p185neu is expressed in yolk sac during rat postimplantation development.

We have shown that the neu oncogene product (p185neu) is not present in the rat embryo before organogenesis. However, coincident with the onset of organogenesis, p185neu was detected in neural and connective tissue as well as in the secretory epithelium as was described by Kokai et al. (1987). In addition, p185neu is also expressed in the rat visceral yolk sac (VYS) endodermal cells but not in the mesenchymal and mesothelial layers of the same structure nor in the amnion. The first detectable sign of p185neu expression in VYS was found at d 11 of gestation and the levels of protein increased towards the end of pregnancy. In the yolk sac carcinoma (YSC), which is considered to be the malignant counterpart of the rat yolk sac, p185neu was observed only within columnar epithelial cells (the visceral component of the neoplasm) while parietal endoderm-like cells were devoid of detectable protein. From d 9 of pregnancy up to delivery some of the trophoblastic giant cells also showed a faint to moderate immunoreactivity. Results are presented which would indicate a possible role of p185neu in rat embryogenesis.

Morphological and immunohistochemical characteristics of axial structures in the transitory human tail.

Ultrastructural relationships between the notochord and neighboring spinal cord were examined during the regression of the human tail. Also, the presence of certain extracellular matrix components in the notochord was immuno-histochemically analysed in the 4th to 12th week old embryos. At the early stages, a close apposition of the notochord to the spinal cord exists in the entire tail region. The external surface of both structures is covered with a continuous basal lamina. The narrow tissue interspace contains interdigitating cell processes and both amorphous and fibrillar extracellular matrix material. With advancing embryonic age, separation of the two structures occurs in craniocaudal direction and the widening interspace becomes occupied by mesenchymal cells. During tail regression and spinal cord retraction, the appearance of large intercellular spaces and cell degeneration takes place in both tissues. With age, the extracellular matrix of the notochord, predominantly the perinotochordal sheath, increases in amount and antigenic complexity. While the intensity of laminin, collagen type IV and type III expression rises continuously during the period examined, the expression of fibronectin begins first at later stages, after the separation of the notochord from the spinal cord. The possible developmental significance of the described phenomena in the regression of the posterior end of the human tail remains to be elucidated.

Morphological evidence for secondary formation of the tail gut in the rat embryo.

The secondary body formation is a developmental mechanism occurring in the caudal part of the embryo in which embryonic structures arise from a mass of mesenchymal cells without previous formation of germ layers. The formation of the tail gut by this mechanism was investigated on transverse serial semithin and ultrathin sections of 12-, 13-, 14- and 15-day rat embryo tails. The tail gut, together with the tail portion of the notochord, originates from an axial mass of condensed mesenchymal cells named tail cord. Formation of the tail gut involves the appearance of large intercellular junctions among tail cord cells, and rearrangement of these cells around a newly formed lumen. Mesenchymal characteristics of these cells are gradually lost, and they simultaneously acquire the morphology of epithelial cells. Some cells of the tail cord, located ventral to the tail gut, do not participate in the tail gut formation and form a separate mass of cells without any definitive morphogenetic fate. This surplus group of cells is first evident in 12-day embryos, and it increases in mass during the following 3 days. In 15-day embryos, after the tail gut has completely disappeared, the surplus cells represent all that remains of the tail cord. The mesenchymal-epithelial transformation of the tail cord cells into the cells of the tail gut, and the appearance of the surplus cells, could be considered as the main morphological arguments for the secondary formation of the tail gut.

Lentoids within sacrococcygeal teratoma: origin by transdifferentiation?

Within a teratoma removed surgically from the sacrococcygeal region of a female newborn, clusters of lens-like cells (lentoids) surrounded by the immature tissue of the neural retina were revealed by routine histologic analysis. Comparison of the cytologic and microtopographic characteristics of lentoids that develop in experimental embryo-derived teratomas suggests that the lentoids within the sacrococcygeal teratoma originate by transdifferentiation (cell-type conversion, metaplasia) of cells of the immature neural retina or the pigmented retinal epithelium. The embryonic origin of sacrococcygeal teratomas is discussed in the context of complex morphogenetic features at the posterior end of the early embryo.

Development of separated germ layers of rodent embryos on ectopic sites: a reappraisal.

