Showing papers in "Development in 1981"

Proliferation and migration of primordial germ cells during compensatory growth in mouse embryos.

Abstract: Primitive-streak-stage mouse embryos were treated with Mitomycin C injected intraperitoneally into pregnant females at 6·75–7·0 days post coitum . The newborn mice developed poorly and mortality was high during the suckling period. Many weaned survivors showed impaired fertility and poor breeding performance. Histological examination revealed a paucity of germ cells in the adult gonads. The deficiency was mainly caused by a severe reduction of the primordial germ cell population in early embryonic life, which was not fully compensated for during the compensatory growth phase of the Mitomycin C-treated embryo. Also contributing to such impaired fertility were retarded migration of the primordial germ cells into the genital ridges, poor development of the foetal gonad and secondary loss of the germ cells during gametogenesis in males.

Staging the metamorphosis of Drosophila melanogaster

Abstract: A sequence of 51 visible changes is described during the course of metamorphosis in Drosophila melanogaster, and a series of 24 convenient stages is defined for use in the experimental analysis and exploitation of this part of the insect life cycle. The duration of each stage is estimated and times are suggested for batch collections of symphasic animals.

Reactivity of monoclonal antibodies against intermediate filament proteins during embryonic development

Abstract: Monoclonal antibodies (mAbs) against a preparation of intermediate filaments from trophoblastoma cells were studied for their reactivity pattern during embryonic development and on adult tissue cells. Up to day 12 of embryonic development, epithelial cells of the three germ layers reacted with these mAbs. Later during development and in adult tissues, positive reactions could be observed only with epithelial cells derived from mesoderm and endoderm. Because of their tissue distribution, the proteins reacting with these mAbs might belong to the keratin family of intermediate filaments or they might represent a new group of intermediate filaments.

X-chromosome activity in foetal germ cells of the mouse

Abstract: A cycle of inactivation and reactivation of one X chromosome in the female (XX) germ line is shown by analysis of gene dosage effects on activity of an X-linked enzyme. The ratio of activities of the X-linked enzyme HPRT and an autosomal enzyme APRT are determined in XX and XY germ cells from embryonic gonads from the 12th to the 17th day of pregnancy. Mitotic stages of XX and XY germ cells on the 12th day have similar HPRT:APRT ratios, but on the 13th day the ratios are significantly higher in XX than XY germ cells. As the XX germ cells enter meiosis they show a marked increase in HPRT:APRT ratio which is primarily due to a rise in X-linked HPRT activity. Comparisons are made with XO germ cells on the 12th and 14th day. On the 12th day, XO do not differ from XX and XY germ cells, suggesting that only one X chromosome is active in XX germ cells at this stage. On the 14th day, on the other hand, the HPRT:APRT ratios in XO and XY germ cells are similar but in XX germ cells the ratio is significantly higher. The twofold difference between the ratio in XX and XO germ cells suggests that by this stage both X chromosomes are active in XX germ cells. The subsequent large increase of the ratio in XX relative to XY germ cells is thought to reflect their differing cell states.

The control of somitogenesis in mouse embryos.

Abstract: Somitogenesis in the mouse embryo commences with the generation of presumptive somitic mesoderm at the primitive streak and in the tail-bud mesenchyme. The presumptive somitic mesoderm is then organized into somite primordia in the presomitic mesoderm. These primordia undergo morphogenesis leading to the segmentation of somites at the cranial end of the presomitic mesoderm. Somite sizes at the time of segmentation vary according to the position of the somite in the body axis: the size of lumbar and sacral somites is nearly twice that of upper trunk somites and of tail somites. The size of the presomitic mesoderm, which is governed by the balance between the addition of cells at the caudal end and the removal of somites at the cranial end, changes during embryonic development. Somitogenesis is disturbed during the compensatory growth of mouse embryos which have suffered a drastic size reduction at the primitive-streak and early-organogenesis stages. The formation of somites is retarded and the upper trunk somites are formed at a smaller size. The embryo also follows an entirely different growth profile, but a normal body size is restored by the early foetal stage. The somite number is regulated to normal and this is brought about by an altered rate of somite formation and the adjustment of somite size in proportion to the whole body size. It is proposed that axis formation and somitogenesis are related morphogenetic processes and that embryonic growth controls the kinetics of somitogenesis, namely by regulating the number of cells allocated to each somite and the rate of somite formation.

