An Introduction to an Evolutionary Tail: EvoDevo, Structure, and Function of Post-Anal Appendages.

Although tails are common and versatile appendages that contribute to evolutionary success of animals in a broad range of ways, a scientific synthesis on the topic is yet to be initiated. For our Society for Integrative and Comparative Biology (SICB) symposium, we brought together researchers from different areas of expertise (e.g., roboticists, biomechanists, functional morphologists, and evolutionary and developmental biologists), to highlight their research but also to emphasise the interdisciplinary nature of this topic. The four main themes that emerged based on the research presented in this symposium are: (1) How do we define a tail?, (2) Development and regeneration inform evolutionary origins of tails, (3) Identifying key characteristics highlights functional morphology of tails, and (4) Tail multi-functionality leads to the development of bioinspired technology. We discuss the research provided within this symposium, in light of these four themes. We showcase the broad diversity of current tail research and lay an important foundational framework for future interdisciplinary research on tails with this timely symposium.

Future Tail Tales: A Forward-Looking, Integrative Perspective on Tail Research.

Synopsis Tails are a defining characteristic of chordates and show enormous diversity in function and shape. Although chordate tails share a common evolutionary and genetic-developmental origin, tails are extremely versatile in morphology and function. For example, tails can be short or long, thin or thick, and feathered or spiked, and they can be used for propulsion, communication, or balancing, and they mediate in predator-prey outcomes. Depending on the species of animal the tail is attached to, it can have extraordinarily multi-functional purposes. Despite its morphological diversity and broad functional roles, tails have not received similar scientific attention as, for example, the paired appendages such as legs or fins. This forward-looking review article is a first step toward interdisciplinary scientific synthesis in tail research. We discuss the importance of tail research in relation to five topics: (1) evolution and development, (2) regeneration, (3) functional morphology, (4) sensorimotor control, and (5) computational and physical models. Within each of these areas, we highlight areas of research and combinations of long-standing and new experimental approaches to move the field of tail research forward. To best advance a holistic understanding of tail evolution and function, it is imperative to embrace an interdisciplinary approach, re-integrating traditionally siloed fields around discussions on tail-related research.

Building divergent body plans with similar genetic pathways.

Deuterostome animals exhibit widely divergent body plans. Echinoderms have either radial or bilateral symmetry, hemichordates include bilateral enteropneust worms and colonial pterobranchs, and chordates possess a defined dorsal-ventral axis imposed on their anterior-posterior axis. Tunicates are chordates only as larvae, following metamorphosis the adults acquire a body plan unique for the deuterostomes. This paper examines larval and adult body plans in the deuterostomes and discusses two distinct ways of evolving divergent body plans. First, echinoderms and hemichordates have similar feeding larvae, but build a new adult body within or around their larvae. In hemichordates and many direct-developing echinoderms, the adult is built onto the larva, with the larval axes becoming the adult axes and the larval mouth becoming the adult mouth. In contrast, indirect-developing echinoderms undergo radical metamorphosis where adult axes are not the same as larval axes. A second way of evolving a divergent body plan is to become colonial, as seen in hemichordates and tunicates. Early embryonic development and gastrulation are similar in all deuterostomes, but, in chordates, the anterior-posterior axis is established at right angles to the animal-vegetal axis, in contrast to hemichordates and indirect-developing echinoderms. Hox gene sequences and anterior-posterior expression patterns illuminate deuterostome phylogenetic relationships and the evolution of unique adult body plans within monophyletic groups. Many genes that are considered vertebrate 'mesodermal' genes, such as nodal and brachyury T, are likely to ancestrally have been involved in the formation of the mouth and anus, and later were evolutionarily co-opted into mesoderm during vertebrate development.

Isolation of genes involved in ascidian metamorphosis: epidermal growth factor signaling and metamorphic competence.

