Tail Muscle Cells Research Articles

Distinct regulation of Snail in two muscle lineages of the ascidian embryo achieves temporal coordination of muscle development.

The transcriptional repressor Snail is required for proper differentiation of the tail muscle of ascidian tadpole larvae. Two muscle lineages (B5.1 and B6.4) contribute to the anterior tail muscle cells, and are consecutively separated from a transcriptionally quiescent germ cell lineage at the 16- and 32-cell stages. Concomitantly, cells of these lineages begin to express Tbx6.b (Tbx6-r.b) at the 16- and 32-cell stages, respectively. Meanwhile, Snail expression begins in these two lineages simultaneously at the 32-cell stage. Here, we show that Snail expression is regulated differently between these two lineages. In the B5.1 lineage, Snail was activated through Tbx6.b, which is activated by maternal factors, including Zic-r.a. In the B6.4 lineage, the MAPK pathway was cell-autonomously activated by a constitutively active form of Raf, enabling Zic-r.a to activate Snail independently of Tbx6.b As a result, Snail begins to be expressed at the 32-cell stage simultaneously in these two lineages. Such shortcuts might be required for coordinating developmental programs in embryos in which cells become separated progressively from stem cells, including germline cells.

Characterization of the compact bicistronic microRNA precursor, miR-1/miR-133, expressed specifically in Ciona muscle tissues

Muscle-specific miR-1/206 and miR-133 families have been suggested to play fundamental roles in skeletal and cardiac myogenesis in vertebrates. To gain insights into the relationships between the divergence of these miRs and muscular tissue types, we investigated the expression patterns of miR-1 and miR-133 in two ascidian Ciona species and compared their genomic structures with those of other chordates. We found that Ciona intestinalis and Ciona savignyi each possess a single copy of the miR-1/miR-133 cluster, which is only 350 nucleotide long. During embryogenesis, Ciona miR-1 and miR-133 are generated as a single continuous primary transcript accumulated in the nuclei of the tail muscle cells, starting at the gastrula stage. In adults, mature miR-133 and miR-1 are differentially expressed in the heart and body wall muscle. Expression of the reporter gene linked to the 850-bp upstream region of the predicted transcription start site confirmed that this region drives the muscle-specific expression of the primary transcript of miR-1/miR-133. In many deuterostome lineages, including that of Ciona, the miR-1/133 cluster is located in the same intron of the mind bomb (mib) gene in reverse orientation. Our results suggest that the origin of genomic organization and muscle-specific regulation of miR-1/133 can be traced back to the ancestor of chordates. Duplication of this miR cluster might have led to the remarkable elaboration in the morphology and function of skeletal muscles in the vertebrate lineage.

Regulation of thyroid hormone sensitivity by differential expression of the thyroid hormone receptor during Xenopus metamorphosis

During amphibian metamorphosis, a series of dynamic changes occur in a predetermined order. Hind limb morphogenesis begins in response to low levels of thyroid hormone (TH) in early prometamorphosis, but tail muscle cell death is delayed until climax, when TH levels are high. It takes about 20 days for tadpoles to grow from early prometamorphosis to climax. To study the molecular basis of the timing of tissue-specific transformations, we introduced thyroid hormone receptor (TR) expression constructs into tail muscle cells of Xenopus tadpoles. The TR-transfected tail muscle cells died upon exposure to a low level of thyroxine (T4). This cell death was suggested to be mediated by type 2 iodothyronine deiodinase (D2) that converts T4 to T3-the more active form of TH. D2 mRNA was induced in the TR-overexpressing cells by low levels of TH. D2 promoter contains a TH-response element (TRE) with a lower affinity for TR. These results show that the TR transfection confers the ability to respond to physiological concentrations of TH at early prometamorphosis to tail muscle cells through D2 activity and promotes TH signaling. We propose the positive feedback loop model to amplify the cell's ability to respond to low levels of T4.

