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.     

Developmental autonomy of muscle fine structure in muscle lineage cells of ascidian embryos

We have observed ultrastructural features of muscle differentiation in the muscle lineage cells of cleavage-arrested whole embryos and partial embryos of ascidians. Whole embryos of Ciona intestinalis and Ascidia ceratodes were cleavage-arrested with cytochalasin B at the 8-cell stage and reared to an age equivalent to several hours after hatching; these embryos formed extensive myofilaments which were often further organized into myofibrils of different sizes and densities in the peripheral cytoplasm of the two muscle lineage blastomeres (B4.1 pair). Developing myofibrils in cleavage-arrested embryos resembled the muscle elements observed in normal hatched larvae, but were less uniformly organized. A similar development of myofilaments and myofibrils occurred in the muscle lineage cells of multicellular partial embryos reared to "hatching" age. These partial embryos resulted from the isolated muscle lineage pair (B4.1) of blastomeres of the 8-cell stage (Ciona and Ascidia), and from a muscle lineage blastomere pair (B5.2) isolated at the 16-cell stage (Ascidia). Muscle lineage cells in the partial embryos were readily identified by the dense aggregates of mitochondria in their cytoplasm. Taken together, these results from the two kinds of partial embryo effectively eliminate inductive interactions with embryonic tissues other than mesodermal as a necessary factor in the onset of self-differentiation in muscle lineage cells. The relative complexity of muscle phenotype expressed in cleavage-arrested and partial embryos attests to an unusually strong developmental autonomy in the ascidian muscle lineages. This autonomy lends further support to the theory that a localized and segregated egg cytoplasmic determinant is responsible for larval muscle development in ascidian embryos.     

The central nervous system, its cellular organisation and development, in the tadpole larva of the ascidian Ciona intestinalis

From its numerical composition, the central nervous system (CNS) of the ascidian larva is one of the simplest known nervous systems having a chordate plan. Fewer than 350 cells together constitute a caudal nerve cord, an interposed visceral ganglion containing motor circuits for swimming and, rostrally, an expanded sensory vesicle containing major sensory and interneuron regions of the CNS. Some cells are ependymal, with ciliated surfaces lining the neural canal, while others are clearly either sensory receptors or motoneurons, but most are distinguishable only on cytological grounds. Although reassignments between categories are still being made, there is evidence for determinancy of total cell number. We have made three-dimensional cell maps either from serial semithin sections, or from confocal image stacks of whole-mounted embryos and larvae stained with nuclear markers. Comparisons between the maps of neural tubes in embryos of successive ages, that is, between cells in one map and their progeny in older maps, enable us to follow the line of mitotic descent through successive maps, at least for the caudal neural tube. Details are clear for the lateral cell rows in the neural tube, at least until the latter contains approximately 320 cells, and somewhat for the dorsal cell row, but the ventral row is more complex. In the hatched larva, serial-EM reconstructions of the visceral ganglion reveal two ventrolateral fibre bundles at the caudalmost end, each of 10-12 axons. These tracts include at least five pairs of presumed motor axons running into the caudal nerve cord. Two pairs of axons decussate. Complementing this vertebrate feature in the CNS of the larval form of Ciona, we confirm that synapses form upon the somata and dendrites of its neurons, and that its motor tracts are ventral.     

High-resolution fate map of the snail Crepidula fornicata: the origins of ciliary bands, nervous system, and muscular elements

The littorinimorph gastropod Crepidula fornicata shows a spiralian cleavage pattern and has been the subject of studies in experimental embryology, cell lineage, and the organization of the larval nervous system. To investigate the contribution of early blastomeres to the veliger larva, we used intracellular cell lineage tracers in combination with high-resolution confocal imaging. This study corroborates many features derived from other spiralian fate maps (such as the origins of the hindgut and mesoderm from the 4d mesentoblast), but also yields new findings, particularly with respect to the origins of internal structures, such as the nervous system and musculature that have never been described in detail. The ectomesoderm in C. fornicata is mainly formed by micromeres of the 3rd quartet (principally 3a and 3b), which presumably represents a plesiomorphic condition for molluscs. The larval central nervous system is mainly formed by the micromeres of the 1st and 2nd quartet, of which 1a, 1c, and 1d form the anterior apical ganglion and nerve tracks to the foot and velum, and 2b and 2d form the visceral loop and the mantle cell. Our study shows that both first and second velar ciliary bands are generated by the same cells that form the prototroch in other spiralians and apparently bear no homology to the metatroch found in annelids.     