The method of separation of germ layers of rodent embryos by treating the embryonic shields with proteolytic enzymes and by microsurgery with the subsequent transplantation to ectopic sites has helped to gain a more detailed insight into what is going on during gastrulation in mammals. The space under the kidney capsule of adult animals seems to be the most appropriate ectopic site for transplantation of early postimplantation rat embryos or separated germ layers. After transplantation the grafts develop into teratomas whose complex histological structure reflects the initial developmental capacities of the graft. At the pre-primitive streak and the early primitive streak stages the primitive ectoderm differentiates into tissue derivatives of all three definitive germ layers, often in complex organotypic combinations. This is indirect evidence that all cells of the embryonic body originate from the primitive embryonic ectoderm. Halves of the primitive ectoderm obtained by a longitudinal or transverse cut through the egg cylinder give the same result. At the head fold stage the capacity for differentiation of the ectoderm is restricted to ectodermal and mesodermal derivatives. One day before gastrulation the isolated primitive ectoderm is not able to differentiate as renal isograft. The mesoderm isolated at the head fold stage and at later stages when its segmentation occurs, differentiates almost exclusively into the brown adipose tissue. The embryonic endoderm differentiates only in combination with the mesoderm. After transplantation the embryonic ectoderm loses its epithelial organization and breaks up into a mass of mesenchyme-like cells in which epithelial structures subsequently appear and differentiate in a way reminiscent of the reaggregation of cells in mixed cell suspension in vitro.

Morphogenetic features in the tail region of the rat embryo.

The secondary (direct) body formation is a mechanism of development in which morphogenesis of various organs occurs directly from a mass of undifferentiated mesenchymal cells (blastema) without previous formation of germ layers. It is characteristic of the posterior end of the embryonic body, i.e. of the tail bud of tailless and the tail of tailed mammals. Development of the neural tube occurring by this mechanism (secondary neurulation) has been previously explained. We investigated the morphogenetic mechanism by which two other axial structures in the rat tail develop: the tail gut and the notochord. Both structures develop from an axial condensation of undifferentiated mesenchymal cells (tail cord) of tail bud origin. The tail gut forms in a similar way to the secondary neural tube: cells in the ventral part of the tail cord elongate, acquire an apicobasal polarity and form a rosette-like structure around a lumen in the centre. The notochord forms by detachment of a group of cells of the tail cord dorsally to the developing tail gut. The peculiarities of this morphogenetic mechanism in comparison with those in other parts of the embryo are discussed. Causal (including evolutionary) explanations of this mechanism are ruled out.

The ability of the epithelium of diencephalic origin to differentiate into cells of the ocular lens.

After the discovery that in adult salamanders following lentectomy a new, functional lens develops by transdifferentiation (cell-type conversion) of previously depigmented epithelial cells of the iris (Wolffian lens regeneration), this phenomenon has been intensively studied by various experimental approaches. During the last two decades it was shown that pleiomorphic aggregates of atypical lens cells (lentoids) differentiated in reaggregates of dissociated cells of the chick neural retina and in spread cell cultures of the pigmented epithelium of the iris and retina, of the neural retina and the pineal gland of the chick embryo. The neural retina of human fetuses and adults also displayed this capacity. We showed that lentoids developed at a low incidence in renal isografts of rat embryonic shields or isolated embryonic ectoderm and of lentectomized eyes of rat fetuses, as well as in organ cultures of rat embryonic shields in chemically defined media. The addition of transferrin significantly increased the incidence of differentiation of lentoids in explants. In both renal isografts and explants in vitro a continuous transformation of retinal epithelial cells into atypical lens cells was observed. In renal isografts lentoids were also observed to originate from the ependyma of the brain ventricle. All tissues having the capacity to convert into lens cells belong to the diencephalon in a broad sense. Evolutionary aspects of this feature are discussed.

Differentiation of the secondary elastic cartilage in the external ear of the rat.