Higher thyroid hormone receptor expression correlates with short larval periods in spadefoot toads and increases metamorphic rate

Spadefoot toad species display extreme variation in larval period duration, due in part to evolution of thyroid hormone (TH) physiology. Specifically, desert species with short larval periods have higher tail tissue content of TH and exhibit increased responsiveness to TH. To address the molecular basis of larval period differences, we examined TH receptor (TR) expression across species. Based on the dual function model for the role of TR in development, we hypothesized that desert spadefoot species with short larval periods would have (1) late onset of TR expression prior to the production of endogenous TH and (2) higher TR levels when endogenous TH becomes available. To test these hypotheses, we cloned fragments of TRα and TRβ genes from the desert spadefoot toads Scaphiopus couchii and Spea multiplicata and their non-desert relative Pelobates cultripes and measured their mRNA levels in tails using quantitative PCR in the absence (premetamorphosis) or presence (natural metamorphosis) of TH. All species express TRα and TRβ from the earliest stages measured (from just after hatching), but S. couchii, which has the shortest larval period, had more TRα throughout development compared to P. cultripes, which has the longest larval period. TRβ mRNA levels were similar across species. Exogenous T3 treatment induced faster TH-response gene expression kinetics in S. couchii compared to the other species, consistent with its higher TRα mRNA expression and indicative of a functional consequence of more TRα activity at the molecular level. To directly test whether higher TRα expression may contribute to shorter larval periods, we overexpressed TRα via plasmid injection into tail muscle cells of the model frog Xenopus laevis and found an increased rate of muscle cell death in response to TH. These results suggest that increased TRα expression evolved in S. couchii and contribute to its higher metamorphic rates.

Expression levels of TRα, LAT1, and PKM2 alter the rate of metamorphic change

Event Abstract Back to Event Expression levels of TRα, LAT1, and PKM2 alter the rate of metamorphic change Jinyoung Choi1* and Daniel R. Buchholz1 1 University of Cincinnati, Department of Biological Science, United States Thyroid hormone (TH) plays a key role for most physiological and developmental changes during metamorphosis. TH exerts its function by binding thyroid hormone receptors (TRs) in the nucleus to initiate TH-dependent gene expression. Intracellular TH levels are regulated by thyroid hormone transporters (THTs) and cytosolic thyroid hormone binding proteins (CTHBPs). Intracellular TH levels and TR expression levels likely regulate the timing of transformation of different tissues during metamorphosis. We hypothesized that altered TR, LAT1 (a THT), and PKM2 (a CTHBP) expression affects the rate of metamorphosis when overexpressed. In this study, we overexpressed TR α, LAT1, and PKM2 in tail muscle cells by intramuscular plasmid DNA injection and quantified the disappearance of tail muscle cells during T3-induced metamorphosis. In cells overexpressing TR, the rate of cell death was significantly faster compared to controls.. Moreover, cell death was accelerated or delayed in muscle cells overexpressing LAT1 or PKM2, respectively, compared to controls. These results show that the expression levels of TR and proteins affecting intracellular TH levels can affect the rate of metamorphic changes. References Shi, Y., Wong, J., Puzianowska-Kuznicka, M., and Stolow, M.A. (1996). Tadpole competence and tissue-specific temporal regulation of amphibian metamorphosis: roles of thyroid hormone and its receptors. BioEssay 18(5), 391-399. Buchholz, D.R., Moskalik, C.L., Kulkarni, S.S., Hollar, A.R.,and Ng, Allison (2011).Hormone regulation and the evolution of frog metamorphic diversity. Mechanisms of Life History Evolution. Oxford publishers (in press). Hollar, A.R., Choi, J., Grimm, A.T., and Buchholz, D.R. (2011). Higher thyroid hormone receptor expression correlates with short 4 larval periods in spadefoot toads and increases metamorphic rate. Submitted to Gen. Comp. Endocrinol Friesema, E.C., Ganguly S., Abdalla, A., Fox, J.E.M., Halestrap, A.P.H. and Visser, T.J. (2003). Identification of monocarboxlyate transporter 8 as a specific thyroid hormone transporter. J. Bio. Chem. 278 (41), 40128-40315. Luze, A.D., Sachs, B., and Demeneix, B.A. (1993). Thyroid hormone-dependent transcriptional regulation of exogenous genes transfected into Xenopus tadpole muscle in vivo. Pro. Natl. Acad. USA 90 (August) 7322-7326. Kinne, A., Kleinau, G., Hoefig, G.S., Gruters, A., Kohrle, J., Krause, G., and Schweizer, U. (2010). Essential molecular determinants for thyroid hormone transport and first structural implications for monocarboxlyate transporter 8. J. Bio. Chem. 285 (36) 28054-28063 Connors, K.A., Korte, J.J., Anderson, G.W., and Degitz, S.J. (2010). Characterization of thyroid hormone transporter expression during tissue-specific metamorphic events in Xenopus tropicalis. Gen. Comp. Endocrinol. 168, 149-159. Keywords: cytosolic thyroid hormone binding protein, LAT1, metamorphosis, PKM2, Thyroid hormone, Thyroid hormone transporter, tissue asynchrony, TRα Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Thyroid Citation: Choi J and Buchholz DR (2011). Expression levels of TRα, LAT1, and PKM2 alter the rate of metamorphic change. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00116 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Miss. Jinyoung Choi, University of Cincinnati, Department of Biological Science, Cincinnati, Ohio, 45221, United States, jinyoung.angela@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jinyoung Choi Daniel R Buchholz Google Jinyoung Choi Daniel R Buchholz Google Scholar Jinyoung Choi Daniel R Buchholz PubMed Jinyoung Choi Daniel R Buchholz Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Mechanism of DNA replication‐dependent transcriptional activation of the acetylcholinesterase gene in the Ciona intestinalis embryo

