Structure of the caudal neural tube in an ascidian larva: Vestiges of its possible evolutionary origin from a ciliated band

Ultrastructural analysis and differential immunocytochemical staining with two antitubulin monoclonal antibodies were used to reexamine the organization and development of the neural tube in the larva of an ascidian, Ciona intestinalis, in appraisal of a theory that the dorsal tubular nervous system of the chordates evolved from two halves of a ciliated band in an auricularia-like larva of the kind found in echinoderms and hemichordates. One of the antibodies stained cilia in the nervous system and elsewhere; the other reacted primarily with neuronal axons. The caudal neural tube consists of four rows of large ciliated ependymal-glial cells enclosing an axial neural canal into which their single cilia extend. Two ventrolateral nerve tracts, containing axons, arise in the posterior brain region and extend along the length of the caudal tube, partially surrounded by the ependymal cells. The nonnervous, ciliated, ependymal neural tube of the ascidian larva with its two associated nerve tracts survives as a primitive early condition that could result from a ciliated band transformation. Tissues in the distal-most part of the ascidian larval tail have cell lineage origins that indicate an evolutionary history different from those in the proximal majority of the tail. The ependymal cells in this presumed later addition to the tail are not ciliated, although all of the others in the caudal ependymal tube appear to be.     

Structure of the caudal neural tube in an ascidian larva: vestiges of its possible evolutionary origin from a ciliated band.

Ultrastructural analysis and differential immunocytochemical staining with two antitubulin monoclonal antibodies were used to reexamine the organization and development of the neural tube in the larva of an ascidian, Ciona intestinalis, in appraisal of a theory that the dorsal tubular nervous system of the chordates evolved from two halves of a ciliated band in an auricularia-like larva of the kind found in echinoderms and hemichordates. One of the antibodies stained cilia in the nervous system and elsewhere; the other reacted primarily with neuronal axons. The caudal neural tube consists of four rows of large ciliated ependymal-glial cells enclosing an axial neural canal into which their single cilia extend. Two ventrolateral nerve tracts, containing axons, arise in the posterior brain region and extend along the length of the caudal tube, partially surrounded by the ependymal cells. The nonnervous, ciliated, ependymal neural tube of the ascidian larva with its two associated nerve tracts survives as a primitive early condition that could result from a ciliated band transformation. Tissues in the distal-most part of the ascidian larval tail have cell lineage origins that indicate an evolutionary history different from those in the proximal majority of the tail. The ependymal cells in this presumed later addition to the tail are not ciliated, although all of the others in the caudal ependymal tube appear to be.     

A conserved role for the MEK signalling pathway in neural tissue specification and posteriorisation in the invertebrate chordate,the ascidian Ciona intestinalis

Ascidians are invertebrate chordates with a larval body plan similar to that of vertebrates. The ascidian larval CNS is divided along the anteroposterior axis into sensory vesicle, neck, visceral ganglion and tail nerve cord. The anterior part of the sensory vesicle comes from the a-line animal blastomeres, whereas the remaining CNS is largely derived from the A-line vegetal blastomeres. We have analysed the role of the Ras/MEK/ERK signalling pathway in the formation of the larval CNS in the ascidian, Ciona intestinalis. We show evidence that this pathway is required, during the cleavage stages, for the acquisition of: (1) neural fates in otherwise epidermal cells (in a-line cells); and (2) the posterior identity of tail nerve cord precursors that otherwise adopt a more anterior neural character (in A-line cells). Altogether, the MEK signalling pathway appears to play evolutionary conserved roles in these processes in ascidians and vertebrates, suggesting that this may represent an ancestral chordate strategy.     

