High-precision morphology: bifocal 4D-microscopy enables the comparison of detailed cell lineages of two chordate species separated for more than 525 million years
© Stach and Anselmi. 2015
Received: 18 June 2015
Accepted: 8 December 2015
Published: 23 December 2015
Understanding the evolution of divergent developmental trajectories requires detailed comparisons of embryologies at appropriate levels. Cell lineages, the accurate visualization of cleavage patterns, tissue fate restrictions, and morphogenetic movements that occur during the development of individual embryos are currently available for few disparate animal taxa, encumbering evolutionarily meaningful comparisons. Tunicates, considered to be close relatives of vertebrates, are marine invertebrates whose fossil record dates back to 525 million years ago. Life-history strategies across this subphylum are radically different, and include biphasic ascidians with free swimming larvae and a sessile adult stage, and the holoplanktonic larvaceans. Despite considerable progress, notably on the molecular level, the exact extent of evolutionary conservation and innovation during embryology remain obscure.
Here, using the innovative technique of bifocal 4D-microscopy, we demonstrate exactly which characteristics in the cell lineages of the ascidian Phallusia mammillata and the larvacean Oikopleura dioica were conserved and which were altered during evolution. Our accurate cell lineage trees in combination with detailed three-dimensional representations clearly identify conserved correspondence in relative cell position, cell identity, and fate restriction in several lines from all prospective larval tissues. At the same time, we precisely pinpoint differences observable at all levels of development. These differences comprise fate restrictions, tissue types, complex morphogenetic movement patterns, numerous cases of heterochronous acceleration in the larvacean embryo, and differences in bilateral symmetry.
Our results demonstrate in extraordinary detail the multitude of developmental levels amenable to evolutionary innovation, including subtle changes in the timing of fate restrictions as well as dramatic alterations in complex morphogenetic movements. We anticipate that the precise spatial and temporal cell lineage data will moreover serve as a high-precision guide to devise experimental investigations of other levels, such as molecular interactions between cells or changes in gene expression underlying the documented structural evolutionary changes. Finally, the quantitative amount of digital high-precision morphological data will enable and necessitate software-based similarity assessments as the basis of homology hypotheses.
The succession of billions of generations of inherited ontogenies with slight modifications from the ontogenies of their predecessors comprises the process of evolution [1, 2]. Comprehension of the evolution of divergent developmental trajectories requires detailed comparisons of embryologies at appropriate levels [3–6]. Cell lineages as detailed and accurate visualizations based on four-dimensional (4D)-microscopy of cleavage patterns, tissue fate restrictions, and morphogenetic movements that occur during the development of individual embryos are currently available for few disparate animal taxa [7–12]. In addition, it is noteworthy that most of the available detailed cell lineages derive from protostomian (sensu ) animals encumbering evolutionarily broader comparisons. Tunicates are marine invertebrates considered to be close relatives of vertebrates  and whose fossil record dates back to 525 million years ago (Mya) [15, 16]. Life-history strategies across this subphylum are radically different, and include biphasic ascidians with free swimming larvae and a sessile adult stage, the directly developing holoplanktonic larvaceans, and the equally holoplanktonic thaliaceans with some of the most complex life-cycles in the animal kingdom . Despite considerable progress, notably on the molecular level, the exact extent of evolutionary conservation and innovation especially on other organismic levels during embryology remain obscure and leave room for speculation [18, 19]. Here, we overcome the limitations in microscopy imposed by the cellular and acellular coverings of ascidian eggs and embryos by using the innovative technique of bifocal 4D-microscopy. In a comparative approach we demonstrate exactly which characteristics in the cell lineages of the ascidian Phallusia mammillata and the previously studied larvacean Oikopleura dioica  were conserved and which were altered during evolution. Our accurate cell lineage trees combined with the exact three-dimensional reconstructions of cell positions identify clearly the conserved correspondences in cell position, identity, cell movements, and fate restrictions in several cell lines while at the same time precisely pinpointing differences observable at all levels of development. These differences comprise fate restrictions, tissue types, complex morphogenetic movement patterns, bilateral asymmetry, and numerous cases of heterochronous acceleration in the larvacean embryo. Our results demonstrate in extraordinary detail the multitude of developmental levels amenable to evolutionary innovation. We anticipate that the detailed cell lineage data combined with the accurate relative spatial representation of cells will moreover serve as a high-precision guide to devise experimental investigation of other levels, such as molecular interactions between cells or changes in gene expression underlying the documented structural evolutionary changes of ontogenetic processes. Finally, the sheer amount of digital high-precision morphological data will enable and necessitate new attempts to formulate software-based, quantifiable similarity assessments as the basis of homology hypotheses.