The acetylcholinesterase-encoding gene in the ascidian Ciona intestinalis (Ci-AChE) is expressed in tail muscle cells from the gastrula stage. When the embryo was continuously treated with aphidicolin from the 32-cell stage, Ci-AChE was not expressed even when control embryos reached the tailbud stage. This result suggests that Ci-AChE acquires the competence to be transcribed after passing through a certain number of DNA replication cycles. A lacZ reporter gene containing the 5' flanking region of Ci-AChE was expressed in the tail muscle cells. Aphidicolin treatment from the 32-cell stage affected, but did not completely suppress, the expression of lacZ. A bisulfite sequencing analysis was carried out to examine the methylation status of four regions within the 5' flanking sequence and the first exon. However, all of these regions remained unmethylated from the 16-cell to 110-cell stages. The results suggested that the DNA of the Ci-AChE locus is not responsible for counting the rounds of replication. We examined the expression of the C. intestinalis MyoD (Ci-MyoD), a transcription factor that activates Ci-AChE. Aphidicolin treatment from the 32-cell stage suppressed the expression of Ci-MyoD, even when control embryos reached the gastrula stage. These results suggest that a lack of Ci-MyoD is critical to the suppression of Ci-AChE in aphidicolin-treated embryos.

Embryology of a planktonic tunicate reveals traces of sessility.

A key problem in understanding deuterostome evolution has been the origin of the chordate body plan. A biphasic life cycle with a sessile adult and a free-swimming larva is traditionally considered ancestral in chordates with subsequent neotenic loss of the sessile adult stage. Molecular phylogenies challenged this view, suggesting that the primitive life cycle in chordates was entirely free-living as in modern day larvaceans. Here, we report the precise cell lineage and fate map in the normal embryo of the larvacean Oikopleura dioica, using 4D microscopy technique and transmission electron microscopy. We document the extraordinary rapidity of cleavage and morphogenetic events until hatching and demonstrate that--compared with ascidians--fate restriction occurs considerably earlier in O. dioica and that clonal organization of the cell lineage is more tightly coupled to tissue fate. We show that epidermal cells in the trunk migrate through 90 degrees, reminiscent of events in ascidian metamorphosis and that the axis of bilateral symmetry in the tail rotates in relation to the trunk. We argue that part of the tail muscle cells are ectomesodermal, because they are more closely associated with prospective epidermis than with other tissues in the cell lineage. Cladistic comparison with other deuterostomes suggests that these traits are derived within tunicates strengthening the hypothesis that the last common ancestor of tunicates had a sessile adult and thus support traditional morphology-derived scenarios. Our results allow hypothesizing that molecular developmental mechanisms known from ascidian models are restricted to fewer, yet identifiable, cells in O. dioica.

