Showing papers in "Anatomy and Embryology in 1989"

Quantitative electron microscopic immunocytochemistry of neuroactive amino acids

Abstract: Amino acids are of crucial importance in brain function, not only as metabolic intermediates and building blocks of proteins, but also as mediators of interneuronal communication. This dual role of the amino acids distinguishes them from other neurotransmitter candidates, and implies that they are unlikely to be restricted to neurons using them as transmitters. This calls for a quantitative approach when attempts are made to analyse the distribution of transmitter amino acids by means of immunocytochemistry. The present review deals with recent methodological developments that have made it possible to utilize specific antisera to explore the cellular and subcellular distribution of neuroactive amino acids in a quantitative manner.

Development of the origin of the coronary arteries, a matter of ingrowth or outgrowth?

Abstract: Inconsistencies still exist with regard to the exact mode of development of proximal coronary arteries and coronary orifices. In this regard 15 quail embryos were investigated using a monoclonal anti-endothelium antibody, enabling a detailed study of the development of endothelium-lined vasculature. Coronary orifices emerged at 7–9 days of incubation (Zacchei stages 24–26) and were invariably present at 10 days of incubation (Zacchei stage 27).

Persistent truncus arteriosus in the Splotch mutant mouse

Abstract: The Splotch mutant mouse shows defects in neural crest-derived cell populations. The septation of the truneus arteriosus and the development of the aortic arch-derived blood vessels was studied in homozygotes of the Splotch mutant allele Sp1H. It is shown that in homozygous mutant embryos, the septation of the truncus arteriosus does not proceed normally, resulting in persistent truncus arteriosus. The ostium of the persistent truncus arteriosus opens to the right ventricle. Frequently, variations of the aortic arch-derived blood vessels are observed. The development of the thymus, the parathyroid and the ultimobranchial bodies are also variably affected in mutants. These results provide indirect evidence, that cells contributing to the aortic arches and the septum of the truncus arteriosus in mice are derived from the neural crest. The Splotch mutant mouse is proposed to be an animal model for persistent truncus arteriosus. The implications of the vascular malformations for the midgestational death of this mutant are discussed.

Monodelphis domestica (grey short-tailed opossum): an accessible model for studies of early neocortical development.

Abstract: The development of the neocortex of the marsupial Monodelphis domestica has been studied from birth until adulthood. Monodelphis is born after a gestational period of 14 days, a time when the neocortex is still at a two-layered “embryonic’ stage of development, that is equivalent to a 13–14 day rat embryo or 6 week human embryo. The cortical plate does not begin to appear until 3 to 5 days postnatal. Thus the whole of neocortical development is a postnatal phenomenon in this species, as has been previously described in other marsupials. The general pattern of development of the characteristic layers of the immature neocortex and the subsequent development of a six-layered adult neocortex is similar to that found in eutherian species. However there are some differences. The depth of the immature cortical plate when compared to the thickness of the neocortical wall is less than in eutherians and the subplate zone is much deeper in Monodelphis; this transient subplate zone consists of widely spaced rows of cells that are aligned parallel to the cortical surface. Unlike eutherians there appears to be no secondary proliferative zone in the subventricular zone of the dorso-lateral neocortical wall. Maturation of the neocortex is apparent by 45 days postnatal and by 60 days (around the time of weaning) the characteristic six-layered adult neocortex is clearly present. The neuronal marker PGP 9.5 was used to define neuronal populations in the adult brain. The density of neurons in Monodelphis appears to be considerably less than in eutherians such as the rat. The suitability of postnatal Monodelphis for studies of neocortical development is discussed.

Immunocytochemical analysis of the dopamine system in the brain and spinal cord of the European eel, Anguilla anguilla.

Abstract: The distribution of dopamine-containing perikarya and fibres in the central nervous system of the eel, Anguilla anguilla, was determined by using a specific dopamine antiserum. Telencephalic dopamine-immunoreactive somato are located in the external cell layer of the olfactory bulb and throughout the rostrocaudal extent of the subpallium; immunoreactive fibres are located primarily in the bulb and in ventral and lateral portions of the hemispheres. Diencephalic dopamine-immunoreactive neurons are associated with the ventricles in the preoptic area and hypothalamus and in the posterior tubercle. Many of the neurons in the hypothalamus are liquor-contacting. Very few immunoreactive neurons are located in the mesencephalon, and no dopamine-containing cells are found in regions that can be homologized with the ventral tegmental area and substantia nigra of amniotes. There is a rich innervation of the medial octavolateralis nucleus and certain layers of the torus semicircularis and of the tectum. dopamine-containing neurons are located in the vagal lobe, by the vagal motor nucleus and in the area postrema, which provides a rich dopaminergic innervation of the brainstem motor column and of the reticular formation. Immunoreactive liquor-contacting neurons line the central canal and another type of labelled neuron lies dorsally in the spinal cord.

The appearance, density and distribution of melanocytes in human embryonic and fetal skin revealed by the anti-melanoma monoclonal antibody, HMB-45.