Results and discussion
In order to facilitate comparisons between trees, we used Conklin’s nomenclature for both species [20, 25]. Conklin also devised a set of rules that assures comparability that we followed in our analyses and applied to the tree [25, 26] depictions (for more information, see “Methods”). To distinguish between the cells from both sides, we again used Conklin’s nomenclature by underlining cell names referring to the right side of the embryo.
While the ascidian cell lineage tree is highly symmetrical (Fig. 1b), the larvacean cell lineage tree shows a marked asymmetry in the lineage of the notochord. On the left side, A8.1 as a descendant of A6.1 is already restricted to an endodermal fate. On the right side, A8.1 gives rise to additional notochordal cells.
The direct side-by-side comparison of the 4D-microscopical cell lineage trees of the ascidian P. mammillata and the larvacean O. dioica (Fig. 1) reveals exact correspondences and differences of various degrees. In the following, we discuss examples of these different relations. It should be noted, that more examples are documented in the extensive supplementary material accompanying this publication online.
Exact correspondences of cell identity and fate
Besides cases of exact correspondences, however, in some other cell lines correspondences are more relaxed. While, for example, the relative positions in the cell lineage trees of muscle cell lines, pericardium cell lines, and germ cells are similar, fate restrictions occur comparatively later in the ascidian (Figs. 1 and 2; Additional files 11, 12 and 13). For example, the germ cell line is the sister lineage to a line that gives rise to pericardium and anterior somatic muscle cells in both species. This separation, however, occurs at the sixth generation in the larvacean (B6.4/B6.4) and at the seventh generation in the ascidian (B7.6/B7.6) (Fig. 1). If the supposition mentioned above that descendants of B5.1/B5.1 might in fact be entirely endodermal, this would be another case of an earlier fate restriction, interestingly with the daughter cells B6.1/B6.1 still being a case of exact correspondence. This general trend towards a relatively earlier fate restriction in the larvacean embryo leads to a tighter coupling of cell lineage and fate restrictions in O. dioica. For example, notochord cells in the left side derive from A6.2 in the larvacean, but from A7.3, A7.7, and B8.6 in the ascidian (Figs. 1 and 2). Similar patterns can be seen in the fate restrictions of nervous system, endoderm, and musculature. These cases where a specific, cohesive larval tissue originates in several separate cell lines indicate in the bifurcating cell lineage tree representation that cell fates might depend on regional cellular interactions in the ascidian, as has been shown in the ascidian Ciona intestinalis . While our data are compatible with the hypothesis that in larvaceans cell lineage determines fate to a large extent, regional inductions are not ruled out, but should be tested in laboratory experiments.
