Lucia Manni

Botryllus schlosseri: a model ascidian for the study of asexual reproduction.

Botryllus schlosseri, a cosmopolitan colonial ascidian reared in the laboratory for more than 50 years, reproduces both sexually and asexually and is used as a model organism for studying a variety of biological problems. Colonies are formed of numerous, genetically identical individuals (zooids) and undergo cyclical generation changes in which the adult zooids die and are replaced by their maturing buds. Because the progression of the colonial life cycle is intimately correlated with blastogenesis, a shared staging method of bud development is required to compare data coming from different laboratories. With the present review, we aim (1) to introduce B. schlosseri as a valuable chordate model to study various biological problems and, especially, sexual and asexual development; (2) to offer a detailed description of bud development up to adulthood and the attainment of sexual maturity; (3) to re‐examine Sabbadins ( 1955 ) staging method and re‐propose it as a simple tool for in vivo recognition of the main morphogenetic events and recurrent changes in the blastogenetic cycle, as it refers to the developmental stages of buds and adults. Developmental Dynamics 236:335–352, 2007.

Mechanism of neurogenesis during the embryonic development of a tunicate.

Ascidian and vertebrate nervous systems share basic characteristics, such as their origin from a neural plate, a tripartite regionalization of the brain, and the expression of similar genes during development. In ascidians, the larval chordate‐like nervous system regresses during metamorphosis, and the adults neural complex, composed of the cerebral ganglion and the associated neural gland is formed. Classically, the homology of the neural gland with the vertebrate hypophysis has long been debated. We show that in the colonial ascidian Botryllus schlosseri, the primordium of the neural complex consists of the ectodermal neurohypophysial duct, which forms from the left side of the anterior end of the embryonal neural tube. The duct contacts and fuses with the ciliated duct rudiment, a pharyngeal dorsal evagination whose cells exhibit ectodermic markers being covered by a tunic. The neurohypophysial duct then differentiates into the neural gland rudiment whereas its ventral wall begins to proliferate pioneer nerve cells which migrate and converge to make up the cerebral ganglion. The most posterior part of the neural gland differentiates into the dorsal organ, homologous to the dorsal strand. Neurogenetic mechanisms in embryogenesis and vegetative reproduction of B. schlosseri are compared, and the possible homology of the neurohypophysial duct with the olfactory/adenohypophysial/hypothalamic placodes of vertebrates is discussed. In particular, the evidence that neurohypophysial duct cells are able to delaminate and migrate as neuronal cells suggests that the common ancestor of all chordates possessed the precursor of vertebrate neural crest/placode cells. J. Comp. Neurol. 412:527–541, 1999. 

Novel, secondary sensory cell organ in ascidians: In search of the ancestor of the vertebrate lateral line

A new mechanoreceptor organ, the “coronal organ,” located in the oral siphon, is described by light and electron microscopy in the colonial ascidians Botryllus schlosseri and Botrylloides violaceus. It is composed of a line of sensory cells (hair cells), accompanied by supporting cells, that runs continuously along the margin of the velum and tentacles of the siphon. These hair cells resemble those of the vertebrate lateral line or, in general, the acoustico‐lateralis system, because they bear a single cilium, located centrally or eccentrically to a hair bundle of numerous stereovilli. In contrast to other sensory cells of ascidians, the coronal hair cells are secondary sensory cells, since they lack axonal processes directed towards the cerebral ganglion. Moreover, at their base they form synapses with nerve fibers, most of which exhibit acetylcholinesterase activity. The absence of axonal extensions was confirmed by experiments with lipophilic dyes. Different kinds of synapses were recognized: usually, each hair cell forms a few afferent synapses with dendrites of neurons located in the ganglion; efferent synapses, both axo‐somatic (between an axon coming from the ganglion and the hair cell) and axo‐dendritic (between an axon coming from the ganglion and an afferent fiber) were occasionally found. The presence of secondary sensory cells in ascidians is discussed in relation to the evolution of sensory cells and placodes in vertebrates. It is proposed that the coronal organ in urochordates is homologous to the vertebrate acoustico‐lateralis system. J. Comp. Neurol. 461:236–249, 2003.

