further suggestion of its possible involvement in patterning the developing nervous system. Moreover, we show that the first pleopod and the endopodite of the second pleopod, whereas present as limb buds in early embryonic stages, are later reduced and actually absent in the first postembryonic stage, although they reappear again in adults. These appendages thus represent an example of Lazarus appendages. Our data show strong plasticity in the use of a key developmental gene and point out the necessity of further research that may end with a revision of the current understanding of its role in animal evolution.

Functional equivalence of Hox gene products in the specification of the tritocerebrum during embryonic brain development of Drosophila.

Hox genes encode evolutionarily conserved transcription factors involved in the specification of segmental identity during embryonic development. This specification of identity is thought to be directed by differential Hox gene action, based on differential spatiotemporal expression patterns, protein sequence differences, interactions with co-factors and regulation of specific downstream genes. During embryonic development of the Drosophila brain, the Hox gene labial is required for the regionalized specification of the tritocerebral neuromere; in the absence of labial, the cells in this brain region do not acquire a neuronal identity and major axonal pathfinding deficits result. We have used genetic rescue experiments to investigate the functional equivalence of the Drosophila Hox gene products in the specification of the tritocerebral neuromere. Using the Gal4-UAS system, we first demonstrate that the labial mutant brain phenotype can be rescued by targeted expression of the Labial protein under the control of CNS-specific labial regulatory elements. We then show that under the control of these CNS-specific regulatory elements, all other Drosophila Hox gene products, except Abdominal-B, are able to efficiently replace Labial in the specification of the tritocerebral neuromere. We also observe a correlation between the rescue efficiency of the Hox proteins and the chromosomal arrangement of their encoding loci. Our results indicate that, despite considerably diverged sequences, most Hox proteins are functionally equivalent in their ability to replace Labial in the specification of neuronal identity. This suggests that in embryonic brain development, differences in Hox gene action rely mainly on cis-acting regulatory elements and not on Hox protein specificity.

The Drosophila proboscis is specified by two Hox genes, proboscipedia and Sex combs reduced, via repression of leg and antennal appendage genes.

The proboscis is one of the most highly modified appendages in Drosophila melanogaster. However, the phenotypes of proboscipedia (pb) mutants, which transform the proboscis into leg or antenna, indicate a basic homology among these limbs. Recent genetic studies have revealed a developmental system for patterning appendages and identified several genes required for limb development. Among these are: extradenticle (exd), homothorax (hth), dachshund (dac), Distal-less (Dll) and spalt (sal). These limb genes have not been well studied in wild-type mouthparts and their role if any in this appendage is not well understood. Here we demonstrate that the homeotic gene products Proboscipedia (Pb) and Sex combs reduced (Scr) regulate the limb genes in the labial disc to give rise to a unique type of appendage, the proboscis. Pb inhibits exd, dac and sal expression and downregulates DLL: This observation explains the ability of Pb to inhibit the effects of ectopically expressed trunk Hox genes in the proboscis, to suppress leg identity in the trunk and to transform antenna to maxillary palp. Scr suppresses sal expression and also downregulates Dll in the labial discs; discs mutant for both pb and Scr give rise to complete antennae, further demonstrating appendage homology. In the labial disc, Pb positively regulates transcription of Scr, whereas in the embryo, Scr positively regulates pb. Additionally, our results suggests a revised fate map of the labial disc. We conclude that the proboscis constitutes a genetically distinct type of appendage whose morphogenesis does not require several important components of leg and/or antennal patterning systems, but retains distal segmental homology with these appendages.

Homeotic Complex (Hox) gene regulation and homeosis in the mesoderm of the Drosophila melanogaster embryo: the roles of signal transduction and cell autonomous regulation.

In this paper we evaluate homeosis and Homeotic Complex (Hox) regulatory hierarchies in the somatic and visceral mesoderm. We demonstrate that both Hox control of signal transduction and cell autonomous regulation are critical for establishing normal Hox expression patterns and the specification of segmental identity and morphology. We present data identifying novel regulatory interactions associated with the segmental register shift in Hox expression domains between the epidermis/somatic mesoderm and visceral mesoderm. A proposed mechanism for the gap between the expression domains of Sex combs reduced (Scr) and Antennapedia (Antp) in the visceral mesoderm is provided. Previously, Hox gene interactions have been shown to occur on multiple levels: direct cross-regulation, competition for binding sites at downstream targets and through indirect feedback involving signal transduction. We find that extrinsic specification of cell fate by signaling can be overridden by Hox protein expression in mesodermal cells and propose the term autonomic dominance for this phenomenon.

Zygotic development without functional mitotic centrosomes.

The centrosome is the dominant microtubule-organizing center in animal cells. At the onset of mitosis, each cell normally has two centrosomes that lie on opposite sides of the nucleus. Centrosomes nucleate the growth of microtubules and orchestrate the efficient assembly of the mitotic spindle. Recent studies in vivo and in vitro have shown that the spindle can form even in the absence of centrosomes and demonstrate that individual cells can divide without this organelle. However, since centrosomes are involved in multiple processes in vivo, including polarized cell divisions, which are an essential developmental mechanism for producing differentiated cell types, it remains to be shown whether or not a complete organism can develop without centrosomes. Here we show that in Drosophila a centrosomin (cnn) null mutant, which fails to assemble fully functional mitotic centrosomes and has few or no detectable astral microtubules, can develop into an adult fly. These results challenge long-held assumptions that the centrosome and the astral microtubules emanating from it are essential for development and are required specifically for spindle orientation during asymmetric cell divisions.

Homologs of Drosophila appendage genes in the patterning of arthropod limbs.

Studies of the genes involved in patterning the appendages of Drosophila melanogaster have revealed a system of signaling and transcriptional regulation that is responsible for specifying the proximo-distal limb axis. Here we report the expression patterns of presumptive homologs of the Drosophila genes extradenticle, dachshund, nubbin, ventral veins lacking (a.k.a. Cf1-a), and Dll in the limbs of the woodlouse Porcellio scaber and the spider Steatoda triangulosa. Although the expression domains of the appendage genes roughly correspond to those of Drosophila, their relative positions and segmental affiliation are distinct. In addition, the expression patterns of the appendage genes allows a resolution of the segmental composition of different appendages within crustacean and spider embryos. We conclude that certain limb types, e.g., mouthparts, appear to be derived from a leg-like ground-plan via the elimination/fusion of the intermediate and distal podomeres. Moreover, we observe just such a modification during the transformation of the anterior legs into mouthparts in P. scaber. Although our data do not unequivocally resolve the question of homology of the arthropod leg segments, they do provide evidence for a single conserved proximo-distal patterning system in the development of noninsect arthropod limbs.

Regulation of proboscipedia in Drosophila by homeotic selector genes.

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. We have identified several of the genes necessary to generate this embryonic pattern of expression. These genes can be roughly split into three categories based on their time of action during development. First, prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. Second, the initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). Lastly, maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. We further show that Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila melanogaster and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct.

The centrosome in Drosophila oocyte development.