FGF9/16/20 and Wnt-5α signals are involved in specification of secondary muscle fate in embryos of the ascidian, Halocynthia roretzi

The tail muscle cells of the ascidian tadpole larva originate from two different lineages, the B- (primary) and A- and b- (secondary) line blastomeres of the eight-cell stage embryo. The primary muscle cells assume muscle fate cell-autonomously with the involvement of a localized muscle determinant, macho-1. On the other hand, fate determination of secondary muscle cells is a non-cell-autonomous process that depends on cellular interactions. In this paper, we investigated the mechanisms underlying fate specification of secondary muscle cells in Halocynthia roretzi. We found that FGF and Wnt5 signals were required. In contrast, the Nodal signal, which is required for specification of A-line muscle cells in another ascidian, Ciona intestinalis, was not necessary for the formation of any secondary muscle cells in Halocynthia embryo. Therefore, Halocynthia and Ciona show distinctly different mechanisms for generation of the secondary lineages, despite the fact that embryogenesis appears very similar between these species. We also found that the mechanisms involved in specification of A- and b-line muscle cells were distinct in that the required timing of the FGF signal for the A-line muscle cells preceded that for the b-line. Moreover, the inducer blastomeres for specification of these two lineages were different.

Neurons of the ascidian larval nervous system in Ciona intestinalis: I. Central nervous system

The tadpole larva of ascidians, basal living relatives of vertebrates, has a chordate body plan. The CNS has many homologies with that of vertebrates yet only about 100 neurons. These few, possibly fixed in number and composition, nevertheless govern a diverse repertoire of behaviors. To elucidate the circuits of the CNS first requires that we recognize each neuron type, for which we used electroporation to transfect precleavage embryos with a plasmid containing green fluorescent protein (GFP) driven by the promoter of the synaptotagmin gene. Hatched larvae were fixed and GFP 3-D reconstructions of confocal image stacks compiled into images of 31 whole or partial larvae, either with many GFP-labelled neurons or with few, each clearly visible. Neuron counts in the sensory vesicle (SV) and visceral ganglion (VG) indicated that between 75% (SV) and 69% (VG) of previously reported numbers of neurons were transfected. Based on their position, shape, and projections, the following neurons were identified in the SV: a prominent eminens neuron, possibly with direct input from papillar neurons, a large ventroposterior interneuron, photoreceptors of the ocellus, and putative antenna cells of the otolith. In the VG, we identified at least four subtypes of motor neuron, including an ovoid cell that may innervate distal tail muscle cells and contrapelo cells with ascending projections, unique among VG neurons. The caudal nerve cord contained the first reported neurons, the somata of planate neurons. These neurons are the first identified types, and will be used to construct a map of the nervous system for this model basal chordate.

Gene regulatory networks for the development and evolution of the chordate heart: Figure 1.

Gene regulatory networks consisting of subcircuits of transcription factors and intercellular signaling molecules are key to an understanding of the complex mechanisms of animal development and evolution (Davidson and Erwin 2006). One of the most intensively studied genetic networks is that for the formation of the animal heart. Since the discovery of the “tinman” gene (Bodmer 1993) and its vertebrate homolog, Nkx2.5 (Lyons et al. 1995), many genes have been found to be involved in heart development in a wide range of animals, from insects to mammals. Actually, it is well established that a gene circuit consisting of GATA, Nkx, and Hand is evolutionarily highly conserved. This gene circuit constitutes a “kernel,” which is evolutionarily inflexible and performs essential regulatory functions in building a body part (Davidson and Erwin 2006). Analyses of vertebrate heart development revealed that the differentiation of cardiac muscle cells and morphogenesis of the heart are governed by this heart kernel gene regulatory network (Cripps and Olson 2002; Harvey 2002; Buckingham et al. 2005). An important question to be answered about the formation of the heart in vertebrates and other chordates is how this kernel is turned on at the earliest stages of the heart cell specification process to establish the heart field. Another intriguing question in chordate heart formation is how the dualor multichambered heart of vertebrates evolved. It is generally believed that the ancestral chordate resembled the present-day ascidian tadpole. The morphogenetic movement of heart precursor cells during ascidian larval development and metamorphosis is reminiscent of those in vertebrates (Davidson and Levine 2003). However, the ascidian tube-like heart lacks chambers. The innovation of the chambered heart was a key event in vertebrate evolution, because the chambered heart generates one-way blood flow with high pressure, a critical requirement for the efficient blood supply of large-body vertebrates. In this issue of Genes & Development, Davidson et al. (2006) addressed these questions by examining the function of an Ets-containing transcription factor in the tunicate, Ciona intestinalis. They found that Ci-Ets1/2 (because this is one of two Ciona orthologs for vertebrate Ets1 and Ets2 and Drosophila pointed, it was originally called Ci-ets/pointed2) (Yagi et al. 2003) establishes the heart field, probably through an FGF signal acting downstream from Ci-Mesp, a basic helix–loop–helix transcription factor gene required for the initial specification of heart precursor cells (Satou et al. 2004). Targeted inhibition of Ci-Ets1/2 or FGF receptor function blocks heart formation. Moreover, targeted expression of a constitutively active Ci-Ets1/2 causes the expansion of the heart field by forced recruitment of larval tail muscle cells that express Ci-Mesp. Interestingly, this heart field expansion, evoked by the subtle alteration of the heart genetic program, caused a morphological change—that is, to a heart with two compartments.

