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

SUMMARY 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.     

Differential Delta expression underlies the diversity of sensory organ patterns among the legs of the Drosophila adult

Many studies have shown that morphological diversity among homologous animal structures is generated by the homeotic (Hox) genes. However, the mechanisms through which Hox genes specify particular morphological features are not fully understood. We have addressed this issue by investigating how diverse sensory organ patterns are formed among the legs of the Drosophila melanogaster adult. The Drosophila adult has one pair of legs on each of its three thoracic segments (the T1-T3 segments). Although homologous, legs from different segments have distinct morphological features. Our focus is on the formation of diverse patterns of small mechanosensory bristles or microchaetae (mCs) among the legs. On T2 legs, the mCs are organized into a series of longitudinal rows (L-rows) precisely positioned along the leg circumference. The L-rows are observed on all three pairs of legs, but additional and novel pattern elements are found on T1 and T3 legs. For example, at specific positions on T1 and T3 legs, some mCs are organized into transverse rows (T-rows). Our studies indicate that the T-rows on T1 and T3 legs are established as a result of Hox gene modulation of the pathway for patterning the L-row mC bristles. Our findings suggest that the Hox genes, Sex combs reduced (Scr) and Ultrabithorax (Ubx), establish differential expression of the proneural gene achaete (ac) by modifying expression of the ac prepattern regulator, Delta (Dl), in T1 and T3 legs, respectively. This study identifies Dl as a potential link between Hox genes and the sensory organ patterning hierarchy, providing insight into the connection between Hox gene function and the formation of specific morphological features.     

Hox genes and the crustacean body plan

The Crustacea present a variety of body plans not encountered in any other class or phylum of the Metazoa. Here we review our current knowledge on the complement and expression of the Hox genes in Crustacea, addressing questions related to the evolution of body architecture. Specifically, we discuss the molecular mechanisms underlying the homeotic transformation of legs into feeding appendages, which occurred in parallel in several branches of the crustacean evolutionary tree. A second issue that can be approached by the comparative study of Hox genes and their expression in the Crustacea bears on the homology of the abdomen. We discuss whether the so-called "abdominal" tagma of the crustaceans is homologous to the abdomen of insects. In addition, the homology of the abdomen between malacostracan and non-malacostracan crustaceans has also been questioned. We also address the question of the molecular developmental basis of the apparent lack of an abdomen in barnacles. We discuss these issues in relation to the problem of constraint versus adaptation in evolution.     

Hox and wings

In many bilaterian phyla, appendages are morphological traits that characterise the identity of the various body parts. In pterygote insects, wings are dorsal appendages on the thorax. The famous "bithorax" fly created by Ed Lewis is the emblematic example of the role of Hox genes.1 Now, Tomoyasu et al.,2 using classical genetics, transgenesis and RNAi, have examined the function of thoracic Hox genes in the beetle Tribolium castaneum. Beetles have rigid elytra in place of the first pair of wings. Instead of the expected transformation of the elytron into a wing, loss of Hox genes' function leads to the homeotic transformation of the second pair of dorsal appendages, the wings, into elytra. This has important consequences for the way that we see the role of Hox genes in development and evolution.     

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.     

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.     

Ectopic gene expression and homeotic transformations in arthropods using recombinant Sindbis viruses

BACKGROUND: The morphological diversity of arthropods makes them attractive subjects for studying the evolution of developmental mechanisms. Comparative analyses suggest that arthropod diversity has arisen largely as a result of changes in expression patterns of genes that control development. Direct analysis of how a particular gene functions in a given species during development is hindered by the lack of broadly applicable techniques for manipulating gene expression. RESULTS: We report that the Arbovirus Sindbis can be used to deliver high levels of gene expression in vivo in a number of non-host arthropod species without causing cytopathic effects in infected cells or impairing development. Using recombinant Sindbis virus, we investigated the function of the homeotic gene Ultrabithorax in the development of butterfly wings and beetle embryos. Ectopic Ultrabithorax expression in butterfly forewing imaginal discs was sufficient to cause the transformation of characteristic forewing properties in the adult, including scale morphology and pigmentation, to those of the hindwing. Expression of Ultrabithorax in beetle embryos outside of its endogenous expression domain affected normal development of the body wall cuticle and appendages. CONCLUSIONS: The homeotic genes have long been thought to play an important role in the diversification of arthropod appendages. Using recombinant Sindbis virus, we were able to investigate homeotic gene function in non-model arthropod species. We found that Ultrabithorax is sufficient to confer hindwing identity in butterflies and alter normal development of anterior structures in beetles. Recombinant Sindbis virus has broad potential as a tool for analyzing how the function of developmental genes has changed during the diversification of arthropods.     