Morphogenetic movements - gastrulation
Morphogenetic movements - neurulation
Loss of sub-lineages and cellular novelties
The juxtaposition of comparable stages in the two tunicates shows that besides the temporal discrepancy of events, the ascidian embryo has undergone eight or nine mitoses in most lineages when neurulation commences, whereas the larvacean has undergone seven. This means that at this stage there are more than twice the number of cells in the ascidian (c = 181) than in the larvacean (c = 63). Concomitant to the higher number of cells, ascidian embryos possess additional tissue types that are not present in larvaceans, such as the trunk lateral cells (TLCs). TLCs are the descendants of cells A7.6/A7.6 and form a conspicuous cluster of lateral mesenchymatic cells in the trunk of tadpole and larval stages of ascidians (Additional file 14). Because other chordates, such as cephalochordates [32–34], hagfishes [35, 36], or ammocoetes [37, 38], possess mesenchymatic cells during early stages in ontogeny, this outgroup comparison indicates that larvaceans are evolutionarily derived in regard to this trait. TLCs, that is, the descendants of cells A7.6/A7.6, have been suggested to be homologous to vertebrate neural crest . Alternatively, Abitua and colleagues hypothesized that cells a9.49/a9.49 and their descendants correspond to vertebrate neural crest cells . Whereas TLCs are absent in larvaceans, a9.49/a9.49 are present but their descendent cells are anterior epidermis cells and not bordering the dorsal anterior neural tube as is the case in the ascidian embryo (Fig. 4). Thus the precise position of the descendants of a9.49/a9.49 in the ascidian tadpole clearly corresponds to the definition of neural crest cells in the zebrafish anatomy ontology (see ), therefore supporting the hypothesis suggested by Abitua and colleagues .
Detailed comparisons of individual cells
Building on the pioneering study by Conklin on Styela partita  that has been considerably expanded by Nishida [42, 43] for Halocynthia roretzi—who used a tedious injection technique to trace cell fates—several modern studies analyzing cell fates in specific tissues of Ciona intestinalis [27, 44–46] documented on the one hand that cell lineages in ascidian species seemed conserved, while stating that “comparative analysis is a difficult exercise due to the current poor anatomical description.” . Our application of bifocal 4D-microscopy for the first time revealed the precise and almost complete cell lineage of an ascidian embryo up to the early tadpole stage (between stage 17 and 18 in the ANISEED database: http://www.aniseed.cnrs.fr/aniseed/anatomy/find_devstage), enabling detailed comparisons with the improved re-analyzed cell lineage of an appendicularian species.
In comparing our detailed documentation of cell lineages of P. mammillata with available information on cell lineages in other ascidian species, we found more similarities to the described cell lineages of C. intestinalis [27, 44–46] and H. roretzi [42, 43] than to the only publication of a cell lineage of P. mammillata itself . In their cell fate tabulation (table 1 in , p. 196), Zalokar and Sardet combined available data from different species and, using injection of a fluorescent marker in P. mammillata, corrected several previous misconceptions [49, 50], notably in the lines becoming muscle cells. For example, these authors realized that cells B7.6/B7.6 do not give rise to muscle cells, but fell short of determining the actual fate of this cell as the germ line cell. This realization came only later in comparative studies using H. roretzi as a model [43, 51, 52]. Because other errors had not been corrected and because the study by Zalokar and Sardet remained the only one on the cell lineage of P. mammillata, our results are closer to the published cell lineages of H. roretzi and C. intestinalis. For example, contrary to the lineage published by Zalokar and Sardet , A7.6/A7.6 do not give rise to notochord but to TLCs as in C. intestinalis  and H. roretzi . Another example are the B8.6/B8.6 cells, which are tabulated as mesenchymal in fate for P. mammillata by Zalokar and Sardet but that we found, in agreement with Nishida  and Lemaire , become notochordal cells. On the other hand, we note minor differences in some lineages to these studies. Both Nishida and Lemaire describe the fate of B7.7/B7.7 cells as mesenchymal, whereas we found the descendants become muscle and mesenchyme. Whether this discrepancy is due to real differences in the lineages of the different species or due to limitations in resolution remains to be verified. The fine grain of our cell lineage observations also allows us to relate these data with the even finer morphological detail of confocal laser scanning microscopy-derived morphological descriptions of certain ascidian embryonic stages that recently became available [53, 54].
