An in vitro model of gonad differentiation in the chick embryo

Summary Embryonic gonads of 6 1/2 to 12 days old chick embryos were enzymatically dissociated. The cell suspensions were cultured in small gas permeable bags of foil (Biofolie Heraeus) in a roller culture apparatus. The cells formed multiple small aggregates, in which sex specific differences developed within two days. In cell suspensions of embryonic testes smooth spheric aggregates formed with well delineated testicular cords in the center and a tunica albuginea-like mesenchymal layer at the outside. Most of the male germ cells were incorporated in the central cords. A number of germ cells were barred from entering the cords by the tunica albuginea-like mesenchymal layer and populated the outer surface of the aggregates. The aggregates of left ovary were irregular in shape and characterized by clusters of germ cells residing in an outer cortical zone. The aggregates of the right ovary, which regresses in vivo, showed poor growth and did not differentiate, thus, indicating that the suppression of right ovary was not removed in culture. In the roller cultures of dissociated embryonic gonads male and female morphogenesis was mimicked in a reproducible manner, so that the system can be used for further experimental studies of gonadal development.     

Kinetics and differentiation of somite cells forming the vertebral column: studies on human and chick embryos

We have studied the kinetics of somite cells with an antibody against proliferating cell nuclear antigen (PCNA/cyclin) in human and chick embryos, and with the BrdU anti-BrdU method in chick embryos, to investigate whether the metameric pattern of the developing vertebral column can be explained by different proliferation rates. Furthermore we applied antibodies against differentiation markers of chondrogenic and myogenic cells of the somites in order to study the correlation between proliferation and differentiation. There are no principal differences in the proliferation pattern of the vertebral column between human and chick embryos. In all stages examined, the cell density is higher in the caudal sclerotome halves than in the cranial halves. Laterally, the caudal sclerotome halves, which give rise to the neural arches, are characterized by a higher proliferative activity than the cranial halves. Although there is a high variability, the labelling indices show significant differences between the two halves with both proliferation markers. With the onset of chondrogenic differentiation, only the perichondrial cells retain a high proliferation rate. During fetal development, the neural arches and their processes grow appositionally. Even at the earliest stages, there is practically no immunostaining for PCNA or BrdU in the desmin-positive myotome cells of human and chick embryos. Axially, a higher proliferation rate is found in the condensed mesenchyme of the anlagen of the intervertebral discs than in the anlagen of the vertebral bodies. During fetal development, cells at the borders between vertebral bodies and intervertebral discs proliferate, indicating appositional growth. Our results show that local differences in the proliferation rates of the paraxial mesoderm exist, and may be an important mechanism for the establishment of the metameric pattern of the vertebral column in human and chick embryos.     

The ventralizing effect of the notochord on somite differentiation in chick embryos

The dorso-ventral pattern formation of the somites becomes manifest by the formation of the epithelially organized dorsal dermomyotome and the mesenchymal ventrally situated sclerotome. While the dermomyotome gives rise to dermis and muscle, the sclerotome differentiates into cartilage and bone of the axial skeleton. The onset of muscle differentiation can be visualized by immunohistochemistry for proteins associated with muscle contractility, e.g. desmin. The sclerotome cells and the epithelial ventral half of the somite express Pax-1, a member of a gene family with a sequence similarity to Drosophila paired-box-containing genes. In the present study, changes of Pax-1 expression were studied after grafting an additional notochord into the paraxial mesoderm region. The influence of the notochord and the floor-plate on dermomyotome formation and myotome differentiation has also been investigated. The notochord is found to exert a ventralizing effect on the establishment of the dorso-ventral pattern in the somites. Notochord grafts lead to a suppression of the formation and differentiation of the dorsal somitic derivatives. Simultaneously, a widening of the Pax-1-expressing domain in the sclerotome can be observed. In contrast, grafted roof-plate and aorta do not interfere with dorso-ventral patterning of the somitic derivatives.     