Abstract: The presence, densities, and patterns of distribution of melanocytes in the epidermis of human embryos and fetuses, ranging in age from 40 d to 140 d estimated gestational age (EGA), were studied using the HMB-45 monoclonal antibody that recognizes an antigen in melanoma cells and fetal melanocytes. Immunostained sections of skin and epidermal sheets revealed dendritic melanocytes within the basal or intermediate layers of 50 d EGA and older skin. Melanocytes could not be identified by immunostaining or electron microscopy in younger (40–50 d EGA) epidermis or in cultured epidermal cells from these specimens. However, skin from a 45 d EGA embryo grown in organ culture for 11 d stained positively with HMB-45, suggesting that melanocytes are present at that age either in the epidermis or dermis of the explant. Double-labeling experiments using ATPase and HMB-45 confirmed the specificity of HMB-45 for melanocytes and demonstrated that melanocytes and Langerhans cells are nonoverlapping populations. Melanocytes were present in the embryonic epidermis in relatively high numbers (mean value of ∼1050 cells/mm2); they increased in density to ∼2300 cells/mm2 during the late first trimester and early second trimester, then declined during later stages of development to a density of ∼800 cells/mm2, within the range of values for the newborn child and young adult. Equivalent numbers of melanocytes were recognized by silver staining and with the HMB-45 antibody in an 87 d EGA test sample, indicating that HMB-45 reacted with the total melanocytic population. Melanocytes appeared to be distributed in epidermal sheets in a regular pattern. Statistical tests used to evaluate the randomness of a population revealed a tendency toward a non-random distribution in specimens younger than 80 d EGA, just prior to appendage formation and epidermal stratification into multiple layers, however there was variability in the degree of randomness for any given age. The results of this study have closed the gap in timing between the conclusion of neural crest formation and migration (around 6 weeks) and the appearance of melanocytes in the skin between 40–50 d EGA.

Genome reactivation in developing early pig embryos: an ultrastructural and autoradiographic analysis.

Abstract: The onset of RNA synthesis in developing early pig embryos from 1-cell to 8-cell and morula stages was studied using high-resolution autoradiography of (5-3H)uridine incorporation. No transcriptional activity was detected in nuclei of 1- and 2- cell stage embryos with this technique. In these embryos nucleolus-like bodies (NLB) consist of sharply delineated, round, electron dense fibrillar masses. In the 4-cell stage embryos, the first uridine-3H incorporation in the nucleoplasm was detected and localized mainly near the regions of condensed chromatin. The first signs or reticulation and chromatin association were observed at the periphery of NLBs. In the next cell cycle (5- to 8-cell embryos) uridine-3H labelling was detected in the nucleoplasm and nucleoli. In these embryos, nucleoli consist of a central dense fibrillar mass without any transcriptional activity and fibrillo-granular cortex over which label was localized. The degree of functional restructure of nucleoli was variable within one blastomere or among different blastomeres, some nucleoli being more reticulated and showing more transcriptional activity than others. Fully developed nucleoli were present in early morulae. Electron dense unidentified structures described here as small dense roundshaped bodies (RDB) often surrounded by blocks of large chromatin granules were observed in intact 2-cell and alpha-amanitin treated 4-cell stage embryos. These structures did not show any transcriptional activity

Ontogeny of the syndesmosis tibiofibularis and the evolution of the bird hindlimb: a caenogenetic feature triggers phenotypic novelty

Abstract: The underlying theme of this study is the contribution of developmental mechanisms to the generation of morphological novelty in evolution. The syndesmosis tibiofibularis, an important structural and functional link between the two zeugopod bones of the bird hindlimb, is used as a model for evolutionary novelty. We analyze the structural, developmental and adaptive aspects of its origin in a combined descriptive, experimental, and comparative approach.The ontogeny of the syndesmosis in the chick embryo involves several developmental steps, including the formation of a separate cartilage rudiment that in turn stimulates the formation of an osseous crest on the tibia, which with eventually replace the cartilage element itself. Some of the epigenetic requirements for the formation of the cartilage element and the osseous crest are demonstrated by experimentally increasing the distance between the two zeugopod bones, an operation that results in the absence of both cartilage and crest. Although a syndesmosis tibiofibularis associated with an osseous crest on the tibiotarsus is unique to birds in extant vertebrates, the presence of a distinct crest at the corresponding location in theropod dinosaurs indicates that a syndesmosis also existed in this group of archosaurs.The results of the study suggest that in the case of the syndesmosis tibiofibularis phenotypic evolutionary novelty is based on a caenogenetic feature, i.e. a feature that initially arose in response to changing developmental conditions. In conclusion we propose a model for the stepwise evolutionary modification of the sauropsid hindlimb, integrating adaptive trends and developmental mechanisms that interactively determine the transformations of skeletal limb morphology. The syndesmosis tibiofibularis and the mechanisms of its formation are not only shown to have played a key-role in this process, but its presence in theropod dinosaurs also points towards the origin of birds.

