Hox cluster organization in the jawless vertebrate Petromyzon marinus

Large-scale gene amplifications may have facilitated the evolution of morphological innovations that accompanied the origin of vertebrates. This hypothesis predicts that the genomes of extant jawless fish, scions of deeply branching vertebrate lineages, should bear a record of these events. Previous work suggests that nonvertebrate chordates have a single Hox cluster, but that gnathostome vertebrates have four or more Hox clusters. Did the duplication events that produced multiple vertebrate Hox clusters occur before or after the divergence of agnathan and gnathostome lineages? Can investigation of lamprey Hox clusters illuminate the origins of the four gnathostome Hox clusters? To approach these questions, we cloned and sequenced 13 Hox cluster genes from cDNA and genomic libraries in the lamprey, Petromyzon marinus. The results suggest that the lamprey has at least four Hox clusters and support the model that gnathostome Hox clusters arose by a two-round-no-cluster-loss mechanism, with tree topology [(AB)(CD)]. A three-round model, however, is not rigorously excluded by the data and, for this model, the tree topologies [(D(C(AB))] and [(C(D(AB))] are most parsimonious. Gene phylogenies suggest that at least one Hox cluster duplication occurred in the lamprey lineage after it diverged from the gnathostome lineage. The results argue against two or more rounds of duplication before the divergence of agnathan and gnathostome vertebrates. If Hox clusters were duplicated in whole-genome duplication events, then these data suggest that, at most, one whole genome duplication occurred before the evolution of vertebrate developmental innovations.     

Surveying phylogenetic footprints in large gene clusters: applications to Hox cluster duplications

Evolutionarily conserved non-coding genomic sequences represent a potentially rich source for the discovery of gene regulatory regions. Since these elements are subject to stabilizing selection they evolve much more slowly than adjacent non-functional DNA. These so-called phylogenetic footprints can be detected by comparison of the sequences surrounding orthologous genes in different species. Therefore the loss of phylogenetic footprints as well as the acquisition of conserved non-coding sequences in some lineages, but not in others, can provide evidence for the evolutionary modification of cis-regulatory elements. We introduce here a statistical model of footprint evolution that allows us to estimate the loss of sequence conservation that can be attributed to gene loss and other structural reasons. This approach to studying the pattern of cis-regulatory element evolution, however, requires the comparison of relatively long sequences from many species. We have therefore developed an efficient software tool for the identification of corresponding footprints in long sequences from multiple species. We apply this novel method to the published sequences of HoxA clusters of shark, human, and the duplicated zebrafish and Takifugu clusters as well as the published HoxB cluster sequences. We find that there is a massive loss of sequence conservation in the intergenic region of the HoxA clusters, consistent with the finding in [Chiu et al., PNAS 99 (2002) 5492]. The loss of conservation after cluster duplication is more extensive than expected from structural reasons. This suggests that binding site turnover and/or adaptive modification may also contribute to the loss of sequence conservation.     

Phylogenetic and chromosomal analyses of multiple gene families syntenic with vertebrate Hox clusters

Background  

Ever since the theory about two rounds of genome duplication (2R) in the vertebrate lineage was proposed, the Hox gene clusters have served as the prime example of quadruplicate paralogy in mammalian genomes. In teleost fishes, the observation of additional Hox clusters absent in other vertebrate lineages suggested a third tetraploidization (3R). Because the Hox clusters occupy a quite limited part of each chromosome, and are special in having position-dependent regulation within the multi-gene cluster, studies of syntenic gene families are needed to determine the extent of the duplicated chromosome segments. We have analyzed in detail 14 gene families that are syntenic with the Hox clusters to see if their phylogenies are compatible with the Hox duplications and the 2R/3R scenario. Our starting point was the gene family for the NPY family of peptides located near the Hox clusters in the pufferfish Takifugu rubripes, the zebrafish Danio rerio, and human.     