Although embryonic development in ascidians has been studied for over a century, the signals involved in coordinating post-larval development and metamorphosis are just beginning to be investigated. In this paper, we demonstrate that transcription is necessary for both the acquisition of metamorphic competence and the completion of the initial events of metamorphosis in Boltenia villosa. Transcripts expressed during metamorphic competence were isolated by a suppressive PCR subtraction of Boltenia villosa larval cDNAs. One of these transcripts is homologous to cornichon. Cornichon has a crucial but undefined role in epidermal growth factor (EGF) signaling during Drosophila embryogenesis. In situ hybridization demonstrates that Boltenia cornichon (Cnib) is expressed in the anterior papillary region of larvae as they gain competence. Our hypothesis is that Cnib acts to potentiate EGF signaling, thereby allowing Boltenia larvae to respond to cues for metamorphosis. Further research into the role of Cnib in urochordate metamorphosis may provide insight into the function of cornichon in other organisms. A better molecular understanding of urochordate metamorphosis will also provide a foundation for exploring the role of metamorphosis in chordate evolution.

p68, a DEAD-box RNA helicase, is expressed in chordate embryo neural and mesodermal tissues.

The p68 DEAD-box RNA helicases have been identified in diverse organisms, including yeast, invertebrates, and mammals. DEAD-box RNA helicases are thought to unwind duplexed RNAs, and the p68 family may participate in initiating nucleolar assembly. Recent evidence also suggests that they are developmentally regulated in chordate embryos. bobcat, a newly described member of this gene family, has been found in eggs and developing embryos of the ascidian urochordate, Molgula oculata. Antisense RNA experiments have implicated this gene in establishing basic chordate features, including the notochord and neural tube in ascidians (Swalla et al. 1999). We have isolated p68 homologs from chick and Xenopus in order to investigate their possible role in vertebrate development. We show that embryonic expression of p68 in chick, frog, and ascidian embryos is high in the developing brain and spinal cord as well as in the sensory vesicles. In frog embryos, p68 expression also marks the streams of migrating cranial neural crest cells throughout neural tube development and in tailbud stages, but neural crest expression is faint in chick embryos. Ascidian embryos also show mesodermal p68 expression during gastrulation and neurulation, and we document some p68 mesodermal expression in both chick and frog. Thus, as shown in these studies, p68 is expressed in early neural development and in various mesodermal tissues in a variety of chordate embryos, including chick, frog, and ascidian. Further functional experiments will be necessary to understand the role(s) p68 may play in vertebrate development.

Tunicates have unusual nuclear lamins with a large deletion in the carboxyterminal tail domain.

Lamins are essential proteins of metazoa. They give rise to the nuclear lamina lining the nucleoplasmic face of the inner nuclear membrane. Here we report the isolation of complete lamin cDNA clones from three urochordate (tunicate) libraries - adult Ciona intestinalis, the tailbud stage of Styela clava and the gastrula stage of Molgula oculata. Lamins L1 and L2 of adult Ciona are derived from two distinct genes. The sequence of the 3' part of the Ciona lamin L1 gene shows that the alpha and beta variants of lamin L1 in Ciona and Styela arise by alternative choice of the 5' splice site at the last intron. Strikingly, all urochordate sequences reveal a 90 residue deletion which removes nearly the entire 105-box. This region is the only long sequence homology segment in the carboxyterminal tail domain of lamins from animals as diverse as Hydra, Drosophila, Priapulus, Caenorhabditis elegans, several echinoderms, the cephalochordate Branchiostoma and various vertebrates. We discuss this unexpected plasticity of lamin sequences as a urochordate specific marker. To increase the database for the chordates we completed the partial sequence of the Branchiostoma lamin by the N-terminal head and central rod domains. The molecular phylogenetic analysis of the metazoan lamin sequences emphasises the monophyletic nature of the chordates in line with the morphological evidence.

Evolution of the chordate body plan: new insights from phylogenetic analyses of deuterostome phyla.