The ultrastructure of sensory nerve endings in human anterior cruciate ligament

Abstract: The sensory innervation of the anterior cruciate ligament (ligamentum cruciatum anterius) of the human knee joint was studied by light-and electron microscopy. The connective tissue between the synovial membrane and the cruciate ligament contains small Ruffini corpuscles and lamellar corpuscles with several inner cores. The connective tissue septa between the individual fascicles of the cruciate ligament contain Ruffini corpuscles and free nerve endings. The free nerve endings are innervated by C-fibres and myelinated A-delta fibres. The afferent axons of Ruffini corpuscles are myelinated and measure 4–6 μm in diameter, those of the lamellar corpuscles with several inner cores measure about 6 μm in diameter.

Topography of the pelvic autonomic nerves in human fetuses between 21-29 weeks of gestation.

Abstract: A topographical study concerning the autonomic nerves in the pelvis of human fetuses was performed by investigating 300-600 microns thick sections through fetal pelves, impregnated with the epoxy resin E 12 and cut with a diamond wire-saw In addition the inferior hypogastric plexus of a 26-week old male fetus was dissected by lateral approach In 21-29-week old fetuses the pelvic autonomic nerves are relatively thick Thus the nerves stand out well against surrounding structures and their topographical relationships can exactly be determined The inferior hypogastric plexus of 21-29-week old fetuses is situated on a curved line between the rectum and the ventrally adjacent structure It constitutes a rectangular plate, which cannot be subdivided into individual plexuses for the different pelvic organs The fetal plexus is heavily ganglionated Large ganglia, forming the so-called ganglion of "Frankenhaeuser", are found in the female as well as in the male fetus In the fetal pelvis the connective tissue compartments are still clearly arranged, because adipose tissue is not yet abundant The greater part of the inferior hypogastric plexus is situated exactly at the border between a dense visceral tissue medially and a loose parietal tissue laterally The plexus does not share a common connective tissue cover with the pelvic blood vessels In fetuses the inferior hypogastric plexus lies in close vicinity to serveral organs, but the pelvic floor is the only region where the nerves can hardly be separated from the surrounding structures

The structure of rabbit retinal Müller (glial) cells is adapted to the surrounding retinal layers.

Abstract: Radial glial (Muller) cells of the rabbit retina were studied by various techniques including Golgi impregnation, scanning electron microscopy, horseradish peroxidase application, and staining of enzymatically isolated cells. This combination of methods produced detailed information on the specialized morphology of the Muller cells within the different topographical regions of the retina, and of the Muller cell processes within the various retinal layers. As a general rule, the retinal periphery contains short thick Muller cells with big endfeet, whereas the thick central retina is occupied by long slender cells with small endfeet. Independent of their location within the retina, Muller cell processes were found to be adapted to the structure of the surrounding retinal layers. Within the outer and inner nuclear layers, Muller cell processes (and somata) extend thin cytoplasmic "bubbles" ensheathing the neuronal somata, as do the "velate" astrocytes in the brain. In the plexiform layers, Muller cells extend many fine side branches between the neuropil, comparable to the protoplasmic astrocytes of the brain. In the thick myelinated nerve fibre layer of the central retina the Muller cell processes are rather smooth, similar to those of fibrous astrocytes. It is concluded that the neuronal microenvironment determines the morphology of a given glial process, or even of a part of a glial process running through a specialized neuronal compartment.

The developing neuroepithelium in human embryonic and fetal brain studied with vimentin-immunocytochemistry.

Abstract: The neuroepithelial cells, which constitute the primordium of the CNS, are potentially capable of generating neuronal and glial cell lineages concomitantly The appearance and morphological development of vimentin-positive neuroepithelial cells in human embryonic and fetal brain (4-16 weeks) were studied with immunocytochemistry In embryos aged 4-6 weeks, vimentin-reactivity was seen in all neuroepithelial cells, including those which exhibited mitotic figures The distribution of reactivity changed according to a general developmental pattern, which commenced and proceeded temporally different in various regions of the CNS All regions exhibited vimentin-positive neuroepithelial cells, the distribution and morphology of which gradually changed, resulting in lamination of the neural wall into two and subsequently three layers The neocortex and midline raphe were the only regions to differ significantly from the general pattern When reactivity to glial fibrillary acidic protein developed at 7-8 weeks, the distribution was very much like that of vimentin at the same stage Reactivity to glial, neuronal and other cellular markers (S-100, neurofilament, neuron specific enolase, desmin, and cytokeratin) revealed different distributions Although cells retaining vimentin beyond the ventricular zone stage are radial glial cells and presumptive fibrous astrocytes, it seems unlikely that vimentin is a marker for a distinct cell lineage during early CNS development It is suggested that all neuroepithelial cells in vivo differentiate to a stage where they express vimentin, and that vimentin may have a functional role in cellular movements and during the interkinetic nuclear migration

Distribution of the neural crest cells in the heart of birds: a three dimensional analysis.

