Notochord degeneration in chick embryo: a particular case of developmental cell death?

To define the type of cell death occurring in notochordal tissue, the cytological features of degenerating notochord were studied by transmission electron-microscope in thirty chick embryos from the 20th hour to the 15th incubation day. During the first two days the notochordal cells show nuclei with large nucleoli and cytoplasm with yolk granules, lipid droplets, phagolysosomes and deposits of glycogen. From the 3rd to the 5th incubation day, besides the peculiar vacuolization, disaggregation of the endoplasmic reticulum, transformation of the mitochondrial morphology, and disintegration of the cell membrane are detectable. Nuclei are normal up to advanced stages of cytoplasmic degeneration. On the 6th day a large number of cells are dying and, later on, the tissue disintegrates at the level of the vertebral bodies. Cell death in the notochord does not seem to be classifiable as one of the types of developmental cell death described in literature: the comparison with similar cytological features referred by pathologists as a consequence of metabolic damage, suggests that the degeneration of the notochord may be related to its morphological isolation and thus to trophic deprivation.

Cytodifferentiation of the notochord in early chick embryo related to the development of the axial and paraxial structures.

The cytology of the notochord was investigated by means of L.M. and T.E.M. in fifty chick embryos from the 7th HH stage to hatching, at different levels of the longitudinal axis and at different levels in each somite. The cytodifferentiation of the notochord was compared with the one of the surrounding axial and paraxial structures (neuro-epithelium and somites). Close segmental relations among notochord, neural groove and sclerotome, i.e. reciprocal contacts of cell processes and filopodia, where observed in 7-9 HH stages embryos. During the following stages in the notochordal cells signs of secretory activity are detectable, but at the same time degenerative changes are evident and they will increase up to the total regression of the notochord. These morphological observations seem to suggest the presence of reciprocal morphogenetic influences among notochord, neuro-epithelium and sclerotome, only before the 9th stage, rather than a determinant role of the notochord in the surrounding structures at later developmental stages.

"Chordoid tissue": biological significance and relevance of the term.

The significance and relevance of the term "chordoid tissue" are discussed on the ground of some ultrastructural features of the notochordal cells, observed in 15 chick embryos from stage 5HH to hatching. The characteristics of the cytoplasmic organelles and of the nucleus, the membrane alterations and in particular the vacuolization show the notochordal cells undergo a process of necrobiosis rather than of apoptosis. The degeneration of the notochord, that not casually starts in the notochordal core, seems to be due to a progressive metabolic isolation, related to the lack of blood vessels and to the formation of a thick perichordal sheath. These findings suggest that the notochordal tissue shows the same cytologic features of any tissue degenerating owing to a metabolic damage. The term "chordoid tissue", in the meaning of a tissue with peculiar structure and function, does not seem hence to be relevant, also on the ground of the fact that in the species in which a nucleous pulposus arises from notochord remnants, the notochordal cells show the same ultrastructural characteristics detectable in chick embryos.

Expression of calcium binding proteins in the developing spine: preliminary report.

Previous ultrastructural studies on the developing spine induced the Authors to analyse the presence of Ca-binding proteins in the vertebral anlagen of the chick embryo, by immunostaining for calretinin. At 7 incubation days, intense positivity for calretinin at level of the intervertebral region is detectable. From the 7th day the positivity is localized in particular to the intervertebral menisci and it appears also in the degenerating chondrocytes of the chondrifying vertebrae and at level of the peripheral zones of vascular erosion in the ossifying vertebrae. The intervertebral positivity for calretinin can be related to the presence at this level of fibroblast-like cells, that for such immunoreactivity are distinguishable from true fibroblasts. Since other Authors showed presence of S-100 protein in fibroblast-like cells during histogenesis of white adipose tissue, the significance of the expression of some Ca-binding proteins in the differentiation of these cell types can be supposed. The meaning of the immunoreactivity in the vertebral bodies is still uncertain and needs further immunohistochemical studies.

Relations between subcellular events in the degenerating notochord and histopathological features of the spinal chordomas.