Expression patterns of the homeotic genes Scr, Antp, Ubx, and abd-A during embryogenesis of the cricket Gryllus bimaculatus

We have studied embryogenesis of the two-spotted cricket Gryllus bimaculatus as an example of a hemimetabolous, intermediate germ insect, which is a phylogenetically basal insect and may retain primitive features. We observed expression patterns of the orthologs of the Drosophila homeotic genes, Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx) and abdominal-A (abd-A) during embryogenesis and compared the expression patterns of these genes with the more basal thysanuran insect, Thermobia domestica (the firebrat), and the derived higher dipteran insect, Drosophila melanogaster. Although Scr is expressed commonly in the presumptive posterior maxillary and labial segment in all three insects, the thoracic expression domains vary. Antp is expressed similarly in the three thoracic segments, the limbs, and the anterior abdominal region among these three insects. The early Antp expression in the firebrat and cricket obeys a segmental register in all three thoracic segments, while in Drosophila its initial expression appears in parasegments 4 and 6. Ubx is expressed in the metathoracic (T3) and abdominal segments similarly in the three insects, whereas the expression pattern in the T3 leg differs among them. abd-A is expressed in the posterior compartment of the first abdominal segment and the remaining abdominal segments in all three insects, although its posterior border varies among them.     

Ectopic expression of homeotic genes caused by the elimination of the Polycomb gene in Drosophila imaginal epidermis

The morphological patterns in the adult cuticle of Drosophila are determined principally by the homeotic genes of the bithorax and Antennapedia complexes. We find that many of these genes become indiscriminately active in the adult epidermis when the Pc gene is eliminated. By using the Pc3 mutation and various BX-C mutant combinations, we have generated clones of imaginal cells possessing different combinations of active homeotic genes. We find that, in the absence of BX-C genes, Pc- clones develop prothoracic patterns; this is probably due to the activity of Sex combs reduced which overrules Antennapedia. Adding contributions of Ultrabithorax, abdominal-A and Abdominal-B results in thoracic or abdominal patterns. We have established a hierarchical order among these genes: Antp less than Scr less than Ubx less than abd-A less than Abd-B. In addition, we show that the engrailed gene is ectopically active in Pc- imaginal cells.     

Direct control of antennal identity by the spineless-aristapedia gene of Drosophila

Loss-of-function mutations in the spineless-aristapedia gene of Drosophila (ssa mutants) cause transformations of the distal antenna to distal second leg, deletions or fusions of the tarsi from all three legs, a general reduction in bristle size, and sterility. Because ssa mutants are pleiotropic, it has been suggested that ss+ has some rather general function and that the ssa antennal transformation is an indirect consequence of perturbations in the expression of other genes that more directly control antennal or second leg identity. Here we test whether the ssa transformation results from aberrant expression of Antennapedia (Antp), a homeotic gene thought to specify directly the identity of the second thoracic segment. We find that Antp-ssa mitotic recombination clones in the distal antenna behave identically to Antp+ ssa clones, and are transformed to second leg. This demonstrates that the ssa antennal transformation is independent of Antp+, and suggests that ss+ may itself directly define distal antennal identity. The results also reveal that Antp+ is not required for the development of distal second leg structures, as these develop apparently normally in Antp- ssa antennal clones. Because Antp- mutations cause deletions or transformations that are restricted to proximal structures, whereas ssa alleles cause similar defects that are distally restricted, we suggest that ss+ and Antp+ may play similar, but complementary, roles in the distal and proximal portions of appendages, respectively.     

Larval legs of mulberry silkworm Bombyx mori are prototypes for the adult legs

Morphological diversity of leg appendages is one of the hallmarks of developmental evolution. Limbs in insects may develop either from their embryonic prototypes or from imaginal discs harbored inside the larva. Bombyx mori (B. mori), a Lepidopteran insect, develops adult wings from larval wing imaginal discs. However, it has been debated whether the adult legs of B. mori arise from imaginal discs or from the larval legs. Here we addressed how the larval legs relate to their adult counterparts. We present the morphological landmarks during early leg development. We used expression of developmental genes like Distalless and extradenticle to mark leg primordia. Finally, we employed classical excision approach to develop a fate map of the adult leg. Excision and ablation of thoracic legs along proximo-distal axis at various times during larval development resulted in the loss of corresponding adult leg segments. Our data suggest that B. mori legs develop from larval appendages rather than leg imaginal discs.     

Ultrastructural study of yolk formation in Porcellio scaber Latr. (Isopoda)

Two types of yolk are formed in the developing oocytes of a terrestrial crustacean, Porcellio scaber. The intra-oocytic yolk, arises through autosynthesis, and the extra-oocytic yolk, is derived from micropinocytosis. the so-called disc shaped bodies, which occur in large numbers within the cisternae of a branched system of endoplasmic reticulum, are precursors of the intra-oocytic uolk. Dictyosomes are not involved in yolk formation in this species. Thus, the vitellogenesis of Porcellio scaber occurs in a manner analogues to that described in the aquatic crustaceans, which indicates that environmental factors have relatively little effect on this process.     

Tissue strategies as developmental constraints: Implications for animal evolution

A consideration of developmental constraints at the tissue level brings into focus the relationship between genes, cell behavior and morphological evolution. This common framework provides a rationale for phenomena as seemingly divergent as the lack of homeotic appendages in humans and the Cambrian explosion.     