Abstract: The distribution of neural crest derived cells (NC) in the heart of quail-chick chimeric embryos was analyzed three-dimensionally after computer reconstruction. During the division of the truncus arteriosus into the aorta and the pulmonary trunk, ventral and dorsal columns of NC-derived cells were found in the truncal swellings. These columns were elongations from the aorticopulmonary (AP) septum. The dorsal column extended more proximally than did the ventral column. Around hatching, NC-derived cells located between the proximal aorta and the pulmonary trunk, differentiated into cartilage and connective tissue. They formed a part of the cardiac skeleton. A small number of NC-derived cells were scattered in the cusps of the arterial valves. Cells derived from the right NC were located around the aorta and the right arch arteries but not around the distal pulmonary trunk and the left arch arteries. At the proximal level, cells derived from the right NC were located in both the dorsal and ventral columns. These results suggest that the AP septum is mainly formed by NC-derived cells, right and left NC cells migrating into assigned areas in the heart. Location of two columns of NC-derived cells may support a translocation hypothesis for the AP septum during truncal division.

Postnatal development of the corticospinal tract in the rat. An ultrastructural anterograde HRP study.

Abstract: Horseradish-peroxidase was used to anterogradely label and thus to trace the growth of corticospinal axons in rats ranging in age from one day to six months. Three to eight HRP-gels were implanted in the left cerebral hemisphere of the cortex. In each spinal cord three levels were studied, the cervical intumescence (C5), the mid-thoracic region (T5) and the lumbar enlargement (L3). The methodology employed for the electron microscopic visualization of HRP has been described previously (Joosten et al. 1987a). The outgrowth of labelled unmyelinated corticospinal tract axons in the rat spinal cord primarily occurs during the first ten postnatal days. The outgrowth of the main weve of these fibres is preceded by a number of pathfinding axons, characterized by dilatations at their distal ends, the growth cones. By contrast, later appearing unmyelinated axons, which presumably grow along the pathfinding axons, do not exhibit such growth cones. The first labelled pioneer axons can be observed in the cervical intumescence at postnatal day one (P1), in the mid-thoracic region at day three (P3) and in the lumbar enlargement at day five (P5). Prior to the entrance of the axons, the prospective corticospinal area or the pre-arrival zone is composed of fascicles consisting of unlabelled, unmyelinated fibres surrounded by lucent amorphous structures. During the outgrowth phase of the corticospinal fibres some myelinated axons could be observed within the outgrowth area even before day 14. These axons, however, were never labelled. These findings strongly suggest that the outgrowth area, which is generally denoted as the pyramidal tract, contains other axons besides the corticospinal fibres (and glial cells). The process of myelination of the labelled corticospinal tract axons in the rat spinal cord starts rostrally (C5) at about day 14 and progresses caudally during the third and fourth postnatal weeks. Although myelination seems to be largely complete at day 28 at all three spinal cord levels, some labelled unmyelinated axons are still present in the adult stage.

Interconnections between hypothalamus and cerebellum

Abstract: The cerebellum and hypothalamus are interconnected through a multitude of direct (monosynaptic) and indirect (polysynaptic) pathways. Direct hypothalamocerebellar fibres are mainly uncrossed and reach all parts of the cerebellar cortex and nuclei. They are neither mossy fibres nor climbing fibres, but appear to terminate in all layers of the cerebellar cortex as multilayered fibres. At least some of the hypothalamocerebellar fibres are histaminergic, and it appears that a small proportion of the hypothalamocerebellar neurons contain GABA. Indirect hypothalamocerebellar connections may be relayed through various brain stem nuclei. The hypothalamo-ponto-cerebellar pathway, which has a contralateral predominance, appears to be the quantitatively most important of these. The direct cerebellohypothalamic projection originates from the cerebellar nuclei and terminates in the posterior hypothalamus, in the same regions where the direct hypothalamocerebellar pathway has its main origin. Indirect cerebellohypothalamic connections with brain stem relays have also been demonstrated. The functions of hypothalamocerebellar circuits are so far unknown. However, these pathways are probably involved in the coordination and integration of somatic as well as non-somatic responses to a given set of inputs.

The fate map of the chick forelimb-bud and its bearing on hypothesized developmental control mechanisms.

Abstract: Carbon particles and isotopic quail grafts were used as markers to study the salient features of the fate map of the chick forelimb between stages 20 and 27. The grafting technique confirmed the reliability of the carbon method: they both revealed striking asymmetries in which apical mesodermal tissue was progressively displaced in a proximal direction (as would be expected on the basis of growth by net apical addition of tissue) but also in a preaxial direction, while postaxial tissue became elongated in the direction of limb outgrowth. Ectoderm showed a similar preaxial-postaxial asymmetry but became displaced from initially underlying mesoderm. In marked contrast to mesoderm, distal ectoderm remained at a constant distance from the apical ectodermal ridge (or became incorporated into it), thus implying that the ectodermal sheet is anchored distally and grows by uniform stretching proximally. Within the ectoderm itself, the outer peridermal layer is displaced distally relative to the underlying epidermal basal layer. Peripheral mesoderm showed patterns of displacement which were intermediate between those of ectoderm and chondrogenic core mesoderm. It is argued that such morphogenetic phenomena are integral components of developmental mechanisms of significance in the control of pattern generation. Implications of the interpretation and use of the fate map in relation to theories of limb development, particularly those based on mechanisms defined in terms of limb axes, are reviewed.

