A Hox class 3 orthologue from the spider Cupiennius salei is expressed in a Hox-gene-like fashion

 The class 3 Hox gene orthologue in insects, zerknüllt (zen), is not expressed along the anterior-posterior axis, but only in extra-embryonic tissues, suggesting that it has lost its function as a normal Hox gene. To analyse whether this loss of Hox gene function has already occurred in a basal arthropod lineage, we have isolated a Hox3 orthologue from the spider Cupiennius salei. In contrast to the insect zen sequences, which have a highly diverged homeobox, the spider Hox3 gene orthologue, Cs-Hox3, shows a high sequence similarity to the class 3 Hox genes of other phyla, including chordates. In situ hybridization in early embryos shows that it is expressed in a continuous region covering the pedipalp segment and the four leg-bearing segments. This expression pattern suggests a Hox-gene-like function for Cs-Hox3. On the other hand, the expression pattern does not strictly follow the colinearity rule, as it overlaps fully with the expression domain of the class 1 orthologue of the spider, Cs-lab. Still, our data suggest that the ancestor of the arthropods must have had a class 3 Hox gene with a function in anterior-posterior axis specification and that this function has been lost in the lineage leading to the insects. Received: 11 February 1998 / Accepted: 20 March 1998     

Organization of the Hox gene cluster of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>: a split of the Hox cluster in a non-<Emphasis Type="Italic">Drosophila</Emphasis> insect

A bacterial artificial chromosome (BAC) contig was constructed by chromosome walking, starting from the Hox genes of the silkworm, Bombyx mori. Bombyx orthologues of the labial (lab) and zerknült (zen) genes were newly identified. The size of the BAC contig containing the Hox gene cluster—except the lab and Hox 2 genes—was estimated to be more than 2 Mb. The Bombyx Hox cluster was mapped to linkage group (LG) 6. The lab gene was mapped on the same LG, but far apart from the cluster. Fluorescence in situ hybridization analysis confirmed that the major Hox gene cluster and lab were at different locations on the same chromosome in B. mori.Edited by M. Akam     

Evolution of <Emphasis Type="Italic">Hox3</Emphasis> and <Emphasis Type="Italic">ftz</Emphasis> in arthropods: insights from the crustacean <Emphasis Type="Italic">Daphnia pulex</Emphasis>

The Drosophila melanogaster genes zerknüllt (zen) and fushi tarazu (ftz) are members of the Hox gene family whose roles have changed significantly in the insect lineage and thus provide an opportunity to study the mechanisms underlying the functional evolution of Hox proteins. We have studied the expression of orthologs of zen (DpuHox3) and ftz (Dpuftz) in the crustacean Daphnia pulex (Branchiopoda), both of which show a dynamic expression pattern. DpuHox3 is expressed in a complex pattern in early embryogenesis, with the most anterior boundary of expression lying at the anterior limit of the second antennal segment as well as a ring of expression around the embryo. In later embryos, DpuHox3 expression is restricted to the mesoderm of mandibular limb buds. Dpuftz is first expressed in a ring around the embryo following the posterior limit of the mandibular segment. Later, Dpuftz is restricted to the posterior part of the mandibular segment. This is the first report of expression of a Hox3 ortholog in a crustacean, and together with Dpuftz data, the results presented here show that Hox3 and ftz have retained a Hox-like expression pattern in crustaceans. This is in accordance with the proposed model of Hox3 and ftz evolution in arthropods and allows a more precise pinpointing of the loss of ftz “Hox-like behaviour”: in the lineage between the Branchiopoda and the basal insect Thysanura.     

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.     

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.     

Purification and properties of theDrosophila zen protein

Summary The zen protein is encoded by the zerknullt gene required for normal early development inDrosophila. Like many regulatory proteins of this type, zen contains a 60 amino acid homeobox sequence. We have purified the zen protein and studied its solution behavior and its interaction with DNA. The zen protein exists as a monomer in solution with a molecular weight of about 40000. It binds specifically to a site about 900 bases upstream from thezen gene. Within this binding site DNase protection experiments indicate that binding is confined to two regions approximately 11 and 14 bases in length that are separated by about 30 base pairs. The protein concentration dependence of the binding curve suggests that protein binding is non cooperative.     

Oncopeltus fasciatus zen is essential for serosal tissue function in katatrepsis

Unlike most Hox cluster genes, with their canonical role in anterior-posterior patterning of the embryo, the Hox3 orthologue of insects has diverged. Here, we investigate the zen orthologue in Oncopeltus fasciatus (Hemiptera:Heteroptera). As in other insects, the Of-zen gene is expressed extraembryonically, and RNA interference (RNAi) experiments demonstrate that it is functionally required in this domain for the proper occurrence of katatrepsis, the phase of embryonic movements by which the embryo emerges from the yolk and adjusts its orientation within the egg. After RNAi knockdown of Of-zen, katatrepsis does not occur, causing embryos to complete development inside out. However, not all aspects of expression and function are conserved compared to grasshopper, beetle, and fly orthologues. Of-zen is not expressed in the extraembryonic tissue until relatively late, suggesting it is not involved in tissue specification. Within the extraembryonic domain, Of-zen is expressed in the outer serosal membrane, but unlike orthologues, it is not detectable in the inner extraembryonic membrane, the amnion. Thus, the role of zen in the interaction of serosa, amnion, and embryo may differ between species. Of-zen is also expressed in the blastoderm, although this early expression shows no apparent correlation with defects seen by RNAi knockdown.     

Maternal expression increases the rate of <Emphasis Type="Italic">bicoid</Emphasis> evolution by relaxing selective constraint

Population genetic theory predicts that maternal effect genes will evolve differently than genes expressed in both sexes because selection is only half as effective on autosomal genes expressed in one sex but not the other. Here, we use sequences of the tandem gene duplicates, bicoid (bcd) and zerknüllt (zen), to test the prediction that, with similar coefficients of purifying selection, a maternal effect gene evolves more rapidly than a zygotic gene because of this reduction in selective constraint. We find that the maternal effect gene, bcd, is evolving more rapidly than zygotically expressed, zen, providing the first direct confirmation of this prediction of maternal effect theory from molecular evidence. Our results extend current explanations for the accelerated rate of bcd evolution by providing an evolutionary mechanism, relaxed selective constraint, that allows bcd the evolutionary flexibility to escape the typical functional constraints of early developmental genes. We discuss general implications of our findings for the role of maternal effect genes in early developmental patterning.     

Hox gene expression in larval development of the polychaetes Nereis virens and Platynereis dumerilii (Annelida, Lophotrochozoa)

The bilaterian animals are divided into three great branches: the Deuterostomia, Ecdysozoa, and Lophotrochozoa. The evolution of developmental mechanisms is less studied in the Lophotrochozoa than in the other two clades. We have studied the expression of Hox genes during larval development of two lophotrochozoans, the polychaete annelids Nereis virens and Platynereis dumerilii. As reported previously, the Hox cluster of N. virens consists of at least 11 genes (de Rosa R, Grenier JK, Andreeva T, Cook CE, Adoutte A, Akam M, Carroll SB, Balavoine G, Nature, 399:772–776, 1999; Andreeva TF, Cook C, Korchagina NM, Akam M, Dondua AK, Ontogenez 32:225–233, 2001); we have also cloned nine Hox genes of P. dumerilii. Hox genes are mainly expressed in the descendants of the 2d blastomere, which form the integument of segments, ventral neural ganglia, pre-pygidial growth zone, and the pygidial lobe. Patterns of expression are similar for orthologous genes of both nereids. In Nereis, Hox2, and Hox3 are activated before the blastopore closure, while Hox1 and Hox4 are activated just after this. Hox5 and Post2 are first active during the metatrochophore stage, and Hox7, Lox4, and Lox2 at the late nectochaete stage only. During larval stages, Hox genes are expressed in staggered domains in the developing segments and pygidial lobe. The pattern of expression of Hox cluster genes suggests their involvement in the vectorial regionalization of the larval body along the antero-posterior axis. Hox gene expression in nereids conforms to the canonical patterns postulated for the two other evolutionary branches of the Bilateria, the Ecdysozoa and the Deuterostomia, thus supporting the evolutionary conservatism of the function of Hox genes in development. Milana Kulakova, Nadezhda Bakalenko and Elena Novikova contributed equally to this work.     

Hox Genes from the Tapeworm Taenia asiatica (Platyhelminthes: Cestoda)

Hox genes are important in forming the anterior-posterior body axis pattern in the early developmental stage of animals. The conserved nature of the genomic organization of Hox genes is well known in diverse metazoans. To understand the Hox gene architecture in human-infecting Taenia tapeworms, we conducted a genomic survey of the Hox gene using degenerative polymerase chain reaction primers in Taenia asiatica. Six Hox gene orthologs from 276 clones were identified. Comparative analysis revealed that T. asiatica has six Hox orthologs, including two lab/Hox1, two Hox3, one Dfd/Hox4, and one Lox2/Lox4. The results suggest that Taenia Hox genes may have undergone independent gene duplication in two Hox paralogs. The failure to detect Post1/2 orthologs in T. asiatica may suggest that sequence divergence or the secondary loss of the posterior genes has occurred in the lineage leading to the cestode and trematode.     