Cell lineage and cis-regulation for a unique GABAergic/glycinergic neuron type in the larval nerve cord of the ascidian Ciona intestinalis

The tunicate Ciona intestinalis larva has a simple central nervous system (CNS), consisting of fewer than 400 cells, which is homologous to the vertebrate CNS. Recent studies have revealed neuronal types and networks in the larval CNS of C. intestinalis, yet their cell lineage and the molecular mechanism by which particular types of neurons are specified and differentiate remain poorly understood. Here, we report cell lineage origin and a cis‐regulatory module for the anterior caudal inhibitory neurons (ACINs), a putative component of the central pattern generator regulating swimming locomotion. The vesicular GABA/glycine transporter gene Ci‐VGAT, a specific marker for GABAergic/glycinergic neurons, is expressed in distinct sets of neurons, including ACINs of the tail nerve cord and others in the brain vesicle and motor ganglion. Comparative genomics analysis between C. intestinalis and Ciona savignyi and functional analysis in vivo identified the cis‐regulatory module responsible for Ci‐VGAT expression in ACINs. Our cell lineage analyses inferred that ACINs derive from A11.116 cells, which have been thought to solely give rise to glial ependymal cells of the lateral wall of the nerve cord. The present findings will provide a solid basis for future studies addressing the molecular mechanism underlying specification of ACINs, which play a critical role in controlling larval locomotion.     

Autonomy of acetylcholinesterase differentiation in muscle lineage cells of ascidian embryos

The two muscle lineage blastomeres were removed surgically from Ciona intestinalis embryos at the eight-cell stage and allowed to develop in isolation. Acetylcholinesterase, an enzyme that occurs only in muscle cells of the developing larva, was detected histochemically in progeny cells of these isolated blastomers. Acetylcholinesterase differentiation in muscle lineage cells is not, therefore, dependent on inductive interactions with embryonic tissues derived from other eight-cell stage blastomeres.     

Developmental autonomy of presumptive notochord cells in partial embryos of an ascidian

Summary

Ultrastructural features of larval notochord cell differentiation, sheath (membrane leaflets and filaments) and vacuoles of intracellular colloid, were found in some cells of certain partial embryos of the ascidian, Ciona intestinalis. As expected from established lineage fate maps, mature quarter-embryos developing from microsurgically isolated anterior-vegetal blastomeres (A4.1 pair) at the 8-cell stage had some cells with the notochord features. Such cells, however, also occurred in quarter-embryos resulting from the posterior-vegetal blastomere pair (B4.1) and in partial embryos derived from the B5.1 cell pair isolated at the next cleavage of the B4.1 blastomeres. These findings confirm a prediction of additional notochord cell fates from a recent revision of the ascidian lineage map based on cell marking with microinjected horseradish peroxidase. Partial embryos obtained from other lineages of the 8- and 16-cell stages did not develop notochord cells.     

Determinative mechanisms in secondary muscle lineages of ascidian embryos: development of muscle-specific features in isolated muscle progenitor cells

Muscle cells of the ascidian larva originate from three different lines of progenitor cells, the B-line, A-line and b-line. Experiments with 8-cell embryos have indicated that isolated blastomeres of the B-line (primary) muscle lineage show autonomous development of a muscle-specific enzyme, whereas blastomeres of the A-line and b-line (secondary) muscle lineage rarely develop the enzyme in isolation. In order to study the mechanisms by which different lines of progenitors are determined to give rise to muscle, blastomeres were isolated from embryos of Halocynthia roretzi at the later cleavage stages when conspicuous restriction of the developmental fate of blastomeres had already occurred. Partial embryos derived from B-line muscle-lineage cells of the 64-cell embryo (B7.4, B7.5 and B7.8) showed autonomous expression of specific features of muscle cells (acetylcholinesterase, filamentous actin and muscle-specific antigen). In contrast, b-line muscle-lineage cells, even those isolated from the 110-cell embryo (b8.17 and b8.19), did not express any muscle-specific features, even though their developmental fate was mainly restricted to generation of muscle. Isolated A-line cells from the 64-cell embryos (A7.8) did not show any features of muscle differentiation, whereas some isolated A-line cells from the 110-cell embryos (A8.16) developed all three above-mentioned features of muscle cells. This transition was shown to occur during the eighth cell cycle. These results suggest that the mechanism involved in the process of determination of the secondary-lineage muscle cells differs from that of the primary-lineage muscle cells. Interaction with cells of other lineages may be required for the determination of secondary precursors to muscle cells. The presumptive b-line and A-line muscle cells that failed to express muscle-specific features in isolation did not develop into epidermal cells. Thus, although interactions between cells may be required for muscle determination in secondary lineages, the process may represent a permissive type of induction and may differ from the processes of induction of mesoderm in amphibian embryos.     