The histogenesis of the isthmic nuclei in chick embryos (Gallus domesticus)

Summary The neuromeric mes-rhombencephalic boundary runs between the oculomotor and trochlear nuclei. During morphogenesis the m ₂-segment is reduced to a cellfree mantle demarcating a morphological mes-rhombencephalic border. The floor plate and glycogen-containing raphe extend rostralwards to terminate at the level of the m ₂-segment ventromedially.The isthmic migration commences within the dorsal and dorsolateral rhombencephalic cell columns caudal to the emergence of the trochlear nerve. The neuroblasts migrate radially out from the synthetic zone of the neural epithelium into the mantle constituting the isthmic migration. The latter migrate longitudinally en masse rostralwards into the mantle below the optic lobe. The m ₂-segment can, however, be identified as a morphological border between the mesencephalic and isthmic (rhombencephalic) mantles throughout the early embryogenesis.The isthmic migration subdivides into a tectal and a tegmental nuclear group. Both groups contribute to the formation of the isthmic nuclei. The caudal portion of the mesencephalic tectal mantle contributes to a mesencephalic isthmic nucleus: Nucleus isthmi principalis mesencephali (magnocellularis).     

Origin of somatic cells and histogenesis in the primordial gonad of the Japanese tree frog Rhacophorus arboreus

Summary Gonadal development in Rhacophorus arboreus, a sexually semidifferentiated type of tree frog, was observed by means of the electron microscope, and cell proliferation kinetics were examined autoradiographically. The genital ridge consisted of coelomic epithelial cells and primordial germ cells. The gonadal medulla was formed by the segregation of epithelial cells within the primordial gonad. Thereafter, the medullary cell mass was well developed and oogenesis began in the gonadal cortex, irrespective of genetic sex. During metamorphosis, the ovarian cavity was formed in the medullary mass. This ovarian structure developed further in females. In males, on the other hand, a layer of medullary cells comprising the epithelium of the ovarian cavity proliferated rapidly and reformed a large cell mass. The degeneration of ovarian follicles and the formation of cell cords (rudimentary seminiferous tubules) were seen in the cortex. These cell cords were separated from the superficial epithelium and continued to the medullary mass (rudimentary testicular rete). These results clearly indicate that both the cortical and medullary cells are derived from the coelomic epithelium and that the development of the cortex and medulla is not always antagonistic in the course of sexual differentiation.     

Behavior of chick primordial germ cells injected into the blood stream of quail embryos

The distribution and behavior of chick primordial germ cells (PGC) injected into quail embryos were examined. PGC from chick embryos at stages 13-14 were injected into the blood stream of quail embryos at stages 15-20. After one day, the quail embryos were examined histologically. The chick PGC in the quail embryos could be readily identified by the histochemical PAS technique, whereas quail PGC were never stained by PAS. When the chick PGC were injected into the quail embryos during stages 15-18, they appeared mostly in the gonadal region of the recipient quail embryos. A few PGC were found at extragonadal sites. When the chick PGC were injected into the quail embryos at stages 19-20, in which the PGC of the recipient quail embryos had finished their migration into the gonads, most of the donor chick PGC were found at ectopic sites, in the head, trunk and limbs. These results indicate that most of the chick PGC, injected at the earlier stages 15-18, migrated to the gonadal anlagen of the recipient, while following later injection (from stage 19), most of the chick PGC migrated to ectopic sites.     

Granulocyte differentiation in the lymphoid organs of chick embryos after antigenic and mitogenic stimulation

Sections of spleen, bursa of Fabricius or thymus glands from 17-day-old chick embryos stimulated with sheep red blood cells, phytohaemagglutinin (PHA-P), concanavalin A or lipopolysaccharide were stained by an "indirect" peroxidase technique that differentiates eosinophils from heterophils and cell counts were carried out. The tissues were also examined with the electron microscope. Whatever stimulus had been used, the light microscopical examination revealed that the predominant granulocytes belonged to the peroxidase-negative heterophil series, outnumbering the peroxidase-positive eosinophils. There were no significant changes in the number of cells between treatments and the saline-injected controls, except in the case of PHA-P stimulation, where a slight reduction in the number of heterophils and a concomitant increase in eosinophils was observed. This study has demonstrated that the heterophil is the main granulocyte present in the lymphoid organs during late embryonic life in the fowl. It challenges previously reported work that the predominant cells are eosinophils and monocytes.     

