Journal of embryology and experimental morphology最新文献

A heat shock (of 15 min at 48 degrees C) given to early embryos of the locust, Schistocerca gregaria, results in localized abnormalities in the segment pattern subsequently formed. Most defects involve two consecutive segments of the thorax or abdomen, and these are analysed in detail. The abdominal defects fall into three main classes each of which involves the absence of a particular region of the segment pair and, in one class, duplication of the region which remains. The thoracic defects similarly involve absence of parts of the segments and the formation of a single limb base from which one, two, or three limbs develop. Heat shock may result in the absence of parts of segments in two distinct ways. It may interfere with the process of segmentation or it may delete parts of already formed segment primordia. These possibilities are discussed although, at present, neither can be excluded. The duplication observed in some abdominal disruptions and the formation of triple limbs indicates that the absence of parts of embryonic segments is followed by pattern regulation similar to that occurring in regeneration studies on larval segments and appendages of other insects. Two out of the three classes of abnormality can be explained in terms of intercalary regeneration restoring pattern continuity, but it is possible that discontinuities persist in the remaining class.

The terminal differentiation of Dictyostelium discoideum cells plated as monolayers with cyclic AMP is dramatically affected by developmental buffer conditions. High pH and addition of weak bases induces spore differentiation while low pH and weak acids favour stalk cell formation. In order to analyse the timing and nature of this regulation we have raised and characterized an anti-stalk serum which we have used together with an anti-spore serum to monitor developmental progression in the monolayer system and to detect the phenotypic effects of pH at earlier stages of development. The stalk serum detects both polysaccharide and protein antigens expressed during the terminal stages of normal development. In monolayer culture, the stalk-specific protein antigen appears precociously, while the timing of prespore vacuole appearance is unaffected. Expression of stalk polysaccharide antigens in monolayer cultures occurs as early as 12 h and is localized in a single subset of cells or region of extracellular space within the small cell clumps that form. The effects of pH (and acid/base) on these phenotype-specific antigens can be detected early in development, shortly after their first appearance. In monolayers of wild-type V12 M2 cells, the low pH regimes appear to act more by suppressing the spore than enhancing the stalk pathway, while the high pH regimes both suppress stalk and enhance spore antigen expression. In monolayers of the sporogenous mutant HM29, low pH regimes both enhance stalk antigen and suppress spore antigen expression. These results show that extracellular pH regulates phenotypic expression during a large part of the differentiation process and is not simply restricted to terminal cytodifferentiation.

Migration of the mesoderm cells in the primitive-streak-stage mouse embryo was directly studied by cinemicrography using whole embryo culture and Nomarski differential interference contrast optics. Relative transparency and small size of the early mouse embryos enabled direct observation of the individual cells and their cell processes. Seven-day-old mouse embryos were isolated and cultured in a small chamber in a medium consisting of 50% rat serum and 50% Dulbecco's modified minimum essential medium. The mesoderm cells move away from the primitive streak in both anterior and antimesometrial (distal) directions at a mean velocity of 46 micron h-1. They extend cell processes and constantly change cell shape. They do not translocate extensively as isolated single cells, but usually maintain attachment to other mesoderm cells. They show frequent cell division preceded by rounding up of the cell bodies, and accompanied by vigorous blebbing before and after cytokinesis. This study shows that it is possible to examine the motility of embryonic cells inside the mammalian embryo by direct observation if the embryo is small and transparent enough for the use of the Nomarski optics.

Branching submandibular glands of 12-day mouse embryos and those cultured in the presence and absence of a collagenase inhibitor from the culture medium of bovine dental pulp or a Clostridial collagenase were examined with the scanning electron microscope. Fracturing of fixed and dried glands with the tip of a fine needle succeeded in exposing the surfaces of the lobules and of their mesenchymal replicas at different stages of branching. At the beginning of branching, corresponding parts of the mesenchyme formed ridges on or in which the fibrillar structures were often found. At the stage forming deeper clefts thicker fibres, 0.5-2.5 micron in diameter, were observed between two adjacent lobules. On the contrary, no apparent differences in the fibrillar structures on the epithelial surfaces were detected between the shallow cleft and noncleft regions at the initial phase of branching. These fibrillar structures were very abundant in glands cultured with collagenase inhibitor and were completely lost in glands cultured with bacterial collagenase, strongly indicating that these materials consisted of collagen. The possible involvement of mesenchyme in epithelial branching is discussed with special reference to mesenchymal traction forces that would be elicited by fibrillar collagens.

The patterns of distribution of both total mesenchymal cells and the ratios of [3H]thymidine-labelled to total cells were mapped during secondary palatal shelf reorientation in vivo and in vitro. Smoothed spatial averaging, a computer-assisted method which takes into account the positions of all cells across an entire histological section of the shelf, was employed. Changes in shelf cross-sectional area and cell size were also measured. Three shelf regions, anterior and posterior presumptive hard and presumptive soft palate, were studied at developmental stages which were 30, 24 and 18 h prior to expected in vivo elevation, after in vivo reorientation and during the course of in vitro reorientation. Region-specific patterns of cell distribution change with shelf reorientation. These changes were observable within 6 h. Increases in cell number by cell division may enhance some high local cell densities, but cannot account for decreases in cell density. Increase in cell size is not a factor in decreasing cell density, nor is cell death. Displacement of cells by expansion of the extracellular matrix may be involved.