Neural crest formation in the head of the mouse embryo as observed using a new histological technique

Abstract: A histological technique is described which results in the differential staining of neural crest cells. This is used to describe the formation and early migration of crest cells in the head of the mouse embryo. The first indications of crest formation are seen in the midbrain/anterior hindbrain at 3–4 somites where crest cells accumulate in the basal surface of the ectodermal epithelium near the future margin of the neural plate. Shortly thereafter (4–6 somites) these cells disrupt the basal surface of the epithelium and escape as mesenchyme. The apical epithelial cells in this region become the surface ectoderm adjacent to the neural plate. Subsequently, crest is formed from neural plate rather than surface ectoderm. In addition, mesenchyme is formed from presumptive surface ectoderm in a groove in the lateral portion of the fold between the forebrain and the midbrain. By 5–7 somites, crest mesenchyme is formed at all levels of the midbrain, hindbrain, and from the margins of the forebrain adjacent to the optic pits. Because of the bending of the embryonic axis, forebrain crest cells appear to migrate dorsally over the presumptive eye where they are met by ventrally migrating midbrain crest cells. Crest formation continues in the region of the midbrain and hindbrain during, and for an undetermined period after closure of the head folds at between 8 and 16 somites. These results demonstrate differences in the origin and timing of crest formation between chick and mouse. From this may be inferred different patterns of crest migration as well. In addition, the ability to directly observe early crest formation should aid in the analysis of the mechanisms by which epithelial cells are converted into mesenchyme.

Embryogenesis of an insect nervous system II: a second class of neuron precursor cells and the origin of the intersegmental connectives.

Abstract: The intersegmental connectives in the locust central nervous system are initiated by the axons of early differentiating neuron trios. Using a combination of electron microscopy and fluorescent dye injection we have shown that the axons of these cells grow out anteriorly and posteriorly in each segment along a basement membrane, and link together at the segment borders to form continuous longitudinal pathways on each side of the developing nervous system. These early neurons are the progeny of a second class of precursor cell, the midline precursors, which are distinct from the segmental neuroblasts. Like the neuroblasts, the midline precursors are arranged in a standard segmentally repeated pattern. This and the standard pattern of axon outgrowth in different segments suggest that the nervous system develops to a common, segmentally repeated programme.

Avian primordial germ cells are of epiblastic origin

Abstract: We have demonstrated that quail PGCs possess a characteristic heterochromatin nuclear marker demonstrable already at stage 6 H & H with Feulgen staining. Chimaeras of stage-XIII E. G & K chick epiblast and quail hypoblast and vice versa have been made. The chimaeras were incubated up to stage 7-8 H & H and checked histologically with Feulgen staining for the presence of the heterochromatin marker in every detectable PGC. It was found that the PGCs are of epiblastic origin, contrary to previous data and speculations concerning avian PGCs.

Distribution of microvilli on dissociated blastomeres from mouse embryos: evidence for surface polarization at compaction

Abstract: Cells of mouse embryos develop a polarization of microvillous distribution at compaction. Cells of the 4-cell embryo show a uniform pattern of fluorescent-ligand binding and an even distribution of microvilli. Each cell of the early 8-cell embryo has a uniform distribution both of microvilli and of fluorescent ligand. During the 8-cell stage, there is a progressive increase in the incidence of cells which show microvilli restricted to a region normally on the exposed surface of the embryo. When late 8-cell embryos were disaggregated to single cells, and these sorted by pattern of fluorescent-ligand binding, each of the four patterns of staining related consistently to a characteristic distribution of microvilli as viewed by scanning electron microscopy. The 16-cell embryo possessed an inside population of uniformly labelled cells with a sparse microvillous distribution, and an outside population of cells, each of which had a microvillous pole.

Muscle nerve branches do not develop in chick wings devoid of muscle

Abstract: If the somitic mesoderm of a 2-day chick embryo is destroyed by X-irradiation, the adjacent limb develops with a normal pattern of connective tissues, but is devoid of muscle. The innervation of muscleless wings produced in this way was examined in silver-stained whole mounts, fixed 3 to 8 days later. The main nerve trunks and their cutaneous branches developed normally; but the nerve branches which in a normal limb would lead to individual muscles were generally absent. In almost all those exceptional cases where muscle nerve branches were present, muscle was found to be present also, despite the X-irradiation. Where there was no muscle, the muscle nerve branches apparently did not even begin to form. As a control for side effects of the X-irradiation, wing buds were grafted from normal to irradiated embryos and vice-versa, and again analysed for their innervation. The results confirmed that the absence of muscle nerve branches was due to the absence of muscle cells in the limb. Thus (1) the routes taken through a limb by the main mixed nerve trunks and by their cutaneous branches are determined by the connective tissues, and not by any mechanisms requiring muscle cells; but (2) muscle cells are necessary to provoke the formation of the side branches leading to the sites of individual muscles.