Although the exact phylogeny of Tunicata remains controversial [15, 55–57], most paleontological studies indicate that the diversification of Tunicata dates back to the early Cambrian (ca. 525 Mya) . Thus, the precision of our cell lineage data opens an entirely new horizon for evolutionary analyses and interpretation. We could reveal detailed similarities with single cell resolution pertaining to different levels of potential homologies, such as cleavage tree pattern, fate restriction, morphogenetic movements, timing, or orientation of planes of cell divisions. We demonstrated that each of these levels is susceptible to changes and conclude that there is no a priori reason to expect one level to be less prone to evolution than another. Indeed the many levels of evolvability of embryonic development are a stark reminder that evolutionary analyses cannot afford to neglect organismal levels.
Cell lineage indicates cellular signaling
Detailed cell lineage data can moreover serve as a high-precision guide to devise experimental approaches to investigate other levels as well, such as molecular interactions between cells, changes in gene expression, but also studies designed to unravel the accompanying molecular changes underlying the documented structural evolutionary changes. For example, from the cell lineage tree we would predict that the determination of nervous system cell fate in several of the comparatively late restricting nervous system cell lines such as a8.17/a8.17 and a8.19/a8.19 (descendants of a4.2/a4.2) depend on regional factor signaling whereas this might not be the case for A7.4/A7.4 (descendants of A4.1/A4.1). And indeed, because neural cell lineages in ascidians were available relatively early on , it could be demonstrated that neural descendants of A4.1/A4.1 develop autonomously, whereas signaling from this source is necessary for the a4.2/a4.2 descendants to assume a neural fate . We predict that regional induction is similarly necessary for neural fate determination in A8.15/A8.15. Moreover, we argue that the restriction to neural fate in a7.9/a7.9 in the larvacean embryo depends on regional signaling as well and that the reduction of such signaling in several cell lines led to the streamlined cell lineage tree documented in the larvacean O. dioica.
Bifocal 4D-microscopy overcomes the limitations imposed on light microscopic observations in ascidian embryos by the extensive extra embryonic covers of a developing embryo. While many molecular studies remove these coverings , removing these covers results in some ontogenetic changes, such as shape of epidermis cells and formation of larval tunic, compared to the wild-type development [59, 60]. Thus bifocal 4D-microscopy can amend these shortcomings and considerably expand our knowledge of tunicate ontogeny at a detailed cellular level. While the present paper focuses on the comparative findings rather than on the methodological advancement, it is obvious that 4D-microscopy in a comparative framework allows for and necessitates new analytical tools and poses new and exciting challenges [26, 61]. An important challenge relates to the notion of homology: with the huge amount of detailed and precise information present in the accumulated data, it becomes obvious that we need to develop software tools that can guide us through the many layers of similarities, including the positional relational information, the movement and cleavage pattern, temporal information of all the observed events, fate restriction patterns, and similarities in underlying gene regulatory networks, and visualize or quantify similarity arguments in support of different homology hypotheses. Because homology is the central concept in evolutionary comparative biology, addressing this challenge with appropriate data mining and analytical tools has to become a priority in the burgeoning field of visual computer analyses.
