Brachyury (T) expression in embryos of a larvacean urochordate, Oikopleura dioica, and the ancestral role of T

The Brachyury, or T, gene is required for notochord development in animals occupying all three chordate subphyla and probably also had this role in the last common ancestor of the chordate lineages. In two chordate subphyla (vertebrates and cephalochordates), T is also expressed during gastrulation in involuting endodermal and mesodermal cells, and in vertebrates at least, this expression domain is required for proper development. In the basally diverging chordate subphylum Urochordata, animals in the class Ascidiacea do not employ T during gastrulation in endodermal or nonaxial mesodermal cells, and it has been suggested that nonnotochordal roles for T were acquired in the cephalochordate-vertebrate lineage after it split with Urochordata. To test this hypothesis, we cloned T from Oikopleura dioica, a member of the urochordate class Appendicularia (or Larvacea), which diverged basally in the subphylum. Investigation of the expression pattern in developing Oikopleura embryos showed early expression in presumptive notochord precursor cells, in the notochord, and in parts of the developing gut and cells of the endodermal strand. We conclude that the ancestral role of T likely included expression in the developing gut and became necessary in chordates for construction of the notochord.     

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.     

Brachyury (T) Expression in Embryos of a Larvacean Urochordate, Oikopleura dioica, and the Ancestral Role of T

The Brachyury, or T, gene is required for notochord development in animals occupying all three chordate subphyla and probably also had this role in the last common ancestor of the chordate lineages. In two chordate subphyla (vertebrates and cephalochordates), T is also expressed during gastrulation in involuting endodermal and mesodermal cells, and in vertebrates at least, this expression domain is required for proper development. In the basally diverging chordate subphylum Urochordata, animals in the class Ascidiacea do not employ T during gastrulation in endodermal or nonaxial mesodermal cells, and it has been suggested that nonnotochordal roles for T were acquired in the cephalochordate–vertebrate lineage after it split with Urochordata. To test this hypothesis, we cloned T from Oikopleura dioica, a member of the urochordate class Appendicularia (or Larvacea), which diverged basally in the subphylum. Investigation of the expression pattern in developing Oikopleura embryos showed early expression in presumptive notochord precursor cells, in the notochord, and in parts of the developing gut and cells of the endodermal strand. We conclude that the ancestral role of T likely included expression in the developing gut and became necessary in chordates for construction of the notochord.     

Development of the central nervous system in the larvacean Oikopleura dioica and the evolution of the chordate brain

In non-vertebrate chordates, central nervous system (CNS) development has been studied in only two taxa, the Cephalochordata and a single Class (Ascidiacea) of the morphologically diverse Urochordata. To understand development and molecular regionalization of the brain in a different deeply diverging chordate clade, we isolated and determined the expression patterns of orthologs of vertebrate CNS markers (otxa, otxb, otxc, pax6, pax2/5/8a, pax2/5/8b, engrailed, and hox1) in Oikopleura dioica (Subphylum Urochordata, Class Larvacea). The three Oikopleura otx genes are expressed similarly to vertebrate Otx paralogs, demonstrating that trans-homologs converged on similar evolutionary outcomes by independent neo- or subfunctionalization processes during the evolution of the two taxa. This work revealed that the Oikopleura CNS possesses homologs of the vertebrate forebrain, hindbrain, and spinal cord, but not the midbrain. Comparing larvacean gene expression patterns to published results in ascidians disclosed important developmental differences and similarities that suggest mechanisms of development likely present in their last common ancestor. In contrast to ascidians, the lack of a radical reorganization of the CNS as larvaceans become adults allows us to relate embryonic gene expression patterns to three subdivisions of the adult anterior brain. Our study of the Oikopleura brain provides new insights into chordate CNS evolution: first, the absence of midbrain is a urochordate synapomorphy and not a peculiarity of ascidians, perhaps resulting from their drastic CNS metamorphosis; second, there is no convincing evidence for a homolog of a midbrain-hindbrain boundary (MHB) organizer in urochordates; and third, the expression pattern of "MHB-genes" in the urochordate hindbrain suggests that they function in the development of specific neurons rather than in an MHB organizer.     