A developmental pathway controlling outgrowth of the Xenopus tail bud

We have developed a new assay to identify factors promoting formation and outgrowth of the tail bud. A piece of animal cap filled with the test mRNAs is grafted into the posterior region of the neural plate of a host embryo. With this assay we show that expression of a constitutively active Notch (Notch ICD) in the posterior neural plate is sufficient to produce an ectopic tail consisting of neural tube and fin. The ectopic tails express the evenskipped homologue Xhox3, a marker for the distal tail tip. Xhox3 will also induce formation of an ectopic tail in our assay. We show that an antimorphic version of Xhox3, Xhox3VP16, will prevent tail formation by Notch ICD, showing that Xhox3 is downstream of Notch signalling. An inducible version of this reagent, Xhox3VP16GR, specifically blocks tail formation when induced in tailbud stage embryos, comfirming the importance of Xhox3 for tail bud outgrowth in normal development. Grafts containing Notch ICD will only form tails if placed in the posterior part of the neural plate. However, if Xwnt3a is also present in the grafts they can form tails at any anteroposterior level. Since Xwnt3a expression is localised appropriately in the posterior at the time of tail bud formation it is likely to be responsible for restricting tail forming competence to the posterior neural plate in our assay. Combined expression of Xwnt3a and active Notch in animal cap explants is sufficient to induce Xhox3, provoke elongation and form neural tubes. Conservation of gene expression in the tail bud of other vertebrates suggests that this pathway may describe a general mechanism controlling tail outgrowth and secondary neurulation.     

Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme: I. Up to the eight-cell stage

Cell lineages during development of ascidian embryos were analyzed by injection of horseradish peroxidase as a tracer enzyme into identified cells at the one-, two-, four-, and eight-cell stages of the ascidians, Halocynthia roretzi, Ciona intestinalis, and Ascidia ahodori. Identical results were obtained with eggs of the three different species examined. The first cleavage furrow coincided with the bilateral symmetry plane of the embryo. The second furrow did not always divide the embryo into anterior and posterior halves as each of the anterior and posterior cell pairs gave rise to different tissues according to their destinies, which became more definitive in the cell pairs at the eight-cell stage. Of the blastomeres constituting the eight-cell stage embryo, the a4.2 pair (the anterior animal blastomeres) differentiated into epidermis, brain, and presumably sense organ and palps. Every descendant cell of the b4.2 pair (the posterior animal blastomeres) has been thought to become epidermis; however, the horseradish peroxidase injection probe revealed that the b4.2 pair gave rise to not only epidermis but also muscle cells at the caudal tip region of the developing tailbud-stage embryos. The A4.1 pair (the anterior vegetal blastomeres) developed into endoderm, notochord, brain stem, spinal cord, and also muscle cells next the caudal tip muscle cells. From the B4.1 pair (the posterior vegetal blastomeres) originated muscle cells of the anterior and middle parts of the tail, mesenchyme, endoderm, endodermal strand, and also notochord at the caudal tip region. These results clearly demonstrate that muscle cells are derived not only from the B4.1 pair, as has hitherto been believed, but also from both the b4.2 and A4.1 pairs.     

Anteroposterior patterning of the epidermis by inductive influences from the vegetal hemisphere cells in the ascidian embryo.

Patterning along the anteroposterior axis is a critical step during animal embryogenesis. Although mechanisms of anteroposterior patterning in the neural tube have been studied in various chordates, little is known about those of the epidermis. To approach this issue, we investigated patterning mechanisms of the epidermis in the ascidian embryo. First we examined expression of homeobox genes (Hrdll-1, Hroth, HrHox-1 and Hrcad) in the epidermis. Hrdll-1 is expressed in the anterior tip of the epidermis that later forms the adhesive papillae, while Hroth is expressed in the anterior part of the trunk epidermis. HrHox-1 and Hrcad are expressed in middle and posterior parts of the epidermis, respectively. These data suggested that the epidermis of the ascidian embryo is patterned anteroposteriorly. In ascidian embryogenesis, the epidermis is exclusively derived from animal hemisphere cells. To investigate regulation of expression of the four homeobox genes in the epidermis by vegetal hemisphere cells, we next performed hemisphere isolation and cell ablation experiments. We showed that removal of the vegetal cells before the late 16-cell stage results in loss of expression of these homeobox genes in the animal hemisphere cells. Expression of Hrdll-1 and Hroth depends on contact with the anterior-vegetal (the A-line) cells, while expression of HrHox-1 and Hrcad requires contact with the posterior-vegetal (the B-line) cells. We also demonstrated that contact with the vegetal cells until the late 32-cell stage is sufficient for animal cells to express Hrdll-1, Hroth and Hrcad, while longer contact is necessary for HrHox-1 expression. Contact with the A-line cells until the late 32-cell stage is also sufficient for formation of the adhesive papillae. Our data indicate that the epidermis of the ascidian embryo is patterned along the anteroposterior axis by multiple inductive influences from the vegetal hemisphere cells and provide the first insight into mechanisms of epidermis patterning in the chordate embryos.     