Fundamentals of 4D-microscopy are described by Schnabel et al. . Individual O. dioica embryos recorded by Stach et al.  were re-analyzed comparatively for the present study. (In contrast to ascidian embryos, O. dioica embryos do not possess follicle or test cells and therefore conventional 4D-microscopy suffices.) Four-cell, eight-cell, or sixteen-cell embryos of P. mammillata were recorded using bifocal 4D-recordings. Bifocal 4D-microscopy was developed by Dr. Ralf Schnabel (Technical University, Braunschweig) in cooperation with Zeiss. Essentially, in a Zeiss Examiner (Zeiss, Jena, Germany) D1 microscope the condenser was replaced with a second optical microscope unit, including a second objective and camera (Additional file 15). The microscope was equipped with an extended internal focus drive (500 μm; (Physical Instruments, Karlsruhe, Germany)) used to move the stage to record a z series with two PCO pixelfly cameras, documenting each focal plane from above and below. Images were compressed with a wavelet function (Lurawave) LuraTech, Remscheid, Germany. The microscope was controlled with a software programmed by K. Schulz and R. Schnabel. Embryos were recorded at 18 °C in a thermocontrolled room; in addition the upper objective was equipped with a thermoconstant cooling ring. Recordings of three different embryos stemming from three different parental pairs were analyzed using SIMI°BioCell software (SIMI) Simi Reality Motion Systems, Unterschleissheim, Germany. Starting with a four-cell or eight-cell embryo, 140 images were recorded every 60 s from 70 planes 1.7 μm apart with the two cameras. A complete scan consisted of 1,000 scans, resulting in a database comprising 140,000 images. Of the approximately 16 h of development recorded, 8 h were analyzed in detail. For cell nomenclature, we used Conklin’s system for both species  (a translation table to the larvacean nomenclature used by Delsman  is given in ). For the determination of a cell’s name and for the tree representation of the cell lineages we also used Conklin’s set of rules. The daughter cell that is situated closer to the vegetal pole after mitosis received the lower number and, in the tree depiction, is represented by the outer branch [25, 26]. In cases where both cells are at the same height along the animal–vegetal axis, the cell closer to the anterior received the lower number and is represented by the outer branch in the anterior half oft he embryo; in the posterior half oft he embryo it is the cell closer to the posterior that received the lower number and is represented by the outer branch. In cases where the daughter cells are also identical in their respective position along the antero-posterior axis, the more distal cell was given the lower number and represented by the outer branch (see figures 133 and 134 in ). To distinguish between the cells from both sides, we again used Conklin’s suggestion and underlined cell names referring to the cells on the right side of the embryo.
P. mammillata adults were obtained through the service Modèles Biologiques (ModBio) from the Centre de Resources Biologiques Marines at the Station Biologique Roscoff (France). In each experiment, two individuals were cross-fertilized in vitro as described  and normally developing embryos were mounted for microscopic analysis. The latter citation reports results from cell lineage tracing in P. mammillata. Our detailed documentation of cell lineages of this very species shows more similarities to the described cell lineages of C. intestinalis and H. roretzi than to the description of the cell lineage of P. mammillata . Complete datasets including complete stacks of differential interference contrast images and SIMI°BioCell-files are deposited on www.morphdbase.de along with Additional files and can be downloaded from there.
Availability of supporting data
The complete datasets supporting the results of this article are available in the MorphDBase repository as zipped archives.
The database consisting of approximately 140,000 images of a recording of the ontogenetic development of a Phallusia mammillata embryo has been separated into two packages, representing the images from the upper and lower camera respectively.
zip-compressed files of the complete scan of the development of P. mammillata, including Simi-Biocell analysis files:
zip-compressed file of complete scan of the development of O. dioica, including Simi-Biocell analysis files:
A supplementary movie at www.morphdbase.de/?T_Stach_20151020-M-13.1 shows the ontogeny and detailed cell lineages of the two tunicate species P. mammillata, an ascidian, and O. dioica, a larvacean. The two species have drastically different life histories and ecologies, yet the cell lineage pattern shows remarkable similarities.
Additional movies of tracings of specific cells can be requested from the first author.
High resolution versions of supplementary figures are available as indicated in the captions to the additional files.