The authors focused their attention on the cytology of the degenerating notochord at the level of the vertebral anlagen and compared it with data of the literature on the histopathology of the spinal chordomas. The purpose of the research was to investigate the notochordal histological features justifying the fact that the remnants of a structure destined to atrophy during prenatal life, maintain a proliferative potential in postnatal life. Therefore, from the earliest stages (24th incubation hour) to the terms of development, the notochord was studied by electron microscope in chick embryo. The data obtained show that, in most notochordal cells, signs of metabolic damage and progressive degeneration coexist with signs of secretory and mitotic activity, from which the proliferative potential of these cells seems to derive. Vacuolar degeneration is also likely due to the progressive metabolic segregation of the notochord, owing to the absence of blood-vessels and the appearance of perichordal sheath.

Notochordal histogenesis: preliminary report of ultrastructural observations in chick embryo.

Transverse, frontal and sagittal notochord sections of chick embryos at different developmental stages were ultrastructurally examined. The notochord tissue organization appears to be more likely to build a stiff but flexible body axis than to have an inducting role on the surrounding embryonic structures.

Chondrogenic gradients in the developing vertebral body.

Since in literature the question of the spatio-temporal sequence of the cartilage maturation in the developing vertebra is still controversial, the authors studied by light and electron microscope, the chondrification of the vertebral body in chick embryo from the 6th to the 13th incubation days, in order to define the correlations between morphology and distribution of the cartilage cells in this phase of vertebral development. The results show that the chondrogenesis follows spatio-temporal gradients, starting at about the 8th incubation day from a zone located between notochord and neural tube, slightly cranially to the midvertebral level. From this starting point the chondrification proceeds with dorso-lateral and radial progression, and at the same time extends towards the cranial and caudal plates of the developing vertebra. These data are compared to the findings obtained by other authors on the ultrastructural and biochemical aspects of the vertebral development.

Differentiation of cardiac conducting cells from the neural crest.

Some of the cells that migrate to the dorsal myocardium of the chick embryo in stage 21 H-H begin to synthesize desmin. They retain this property even when they reach the subendothelial layers of the heart bud, i.e. the characteristic site of the cardiac conducting cells.

Embryogenesis of the avian iris sphincter muscle: in vivo and in vitro studies.

The histogenesis of iris sphincter muscle was studied in birds. Chick embryo iris "anlagen", ages from 3 days (st. 18 H.H.) to hatching, were examined. At the 4th day (st. 24 H.H.), nerve fibers were observed in the mesenchyme of the inferotemporal quadrant of the optic cup near the colobomic fissue. Among the mesenchymal cells, there were cells characterized by AChE activity, presence of desmin filaments, exhibiting ACh receptors, and ultrastructurally similar to the presumptive skeletal myoblasts. One day later (st. 27 H.H.), these myoblasts could be cultivated. The formation of myotubes began between 10 and 12 days. From 9 to 14 days, cells left the anterior epithelium of the iris to give rise to the smooth iris muscle; during this evolution some epithelial cells fused with the myotubes taking part in the histogenesis of striated muscle. The possibility of a neurogenic determination for the iris skeletal muscle is discussed.

Dynamics of soleplate formation.

Immunohistochemical techniques with anti-desmin, anti-acetylcholine receptor and anti-fibronectin antisera and autohistoradiography were used to determine the dynamics of neuromuscular synaptogenesis. Fast twitching muscles were taken from chick embryos at 5 to 14 days of incubation. "Primitive eminences" at terminal arborizations of motor neurons were composed of Karnowsky positive, anti-desmin and anti-acetylcholine receptor positive cells which contained sites bound to alpha-bungarotoxin. These cells, characterized as myoblasts, fused with the myotubes during formation of neuromuscular junctions in the sites of contact with terminal arborizations of motor neurons. Their nuclei and cytoplasmic organelles become the nuclei and organelles in the soleplate.

Distribution of substance P immunoreactive cell bodies and fibers in cranial sensory and autonomic ganglia of the chick.

Substance P-immunoreactive neurons were demonstrated in chick embryonic and adult trigeminal ganglion and jugular-superior ganglionic complex using FITC-immunohistochemical methods. Both small-size and large ganglion cells exhibited SP immunoreactivity, without apparent changes during embryonic and post-hatching development. SP-positive fibers could be detected in a good number in the sympathetic cranial cervical ganglion, either during embryonic development or in adult chick. No immunoreactive perikarya were observed in this ganglion. In the ciliary ganglion, both choroidal and ciliary neurons were SP-negative, whereas SP immunoreactive fibers surrounded the perikarya of both cell populations.

Distribution and development of substance P immunoreactive axons in the chick cornea and uvea.

