A Gbx homeobox gene in amphioxus: insights into ancestry of the ANTP class and evolution of the midbrain/hindbrain boundary

In the vertebrate central nervous system (CNS), mutual antagonism between posteriorly expressed Gbx2 and anteriorly expressed Otx2 positions the midbrain/hindbrain boundary (MHB), but does not induce MHB organizer genes such as En, Pax2/5/8 and Wnt1. In the CNS of the cephalochordate amphioxus, Otx is also expressed anteriorly, but En, Pax2/5/8 and Wnt1 are not expressed near the caudal limit of Otx, raising questions about the existence of an MHB organizer in amphioxus. To investigate the evolutionary origins of the MHB, we cloned the single amphioxus Gbx gene. Fluorescence in situ hybridization showed that, as in vertebrates, amphioxus Gbx and the Hox cluster are on the same chromosome. From analysis of linked genes, we argue that during evolution a single ancestral Gbx gene duplicated fourfold in vertebrates, with subsequent loss of two duplicates. Amphioxus Gbx is expressed in all germ layers in the posterior 75% of the embryo, and in the CNS, the Gbx and Otx domains abut at the boundary between the cerebral vesicle (forebrain/midbrain) and the hindbrain. Thus, the genetic machinery to position the MHB was present in the protochordate ancestors of the vertebrates, but is insufficient for induction of organizer genes. Comparison with hemichordates suggests that anterior Otx and posterior Gbx domains were probably overlapping in the ancestral deuterostome and came to abut at the MHB early in the chordate lineage before MHB organizer properties evolved.     

Molecular Evolution and Structural Features of IRAK Family Members

The interleukin-1 receptor-associated kinase (IRAK) family comprises critical signaling mediators of the TLR/IL-1R signaling pathways. IRAKs are Ser/Thr kinases. There are 4 members in the vertebrate genome (IRAK1, IRAK2, IRAKM, and IRAK4) and an IRAK homolog, Pelle, in insects. IRAK family members are highly conserved in vertebrates, but the evolutionary relationship between IRAKs in vertebrates and insects is not clear. To investigate the evolutionary history and functional divergence of IRAK members, we performed extensive bioinformatics analysis. The phylogenetic relationship between IRAK sequences suggests that gene duplication events occurred in the evolutionary lineage, leading to early vertebrates. A comparative phylogenetic analysis with insect homologs of IRAKs suggests that the Tube protein is a homolog of IRAK4, unlike the anticipated protein, Pelle. Furthermore, the analysis supports that an IRAK4-like kinase is an ancestral protein in the metazoan lineage of the IRAK family. Through functional analysis, several potentially diverged sites were identified in the common death domain and kinase domain. These sites have been constrained during evolution by strong purifying selection, suggesting their functional importance within IRAKs. In summary, our study highlighted the molecular evolution of the IRAK family, predicted the amino acids that contributed to functional divergence, and identified structural variations among the IRAK paralogs that may provide a starting point for further experimental investigations.     

Reconciling extreme branch length differences: decoupling time and rate through the evolutionary history of filmy ferns

The rate of molecular evolution is not constant across the Tree of Life. Characterizing rate discrepancies and evaluating the relative roles of time and rate along branches through the past are both critical to a full understanding of evolutionary history. In this study, we explore the interactions of time and rate in filmy ferns (Hymenophyllaceae), a lineage with extreme branch length differences between the two major clades. We test for the presence of significant rate discrepancies within and between these clades, and we separate time and rate across the filmy fern phylogeny to simultaneously yield an evolutionary time scale of filmy fern diversification and reconstructions of ancestral rates of molecular evolution. Our results indicate that the branch length disparity observed between the major lineages of filmy ferns is indeed due to a significant difference in molecular evolutionary rate. The estimation of divergence times reveals that the timing of crown group diversification was not concurrent for the two lineages, and the reconstruction of ancestral rates of molecular evolution points to a substantial rate deceleration in one of the clades. Further analysis suggests that this may be due to a genome-wide deceleration in the rate of nucleotide substitution.     

Dynamic expression of Pax6 in the shark olfactory system: evidence for the presence of Pax6 cells along the olfactory nerve pathway

Pax6 is involved in the control of neuronal specification, migration, and differentiation in the olfactory epithelium and in the generation of different interneuron subtypes in the olfactory bulb. Whether these roles are conserved during evolution is not known. Cartilaginous fish are extremely useful models for assessing the ancestral condition of brain organization because of their phylogenetic position. To shed light on the evolution of development of the olfactory system in vertebrates and on the involvement of Pax6 in this process, we analyzed by in situ hybridization and immunohistochemistry the expression pattern of Pax6 in the developing olfactory system in a basal vertebrate, the lesser spotted dogfish Scyliorhinus canicula. This small shark is becoming an important fish model in studies of vertebrate development. We report Pax6 expression in cells of the olfactory epithelium and olfactory bulb, and present the first evidence in vertebrates of strings of Pax6-expressing cells extending along the developing olfactory nerve. The results indicate the olfactory epithelium as the origin of these cells. These data are compatible with a role for Pax6 in the development of the olfactory epithelium and fibers, and provide a basis for future investigations into the mechanisms that regulate development of the olfactory system throughout evolution.     