Matrix-mediated epitheliomesenchymal interactions control dental cytodifferentiations. Experiments were performed in order to study the effects of noncollagenous proteins extracted from dentin on cultured enamel organs and dental papillae. Seven noncollagenous protein fractions were prepared from rabbit incisor dentin and used as substrates to coat Millipore filters. Embryonic mouse tooth germs were dissociated and the isolated tissues were cultured for 4 days on these different substrates as well as on noncoated Millipore filters. When compared to control cultures, only two protein fractions affected the behaviour of epithelial cells. A slight elongation of the cell body and a preferential localization of the nuclei at the basal pole of the cells in contact with the filter was observed with protein fractions 5 and 6. When dental papillae were cultured on Millipore filters coated either with protein fraction 2 or fraction 6, the mesenchymal cells in contact with the filter elongated, polarized and demonstrated a high metabolic activity. Such modifications in the cell organization, implying changes in the cytoskeleton organization and, or, activity, never occurred spontaneously or in the presence of isolated collagens (I-V), laminin or fibronectin.

Rat mammary ducts, free of buds, can alone regenerate complete mammary trees when transplanted into the interscapular fat pads of syngeneic host rats. All the main mammary cell types are identified within such outgrowths. Epithelial cells, which show the presence of milk fat globule membrane antigens and microvilli on their luminal surfaces, line the ducts. Basal cells surrounding the ducts show characteristic features of myoepithelial cells: immunoreactive actin and keratin within the cytoplasm, myofilaments, pinocytotic vesicles and hemidesmosomal attachments to the basement membrane. Cells within the end buds and lateral buds, however, show few if any cytoplasmic myofilaments and are relatively undifferentiated in appearance. Intermediate morphologies between these cells and myoepithelial cells are seen nearer the ducts. In this respect they exactly resemble the cap cells found in terminal end buds (TEBs) of normal mammary glands. Occasional epithelial cells within alveolar buds show the presence of immunoreactive casein, which is a product of secretory alveolar cells in the normal rat mammary gland. Dissected terminal end buds can regenerate similar ductal outgrowths. Thus, ductal tissue alone can generate all the major mammary cell types seen in the normal gland, including the cap cells.

The effect of heat shock (15 min at 48 degrees C) on segmentation has been investigated in the short germ embryo of the locust (Schistocerca gregaria). Prior to formation of the germ anlage and at the disc stage heat shock considerably reduced the survival of eggs but appeared to have little effect upon segmentation. At later stages heat shock had no effect on survival but resulted in disruptions of the segmental pattern. The location of abnormal segments depended upon the stage at heat shock and the number affected depended on its severity. A constant number of normal segments developed between the last segment visible at the time of heat shock and the first abnormal segment. These results are similar to the disruptions observed in amphibian somites following heat shock. However, different parts of the segment pattern varied in their response; the head segments were very rarely affected, and disrupted regions rarely started in the middle abdomen (segments A5 and A6). The results are discussed in relation to two models (the clock and wavefront and progress zone models) that have been proposed as an explanation for the specification of the somite pattern in amphibians.

The postnatal maturation of the epithelium and tubule wall of efferent tubules in the rat was investigated by light and transmission electron microscopy, from birth to 50 days of age, when sperms were released from the seminiferous tubules and appeared in the genital duct. At the end of the first week of life, an endocytotic apparatus is differentiated in the epithelial cells. During the third week of life, efferent tubules developed specializations for the transport of sperms and fluids, namely the appearance of ciliated elements interspersed among the principal cells of the epithelium, and differentiation of myoid elements in the tubule wall. The appearance of specializations related to endocytosis and fluid transport across the epithelium preceded the canalization of the seminiferous cords which, in fact, is reported to appear at the end of the second week of life in the rat, along with the initial secretion of testicular fluid. This suggested that the maturation of efferent tubules is not triggered by the passage of testicular fluid, as surmised for the postnatal differentiation of caput epididymis. The postnatal maturation of efferent tubules was almost complete 35 days after birth. The appearance of sperms in the genital duct of 50-day-old animals was not associated with any remarkable structural change.

The influence of innervation on the initial differentiation of muscle fibre types was investigated by using the trochlear nucleus-superior oblique muscle system of duck. The adult muscle is composed of three types of fibres (designated as type I, II, III) as identified with the histochemical techniques for ATPase pH sensitivity. Type I fibre ATPase activity was acid-stable, alkali-labile; type II alkali-stable, acid-labile; and type III both acid- and alkali-stable. These types showed variable mitochondrial alpha-glycerophosphatase dehydrogenase, nicotinamide adenine dinucleotide tetrazolium reductase, and phosphorylase activity. Type I and II fibres are primarily located in the portion of the muscle adjacent to the orbit whereas the rest of the muscle is primarily composed of type III fibres. In the normally developing muscle, type II and III fibres are present as early as embryonic day 9; one day prior to the arrival of nerve fibres in the muscle. Type I fibres are first observed on embryonic day 17. On day 22 the percentages of type I, II and III fibres are 29, 53 and 18, respectively. As the development progressed the percentages of type I and II fibres decrease and after hatching 76% of the fibres belong to type III, 17% of type II and only 7% to type I. In embryos paralysed with daily application of 3 mg d-tubocurarine (d-TC) from day 9 onwards the differentiation of type II and III fibres occurs, but type I fibres were never observed in the paralysed muscles. These muscles also contained significantly fewer myotubes than the normal muscle. By contrast, when the muscle was made aneural by permanent destruction of motor neurones on embryonic day 7 all three types of fibres differentiated. When embryos with aneural muscles were also subjected to d-TC treatment the type I fibres failed to differentiate. It is concluded that the initial differentiation of fibre types is independent of innervation and that primary myotubes are capable of differentiating into all three types of fibres. The absence of type I fibres in curarized muscles may be due to some unique effect of d-TC on the muscle itself.