A study was made of the distribution of Substance P-immunoreactive fibers in chick cornea and uvea in whole mount preparation, using the indirect immunofluorescence technique. The development of these fibers during embryogenesis was also investigated. SP-fibers were present in all chick eye structures, in the various prenatal and postnatal stages examined. Their distribution was comparable with that observed by other workers in mammals. Transformation of the iris musculature from smooth to striated, during development, is not accompanied by significant changes in SP-ergic innervation.

Chick heart peptidergic innervation: localization and development.

Localization and development of chick heart peptidergic innervation (Substance P, VIP and Somatostatin) were investigated by means of immunofluorescence technique. The peptidergic component of the heart innervation was observed, for the first time, in older than 11 day chick embryos, i.e., subsequently to the appearance of the cholinergic component. The peptidergic structures achieve nearly full development in about 16-17 day embryos. Substance P is the most represented of the three peptides. It is localized both in nerve bundle fibers and in isolated fibers within the myocardium, the pericardium, the vessel walls; it is also present in fibers of some heart base ganglia. VIP is mostly contained in some thick single fibers travelling along the vessel walls of the heart base, the myocardium and the pericardium. Some VIP immunoreactive cells were also observed in the base ganglia. Somatostatin is mostly contained in some ganglia cells, whilst thin Somatostatin-immunoreactive fibers form a rich plexus among the atrial and ventricular myofibers, without contacting the vessel walls.

Origin of the iris sphincter muscle in chick embryo.

The origin of the iridial sphincter muscle in chick embryo was investigated by means of immunohistochemistry. Desmin immunoreactive cells are shown in the mesenchymal stroma overlying the anterior epithelial layer of the iris in 4 1/2-day chick embryos. In 9-11-day chick embryos also some cells of the posterior epithelium near the pupillary margin, and of the iridial lamella show a slighter desmin-immunoreactivity. This finding agrees with a double origin of the iridial sphincter muscle: an early mesenchymal one and a later epithelial other.

Early myoblast differentiation in fast and slow types.

Serial sections of stage 18-27 HH (3-5 days of incubation) chick embryo myotomes were investigated by electron microscope. Two morphologically different types of contractile elements (myoblasts and myotubes) with an exact and constant localization were identified. Myotome sections of other chick embryos in the same stages were examined with the immunofluorescence technique, after treatment with antisera directed against fast and slow adult myosins. As from stage 24 HH, some contractile elements react positively with anti-fast adult myosin antiserum, others with anti-slow antiserum. A very precise correspondence was constantly found between the ultrastructural and immunohistochemical findings. An identical pattern was observed in the buds of the fast-twitching pectoralis major and posterior latissimus dorsi and slow-twitching anterior latissimus dorsi muscles.

[Displacement of myotomic cells in muscle buds. Autoradiography study]. / Le déplacement des cellules myotomales dans les ébauches musculaires. Etudié par autordiographie.

Autoradiographic investigation of the migration of myotomal cells into the muscle buds. Autoradiography following the administration of 3H-thymidine in chick embryos showed that myogenically differentiated muscle plate cells leave the mitotic cycle, and remain in that state even after their migration to muscle buds derived from the myotome itself.

[Acetylcholine receptors in chick embryo somites]. / Le récepteur pour l'acétylcholine dans les somites chez l'embryon de poulet.

Autoradiography was used to demonstrate the presence of alpha-bungarotoxin binding sites in the medial wall cells of thoracic somites from 44th hr incubation chick embryos. It is suggested that these cells are presumptive myoblasts about to migrate to form the myotome.

Immunohistochemical localization of fibronectin in structures of the human oral cavity: dental pulp and gingiva.

The distribution of fibronectin (FN) in the dental pulp and gingiva of the human adult was investigated by indirect immunofluorescence and indirect immunoperoxidase techniques. FN could not be demonstrated in the connective tissue of the dental pulp even in the blood vessels. On the contrary, a positive stain was obtained in the ground substance at the base of the odontoblast cell bodies and in various structures of a denticle which was accidentally found in the pulp of one of the examined teeth. The lack of FN in the dental pulp could mean that this tissue, sometimes considered to be of an immature and undifferentiated type, is instead a mature one. The subepithelial connective tissue and the epithelium of the gingiva were examined. The distribution of FN in the connective tissue of the gingiva was found to be similar to that described in other types of connective tissue; high concentrations were found both in the epithelial cells and around them. This finding is described here for the first time and remains to be verified in other types of stratified epithelia.