The deuterostome phyla include Echinodermata, Hemichordata, and Chordata. Chordata is composed of three subphyla, Vertebrata, Cephalochordata (Branchiostoma), and Urochordata (Tunicata). Careful analysis of a new 18S rDNA data set indicates that deuterostomes are composed of two major clades: chordates and echinoderms + hemichordates. This analysis strongly supports the monophyly of each of the four major deuterostome taxa: Vertebrata + Cephalochordata, Urochordata, Hemichordata, and Echinodermata. Hemichordates include two distinct classes, the enteropneust worms and the colonial pterobranchs. Most previous hypotheses of deuterostome origins have assumed that the morphology of extant colonial pterobranchs resembles the ancestral deuterostome. We present a molecular phylogenetic analysis of hemichordates that challenges this long-held view. We used 18S rRNA to infer evolutionary relationships of the hemichordate classes Pterobranchia and Enteropneusta. Our data show that pterobranchs may be derived within enteropneust worms rather than being a sister clade to the enteropneusts. The nesting of the pterobranchs within the enteropneusts dramatically alters our view of the evolution of the chordate body plan and suggests that the ancestral deuterostome more closely resembled a mobile worm-like enteropneust than a sessile colonial pterobranch.

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.

Urochordates are monophyletic within the deuterostomes.

Understanding the phylogenetic relationships of the three major urochordate groups within the deuterostomes is central to understanding the evolution of the chordates. We have prepared a detailed phylogenetic analysis of urochordates based on comparisons of 10 new urochordate 18S ribosomal DNA sequences with other urochordate sequences in GenBank. Maximum parsimony, neighbor-joining, minimum evolution, and maximum likelihood analyses of this large urochordate data set are consistent with a topology in which the urochordates are monophyletic within the deuterostomes and there are four separate clades of urochordates. These four distinct clades--styelid + pyurid ascidians, molgulid ascidians, phlebobranch ascidians + thaliaceans, and larvaceans--are mostly consistent with traditional morphological hypotheses and classifications. However, we find that the ascidians may not be a monophyletic group (as they have been considered traditionally) but instead appear paraphyletic. Another disparity with traditional classification is that the thaliaceans do not form a separate urochordate clade but rather cluster with the phlebobranch ascidians. Larvaceans have long branch lengths, which can be problematic for molecular phylogenetic methods, and their position within the urochordates cannot be unequivocally determined with 18S rDNA. This is important because the tadpole morphology of larvacean and ascidian larvae is the key trait of interest that distinguishes urochordates as chordates. Nevertheless, the present data set resolves at least three clades of urochordates and suggests strongly that urochordates form a monophyletic clade within the deuterostomes.

Evolution of the ascidian anural larva: evidence from embryos and molecules.

Most ascidians pass through a tadpole (urodele) larval stage, although some species have derived a tailless (anural) larva. New insights into the evolution of anural larvae in the Roscovita clade of molgulid ascidians were obtained from studing embryonic development of the transitional anural species Molgula bleizi and from phylogenetic analysis based on muscle and cytoskeletal actin gene sequences. By observing in vitro fertilized eggs, we found that M. bleizi, previously described as a typical anural developer, actually forms a short immotile tail during embryogenesis. The short tail contains notochord lineage cells, which undergo abbreviated morphogenetic movements but eventually arrest in development. Molgula bleizi tail muscle lineage cells produce the muscle enzyme acetylcholinesterase (AChE) but do not express muscle actin genes. The presence of a short tail, a vestigial notochord, and AChE-positive muscle cells suggest that M. bleizi is a recently derived anural species. An M. bleizi larval muscle actin gene (MbMA1) was isolated, sequenced, and shown to be a pseudogene based on critical deletions in its coding region that would result in a nonfunctional actin protein. The mutations in MbMA1 are distinct from and have evolved independent of the larval muscle actin pseudogenes MoccMA1a and MoccMA1b in Molgula occulta, another anural developer in the Roscovita clade. Pseudogene formation explains the absence of muscle actin mRNA in M. bleizi embryos. The 3' untranslated region of an M. bleizi cytoskeletal actin gene was also isolated and sequenced. Phylogenetic trees reconstructed using muscle and cytoskeletal actin sequences suggest that the anural developer M. bleizi evolved prior to the divergence of the urodele developer Molgula oculata and the anural developer M. occulta in the Roscovita clade. Since M. bleizi lives attached to hard substrata in the tidal zone, whereas M. oculata and M. occulta live buried in subtidal sand flats, our results suggest that the anural larva evolved at least twice in the Roscovita clade of molgulid ascidians as an adaptation to different habitats.