Selective effect of gonadotrophins on cell coupling, nuclear maturation and protein synthesis in mammalian oocytes.

Abstract: Individual gonadotrophic hormones were used to examine the; degree to which changes in intercellular coupling between somatic and germ cells initiate rneiotic maturation, regulate protein synthesis or alter the ultrastructure of the ovine oocyte. Follicle Stimulating Hormone (FSH; 50 ng ml−1) suppressed intercellular coupling to the same extent as that observed during oocyte maturation in vivo . At low concentrations FSH did not, however, initiate resumption of meiosis. By contrast, luteinising hormone (LH; 100–500 ng ml−1) invariably initiated meiosis in oocytes cultured within the follicle but did not disrupt intercellular coupling. We conclude that nuclear maturation is not dependent upon the disruption of cell contact between the oocyte and the surrounding follicle cells. The profile of proteins synthesized by untreated oocytes differed greatly from that of oocytes matured for 18 h in follicles treated with a combination of FSH and LH. Pretreatment of follicles with either FSH or LH at low concentrations resulted in the synthesis of an intermediate and more variable pattern of proteins. No correlation was found between changes in protein synthesis and the extent of junctional communication between the cumulus cells and oocyte. Membrane vesiculation and lysosomal change in the transzonal processes are early structural changes associated with the suppression of intercellular coupling in oocytes. These changes in coupling probably result in the relocation of intracellular organelles in the final stages of oocyte maturation.

The developmental capacity of blastomeres from 4- and 8-cell sheep embryos

Abstract: The developmental capacity of ( a ) single blastomeres of 4-cell embryos, ( b ) pairs of blastomeres of 8-cell embryos and ( c ) single blastomeres of 8-cell embryos was studied. The blastomeres were inserted into evacuated foreign zonae pellucidae, embedded in agar, transferred to ligated oviducts of dioestrous ewes, and recovered when their total age was 5½ to 6½ days. At lecovery the majority of embryos belonging to categories ( a ) and ( b ) had developed into small blastocysts, whereas most embryos of category ( c ) were vesicular forms with no clearly observable inner cell mass. The viability of embryos which had continued their development during culture in the sheep oviduct was tested by their transfer to the uterine horns of recipient ewes which had come into oestrus on the same day as the respective embryo donors. Three lambs were produced after transfer of eight embryos of category ( a ) deriving from two parent embryos. These three lambs derived from a single 4-cell embryo. Nine lambs were produced after transfer of 16 embryos of category ( b ) deriving from four parent embryos. Four of these lambs derived from a single 8-cell embryo. When ewes which had come into oestrus one or two days after the respective embryo donors were used as recipients for 16 embryos of category ( a ) and 16 embryos of category (b) one and five lambs, respectively, were produced. Finally, two lambs were produced after transfer of 31 embryos of category ( c ). These lambs derived from two different parent embryos. The principal conclusions drawn from the study are that at least some individual blastomeres from 4- and 8-cell sheep embryos can give rise to an entire conceptus, and that the viability of embryos produced by blastomere separation depends more on the degree of reduction in cell number than on the stage of development at which separation is performed.

The role of the visceral yolk sac in mediating protein utilization by rat embryos cultured in vitro.

Abstract: Conceptuses from 9·5-day pregnant rats have been cultured for 48 h in heat-inactivated homologous serum. Embryonic development was normal. The protein contents of embryos and visceral yolk sacs after different periods of culture were recorded. When 125-labelled polyvinylpyrrolidone or [3H]dextran were added to the culture serum, radioactivity was accumulated by the yolk sac, but only background levels were detected in the embryo itself. The amount of radioactivity found in the yolk sac varied with the length of the interval before harvesting during which 125 I-labelled PVP or [3H]dextran was present. When formaldehyde-denatured 125 I-labelled bovine serum albumin was added to the culture serum, little radioactivity accumulated in the yolk sac and only background levels were found in the embryo. Trichloroacetic acid-soluble radioactivity steadily appeared in the culture serum, however. When conceptuses were cultured in glucose- and vitamin-supplemented dialysed serum from rats injected 2 h previously with [3H]leucine, radioactivity was found in both embryos and yolk sacs. The amount of radioactivity in these tissues increased with duration of exposure to 3H-labelled serum proteins. After short exposures little of the yolk sac and embryonic radioactivity was acid-insoluble, but this proportion increased with duration of exposure. These results are interpreted as follows. Intact macromolecules cannot enter the cells of the embryo itself, but are captured by pinocytosis into the cells of the visceral yolk-sac endoderm. Indigestible macromolecules such as 125 I-labelled polyvinylpyrrolidone and [3H]- dextran accumulate within the yolk-sac lysosomes, but proteins are digested there by the lysosomal enzymes. The radiolabelled digestion product of 125 I-labelled bovine albumin is [125 I]iodotyrosine, which cells cannot utilize and so is excreted into the culture serum. The labelled digestion product of the 3H-labelled rat serum proteins is [3H]leucine, which is used for protein synthesis in both embryo and yolk sac. The experiments provide direct evidence for the long-suspected role of the yolk sac in mediating embryonic nutrition in the period of development prior to the establishment of a functional chorioallantoic placenta.