Characterization of Brachyury-downstream notochord genes in the Ciona intestinalis embryo

The notochord has two major roles during chordate embryogenesis, as a source of inductive signals for the patterning of neural tube and paraxial mesoderm and as a supportive organ of the larval tail. Despite the recent identification of mutations that affect the notochord development in vertebrate embryos, little is known about genes that are expressed in the differentiating notochord itself. In the urochordate ascidian Ciona intestinalis, Brachyury (Ci-Bra) plays a key role in notochord differentiation. In a previous study, we isolated cDNA clones for nearly 40 potential Ci-Bra target genes that are expressed in notochord cells (H. Takahashi et al., 1999, Genes Dev. 13, 1519-1523). Here we characterized 20 of them by determining the complete nucleotide sequences of the cDNAs. These genes encode a broad spectrum of divergent proteins associated with notochord formation and function. Two genes encode ascidian homologs of the Drosophila Prickle LIM domain proteins and another encodes the ERM protein, all 3 of which appear to be involved in the control of cytoskeletal architecture. In addition, genes for netrin, leprecan, cdc45, ATP:citrate lyase, ATP sulfurylase/APS kinase, protein tyrosine phosphatase, beta4-galactosyltransferase, fibrinogen-like protein, divergent tropomyosin-like proteins, and Drosophila Pellino-like protein were identified. The observation of the netrin gene expression in the notochord may provide the first molecular evidence that the ascidian notochord is a source of signals as in vertebrates. In addition, the present information should be used to identify nonchordate deuterostome tissues homologous to the notochord as well as genes which are expressed in the notochord cells of vertebrate embryos.     

Ascidian larva reveals ancient origin of vertebrate-skeletal-muscle troponin I characteristics in chordate locomotory muscle

Ascidians are protochordates related to vertebrate ancestors. The ascidian larval tail, with its notochord, dorsal nerve cord, and flanking rows of sarcomeric muscle cells, exhibits the basic chordate body plan. Molecular characterization of ascidian larval tail muscle may provide insight into molecular aspects of vertebrate skeletal muscle evolution. We report studies of the Ci-TnI gene of the ascidian Ciona intestinalis, which encodes the muscle contractile regulatory protein troponin I (TnI). Previous studies of a distantly related ascidian, Halocynthia roretzi, showed that different TnI genes were expressed in larval and adult muscles, the larval TnI isoforms having an unusual C-terminal truncation not seen in any vertebrate TnI. Here we show that, in contrast with Halocynthia, Ciona does not have a specialized larval TnI; the same TnI gene that is expressed in the heart and body-wall muscle of the sessile adult is also expressed in embryonic/larval tail muscle cells. Moreover the TnI isoform produced in embryonic/larval muscle is identical to that produced in adult body-wall muscle, i.e., a 182-residue protein with the characteristic chain length and overall structure of vertebrate skeletal muscle TnI isoforms. Phylogenetic analyses indicate that the unique features of Halocynthia larval TnI likely represent derived features, and hence that the vertebrate-skeletal-muscle -like TnI of Ciona is a closer reflection of the ancestral ascidian larval TnI. Our results indicate that characteristics of vertebrate skeletal muscle TnI emerged early in the evolution of chordate locomotory muscle, before the ascidian/vertebrate divergence. These features could be related to a basal chordate locomotory innovation-e.g., swimming by oscillation of an internal notochord skeleton-or they may be of even greater antiquity within the deuterostomes.     

Long-distance cell migration during larval development in the appendicularian, Oikopleura dioica

The appendicularian, Oikopleura dioica, is a planktonic chordate. Its simple and transparent body, invariant cell lineages and short life cycle of 5 days make it a promising model organism for studies of chordate development. Here we describe the cell migration that occurs during development of the O. dioica larva. Using time-lapse imaging facilitated by florescent labeling of cells, three cell populations exhibiting long-distance migration were identified and characterized. These included (i) a multinucleated oral gland precursor that migrates anteriorly within the trunk region and eventually separates into the left and right sides, (ii) endodermal strand cells that are collectively retracted from the tail into the trunk in a tractor movement, and (iii) two subchordal cell precursors that individually migrate out from the trunk to the tip of the tail. The migration of subchordal cell precursors starts when all of the endodermal strand cells enter the trunk, and follows the same path but in a direction opposite to that of the latter. Labeling of these cells with a photoconvertible fluorescent protein, Kaede, demonstrated that the endodermal strand cells and subchordal cell precursors have distinct origins and eventual fates. Surgical removal of the trunk from the tail demonstrated that the endodermal strand cells do not require the trunk for migration, and that the subchordal cell precursors would be attracted by the distal part of the tail. This well-defined, invariant and traceable long-distance cell migration provides a unique experimental system for exploring the mechanisms of versatile cell migration in this simple organism with a chordate body plan.     