Neurogenic role of the neural gland in the development of the ascidian, Botryllus schlosseri (Tunicata, Urochordata)

In adult ascidians, the neural complex consists of a cerebral ganglion (the brain) and the associated neural gland. We have studied the development of the neural complex during the vegetative reproduction of the colonial ascidian Botryllus schlosseri, the buds of which arise from the atrial mantle of the parental zooid. Each bud develops into a new organism within which a neural complex becomes differentiated. We found that the presumptive (pioneer) nerve cells that ultimately form the cerebral ganglion of the adult arise as migratory cells from a primordial cluster of rudimentary gland cells. Hence, the neural gland appears to be neurogenic in that it serves as the cellular source of components that differentiate into conventional nerve cells. In the adult, these cells take on the form of a typical invertebrate ganglion with an outer cortex of nerve cell bodies and an internal medulla. This medulla consists of a neuropile of neuronal processes making classical synaptic contacts. The adult neural gland differentiates into a structure with a ciliated duct that opens into the branchial chamber, the body of the gland, and the dorsal organ, which is quite distinct from the dorsal strand of other ascidians. The rudimentary neural gland cells, therefore, differentiate into one of two distinct pathways: the first, glandular, is possibly involved in the evaluation of environmental signals, and the other, nervous, leads to brain formation. This compares with the vertebrate situation in which the olfactory–pituitary placodes are thought to originate from a common cellular source. Thus, these data support the earlier contention of a homology between the tunicate neural gland and the vertebrate adenohypophysis. J. Comp. Neurol. 394:230–241, 1998.

Embryonic versus blastogenetic development in the compound ascidian Botryllus schlosseri: insights from Pitx expression patterns.

The colonial ascidians reproduce either sexually or asexually, having evolved a rich variety of modes of propagative development. During embryogenesis, the fertilized egg develops into a swimming tadpole larva that subsequently metamorphoses into a sessile oozooid. Clonal individuals (blastozooids), resembling oozooids, are formed from few bud‐forming multipotent somatic cells, following a wide range of ways that seem to characterize each family of this class. Here, we compare these two developmental processes in the compound ascidian species Botryllus schlosseri to determine whether similar gene activities are used during embryogenesis/metamorphosis and recruited in the asexual development. We analyzed expression of Pitx, a Paired‐related homeobox gene. Pitx genes are key developmental genes in vertebrates, and their expression is reported to be conserved in chordate stomodea and in the establishment of left/right asymmetries. Here, we report full‐length cDNA cloning of a B. schlosseri Pitx ortholog (Bs‐Pitx) and expression analysis during both embryo/metamorphosis and blastogenesis. During organogenesis of both developmental sequences, Bs‐Pitx was detected in identical domains: the stomodeum/neural complex and asymmetrically in the left digestive system. In striking contrast, expression patterns at early stages differ deeply. These observations provide the first evidence for a key developmental gene being deployed in essentially similar ways in two different developmental sequences that eventually give rise to similar zooids. Developmental Dynamics 232:468–478, 2005.

Morphological Differences between Larvae of the Ciona intestinalis Species Complex: Hints for a Valid Taxonomic Definition of Distinct Species.

The cosmopolitan ascidian Ciona intestinalis is the most common model species of Tunicata, the sister-group of Vertebrata, and widely used in developmental biology, genomics and evolutionary studies. Recently, molecular studies suggested the presence of cryptic species hidden within the C. intestinalis species, namely C. intestinalis type A and type B. So far, no substantial morphological differences have been identified between individuals belonging to the two types. Here we present morphometric, immunohistochemical, and histological analyses, as well as 3-D reconstructions, of late larvae obtained by cross-fertilization experiments of molecularly determined type A and type B adults, sampled in different seasons and in four different localities. Our data point to quantitative and qualitative differences in the trunk shape of larvae belonging to the two types. In particular, type B larvae exhibit a longer pre-oral lobe, longer and relatively narrower total body length, and a shorter ocellus-tail distance than type A larvae. All these differences were found to be statistically significant in a Discriminant Analysis. Depending on the number of analyzed parameters, the obtained discriminant function was able to correctly classify > 93% of the larvae, with the remaining misclassified larvae attributable to the existence of intra-type seasonal variability. No larval differences were observed at the level of histology and immunohistochemical localization of peripheral sensory neurons. We conclude that type A and type B are two distinct species that can be distinguished on the basis of larval morphology and molecular data. Since the identified larval differences appear to be valid diagnostic characters, we suggest to raise both types to the rank of species and to assign them distinct names.

Coronal organ of ascidians and the evolutionary significance of secondary sensory cells in chordates