Influence of the entoderm on mesodermal expansion in the area vasculosa of the chick.

Abstract: To determine whether expansion of the splanchnic mesoderm of the area vasculosa is influenced by the entodermal substratum on which it occurs, entoderm was separated from a small area of splanchnic mesoderm. The splanchnic mesoderm then contracted and thickened, decreasing to 7% of its original area in 16 h. By then entoderm had reattached to most of it, and it expanded, reaching 11% of its original area by 24 h. It was concluded that attachment to entoderm may be required for expansion of the splanchnic mesoderm, but the small amount of expansion obtained made this conclusion tentative. For technical reasons subsequent investigation was done on mesodermal transplants, which attached to the host9s entoderm in 6 h, by which time they had contracted to 15% of their original area. They then expanded, reaching 30% by 16 h and 49% by 24 h. The onset of their expansion was also accompanied by the formation of connexions between their blood vessels and those of the host, and by the resumption of blood flow in them. To see whether their expansion was due to resumption of blood flow or to attachment to entoderm, other transplants were made in which the middle one-third was separated from the host9s entoderm by a piece of Millipore filter. This portion failed to expand although it became connected to the host9s blood vessels and flow of blood resumed in it, while the two lateral thirds, which regained attachment to entoderm, expanded. Transplants were also rotated so that their splanchnic mesoderm attached to ectoderm instead of entoderm. These transplants also formed connexions with the host9s vessels and blood flow resumed in them, but they expanded only slightly compared to non-rotated controls, in which the splanchnic mesoderm attached to entoderm. It was concluded that while flow of blood undoubtedly promotes splanchnic mesodermal expansion as others have shown, attachment of the splanchnic mesoderm to entoderm is also important, and without it the promotive effect of blood flow does not occur. Evidence was also obtained that attachment to entoderm maintains the thinness of the splanchnic mesoderm, and that a vascular growth stimulus may be produced by the unvascularized entoderm distal to the mesoderm.

An autoradiographic analysis of the potency of embryonic ectoderm in the 8th day postimplantation mouse embryo

Abstract: The potency of 8th day mouse embryonic ectoderm cells has been studied by injecting them into synchronous embryos which were subsequently cultured for 36 h. The development of injected embryos in vitro was comparable to that of embryos maintained in vivo. Tritiated thymidine was used to label the donor cells so that chimaerism could be analysed histologically. The results demonstrate the pluripotency of embryonic ectoderm in situ in the late primitive-streak-stage embryo. In addition, the patterns of donor cell colonization vary according to the site of origin and injection of the donor tissue.

Cell interactions and endoderm differentiation in cultured mouse embryos.

Abstract: Morphological and biochemical evidence is presented that the visceral extraembryonic endoderm of the 6·5-day mouse embryo will differentiate into parietal endoderm when cultured in contact with extraembryonic ectoderm undergoing transition into trophoblast giant cells. Egg cylinders from 6·5-day embryos were dissected into embryonic and extraembryonic halves and cultured in suspension in vitro for up to 7 days. After 4 days, the endoderm cells of the extraembryonic fragments morphologically resemble parietal endoderm, are associated with a thick basement membrane and synthesize large amounts of the matrix proteins laminin and Type IV procollagen. A similar transition in phenotype is not seen in the endoderm of embryonic fragments, nor in visceral extraembryonic endoderm cells cultured in isolation. In another series of experiments, complete egg cylinders were dissected free of visceral endoderm ovei lying the extraembryonic ectoderm and then cultured in vitro . The visceral endoderm cells which recolonize the surface of the extraembryonic ectoderm develop a parietal endoderm phenotype and lay down a thick basement membrane. These results suggest that the differentiation of the extraembryonic endoderm of the early mouse embryo into visceral and parietal phenotypes can be influenced by local cell—cell or cell—substrate interactions, and is not determined solely by cell lineage.

