Neurogenic and non-neurogenic placodes in ascidians.

The late differentiation of the ectodermal layer is analysed in the ascidians Ciona intestinalis and Botryllus schlosseri, by means of light and electron microscopy, in order to verify the possible presence of placodal structures. Cranial placodes, ectodermal regions giving rise to nonepidermal cell types, are classically found exclusively in vertebrates; however, data are accumulating to demonstrate that the nonvertebrate chordates possess both the genetic machinery involved in placode differentiation, and ectodermal structures that are possible homologues of vertebrate placodes. Here, the term "placode" is used in a broad sense and defines thickenings of the ectodermal layer that can exhibit an interruption of the basal lamina where cells delaminate, and so are able to acquire a nonepidermal fate. A number of neurogenic placodes, ones capable of producing neurons, have been recognised; their derivatives have been analysed and their possible homologies with vertebrate placodes are discussed. In particular, the stomodeal placode may be considered a multiple placode, being composed of different sorts of placodes: part of it, which differentiates hair cells, is discussed as homologous to the octavo-lateralis placodes, while the remaining portion, giving rise to the ciliated duct of the neural gland, is considered homologous to the adenohypophyseal placode. The neurohypophyseal placode may include the homologues of the hypothalamus and vertebrate olfactory placode; the rostral placode, producing the sensorial papillae, may possibly be homologous to the placodes of the adhesive gland of vertebrates.

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.

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.

Development of the motor nervous system in ascidians.

The motor nervous system of adult ascidians consists of neurons forming the cerebral ganglion from which axons run out directly to the effectors, i.e., muscular and ciliary cells. In this study, we analyzed the development of the motor fibers, correlating this with organ differentiation during asexual reproduction in Botryllus schlosseri. We used a staining method for acetylcholinesterase, whose reaction product is visible with both light and electron microscopy and which labels entire nerves, including their thin terminals, making them identifiable between tissues. While the cerebral ganglion is forming, the axons elongate and follow stereotypical pathways to reach the smooth muscle cells of the body, the striated muscle of the heart, and the ciliated cells of the branchial stigmata and the gut. A strict temporal relation links the development of the local neural network with its target organ, which is approached by nerves before the effector cells are fully differentiated. This process occurs for oral and cloacal siphons, branchial basket, gut, and heart. Axons grow through the extracellular matrix and arrive at their targets from different directions. In some cases, the blood sinuses constitute the favorite roads for growing axons, which seem to be guided by a mechanism involving contact guidance or stereotropism. The pattern of innervation undergoes dynamic rearrangements and a marked process of elimination of axons, when the last stages of blastogenesis occur. The final pattern of motor innervation seems to be regulated by axon withdrawal, rather than apoptosis of motor neurons.

Puffs and salivary gland function: The fine structure of the larval and prepupal salivary glands ofDrosophila melanogaster.

The salivary glands ofDrosophila melanogaster have been examined by electron microscopy for fine structural alterations occurring during larval and prepupal stages. The changes observed in the glands have been correlated with the puffing patterns of the polytene chromosomes at corresponding stages. In early third instar larvae, the lumen of the salivary gland appears empty, and no signs of secretory activity are visible in the glandular cytoplasm. From puff stages 1 to 6 the endoplasmic reticulum becomes reorganized and increases in volume. Electron dense material appears within its cisternae and subsequently within the Golgi saccules. Dense secretory granules then appear to be elaborated from the Golgi by terminal budding; these granules represent the 'glue' for adhering the pupa to its substrate, and gradually increase in size and complexity. By puff stage 6 their contents have been liberated into the glandular lumen. Following puparium formation, those granules which are not extruded coalesce to form larger granules. Other dense bodies and autophagic vacuoles, considered to be lysosomes, appear, and the 'surplus' secretory granules begin to display myelination at their peripheries; ultimately they are reduced to dense residual bodies. At puparium formation, the lumen is depleted of the glue and contains flocculent material. Histolysis commences after puff stage 11, and the cytoplasm becomes vacuolated and opaque; the nucleus becomes reduced in volume and crenated in outline. Nuclear blebbing occurs after puff stage 12, and material seemingly moves from the nucleus into the cytoplasm; the glandular lumen now becomes empty. An attempt has been made to ascertain how the chromosomal puffing activity relates to these cytoplasmic developments.