Journal of embryology and experimental morphology最新文献

The effects of inhibitors of follicular steroidogenesis on biochemical changes occurring in oocytes maturing in vitro were studied using radiolabelling and polyacrylamide gel electrophoresis. These effects were correlated with previously investigated developmental abnormalities induced by the same inhibitors. The most severe effects were generated by inhibition of 17 alpha-hydroxylase with the drug SU10603 which resulted in a greatly increased ratio of progesterone to testosterone and oestrogen. Such treatment halved the rate of meiotic maturation. Treated oocytes were analysed individually on SDS-PAGE gels and quantitative analysis showed that the drug had induced synthetic abnormalities even in those oocytes that resumed meiosis. This conclusion was confirmed by separation of oocyte proteins on two-dimensional gels. The effects of SU were reduced by delaying addition of the drug until 6h after the beginning of maturation but were not alleviated by the addition of exogenous oestrogen to the culture medium. When oocytes from SU-treated follicles were transferred to inseminated, recipient ewes and recovered 24h later, two-dimensional electrophoresis again revealed abnormalities in their protein synthetic patterns. Almost total abolition of steroid secretion by aminoglutethimide (AG) had much less effect on oocyte protein synthesis, although the proportion of oocytes maturing was reduced from 65% to 46%. The aromatase inhibitor, androstatriendione (AST) although eliminating follicular oestrogen secretion, had no effect on the rate of maturation and very little effect on protein synthesis. These results correlate well with the effects of steroid inhibitors on fertilization and early cleavage.

Day 9 rat embryos (late presomite stage with cranial neural plate or very early neural folds) were cultured for various periods of time from 6-48 h in medium containing 20 TRU ml-1 Streptomyces hyaluronidase. Exposure to the enzyme resulted in considerable reduction of mesenchymal extracellular matrix. Access of the enzyme to the embryo was confirmed by alcian blue staining which indicated considerable reduction of extracellular and cell surface hyaluronate. Cranial neurulation was retarded, but not inhibited, and migration of both neural crest and primary mesenchyme cells occurred. In general, morphology was normal at 48 h. The major effect was on growth: embryos were smaller, with slightly reduced neuroepithelial cell number and greatly reduced mesenchymal cell number. Neuroepithelial cell cycle time was slightly prolonged, and that of the mesenchyme more than doubled. This differential effect on the growth rates of these two tissues reflects the normal distribution of hyaluronate, which is particularly abundant in the mesenchymal extracellular matrix.

After removal of a transverse strip of ventral thorax from the beetle, Tenebrio molitor, interaction occurred between epidermis posterior to the mesothoracic leg and that anterior to the metathoracic leg. Depending on the size and position of the excision, this interaction resulted in either the regeneration of the extirpated tissue or its replacement by an A/P reversed pattern of sclerites and supernumerary leg. By either route, local pattern continuity was restored between the normal meso- and metathoracic legs. Similarly, when a leg plus adjacent tissue was extirpated, continuity was restored by leg regeneration or by formation of an A/P reversed duplication of sclerites. The results of these strip excisions can be understood in terms of two current models of the ventral thorax (the Boundary Model and the Polar Coordinate Model), each of which postulates a distinct compartment or region intervening between the epidermis surrounding the bases of successive legs. However, the models do not explain the large differences in the frequency of formation of the duplication/deletion pattern after excisions of different widths. The results are also compatible with a different model, involving an A-P sequence of positional values similar to that proposed for the abdominal segment. Regeneration would restore continuity within the sequence by the shortest route, forming either the midsegment (including the leg) or the intersegmental region. The meso- and metathorax differ in the structure of the ventral sclerites and in the segmentation of the tarsus of the leg. The structures regenerated after the various excisions show that the segment border is not crossed during regeneration and indicate that an A/P compartment border running through the leg is usually also respected. There is no sign, however, of a third line of lineage restriction that would indicate a subdivision of the segment into three compartments (as proposed in the Boundary Model).

The earliest stage of neural crest cell (NCC) migration is characterized by an epitheliomesenchymal transformation, as the cells leave the neural tube. There is evidence that in a number of cell systems this transformation is accompanied by alteration or depletion of associated basement membranes. This study examines the ultrastructural relationship between mouse NCCs and adjacent basement membranes during the earliest stages of migration from the neural tube. Basement membranes were identified by transmission electron microscopy (TEM) and immunofluorescence using antibodies to type-IV collagen. The ultrastructural features of NCCs and their relationship with surrounding tissues were also examined using TEM. In the dorsal region of the neural tube, from which NCCs originate, the basement membrane was depleted or absent, and with the immunofluorescence technique it was shown that this pattern was reflected in a deficit of type-IV collagen. TEM observations indicated that ultrastructurally NCCs differ from their neuroepithelial neighbours only in overall cell shape and their relationship to other cells and the extracellular matrix.

The amount of histone H4 mRNA per embryo was followed during early development of Xenopus laevis by Northern blot analyses using a cloned histone H4 cDNA as the probe. The H4 mRNA content was nearly constant until the blastula stage, increased greatly at the gastrula stage and then decreased at the neurula stage. Experiments with actinomycin D suggested that most H4 mRNA molecules detected at the late gastrula and neurula stages were maintained depending on new transcription of H4 genes during these stages. To see if the H4 mRNA level is affected by cell adhesion, we prepared dissociated cells and measured H4 mRNA content under conditions that inhibit cellular reaggregation. It was found that the amount of H4 mRNA per embryo in dissociated and reaggregation-inhibited cells was nearly equal to that of the control embryo at the neurula stage. Therefore, we conclude that the synthetic activity of histone H4 mRNA is not dependent on the cellular adhesion during development.

