Origins of anteroposterior patterning and Hox gene regulation during chordate evolution

All chordates share a basic body plan and many common features of early development. Anteroposterior (AP) regions of the vertebrate neural tube are specified by a combinatorial pattern of Hox gene expression that is conserved in urochordates and cephalochordates. Another primitive feature of Hox gene regulation in all chordates is a sensitivity to retinoic acid during embryogenesis, and recent developmental genetic studies have demonstrated the essential role for retinoid signalling in vertebrates. Two AP regions develop within the chordate neural tube during gastrulation: an anterior 'forebrain-midbrain' region specified by Otx genes and a posterior 'hindbrain-spinal cord' region specified by Hox genes. A third, intermediate region corresponding to the midbrain or midbrain-hindbrain boundary develops at around the same time in vertebrates, and comparative data suggest that this was also present in the chordate ancestor. Within the anterior part of the Hox-expressing domain, however, vertebrates appear to have evolved unique roles for segmentation genes, such as Krox-20, in patterning the hindbrain. Genetic approaches in mammals and zebrafish, coupled with molecular phylogenetic studies in ascidians, amphioxus and lampreys, promise to reveal how the complex mechanisms that specify the vertebrate body plan may have arisen from a relatively simple set of ancestral developmental components.     

Characterization of microRNAs in cephalochordates reveals a correlation between microRNA repertoire homology and morphological similarity in chordate evolution

SUMMARY Cephalochordates, urochordates, and vertebrates comprise the three extant groups of chordates. Although higher morphological and developmental similarity exists between cephalochordates and vertebrates, molecular phylogeny studies have instead suggested that the morphologically simplified urochordates are the closest relatives to vertebrates. MicroRNAs (miRNAs) are regarded as the major factors driving the increase of morphological complexity in early vertebrate evolution, and are extensively characterized in vertebrates and in a few species of urochordates. However, the comprehensive set of miRNAs in the basal chordates, namely the cephalochordates, remains undetermined. Through extensive sequencing of a small RNA library and genomic homology searches, we characterized 100 miRNAs from the cephalochordate amphioxus, Branchiostoma japonicum , and B. floridae . Analysis of the evolutionary history of the cephalochordate miRNAs showed that cephalochordates possess 54 miRNA families homologous to those of vertebrates, which is threefold higher than those shared between urochordates and vertebrates. The miRNA contents demonstrated a clear correlation between the extent of miRNA overlapping and morphological similarity among the three chordate groups, providing a strong evidence of miRNAs being the major genetic factors driving morphological complexity in early chordate evolution.     

Ascidians and the plasticity of the chordate developmental program

Little is known about the ancient chordates that gave rise to the first vertebrates, but the descendants of other invertebrate chordates extant at the time still flourish in the ocean. These invertebrates include the cephalochordates and tunicates, whose larvae share with vertebrate embryos a common body plan with a central notochord and a dorsal nerve cord. Tunicates are now thought to be the sister group of vertebrates. However, research based on several species of ascidians, a diverse and wide-spread class of tunicates, revealed that the molecular strategies underlying their development appear to diverge greatly from those found in vertebrates. Furthermore, the adult body plan of most tunicates, which arises following an extensive post-larval metamorphosis, shows little resemblance to the body plan of any other chordate. In this review, we compare the developmental strategies of ascidians and vertebrates and argue that the very divergence of these strategies reveals the surprising level of plasticity of the chordate developmental program and is a rich resource to identify core regulatory mechanisms that are evolutionarily conserved in chordates. Further, we propose that the comparative analysis of the architecture of ascidian and vertebrate gene regulatory networks may provide critical insight into the origin of the chordate body plan.     

The caspase family in urochordates: distinct evolutionary fates in ascidians and larvaceans

BACKGROUND INFORMATION: Caspases are cysteine proteases that mediate apoptosis (programmed cell death) initiation and execution. Apoptosis is a conserved mechanism shared by all metazoans, although its physiological function and complexity show considerable taxon-dependent variations. To gain insight into the caspase repertoire of putative ancestors to vertebrates, we performed exhaustive genomic searches in urochordates, a sister taxon to vertebrates in which ascidians and appendicularians display chordate characters at early stages of their development. RESULTS: We identified the complete caspase families of two ascidians (Ciona intestinalis and C. savignyi) and one larvacean (Oikopleura dioica). We found in ascidian species an extremely high number of caspase genes (17 for C. intestinalis and 22 for C. savignyi), deriving from five founder gene orthologues to human pro-inflammatory, initiator and executioner caspases. Although considered to be sibling species, C. intestinalis and C. savignyi only share 11 orthologues, most of the additional genes resulting from recent mass duplications. A sharply contrasted picture was found in O. dioica, which displayed only three caspase genes deriving from a single founder gene distantly related to caspase 3/7. The difference between ascidian and larvacean caspase repertoires is discussed in the light of their developmental patterns and life cycles. CONCLUSIONS: The identification of caspase members in two ascidian species delineates five founder genes that bridge the gap between vertebrates and Ecdysozoa (arthropods and nematodes). Given the amazing diversity among urochordates, determination and comparison of the caspase repertoires in species from orders additional to Enterogona (ascidians) and Oikopleuridae might be highly informative on the evolution of caspase-dependent physiological processes.     