A new mechanoreceptor organ, the coronal organ, in the oral siphon of some ascidians belonging to the order Pleurogona has recently been described. In contrast to the known mechanoreceptor organs of ascidian atrium that consist of sensory neurons sending their own axons to the cerebral ganglion, coronal sensory cells are secondary mechanoreceptors, i.e., axonless cells forming afferent and efferent synapses with neurites of neurons located in the ganglion. Moreover, coronal cells exhibit an apical apparatus composed of a cilium accompanied or flanked by rod‐like microvilli (stereovilli). Because of the resemblance of these cells to vertebrate hair cells, their ectodermal origin and location in a linear array bordering the bases of the oral tentacles and velum, the coronal organ has been proposed as a homologue to the vertebrate acousticolateralis system. Here we describe the morphology of the coronal organs of six ascidians belonging to the suborders Phlebobranchia and Aplousobranchia (order Enterogona). The sensory cells are ciliated, lack typical stereovilli, and at their bases form synapses with neurites. In two species, the sensory cells are accompanied by large cells involved in synthesis and secretion of protein. We hypothesize that the coronal organ with its secondary sensory cells represents a plesiomorphic feature of ascidians. We compare the coronal organ with other chordate sensory organs formed of secondary sensory cells, i.e., the ventral lip receptors of appendicularians, the oral secondary sensory cells of cephalochordates, and the acousticolateralis system of vertebrates, and we discuss their homologies at different levels of organization. J. Comp. Neurol. 495:363–373, 2006.

Hair cells in ascidians and the evolution of lateral line placodes

The vertebrate hair cells are ciliary highly differentiated mechanoreceptors whose name derives from the peculiar microvilli, called stereovilli, that protrude into the fluid-filled cavities of the inner ear or lateral line organs. They differ from the primary sensory cells found in most invertebrates in that they are axonless, thus being secondary sensory cells that synapse with the dendrites of neurons whose cell bodies are located in the central nervous system (CNS). Although their morphology varies in different vertebrate species, hair cells typically have a single eccentric cilium and a collar of stereovilli graded in length from one side to the other. Fibrillar links between the stereovilli represent a structural device for transduction of the stimulatory force at mechanosensitive ion channels on the membranes of the stereovilli. Excitation is transmitted by synapses between the hair cells and afferent neurites going to the CNS; sensitivity of the hair cells is regulated by efferent synapses from neurites, providing input directly to the hair cells or to the hair cells’ afferent neurites. Hair cells derive from placodes of the acustico-lateralis system that, together with the other neurogenic placodes, are generally believed to originate ontogenetically from a wide panplacodal field (Baker and Bronner-Fraser 2001; Schlosser 2002) and are usually considered exclusive to craniates. However, recent molecular and morphological data suggest that cell populations with the properties of neurogenic placodes

Cell reorganisation during epithelial fusion and perforation: the case of ascidian branchial fissures.

In this study, we have analysed ultrastructurally the mechanism of epithelial fusion and perforation during the development of branchial fissures in the larva and bud of the colonial urochordate Botryllus schlosseri. Perforation of membranes represents an important process during embryogenesis, occurring to create communication between two separate compartments. For example, all chordate embryos share the formation of pharyngeal plates, which are constituted of apposed endodermal and ectodermal epithelia, which have the capacity to fuse and perforate. Although the process of perforation is extremely common, its cellular mechanism remains little understood in detail, because of the complexity of the structures involved. In B. schlosseri, two epithelial monolayers, the peribranchial and the branchial ones, with no interposed mesenchymal cells, participate in pharyngeal perforation. Blood flows in the interspace between the two cellular leaflets. Apico‐lateral zonulae occludentes seal the cells of each epithelium, so that the blood compartment is separated from the environment of the peribranchial and branchial chambers; here, sea water will flow when the zooid siphons open. Stigmata primordia appear as contiguous thickened discs of palisading cells of branchial and peribranchial epithelia. The peribranchial component invaginates to contact the branchial one. Here, the basal laminae intermingle, compact, and are degraded, while the intercellular space between the two epithelia is reduced to achieve the same width as that found between the lateral membranes of adjacent cells. Cells involved in this fusion rapidly change their polarity: they acquire a new epithelial axis, because part of the adhering basal membrane becomes a new lateral surface, whereas the original lateral membranes become new apical surfaces. Before disassembling the old tight junctions and establishing communication between branchial and peribranchial chambers, cells of the stigmata rudiments form new tight junctions organised as distinct entities, so that the structural continuum of the epithelial layers is maintained throughout the time of fusion and perforation.

Germline cell formation and gonad regeneration in solitary and colonial ascidians

The morphology of ascidian gonad is very similar among species. The testis consists of variable number of testicular follicles; the ovary consists of ovarian tubes that are thickened forming the germinal epithelium with stem cells for female germ cells with the exception of botryllid ascidians. Peculiar accessory cells that would be germline in origin accompany the oocytes. Using vasa homologues as a molecular marker, germline precursor cells can be traced back to the embryonic posterior‐most blastomeres and are found in the tail of tailbud embryo in some solitary and colonial ascidians. In Ciona, they are subsequently located in the larval tail, while in colonial botryllid ascidians vasa‐expressing cells become obscure in the tail. Recent evidence suggests that ascidian germ cells can regenerate from cells other than embryonic germline. An ensemble of the embryonic stringency of germ cell lineage and the postembryonic flexibility of gonad formation is discussed. Developmental Dynamics 240:299–308, 2011.