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 Sabbadin's (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.

Programmed cell death in vegetative development: apoptosis during the colonial life cycle of the ascidian Botryllus schlosseri.

Programmed cell death (PCD) by apoptosis is a physiological mechanism by which cells are eliminated during embryonic and post-embryonic stages of animal life cycle. During asexual reproduction, the zooids of colonial ascidians originate from an assorted cell population instead of a single zygote, so that we assume that regulation of the equilibrium among proliferation, differentiation and cell death may follow different pathways in comparison to the embryonic development. Here we investigate the presence of apoptotic events throughout the blastogenetic life cycle of the colonial ascidian Botryllus schlosseri, by means of terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) coupled with histochemical and electron microscopy techniques. The occurrence of low levels of morphogenetic cell death suggests that, in contrast to what happens during sexual development (embryogenesis and metamorphosis), apoptosis does not play a pivotal role during asexual propagation in botryllid ascidian. Nevertheless, PCD emerges as a key force to regulate homeostasis in adult zooids and to shape and modulate the growth of the whole colony.

Macrophage-secreted myogenic factors: a promising tool for greatly enhancing the proliferative capacity of myoblasts in vitro and in vivo.

In this work we set out to determine if the murine macrophage J774 cell line can be used to produce myogenic growth factors. Activated J774 macrophages were grown in serum-free conditions. The macrophage-conditioned medium (MCM) was then used to treat cultures of primary myoblasts and regenerating muscle tissue, in vitro and in vivo respectively. MCM activity in vitro was tested by analyzing the expression of muscle-specific transcription factors, in parallel with the proliferation and differentiation rates of the cells. The macrophage-secreted factors greatly enhanced the proliferative potential of both rat and human primary myoblasts and were found to be highly muscle-specific. In vivo, MCM administration markedly enhanced the regenerative processes in damaged muscles. The ability to produce large amounts of macrophage-secreted myogenic factor(s) in the absence of serum holds great promise for its biochemical characterization and successive application in therapeutic protocols, both for ex vivo gene therapy and for muscle repair.

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 adult's 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.

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.

An unusual membrane system in the oocyte of the ascidian Botryllus schlosseri.

During vitellogenesis, oocytes of Botryllus schlosseri always exhibit an unusual system scattered in the cytoplasm. It consists of an association between a single fenestrated endoplasmic reticulum cisterna and one or a few smooth vesicles (cisterna vesicle association: CVA) containing a dense core facing the cisterna itself. The latter is smooth and perforated by numerous small pores (about 25 nm in diameter) in the area of association; towards the periphery, it extends into several branches with ribosomes bound to their membranes. In the vesicles, fibrillar material radiates from the dense core and is sometimes organized into a long, dense lamina. The membranes of both cisterna and vesicles appear to be coupled, but are in fact separated by a constant narrow space occupied by short densities. The presence in B. schlosseri of this unusual fenestrated membrane system contrasts with the absence of a typical porous cytoplasmic organelle, the annulate lamellae (ALs), which is widely distributed in female gametes. However, as in other animals, B. schlosseri oocytes possess intranuclear annulate lamellae (IALs) and vesicles. Comparative observations extended to the oocytes of the ascidian Ciona intestinalis have shown that the latter species exhibits typical ALs and IALs, but not the CVA. The morphology of the CVA is analysed here in detail, and similarities and differences with ALs are pointed out. Hypotheses regarding CVA function are discussed in terms of possible relations with ALs.