A multigene locus containing the Manx and bobcat genes is required for development of chordate features in the ascidian tadpole larva.

The Manx gene is required for the development of the tail and other chordate features in the ascidian tadpole larva. To determine the structure of the Manx gene, we isolated and sequenced genomic clones from the tailed ascidian Molgula oculata. The Manx gene contains 9 exons and encodes both major and minor Manx mRNAs, which differ in the length of their 5' untranslated regions. The coding region of the single-copy bobcat gene, which encodes a DEAD-box RNA helicase, is embedded within the first Manx intron. The organization of the bobcat and Manx transcription units was determined by comparing genomic and cDNA clones. The Manx-bobcat gene locus has an unusual organization in which a non-coding first exon is alternatively spliced at the 5' end of two different mRNAs. The bobcat and Manx genes are expressed coordinately during oogenesis and embryogenesis, but not during spermatogenesis, in which bobcat mRNA accumulates independently of Manx mRNA. Similar to Manx, zygotic bobcat transcripts accumulate in the embryonic primordia responsible for generating chordate features, including the dorsal neural tube and notochord, are downregulated during embryogenesis in the tailless species Molgula occulta and are upregulated in M. occulta X M. oculata hybrids, which restore these chordate features. Antisense experiments indicate that zygotic bobcat expression is required for development of the same suite of chordate features as Manx. The results show that the Manx-bobcat gene complex has a role in the development of chordate features in ascidian tadpole larvae.

Cytoskeletal actin genes function downstream of HNF-3beta in ascidian notochord development.

We have examined the expression and regulation of cytoskeletal actin genes in ascidians with tailed (Molgula oculata) and tailless larvae (Molgula occulta). Four cDNA clones were isolated representing two pairs of orthologous cytoskeletal actin genes (CA1 and CA2), which encode proteins differing by five amino acids in the tailed and tailless species. The CA1 and CA2 genes are present in one or two copies, although several related genes may also be present in both species. Maternal CA1 and CA2 mRNA is present in small oocytes but transcript levels later decline, suggesting a role in early oogenesis. In the tailed species, embryonic CA1 and CA2 mRNAs first appear in the presumptive mesenchyme and muscle cells during gastrulation, subsequently accumulate in the presumptive notochord cells, and can be detected in these tissues through the tadpole stage. CA1 mRNAs accumulate initially in the same tissues in the tailless species but subsequently disappear, in concert with the arrest of notochord and tail development. In contrast, CA2 mRNAs were not detected in embryos of the tailless species. Fertilization of eggs of the tailless species with sperm of the tailed species, which restores the notochord and the tail, also results in the upregulation of CA1 and CA2 gene expression in hybrid embryos. Antisense oligodeoxynucleotide experiments suggest that CA1 and CA2 expression in the notochord, but not in the muscle cells, is dependent on prior expression of Mocc FHI, an ascidian HNF-3beta-like gene. The expression of the CA1 and CA2 genes in the notochord in the tailed species, downregulation in the tailless species, upregulation in interspecific hybrids, and dependence on HNF-3beta activity is consistent with a role of these genes in development of the ascidian notochord.

The origin and evolution of animal appendages.