Development of the appendicularian Oikopleura dioica: Culture, genome, and cell lineages

Appendicularians are planktonic tunicates (urochordates), and retain a swimming tadpole shape throughout their life. Together with ascidians, they are the closest relatives of the vertebrates. Oikopleura dioica is characterized by its simplified life habit and anatomical organization. It has a tiny genome, the smallest ever found in a chordate. Its life cycle is extremely short – about 5 days – and it can be maintained in the laboratory over many generations. Embryos and adults are transparent and consist of a small number of cells. The anatomy of juveniles and adults has been described in detail. Cleavage pattern, cell lineages, and morphogenetic movements during embryogenesis have also been comprehensively documented. A draft genome sequence is now available. These features make this organism a suitable experimental model animal in which genetic manipulations would be feasible, as in Drosophila and Caenorhabditis elegans . In this review, I summarize a hundred years' knowledge on the development throughout the life cycle of this organism. Oikopleura is an attractive organism for developmental and evolutionary studies of chordates. It offers considerable promise for future genetic approaches.     

A novel amphioxus cadherin that localizes to epithelial adherens junctions has an unusual domain organization with implications for chordate phylogeny

Although data are available from only vertebrates, urochordates, and three nonchordate animals, there are definite differences in the structures of classic cadherins between vertebrates plus urochordates and nonchordates. In this study we examined structural diversity of classic cadherins among bilaterian animals by obtaining new data from an amphioxus (Cephalochordata, Chordata), an acorn worm (Hemichordata), a sea star (Echinodermata), and an oyster (Mollusca). The structures of newly identified nonchordate cadherins are grouped together with those of the known sea urchin and Drosophila cadherins, whereas the structure of an amphioxus (Branchiostoma belcheri) cadherin, designated BbC, is differently categorized from those of other known chordate cadherins. BbC is identified as a cadherin by its cytoplasmic domain whose sequence is highly related to the cytoplasmic sequences of all known classic cadherins, but it lacks all of the five repeats constituting the extracellular homophilic-binding domain of other chordate cadherins. The ectodomains of BbC match the ectodomains found in nonchordate cadherins but not present in other chordate cadherins. We show that the BbC functions as a cell-cell adhesion molecule when expressed in Drosophila S2 cells and localizes to adherens junctions in the ectodermal epithelia in amphioxus embryos. We argue that BbC is the amphioxus homologue of the classic cadherins involved in the formation of epithelial adherens junctions. The structural relationships of the cadherin molecules allow us to propose a possibility that cephalochordates might be basal to the sister-groups vertebrates and urochordates.     

Mutations affecting tail and notochord development in the ascidian Ciona savignyi.

Ascidians are among the most distant chordate relatives of the vertebrates. However, ascidians share many features with vertebrates including a notochord and hollow dorsal nerve cord. A screen for N-ethyl-N-nitrosourea (ENU)-induced mutations affecting early development in the ascidian Ciona savignyi resulted in the isolation of a number of mutants including the complementing notochord mutants chongmague and chobi. In chongmague embryos the notochord fails to develop, and the notochord cells instead adopt a mesenchyme-like fate. The failure of notochord development in chongmague embryos results in a severe truncation of tail, although development of the tail muscles and caudal nerve tracts appears largely normal. Chobi embryos also have a truncation of the tail stemming from a disruption of the notochord. However, in chobi embryos the early development of the notochord appears normal and defects occur later as the notochord attempts to extend and direct elongation of the tail. We find in chobi tailbud embryos that the notochord is often bent, with cells clumped together, rather than extended as a column. These results provide new information on the function and development of the ascidian notochord. In addition, the results demonstrate how the unique features of ascidians can be used in genetic analysis of morphogenesis.     

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.     