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.     

Cleavage pattern,gastrulation, and neurulation in the appendicularian, <Emphasis Type="Italic">Oikopleura dioica</Emphasis>

The appendicularian, Oikopleura dioica is a chordate. Its life cycle is extremely short—approximately 5 days—and its tadpole shape with a beating tail is retained throughout entire life. The tadpole hatches after 3 h of development at 20°C. Here, we describe the cleavage pattern and morphogenetic cell movements during gastrulation and neurulation. Cleavage showed an invariant pattern. It is basically bilateral but also shows various minor left–right asymmetries starting from the four-cell stage. We observed two rounds of unequal cleavage of the posterior-vegetal B-line cells at the posterior pole. The nature of the unequal cleavages is reminiscent of those in ascidian embryos and suggests the presence of a centrosome-attracting body, a special subcellular structure at the posterior pole. The representation of the cell division pattern in this report will aid the identification of each cell, a prerequisite for clarifying the gene expression patterns in early embryos. Gastrulation started as early as the 32-cell stage and progressed in three phases. By the end of the second phase at the 64-cell stage, every vegetal cell had ingressed into the embryo, and animal cells had covered the entire embryo by epiboly. There was no archenteron formation. In the anterior region, eight A-line cells were aligned as a 2 × 4 array along the anterior–posterior axis and become internalized during the 64-cell stage. This process was considered to correspond to neurulation. The simple and accelerated development of Oikopleura, nevertheless giving rise to a conserved chordate body plan, is advantageous for studying developmental mechanisms using molecular and genetic approaches and makes this animal the simplest model organism in the phylum Chordata. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The forkhead gene FH1 is involved in evolutionary modification of the ascidian tadpole larva.

The forkhead gene FH1 encodes a HNF-3beta protein required for gastrulation and development of chordate features in the ascidian tadpole larva. Although most ascidian species develop via a tadpole larva, the conventional larva has regressed into an anural (tailless) larva in some species. Molgula oculata (the tailed species) exhibits a tadpole larva with chordate features (a dorsal neural sensory organ or otolith, a notochord, striated muscle cells, and a tail), whereas its sister species Molgula occulta (the tailless species) has evolved an anural larva, which has lost these features. Here we examine the role of FH1 in modifying the larval body plan in the tailless species. We also examine FH1 function in tailless speciesxtailed species hybrids, in which the otolith, notochord, and tail are restored. The FH1 gene is expressed primarily in the presumptive endoderm and notochord cells during gastrulation, neurulation, and larval axis formation in both species and hybrids. In the tailless species, FH1 expression is down-regulated after neurulation in concert with arrested otolith, notochord, and tail development. The FH1 expression pattern characteristic of the tailed species is restored in hybrid embryos prior to the development of chordate larval features. Antisense oligodeoxynucleotides (ODNs) shown previously to disrupt FH1 function were used to compare the developmental roles of this gene in both species and hybrids. As described previously, antisense FH1 ODNs inhibited endoderm invagination during gastrulation, notochord extension, and larval tail formation in the tailed species. Antisense FH1 ODNs also affected gastrulation in the tailless species, although the effects were less severe than in the tailed species, and an anural larva was formed. In hybrid embryos, antisense FH1 ODNs blocked restoration of the otolith, notochord, and tail, reverting the larva back to the anural state. The results suggest that changes in FH1 expression are involved in re-organizing the tadpole larva during the evolution of anural development.     

Polarized basolateral cell motility underlies invagination and convergent extension of the ascidian notochord.