Embryogenesis in C. elegans after elimination of individual blastomeres or induced alteration of the cell division order

Summary Our earlier studies on embryonic arrest mutants of C. elegans had indicated that early deviations from the normal temporal and spatial pathway of development lead to monstrous terminal phenotypes with little resemblance to a hatched juvenile. To analyze more directly the roles of different parameters for cellular pattern formation, various experiments with a laser microbeam have now been performed and are described in this and the accompanying paper. By ablating early blastomeres we demonstrate here that the establishment of certain cell lineages is not necessary for the generation of a hatching juvenile. However, no replacement of missing cells was observed in these cases, and the resultant animals lacked those structures which are normally produced by the ablated cells. We found that retardation of cell cycle periods in certain cell lineages and thus a change in the normal order of cell divisions is compatible with development to a hatching juvenile. This is also true when, after irradiation of gut precursor cells, their inward migration is considerably delayed. Our results demonstrate that the invariant pattern of early nematode embryogenesis is not a necessary prerequisite for normal development. Studying parameters necessary for gastrulation we found that after irradiation leading to prolonged cell cycle periods the undivided gut founder cell itself rather than its two daughters moves into the center of the embryo. We removed individual early blastomeres and tested whether the typical inward movement of gut precursors still took place. Our results show that the presence of specific neighboring founder cells is not required, indicating that prospective gut cells reduce their cohesive contacts with adjacent blastomeres prior to the onset of gastrulation. Correspondence to: E. Schierenberg     

Cell lineage and determination of cell fate in ascidian embryos

A detailed cell lineage of ascidian embryos has been available since the turn of the century. This cell lineage was deduced from the segregation of pigmented egg cytoplasmic regions into particular blastomeres during embryogenesis. The invariant nature of the cell lineage, the segregation of specific egg cytoplasmic regions into particular blastomeres, and the autonomous development of most embryonic cells suggests that cell fate is determined primarily by cytoplasmic determinants. Modern studies have provided strong evidence for the existence of cytoplasmic determinants, especially in the primary muscle cells, yet the molecular identity, localization, and mode of action of these factors are still a mystery. Recent revisions of the classic cell lineage and demonstrations of the lack of developmental autonomy in certain embryonic cells suggest that induction may also be an important mechanism for the determination of cell fate in ascidians. There is strong evidence for the induction of neural tissue and indirect evidence for inductive interactions in the development of the secondary muscle cells. In contrast to the long-accepted dogma, specification of cell fate in ascidians appears to be established by a combination of cytoplasmic determinants and inductive cell interactions.     

Neuronal form in the central nervous system of the tadpole larva of the ascidian Ciona intestinalis.

The dorsal tubular central nervous system (CNS) of the ascidian tadpole larva is a diagnostic feature by which the chordate affinities of this group, as a whole, are recognized. We have used two methods to identify larval neurons of Ciona intestinalis. The first is serial electron microscopy (EM), as part of a dedicated study of the visceral ganglion (1), and the second is the transient transfection of neural plate progeny with green fluorescent protein (GFP) (2), to visualize the soma and its neurites of individual neurons in whole-mounted larvae of C. intestinalis. Our observations reveal that ascidian larval neurons are simple inform, with a single axonal neurite arising from a soma that is either monopolar or has only very few, relatively simple neurites arising from it, as part of a presumed dendritic arbor. Somata in the visceral ganglion giving rise to axons descending in the caudal nerve cord are presumed to be those of motor neurons.     

Differentiation of histospecific ultrastructural features in cells of cleavage-arrested early ascidian embryos

Summary Ultrastructural features of histospecific differentiation were found in early cleavage stage ascidian embryos treated with cytochalasin B and held thereby in cleavagearrest until hatching time. Markers characteristic of tissue differentiation during normal embryonic and larval stages ofCiona intestinalis were expressed in muscle and two brain cell lineages of cleavage-arrested whole embryos and in epidermal and notochordal cell lineages of cleavage-arrested partial embryos. These features were muscle myofilaments and myofibrils, melanosomes of the brain pigment cells, cilium-derived structures present in a proprioceptive brain cell, extracellular test material of epidermal cell origin, and the sheath filaments, membrane leaflets, and vacuolar colloid associated with notochord cells. All of these ultrastructural markers of differentiation were blocked in their development by treatment of gastrula stage embryos with actinomycin D, an inhibitor of RNA synthesis, and presumably result from the expression of new gene activity. At the time of cleavage-arrest the five cell lineages studies still contained two or more unsegregated lineage pathways. Subsequent developmental autonomy within the lineages is consistent with the hypothesis of segregation during early development of functionally independent gene regulatory factors.     