Common and divergent pathways in alternative developmental processes of ascidians

Colonial ascidians offer opportunities to investigate how developmental events are integrated to generate the animal form, since they can develop similar individuals (oozooids from eggs, blastozooids from pluripotent somatic cells) through very different reproductive processes, i.e. embryogenesis and blastogenesis. Moreover, thanks to their key phylogenetic position, they can help in the understanding of the molecular mechanisms of morphogenesis and their evolution in chordates. We review organogenesis of the ascidian neural complex comparing embryos and buds in terms of topology, developmental mechanisms and terminology. We propose a new interpretation of bud territories, and reconsider nervous system development based on recent results suggesting that ascidians have vertebrate placodal and neural-crest-like cells. Comparing embryonic and blastogenic development in Botryllus schlosseri, we propose that the bud has territories with a placodal potentiality, suggesting that chordate ancestors possessed neurogenic placodes, and that the genetic pathways regulating neurogenic placode formation were co-opted for new developmental processes, such as blastogenesis.     

Decoding cis-regulatory systems in ascidians

Ascidians, or sea squirts, are lower chordates, and share basic gene repertoires and many characteristics, both developmental and physiological, with vertebrates. Therefore, decoding cis-regulatory systems in ascidians will contribute toward elucidating the genetic regulatory systems underlying the developmental and physiological processes of vertebrates. cis-Regulatory DNAs can also be used for tissue-specific genetic manipulation, a powerful tool for studying ascidian development and physiology. Because the ascidian genome is compact compared with vertebrate genomes, both intergenic regions and introns are relatively small in ascidians. Short upstream intergenic regions contain a complete set of cis-regulatory elements for spatially regulated expression of a majority of ascidian genes. These features of the ascidian genome are a great advantage in identifying cis-regulatory sequences and in analyzing their functions. Function of cis-regulatory DNAs has been analyzed for a number of tissue-specific and developmentally regulated genes of ascidians by introducing promoter-reporter fusion constructs into ascidian embryos. The availability of the whole genome sequences of the two Ciona species, Ciona intestinalis and Ciona savignyi, facilitates comparative genomics approaches to identify cis-regulatory DNAs. Recent studies demonstrate that computational methods can help identify cis-regulatory elements in the ascidian genome. This review presents a comprehensive list of ascidian genes whose cis-regulatory regions have been subjected to functional analysis, and highlights the recent advances in bioinformatics and comparative genomics approaches to cis-regulatory systems in ascidians.     

Development of a chordate anterior-posterior axis without classical retinoic acid signaling

Developmental signaling by retinoic acid (RA) is thought to be an innovation essential for the origin of the chordate body plan. The larvacean urochordate Oikopleura dioica maintains a chordate body plan throughout life, and yet its genome appears to lack genes for RA synthesis, degradation, and reception. This suggests the hypothesis that the RA-machinery was lost during larvacean evolution, and predicts that Oikopleura development has become independent of RA-signaling. This prediction raises the problem that the anterior-posterior organization of a chordate body plan can be developed without the classical morphogenetic role of RA. To address this problem, we performed pharmacological treatments and analyses of developmental molecular markers to investigate whether RA acts in anterior-posterior axial patterning in Oikopleura embryos. Results revealed that RA does not cause homeotic posteriorization in Oikopleura as it does in vertebrates and cephalochordates, and showed that a chordate can develop the phylotypic body plan in the absence of the classical morphogenetic role of RA. A comparison of Oikopleura and ascidian evidence suggests that the lack of RA-induced homeotic posteriorization is a shared derived feature of urochordates. We discuss possible relationships of altered roles of RA in urochordate development to genomic events, such as rupture of the Hox-cluster, in the context of a new understanding of chordate phylogeny.     

The evolution of anural larvae in molgulid ascidians

Ascidians are urochordates, marine invertebrates with non-feeding motile chordate tadpole larvae, except in the family Molgulidae. Urodele, or tailed, Molgulids have typical ascidian chordate tadpole larvae possessing tails with muscle cells, a notochord, and a dorsal hollow nerve cord. In contrast, anural (or tail-less) Molgulids lack a tail and defining chordate features. Molecular phylogenies generated with 18S and 28S ribosomal sequences indicate that Molgulid species fall into at least four distinct clades, three of which have multiple anural members. This refined and expanded phylogeny allows careful examination of the factors that may have influenced the evolution of tail-less ascidians.     