Growth and development of pattern in the cranial neural epithelium of rat embryos during neurulation.

Abstract: The pattern of growth and morphogenesis of the cranial neural epithelium of rat embryos during neurulation is described. Transverse sections of the midbrain/hindbrain neural epithelium at different stages (0-14 somites) show a constant area and cell number throughout neurulation, even though there is a high level of mitosis. Mitotic spindles are orientated parallel to the long axis of the embryo, so that increase in cell number occurs in this direction only. Growth is expressed only as an increase in size of the forebrain, which projects rostrad to the tip of the notochord. In the midbrain/upper hindbrain regions, cellular organization of the neural epithelium changes from columnar to cuboidal to pseudostratified, while its shape changes from flat to biconvex to V shaped. Closure is immediately preceded by neural crest cell emigration from the lateral edges. Throughout neurulation the cranial notochord develops an increasingly convex curvature in the rostrocaudal plane. The attached neural epithelium curves with the notochord (forming the primary cranial flexure) so that as its lateral edges move dorsomedially they form a more distant concentric arc with that of the notochord, and are hence stretched during the final closure period. The whole rat embryo culture technique was used to investigate the morphogenetic role of proteoglycans during neurulation, neural crest cell emigration and other events in the lateral edge region prior to closure, and the importance of microfilament contraction during concave curvature of the neural epithelium.

Cytoplasmic polarity develops at compaction in rat and mouse embryos

Abstract: Cells of both rat and mouse morulae can be stained vitally to reveal an asymmetry in the organization of their cytoplasm. In each cell of the rat 8-cell-embryo a column of organelles develops between the nucleus and the embryo periphery as revealed by toluidine blue, acridine orange and horseradish peroxidase (HRP). Although cells of the mouse morula lack the blatant asymmetric distribution of organelles observed in rat cells, a long pulse (greater than 3 h) of HRP to compact 8-cell mouse embryos revealed a distinct restricted localization of the enzyme not evident at earlier pre-compaction stages. The cytoplasmic polarity generated in these embryos can be demonstrated in cells of intact embryos, and also in cells disaggregated from embryos before vital staining.

Temporal pattern of cleavage and the onset of gastrulation in amphibian embryos developed from eggs with the reduced cytoplasm

Abstract: Fertilized eggs of the Japanese newt, Cynops pyrrhogaster , were divided into two or four equal-sized parts with fine glass rods before the first cleavage. In such cases one of the egg fragments, at least, proceeds to cleavage and gastrulates. The temporal pattern of cell division and the onset of gastrulation in such half or quarter embryos were investigated and compared with normal development. The following results were obtained: (1) desynchronization starts two divisions earlier in quarter embryos and one division earlier in half embryos compared with whole embryos, (2) the time from the first cleavage to the onset of gastrulation does not widely vary among quarter, half and whole embryos and (3) the numbers of blastomeres which constitute embryos at the pigment stage decrease in proportion to the diminution of egg volume.

Hepatic induction in the avian embryo: specificity of reactive endoderm and inductive mesoderm.

Abstract: Mesoderm of precardiac and cardiac region (‘cardiac’ mesoderm) of chick, quail and mouse embryos could induce hepatic epithelium in the endoderm of the anterior half of young quail or chick embryos (anterior endoderm) in vitro as well as in vivo . No species specificity in the induction of hepatic epithelium by the ‘cardiac’ mesoderm could be observed. The hepatic induction, was controlled strictly by tissue specificity of both endoderm and mesoderm. Replacement of the ‘cardiac’ mesoderm or the anterior endoderm by noncardiac mesoderms or endoderms other than the anterior endoderm resulted in failure of hepatic induction. Only the anterior endoderm was found to have competence for hepatic induction, indicating that it was committed, in unknown ways, to react with ‘cardiac’ mesoderm, and can properly be called pre-hepatic endoderm. Comparison between the development of hepatic endoderm and the hepatic induction potency of ‘cardiac’ mesoderm, which was most intense during 1- to 1·5- incubation days and decreased gradually with the increase of the stage, suggests that in normal development the ‘cardiac’ mesoderm actually induces hepatic epithelium in the competent endoderm. Hepaticinduction potency remained up to 6 days, and was found in truncus arteriosus, ventricle and auricle areas and in endocardial and myocardial layers of the heart.

Enzymo- and immunocytochemical analyses of the differentiation of liver cells in the prenatal mouse.