An evolutionary change in the muscle lineage of an anural ascidian embryo is restored by interspecific hybridization with a urodele ascidian

Anural ascidians do not develop into a conventional tailed larva with differentiated muscle cells, however, embryos of some anural ascidian species retain the ability to express acetylcholinesterase (AChE) in a vestigial muscle cell lineage. This study examines the number of AChE-positive cells that develop in the anural ascidian Molgula occulta relative to that in the closely related urodele (tailed) species, Molgula oculata. Histochemical assays showed that M. oculata embryos develop 36 to 38 AChE-positive cells, consistent with the number of tail muscle cells expressed in other urodele ascidians. In contrast, M. occulta embryos develop a mean of only 20 AChE-positive cells in their vestigial muscle lineage. Cleavage-arrested embryos of the anural species express AChE only in B-line blastomeres, showing that the vestigial muscle lineage cells are derived from the primary muscle lineage. Less than the expected number of AChE-positive B-line cells develop in cleavage-arrested anural embryos, however, implying that the allocation of primary muscle lineage cells is decreased. Eggs of the anural species can be fertilized with sperm of the urodele species resulting in the development of some larvae that contain a short tail and/or a brain melanocyte, specific features of urodele larvae. The typical urodele number of AChE-positive cells is restored in some of these hybrid embryos. Both primary and secondary muscle lineages are restored because cleavage-arrested hybrid embryos develop more AChE-positive cells in the B-line blastomeres and supernumerary AChE-positive cells in the A-line blastomeres. Hybrid embryos that develop the urodele complement of AChE-positive cells also form a tail and/or a brain melanocyte showing that restoration of muscle lineage cells is coupled to the development of other urodele features. AChE expression occurred in anural embryos with disorganized or dissociated blastomeres, indicating that AChE expression is determined autonomously. It is concluded that an evolutionary change in the allocation of larval muscle lineage cells occurs during development of the anural ascidian M. occulta which can be restored by interspecific hybridization with the urodele ascidian M. oculata.     

Distinct Neuronal Lineages of the Ascidian Embryo Revealed by Expression of a Sodium Channel Gene

The ascidian larva contains tubular neural tissue, one of the prominent anatomical features of the chordates. The cell-cleavage pattern and cell maps of the nervous system have been described in the ascidian larva in great detail. Cell types in the neural tube, however, have not yet been defined due to the lack of a suitable molecular marker. In the present work, we identified neuronal cells in the caudal neural tube of theHalocynthiaembryo by utilizing a voltage-gated Na⁺channel gene, TuNa I, as a molecular marker. Microinjection of a lineage tracer revealed that TuNa I-positive neurons in the brain and in the trunk epidermis are derived from the a-line of the eight-cell embryo, which includes cell fates to epidermal and neural tissue. On the other hand, TuNa I-positive cells in the more caudal part of the neural tissue were not stained by microinjection into the a-line. These neurons are derived from the A-line, which contains fates of notochord and muscle, but not of epidermis. Electron microscopic observation confirmed that A-line-derived neurons consist of motor neurons innervating the dorsal and ventral muscle cells. Isolated A-line blastomeres have active membrane excitability distinct from those of the a-line-derived neuronal cells after culture under cleavage arrest, suggesting that the A-line gives rise to a neuronal cell distinct from that of the a-lineage. TuNa I expression in the a-line requires signals from another cell lineage, whereas that in the A-line occurs without tight cell contact. Thus, there are at least two distinct neuronal lineages with distinct cellular behaviors in the ascidian larva: the a-line gives rise to numerous neuronal cells, including sensory cells, controlled by a mechanism similar to vertebrate neural induction, whereas A-line cells give rise to motor neurons and ependymal cells in the caudal neural tube that develop in close association with the notochord or muscle lineage, but not with the epidermal lineage.     

Trunk lateral cells are neural crest-like cells in the ascidian Ciona intestinalis: insights into the ancestry and evolution of the neural crest

Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.     

Ontogenetic changes of circulating erythroid cells and haemoglobin components in Esox lucius

Using light microscopy the morphology, the mitotic index and levels of erythroid cell types were detected from 48 h pike Esox lucius embryos before hatching to adult specimens. At the same developmental stages, the haemoglobins and globin chains expressed were electrophoretically characterized. The erythroid cells of the primitive generation were the most abundant from 48 h before hatching until 15–20 days after hatching, then their number decreased and only rare cells remained in the 3 month‐old juvenile specimens. These cells divided and differentiated in the blood and were substituted by the definitive erythrocyte series. As in other vertebrates, the immature cells of the two generations differed in morphological properties and in the synthetized haemoglobin. The circulating erythroid cells of the definitive population cell lineage were, at all differentiation stages, smaller than those of the primitive generation. The definitive erythrocytes appeared in blood smears of 7 days post‐hatching larvae, they increased rapidly and at 20 days they represented the predominant red blood cell population in the circulation of young pike. Electrophoretic analysis of haemolysates obtained from different developmental stages indicated the presence of distinct embryonic, larval and adult haemoglobins. The embryonic haemoglobins differed from those of the older larva and juvenile specimens and were detectable within the first week of post‐hatching development when only primitive erythrocytes were present in the blood.