Corticotropin-Releasing Factor Is Cytoprotective in Xenopus Tadpole Tail: Coordination of Ligand, Receptor, and Binding Protein in Tail Muscle Cell Survival

Upon metamorphosis, amphibian tadpoles lose their tails through programmed cell death induced by thyroid hormone (T3). Before transformation, the tail functions as an essential locomotory organ. The binding protein for the stress neuropeptide corticotropin-releasing factor (CRF; CRF-BP) is strongly up-regulated in the tail of Xenopus tadpoles during spontaneous or T3-induced metamorphosis. This finding led us to investigate physiological roles for CRF and CRF-BP in tadpole tail. We found CRF, CRF-BP, and functional CRF1 receptor in tail and CRF and functional CRF1 receptors, but not CRF-BP, in the tail muscle-derived cell line XLT-15. CRF, acting via the CRF1 receptor, slowed spontaneous tail regression in explant culture and caused a reduction in caspase 3/7 activity. CRF increased, but stable CRF-BP overexpression decreased, [3H]thymidine incorporation in XLT-15 cells. Overexpression of CRF-BP in vivo accelerated the loss of tail muscle cells during spontaneous metamorphosis. Lastly, exposure of tail explants to hypoxia increased CRF and urocortin 1 but strongly decreased CRF-BP mRNA expression. We show that CRF is expressed in tadpole tail, is up-regulated by environmental stressors, and is cytoprotective. The inhibitory binding protein for CRF is regulated by hormones or by environmental stressors and can modulate CRF bioactivity.

Promoter activity of the retinoic acid receptor gene in the Ciona intestinalis embryo

Retinoic acid regulates development of many chordate-specific characteristics. The genes encoding retinoic acid receptors (RARs) were isolated only from chordates. In the present study, a genomic DNA fragment containing the promoter region of the RAR gene (Ci-RAR) in the protochordate Ciona intestinalis was isolated. The transgene containing 1,326 bp of the Ci-RAR 5' flanking region was expressed in the epidermis, central nervous system, and muscle. The sequence element necessary for expression in the epidermis seemed to be subdivided into two elements. One was required for activation in the dorsal midline epidermis and the other for the rest of the epidermis. The element responsible for activation in the central nervous system was characterized by putative binding sites for Fox, Ets, and GATA transcription factors. An E-box sequence was required for the transgene expression in the tail muscle cells.

Energy storage during the transition from endogenous to exogenous feeding in Australian redclaw crayfish Cherax quadricarinatus (Von Martens, 1898)

Summary The Australian redclaw crayfish Cherax quadricarinatus hatches at an advanced stage. Two post-hatching stages rely on yolk reserves before the organism becomes a juvenile. After a juvenile appears, the yolk is depleted and young crayfish might spend a few days among female pleopods before starting to feed independently. During this phase, the juvenile organism needs alternative energy sources. Offspring from three females were analyzed with histological and histochemical techniques, from hatching until reaching the 2-week juvenile stage, to trace digestive system development and internal organ lipid and glycogen accumulation. Neutral lipids are stored in specialized cells of the hepatopancreatic tissue until the juvenile stage is reached. An increasing rate of accumulation was found. Glycogen was stored in hepatopancreas and tail muscle cells until post-embryo II, and no increasing or decreasing rate was found in storage functions.

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.

Central nervous system-specific expression of G protein alpha subunits in the ascidian Ciona intestinalis.