The cartilage in the external ear of the rat belongs to the group of secondary cartilages and it has a unique structural organization. The chondrocytes are transformed into typical adipose cells, the proteoglycan cartilage matrix is reduced to thin capsules around the cells and the rest of the extracellular matrix is occupied by a network of coarse elastic fibers. It appears late in development (16-day fetus) and needs more than one month for final development. The differentiation proceeds in several steps which partly overlap: the appearance of collagen fibrils, elastin fibers, the proteoglycan matrix, and the adipose transformation of chondrocytes. The phenotype of this cartilage and the course of its differentiation are very stable, even in very atypical experimental environmental conditions. The only exceptions are explants in organ culture in vitro and perichondrial regenerates. In these conditions the development of elastic fibers is slow and poor while the production of the proteoglycan matrix is abundant. The resulting cartilage then displays structural characteristics of hyaline cartilage rather than those of the initial elastic one.

Transferrin enhances lentoid differentiation in rat egg cylinders cultivated in a chemically defined medium.

Rat egg cylinders at the primitive streak stage were grown in modified organ culture for 2 weeks using a chemically-defined medium. The purpose of the experiment was to determine whether the terminal tissue differentiation is modified by human transferrin. The control sets were grown in medium with or without rat serum. In explants treated with transferrin, groups of atypical cells of the ocular lens (lentoids) appeared more frequently than in both control sets; however neuroblasts were observed as often as in the serum-supplemented medium. Bovine serum albumin (BSA) stimulated the differentiation of neuroblasts but did not promote lentoid formation. We conclude that human transferrin does stimulate the differentiation of lentoids in rat embryonic explants, but the mechanism of its action remains unknown.

Lentoid formation in ectopic grafts of lentectomized eyes of rat foetuses.

Enucleated and lentectomized eyeballs of 14- and 18-day rat foetuses were grafted under the kidney capsule of adult syngeneic rats for a period of 5-66 days. The grafts were then analysed by routine histology for the presence of lentoids. These developed only in 4 out of 41 grafts from the 18-day foetuses. Their origin from the retinal epithelium was evident from the cellular continuity via gradual transitional cellular forms. Lentoids did not develop in grafts from 14-day rat foetuses.

Origin of the notochord in the rat embryo tail.

The origin of the notochord in the rat tail was investigated on transverse serial semi-thin and ultra-thin sections of 12- and 13-day embryo tails. It was found that the notochord develops from a mass of condensed mesenchymal cells which is located ventrally to the secondary neural tube, and which subsequently splits into a) a thin cord which becomes notochord and b) a thick portion which gives rise to the tail gut. By analogy with the secondary neurulation and the secondary gut formation, one might therefore speak of a secondary notochord formation in the tail. It occurs in close relationship with the formation of the tail gut.

The effect of intermittent cold treatment on the adipose tissue of the cat. Apparent transformation from white to brown adipose tissue.

Young cats (Felis domestica), aged 10-13 weeks, were intermittently exposed to a temperature of -30 degrees C for two periods of 1 hr per day. Animals were sacrificed on the 7th day and adipose tissue from the perirenal, pericardial, axillary, interscapular, and subcutaneous-inguinal depots was examined by electron microscopy and analysed stereologically. All examined depots were morphologically changed after cold treatment. Adipose tissue of perirenal, pericardial, and axillary depots showed a greater decrease in lipid content than the interscapular and subcutaneous-inguinal depots, but other changes were similar. Compared to the control group, which consisted of typical white adipose tissue, the diameter of adipose cells examined after cold treatment was diminished, in extreme cases to 18 micron (from 75 micron in the control group). The number of capillaries per cell was doubled (as evaluated on semithin sections). The most dramatic changes were observed in the mitochondria. Their volume increased to 0.48 micron 3 (from 0.13 micron 3 in the control), and the surface density of mitochondrial cristae per mitochondrial volume increased to 50 micron 2/micron 3 (from 32 in the control). Pleomorphism in mitochondrial size and inner structure and the presence of intramitochondrial electron-dense bodies and crystalline structures led us to conclude that the cold stress induced an increase in the absolute number of mitochondria in the adipose cells. The adipose tissue after cold treatment thus morphologically resembled the brown adipose tissue of cold-acclimated rodents. This implies that the adipose tissue of young cats can change its morphology and function, depending on the requirements of the organism.

On the ultrastructure of the developing elastic cartilage in the rat external ear.

Selected ultrastructural features of chondrocytes and the extracellular matrix in the developing elastic cartilage of the external ear were studied in rat fetuses and young animals. The cytoplasmic lipid droplets were first observed in the 19-day fetus. They increase in number and size during the first post-natal week. The elastogenesis proceeds in the sequence: oxytalan fibers (17-day fetus), elaunin fibers (1-day rat), elastic fibers (5-day rat). Intermediary stages between the randomly oriented individual microfibrils and bundles of microfibrils (oxytalan fibers) were also observed.