Novel interactions between vertebrate Hox genes

Understanding why metazoan Hox/HOM-C genes are expressed in spatiotemporal sequences showing colinearity with their genomic sequence is a central challenge in developmental biology. Here, we studied the consequences of ectopically expressing Hox genes to investigate whether Hox-Hox interactions might help to order gene expression during very early vertebrate embryogenesis. Our study revealed conserved autoregulatory loops for the Hox4 and Hox7 paralogue groups, detected following ectopic expression Hoxb-4 or HOXD4, and Hoxa-7, respectively. We also detected specific induction of 5' posterior Hox genes; Hoxb-5 to Hoxb-9, following ectopic expression of Hoxb-4/HOXD4; Hoxb-8 and Hoxb-9 following ectopic expression of Hoxa-7. Additionally, we observed specific repression of 3' anterior genes, following ectopic expression of Hox4 and Hox7 paralogues. We found that induction of Hoxb-4 and Hoxb-5 by Hoxb-4 can be direct, whereas induction of Hoxb-7 is indirect, suggesting the possibility of an activating cascade. Finally, we found that activation of Hoxb-4 itself and of posterior Hox genes by Hoxb-4 can be both non-cell-autonomous, as well as direct. We believe that our findings could be important for understanding how a highly ordered Hox expression sequence is set up in the early vertebrate embryo.     

Hox patterning of the vertebrate rib cage

Unlike the rest of the axial skeleton, which develops solely from somitic mesoderm, patterning of the rib cage is complicated by its derivation from two distinct tissues. The thoracic skeleton is derived from both somitic mesoderm, which forms the vertebral bodies and ribs, and from lateral plate mesoderm, which forms the sternum. By generating mouse mutants in Hox5, Hox6 and Hox9 paralogous group genes, along with a dissection of the Hox10 and Hox11 group mutants, several important conclusions regarding the nature of the ;Hox code' in rib cage and axial skeleton development are revealed. First, axial patterning is consistently coded by the unique and redundant functions of Hox paralogous groups throughout the axial skeleton. Loss of paralogous function leads to anterior homeotic transformations of colinear regions throughout the somite-derived axial skeleton. In the thoracic region, Hox genes pattern the lateral plate-derived sternum in a non-colinear manner, independent from the patterning of the somite-derived vertebrae and vertebral ribs. Finally, between adjacent sets of paralogous mutants, the regions of vertebral phenotypes overlap considerably; however, each paralogous group imparts unique morphologies within these regions. In all cases examined, the next-most posterior Hox paralogous group does not prevent the function of the more-anterior Hox group in axial patterning. Thus, the ;Hox code' in somitic mesoderm is the result of the distinct, graded effects of two or more Hox paralogous groups functioning in any anteroposterior location.     

Hox gene duplications correlate with posterior heteronomy in scorpions

The evolutionary success of the largest animal phylum, Arthropoda, has been attributed to tagmatization, the coordinated evolution of adjacent metameres to form morphologically and functionally distinct segmental regions called tagmata. Specification of regional identity is regulated by the Hox genes, of which 10 are inferred to be present in the ancestor of arthropods. With six different posterior segmental identities divided into two tagmata, the bauplan of scorpions is the most heteronomous within Chelicerata. Expression domains of the anterior eight Hox genes are conserved in previously surveyed chelicerates, but it is unknown how Hox genes regionalize the three tagmata of scorpions. Here, we show that the scorpion Centruroides sculpturatus has two paralogues of all Hox genes except Hox3, suggesting cluster and/or whole genome duplication in this arachnid order. Embryonic anterior expression domain boundaries of each of the last four pairs of Hox genes (two paralogues each of Antp, Ubx, abd-A and Abd-B) are unique and distinguish segmental groups, such as pectines, book lungs and the characteristic tail, while maintaining spatial collinearity. These distinct expression domains suggest neofunctionalization of Hox gene paralogues subsequent to duplication. Our data reconcile previous understanding of Hox gene function across arthropods with the extreme heteronomy of scorpions.     

Patterns and consequences of vertebrate Emx gene duplications

SUMMARY We have cloned and analyzed two Emx genes from the lamprey Petromyzon marinus and our findings provide insight into the patterns and developmental consequences of gene duplications during early vertebrate evolution. The Emx gene family presents an excellent case for addressing these issues as gnathostome vertebrates possess two or three Emx paralogs that are highly pleiotropic, functioning in or being expressed during the development of several vertebrate synapomorphies. Lampreys are the most primitive extant vertebrates and characterization of their development and genomic organization is critical for understanding vertebrate origins. We identified two Emx genes from P. marinus and analyzed their phylogeny and their embryological expression relative to other chordate Emx genes. Our phylogenetic analysis shows that the two lamprey Emx genes group independently from the gnathostome Emx1, Emx2 , and Emx3 paralogy groups. Our expression analysis shows that the two lamprey Emx genes are expressed in distinct spatial and temporal patterns that together broadly encompass the combined sites of expression of all gnathostome Emx genes. Our data support a model wherein large-scale regulatory evolution of a single Emx gene occurred after the protochordate/vertebrate divergence, but before the vertebrate radiation. Both the lamprey and gnathostome lineages then underwent independent gene duplications followed by extensive paralog subfunctionalization. Emx subfunctionalization in the telencephalon is remarkably convergent and refines our understanding of lamprey forebrain patterning. We also identify lamprey-specific sites of expression that indicate either neofunctionalization in lampreys or sites-specific nonfunctionalization of all gnathostome Emx genes. Overall, we see only very limited correlation between Emx gene duplications and the acquisition of novel expression domains.     

Two more Posterior Hox genes and Hox cluster dispersal in echinoderms

Background

Hox genes are key elements in patterning animal development. They are renowned for their, often, clustered organisation in the genome, with supposed mechanistic links between the organisation of the genes and their expression. The widespread distribution and comparable functions of Hox genes across the animals has led to them being a major study system for comparing the molecular bases for construction and divergence of animal morphologies. Echinoderms (including sea urchins, sea stars, sea cucumbers, feather stars and brittle stars) possess one of the most unusual body plans in the animal kingdom with pronounced pentameral symmetry in the adults. Consequently, much interest has focused on their development, evolution and the role of the Hox genes in these processes. In this context, the organisation of echinoderm Hox gene clusters is distinctive. Within the classificatory system of Duboule, echinoderms constitute one of the clearest examples of Disorganized (D) clusters (i.e. intact clusters but with a gene order or orientation rearranged relative to the ancestral state).

Results

Here we describe two Hox genes (Hox11/13d and e) that have been overlooked in most previous work and have not been considered in reconstructions of echinoderm Hox complements and cluster organisation. The two genes are related to Posterior Hox genes and are present in all classes of echinoderm. Importantly, they do not reside in the Hox cluster of any species for which genomic linkage data is available.

Conclusion

Incorporating the two neglected Posterior Hox genes into assessments of echinoderm Hox gene complements and organisation shows that these animals in fact have Split (S) Hox clusters rather than simply Disorganized (D) clusters within the Duboule classification scheme. This then has implications for how these genes are likely regulated, with them no longer covered by any potential long-range Hox cluster-wide, or multigenic sub-cluster, regulatory mechanisms.

     

The amphioxus Hox cluster: deuterostome posterior flexibility and Hox14

SUMMARY The amphioxus ( Branchiostoma floridae ) Hox cluster is a model for the ancestral vertebrate cluster, prior to the hypothesized genome-wide duplications that may have facilitated the evolution of the vertebrate body plan. Here we describe the posterior (5') genes of the amphioxus cluster, and report the isolation of four new homeobox genes. Vertebrates possess 13 types of Hox gene (paralogy groups), but we show that amphioxus possesses more than 13 Hox genes. Amphioxus is now the first animal in which a Hox14 gene has been found. Our mapping and phylogenetic analysis of amphioxus "Posterior Class" Hox genes reveals that these genes are evolving at a faster rate in deuterostomes than in protostomes, a phenomenon we term Posterior Flexibility.     

Sea urchin Hox genes: insights into the ancestral Hox cluster

We describe the Hox cluster in the radially symmetric sea urchin and compare our findings to what is known from clusters in bilaterally symmetric animals. Several Hox genes from the direct-developing sea urchin Heliocidaris erythrogramma are described. CHEF gel analysis shows that the Hox genes are clustered on a < or = 300 kilobase (kb) fragment of DNA, and only a single cluster is present, as in lower chordates and other nonvertebrate metazoans. Phylogenetic analyses of sea urchin, amphioxus, Drosophila, and selected vertebrate Hox genes confirm that the H. erythrogramma genes, and others previously cloned from other sea urchins, belong to anterior, central, and posterior groups. Despite their radial body plan and lack of cephalization, echinoderms retain at least one of the anterior group Hox genes, an orthologue of Hox3. The structure of the echinoderm Hox cluster suggests that the ancestral deuterostome had a Hox cluster more similar to the current chordate cluster than was expected Sea urchins have at least three Abd-B type genes, suggesting that Abd-B expansion began before the radiation of deuterostomes.      

Hox clusters as models for vertebrate genome evolution

The surprising variation in the number of Hox clusters and the genomic architecture within vertebrate lineages, especially within the ray-finned fish, reflects a history of duplications and subsequent lineage-specific gene loss. Recent research on the evolution of conserved non-coding sequences (CNS) in Hox clusters promises to reveal interesting results for functional and phenotypic diversification.     

The role of Hox genes during vertebrate limb development

The potential role of Hox genes during vertebrate limb development was brought into focus by gene expression analyses in mice (P Dolle, JC Izpisua-Belmonte, H Falkenstein, A Renucci, D Duboule, Nature 1989, 342:767-772), at a time when limb growth and patterning were thought to depend upon two distinct and rather independent systems of coordinates; one for the anterior-to-posterior axis and the other for the proximal-to-distal axis (see D Duboule, P Dolle, EMBO J 1989, 8:1497-1505). Over the past years, the function and regulation of these genes have been addressed using both gain-of-function and loss-of-function approaches in chick and mice. The use of multiple mutations either in cis-configuration in trans-configuration or in cis/trans configurations, has confirmed that Hox genes are essential for proper limb development, where they participate in both the growth and organization of the structures. Even though their molecular mechanisms of action remain somewhat elusive, the results of these extensive genetic analyses confirm that, during the development of the limbs, the various axes cannot be considered in isolation from each other and that a more holistic view of limb development should prevail over a simple cartesian, chess grid-like approach of these complex structures. With this in mind, the functional input of Hox genes during limb growth and development can now be re-assessed.     

Ancient origin of the Hox gene cluster

The Hox gene cluster has a crucial function in body patterning during animal development. How and when this gene cluster originated is being clarified by recent data from Cnidaria, a basal animal phylum. The characterization of Hox-like genes from Hydra, sea anemones and jellyfish has revealed that a Hox gene cluster is extremely ancient, having originated even before the divergence of these basal animals.     

Evolution of the echinoderm Hox gene cluster

SUMMARY Extant echinoderms are members of an ancient and highly derived deuterostome phylum. The composition and arrangement of their Hox gene clusters are consequently of interest not only from the perspective of evolution of development, but also in terms of metazoan phylogeny and body plan evolution. Over the last decade numerous workers have reported partial Hox gene sequences from a variety of echinoderms. In this paper we used a combined methods approach to analyze phylogenetic relationships between 68 echinoderm Hox homeodomain fragments, from species of five extant classes—two asteroids, one crinoid, one ophiuroid, one holothuroid, and three echinoids. This analysis strengthens Mito and Endo's (2000) proposition that the ancestral echinoderm's Hox gene cluster contained at least eleven genes, including at least four posterior paralogous group genes. However, representatives of all paralogous groups are not known from all echinoderm classes. In particular, these data suggest that echinoids may have lost a posterior group Hox gene subsequent to the divergence of the echinoderm classes. Evolution of the highly derived echinoderm body plan may have been accompanied by class-specific duplication, diversification and loss of Hox genes.     

Phylogenetic analysis of vertebrate kininogen genes

Kininogens, the precursors of bradykinins, vary extremely in both structure and function among different taxa of animals, in particular between mammals and amphibians. This includes even the most conserved bradykinin domain in terms of biosynthesis mode and structure. To elucidate the evolutionary dynamics of kininogen genes, we have identified 19 novel amino acid sequences from EST and genomic databases (for mammals, birds, and fishes) and explored their phylogenetic relationships using combined amino acid sequence and gene structure as markers. Our results show that there were initially two paralogous kininogen genes in vertebrates. During their evolution, the original gene was saved with frequent multiplication in amphibians, but lost in fishes, birds, and mammals, while the novel gene was saved with multiple functions in fishes, birds, and mammals, but became a pseudogene in amphibians. We also propose that the defense mechanism against specific predators in amphibian skin secretions has been bradykinin receptor dependent. Our findings may provide a foundation for identification and structural, functional, and evolutionary analyses of more kininogen genes and other gene families.     

Extensive duplications of phototransduction genes in early vertebrate evolution correlate with block (chromosome) duplications

Many gene families in mammals have members that are expressed more or less uniquely in the retina or differentially in specific retinal cell types. We describe here analyses of nine such gene families with regard to phylogenetic relationships and chromosomal location. The families are opsins, G proteins (alpha, beta, and gamma subunits), phosphodiesterases type 6, cyclic nucleotide-gated channels, G-protein-coupled receptor kinases, arrestins, and recoverins. The results suggest that multiple new gene copies arose in all of these families very early in vertebrate evolution during a period with extensive gene duplications. Many of the new genes arose through duplications of large chromosome regions (blocks of genes) or even entire chromosomes, as shown by linkage with other gene families. Some of the phototransduction families belong to the same duplicated regions and were thus duplicated simultaneously. We conclude that gene duplications in early vertebrate evolution probably helped facilitate the specialization of the retina and the subspecialization of different retinal cell types.