The history of a developmental stage: metamorphosis in chordates

Metamorphosis displays a striking diversity in chordates, a deuterostome phylum that comprises vertebrates, urochordates (tunicates), and cephalochordates (amphioxus). In anuran amphibians, the tadpole loses its tail, develops limbs, and undergoes profound changes at the behavioral, physiological, biochemical, and ecological levels. In ascidian tunicates, the tail is lost and the head tissues are drastically remodeled into the adult animal, whereas in amphioxus, the highly asymmetric larva transforms into a relatively symmetric adult. This wide diversity has led to the proposal that metamorphosis evolved several times independently in the different chordate lineages during evolution. However, the molecular mechanisms involved in metamorphosis are largely unknown outside amphibians and teleost fishes, in which metamorphosis is regulated by the thyroid hormones (TH) T3 and T4 binding to their receptors (thyroid hormone receptors). In this review, we compare metamorphosis in chordates and then propose a unifying definition of the larva-to-adult transition, based on the conservation of the role of THs and some of their derivatives as the main regulators of metamorphosis. According to this definition, all chordates (if not, all deuterostomes) have a homologous metamorphosis stage during their postembryonic development. The intensity and the nature of the morphological remodeling varies extensively among taxa, from drastic remodeling like in some ascidians or amphibians to more subtle events, as in mammals.     

Suppression of macho-1-directed muscle fate by FGF and BMP is required for formation of posterior endoderm in ascidian embryos

Specification of germ layers is a crucial event in early embryogenesis. In embryos of the ascidian, Halocynthia roretzi, endoderm cells originate from two distinct lineages in the vegetal hemisphere. Cell dissociation experiments suggest that cell interactions are required for posterior endoderm formation, which has hitherto been thought to be solely regulated by localized egg cytoplasmic factors. Without cell interaction, every descendant of posterior-vegetal blastomeres, including endoderm precursors, assumed muscle fate. Cell interactions are required for suppression of muscle fate and thereby promote endoderm differentiation in the posterior endoderm precursors. The cell interactions take place at the 16- to 32-cell stage. Inhibition of cell signaling by FGF receptor and MEK inhibitor also supported the requirement of cell interactions. Consistently, FGF was a potent signaling molecule, whose signaling is transduced by MEK-MAPK. By contrast, such cell interactions are not required for formation of the anterior endoderm. Our results suggest that another redundant signaling molecule is also involved in the posterior endoderm formation, which is likely to be mediated by BMP. Suppression of the function of macho-1, a muscle determinant in ascidian eggs, by antisense oligonucleotide was enough to allow autonomous endoderm specification. Therefore, the cell interactions induce endoderm formation by suppressing the function of macho-1, which is to promote muscle fate. These findings suggest the presence of novel mechanisms that suppress functions of inappropriately distributed maternal determinants via cell interactions after embryogenesis starts. Such cell interactions would restrict the regions where maternal determinants work, and play a key role in marking precise boundaries between precursor cells of different tissue types.     

Analysis of ascidian <Emphasis Type="Italic">Not</Emphasis> genes highlights their evolutionarily conserved and derived features of structure and expression in development

The ascidian larva is often regarded as an organism close to the ancestral form of chordates, while it is generally accepted that the Spemanns organizer is absent from ascidian embryos. Not is one of the genes expressed in the organizer to execute functions in vertebrate embryos. To address the extent of conservation of Not gene expression among ascidians and vertebrates, we examined the structure and developmental expression of Not of the two distantly related ascidian species, Halocynthia and Ciona. Putative ascidian Not proteins were noted by the absence of one of the two motifs conserved among Not proteins of sea urchin and vertebrates. Analysis by in situ hybridization revealed that Not gene expression of ascidians could be categorized into three types: expression likely to be conserved between ascidians and vertebrates, that probably unique to ascidians, and that specific to ascidian species. Expression of ascidian Not in the posterior end of the tail as well as the notochord and a small part of the anterior neural tube at the tailbud stage is reminiscent of the expression of the vertebrate counterparts in the tailbud, which is regarded as a continuation of the organizer and the pineal gland, respectively. The expression of Not in the epidermis precursors during cleavage stage may be unique to ascidians. In the light of the present findings, evolutionary aspects of Not genes are discussed.Electronic Supplementary Material Supplementary material is available for this article at Edited by N. Satoh     