Changes in the arrangement of actin bundles during heart looping in the chick embryo.

Abstract: We assessed the arrangement of actin bundles in the looping chick heart. Actin filaments were stained with rhodamine-labeled phalloidin, and their total arrangement was observed in whole mount specimens. Before the straight heart tube was formed, actin bundles were in a net-like arrangement as if to indicate the cell borders. With progress of the heart tube formation, actin bundles were gradually arranged in a circumferential direction. In the looped heart, regional differences in actin arrangements were observed. In the truncus arteriosus, actin bundles ran in a net-like arrangement. In the bulbus cordis, actin bundles ran in random directions. In the ventricle, actin bundles were roughly arranged in a circumferential direction. Between these three regions, actin bundles ran in a circumferential direction especially on the concave side. Near the right contour on the ventral face, some actin bundles ran in a longitudinal direction along the axis of the tubular heart. In the bulbus cordis and the ventricle at the looped stage, there was another group of actin bundles in the inner layer of the myocardium which ran in a circumferential direction. We presume that the arrangement of actin bundles is related to heart looping.

The special status of the pulmonary arch artery in the branchial arch system of the rat.

Abstract: A renewed study of the development of the branchial arch system was essential in view of the special morphologic characteristics of the ductus arteriosus, which derives from the pulmonary arch artery or sixth branchial arch artery. In congenital heart disease certain aorto-pulmonary collateral arteries have a marked histological similarity to the ductus arteriosus. To gain a better insight into the development of these vessels, 27 rat embryos, with the number of somites ranging between 19 and 41, were studied. Most embryos were collected after shortterm in vitro-culture, allowing precise staging of age and development. The vascular system of these embryos was injected with Indian ink, to enable easy recognition of even the smallest endotheliumlined vessels. The embryos were serially sectioned (3–5 μm) and reconstructed using a graphic method.

Mitosis and cell death in the tail of the chick embryo.

Abstract: Although somites develop from the mesoderm in the tail of the chick embryo, they do not form to the tip of the tail. Previous work has shown that this terminal mesoderm possesses many of the characteristics of the segmental plate mesoderm which gives rise to the somites in the trunk. This investigation is aimed therefore at understanding why the terminal mesoderm fails to form somites.

Morphologic and regulatory aspects of prostatic function.

Abstract: Current concepts of the structural and functional organization of the human prostate are presented and are related to endocrine principles which have been studied in experimental animals. Based on embryological and histological studies, the internal structure of the human prostate gland is divided into four subdivisions: 1. the anterior nonglandular fibromuscular stroma, 2. the periurethral portion, 3. the peripheral zone, and 4. the central zone. The central zone which accounts for 25% of the gland, is formed by a wedge-shaped group of ducts, arising close to the orifices of the ejaculatory ducts and is surrounded by the peripheral zone (75% of the gland). The functional interdependence and relationship between the stroma and the epithelium observed during embryological development, postnatal maturation and under certain pathological conditions, has led to the concept of a functional prostatic unit, which is useful for the explanation of prostatic growth and the expression of specific genes. There is growing evidence of a functional heterogeneity within the prostatic secretory duct system, with a concentration of estrogen-sensitive cells close to the urethra, and a relatively long persistence of undifferentiated nonsecretory acini at the peripheral tips of the gland ducts close to the dorsal capsule until late puberty. Secretory and proliferative activities of the gland are strictly androgen-dependent. Of particular importance with respect to glandular and stromal proliferation are the recent reports on the presence of different growth factors in the prostate. Hormonally induced imbalances in the system of growth factor production, androgen- and estrogen-dependence and general ageing of the cells have to be taken into consideration in understanding various prostatic pathologies such as benign prostatic hyperplasia and prostate cancer.

Changes in epithelial secretory cells and potentiation of neurogenic inflammation in the trachea of rats with respiratory tract infections

