HOM/Hox genes of Artemia: implications for the origin of insect and crustacean body plans

BACKGROUND: Insects and crustaceans are generally assumed to derive from a segmented common ancestor that had a distinct head but uniform, undifferentiated trunk segments. The subdivision of the body into functionally distinct regions (e.g. thorax and abdomen) is thought to have evolved independently in these two lineages. In insects, the differences between segments in the trunk are controlled by the Antennapedia-like genes of the homeotic gene clusters. Study of these genes in crustaceans should provide a basis for comparing body plans and assessing their evolutionary origin. RESULTS: Using a polymerase chain reaction (PCR) / inverse PCR strategy, we have isolated six genes of the HOM/Hox family from the crustacean Artemia franciscana. Five of these are clearly identifiable as specific homologues of the insect homeotic genes Dfd, Scr, Antp, Ubx and abdA. The sixth appears to have no close counterpart in insects. CONCLUSION: All the homeotic genes that specify middle body regions in insects originated before the divergence of the insect and crustacean lineages, probably not later than the Cambrian (about 500 million years ago). A commonly derived groundplan may underlie segment diversity in these two groups.     

Knockdown of spalt function by RNAi causes de-repression of Hox genes and homeotic transformations in the crustacean Artemia franciscana

Hox genes play a central role in the specification of distinct segmental identities in the body of arthropods. The specificity of Hox genes depends on their restricted expression domains, their interaction with specific cofactors and selectivity for particular target genes. spalt genes are associated with the function of Hox genes in diverse species, but the nature of this association varies: in some cases, spalt collaborates with Hox genes to specify segmental identities, in others, it regulates Hox gene expression or acts as their target. Here we study the role of spalt in the branchiopod crustacean Artemia franciscana. We find that Artemia spalt is expressed in the pre-segmental 'growth zone' and in stripes in each of the trunk (thoracic, genital and post-genital) segments that emerge from this zone. Using RNA interference (RNAi), we show that knocking down the expression of spalt has pleiotropic effects, which include thoracic to genital (T-->G), genital to thoracic (G-->T) and post-genital to thoracic (PG-->T) homeotic transformations. These transformations are associated with a stochastic de-repression of Hox genes in the corresponding segments of RNAi-treated animals (AbdB for T-->G and Ubx/AbdA for G-->T and PG-->T transformations). We discuss a possible role of spalt in the maintenance of Hox gene repression in Artemia and in other animals.     

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.     

Expression of a homologue of the fushi tarazu (ftz) gene in a cirripede crustacean

In Metazoa, Hox genes control the identity of the body parts along the anteroposterior axis. In addition to this homeotic function, these genes are characterized by two conserved features: They are clustered in the genome, and they contain a particular sequence, the homeobox, encoding a DNA-binding domain. Analysis of Hox homeobox sequences suggests that the Hox cluster emerged early in Metazoa and then underwent gene duplication events. In arthropods, the Hox cluster contains eight genes with a homeotic function and two other Hox-like genes, zerknullt (zen)/Hox3 and fushi tarazu (ftz). In insects, these two genes have lost their homeotic function but have acquired new functions in embryogenesis. In contrast, in chelicerates, these genes are expressed in a Hox-like pattern, which suggests that they have conserved their ancestral homeotic function. We describe here the characterization of Diva, the homologue of ftz in the cirripede crustacean Sacculina carcini. Diva is located in the Hox cluster, in the same position as the ftz genes of insects, and is not expressed in a Hox-like pattern. Instead, it is expressed exclusively in the central nervous system. Such a neurogenic expression of ftz has been also described in insects. This study, which provides the first information about the Hoxcluster in Crustacea, reveals that it may not be much smaller than the insect cluster. Study of the Diva expression pattern suggests that the arthropod ftz gene has lost its ancestral homeotic function after the divergence of the Crustacea/Hexapoda clade from other arthropod clades. In contrast, the function of ftz during neurogenesis is well conserved in insects and crustaceans.     

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.     