Activated mutants of SHP-2 preferentially induce elongation of Xenopus animal caps

In Xenopus ectodermal explants (animal caps), fibroblast growth factor (FGF) evokes two major events: induction of ventrolateral mesodermal tissues and elongation. The Xenopus FGF receptor (XFGFR) and certain downstream components of the XFGFR signal transduction pathway (e.g., members of the Ras/Raf/MEK/mitogen-activated protein kinase [MAPK] cascade) are required for both of these processes. Likewise, activated versions of these signaling components induce mesoderm and promote animal cap elongation. Previously, using a dominant negative mutant approach, we showed that the protein-tyrosine phosphatase SHP-2 is necessary for FGF-induced MAPK activation, mesoderm induction, and elongation of animal caps. Taking advantage of recent structural information, we now have generated novel, activated mutants of SHP-2. Here, we show that expression of these mutants induces animal cap elongation to an extent comparable to that evoked by FGF. Surprisingly, however, activated mutant-induced elongation can occur without mesodermal cytodifferentiation and is accompanied by minimal activation of the MAPK pathway and mesodermal marker expression. Our results implicate SHP-2 in a pathway(s) directing cell movements in vivo and identify potential downstream components of this pathway. Our activated mutants also may be useful for determining the specific functions of SHP-2 in other signaling systems.     

Mutations affecting tail and notochord development in the ascidian Ciona savignyi.

Ascidians are among the most distant chordate relatives of the vertebrates. However, ascidians share many features with vertebrates including a notochord and hollow dorsal nerve cord. A screen for N-ethyl-N-nitrosourea (ENU)-induced mutations affecting early development in the ascidian Ciona savignyi resulted in the isolation of a number of mutants including the complementing notochord mutants chongmague and chobi. In chongmague embryos the notochord fails to develop, and the notochord cells instead adopt a mesenchyme-like fate. The failure of notochord development in chongmague embryos results in a severe truncation of tail, although development of the tail muscles and caudal nerve tracts appears largely normal. Chobi embryos also have a truncation of the tail stemming from a disruption of the notochord. However, in chobi embryos the early development of the notochord appears normal and defects occur later as the notochord attempts to extend and direct elongation of the tail. We find in chobi tailbud embryos that the notochord is often bent, with cells clumped together, rather than extended as a column. These results provide new information on the function and development of the ascidian notochord. In addition, the results demonstrate how the unique features of ascidians can be used in genetic analysis of morphogenesis.     

Cross-phylum regulatory potential of the ascidian Otx gene in brain development in Drosophila melanogaster

The origin of molecular mechanisms of cephalic development is an intriguing question in evolutionary and developmental biology. Ascidians, positioned near the origin of the phylum Chordata, share a conserved set of anteroposterior patterning genes with vertebrates. Here we report the cross-phylum regulatory potential of the ascidian Otx gene in the development of the Drosophila brain and the head vertex structures. The ascidian Otx gene rescued the embryonic brain defect caused by a null mutation of the Drosophila orthodenticle (otd) gene and enhanced rostral brain development while it suppressed trunk nerve cord formation. Furthermore, the ascidian Otx gene restored the head vertex defects caused by a viable otd mutation, ocelliless, via specific activation and repression of downstream regulatory genes. These cross-phylum regulatory potentials of the ascidian Otx gene are equivalent to the activities of the Drosophila and human otd/Otx genes in these developmental processes. These results support the notion that basal chordates such as ascidians have the same molecular patterning mechanism for the anterior structures found in higher chordates, and suggest a common genetic program of cephalic development in invertebrate, protochordate and vertebrate.     