Animals have evolved diverse appendages adapted for locomotion, feeding and other functions. The genetics underlying appendage formation are best understood in insects and vertebrates. The expression of the Distal-less (Dll) homeoprotein during arthropod limb outgrowth and of Dll orthologs (Dlx) in fish fin and tetrapod limb buds led us to examine whether expression of this regulatory gene may be a general feature of appendage formation in protostomes and deuterostomes. We find that Dll is expressed along the proximodistal axis of developing polychaete annelid parapodia, onychophoran lobopodia, ascidian ampullae, and even echinoderm tube feet. Dll/Dlx expression in such diverse appendages in these six coelomate phyla could be convergent, but this would have required the independent co-option of Dll/Dlx several times in evolution. It appears more likely that ectodermal Dll/Dlx expression along proximodistal axes originated once in a common ancestor and has been used subsequently to pattern body wall outgrowths in a variety of organisms. We suggest that this pre-Cambrian ancestor of most protostomes and the deuterostomes possessed elements of the genetic machinery for and may have even borne appendages.

Requirement of the Manx gene for expression of chordate features in a tailless ascidian larva.

An evolutionary change in development was studied in two closely related ascidian species, one exhibiting a conventional tadpole larva and the other a modified tailless larva. Interspecific hybridization restores chordate features to the tailless larva. The zinc finger gene Manx is expressed in cells that generate chordate features in the tailed species but is down-regulated in the tailless species. Manx expression is restored in hybrid embryos. Antisense oligodeoxynucleotide treatment inhibited Manx expression and chordate features in hybrid embryos, which suggests that Manx is required for development of the chordate larval phenotype in ascidians.

PCNA mRNA has a 3'UTR antisense to yellow crescent RNA and is localized in ascidian eggs and embryos.

The myoplasm is a localized cytoplasmic region that is involved in axis determination, gastrulation, muscle cell specification, and the pattern of cell divisions during ascidian development. The noncoding yellow crescent (YC) RNA is localized in the myoplasm, but the function of this transcript is unknown. Probes containing the 3' region of YC RNA hybridize to other RNAs in ascidian eggs. A cDNA library from the ascidian Styela clava was screened with a YC probe to identify maternal YC-related RNAs. This screen resulted in isolation of ScYC26b, a cDNA clone encoding the ascidian proliferating cell nuclear antigen (PCNA). The PCNA mRNA has a long 3' untranslated region containing a 521-nucleotide sequence with antisense complementarity to part of the 3' region of YC RNA. The PCNA and YC genes appear to be single copy and may overlap in their 3' regions on opposite DNA strands. The ascidian PCNA protein has 61, 69, and 71% amino acid identity to the Drosophila, Xenopus, and human PCNAs, respectively. S. clava embryos contain maternal and zygotic PCNA mRNAs. Maternal PCNA mRNA is localized in the ectoplasm, a cytoplasmic region that is segregated to cell lineages that proliferate extensively during embryogenesis, and is depleted in the myoplasm, which is segregated to cell lineages that undergo fewer divisions. Zygotic PCNA mRNA is confined to the developing nervous system and is still abundant after the neural cells have ceased to proliferate. PCNA protein, detected with PC10 monoclonal antibody, is also excluded from the myoplasm. These results show that the 3' UTR of PCNA mRNA is antisense and complementary to YC RNA and suggest that differential cell proliferation in the embryo may be limited by localization of maternal PCNA mRNA and protein. Furthermore, zygotic PCNA may have a novel role in neural development in the tadpole larva.

Expression of an Msx homeobox gene in ascidians: insights into the archetypal chordate expression pattern.