The Drosophila oocyte is a highly specialized cell type whose development utilizes MTOCs in various contexts. Figure 4 (see color insert) summarizes the characteristics of the MTOCs at different stages of oogenesis. Polarized mitoses are required to achieve oocyte determination. In the asymmetric germ-cell divisions that culminate in the egg chamber, the mitotic centrosomes are anchored to the spectrosome or fusome in order to produce the regular branching pattern of the cyst cells. It appears that the primary role of the fusome is to orchestrate the polarity and synchrony of oogenic mitoses. In the absence of fusomes or anchored spindles, the regular interconnected cyst network is lost and the oocyte does not differentiate. It is not known if the spindle itself is asymmetric, or whether either centrosome has equal potential to interact with the fusome. Several models can explain the need for polarized mitoses for oocyte differentiation. In one, an unequal distribution of unknown oocyte differentiation factors occurs from as early as the first cystoblast division. Here, the fusome may be required for the distribution of the factors. In another model, there is a mechanism that measures the number of ring canals in the cell, limiting the choice of oocyte to two potential pro-oocytes. In this model, polarized, synchronous divisions must occur to produce only two cells with the highest number of ring canals. In both of these models the centrosome plays an indirect role. A critical event in the determination of the oocyte is the formation of the MTOC. The oocyte MTOC forms shortly after completion of the germ cell mitoses and establishes a microtubule array along which factors required for oocyte determination are transported. It is unclear how this single MTOC forms in the 16-cell cyst, how the centrosomes become inactivated in the adjoining 15 nurse cells, or why the inactivated centrioles are transported into the oocyte. No molecular components of the MTOC are known except for centrosomin, which accumulates at the MTOC relatively late, at approximately stage 5 or 6 of oogenesis. The MTOC plays a central role in establishing the oocyte's polar coordinates. The oocyte microtubule array is required for the polar localization of axis-determining factors. At midoogenesis the MTOC appears to mediate the reversal of the microtubule array and the migration of the nucleus in the oocyte. The posterior follicle cells signal this reversal after receiving the gurken signal. What changes occur at the MTOC to trigger this cytoskeletal rearrangement? A better understanding of the MTOC's molecular components is necessary before we can begin to unravel the mechanisms underlying these events. The morphology of the MTOC changes after it shifts to the oocyte anterior. Staining with anti-centrosomin antibodies shows that the MTOC changes from discrete nucleus-associated bodies into a broad structure associated with the anterior cortex. The molecular mechanisms underlying this structural rearrangement of the MTOC at midoogenesis are presently unknown. Meiosis I occurs in the absence of centrosomes, but meiosis II spindles are linked by a shared, acentriolar, astral MTOC. The organization of the meiosis I spindle poles requires the NCD motor protein; however, the meiosis I spindle poles are acentriolar and contain no known centrosomal core proteins. The meiosis II astral spindle pole has a unique ring-shaped morphology and contains centrosomal proteins, such as gamma-tubulin. Strong mutations in the maternal gamma Tub37C gene do not block meiosis I, but prevent the progression of meiosis II.

RNAi analysis of Deformed, proboscipedia and Sex combs reduced in the milkweed bug Oncopeltus fasciatus: novel roles for Hox genes in the hemipteran head.

Insects have evolved a large variety of specialized feeding strategies, with a corresponding variability in mouthpart morphology. We have, however, little understanding of the developmental mechanisms that underlie this diversity. Until recently it was difficult to perform any analysis of gene function outside of the genetic model insects Drosophila melanogaster and Tribolium castaneum. In this paper, we report the use of dsRNA-mediated interference (RNAi) to dissect gene function in the development of the milkweed bug Oncopeltus fasciatus, which has specialized suctorial mouthparts. The Hox genes Deformed (Dfd), proboscipedia (pb) and Sex combs reduced (Scr) have previously been shown to be expressed in the gnathal appendages of this species. Strikingly, the milkweed bug was found to have an unusual expression pattern of pb. Here, by analyzing single and combination RNAi depletions, we find that Dfd, pb and Scr are used in the milkweed bug to specify the identity of the mouthparts. The exact roles of the genes, however, are different from what is known in the two genetic model insects. The maxillary appendages in the bug are determined by the activities of the genes Dfd and Scr, rather than Dfd and pb as in the fly and beetle. The mandibular appendages are specified by Dfd, but their unique morphology in Oncopeltus suggests that Dfd's target genes are different. As in flies and beetles, the labium is specified by the combined activities of pb and Scr, but again, the function of pb appears to be different. Additionally, the regulatory control of pb by the other two genes seems to be different in the bug than in either of the other species. These novelties in Hox function, expression pattern and regulatory relationships may have been important for the evolution of the unique Hemipteran head.

Crustacean (malacostracan) Hox genes and the evolution of the arthropod trunk.