Financial support by the German Research Foundation (DFG-grants STA655/4 & STA655/5) and by ERASMUS is gratefully acknowledged.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Garstang W. The theory of recapitulation: a critical re-statement of the biogenetic law. Linnean J Zool. 1921;35:81–101.View ArticleGoogle Scholar
- Kalinka AT, Tomancak P. The evolution of early animal embryos: conservation or divergence? Trends Ecol Evol. 2012;27:385–93.PubMedView ArticleGoogle Scholar
- Nielsen C. Larval nervous systems: true larval and precocious adult. J Exp Biol. 2015;218(4):629–36.PubMedView ArticleGoogle Scholar
- Richter S, Wirkner CS. A research program for evolutionary morphology. J Zool Syst Evol Res. 2014;52(4):338–50.View ArticleGoogle Scholar
- Scholtz G. Versuch einer analytischen Morphologie. Bildwelten des Wissens. 2013;9(2):30–44.Google Scholar
- Wray GA. The evolution of cell lineage in echinoderms. Am Zool. 1994;34:353–63.View ArticleGoogle Scholar
- Hejnol A, Martindale M. Acoel development supports a simple planula-like urbilaterian. Philos T R Soc B. 2008;363:1493–501.View ArticleGoogle Scholar
- Alwes F, Scholtz G. Cleavage and gastrulation of the euphausiacean Meganyctiphanes norvegica (Crustacea, Malacostraca). Zoomorphology 2004;123(3):125–37.Google Scholar
- Hejnol A, Martindale MQ, Henry JQ. High-resolution fate map of the snail Crepidula fornicata: the origins of ciliary bands, nervous system, and muscular elements. Dev Biol. 2007;305(1):63–76.PubMedView ArticleGoogle Scholar
- Hejnol A, Schnabel R. The eutardigrade Thulinia stephaniae has an indeterminate development and the potential to regulate early blastomere ablations. Development. 2004;132:1349–61.View ArticleGoogle Scholar
- Schnabel R, Hutter H, Moermann D, Schnabel H. Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of development and regional specification. Dev Biol. 1997;184:234–65.PubMedView ArticleGoogle Scholar
- Schulze J, Schierenberg E. Evolution of embryonic development in nematodes. EvoDevo. 2011;2:18.PubMedPubMed CentralView ArticleGoogle Scholar
- Dunn CW, Giribet G, Edgecombe GD, Hejnol A. Animal phylogeny and its evolutionary implications. Annu Rev Ecol Evolut Syst. 2014;45:371–95.View ArticleGoogle Scholar
- Delsuc F, Brinkmann H, Chourrout D, Philippe H. Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature. 2006;439:965–8.PubMedView ArticleGoogle Scholar
- Fedonkin MA, Vickers-Rich P, Swalla BJ, Trusler P, Hall M. A new metazoan from the Vendian of the White Sea, Russia, with possible affinities to the ascidians. Paleontol J. 2012;46(1):1–11.View ArticleGoogle Scholar
- Shu D, Isozaki Y, Zhang X, Han J, Maruyama S. Birth and early evolution of metazoans. Gondwana Res. 2014;25(3):884–95.View ArticleGoogle Scholar
- Lemaire P. Evolutionary crossroads in developmental biology: the tunicates. Development. 2011;138(11):2143–52.PubMedView ArticleGoogle Scholar
- Onai T, Irie N, Kuratani S. The evolutionary origin of the vertebrate body plan: the problem of head segmentation. Annu Rev Genomics Hum Genet. 2014;15(1):443–59.PubMedView ArticleGoogle Scholar
- Satoh N, Tagawa K, Lowe CJ, Yu J-K, Kawashima T, Takahashi H, et al. On a possible evolutionary link of the stomochord of hemichordates to pharyngeal organs of chordates. Genesis. 2014;52(12):925–34.PubMedView ArticleGoogle Scholar
- Stach T, Winter J, Bouquet J-M, Chourrout D, Schnabel R. Embryology of a planktonic tunicate reveals traces of sessility. Proc Natl Acad Sci. 2008;105(20):7229–34.PubMedPubMed CentralView ArticleGoogle Scholar