The Msx homeobox genes are expressed in complex patterns during vertebrate development in conjunction with inductive tissue interactions. As a means of understanding the archetypal role of Msx genes in chordates, we have isolated and characterized an Msx gene in ascidians, protochordates with a relatively simple body plan. The Mocu Msx-a and McMsx-a genes, isolated from the ascidians Molgula oculata and Molgula citrina, respectively, have homeodomains that place them in the msh-like subclass of Msx genes. Therefore, the Molgula Msx-a genes are most closely related to the msh genes previously identified in a number of invertebrates. Southern blot analysis suggests that there are one or two copies of the Msx-a gene in the Molgula genome. Northern blot and RNase protection analysis indicate that Msx-a transcripts are restricted to the developmental stages of the life cycle. In situ hybridization showed that Msx-a mRNA first appears just before gastrulation in the mesoderm (presumptive notochord and muscle) and ectoderm (neural plate) cells. Transcript levels decline in mesoderm cells after the completion of gastrulation, but are enhanced in the folding neural plate during neurulation. Later, Msx-a mRNA is also expressed in the posterior ectoderm and in a subset of the tail muscle cells. The ectoderm and mesoderm cells that express Msx-a are undergoing morphogenetic movements during gastrulation, neurulation, and tail formation. Msx-a expression ceases after these cells stop migrating. The ascidian M. citrina, in which adult tissues and organs begin to develop precociously in the larva, was used to study Msx-a expression during adult development. Msx-a transcripts are expressed in the heart primordium and the rudiments of the ampullae, epidermal protrusions with diverse functions in the juvenile. The heart and ampullae develop in regions where mesenchyme cells interact with endodermal or epidermal epithelia. A comparison of the expression patterns of the Molgula genes with those of their vertebrate congeners suggests that the archetypal roles of the Msx genes may be in morphogenetic movements during embryogenesis and in mesenchymal-epithelial interactions during organogenesis.

Mechanism of an evolutionary change in muscle cell differentiation in ascidians with different modes of development.

We have investigated the mechanism of an evolutionary change in ascidian muscle cell differentiation. The ascidians Molgula oculata and Molgula occulta are closely related species with different modes of development. M. oculata embryos develop into conventional tadpole larvae with a tail containing striated muscle cells, whereas M. occulta embryos develop into tailless larvae with undifferentiated vestigial muscle cells. The muscle actin gene MocuMA1 was isolated from an M. oculata genomic library. MocuMA1 is a single-copy, larval-type muscle actin gene which appears to lack introns. However, the 5' upstream region of MocuMA1 is sufficient to drive expression of a lacZ fusion construct in the larval muscle cells, implying that it is a functional gene. MocuMA1 mRNA first appears in the prospective muscle cells of M. oculata embryos during gastrulation, and transcripts continue to be present throughout embryogenesis. Muscle actin mRNA was not detected during M. occulta embryogenesis, although the same probe was capable of detecting muscle actin mRNA in more distantly related ascidian species with tail muscle cells. Interspecific hybrids produced by fertilizing M. occulta eggs with M. oculata sperm recover the ability to express muscle actin mRNA in the vestigial muscle cells, suggesting that trans-acting factors responsible for muscle actin gene expression are conserved in M. occulta. The presence of these trans-acting factors was confirmed by showing that the MocuMA1/lacZ fusion construct is expressed in the vestigial muscle cells of M. occulta larvae. The orthologous larval muscle actin genes MoccMA1a and MoccMA1b were isolated from a M. occulta genomic library. The coding regions of these genes contain deletions, insertions, and codon substitutions that would make their products nonfunctional. Although the 5' upstream regions of the M. occulta muscle actin genes also contain numerous changes, expression of MoccMA1a/lacZ and MoccMA1b/lacZ fusion constructs showed that they both retain specific promoter activity, although it is reduced in MoccMAlb. The results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression.

Localization of ribosomal protein L5 mRNA in myoplasm during ascidian development.