Representatives of the Insecta and the Malacostraca (higher crustaceans) have highly derived body plans subdivided into several tagma, groups of segments united by a common function and/or morphology. The tagmatization of segments in the trunk, the part of the body between head and telson, in both lineages is thought to have evolved independently from ancestors with a distinct head but a homonomous, undifferentiated trunk. In the branchiopod crustacean, Artemia franciscana, the trunk Hox genes are expressed in broad overlapping domains suggesting a conserved ancestral state (Averof, M. and Akam, M. (1995) Nature 376, 420-423). In comparison, in insects, the Antennapedia-class genes of the homeotic clusters are more regionally deployed into distinct domains where they serve to control the morphology of the different trunk segments. Thus an originally Artemia-like pattern of homeotic gene expression has apparently been modified in the insect lineage associated with and perhaps facilitating the observed pattern of tagmatization. Since insects are the only arthropods with a derived trunk tagmosis tested to date, we examined the expression patterns of the Hox genes Antp, Ubx and abd-A in the malacostracan crustacean Porcellio scaber (Oniscidae, Isopoda). We found that, unlike the pattern seen in Artemia, these genes are expressed in well-defined discrete domains coinciding with tagmatic boundaries which are distinct from those of the insects. Our observations suggest that, during the independent tagmatization in insects and malacostracan crustaceans, the homologous 'trunk' genes evolved to perform different developmental functions. We also propose that, in each lineage, the changes in Hox gene expression pattern may have been important in trunk tagmatization.

Evolution of distinct expression patterns for engrailed paralogues in higher crustaceans (Malacostraca).

The segment-polarity gene engrailed of Drosophila melanogaster and its homologues in other arthropods possess a highly conserved expression domain in the posterior portion of each segment. We report here that the two pan-specific antibodies, Mab4D9 and Mab4F11, reveal strikingly different accumulation patterns in both of the malacostracan crustaceans Porcellio scaber (Isopoda) and Procambarus clarkii (Decapoda), compared with insects. The signal detected with Mab4D9 resides in the posterior part of each segment, including the appendages, the ventral and lateral sides of the trunk and the CNS. However, Mab4F11 reveals a signal only in small groups of neurons in the CNS and PNS, primarily localized in the pereon. We observe similar Mab4D9 and Mab4F11 patterns in the crayfish P. clarkii, except that no Mab4F11 signal is detected in the pleon. To address the possibility of multiple engrailed paralogues, we cloned partial cDNAs of two engrailed genes, Ps-en1 and Ps-en2, from P. scaber, and studied their expression patterns using whole-mount in situ hybridization. Although the Ps-en1 and Ps-en2 patterns are comparable in early development, they become distinct in late embryogenesis. Ps-en1 is expressed in the CNS, where Mab4F11 stains, but also accumulates in the epidermis. In contrast, Ps-en2 is expressed in the lateral aspect and limbs of all segments. Phylogenetic analysis of en sequences from crustaceans and insects suggests that the two en genes from the apterygote insect Thermobia domestica (Thysanura) may be related to en1 and en2 of higher crustaceans.

Characterization of the Hox cluster from the mosquito Anopheles gambiae (Diptera: Culicidae).

The Hox genes have been found to encode transcription factors, which specify the morphological identity of structures along the anteroposterior axis of animals ranging from worms to mice. The canonical set of nine genes is organized in a cluster in the genome of several protostomes and deuterostomes. However, within insects, whereas the Hox genes are organized in a single cluster in the beetle Tribolium castaneum, they are split into two separate groups in the flies Drosophila melanogaster and Drosophila virilis. The significance of a split Hox cluster is unknown and has been observed in only one organism outside the Drosophila lineage: the nematode Caenorhabditis elegans. We have cloned a majority of the Hox genes from the mosquito Anopheles gambiae (Diptera: Culicidae) and compared their genomic organization with that of Tribolium and Drosophila to determine if a split Hox cluster is found in dipterans aside from the Drosophilidae. We find that the Hox genes in Anopheles, as in Tribolium, are organized in a single cluster that spans a genomic region of at least 700 kb. This finding suggests that, within the insect genome, the partition of the Hox cluster may have evolved exclusively within the Drosophila lineage. The genomic structures of the resident genes, however, appear to be largely conserved between A. gambiae and D. melanogaster.

Embryonic expression patterns of the Hox genes of the crayfish Procambarus clarkii (Crustacea, Decapoda).