Phylogenetic and evolutionary analysis of the septin protein family in metazoan

Septins, a conserved family of cytoskeletal GTP-binding proteins, were presented in diverse eukaryotes. Here, a comprehensive phylogenetic and evolutionary analysis for septin proteins in metazoan was carried out. First, we demonstrated that all septin proteins in metazoan could be clustered into four subgroups, and the representative homologue of every subgroup was presented in the non-vertebrate chordate Ciona intestinalis, indicating that the emergence of the four septin subgroups should have occurred prior to divergence of vertebrates and invertebrates, and the expansion of the septin gene number in vertebrates was mainly by the duplication of pre-existing genes rather than by the appearance of new septin subgroup. Second, the direct orthologues of most human septins existed in zebrafish, which suggested that human septin gene repertoire was mainly formed by as far as before the split between fishes and land vertebrates. Third, we found that the evolutionary rate within septin family in mammalian lineage varies significantly, human SEPT1, SEPT 10, SEPT 12, and SEPT 14 displayed a relative elevated evolutionary rate compared with other septin members. Our data will provide new insights for the further function study of this protein family.     

Origin of the paired domain

Pax proteins play a diverse role in early animal development and contain the characteristic paired domain, consisting of two conserved helix-turn-helix motifs. In many Pax proteins the paired domain is fused to a second DNA binding domain of the paired-like homeobox family. By amino acid sequence alignments, secondary structure prediction, 3D-structure comparison, and phylogenetic reconstruction, we analyzed the relationship between Pax proteins and members of the Tc1 family of transposases, which possibly share a common ancestor with Pax proteins. We suggest that the DNA binding domain of an ancestral transposase (proto-Pax transposase) was fused to a homeodomain shortly after the emergence of metazoans about one billion years ago. Using the transposase sequences as an outgroup we reexamined the early evolution of the Pax proteins. Our novel evolutionary scenario features a single homeobox capturing event and an early duplication of Pax genes before the divergence of porifera, indicating a more diverse role of Pax proteins in primitive animals than previously expected. Received: 16 February 2000 / Accepted: 13 August 2000     

Functional conservation of the Drosophila gooseberry gene and its evolutionary alleles

The Drosophila Pax gene gooseberry (gsb) is required for development of the larval cuticle and CNS, survival to adulthood, and male fertility. These functions can be rescued in gsb mutants by two gsb evolutionary alleles, gsb-Prd and gsb-Pax3, which express the Drosophila Paired and mouse Pax3 proteins under the control of gooseberry cis-regulatory region. Therefore, both Paired and Pax3 proteins have conserved all the Gsb functions that are required for survival of embryos to fertile adults, despite the divergent primary sequences in their C-terminal halves. As gsb-Prd and gsb-Pax3 uncover a gsb function involved in male fertility, construction of evolutionary alleles may provide a powerful strategy to dissect hitherto unknown gene functions. Our results provide further evidence for the essential role of cis-regulatory regions in the functional diversification of duplicated genes during evolution.     

Life-history specialization was not an evolutionary dead-end in Pyrenean cave beetles

Research on subterranean organisms has focused on the colonization process and some of the associated phenotypic changes, but little is known on the long-term evolutionary dynamics of subterranean lineages and the origin of some highly specialized complex characters. One of the most extreme modifications is the reduction of the number of larval instars in some Leptodirini beetles from the ancestral 3 to 2 and ultimately a single instar. This reduction is usually assumed to have occurred independently multiple times within the same lineage and geographical area, but its evolution has never been studied in a phylogenetic framework. Using a comprehensive molecular phylogeny, we found a low number of independent origins of the reduction in the number of instars, with a single transition, dated to the Oligocene–Miocene, from 3 to 2 and then 1 instar in the Pyrenees, the best-studied area. In the Pyrenees, the 1-instar lineage had a diversification rate (0.22 diversification events per lineage per million years) significantly higher than that of 3- or 2-instar lineages (0.10), and similar to that seen in other Coleopteran radiations. Far from being evolutionary dead-ends, ancient lineages fully adapted to subterranean life seem able to persist and diversify over long evolutionary periods.     

Evolution of developmental roles of Pax2/5/8 paralogs after independent duplication in urochordate and vertebrate lineages

Background

Gene duplication provides opportunities for lineage diversification and evolution of developmental novelties. Duplicated genes generally either disappear by accumulation of mutations (nonfunctionalization), or are preserved either by the origin of positively selected functions in one or both duplicates (neofunctionalization), or by the partitioning of original gene subfunctions between the duplicates (subfunctionalization). The Pax2/5/8 family of important developmental regulators has undergone parallel expansion among chordate groups. After the divergence of urochordate and vertebrate lineages, two rounds of independent gene duplications resulted in the Pax2, Pax5, and Pax8 genes of most vertebrates (the sister group of the urochordates), and an additional duplication provided the pax2a and pax2b duplicates in teleost fish. Separate from the vertebrate genome expansions, a duplication also created two Pax2/5/8 genes in the common ancestor of ascidian and larvacean urochordates.