We have isolated and characterized the cDNA clone ScYC26a from the ascidian Styela clava based on its relationship to the non-coding yellow crescent (YC) RNA. The ScYC26a mRNA has a long 5' non-coding sequence that is complementary to YC RNA. The deduced amino acid sequence indicates that ScYC26a encodes the ribosomal protein L5. The ScYC26a mRNA is probably encoded by a single copy gene, which shares genomic DNA restriction fragments with the gene encoding YC RNA, suggesting that the ScYC26a and YC genes are closely linked in the S. clava genome. Northern blot hybridization showed that S. clava eggs and embryos contain maternal ScYC26a mRNA and that zygotic ScYC26a transcripts do not accumulate until after metamorphosis. In situ hybridization showed that maternal ScYC26a mRNA is localized in the myoplasm and is segregated primarily to the muscle cell lineages during embryogenesis. The interaction of YC and ScYC26a transcripts may be involved in translational control or localization of L5 mRNA in the myoplasm.

A maternal RNA localized in the yellow crescent is segregated to the larval muscle cells during ascidian development.

A cDNA library prepared from one-cell zygotes of the ascidian Styela clava was screened with probes from isolated cellular fractions to identify clones encoding RNAs localized in the yellow crescent or myoplasm, a cytoskeletal domain with multiple developmental roles. The differential screen yielded five overlapping cDNA (Styela clava yellow crescent or ScYC) clones encoding a 1.2-kb polyadenylated RNA (yellow crescent or YC RNA) which is present throughout embryonic development. In situ hybridization confirmed that YC RNA is localized in the yellow crescent. Antisense probes containing the 3' region of YC RNA hybridize with multiple maternal and zygotic RNAs, suggesting sequence homologies with other transcripts. YC RNA was first detected during oogenesis when transcripts accumulate in the perinuclear region of vitellogenic oocytes and are gradually translocated to the cortex. The YC transcripts are localized in the cortex of unfertilized eggs but after fertilization segregate with the myoplasm to the yellow crescent. During cleavage most YC transcripts enter the primary muscle cell lineage. YC RNA is also present in the secondary muscle cells. The YC transcripts are retained in the myoplasm of oocytes and eggs extracted with the non-ionic detergent Triton X-100, suggesting that they are associated with the cytoskeleton. The nucleotide sequence of the longest ScYC clone contains a short open reading frame (ORF). The YC ORF would encode a putative polypeptide of 49 amino acids, which shows no significant homology to known proteins. Several features of the YC RNA, however, suggest that it functions as an RNA rather than as a protein coding molecule. We conclude that the myoplasm contains a novel maternal RNA which is associated with the cytoskeleton and segregated to the muscle cells during ascidian embryogenesis. The YC RNA may be a new member of a growing family of noncoding RNAs that play important roles in growth and development.

Multiple origins of anural development in ascidians inferred from rDNA sequences.

Ascidians exhibit two different modes of development. A tadpole larva is formed during urodele development, whereas the larval phase is modified or absent during anural development. Anural development is restricted to a small number of species in one or possibly two ascidian families and is probably derived from ancestors with urodele development. Anural and urodele ascidians constitute a model system in which to study the evolution of development, but the phylogeny of anural development has not been resolved. Classification based on larval characters suggests that anural species are monophyletic, whereas classification according to adult morphology suggests they are polyphyletic. In the present study, we have inferred the origin of anural development using rDNA sequences. The central region of 18S rDNA and the hypervariable D2 loop of 28S rDNA were amplified from the genomic DNA of anural and urodele ascidian species by the polymerase chain reaction and sequenced. Phylogenetic trees inferred from 18S rDNA sequences of 21 species placed anural developers into two discrete groups corresponding to the Styelidae and Molgulidae, suggesting that anural development evolved independently in these families. Furthermore, the 18S rDNA trees inferred at least four independent origins of anural development in the family Molgulidae. Phylogenetic trees inferred from the D2 loop sequences of 13 molgulid species confirmed the 18S rDNA phylogeny. Anural development appears to have evolved rapidly because some anural species are placed as closely related sister groups to urodele species. The phylogeny inferred from rDNA sequences is consistent with molgulid systematics according to adult morphology and supports the polyphyletic origin of anural development in ascidians.