A signalling relay involving Nodal and Delta ligands acts during secondary notochord induction in Ciona embryos

The notochord is one of the defining features of chordates. The ascidian notochord is a rod like structure consisting of a single row of 40 cells. The anterior 32 ;primary' notochord cells arise from the A-line (anterior vegetal) blastomeres of the eight-cell stage embryo, whereas the posterior 8 ;secondary' notochord cells arise from the B-line (posterior vegetal) blastomeres of the eight-cell stage embryo. Specification of notochord precursors within these two lineages occurs in a spatially and temporally distinct manner. We show that specification of the secondary but not the primary notochord in Ciona intestinalis requires a relay mechanism involving two signalling pathways. First, we show evidence that acquisition of secondary notochord fate is dependent upon lateral Nodal signalling sources, situated in the adjacent b-line animal cells. Expression of the notochord specific gene Ci-Brachyury in the secondary notochord precursor was downregulated following selective inhibition of Nodal signal reception in B-line derivatives and also, strikingly, following selective inhibition of Nodal signal reception in A-line cell derivatives. Within the A-line, Nodal signals are required for localised expression of Delta2, which encodes a divergent form of Delta ligand. Using four distinct reagents to inhibit Delta2/Notch signals, we showed that Delta2 signalling from A-line cells, which activates the Notch/Su(H) pathway in adjacent B-line cells, is required for specification of the secondary notochord precursor. We propose a model whereby laterally produced Nodal acts to specify the secondary notochord precursor both directly in the B-line cells and via Delta2 induction in adjacent A-line cells.     

Tail gut endoderm and gut/genitourinary/tail development: a new tissue-specific role for Hoxa13

Hoxa13 is expressed early in the caudal mesoderm and endoderm of the developing hindgut. The tissue-specific roles of Hoxa13 function have not been described. Hand-foot-genital syndrome, a rare dominantly inherited human malformation syndrome characterized by distal extremity and genitourinary anomalies, is caused by mutations in the HOXA13 gene. We show evidence that one specific HOXA13 mutation likely acts as a dominant negative in vivo. When chick HFGa13 is overexpressed in the chick caudal endoderm early in development, caudal structural malformations occur. The phenotype is specific to HFGa13 expression in the posterior endoderm, and includes taillessness and severe gut/genitourinary (GGU) malformations. Finally, we show that chick HFGa13 negatively regulates expression of Hoxd13 and antagonizes functions of both endogenous Hoxa13 and Hoxd13 proteins. We suggest a fundamental role for epithelial specific expression of Hoxa13 in the epithelial-mesenchymal interaction necessary for tail growth and posterior GGU patterning.     

The developing dorsal ganglion of the salp Thalia democratica,and the nature of the ancestral chordate brain

The development of the dorsal ganglion of the salp, Thalia democratica, is described from electron microscope reconstructions up to the stage of central neuropile formation. The central nervous system (CNS) rudiment is initially tubular with an open central canal. Early developmental events include: (i) the formation of a thick dorsal mantle of neuroblasts from which paired dorsal paraxial neuropiles arise; (ii) the differentiation of clusters of primary motor neurons along the ventral margin of the mantle; and (iii) the development from the latter of a series of peripheral nerves. The dorsal paraxial neuropiles ultimately connect to the large central neuropile, which develops later. Direct contact between neuroblasts and muscle appears to be involved in the development of some anterior nerves. The caudal nerves responsible for innervating more distant targets in the posterior part of the body develop without such contacts, which suggests that a different patterning mechanism may be employed in this part of the neuromuscular system. The results are compared with patterns of brain organization in other chordates. Because the salp CNS is symmetrical and generally less reduced than that of ascidian larvae, it is more easily compared with the CNS of amphioxus and vertebrates. The dorsal paraxial centres in the salp resemble the dorsolateral tectal centres in amphioxus in both position and organization; the central neuropile in salps likewise resembles the translumenal system in amphioxus. The neurons themselves are similar in that many of their neurites appear to be derived from the apical surface instead of the basal surface of the cell. Such neurons, with extensively developed apical neurites, may represent a new cell type that evolved in the earliest chordates in conjunction with the formation of translumenal or intralumenal integrative centres. In comparing the salp ganglion with vertebrates, we suggest that the main core of the ganglion is most like the mes-metencephalic region of the vertebrate brain, i.e. the zone occupied by the midbrain, isthmus, and anterior hindbrain. Counterparts of more anterior regions (forebrain) and posterior ones (segmented hindbrain) appear to be absent in salps, but are found in other tunicates, suggesting that evolution has acted quite differently on the main subdivisions of the CNS in different types of tunicates.