Evolutionary crossroads in developmental biology: the tunicates

The tunicates, or urochordates, constitute a large group of marine animals whose recent common ancestry with vertebrates is reflected in the tadpole-like larvae of most tunicates. Their diversity and key phylogenetic position are enhanced, from a research viewpoint, by anatomically simple and transparent embryos, compact rapidly evolving genomes, and the availability of powerful experimental and computational tools with which to study these organisms. Tunicates are thus a powerful system for exploring chordate evolution and how extreme variation in genome sequence and gene regulatory network architecture is compatible with the preservation of an ancestral chordate body plan.     

Enhancer evolution in chordates: Lessons from functional analyses of cephalochordate cis-regulatory modules

Chordates comprise three major groups, cephalochordates (amphioxus), tunicates (urochordates), and vertebrates. Since cephalochordates were the early branching group, comparisons between amphioxus and other chordates help us to speculate about ancestral chordates. Here, I summarize accumulating data from functional studies analyzing amphioxus cis-regulatory modules (CRMs) in model systems of other chordate groups, such as mice, chickens, clawed frogs, fish, and ascidians. Conservatism and variability of CRM functions illustrate how gene regulatory networks have evolved in chordates. Amphioxus CRMs, which correspond to CRMs deeply conserved among animal phyla, govern reporter gene expression in conserved expression domains of the putative target gene in host animals. In addition, some CRMs located in similar genomic regions (intron, upstream, or downstream) also possess conserved activity, even though their sequences are divergent. These conservative CRM functions imply ancestral genomic structures and gene regulatory networks in chordates. However, interestingly, if expression patterns of amphioxus genes do not correspond to those of orthologs of experimental models, some amphioxus CRMs recapitulate expression patterns of amphioxus genes, but not those of endogenous genes, suggesting that these amphioxus CRMs are close to the ancestral states of chordate CRMs, while vertebrates/tunicates innovated new CRMs to reconstruct gene regulatory networks subsequent to the divergence of the cephalochordates. Alternatively, amphioxus CRMs may have secondarily lost ancestral CRM activity and evolved independently. These data help to solve fundamental questions of chordate evolution, such as neural crest cells, placodes, a forebrain/midbrain, and genome duplication. Experimental validation is crucial to verify CRM functions and evolution.     

Ascidians as excellent chordate models for studying the development of the nervous system during embryogenesis and metamorphosis

The swimming larvae of the chordate ascidians possess a dorsal hollowed central nervous system (CNS), which is homologous to that of vertebrates. Despite the homology, the ascidian CNS consists of a countable number of cells. The simple nervous system of ascidians provides an excellent experimental system to study the developmental mechanisms of the chordate nervous system. The neural fate of the cells consisting of the ascidian CNS is determined in both autonomous and non-autonomous fashion during the cleavage stage. The ascidian neural plate performs the morphogenetic movement of neural tube closure that resembles that in vertebrate neural tube formation. Following neurulation, the CNS is separated into five distinct regions, whose homology with the regions of vertebrate CNS has been discussed. Following their larval stage, ascidians undergo a metamorphosis and become sessile adults. The metamorphosis is completed quickly, and therefore the metamorphosis of ascidians is a good experimental system to observe the reorganization of the CNS during metamorphosis. A recent study has shown that the major parts of the larval CNS remain after the metamorphosis to form the adult CNS. In contrast to such a conserved manner of CNS reorganization, most larval neurons disappear during metamorphosis. The larval glial cells in the CNS are the major source for the formation of the adult CNS, and some of the glial cells produce adult neurons.     

Evolution of the chordate muscle actin gene

Summary The ascidians Styela plicata, S. clava, and Mogula citrina are urochordates. The larvae of urochordates are considered to morphologically resemble the ancestral vertebrate. We asked whether larval and adult ascidian muscle actin sequences are nonmusclelike as in lower invertebrates, musclelike as in vertebrates, or possess characteristics of both. Nonmuscle and muscle actin cDNA clones from S. plicata were sequenced. Based on 27 diagnostic amino acids, which distinguish vertebrate muscle actin from other actins, we found that the deduced protein sequences of ascidian muscle actins exhibit similarities to both invertebrate and vertebrate muscle actins. A comparison to muscle actins from different vertebrate and invertebrate phylogenetic groups suggested that the urochordate muscle actins represent a transition from a nonmusclelike sequence to a vertebrate musclelike sequence. The ascidian adult muscle actin is more similar to skeletal actin and the larval muscle actin is more similar to cardiac actin, which indicates that the divergence of the skeletal and cardiac isoforms occurred before the emergence of urochordates. The muscle actin gene may be a powerful probe for investigating the chordate lineage. Offprint requests to: C.R. Tomlinson