Heterotrimeric G proteins play crucial roles as mediators of signaling by many extracellular stimuli. The receptors that activate G proteins constitute the largest and most diverse family of cell surface molecules involved in signal transmission of metazoan cells. To investigate G protein signaling in the central nervous system (CNS) of chordates, we isolated cDNA fragments encoding five different G protein alpha subunits (CiGalpha(x), CiGalpha(q), CiGalpha(i1a), CiGalpha(i1b), and CiGalpha(i2)) from larvae of the ascidian, a simple chordate, Ciona intestinalis. In situ hybridization analysis revealed that each isoform had distinct patterns of spatial distribution in embryos. Among them, CiGalpha(i1a) and CiG alpha(i1b) mRNAs were specifically expressed in the CNS of the larva, whereas CiGalpha(q) transcripts were expressed in small parts of the trunk epidermis and the tip of the tail, but not in the CNS. The CiGalpha(x) expression was widely observed throughout the trunk and tail of the embryos, and the signals were stronger in the epidermis, mesenchyme, and tail muscle cells. Comparison of cDNA sequences and the exon-intron organization indicate that CiGalpha(i1a) and CiGalpha(i1b) are produced by alternative splicing of transcripts from a single gene, CiGalpha(i1). In the cleavage and gastrula stages, transcripts of CiGalpha(i1) were widely distributed in embryos, and the expression then became restricted to the CNS of tailbud embryos and larvae. An exhaustive search has failed to find transducin-type alpha subunits in C. intestinalis. Since CiGalpha(i1) is expressed in the ocellus, CiGalpha(i1) may mediate signals from Ci-opsin1, a visual pigment of the ocellus photoreceptor cells.

Regulation of metamorphosis in ascidians involves NO/cGMP signaling and HSP90.

Treatment of larvae of the ascidians Boltenia villosa (Family: Pyuridae) and Cnemidocarpa finmarkiensis (Family: Styelidae) with drugs that inhibit the function of the molecular chaperone HSP90 increased the frequency of tail resorption, the primary morphogenetic event of metamorphosis. If treatment was initiated at hatching, metamorphic events subsequent to tail resorption failed to occur, indicating an ongoing role for HSP90 during morphogenesis. Removal of tails from heads of mature, but not newly hatched larvae, induced metamorphosis of the head. Decapitation experiments indicate that the capacity of tails to shorten in response to inhibition of HSP90 function requires communication with heads. To identify candidate proteins with which HSP90 may interact to regulate metamorphosis, we noted that in mammalian cells, nitric oxide synthase (NOS) interacts with HSP90 and its activity is sensitive to drugs that inhibit HSP90 function. In addition, nitric oxide (NO) signaling in the marine snail Ilyanassa obsoleta is an important regulator of metamorphosis. Inhibition of NOS activity in these ascidian larvae with L-NAME increased the frequency of metamorphosis, consistent with a putative interaction of NOS and HSP90. NOS is present in tail muscle cells, implicating them as targets for the drug treatments, consistent with the decapitation experiments. Inhibition of soluble guanylyl cyclase, the most common effector of NO signaling, also increased the frequency of metamorphosis. In contrast to treatment with anti-HSP90 drugs, metamorphosis induced with L-NAME or ODQ was complete. The results presented suggest that an HSP90-dependent, NO-based regulatory mechanism localized in tails represses ascidian metamorphosis. We discuss these results in relation to the induction of ascidian metamorphosis by several unrelated agents.

Muscle actin genes and muscle cells in the appendicularian,Oikopleura longicauda: Phylogenetic relationships among muscle tissues in the urochordates

Appendicularians (larvaceans) are planktonic tunicates. They possess a tail throughout their life, which marks a distinct difference between appendicularians and the other tunicate groups, including salps, doliolids, pyrosomes, and ascidians. We isolated cDNA clones encoding muscle-type and cytoplasmic-type actin isoforms from the appendicularian, Oikopleura longicauda. The types of the actin isoforms were confirmed by amino acid identities in the diagnostic residues compared to those of the other chordate muscle and cytoplasmic actins. Interestingly the appendicularian muscle actin isoform sequence has an intermediate feature between the ascidian tail (larval) muscle actin isoform and the body-wall (adult) muscle actin isoform. Analysis of a genomic clone from a muscle actin gene revealed that it contains only one intron in the coding region. It is located at a position that does not correspond to those of any introns reported in other deuterostome actin genes. Whole-mount in situ hybridization shows that the muscle actin transcript is detected specifically in the tail muscle cells of late tailbud-stage embryos. A signal is also found transiently in the region where the heart will form. In adults, the gene is expressed in tail muscle cells but not in the heart. Together with results of cytochemical and histochemical studies demonstrating the structure of muscle tissue, the present study highlights characteristics of appendicularian muscle organization, which are compared with those of the larval and adult muscle of ascidians.

