Paolo Burighel

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.

Degenerative regression of the digestive tract in the colonial ascidian Botryllus schlosseri (Pallas).

SummaryDegenerative changes in the digestive tract of zooids of Botryllus schlosseri were studied by light and electron microscopy. Three main processes occurred in the tissues: contraction, involution and phagocytosis. The contraction of epidermis and peribranchial epithelium in which cytoplasmic microfilaments probably participate, seemed to have a special role in compressing the underlying organs. During contraction most of the body cavities collapsed, the branchial walls disintegrated and the fragments were rapidly taken up by large phagocytes. The gut epithelium retained its apparent continuity longer, though isolated phagocytes infiltrated it to eliminate single cells. Cell degeneration came about chiefly either through swelling and lysis of cells or through loss of water and condensation of cytoplasm and nucleus.The fate of all regressed tissues was to be engulfed and digested by wandering phagocytes. However, it was also observed that numerous cells of different epithelia could act as fixed phagocytes by engulfing cell debris and entire cells into heterophagic vacuoles.

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.

Fine structure of the testicular gland of the black goby, Gobius jozo L.

SummaryThe homologue of mammalian Leydig cells in the testis of fish of the genus Gobius is believed to be a glandular mass lying along the mesorchium and quite distinct from the seminiferous region. This gland was studied in Gobius jozo by light and electron microscopy. Histologically, the gland is composed of cords of polyhedral cells located between the main vessels of the testis and the deferent duct. Dark and light cell varieties were observed both after staining with toluidine blue and with respect to their cytoplasmic electron density. The fine structure of the glandular cells is characterized by a well developed agranular endoplasmic reticulum in the form of random anastomosing tubules and vesicles and by very numerous mitochondria with tubulo-vesicular cristae. In some mitochondria the internal organization is paracrystalline. One or two bodies of unknown nature are Found in the Golgi region. They are partially limited by an unusually thick membrane and contain a material subdivided mostly into small vesicles. Liposomes and lipofuscin pigment granules are also present.The ultrastructural features of the glandular cells of the testis of Gobius jozo confirm a specialization in steroid hormone production.

Haemocytes and blastogenetic cycle in the colonial ascidian Botryllus schlosseri: a matter of life and death

A recurrent blastogenetic cycle characterizes colonies of the ascidian Botryllus schlosseri. This cycle starts when a new zooid generation opens its siphons and ends with take-over, when adult zooids cease filtering and are progressively resorbed and replaced by a new generation of buds, reaching functional maturity. During the generation change, massive apoptosis occurs in the colony, mainly in the tissues of old zooids. In the present study, we have investigated the behaviour of haemocytes during the colonial blastogenetic cycle, in terms of the occurrence of cell death and the expression of molecules involved in the induction of apoptosis. Our results indicate that, during take-over, caspase-3 activity in haemocyte lysates increases. In addition, about 20%–30% of haemocytes express phosphatidylserine on the outer leaflet of their plasma membrane, show DNA fragmentation and are immunopositive for caspase-3. Senescent cells are quickly ingested by circulating phagocytes that frequently, having once engulfed effete cells, in turn enter apoptosis. Dying cells and corpses are replaced by a new generation of cells that appear in the circulation during the generation change.

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