Abstract: In rats respiratory tract infections due to Sendai virus and coronavirus usually are transient, but they can have long-lasting consequences when accompanied by Mycoplasma pulmonis infections. Morphological alterations in the tracheal epithelium and a potentiation of the inflammatory response evoked by sensory nerve stimulation ("neurogenic inflammation") are evident nine weeks after the infections begin, but the extent to which these changes are present at earlier times is not known. In the present study we characterized these abnormalities in the epithelium and determined the extent to which they are present 3 and 6 weeks after the infections begin. We also determined the magnitude of the potentiation of neurogenic inflammation at these times, whether the potentiation can be reversed by glucocorticoids, and whether a proliferation of blood vessels contributes to the abnormally large amount of plasma extravasation associated with this potentiation. To this end, we studied Long-Evans rats that acquired these viral and mycoplasmal infections from other rats. We found that the tracheal epithelium of the infected rats had ten times as many Alcian blue-PAS positive mucous cells as did that of pathogen-free rats; but it contained none of the serous cells typical of pathogen-free rats, so the total number of secretory cells was not increased. In addition, the epithelium of the infected rats had three times the number of ciliated cells and had only a third of the number of globule leukocytes. In response to an injection of capsaicin (150 micrograms/kg i.v.), the tracheas of the infected rats developed an abnormally large amount of extravasation of two tracers, Evans blue dye and Monastral blue pigment, and had an abnormally large number of Monastral blue-labeled venules, particularly in regions of mucosa overlying the cartilaginous rings. This abnormally large amount of extravasation was blocked by dexamethasone (1 mg/day i.p. for 5 days). We conclude that M. pulmonis infections, exacerbated at the outset by viral infections, result within three weeks in the transformation of epithelial serous cells into mucous cells, the proliferation of ciliated cells, and the depletion of globule leukocytes. They also cause a proliferation of mediator-sensitive blood vessels in the airway mucosa, which is likely to contribute to the potentiation of neurogenic inflammation that accompanies these infections.

A quantitative analysis of the development of the pyramidal tract in the cervical spinal cord in the rat.

Abstract: A quantitative electron microscopic analysis was undertaken of the development of the pyramidal tract, at the level of the third cervical spinal segment, in rats ranging in age from the day of birth to three months old. The axon number was calculated as the product of axon density, determined in a systematic random sample of electron micrographs, and tract area. During the first postnatal week the tract contains thin unmyelinated axons and growth cones. Growth cones are abundant in neonatal rats, but can still be observed occasionally at the end of the first postnatal week, indicating a continuous addition of pyramidal tract axons during the first postnatal week. Myelination starts around P10. By the end of the first postnatal month approximately 50% of the axons have already been myelinated. Myelination proceeds during further maturation, but in the three month old rat 28% of the axons are still unmyelinated. The total number of axons increases rapidly after birth up to 153 000 at the fourth postnatal day. Subsequently, the number of axons is reduced by nearly 50% to 79 000 in the adult rat. The axon loss is most prominent during the second postnatal week, when 32 000 axons are climinated, but continues for several weeks at a slower rate.

The binding pattern of peanut lectin associated with sclerotome migration and the formation of the vertebral axis in the chick embryo.

Abstract: Lectins have been used extensively to detect changes in carbohydrate moieties on the surface of embryonic cells during early development. Peanut agglutinin (PNA) in particular has been used to investigate changes related to cell differentiation. PNA has also been used to differentiate between the rostral and caudal sclerotome halves which have been shown to be functionally different, with neural crest cells and neurites traversing only the rostral half during their migration. In this study, we have sectioned and stained chick embryos between 3 and 8 days of age with PNA to examine the distribution of PNA binding sites associated with the vertebral column during this period and also to determine the fates of the rostral and caudal sclerotome halves. Ultrastructural localisation of PNA-gold conjugate showed that binding sites for this lectin were present intracellularly and extracellularly both on cell surfaces and in the matrix. At the light microscope level, a clear banding pattern emerged after staining with PNA which consisted of alternating light and dark staining along the entire length of the vertebral axis of the embryo. In the younger embryos, a simple banding pattern emerged where the rostral sclerotome half of each segment stained only lightly while the caudal half stained darkly. This banding pattern was present throughout the 6 day period of development and could be traced continuously but grew more complex as the sclerotome cells migrated to surround the notochord and neural tube and as the dorsal root ganglia developed. The rostral sclerotome half was found to contribute to the caudal part of one vertebral body and its neural arch, while the caudal sclerotome half was found to contribute to the intervertebral disc, the rostral half of the next caudal vertebra, and part of its neural arch.

Is extraembryonic angiogenesis in the chick embryo controlled by the endoderm? A morphology study.