Inhibition of settlement and metamorphosis of the ascidian Herdmania curvata by non-geniculate coralline algae

The surfaces of non-geniculate coralline algae (NCA) are known to induce the settlement and metamorphosis of disparate marine taxa. In this study we investigate the responsiveness of larvae of Herdmania curvata (Ascidiacea: Stolidobranchia) to three species of NCA (Neogoniolithon brassica-florida, Hydrolithon onkodes, and Lithothamnium prolifer) that cohabit the slope and crest of Heron Reef, Great Barrier Reef. H. curvata larvae were first exposed to these NCA at or within 2 h of hatching, which is 1 to 2 h prior to attaining competence, and then cultured continuously with the NCA for 12 to 14 h. Rates of settlement and metamorphosis of H. curvata cultured in laboratory chambers in the presence of the different NCA were significantly lower than spontaneous rates in seawater. The limited settlement in treatments containing NCA were confined entirely to the chamber periphery, and settlement never occurred on the surface of the NCA. The inhibitory effect was dose-dependent and was stronger in N. brassica-florida and H. onkodes than in L. prolifer. Larvae that did not settle in treatments with NCA had rounded anterior trunks and, in extreme cases, kinked tails with rounded and dissociated tail muscle cells. In some individuals, we observed the anterior chemosensory papillae being sloughed off the larval body. Morphological analysis of trunk ectodermal and mesenchymal nuclei of larvae cultured in the presence of the NCA revealed that general necrotic cell death was occurring. Importantly, H. curvata larvae that were exposed to NCA could not subsequently be induced to metamorphose in KCl-elevated seawater, whereas larvae not exposed to NCA metamorphosed at high rates in KCl-elevated seawater.

An alternatively spliced gene encoding a Y-box protein showing maternal expression and tissue-specific zygotic expression in the ascidian embryo.

An ascidian Y-box protein gene was cloned, designated as CiYB, which consists of a highly conserved cold shock domain and an auxiliary tail domain with alternating modules of acidic and basic amino acids. CiYB is a single copy gene in the ascidian genome. During oogenesis and early development, CiYB produces three different transcripts (CiYB1, CiYB2 and CiYB3) by alternate splicing. CiYB1 and CiYB2 were expressed during oogenesis, suggesting that they are recruited into maternal ribonucleoprotein particles. According to gel mobility shift assay, the CiYB1 protein has the ability to bind RNA. The sequence preference of RNA binding is similar to that of the Xenopus Y-box protein (FRGY2), which is a major component of the maternal messenger ribonucleoprotein particles (mRNP) in the oocyte. These results suggest that the ascidian Y-box protein may have an important role for masking and translational regulation of maternal mRNA. Furthermore, CiYB1, CiYB2 and CiYB3 were expressed zygotically in a tissue restricted manner. CiYB1 was expressed specifically in muscle precursor blastomeres and tail muscle cells suggesting its important role in muscle differentiation.

Localization of constitutive heat shock proteins in developing ascidians.

Heat shock proteins (HSP) are a group of highly conserved proteins that regulate protein folding and ameliorate the effects of environmental stress. In the present study, the question of whether or not ascidian oocytes, embryos and larvae constitutively synthesize HSP was studied using HSP 60 and HSP 70 antibodies. Developmental stages obtained from Boltenia villosa, Cnemidocarpa finmarkiensis, Styela montereyensis and Corella willmeriana were examined for HSP using indirect immunocytochemistry. Myoplasm in oocytes and unfertilized eggs reacted with HSP 60 and 70 antibodies. HSP signals dramatically moved into the vegetal egg cytoplasm during ooplasmic segregation and colocalized with the myoplasm. In cleavage-stage embryos, HSP signals were partitioned with the myoplasm into muscle progenitor blastomeres and HSP signals were evident in the tail muscle cells of larvae. Immunoblots of proteins extracted from oocytes, eggs, embryos and larvae indicate that anti-HSP 60 recognizes a single band having an estimated molecular weight of 60 kDa. Egg centrifugation experiments suggest that most of the ascidian myoplasmic HSP are mitochondrial proteins. These results raise an intriguing possibility that mitochondria associated with the myoplasm perform biochemical functions that are unique to the embryonic muscle cell lineage.