Morphogenetic behaviour of the rat embryonic ectoderm as a renal homograft.

Halves of transversely or longitudinally cut primary ectoderm of the pre-primitive streak and the early primitive streak rat embryonic shield developed after 15-30 days in renal homografts into benign teratomas composed of various adult tissues, often in perfect organ-specific associations. No clear difference exists in histological composition of grafted halves of the same embryonic ectoderm. The primary ectoderm of the pre-primitive streak rat embryonic shield grafted under the kidney capsule for 2 days displayed an atypical morphogenetic behaviour, characterized by diffuse breaking up of the original epithelial layer into mesenchyme. Some of these cells associated into cystic or tubular epithelial structures. The definitive ectoderm of the head-fold-stage rat embryo grown as renal homograft for 1-3 days gave rise to groups of mesenchymal cells. These migrated from the basal side of the ectoderm in a manner which mimicked either the formation of the embryonic mesoderm or the initial migration of neural crest cells. This latter morphogenetic activity was retained in the entire neural epithelium of the early somite embryo but was only seen in the caudal open portion of the neural groove at the 10- to 12-somite stage. The efficient histogenesis in grafts of dissected primary ectoderm and the atypical morphogenetic behaviour of grafted primary and definitive rat embryonic ectoderm were discussed in the light of current concepts on mosaic and regulative development, interactive events during embryogenesis and positioning and patterning of cells by controlled morphogenetic cell displacement.

Ultrastructure of elastic cartilage in the rat external ear.

The structure of elastic cartilage in the external ear of the rat was investigated by transmission and scanning electron microscopy. The narrow subperichondrial, boundary zone contains predominantly ovoid cells rich in cell organelles: mitochondria, Golgi complex, granular endoplasmic reticulum and small (40--100 nm) vesicles. Scarce glycogen granules and bundles of 6--7 nm cytoplasmic filaments are also present. Deeper in the boundary zone, one or more cytoplasmic lipid droplets appear and cytofilaments become more abundant. Fully differentiated chondrocytes in the central zone of the cartilage plate resemble white adipose cells. They are globular and contain a single, large cytoplasmic lipid droplet. The cytoplasm is reduced to a thin peripheral rim; it contains a flattened nucleus, few cytoplasmic organelles and abundant, densely packed, cytoplasmic filaments. The intercellular matrix is very sparse. The pericellular ring consists of collagen fibrils about 20 nm in diameter and a proteoglycan cartilage matrix in the form of a "stellate reticulum". The complex of these two structures appears in the scanning electron micrographs as a a network of randomly oriented, ca 100 nm thick fibrils. Spaces between pericellular rings of matrix also contain thick elastic fibers or plates, apparently devoid of microfibrils. In scanning electron micrographs elastic fibers could be detected only in a few areas, in which they were not obscured by other constituents of the matrix. Immature forms of elastic fibers, oxytalan (pre-elastic) and elaunin fibers, were found in the perichondrial and boundary zones.

Histological differentiation and organogenesis of rat fetal kidneys after isotransplantation under the kidney capsule of adult rats.

Developing kidneys of 14- and 15-day rat embryos were grafted under the kidney capsule of adult syngeneic rats and examined histologically after 7 and 14 days. Although the grafted fetal kidney developed in a relatively hypopotassemic environment, no cystic dilatations or other anomalies of the renal tubules were found.

Course of development of isolated rat embryonic ectoderm as renal homograft.

When the isolated head-fold stage rat embryonic ectoderm is grafted under the kidney capsule, it gives rise to a new mesenchyme with the capacity to differentiate into mesodermal tissues.

Pre-elastic (oxytalan) fibres in the developing elastic cartilage of the external ear of the rat.

During chondrogenesis in the external ear of the rat oxytalan fibres precede the appearance of mature elastic fibres by 6-7 days. The spatial distribution of oxytalan fibres in fetal and neonatal pre-cartilage corresponds to that of the elastic fibres in mature cartilage. These findings support the hypothesis that oxytalan fibres in the pre-cartilage of the external ear of the rat are pre-elastic in nature.