Higher crustaceans (class Malacostraca) represent the most species-rich and morphologically diverse group of non-insect arthropods. The superorders Eucarida and Peracarida, two large groups that separated over 350 million years ago, encompass most malacostracan diversity. Recently, the Hox genes of the peracarid woodlouse Porcellio scaber(Isopoda) were shown to be expressed in domains that coincide with morphological boundaries of body tagmata, which differ from those in insects (Abzhanov and Kaufman 1999a,b). Moreover, observed changes in Hox expression domains during ontogeny correlate with morphological remodeling, such as a transformation of the first thoracic leg into mouthpart maxillipeds, which occurs in the trunk of the embryo. Decapods have a different modification of the malacostracan bodyplan, with up to three pairs of maxillipeds and extensive fusion and cephalization of the thorax. Here we describe expression patterns of the trunk Hox genes Scr, Antp, Ubx, abd-A and cad in the eucarid crayfish Procambarus clarkii (Decapoda). We find that the crayfish expression patterns, for the most part, resemble those of the woodlouse Porcellio scaber(Isopoda), but are more modulated and complex. Nevertheless, as in Porcellio the boundaries of the Hox expression domains do correlate with morphological features and their modulations to transformations in the embryo. Thus we propose that the trunk Hox genes were likely important in the evolution of and currently play an essential role in the development of the complex decapod bodyplan.

Homeotic genes and the arthropod head: expression patterns of the labial, proboscipedia, and Deformed genes in crustaceans and insects.

cDNA fragments of the homologues of the Drosophila head homeotic genes labial (lab), proboscipedia (pb), and Deformed (Dfd) have been isolated from the crustacean Porcellio scaber. Because the accumulation domains of the head homeotic complex (Hox) genes had not been previously reported for crustaceans, we studied the expression patterns of these genes in P. scaber embryos by using in situ hybridization. The P. scaber lab homologue is expressed in the developing second antennal segment and its appendages. This expression domain in crustaceans and in the homologous intercalary segment of insects suggests that the lab gene specified this metamere in the last common ancestor of these two groups. The expression domain of the P. scaber pb gene is in the posterior part of the second antennal segment. This domain, in contrast to that in insects, is colinear with the domains of other head genes in P. scaber, and it differs from the insect pb gene expression domain in the posterior mouthparts, suggesting that the insect and crustacean patterns evolved independently from a broader ancestral domain similar to that found in modern chelicerates. P. scaber Dfd is expressed in the mandibular segment and paragnaths (a pair of ventral mouthpart structures associated with the stomodeum) and differs from insects, where expression is in the mandibular and maxillary segments. Thus, like pb, Dfd shows a divergent Hox gene deployment. We conclude that homologous structures of the mandibulate head display striking differences in their underlying developmental programs related to Hox gene expression.

The centrosomin protein is required for centrosome assembly and function during cleavage in Drosophila.

Centrosomin is a 150 kDa centrosomal protein of Drosophila melanogaster. To study the function of Centrosomin in the centrosome, we have recovered mutations that are viable but male and female sterile (cnnmfs). We have shown that these alleles (1, 2, 3, 7, 8 and hk21) induce a maternal effect on early embryogenesis and result in the accumulation of low or undetectable levels of Centrosomin in the centrosomes of cleavage stage embryos. Hemizygous cnn females produce embryos that show dramatic defects in chromosome segregation and spindle organization during the syncytial cleavage divisions. In these embryos the syncytial divisions proceed as far as the twelfth cycle, and embryos fail to cellularize. Aberrant divisions and nuclear fusions occur in the early cycles of the nuclear divisions, and become more prominent at later stages. Giant nuclei are seen in late stage embryos. The spindles that form in mutant embryos exhibit multiple anomalies. There is a high occurrence of apparently linked spindles that share poles, indicating that Centrosomin is required for the proper spacing and separation of mitotic spindles within the syncytium. Spindle poles in the mutants contain little or no detectable amounts of the centrosomal proteins CP60, CP190 and (gamma)-tubulin and late stage embryos often do not have astral microtubules at their spindle poles. Spindle morphology and centrosomal composition suggest that the primary cause of these division defects in mutant embryos is centrosomal malfunction. These results suggest that Centrosomin is required for the assembly and function of centrosomes during the syncytial cleavage divisions.