Influence of partial decerebration and hypophyseal allograft on differentiation of thymic epithelial cells in chick embryos: an ultrastructural study

The thymus of 18-day-old normal-chick embryos, partially decerebrated chick embryos, and partially decerebrated embryos bearing hypophyseal allografts were analysed by light and transmission electron microscopy. The hypophyseal influence on the cytological differentiation of epithelial components has been studied. The thymus of partially decerebrated embryos showed a delayed differentiation of some types of epithelial cells and a marked decrease in number of lymphoid cells. Partially decerebrated embryos with hypophyseal implants showed a consistent recovery in the degree of differentiation of epithelial components. These findings indicate the influence of the hypophysis in establishing a correct environment for stromal cell differentiation.     

Relationship between genital ridge formation and settlement site of primordial germ cells in chick embryos

Chick embryos from stage 15 to stage 18, which is the most frequent extravasation period, were investigated by means of serial sections and light microscopy in order to learn the detailed relationship between the settlement sites of the primordial germ cells (PGCs) and the forming genital ridge. PGCs circulating in the vascular system came out of the small vessels in the splanchnopleure posterior to the vitelline artery. This PGC extravasation was limited to an area about 1.2 mm caudal to the vitelline artery. After the extravasation, the PGCs entered the neighboring thickened coelomic epithelium of the splanchnopleure. This thickened epithelium, which had incorporated the PGCs, changed the location toward the future gonadal site with the advance of development. At stage 16, the thickened portion of the epithelium was located in the splanchnopleure; then it moved toward the somatopleure via the coelomic angle; finally, at stage 18, this epithelium occupied the region between coelomic angle and the mesonephros which corresponded to the future genital ridge. This means that the thickened epithelium of the splanchnopleure which initially incorporated the PGCs becomes the superficial epithelium of the genital ridge in more advanced stages. The thickened portion of the epithelium of the splanchnopleure at stage 16 is thought to be the definitive gonadal anlage.     

Analysis of nuclear ribonucleoproteic structures during notochordal cell differentiation and maturation in chick embryos

The ultrastructure of notochordal cells and the quantitative changes of nuclear mRNA-containing particles were studied in several stages of the development of the chick embryo. The modifications in the frequency of perichromatin granules (PCG) were analyzed in embryos at 24 hr to 10 days of incubation (stages 6-36 of Hamburger and Hamilton). The ultrastructural and morphometric data show that notochordal cells undergo changes that can be systematized in four periods. Very early notochordal cells (stages 6-11), are characterized by the presence of large nucleoli and abundant PCG, traits probably related to the frequent mitotic division and the expression of inductive signals reported in numerous papers. During the second period (stages 16-21) the number of PCG and the size of the nucleolus decrease. These changes are coincident with the beginning of vacuolization. In the third period (stages 21-30), the notochordal cells undergo a second cytodifferentiation characterized by a large increase of cytoplasmic vacuolization and secretion of materials that thicken the perichordal sheath. During this period, the nucleolus becomes smaller and the number of PCG increases. Similar features were previously described during functional maturation of embryonic neurons and striated fibers at synaptogenesis, and epidermal cells. The fourth period, beginning at stage 30, is characterized by the decrease of the density of PCG and of the nucleolar volume and corresponds to cessation of mitosis and cell degeneration.     

Ontogeny of spontaneous antigen-binding cells in developing chick embryos.

Bursal and splenic lymphocytes in developing chick embryos were examined for the number of antigen binding cells (ABC) which could form rosettes with red blood cells from sheep, guinea-pig, rabbit or dinitrophenylated sheep red blood cells (DNP-SRBC). The kinetics of the development of bursal ABC to rabbit red blood cells (RRBC) did not vary significantly with age of embryo examined, and its frequency was constantly high. The number of cells which bind to SRBC, guinea-pig red blood cells (GPRBC) or DNP-SRBC was low in the bursa and increased in an approximately linear fashion with the age of embryo. With the exception of RRBC-ABC, similar results were obtained with splenic ABC. Furthermore, the frequencies of ABC were higher in the spleen than in the bursa. These events suggest the antigen-independent generation of clonal diversity during the developmental stage of chick embryos.