Temporal and spatial expression of homeotic genes is important for segment-specific neuroblast 6-4 lineage formation in Drosophila

Different proliferation of neuroblast 6-4 (NB6-4) in the thorax and abdomen produces segmental specific expression pattern of several neuroblast marker genes. NB6-4 is divided to form four medialmost cell body glia (MM-CBG) per segment in thorax and two MM-CBG per segment in abdomen. As homeotic genes determine the identities of embryonic segments along theA/P axis, we investigated if temporal and specific expression of homeotic genes affects MM-CBG patterns in thorax and abdomen. A Ubx loss-of-function mutation was found to hardly affect MM-CBG formation, whereas abd-A and Abd-B caused the transformation of abdominal MM-CBG to their thoracic counterparts. On the other hand, gain-of-function mutants of Ubx, abd-A and Abd-B genes reduced the number of thoracic MM-CBG, indicating that thoracic MM-CBG resembled abdominal MM-CBG. However, mutations in Polycomb group (PcG) genes, which are negative transregulators of homeotic genes, did not cause the thoracic to abdominal MM-CBG pattern transformation although the number of MM-CBG in a few per-cent of embryos were partially reduced or abnormally patterned. Our results indicate that temporal and spa-tial expression of the homeotic genes is important to determine segmental-specificity of NB6-4 daughter cells along the anterior-posterior (A/P) axis.     

Homeotic genes have specific functional roles in the establishment of the Drosophila embryonic peripheral nervous system.

The Drosophila embryonic peripheral nervous system (PNS) contains segment-specific spatial patterns of sensory organs which derive from the ectoderm. Many studies have established that the homeotic genes of Drosophila control segment specific characteristics of the epidermis, and more recently these genes have also been shown to control gut morphogenesis through their expression in the visceral mesoderm (Tremml, G. and Bienz, M. (1989), EMBO J. 8, 2677-2685). We report here the roles of homeotic genes in establishing the spatial patterns of sensory organs in the embryonic PNS. The PNS was examined in embryos homozygous for mutations in the homeotic genes Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B) with antibodies that label specific subsets of sensory organs. Our results suggest that the homeotic genes have specific roles in establishing the correct spatial patterns of sensory organs in their normal domains of expression. In addition, we also report the effects of ectopic expression of the homeotic genes labial (lab), Deformed (Dfd), Scr, Antp or Ubx on the normal development of sensory organs in the embryonic PNS. Interestingly, while previous studies have concluded that ectopic expression of the homeotic genes Dfd, Scr and Antp has no effect on the segmental identity of the abdominal segments, our results demonstrate that this is not true. We show that ectopic expression of these genes does result in the disruption of the developing PNS in the abdomen. Our results are suggestive of a role for the homeotic gene products in regulating genes which are necessary for generating sensory progenitor cells in the developing PNS.     

Projection of sensory neurons from a homeotic mutant appendage,Antennapedia, in Drosophila melanogaster

Central projections of sensory neurons from homeotic mutant appendages (Antennapedia) of Drosophila melanogaster were compared with those of wild-type antennae and wild-type legs by means of degeneration and cobalt backfilling methods. Sensory axons originating from wild-type thoracic legs terminate within the ventral ipsilateral half of the corresponding neuropile segment and do not project to the brain. Sensory fibers from the third antennal segment (AIII) of wild-type animals project into the ipsilateral antennal glomerulus (AG) and to a lesser extent into the contralateral AG, whereas those from the second antennal segment terminate principally within the ipsilateral posterior antennal center. The sensory terminals of femur, tibia, and tarsi of the homeotic leg show a distribution very similar to that of the homologous wild-type antennal segment AIII, differing to a minor degree only in the size and precise localization of terminals within the antennal glomeruli. No degenerating axons were evident in ultrastructural examination of neck connectives after removal of homeotic legs. It is thus very improbable that any sensory fibers of the homeotic leg project to normal leg projection areas in the thoracico-abdominal ganglion. Several alternative explanations are offered for the apparent retention of antennal specificity by axons from the transformed appendage.     

Molecular genetics of crustacean feeding appendage development and diversification

Arthropods dominate our seas, land, and air and have done so for hundreds of millions of years. Among the arthropods, crustaceans present us with a rich history of morphological change, much of which is still represented among extant forms. Crustacea largely interact with their environment via their appendages; thus vast amounts of variation exist among the different appendages of a single individual and between appendages from different species. Comparative studies of crustacean appendage development present us with an important story regarding the evolution of morphology over both relatively short (a few million years) and relatively long (a few hundred million years) evolutionary time scales. Recent studies have used the genetic and molecular data from Drosophila development to try to understand the molecular basis for some of the variations seen in crustacean limbs. Here we review some of these data based on the expression patterns of the genes Ultrabithorax, abdominal - A, Sex combs reduced, and Distal-less.