- Burighel P, Cloney RA. Urochordata: Ascidiacea. In: Harrison FW, Ruppert EE, editors. Microscopic anatomy of Invertebrates Hemichordata, Chaetognatha, and the invertebrate chordates, vol. 15. New York: Willey-Liss, Incorporation; 1997. p. 221–347.Google Scholar
- Satoh N. Developmental biology of ascidians. Cambridge: Cambridge University Press; 1994.Google Scholar
- Stach T. Anatomy of the trunk mesoderm in tunicates: homology considerations and phylogenetic interpretation. Zoomorphology. 2009;128:97–109.View ArticleGoogle Scholar
- Lohmann H. Erste Klasse der Tunicaten. Appendiculariae. In: Krumbach T, editor. Handbuch der Zoologie, vol. 5.2. Tunicata. Berlin: Walter de Gruyter & Co; 1956. p. 15–202.Google Scholar
- Conklin EG. Organization and cell lineage of the ascidian egg. J Acad Natl Sci Phila. 1905;XIII(Part 1):1–119. plates I-XII.Google Scholar
- Nishida H, Stach T. Cell lineages and fate maps in tunicates: conservation and modification. Zool Sci. 2014;31(10):645–52.PubMedView ArticleGoogle Scholar
- Lemaire P. Unfolding a chordate developmental program, one cell at a time: invariant cell lineages, short-range inductions and evolutionary plasticity in ascidians. Dev Biol. 2009;332(1):48–60.PubMedView ArticleGoogle Scholar
- Lowery LA, Sive H. Strategies of vertebrate neurulation and a re-evaluation of teleost neural tube formation. Mech Devel. 2004;121:1189–97.View ArticleGoogle Scholar
- Harrington MJ, Hong E, Brewster R. Comparative analysis of neurulation: first impressions do not count. Mol Reprod Dev. 2009;76:954–65.PubMedView ArticleGoogle Scholar
- Hirakow R, Kajita N. Electron microscopic study of the development of amphioxus, Branchiostoma belcheri tsingtauense, the neurula and larva. Acta Anat Nippon. 1994;69:1–13.PubMedGoogle Scholar
- Stach T. Microscopic anatomy of developmental stages of Branchiostoma lanceolatum (Cephalochordata, Chordata). Bonn Zool Monogr. 2000;47:1–111.Google Scholar
- Stach T. Chordate phylogeny and evolution: a not so simple three-taxon problem. J Zool. 2008;276(2):117–41.View ArticleGoogle Scholar
- Oisi Y, Ota KG, Kuraku S, Fujimoto S, Kuratani S. Craniofacial development of hagfishes and the evolution of vertebrates. Nature. 2013;493(7431):175–80.PubMedView ArticleGoogle Scholar
- Ota KG, Kuraku S, Kuratani S. Hagfish embryology with reference to the evolution of the neural crest. Nature. 2007;446:672–5.PubMedView ArticleGoogle Scholar
- Kuratani S, Kuraku S, Murakami Y. Lamprey as an evo-devo model: lessons from comparative embryology and molecular phylogenetics. Genesis. 2002;34:175–83.PubMedView ArticleGoogle Scholar
- Kuratani S, Murakami Y, Nobusada Y, Kusakabe R, Hirano S. Developmental fate of the mandibular mesoderm in the lamprey, Lethenteron japonicum: comparative morphology and development of the gnathostome jaw with special reference to the nature of the trabecula cranii. J Exp Zool. 2004;302B:458–68.View ArticleGoogle Scholar
- Jeffery WR, Chiba T, Krajka FR, Deyts C, Satoh N, Joly J-S. Trunk lateral cells are neural crest-like cells in the ascidian Ciona intestinalis: insights into the ancestry and evolution of the neural crest. Dev Biol. 2008;324(1):152–60.PubMedView ArticleGoogle Scholar
- Abitua PB, Wagner E, Navarrete IA, Levine M. Identification of a rudimentary neural crest in a non-vertebrate chordate. Nature. 2012;492:104–8.PubMedPubMed CentralView ArticleGoogle Scholar
- Nishida H. Cell division pattern during gastrulation of the ascidian. Halocynthia roretzi Dev Growth Differ. 1986;28:191–201.View ArticleGoogle Scholar
- Nishida H. Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. Dev Biol. 1987;121:526–41.PubMedView ArticleGoogle Scholar