Abstract: Differentiation of the endodermal cells of the mouse liver was studied enzymo- and immunocytochemically by analyzing the cellular localization of alphafoetoprotein (AFP), glycogen, and alkaline phosphatase (ALP) and 5'-nucleotidase (5'-Nase) activities. 1. In 8.5-day foetuses, AFP appears in some endodermal cells of the anterior intestinal portal region. The cells of the cranial diverticulum contain much AFP at 9.5 days, while those of the caudal diverticulum contain less AFP. 2. In 9.5- to 15.5-day foetuses, hepatocytes are intensely fluorescent for AFP. After 16.5 days less-positive hepatocytes increase in number. AFP is still present in a few hepatocytes of 14-day-old postnatal mice. ALP and 5'-Nase activities appear in a small proportion of hepatocytes at 13.5 and 14.5 days of embryonal life, respectively. At 15.5 days, many hepatocytes possess these enzyme activities, and initiate accumulation of glycogen. AFP-containing hepatocytes type I (gestation day 9.5-16.5) successively acquire ALP and 5'-Nase activities and accumulate glycogen, and then differentiate into hepatocytes type II after gestation day 17.5. 3. Endodermal cells constituting lumen structures in the liver trabeculae are the precursor of the intrahepatic bile duct cells. They possess much AFP, but no glycogen and no ALP activity, and are similar to hepatocytes type I. Since immature hepatic duct cells also possess much AFP, but no glycogen, and no ALP and 5'-Nase activities, they are similar to endodermal cells of the lumen structures. Therefore, that the endodermal cells of the lumen structures are the intermediate cells between hepatocytes type I and hepatic duct cells may be conceivable.

Autonomous development of parts isolated from primitive-streak-stage mouse embryos. Is development clonal?

Abstract: The relationship between growth rate and regionalization of amphibian, bird and mammalian embryos is briefly reviewed. In contrast to the others, mammals start gastrulation with few cells but accelerate cell proliferation coincidentally. Experiments are described which demonstrate (1) autonomous development of pieces isolated surgically from such mouse embryos, and (2) an absence of regeneration or regulation. Since such embryos regulate completely after chemically induced random cell death it is postulated that these results reflect developmental determination and a resulting mosaicism that suggests development may have a clonal basis. Maps are drawn, allocating positions to various tissues in the embryo.

Interaction of epiblast and hypoblast in the formation of the primitive streak and the embryonic axis in chick, as revealed by hypoblast-rotation experiments.

Abstract: Three types of experiments were performed to determine the interaction between the epiblast and hypoblast for primitive streak formation: (1) Hypoblasts of blastoderms from stages XIII E.G & K to 3 H & H were separated from the epiblasts and rotated by 90 degrees counterclockwise; (2) hypoblasts from stages XIII E.G & K to 3 H & H blastoderms were rotated by 180 degrees; (3) hypoblasts were exchanged between blastoderms of different developmental stages and placed at 90 degrees counterclockwise to the axis of the recipient epiblast. In all blastoderms studied only a single PS developed. After rotation of the hypoblast by 90 degrees, the direction of the PS was according to the orientation of the hypoblast at stage XIII, whereas at older stages it gradually shifted towards the axis of the epiblast. At stage 3- H & H the PS is already imprinted in the epiblast and cannot be shifted. After rotation of the hypoblast by 180 degrees the PS originated at the point near the marginal zone at which the inductive part of the hypoblast interacted with a component epiblast. Conclusions are drawn about the dynamics of the inductiveness of the hypoblast and the competence of the epiblast for the PS formation and orientation.

The induction of neural crest-derived cartilage and bone by embryonic epithelia: an analysis of the mode of action of an epithelialmesenchymal interaction

Abstract: The formation of membrane bone from neural crest-derived mesenchyme of the maxillary and mandibular processes of the embryonic chick depends upon prior interactions between the mesenchyme and maxillary or mandibular epithelia. The present study explores the specificity of these interactions using tissue recombinations between heterotypic epithelia and mesenchyme. Mandibular and maxillary mesenchyme responded to maxillary and mandibular epithelia by forming bone. A third osteogenically inductive epithelium, the scleral epithelium with its specialized scleral papillae, also allowed mandibular mesenchyme to form bone, indicating that mesenchyme can form bone in response to osteogenic epithelia other than its own. Epithelia which normally do not induce membrane bone formation in situ (wing and leg bud, back and abdominal epithelia) also allowed mandibular epithelia to ossify as did mandibular epithelia from the 10-day-old foetal mouse. Thus this tissue interaction is neither site nor species specific. Mandibular epithelium allowed bone to form in osteogenic mesenchyme from the maxilla and the sclera of the chick and from the mouse mandible but would not induce bone formation from normally non-osteogenic mesenchyme of the limb buds, chorioallantoic membrane or trunk neural crest. The results obtained with all of the tissue recombinations were consistent with the epithelial- mesenchyme interactions that initiate osteogenesis in both the mandibular and the maxillary processes being permissive interactions. The distinction between permissive and instructive interactions is discussed.