Rat embryos were grown in vitro during the period of cranial neural crest cell migration. In order to study the pathways and positional fates of cells from different regions of the neural crest, labelled premigratory crest cells from donor embryos were microinjected orthotopically into host embryos of the same developmental stage except for area 1 (forebrain) grafts which were, for technical reasons, injected into area 2. After various periods of time in whole embryo culture, the embryos were examined by immunohistochemical staining in order to determine the new positions of the labelled cells, and a map of their migration pathways was constructed. The observed patterns of migration were consistent with predictions from morphological studies in mammals and with extrapolations from transplantation studies in birds. However, crest cell migratory behaviour in rat and chick embryos was not identical; possible reasons for this are discussed.

Evidence from studies of craniofacial anomalies and the evolutionary transition from reptiles to mammals suggests that the temporomandibular joint (TMJ), bony zygomatic arch, middle ear ossicles and mandibular muscle pattern may form a correlated suite of characters. To test the degree of phenotypic interdependence among these features, mandibular arch defects were analysed in prenatal mice. Retinoic palmitate was administered to pregnant mice on day 8.7 to produce test foetuses with malformations of the mandibular arch. A rating scale was developed for each of the four characters so that numerical values could be assigned to each phenotype encountered. Control animals were used to establish normal phenotypes for each character which were assigned a value of 1. Data from each test age, 16, 18 and 19 days postconception, were pooled and Spearmann rank correlation coefficients between each of the traits were calculated. Coefficients (R) range from a high of 0.87, between the TMJ and zygomatic arch, to a low of 0.67 between the zygomatic arch and the mandibular musculature showing highly significant correlations (P less than 0.0001) among all characters. Therefore, the data suggest that the musculoskeletal features of the mandibular arch are phenotypically interdependent during development.

Mesonephric agenesis was achieved by microsurgical excision of the left Wolffian duct and the underlying intermediate mesoderm of different regions between somites 16 and 23 in chickens after 50-52 h of incubation (stage 14 HH). Quail-chick chimaeras were produced by transplantation of corresponding quail tissue in the region of somites 18-21. A morphometrical analysis of the mesonephric and gonadal area in cross sections shows that the intermediate mesoderm from somites 16 to 23 develops into the mesonephros. A partial agenesis of the mesonephros brought about by removal of the intermediate mesoderm at the level of somites 18 to 21 at stage 14 leads to a mean reduction of the gonadal volume of 37.8% compared to the volume of the untreated side at stage 30. Transplantation of quail intermediate mesoderm in this region of the excision results in development of a hybrid mesonephros. Consequently, the gonads are invaded and colonized by quail cells mobilized from mesonephric corpuscles examined at stage 30, 35 and 36. These results are discussed in terms of the origin of the gonadal stroma during this developmental period; they show that in the region from the third to the sixth segment the ventromedial part of the differentiating mesonephros participates in the contribution of stromal cells to the gonad.

Head skin was used to replace different halves of limb skin from the upper and lower arms of axolotls. Replacement of upper arm posterior skin caused the regeneration of a high proportion of single-digit limbs while replacement of dorsal, ventral or anterior skin caused only minor defects to the normal skeletal pattern. When dorsal or ventral skin was replaced, however, regenerates often lacked dorsal or ventral muscle. Results from the lower arm were different in that replacement of any half of limb skin failed to cause defects either in the skeletal or muscular pattern. These results are used in conjunction with previous work (Wigmore & Holder, 1985; Wigmore, 1986) to suggest that posterior skin is essential for regeneration of the anteroposterior axis and dorsal and ventral skin is necessary for the differentiation of the muscle pattern in regenerates from the upper arm. In the lower arm no localized region of skin appears to be essential for regeneration of the normal pattern and the patterning mechanism may have a different spatial organization.

In the trunk of higher vertebrates, the neural crest (NC) cells remain temporarily within the dorsal portion of the neural tube after fusion of the neural folds; shortly thereafter they emigrate, invading surrounding spaces and tissues. One of the factors postulated to be important in the initiation of migration of NC cells is the disruption of the basal lamina (BL) over the dorsal portion of the neural tube. It has been assumed by many that the BL must be discontinuous in order that the NC cells can leave the neural tube; and indeed, experiments performed in our laboratory, and by others, have shown that NC cells cannot penetrate an intact BL. Therefore, we have undertaken a systematic ultrastructural study to evaluate the condition of the BL during neural fold elevation and NC cell emigration. Our results show that: (i) BL surrounding the neural epithelium (NE) becomes progressively more extensive from neural fold to migratory stages. It first forms on the lateral portion of the neuroepithelium of the neural folds and then extends ventrally into the region adjacent to the notochord; (ii) BL becomes continuous beneath the epidermal ectoderm (EE) that overlies the NC cell region only during the terminal stages of NC cell emigration; (iii) BL does not form over the dorsal portion of the neural tube until NC emigration is terminated; and (iv) the morphology of the BL changes as development proceeds. We conclude that absence of a BL over the premigratory NC cell population in the trunk of mouse embryos is a necessary but not a sufficient condition for emigration to take place.