- Davidson B, Shi W, Levine M. Uncoupling heart cell specification and migration in the simple chordate Ciona intestinalis. Development. 2005;132(21):4811–8.PubMedView ArticleGoogle Scholar
- Hudson C, Ba M, Rouvière C, Yasuo H. Divergent mechanisms specify chordate motoneurons: evidence from ascidians. Development. 2011;138(8):1643–52.PubMedView ArticleGoogle Scholar
- Cole AG, Meinertzhagen IA. The central nervous system of the ascidian larva: mitotic history of cells forming the neural tube in late embryonic Ciona intestinalis. Dev Biol. 2004;271(2):239–62.PubMedView ArticleGoogle Scholar
- Lemaire P, Bertrand V, Hudson C. Early steps in the formation of neural tissue in ascidian embryos. Dev Biol. 2002;252:151–69.PubMedView ArticleGoogle Scholar
- Zalokar M, Sardet C. Tracing of cell lineage in embryonic development of Phallusia mammillata (Ascidia) by vital staining of mitochondria. Dev Biol. 1984;102:195–205.PubMedView ArticleGoogle Scholar
- Swalla BJ, Smith AB. Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives. Philos Trans R Soc Lond B. 2008;363:1557–68.View ArticleGoogle Scholar
- García-Bellido DC, Lee MSY, Edgecombe GD, Jago JB, Gehling JG, Paterson JR. A new vetulicolian from Australia and its bearing on the chordate affinities of an enigmatic Cambrian group. BMC Evol Biol. 2014;14:214.PubMedPubMed CentralView ArticleGoogle Scholar
- Christiaen L, Wagner E, Shi W, Levine M. Isolation of sea squirt (Ciona) gametes, fertilization, dechorionation, and development. Cold Spring Harb Protoc. 2009;2009(12):pdb.prot5344.PubMedGoogle Scholar
- Thompson H, Shimeld SM. Transmission and scanning electron microscopy of the accessory cells and chorion during development of Ciona intestinalis type B embryos and the impact of their removal on cell morphology. Zool Sci. 2015;32:217–22.PubMedView ArticleGoogle Scholar
- Cloney RA, Cavey MJ. Ascidian larval tunic - extra-embryonic structures influence morphogenesis. Cell Tissue Res. 1982;222:547–62.PubMedView ArticleGoogle Scholar
- Whittaker JR. Cytoplasmic determinants of tissue differentiation in the Ascidian egg. In: Subtelny S, Konigsberg IR, editors. Determinants of spatial organization. New York: Academic; 1979. p. 29–51.Google Scholar
- Mancuso V. L’uovo di Ciona intestinalis (Ascidia) osservato al microscopio electronico. I. Il cell-lineage. Acta Embryologica Experimentalia. 1969;12:231–255Google Scholar
- Iseto T, Nishida H. Ultrastructural studies on the centrosome-attracting body: electron-dense matrix and its role in unequal cleavages in ascidian embryos. Develop Growth Differ. 1999;41:601–9.View ArticleGoogle Scholar
- Nakamura MJ, Terai J, Okubo R, Hotta NK, Oka K. Three-dimensional anatomy of the Ciona intestinalis tail bud embryo at single-cell resolution. Dev Biol. 2012;372:274–84.PubMedView ArticleGoogle Scholar
- Veeman M, Reeves W. Quantitative and in toto imaging in ascidians: working toward an image-centric systems biology of chordate morphogenesis. Genesis. 2015;53:143–59.PubMedView ArticleGoogle Scholar
- Tsagkogeorga G, Turon X, Hopcroft R, Tilak M, Feldstein T, Shenkar N, et al. An updated 18S rRNA phylogeny of tunicates based on mixture and secondary structure models. BMC Evol Biol. 2009;9:187.PubMedPubMed CentralView ArticleGoogle Scholar
- Govindarajan AF, Bucklin A, Madin LP. A molecular phylogeny of the Thaliacea. J Plankton Res. 2011;33:843–53.View ArticleGoogle Scholar
- Vogt L, Nickel M, Jenner RA, Deans AR. The need for data standards in zoomorphology. J Morphol. 2013;274(7):793–808.PubMedView ArticleGoogle Scholar
- Delsman HC. Beiträge zur Entwicklungsgeschichte von Oikopleura dioica. Verhandelingen uit het Rijksinstituut voor het Onderzoek der Zee. 1910;3:1–24.Google Scholar