Effect of culture conditions on diploid to giant-cell transformation in postimplantation mouse trophoblast.

Abstract: Diploid extraembryonic ectoderm and ectoplacental cone from the 7.5-day mouse embryo were grown in vitro under a variety of culture conditions in an attempt to discover conditions which maintain trophoblast in a diploid state and prevent giant-cell formation. It was found that maintenance of tissue integrity was not enough to keep the tissues dividing and diploid, but that the presence of inner-cell-mass derivatives did have some effect. This effect was only apparent when trophoblast cells were entirely enclosed by embryonic tissues. Monolayers of embryonic or embryonal carcinoma cells did not prevent giant-cell formation. Diploid extraembryonic ectoderm and ectoplacental cone responded differently: ectoplacental cells eventually formed trophoblast giant cells even when enclosed by embryonic cells whereas extraembryonic ectoderm cells apparently could be maintained in a diploid condition. This and other differences in properties between extraembryonic ectoderm and ectoplacental cone are discussed with reference to a new model for the postimplantation trophoblast lineage in the mouse.

On the ‘clock’ mechanism determining the time of tissue-specific enzyme development during ascidian embryogenesis: II. Evidence for association of the clock with the cycle of DNA replication

Abstract: Acetylcholinesterase (AChE) is a tissue-specific enzyme of the muscle cells of ascidian embryos and its synthesis begins at the neurula stage. Embryos which had been permanently cleavage-arrested with cytochalasin B could develop AChE activity. The time of first AChE occurrence in embryos which had been arrested in the 32-cell stage with cytochalasin was about the same as in normal embryos. The nucleus in the cell of cytochalasin-arrested embryos divided in good synchrony with that of normal embryos. Embryos which had been continuously arrested with colchicine could also produce AChE activity at nearly the same time as did normal embryos. In the cell of colchicine-arrested embryos normal nuclear divisions did not occur, but the cell showed repeated cycles of nuclear envelope breakdown and nuclear envelope reformation in almost parallel with cell cycles of normal embryos. The cell of colchicine-arrested embryos incorporated [3H]thymidine. Aphidicolin, a specific inhibitor of DNA synthesis, prevented cleavages of ascidian eggs. Embryos which had been permanently arrested with aphidicolin in the cleavage stages up to the 64-cell stage did not develop AChE activity, while embryos which had been treated with it from the 76-cell stage onwards were found to be able to differentiate AChE activity. Based on these findings it was proposed that DNA replication is prerequisite for development of the histospecific protein and that the cycle of DNA replication is closely associated with the clock mechanism which is determining the time of initiation of the enzyme development.

A definite number of aphidicolin-sensitive cell-cyclic events are required for acetylcholinesterase development in the presumptive muscle cells of the ascidian embryos.

Abstract: In order to determine whether or not a crucial number of DNA replications are prerequisite for cellular differentiation, we have studied development of a tissue-specific enzyme, muscle acetylcholinesterase (AChE) in the presumptive muscle cells of cleavage-arrested ascidian embryos. Embryos were cleavage-arrested with cytochalasin B (an inhibitor of cytokinesis) and aphidicolin (an inhibitor of DNA synthesis). The 64-ceIl-stage embryos which had been permanently cleavage-arrested with cytochalasin B developed AChE in all the eight presumptive muscle cells, but the same stage embryos which had been prevented from undergoing further divisions by simultaneous treatment with aphidicolin and cytochalasin did not produce AChE at all. Cytochalasin-arrested 76-cell-stage embryos were able to differentiate AChE in the ten presumptive muscle cells, while aphidicolin-cytochalasin-arrested 76-cell stages in as many as four cells. The early gastrulae which had been arrested with cytochalasin B produced AChE in all the sixteen presumptive muscle cells, while the same stage embryos arrested with aphidicolin and cylochalasin in as many as twelve cells. Cytochalasin-arrested late gastrulae developed AChE in twenty blastomeres, while aphidicolin-cytochalasinarrested late gastrulae in eighteen cells. The presumptive muscle cells at these four stages consist of those of three different (seventh, eighth, and ninth) generations, and the relative positions of the blastomeres in the cleavagearrested embryos remained fixed. Judging from the relative positions of the blaslomeres, the AChE-producing cells in aphidicolin-cytochalasin-arrested embryos were always at eighth or ninth generation, while those with no AChE activity were certainly at seventh generation. Based on these findings it was supposed that aphidicolin-sensitive cell-cyclic events, presumably DNA replication, are closely associated with AChE development and that the eighth cleavage cycle may be ‘quantal’ for the histospecific enzyme development.