Abstract: The area vasculosa of the chick embryo is subdivided into two concentric zones: the inner transparent area pellucida vasculosa (AVP) and the less transparent surrounding area opaca vasculosa (AOV). The different optical properties of these zones are caused by the different morphology of the endoderm, which consists of flat cells in the APV and of high-prismatic cells containing large yolk vacuoles in the AOV. The present study describes how this endodermal subdivision of the area vasculosa is related to the development of the extraembryonic vascular pattern. By injection of ink into the vascular system of chick embryos at stages 12 to 20 (Hamburger and Hamilton 1951 “HH”), it has been demonstrated that the vascular net of the area vasculosa from stage 14 (HH) onwards develops into different patterns in APV and AOV. The small loops of uniform capillary vessels of stage 13 (HH) are widened due to the rapid expansion of the extraembryonic mesoderm. In the AOV from stage 14 (HH) onwards numerous small blood vessels sprout into the enlarged intervascular spaces. This process is maximal at stage 17 (HH). In contrast, the blood vessels of the APV remain largely unbranched. These findings suggest that the development of the extraembryonic vascular pattern is controlled by the endodermal pattern. To test this hypothesis, both zones (APV and AOV) were examined by light microscopy, transmission and scanning electron microscopy, in vivo observations and by treatment with bromodeoxyuridine (BrdU). TEM examinations show that the ultrastructural organization of the APV mesoderm is different from that of the AOV: The splanchnopleuric cells of the APV form a continuous cover around the endothelial cells connected by numerous desmosomes, whereas the splanchnopleuric cells of the AOV are frequently separated by gaps. The largest gaps are seen in the small blood vessels at stage 17 (HH). These results should be considered in relation to the dynamic changes in the vascular pattern of the AOV. The endodermal cells of APV and AOV are two different populations. In vivo observation of the endodermal transition from APV to AOV detected no transformations of APV cells into AOV cells or vice versa. The borderline between the zones is stable. The AOV endoderm, having been overgrown by the expanding mesoderm, stops proliferating almost completely, whereas the proliferation of the APV endoderm is unaffected by contact with the mesoderm. The rate of its proliferation is approximately as high as that of the AOV prior to contact with the expanding mesoderm (results after treatment with BrdU). The contact of the basal side of the AOV endoerm with mesoderm is closer than that of the APV. In the AOV the basal compartments of endodermal cells show numerous small coated vesicles, probably exocytotic in nature. The transition between zones in the endoderm was found to be formed by small vaulted cells bearing microvilli on their surface. These cells probably are daughter cells of the primary hypoblast cells, which have been withdrawn to the margin of the APV by the invading endoblast.

Elastoidin turn-over during tail fin regeneration in teleosts. A morphometric and radioautographic study.

Abstract: During teleostean fin regeneration the actinotrichia, rods of a collagen-like protein, the elastoidin, are immersed in the blastema, maintaining their apical position. In this epimorphic event the latter fact might be achieved by either a cellular carriage or a continuous turn-over of these hyperpolimerized fibrils. By means of a 3H-proline pulse and radioautographic chase experiment of the isolated actinotrichia we have found a turn-over of collagen within the structure. From these and additional morphometric results, we present in this work an operational hypothesis of how gradually differentiating blastemal cells and an appropriately shaped basal lamina, can control the number and distribution of actinotrichia which might be under the balanced control of their synthesis and degradation.

Development of smooth muscle: ultrastructural study of the chick embryo gizzard

Abstract: The growth and differentiation of smooth muscle in the chicken gizzard were studied by electron microscopy from the 10th day in ovo to 6 months after hatching; during this period the organ grows 1000-fold in weight. At the earliest stage studied, smooth muscle cells, interstitial cells, and fibroblasts are immature but can already be clearly distinguished. The structural components of muscle cells develop in a characteristic sequence. Mitochondria are more abundant in immature muscle cells (8% in 14 days embryos and 7% in 19 days embryos) than in the adult (5%). Caveolae are virtually absent in the 11 day embryo; they become more common at the end of embryonic life, but continue to increase in relative frequency after hatching. Gap junctions appear around the 16th day in ovo as minute aggregates of connexons, which then grow in size, probably by addition of new connexons. In the earliest stages studied, myofilaments occupy 25% of the cell profile and are assembled into bundles accompanied by dense bodies and surrounded by loosely arranged intermediate filaments. By contrast, membrane-bound dense bands are scarce until the latter part of embryonic life, an observation suggesting that myofilament formation and alignment is not a process initiated near the cell membrane or directed by the cell membrane, and that only late in development bundles of myofilaments become extensively anchored to dense bands over the entire cell surface: at that time myofilaments occupy more than 75% of the cell volume. The muscle cells increase about four-fold in volume over the period studied; the 1000-fold increase in muscle volume is mainly accounted for by an increase in muscle cell number. Mitoses are found in the gizzard musculature at all embryonic ages with a peak at 17-19 days; they occur in muscle cells with a high degree of differentiation. These cells divide at a stage when they are packed with myofilaments and form junctions with neighbouring cells: the mitotic process affects the middle portion of the cell, which takes up an ovoid shape and eventually divides, whereas the remaining portions of the cell do not differ in appearance from the surrounding muscle cells. At all stages of development the population of muscle cells has a uniform appearance (apart from the cells in mitosis), and the growth and differentiation seem to proceed at the same pace in all the cells. There are no undifferentiated cells left behind in the tissue for later development.