Ancient Expansion of the Hox Cluster in Lepidoptera Generated Four Homeobox Genes Implicated in Extra-Embryonic Tissue Formation

Gene duplications within the conserved Hox cluster are rare in animal evolution, but in Lepidoptera an array of divergent Hox-related genes (Shx genes) has been reported between pb and zen. Here, we use genome sequencing of five lepidopteran species (Polygonia c-album, Pararge aegeria, Callimorpha dominula, Cameraria ohridella, Hepialus sylvina) plus a caddisfly outgroup (Glyphotaelius pellucidus) to trace the evolution of the lepidopteran Shx genes. We demonstrate that Shx genes originated by tandem duplication of zen early in the evolution of large clade Ditrysia; Shx are not found in a caddisfly and a member of the basally diverging Hepialidae (swift moths). Four distinct Shx genes were generated early in ditrysian evolution, and were stably retained in all descendent Lepidoptera except the silkmoth which has additional duplications. Despite extensive sequence divergence, molecular modelling indicates that all four Shx genes have the potential to encode stable homeodomains. The four Shx genes have distinct spatiotemporal expression patterns in early development of the Speckled Wood butterfly (Pararge aegeria), with ShxC demarcating the future sites of extraembryonic tissue formation via strikingly localised maternal RNA in the oocyte. All four genes are also expressed in presumptive serosal cells, prior to the onset of zen expression. Lepidopteran Shx genes represent an unusual example of Hox cluster expansion and integration of novel genes into ancient developmental regulatory networks.     

A Combination of Activation and Repression by a Colinear Hox Code Controls Forelimb-Restricted Expression of Tbx5 and Reveals Hox Protein Specificity

Tight control over gene expression is essential for precision in embryonic development and acquisition of the regulatory elements responsible is the predominant driver for evolution of new structures. Tbx5 and Tbx4, two genes expressed in forelimb and hindlimb-forming regions respectively, play crucial roles in the initiation of limb outgrowth. Evolution of regulatory elements that activate Tbx5 in rostral LPM was essential for the acquisition of forelimbs in vertebrates. We identified such a regulatory element for Tbx5 and demonstrated Hox genes are essential, direct regulators. While the importance of Hox genes in regulating embryonic development is clear, Hox targets and the ways in which each protein executes its specific function are not known. We reveal how nested Hox expression along the rostro-caudal axis restricts Tbx5 expression to forelimb. We demonstrate that Hoxc9, which is expressed in caudal LPM where Tbx5 is not expressed, can form a repressive complex on the Tbx5 forelimb regulatory element. This repressive capacity is limited to Hox proteins expressed in caudal LPM and carried out by two separate protein domains in Hoxc9. Forelimb-restricted expression of Tbx5 and ultimately forelimb formation is therefore achieved through co-option of two characteristics of Hox genes; their colinear expression along the body axis and the functional specificity of different paralogs. Active complexes can be formed by Hox PG proteins present throughout the rostral-caudal LPM while restriction of Tbx5 expression is achieved by superimposing a dominant repressive (Hoxc9) complex that determines the caudal boundary of Tbx5 expression. Our results reveal the regulatory mechanism that ensures emergence of the forelimbs at the correct position along the body. Acquisition of this regulatory element would have been critical for the evolution of limbs in vertebrates and modulation of the factors we have identified can be molecular drivers of the diversity in limb morphology.     

Genomic organization of Hox and ParaHox clusters in the echinoderm,Acanthaster planci

The organization of echinoderm Hox clusters is of interest due to the role that Hox genes play in deuterostome development and body plan organization, and the unique gene order of the Hox complex in the sea urchin Strongylocentrotus purpuratus, which has been linked to the unique development of the axial region. Here, it has been reported that the Hox and ParaHox clusters of Acanthaster planci, a corallivorous starfish found in the Pacific and Indian oceans, generally resembles the chordate and hemichordate clusters. The A. planci Hox cluster shared with sea urchins the loss of one of the medial Hox genes, even‐skipped (Evx) at the anterior of the cluster, as well as organization of the posterior Hox genes. genesis 52:952–958, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

Evidence for the derivation of the Drosophila fushi tarazu gene from a Hox gene orthologous to lophotrochozoan Lox5

The DNA-binding homeobox motif was first identified in several Drosophila homeotic genes but also in fushi tarazu, a gene found in the Hox cluster yet involved in segmentation, not anteroposterior patterning [1]. Homeotic transformations are not seen in insect ftz mutants, and insect ftz genes do not have Hox-like expression except within the nervous system [2] [3]. Insect ftz homeobox sequences link them to the Antp-class genes and Tribolium and Schistocerca orthologs have Antp-class YPWM motifs amino-terminal to the homeobox [2] [3]. Orthologs of ftz cloned from a centipede and an onychophoran [4] show that it predates the emergence of the arthropods, but the inability to pinpoint non-arthropodan orthologs suggested that ftz is the product of a Hox gene duplication in the arthropod ancestor [4] [5]. I have cloned ftz orthologs from a mite and a tardigrade, arthropod outgroups of the insects [6]. Mite ftz is expressed in a Hox-like pattern, confirming its ancestral role in anteroposterior patterning. Phylogenetic analyses indicate that arthropod ftz genes are orthologous to the Lox5 genes of lophotrochozoans (a group that includes molluscs) [7] and, possibly, with the Mab-5 genes of nematodes and Hox6 genes of deuterostomes and would therefore have been present in the triploblast ancestor.