A gradation of hyaluronate accumulation along the proximodistal axis of the embryonic chick limb bud.

Abstract: We are currently investigating the mechanism by which the apical ectodermal ridge (AER) of the embryonic chick limb bud exerts its negative effect on the cytodifferentiation of limb mesenchymal cells directly subjacent to it, and the mechanism by which cytodifferentiation is triggered when the cells leave the influence of the AER. Since there is a gradation of differentiation along the proximodistal axis of the limb bud, we have dissected limb buds into discrete segments along the proximodistal axis, and biochemically examined the accumulation of hyaluronate (HA) and other glycosaminoglycans (GAG) in each segment. The unspeciajized subridge region of stage-25 limb buds was separated into distal (segment 1) and proximal (segment 2) regions, and the remaining proximal portion of the limb was separated into four segments (segments 3, 4, 5 and 6) along the proximodistal axis. Stage-24 limb buds were separated into corresponding regions. Since in the proximal regions of the limb (segment 3 through 6), only the cells comprising the central cores of the limb are involved in chondrogenic differentiation, the central core tissue was surgically separated from the peripheral tissue. We have found that HA is by far the predominant GAG accumulated by cells comprising the distal subridge region (representing greater than 50% of the total GAG accumulated during a 90-min labelling period with [3H]glucosamine), and that there is a progressive decline in HA accumulation along the proximodistal axis. The relative and total amount of HA accumulated is highest in the distal subridge region (segment 1), intermediate i(i the proximal subridge region (segment 2) and lowest in the proximal central core regions Of the limb (segments 3 through 6). The striking decrease in HA accumulation in the central core of segment 3 is accompanied by a striking increase in the accumulation of chondroitin sulphate, one of the major constituents of cartilage matrix. In contrast to the central core regions of segments 3 through 6, the relative and total amount of HA accumulated by the peripheral non-chondrogenic regions of these segments remains relatively high, being similar to the accumulation observed in the proximal subridge region. These results indicate that there is a gradation of HA accumulation along the proximodistal axis of both stages-24 and -25 limb buds which correlates with distance of cells from the AER and the state of differentiation of the cells.

Somitogenesis in the amphibian Xenopus laevis: scanning electron microscopic analysis of intrasomitic cellular arrangements during somite rotation.

Abstract: The intrasomitic changes in cell arrangement which accompany somite rotation during somitogenesis in Xenopus laevis were analysed with the scanning electron microscope (SEM). Longitudinal, horizontal fractures of whole embryos were examined at various dorsoventral levels of stage-22 to -24 embryos. Observations of the gross morphological features of somitogenesis, and the cellular changes which accompany somite segmentation and somite rotation were made. Several of these observations lead to modifications of previous models for the cellular basis of somitogenesis in Xenopus. Individual cellular rearrangements, rather than simultaneous block rotation of a whole somite, appear to be responsible for the 90 degrees rotation of myotomal cells within a single somite. Cellular arrangements in fused somites were also examined. Some ultraviolet-irradiated embryos displayed a complete lack of a notochord. The somites in those embryos were fused across the midline beneath the neural tube. The dorsal and ventral arms of the somites are not fused. Normal rotation occurs only in the dorsal and ventral arms while, in the majority of cases, cells in the fused region fail to rotate normally. In some cases, individual cells in the fused region undergo partial rearrangement. Those observations support the notion that individual cellular rearrangements account for the rotation of the whole somite.

Evidence for a metameric pattern in the development of the chick humerus

Abstract: It has been proposed that the wing bud is induced by some axial influence at a specific confined location and that the ZPA is the residual influence of such induction. The purpose of the present investigation was to test this hypothesis. Tantalum foil barriers were placed lateral to the mesonephric duct and parallel to the long axis of the embryo in the wing field of stage-12 to -15 chick embryos. These barriers blocked the somatopleure's communication with more medial tissues at specific somitic levels. The results of these experiments demonstrate that (1) the limb is not induced at one specific point, (2) portions of the humerus appear to be induced segmentally along the entire limb field and (3) the ZPA is not induced by axial structures. We propose a model of wing development suggesting that the humerus is induced as several separate components which then fuse to form the definitive bone.