We use 3D time-lapse analysis of living embryos and laser scanning confocal reconstructions of fixed, staged, whole-mounted embryos to describe three-dimensional patterns of cell motility, cell shape change, cell rearrangement and tissue deformation that accompany formation of the ascidian notochord. We show that notochord formation involves two simultaneous processes occurring within an initially monolayer epithelial plate: The first is invagination of the notochord plate about the axial midline to form a solid cylindrical rod. The second is mediolaterally directed intercalation of cells within the plane of the epithelial plate, and then later about the circumference of the cylindrical rod, that accompanies its extension along the anterior/posterior (AP) axis. We provide evidence that these shape changes and rearrangements are driven by active extension of interior basolateral notochord cell edges directly across the faces of their adjacent notochord neighbors in a manner analogous to leading edge extension of lamellapodia by motile cells in culture. We show further that local edge extension is polarized with respect to both the AP axis of the embryo and the apicobasal axis of the notochord plate. Our observations suggest a novel view of how active basolateral motility could drive both invagination and convergent extension of a monolayer epithelium. They further reveal deep similarities between modes of notochord morphogenesis exhibited by ascidians and other chordate embryos, suggesting that cellular mechanisms of ascidian notochord formation may operate across the chordate phylum.     

Cellular features of an apoptotic form of programmed cell death during the development of the ascidian, Boltenia villosa

The phylogenetic position of ascidians near the base of the chordate tree makes them ideal organisms for evolutionary developmental studies of programmed cell death (PCD). In the present study, the following key features of an apoptotic form of PCD are described in Boltenia villosa: fragmentation of DNA, increases in plasma membrane permeability, decreases in mitochondrial activity, production of reactive oxygen species (ROS), and caspase activation. First, evidence is presented for apoptosis of cells within the ovary. Later in development, during the early phase of larval tail resorption at the beginning of metamorphosis, some notochord nuclei showed DNA fragmentation and their cell corpses were rapidly eliminated from the larval body. In striking contrast to the rapid demise of notochord cells, larval muscle cells persisted for more than a week within developing juveniles. Rhodamine 123 and MTT experiments suggest that mitochondria within some of the resorbed larval tail muscle cells were metabolically active for more than a week. Furthermore, resorbed tail muscle cells contained a muscle-specific intermediate filament, termed p58, despite relatively high levels of ROS activity and the ubiquitination of their plasma membranes at day two. Corpses of larval tail muscle cells containing aggregated pigment granules survived within juveniles for more than a month, in contrast to the rapid elimination of notochord cells. Evidence consistent with the formation of larval muscle cell apoptotic bodies is presented. The most surprising result of the present study was that caspase-8, usually associated with apoptotic signaling, was activated in larval endoderm cells that develop into adult structures. When the present results were compared to features of PCD previously reported in other ascidians, significant species differences in PCD were revealed.     

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.     

Tail morphogenesis in the ascidian, Ciona intestinalis, requires cooperation between notochord and muscle

We present evidence that notochord and muscle differentiation are crucial for morphogenesis of the ascidian tail. We developed a novel approach for embryological manipulation of the developing larval tissues using a simple method to introduce DNA into Ciona intestinalis and the several available tissue-specific promoters. With such promoters, we misexpressed the Xenopus homeobox gene bix in notochord or muscle of Ciona embryos as a means of interfering with development of these tissues. Ciona embryos expressing bix in the notochord from the 64-cell stage develop into larvae with very short tails, in which the notochord precursors fail to intercalate and differentiate. Larvae with mosaic expression of bix have intermediate phenotypes, in which a partial notochord is formed by the precursor cells that did not receive the transgene while the precursors that express the transgene cluster together and fail to undergo any of the cell-shape changes associated with notochord differentiation. Muscle cells adjacent to differentiated notochord cells are properly patterned, while those next to the notochord precursor cells transformed by bix exhibit various patterning defects. In these embryos, the neural tube extends in the tail to form a nerve cord, while the endodermal strand fails to enter the tail region. Similarly, expression of bix in muscle progenitors impairs differentiation of muscle cells, and as a result, notochord cells fail to undergo normal extension movements. Hence, these larvae have a shorter tail, due to a block in the elongation of the notochord. Taken together, these observations suggest that tail formation in ascidian larvae requires not only signaling from notochord to muscle cells, but also a "retrograde" signal from muscle cells to notochord.     

Interaction of notochord-derived fibrinogen-like protein with Notch regulates the patterning of the central nervous system of Ciona intestinalis embryos

The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS.