Molecular evidence from ascidians for the evolutionary origin of vertebrate cranial sensory placodes

Cranial sensory placodes are specialised areas of the head ectoderm of vertebrate embryos that contribute to the formation of the cranial sense organs and associated ganglia. Placodes are often considered a vertebrate innovation, and their evolution has been hypothesised as one key adaptation underlying the evolution of active predation by primitive vertebrates. Here, we review recent molecular evidence pertinent to understanding the evolutionary origin of placodes. The development of vertebrate placodes is regulated by numerous genes, including members of the Pax, Six, Eya, Fox, Phox, Neurogenin and Pou gene families. In the sea squirt Ciona intestinalis (a basal chordate and close relative of the vertebrates), orthologues of these genes are deployed in the development of the oral and atrial siphons, structures used for filter feeding by the sessile adult. Our interpretation of these findings is that vertebrate placodes and sea squirt siphon primordia have evolved from the same patches of specialised ectoderm present in the common ancestor of the chordates.     

Regeneration of oral siphon pigment organs in the ascidian Ciona intestinalis

Ascidians have powerful capacities for regeneration but the underlying mechanisms are poorly understood. Here we examine oral siphon regeneration in the solitary ascidian Ciona intestinalis. Following amputation, the oral siphon rapidly reforms oral pigment organs (OPO) at its distal margin prior to slower regeneration of proximal siphon parts. The early stages of oral siphon reformation include cell proliferation and re-growth of the siphon nerves, although the neural complex (adult brain and associated organs) is not required for regeneration. Young animals reform OPO more rapidly after amputation than old animals indicating that regeneration is age dependent. UV irradiation, microcautery, and cultured siphon explant experiments indicate that OPOs are replaced as independent units based on local differentiation of progenitor cells within the siphon, rather than by cell migration from a distant source in the body. The typical pattern of eight OPOs and siphon lobes is restored with fidelity after distal amputation of the oral siphon, but as many as 16 OPOs and lobes can be reformed following proximal amputation near the siphon base. Thus, the pattern of OPO regeneration is determined by cues positioned along the proximal distal axis of the oral siphon. A model is presented in which columns of siphon tissue along the proximal-distal axis below pre-existing OPO are responsible for reproducing the normal OPO pattern during regeneration. This study reveals previously unknown principles of oral siphon and OPO regeneration that will be important for developing Ciona as a regeneration model in urochordates, which may be the closest living relatives of vertebrates.     

Developmental fates of larval tissues after metamorphosis in the ascidian, Halocynthia roretzi

Cell lineages during ascidian embryogenesis are invariant. Developmental fates of larval mesodermal cells after metamorphosis are also invariant with regard to cell type of descendants. The present study traced developmental fates of larval endodermal cells after metamorphosis in Halocynthia roretzi by labeling each endodermal precursor blastomere of larval endoderm. Larval endodermal cells gave rise to various endodermal organs of juveniles: endostyle, branchial sac, peribranchial epithelium, digestive organs, peripharyngeal band, and dorsal tubercle. The boundaries between clones descended from early blastomeres did not correspond to the boundaries between adult endodermal organs. Although there is a regular projection from cleavage stage and larval stage to juvenile stage, this varies to some extent between individuals. This indicates that ascidian development is not entirely deterministic. We composed a fate map of adult endodermal organs in larval endoderm based on a statistical analysis of many individual cases. Interestingly, the topographic position of each prospective region in the fate map was similar to that of the adult organ, indicating that marked rearrangement of the positions of endodermal cells does not occur during metamorphosis. These findings suggest that fate specification in endoderm cells during metamorphosis is likely to be a position-dependent rather than a deterministic and lineage-based process. Received: 16 June 1999 / Accepted: 16 August 1999     

Tissue-specific profile of DNA replication in the swimming larvae of Ciona intestinalis

The cell cycle is strictly regulated during development and its regulation is essential for organ formation and developmental timing. Here we observed the pattern of DNA replication in swimming larvae of an ascidian, Ciona intestinalis. Usually, Ciona swimming larvae obtain competence for metamorphosis at about 4-5 h after hatching, and these competent larvae initiate metamorphosis soon after they adhere to substrate with their papillae. In these larvae, three major tissues (epidermis, endoderm and mesenchyme) showed extensive DNA replication with distinct pattern and timing, suggesting tissue-specific cell cycle regulation. However, DNA replication did not continue in aged larvae which kept swimming for several days, suggesting that the cell cycle is arrested in these larvae at a certain time to prevent further growth of adult organ rudiments until the initiation of metamorphosis. Inhibition of the cell cycle by aphidicolin during the larval stage affects only the speed of metamorphosis, and not the formation of adult organ rudiments or the timing of the initiation of metamorphosis. However, after the completion of tail resorption, DNA replication is necessary for further metamorphic events. Our data showed that DNA synthesis in the larval trunk is not directly associated with the organization of adult organs, but it contributes to the speed of metamorphosis after settlement.     

Isolation and characterization of genes that are expressed during Ciona intestinalis metamorphosis

In ascidians, the events of metamorphosis transform the non-feeding, mobile tadpole larva into a filter-feeding, fixed juvenile, and the process involves rearrangements of cells, two organs and physiological changes. Differential screening was used to isolate two genes that are not expressed in swimming larvae but are expressed immediately after the initiation of metamorphosis in Ciona intestinalis. One of the genes, Ci-meta1, encodes a polypeptide with a putative secretion signal sequence, 6 epidermal growth factor (EGF)-like repeats and 13 calcium-binding EGF-like repeats. The gene begins to be expressed immediately after the beginning of metamorphosis in the adhesive organ and is likely to be associated with the signal response for metamorphosis. Another gene named Ci-meta2 encodes a protein with a putative secretion signal and three thrombospondin type-1 repeats. Ci-meta2 gene expression begins at the larval stage and is upregulated in the metamorphosing juveniles. Ci-meta2 expression is found in three regions; the adhesive organ which is also associated with settlement, the neck region between the trunk and the tail of the larva which is associated with tail resorption, and dorsal regions of the trunk which correspond to the location of the siphon primordium. This gene may be involved in the dynamic arrangement of cells during ascidian metamorphosis.     

Loss of test cells leads to the formation of new tunic surface cells and abnormal metamorphosis in larvae of Ciona intestinalis (Chordata, Ascidiacea)

The larvae of the ascidian Ciona intestinalis from which the chorion with the test cells and follicle cells were removed developed normally without the test cells until the early tailbud stage. A number of round-shaped cells morphologically similar to the test cells but with different lectin affinities and autofluorescence, then appeared on the neck region of the demembranated embryos. The new cells had three different types: round, particulate, and granular, and these cells increased in number after the late tailbud stage. The morphology of the adhesive papillae, tunic layers and epidermis of the demembranated larvae was similar to that of control larvae; however, the affinity to lectins was different in the swimming period. Control larvae attached to the substratum after the swimming period, resorbed the tail completely and underwent rotation of the visceral organs. Conversely, rotation occurred before completion of tail resorption in the demembranated larvae. Furthermore, the metamorphic events progressed more slowly in the demembranated larvae. These results suggest that the test cells play important roles in normal development and morphogenesis of ascidian larvae. Received: 4 December 1998 / Accepted: 9 April 1999     

A conserved role for FGF signaling in chordate otic/atrial placode formation

The widely held view that neurogenic placodes are vertebrate novelties has been challenged by morphological and molecular data from tunicates suggesting that placodes predate the vertebrate divergence. Here, we examine requirements for the development of the tunicate atrial siphon primordium, thought to share homology with the vertebrate otic placode. In vertebrates, FGF signaling is required for otic placode induction and for later events following placode invagination, including elaboration and patterning of the inner ear. We show that results from perturbation of the FGF pathway in the ascidian Ciona support a similar role for this pathway: inhibition with MEK or Fgfr inhibitor at tailbud stages in Ciona results in a larva which fails to form atrial placodes; inhibition during metamorphosis disrupts development of the atrial siphon and gill slits, structures which form where invaginated atrial siphon ectoderm apposes pharyngeal endoderm. We show that laser ablation of atrial primordium ectoderm also results in a failure to form gill slits in the underlying endoderm. Our data suggest interactions required for formation of the atrial siphon and highlight the role of atrial ectoderm during gill slit morphogenesis.     

Primordial germ cells originate from the endodermal strand cells in the ascidian Ciona intestinalis

The origin of germ cells in the ascidian is still unknown. Previously, we cloned a vasa homologue (CiVH) of Ciona intestinalis from the cDNA library of ovarian tissue by polymerase chain reaction and showed that its expression was specific to germ cells in adult and juvenile gonads. In the present study, we prepared a monoclonal antibody against CiVH protein and traced the staining for this antibody from the middle tailbud stage to young adulthood. Results showed that positive cells are present in the endodermal strand in middle tailbud embryos and larvae. When the larval tail was absorbed into the trunk during metamorphosis, the CiVH-positive cells migrated from the debris of the tail into the developing gonad rudiment, and appeared to give rise to a primordial germ cell (PGC) in the young juvenile. The testis rudiment separated from the gonad rudiment, the remainder of which differentiated into the ovary. PGCs of the testis rudiment and the ovary rudiment differentiated into spermatogenic and oogenic cells, respectively. When the larval tail containing the antibody-positive cells was removed, the juveniles did not contain any CiVH-positive cells after metamorphosis, indicating that the PGCs in the juvenile originated from part of the larval tail. However, even in such juveniles, positive cells newly appeared in the gonad rudiment at a later stage. This observation suggests that a compensatory mechanism regulates germline formation in C. intestinalis.     

Lineage segregation and developmental autonomy in expression of functional muscle acetylcholinesterase mRNA in the ascidian embryo

Acetylcholinesterase is a histospecific marker of cell differentiation occurring only in the muscle and mesenchyme tissues of the ascidian embryo. The distribution of functional mRNA coding for this enzyme has been investigated and it is shown here that only cells of muscle and mesenchyme lineages possess such a template. Blastomeres of four cell lineage quadrants were separated microsurgically from eight-cell-stage embryos of Ciona intestinalis and raised in isolation until muscle development was well advanced. Measurement of enzyme activity in the resulting partial embryos revealed that acetylcholinesterase was limited to descendants of one blastomere pair, the B4.1 blastomeres containing muscle and mesenchyme lineages. To study the tissue distribution of acetylcholinesterase mRNA, RNA from partial embryos was translated in Xenopus laevis oocytes. When oocytes were injected with an appropriate template, they synthesized a biologically active acetylcholinesterase that could be selectively immunopurified with an antiserum to the ascidian enzyme. Under the conditions used the quantity of acetylcholinesterase mRNA was directly related to the enzyme activity in immunoprecipitates. Acetylcholinesterase mRNA was found only in B4.1 lineage partial embryos where it occurred in approximately the same amount as in whole embryos of the same age. Since there is a limited period from gastrulation until the middle tail-formation stage when functional acetylcholinesterase mRNA accumulates, the results of our mRNA distribution experiments strongly suggest that the gene for ascidian acetylcholinesterase is active only in muscle and mesenchyme tissues. The histospecific occurrence of this enzyme apparently does not involve selective, cell-specific control of translation.     

Expression and functional analysis of Cititf1, an ascidian NK-2 class gene, suggest its role in endoderm development.

In solitary ascidians the fate of endoderm is determined at a very early stage of development and depends on cytoplasmic factors whose nature has not been determined. We have isolated a member of the NK-2 gene family, Cititf1, from the ascidian Ciona intestinalis, showing high sequence homology to mammalian TITF1. The Cititf1 gene was expressed in all endodermal precursors at the pregastrula and gastrula stages, and is thus the first specific regulatory endodermal marker to be isolated from an ascidian. Cititf1 expression was downregulated at the end of gastrulation to reappear at middle tailbud and larval stages in the most anterior and ventral parts of head endoderm, regions which give rise, after metamorphosis, to the adult endostyle, where Cititf1 mRNA was still present. Microinjection of Cititf1 mRNA into fertilized eggs resulted in tadpole larvae with abnormalities in head-trunk development consequent to the formation of excess endoderm, perhaps due to recruitment of notochord precursors to an endodermal fate. These data suggest that Cititf1 plays an important role in normal endoderm differentiation during ascidian embryogenesis.     

An ascidian homologue of the gonadotropin-releasing hormone receptor is a retinoic acid target gene

Transdifferentiation of the multipotent atrial epithelium is a key event during budding of the ascidian Polyandrocarpa misakiensis. The transdifferentiation is induced by mesenchyme cells that were stimulated by retinoic acid. The fluorescent differential display identified a few cDNA fragments for retinoic acid-inducible genes. One of the cDNA clones, named Pm-GnRHR, encoded a seven-pass transmembrane receptor similar to gonadotropin-releasing hormone receptors. Putative amino acid sequence showed high similarity to Ciona intestinalis GnRHRs and formed a cluster with other GnRHR proteins in a phylogenetic tree. The level of expression of the Pm-GnRHR mRNA increased during the early stage of bud development, suggesting that the Pm-GnRHR function is involved in some aspects of bud development.     

Vestigial Brain Melanocyte Development During Embryogenesis of an Anural Ascidian

Anural development was examined in the ascidian Bostrichobranchus digonas using specific markers for differentiated urodele ascidian larval cells and tissues. In this ovoviviparous anural ascidian, eggs, embryos and developing juveniles were present in the gonads, brood sacs, and atrial cavity, respectively. Morphological studies indicated that B. digonas embryos do not develop into tailed larvae with an extended notochord and differentiated muscle cells. In addition, these embryos lack detectable expression of the muscle-specific markers acetylcholinesterase, alpha actin, and myosin heavy chain. In striking contrast to other anural ascidian embryos, however, B. digonas embryos can develop tyrosinase in several melanocyte precursor cells and eventually form a brain pigment cell. The melanocyte does not become part of a definitive brain sensory organ (otolith) and subsequently disappears during metamorphosis. A period of tyrosinase expression was also observed following metamorphosis in which many tyrosinase-positive cells appear in the body of the developing juvenile. The results demonstrate that different urodele features can be uncoupled during the evolution of anural development. The development of a vestigial brain melanocyte also suggests that B. digonas evolved from a urodele ancestor rather than from another anural ascidian lacking a brain pigment cell.     

Gene expression profile of Ci-VSP in juveniles and adult blood cells of ascidian

VSP is a transmembrane protein whose cytoplasmic region shows significant similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Notably, VSP exhibits a unique ability to transduce electrical signals into phosphoinositide turnover by coupling a transmembrane voltage sensor domain to the PTEN-like phosphoinositide phosphatase domain. Moreover, VSP gene is known to be widely conserved among deuterostome genomes, though the function of VSP in vivo remains largely unknown. In the present study, the expression pattern of ascidian VSP(Ci-VSP) was examined in embryos and juveniles of a marine invertebrate chordate, Ciona intestinalis. RT-PCR showed that Ci-VSP is expressed at the larval stage and that expression persists in juveniles. Whole mount in situ hybridization showed that Ci-VSP is expressed in cells of the stomach, intestine and blood cells of 2- to 3-week-old juveniles. Moreover, double staining blood cells from 2-month-old adults with Ci-VSP and Ci-PTEN probes showed that Ci-VSP-positive cells are a distinct population, separate from cells expressing Ci-PTEN. These findings suggest that in addition to its previously suggested roles in testis or sperm, Ci-VSP plays a key role in voltage-induced signal transduction in cells of the digestive system and blood.     

Differentiation of tropomyosin-containing myofibrils in cleavage-arrested ascidian zygotes expressing acetylcholinesterase

Two muscle differentiation programs, acetylcholinesterase and tropomyosin-containing filaments and fibrils, occur together in the same cleavage-arrested zygotes (1-celled) of the ascidian Ciona intestinalis. Coexpression in such undivided but developing 'embryos' is consistent with the idea that separate elements of muscle differentiation are related at some regulatory level, perhaps through a single multi-gene regulatory factor. Fertilized Ciona eggs were exposed to cytochalasin B for 20 h and then briefly reacted histochemically for acetylcholinesterase activity. Strongly reacting specimens were selected and processed for transmission electron microscopy to reveal regions of muscle ultrastructure. Every acetylcholinesterase-reactive zygote tested contained muscle contractile elements; no example lacking acetylcholinesterase was found with myofilaments and myofibrils. As demonstrated by immunogold labelling, a polyclonal antibody to tropomyosin from Ciona adult body wall reacted differentially with the presumed ultrastructural muscle elements in cleavage-arrested zygotes. Site-specific reactions were also observed in larval tail muscle and the siphon muscles of postmetamorphic zooids.     

Retinoic acid-driven Hox1 is required in the epidermis for forming the otic/atrial placodes during ascidian metamorphosis

Retinoic acid (RA)-mediated expression of the homeobox gene Hox1 is a hallmark of the chordate central nervous system (CNS). It has been suggested that the RA-Hox1 network also functions in the epidermal ectoderm of chordates. Here, we show that in the urochordate ascidian Ciona intestinalis, RA-Hox1 in the epidermal ectoderm is necessary for formation of the atrial siphon placode (ASP), a structure homologous to the vertebrate otic placode. Loss of Hox1 function resulted in loss of the ASP, which could be rescued by expressing Hox1 in the epidermis. As previous studies showed that RA directly upregulates Hox1 in the epidermis of Ciona larvae, we also examined the role of RA in ASP formation. We showed that abolishment of RA resulted in loss of the ASP, which could be rescued by forced expression of Hox1 in the epidermis. Our results suggest that RA-Hox1 in the epidermal ectoderm played a key role in the acquisition of the otic placode during chordate evolution.