Posterior patterning genes and the identification of a unique body region in the brine shrimp Artemia franciscana

All arthropods share the same basic set of Hox genes, although the expression of these genes differs among divergent groups. In the brine shrimp Artemia franciscana, their expression is limited to the head, thoracic/trunk and genital segments, but is excluded from more posterior parts of the body which consist of six post-genital segments and the telson (bearing the anus). Nothing is currently known about the genes that specify the identity of these posterior structures. We examine the expression patterns of four candidate genes, Abdominal-B, caudal/Cdx, even-skipped/Evx and spalt, the homologues of which are known to play an important role in the specification of posterior structures in other animals. Abdominal-B is expressed in the genital segments of Artemia, but not in the post-genital segments at any developmental stage. The expression of caudal, even-skipped and spalt in the larval growth-zone suggests they may play a role in the generation of body segments (perhaps comparable with the role of gap and segmentation genes in insects), but not a direct role in defining the identity of post-genital segments. The expression of caudal at later stages suggests a role in the specification of anal structures. A PCR screen designed to isolate Hox genes expressed specifically in the posterior part of the body failed to identify any new Hox genes. We conclude that the post-genital segments of Artemia are not defined by any of the genes known to play a role in the specification of posterior segments in other arthropods. We argue that these segments constitute a unique body region that bears no obvious homology to previously characterised domains of Hox gene activity.     

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.     

Expression of hunchback during trunk segmentation in the branchiopod crustacean Artemia franciscana

Comparative studies have shown that some aspects of segmentation are widely conserved among arthropods. Yet, it is still unclear whether the molecular prepatterns that are required for segmentation in Drosophila are likely to be similarly conserved in other arthropod groups. Homologues of the Drosophila gap genes, like hunchback, show regionally restricted expression patterns during the early phases of segmentation in diverse insects, but their expression patterns in other arthropod groups are not yet known. Here, we report the cloning of a hunchback orthologue from the crustacean Artemia franciscana and its expression during the formation of trunk segments. Artemia hunchback is expressed in a series of segmental stripes that correspond to individual thoracic/trunk, genital, and postgenital segments. However, this expression is not associated with the segmenting ectoderm but is restricted to mesodermal cells that associate with the ectoderm in a regular metameric pattern. All cells in the early segmental mesoderm appear to express hunchback. Later, mesodermal expression fades, and a complex expression pattern appears in the central nervous system (CNS), which is comparable to hunchback expression in the CNS of insects. No regionally restricted expression, reminiscent of gap gene expression, is observed during trunk segmentation. These patterns suggest that the expression patterns of hunchback in the mesoderm and in the CNS are likely to be ancient and conserved among crustaceans and insects. In contrast, we find no evidence for a conserved role of hunchback in axial patterning in the trunk ectoderm.     

Drosophila fushi tarazu. a gene on the border of homeotic function

BACKGROUND: Hox genes specify cell fate and regional identity during animal development. These genes are present in evolutionarily conserved clusters thought to have arisen by gene duplication and divergence. Most members of the Drosophila Hox complex (HOM-C) have homeotic functions. However, a small number of HOM-C genes, such as the segmentation gene fushi tarazu (ftz), have nonhomeotic functions. If these genes arose from a homeotic ancestor, their functional properties must have changed significantly during the evolution of modern Drosophila. RESULTS: Here, we have asked how Drosophila ftz evolved from an ancestral homeotic gene to obtain a novel function in segmentation. We expressed Ftz proteins at various developmental stages to assess their potential to regulate segmentation and to generate homeotic transformations. Drosophila Ftz protein has lost the inherent ability to mediate homeosis and functions exclusively in segmentation pathways. In contrast, Ftz from the primitive insect Tribolium (Tc-Ftz) has retained homeotic potential, generating homeotic transformations in larvae and adults and retaining the ability to repress homothorax, a hallmark of homeotic genes. Similarly, Schistocerca Ftz (Sg-Ftz) caused homeotic transformations of antenna toward leg. Primitive Ftz orthologs have moderate segmentation potential, reflected by weak interactions with the segmentation-specific cofactor Ftz-F1. Thus, Ftz orthologs represent evolutionary intermediates that have weak segmentation potential but retain the ability to act as homeotic genes. CONCLUSIONS: ftz evolved from an ancestral homeotic gene as a result of changes in both regulation of expression and specific alterations in the protein-coding region. Studies of ftz orthologs from primitive insects have provided a "snap-shot" view of the progressive evolution of a Hox protein as it took on segmentation function and lost homeotic potential. We propose that the specialization of Drosophila Ftz for segmentation resulted from loss and gain of specific domains that mediate interactions with distinct cofactors.     

Trilobite body patterning and the evolution of arthropod tagmosis

Preservation permitting patterns of developmental evolution can be reconstructed within long extinct clades, and the rich fossil record of trilobite ontogeny and phylogeny provides an unparalleled opportunity for doing so. Furthermore, knowledge of Hox gene expression patterns among living arthropods permit inferences about possible Hox gene deployment in trilobites. The trilobite anteroposterior body plan is consistent with recent suggestions that basal euarthropods had a relatively low degree of tagmosis among cephalic limbs, possibly related to overlapping expression domains of cephalic Hox genes. Trilobite trunk segments appeared sequentially at a subterminal generative zone, and were exchanged between regions of fused and freely articulating segments during growth. Homonomous trunk segment shape and gradual size transition were apparently phylogenetically basal conditions and suggest a single trunk tagma. Several derived clades independently evolved functionally distinct tagmata within the trunk, apparently exchanging flexible segment numbers for greater regionally autonomy. The trilobite trunk chronicles how different aspects of arthropod segmentation coevolved as the degree of tagmosis increased.     

Neurogenesis in the crustacean ventral nerve cord: homology of neuronal stem cells in Malacostraca and Branchiopoda?

SUMMARY In Insecta and malacostracan Crustacea, neurons in the ventral ganglia are generated by the unequal division of neuronal stem cells, the neuroblasts (Nbs), which are arranged in a stereotyped, grid‐like pattern. In malacostracans, however, Nbs originate from ectoteloblasts by an invariant lineage, whereas Nbs in insects differentiate without a defined lineage by cell‐to‐cell interactions within the neuroectoderm. As the ventral ganglia in entomostracan crustaceans were thought to be generated by a general inward proliferation of ectodermal cells, the question arose as to whether neuroblasts in Euarthropoda represent a homologous type of stem cell. In the current project, neurogenesis in metanauplii of the entomostracan crustaceans Triops cancriformis Fabricius, 1780 (Branchiopoda, Phyllopoda) and Artemia salina Linné, 1758 (Branchiopoda, Anostraca) was examined by in vivo incorporation of the mitosis marker bromodeoxyuridine (BrdU) and compared to stem cell proliferation in embryos of the malacostracan Palaemonetes argentinus Nobili, 1901 (Eucarida, Decapoda). The developmental expression of synaptic proteins (synapsins) was studied immunohistochemically. Results indicate that in the ventral neurogenic zone of Branchiopoda, neuronal stem cells with cellular characteristics of malacostracan neuroblasts are present. However, a pattern similar to the lineage‐dependent, grid‐like arrangement of the malacostracan neuroblasts was not found. Therefore, the homology of entomostracan and malacostracan neuronal stem cells remains uncertain. It is now well established that during arthropod development, identical and most likely homologous structures can emerge, although the initiating steps or the mode of generation of these structures are different. Recent evidence suggests that adult Entomostraca and Malacostraca share corresponding sets of neurons so that the present report provides an example that those homologous neurons may be generated via divergent developmental pathways. In this perspective, it remains difficult at this point to discuss the question of common patterns of stem cell proliferation with regard to the phylogeny and evolution of Atelocerata and Crustacea.     

Conservation of a functional hierarchy between mammalian and insect Hox/HOM genes.

We have generated several transgenic Drosophila strains containing different mouse Hox genes under heat shock control and studied how their generalized expression affects Drosophila larval patterns. We find that they have spatially restricted effects which correlate with their genetic order and expression pattern in the mouse; as they are expressed more posteriorly in the mouse, they have more extensive effects in Drosophila. The generalized expressions of Hoxd-8 and d-9 modify Drosophila anterior head segment(s), but have no effect in the rest of the body. Hoxd-10 expression affects head and thorax, but not the abdomen. Finally, Hoxd-11 alters head, thorax not the abdomen. Finally, Hoxd-11 alters head, thorax and abdomen. The developmental effect of the Hox genes consists of a homeotic transformation of the affected segment(s), which exhibit a 'ground' pattern similar to that obtained in the absence of homeotic information, suggesting that Hox genes are able to inactivate Drosophila homeotic genes, but do not specify a pattern of their own. A partial exception is Hoxd-11 which, even though it has a general suppressing effect, can also activate the resident Abdominal-B and empty spiracles genes in ectopic positions. Our results strongly suggest a general conservation of the functional hierarchy of homeotic genes that correlates with genetic order and expression patterns.     

Expression patterns of the rogue Hox genes Hox3/zen and fushi tarazu in the apterygote insect Thermobia domestica

Many embryonic patterning genes are remarkably conserved between vertebrates and invertebrates, and the Hox genes are paradigmatic examples of this conservation. Yet even Hox genes can change dramatically in evolution. Two genes in particular--Hox3 and fushi tarazu--lost their ancestral roles as homeotic genes and play very different developmental roles in the fruit fly Drosophila melanogaster. The Drosophila Hox3 homologs zerknullt and bicoid act in extraembryonic tissues and in establishment of the anteroposterior axis, respectively, whereas fushi tarazu acts in segmentation and neurogenesis. It would be valuable to know what mechanisms allowed Hox3 and ftz to abandon their ancestral roles as homeotic genes and take on new roles. To explore the evolutionary transition of these genes, we analyzed their expression in a primitive insect, the firebrat Thermobia domestica. The expression patterns seem to represent a stage of evolution intermediate between the ancestral state seen in basal arthropods and the derived expression patterns in Drosophila. These expression data help us to narrow the period in which the gene transitions took place. Hox3 appears to have evolved directly into zen within the insects, whereas ftz seems to have adopted the expression patterns of a segmentation and neurogenesis gene earlier in the mandibulate arthropods.     

Conservation of arthropod midline netrin accumulation revealed with a cross-reactive antibody provides evidence for midline cell homology

SUMMARY Although many similarities in arthropod CNS development exist, differences in axonogenesis and the formation of midline cells, which regulate axon growth, have been observed. For example, axon growth patterns in the ventral nerve cord of Artemia franciscana differ from that of Drosophila melanogaster . Despite such differences, conserved molecular marker expression at the midline of several arthropod species indicates that midline cells may be homologous in distantly related arthropods. However, data from additional species are needed to test this hypothesis. In this investigation, nerve cord formation and the putative homology of midline cells were examined in distantly related arthropods, including: long- and short-germ insects ( D. melanogaster, Aedes aeygypti , and Tribolium castaneum ), branchiopod crustaceans ( A. franciscana and Triops longicauditus ), and malacostracan crustaceans ( Porcellio laevis and Parhyale hawaiensis ). These comparative analyses were aided by a cross-reactive antibody generated against the Netrin (Net) protein, a midline cell marker and regulator of axonogenesis. The mechanism of nerve cord formation observed in Artemia is found in Triops , another branchiopod, but is not found in the other arthropods examined. Despite divergent mechanisms of midline cell formation and nerve cord development, Net accumulation is detected in a well-conserved subset of midline cells in branchiopod crustaceans, malacostracan crustaceans, and insects. Notably, the Net accumulation pattern is also conserved at the midline of the amphipod P. hawaiensis , which undergoes split germ-band development. Conserved Net accumulation patterns indicate that arthropod midline cells are homologous, and that Nets function to regulate commissure formation during CNS development of Tetraconata.     

Hox基因与昆虫翅的特化

自从1978年E.B. Lewis描述了著名的果蝇双胸突变体（bithorax）以来,大量的比较发育遗传学研究为我们揭示了形态进化的遗传基础,从而使形态进化研究进入了一个新的时代。同时,Hox基因的研究也成为这一领域的焦点。本文综述了昆虫翅的起源及其特化类群翅的发育遗传学研究的最新进展。一般认为,原始的有翅昆虫胸腹部多附肢（包括翅）; 之后不同的体节受到了不同Hox的抑制,形成两对翅以及前后翅的分化; Ubx的不同表达导致了前后翅的分化,并且Ubx负责识别后翅。我们选择翅特化最为显著的3个类群——鞘翅目（T2鞘翅）、双翅目（T3平衡棒）和捻翅目（T2平衡棒）,结合Hox的表达情况讨论了翅的特化机理。目前已知双翅目和鞘翅目的翅的控制模式存在巨大差异,两种模式的比较研究对于理解翅的形态进化具有重要的意义。但是对捻翅目昆虫的研究则很少。     

Exploring the myriapod body plan: expression patterns of the ten Hox genes in a centipede

The diversity of the arthropod body plan has long been a fascinating subject of study. A flurry of recent research has analyzed Hox gene expression in various arthropod groups, with hopes of gaining insight into the mechanisms that underlie their evolution. The Hox genes have been analyzed in insects, crustaceans and chelicerates. However, the expression patterns of the Hox genes have not yet been comprehensively analyzed in a myriapod. We present the expression patterns of the ten Hox genes in a centipede, Lithobius atkinsoni, and compare our results to those from studies in other arthropods. We have three major findings. First, we find that Hox gene expression is remarkably dynamic across the arthropods. The expression patterns of the Hox genes in the centipede are in many cases intermediate between those of the chelicerates and those of the insects and crustaceans, consistent with the proposed intermediate phylogenetic position of the Myriapoda. Second, we found two 'extra' Hox genes in the centipede compared with those in DROSOPHILA: Based on its pattern of expression, Hox3 appears to have a typical Hox-like role in the centipede, suggesting that the novel functions of the Hox3 homologs zen and bicoid were adopted somewhere in the crustacean-insect clade. In the centipede, the expression of the gene fushi tarazu suggests that it has both a Hox-like role (as in the mite), as well as a role in segmentation (as in insects). This suggests that this dramatic change in function was achieved via a multifunctional intermediate, a condition maintained in the centipede. Last, we found that Hox expression correlates with tagmatic boundaries, consistent with the theory that changes in Hox genes had a major role in evolution of the arthropod body plan.     

Novel patterns of homeotic protein accumulation in the head of the Drosophila embryo

Antibodies that specifically recognize proteins encoded by the homeotic genes: Sex combs reduced, Deformed, labial and proboscipedia, were used to follow the distribution of these gene products during embryogenesis. The position of engrailed-expressing cells was used as a reference and staining conditions were established that could distinguish, among cells expressing engrailed, one of the homeotic proteins or both. Our observations demonstrate two important facts about establishing identity in the head segments. First, in contrast to the overlapping pattern of homeotic gene expression in the trunk segments, we observe a non-overlapping pattern in the head for those homeotic proteins required during embryogenesis. In contrast, the spatial accumulation of the protein product of the non-vital proboscipedia locus overlaps partially with the distribution of the Deformed and Sex combs reduced proteins in the maxillary and labial segments, respectively. Second, two of the proteins, Sex combs reduced and Deformed, have different dorsal and ventral patterns of accumulation. Dorsally, these proteins are expressed in segmental domains while, within the ventral region, a parasegmental register is observed. The boundary where this change in pattern occurs coincides with the junction between the ventral neurogenic region and the dorsal epidermis. After contraction of the germ band, when the nerve cord has completely separated from the epidermis, the parasegmental pattern is observed only within the ventral nerve cord while a segmental register is maintained throughout the epidermis.