Somatostatin expression in the cerebellar cortex during postnatal development. An immunohistochemical study in the rat

Abstract: The distribution of somatostatin-immunoreactive (SOM-IR) elements in the cerebellar cortex of the rat has been studied at different stages of postnatal development (from birth to day 30) and in adult animals using immunohistochemistry. The results showed that in vermis of new born animals there are three main groups of SOM-IR structures within the cortex which subsequently spread along the Purkinje cell layer. In addition, both in the vermis and in the lateral lobes, numerous more evenly distributed SOM-positive cells and fibers could be seen. SOM-IR Golgi cells, Purkinje cells and climbing fibers could then be recognized during the subsequent developmental stages. In the vermal zone, SOM-IR Purkinje cells formed patches, which seemed to be part of a sagittal columnar or band-like organization. This was most obvious between days 5 and 21 of postnatal development. Subsequently there was a reduction in the number of immunoreactive Purkinje cells but a patchy disposition remained. In addition high numbers of SOM-IR Purkinje and Golgi cells and also climbing fibers were identified in the flocculus and paraflocculus at all stages of development studied, and they were also seen in the adult rats in these regions. In the lateral lobes expression of SOM-like immunoreactivity (LI) decreased and almost completely disappeared in adult animals. The present results demonstrate that a SOM or a SOM-LI peptide can be transiently detected in many Purkinje and Golgi cells in the cerebellar cortex, suggesting a role in events related to developmental processes. However, in some regions and structures SOM-LI can be seen also in adult animals.

NCAM and Thy-1 in special sense organs of the developing mouse

Abstract: The distribution of the neural cell adhesion molecule (NCAM) and Thy-1 in the olfactory mucosa and olfactory bulb, the eye and the inner ear was examined with immunocytochemistry in mouse embryos from embryonic day 12 (E 12) to embryonic day 19 (E 19). In general, neurons are completely outlined with NCAM, whereas Thy-1 outlines only dendrites and axons. A variable cytoplasmic staining for Thy-1 is present in the perikarya. Neurons directly associated with special sense organs express NCAM and Thy-1 already from the earliest stage and throughout the period investigated, apart from the olfactory neurons in which Thy-1 disappears at E 19. The mitral cells in the olfactory bulb show Thy-1 but no NCAM reactivity. In the eye, lens fibers express Thy-1 and the pigmented layer expresses NCAM; neither of the two molecules can be detected at E 19. In the inner ear, hair cells express NCAM at E 19. Based on the distribution during the developmental period studied and on the cellular localisation of reaction products, it is suggested that the NCAM adhesion function could be of a more general nature by keeping appropriate cell membranes in close contact and thereby allowing more specific molecular interactions to take place. Thy-1, which is located on dendrites and axons, could be such a specific factor and function as recognition molecule in the developing nervous system.

The effect of fluoride on the patterns of adherence of osteoclasts cultured on and resorbing dentine: a 3-D assessment of vinculin-labelled cells using confocal optical microscopy

Abstract: Osteoclasts were isolated mechanically from chick long bones and cultured on dentine for three days in MEM/10% FCS with or without 1 mg/l NaF, (near to therapeutic ranges for serum fluoride levels) or 15 mg/l NaF (which has been found to reduce the volume: plan-area ratios of resorption pits in vitro). The distribution of close contacts with the substratum was determined by immunolabelling of vinculin and confocal fluorescence microscopy. Four characteristic patterns of contacts were observed: infilled discs, rings, crescents and patches: these reflect the progress of the cell in the process of pit formation. Cellsubstratum adherence was more extensive than previously reported with contacts often extending down the sides of pits and sometimes across the floor. This distribution explains the curved shape of unilocular pits and the kidneyshaped extensions of multilocular pits. The peripheral creeping of the contact region was more marked in the osteoclasts of the 15 mg/l NaF cultures, and these cultures showed a decrease in pit depth and inhibition of osteoclastic movement in the presence of fluoride.

The human brain at stage 16, including the initial evagination of the neurohypophysis

Abstract: Thirty-nine sectioned embryos of stage 16 were studied. Up to this stage the amygdaloid body is derived entirely from the medial eminence, which was purely diencephalic in stage 14, but now extends also to the telencephalon. The area of the future olfactory bulb is indicated by the presence of olfactory fibres entering the brain wall; the future olfactory tubercle is characterized by cellular islands. The presence of the hippocampal thickening and various histological features make it possible to outline the main, future cortical areas already at this early stage: archi-, paleo-, and neopallium. Hippocampus and area dentata correspond to the areas identified by Hines (1922) and Bartelmez and Dekaban (1962) but not to those identified by Humphrey (1966). The interventricular foramen is wide. The cerebral hemispheres grow rostrally and dorsally, thereby forming the beginning of the longitudinal fissure. Apart from the commissure of the superior colliculi, which began to appear in advanced embryos of stage 14, fibres of the posterior commissure are now present in some specimens. The neurohypophysis is apparent in fewer than half of the embryos. The marginal ridge (zona limitans intrathalamica) separates the dorsal from the ventral thalamus. Cranial nerve 3 emerges from M2. M1 has become shorter. Important pathways are beginning: the olfactory route by the olfactory fibres and the medial forebrain bundle; the vestibular by vestibulocerebellar and vestibulospinal fibres; gustatory by chorda tympani, nervus intermedius, and tractus solitarius. Fibres of the cochlear nerve are noted. The first parasympathetic ganglia, submandibular and ciliary, are identifiable. Asymmetry of the cerebral hemispheres was noted in one specimen.