Results

To better understand mechanisms underlying the evolution of duplicated genes, we investigated, in the larvacean urochordate Oikopleura dioica, the embryonic gene expression patterns of Pax2/5/8 paralogs. We compared the larvacean and ascidian expression patterns to infer modular subfunctions present in the single pre-duplication Pax2/5/8 gene of stem urochordates, and we compared vertebrate and urochordate expression to infer the suite of Pax2/5/8 gene subfunctions in the common ancestor of olfactores (vertebrates + urochordates). Expression pattern differences of larvacean and ascidian Pax2/5/8 orthologs in the endostyle, pharynx and hindgut suggest that some ancestral gene functions have been partitioned differently to the duplicates in the two urochordate lineages. Novel expression in the larvacean heart may have resulted from the neofunctionalization of a Pax2/5/8 gene in the urochordates. Expression of larvacean Pax2/5/8 in the endostyle, in sites of epithelial remodeling, and in sensory tissues evokes like functions of Pax2, Pax5 and Pax8 in vertebrate embryos, and may indicate ancient origins for these functions in the chordate common ancestor.

Conclusion

Comparative analysis of expression patterns of chordate Pax2/5/8 duplicates, rooted on the single-copy Pax2/5/8 gene of amphioxus, whose lineage diverged basally among chordates, provides new insights into the evolution and development of the heart, thyroid, pharynx, stomodeum and placodes in chordates; supports the controversial conclusion that the atrial siphon of ascidians and the otic placode in vertebrates are homologous; and backs the notion that Pax2/5/8 functioned in ancestral chordates to engineer epithelial fusions and perforations, including gill slit openings.     

Pituitary glycoprotein hormone beta subunits in the Australian lungfish and estimation of the relative evolution rate of these subunits within vertebrates

The beta subunits of the two pituitary gonadotropins LH and FSH and of thyroid-stimulating hormone (TSH) were cloned from Australian lungfish (Neoceratodus forsteri) pituitary glands. These three glycoprotein hormone beta subunits possess the main characteristics common to their counterparts in other vertebrates. Taking advantage of the phylogenetic position of the lungfish, close to the root of tetrapods, a maximum parsimony tree was inferred from these new sequences and sequences from representatives of the diversity of vertebrates. The topology of the tree was imposed so that it reflected as closely as possible the real evolutionary history of the subunits. This tree was used to estimate the relative evolution rate of the three subunits in vertebrates. Cumulated amino acid substitutions from the basal subunit node (ancestral subunit sequence) to the species node were calculated and compared. It showed that a burst in evolutionary rate occurred for the LHbeta subunit in the tetrapod lineage sometime after the emergence of amphibians. The rate of evolution of the LHbeta subunit was particularly high throughout the radiation of mammals while FSH and TSHbeta subunits kept quite stable in this lineage. A burst in evolutionary rate was also observed for the FSHbeta subunit in the lineage leading to teleosts sometime after the emergence of chondrosteans and the dynamic of evolution was high throughout the radiation of teleosts. These results were consistent with data obtained from pairwise comparisons.     

Rapid evolutionary emergence of the combinatorial recognition repertoire

Although the capacity of cells to respond to environmental challengessuch as oxidative damage are ancient evolutionary developmentsthat have been carried through to modern higher vertebratesas "innate" immunity, the characteristic immune response ofvertebrates is a relatively recent evolutionary developmentthat is present only in jawed vertebrates. The vertebrate "combinatorial"response is defined by the presence of lymphocytes as specificantigen recognition cells and by the complete panel of antibodies,T cell receptors, and major histocompatibility complex moleculesall of which are members of the immunoglobulin family. Its emergencein evolution was an extremely rapid event (approximately 10million years) that was catalyzed by the horizontal transferof recombinase activator genes (RAG) from microbes to an ancestraljawed vertebrate. RAGs occur in jawed vertebrates, but havenot been found in invertebrates and other intermediate species.We propose that antigen recognition capacity contributed bythis novel combinatorial mechanism gave jawed vertebrates theability to recognize the entire range of potential antigenicmolecular structures, including self components and moleculesof infectious microbes not shared with vertebrates. The contrastwithin the vertebrates is striking because the most ancientextant jawed vertebrates, sharks and their kin, have the completepanoply of T-cell receptors, antibodies, MHC products and RAGgenes, whereas agnathans possess cells resembling lymphocytesbut ostensibly lack all of the molecules definitive of combinatorialimmunity. Another vertebrate innovation may have been the utilizationof nuclear receptor superfamily, in the regulation of lymphocytesand other cells of the immune lineage. Unlike, RAG, however,this superfamily occurs in all metazoans with the exceptionof sponges.