Novel regulation of the homeotic gene Scr associated with a crustacean leg-to-maxilliped appendage transformation.

Homeotic genes are known to be involved in patterning morphological structures along the antero-posterior axis of insects and vertebrates. Because of their important roles in development, changes in the function and expression patterns of homeotic genes may have played a major role in the evolution of different body plans. For example, it has been proposed that during the evolution of several crustacean lineages, changes in the expression patterns of the homeotic genes Ultrabithorax and abdominal-A have played a role in transformation of the anterior thoracic appendages into mouthparts termed maxillipeds. This homeotic-like transformation is recapitulated at the late stages of the direct embryonic development of the crustacean Porcellio scaber (Oniscidea, Isopoda). Interestingly, this morphological change is associated with apparent novelties both in the transcriptional and post-transcriptional regulation of the Porcellio scaber ortholog of the Drosophila homeotic gene, Sex combs reduced (Scr). Specifically, we find that Scr mRNA is present in the second maxillary segment and the first pair of thoracic legs (T1) in early embryos, whereas protein accumulates only in the second maxillae. In later stages, however, high levels of SCR appear in the T1 legs, which correlates temporally with the transformation of these appendages into maxillipeds. Our observations provide further insight into the process of the homeotic leg-to-maxilliped transformation in the evolution of crustaceans and suggest a novel regulatory mechanism for this process in this group of arthropods.

The embryonic expression pattern of labial, posterior homeotic complex genes and the teashirt homologue in an apterygote insect.

During embryogenesis of the fruit fly, Drosophila melanogaster, the homeotic genes are required to specify proper cell fates along the anterior-posterior axis of the embryo. We cloned partial cDNAs of homologues of the Drosophila homeotic gene teashirt and five of the homeotic-complex (HOM-C) genes from the thysanuran insect, Thermobia domestica, and assayed their embryonic expression patterns. The HOM-C genes we examined were labial, Antennapedia, Ultrabithorax, abdominal-A and Abdominal-B. As the expression pattern of these HOM-C genes is largely conserved among insects and as Thermobia is a member of a phylogenetically basal order of insects, we were able to infer their ancestral expression patterns in insects. We compare the expression patterns of the Thermobia HOM-C genes with their expression in Drosophila and other insects and discuss the potential roles these genes may have played in insect evolution. Interestingly, the teashirt homologue shows greater variability between Thermobia and Drosophila than any of the HOM-C genes. In particular, teashirt is not expressed strongly in the Thermobia abdomen, unlike Drosophila teashirt. We propose that teashirt expression has expanded posteriorly in Drosophila and contributed to a homogenization of the Drosophila larval thorax and abdomen.

Chelicerate Hox genes and the homology of arthropod segments.

Genes of the homeotic complex (HOM-C) in insects and vertebrates are required for the specification of segments along the antero-posterior axis. Multiple paralogues of the Hox genes in the horseshoe crab Limulus poliphemus have been used as evidence for HOM-C duplications in the Chelicerata. We addressed this possibility through a limited PCR survey to sample the homeoboxes of two spider species, Steatoda triangulosa and Achaearanea tepidariorum. The survey did not provide evidence for multiple Hox clusters although we have found apparent duplicate copies of proboscipedia (pb) and Deformed (Dfd). In addition, we have cloned larger cDNA fragments of pb, zerknullt (zen/Hox3) and Dfd. These fragments allowed the determination of mRNA distribution by in situ hybridization. Our results are similar to the previously published expression patterns of Hox genes from another spider and an oribatid mite. Previous studies compared spider/mite Hox gene expression patterns with those of insects and argued for a pattern of segmental homology based on the assumption that the co-linear anterior boundaries of the Hox domains can be used as markers. To test this assumption we performed a comparative analysis of the expression patterns for UBX/ABD-A in chelicerates, myriapods, crustaceans, and insects. We conclude that the anterior boundary can be and is changed considerably during arthropod evolution and, therefore, Hox expression patterns should not be used as the sole criterion for identifying homology in different classes of arthropods.