Showing papers in "Development in 1971"

The growth of the retina in Xenopus laevis: an autoradiographic study.

Abstract: The growth of the retina has been studied in Xenopus by use of autoradiography with tritiated thymidine. At the time when retinal polarization first occurs (around stage 30) there are only some 20 ganglion cells across the retinal equator and the rest of the retina develops later, by annular addition of cells at the ciliary margin. This process continues beyond metamorphosis.

An ultrastructural study of primordial germ cells, oogonia and early oocytes in Xenopus laevis.

Abstract: SUMMARY Electron-microscope observations on the differentiation of germ cells in Xenopus laevis have revealed that the Balbiani body, cytoplasmic nucleolus-like bodies and groups of mitochondria associated with granular material previously reported only in older amphibian oocytes, are also present in the primordial germ cells, oogonia and early meiotic (prediplotene) oocytes of this species. Although there is considerable morphological reorganization of the gonad as a whole at the time of sex determination, little visible change in the ultrastructure of the primordial germ cells appears to take place during their transition to oogonia. Both primordial germ cells and oogonia have highly lobed nuclei and their cytoplasm contains a conspicuous, juxtanuclear organelle aggregate (consisting for the most part of mitochondria), which is considered to represent the precursor of the Balbiani body. In marked contrast, the transition from oogonium to oocyte in Xenopus is characterized by a distinctive change in nuclear shape (from lobed to round) associated with the onset of meiosis. During leptotene the oocyte chromatin becomes visibly organized into electron-dense axial elements (representing the single unpaired chromosomes) which are surrounded by a fibrillar network. Towards the end of leptotene, these axial elements become attached to the inner surface of the nuclear membrane in a localized region adjacent to the juxtanuclear mitochondrial aggregate. Zygotene is marked by the initiation of axial element pairing over short regions, resulting in the typical synaptonemal complex configuration of paired homologous chromosomes. The polarization of these tripartite ribbons within the nucleus becomes more pronounced in late zygotene, producing the familiar Bouquet arrangement. The synaptonemal complexes are more extensive as synapsis reaches a climax during pachytene, whereas the polarization is to some extent lost. The fine structure of synaptonemal complexes in the Xenopus oocyte is essentially the same as that described in numerous other plant and animal meiocytes. It is not until the beginning of the extended diplotene phase that any appreciable increase in cell diameter takes place. During early diplotene (oocyte diameter approximately 50 /*m), the compact Balbiani body characteristic of the pre-vitellogenic anuran oocyte is formed by condensation of the juxtanuclear mitochondrial aggregate. Electron-dense, granular material appears to pass between nucleus and cytoplasm via nuclear pores in all stages of Xenopus germ cell differentiation studied. There is a distinct similarity in electron density and granular content between this 'nuage material' associated with the nuclear pores and the cytoplasmic aggregates of granular material in association with mitochondria or in the form of nucleolus-like bodies.

The glandular aspects of the tabby syndrome in the mouse.

Abstract: The tabby syndrome includes abnormalities of the coat and of the sinus hairs, of the teeth, of a multitude of exocrine glands and of some surface structures like tail rings, plicae digitales and the papilla vallata of the tongue. All structures known to be affected by the tabby gene arise by the downgrowth of a surface epithelium into the membrana propria. The involvement of some structures but not of others cannot be accounted for by common origin from one germ layer (as both ectodermal and endodermal derivatives are involved), nor by common function of the structures affected, nor by a time concept such that structures formed before a critical period escape unharmed. The fault may lie either with the surface epithelium, or with the stimuli from the membrana propria which elicit local downgrowth, or with the interaction between the two. It seems least likely that the fault lies with the membrana propria, but a decision cannot be made on the information so far available. New facts concerning the manifestation of tabby in Ta /+ ♀♀ either lend no support to the ‘inactive X -chromosome’ hypothesis or are at variance with it. The tabby syndrome is also found in the autosomal genes for crinkled and (presumably) downless.

Cell proliferation patterns during cytodifferentiation in embryonic chick tissues: liver, heart and erythrocytes

Abstract: Tritiated thymidine was administered to four groups of embryos at various stages of development. The first group was killed 45 min after isotope administration. The second group received the isotope three times over a 12 h period, the third group four times over a 24 h period, and the last group eight times over a 48 h period. Sagittal histological sections of the embryos were prepared for radioautography. In the radioautographs the percentage labeled nuclei were scored as an index of cell proliferation. The following observations were made. From stage 10 to 23 (45 min series) there is a progressive increase in the proliferative index of the labeled erythrocytes. This was followed by a precipitous drop as embryonic age increased. At stage 23 of the .12 h series, 97% of the cells were labeled. The 24 and 48 h series of embryos also exhibit a high labeling index, 97% and 95% respectively, by stages 20–24. This indicates that at these stages most of the cells were in the proliferative pool. In correlation with the demonstrated presence of hemoglobin as early as stage 10, it was concluded that cell proliferation and cytodifferentiation in chick primary erythrocytes are not mutually exclusive. A random pattern of proliferative activity existed in the liver of the early embryos; however, at stage 29 the periphery began to show a higher labeling index than was found in the center. This indicates that liver growth was primarily appositional. From stage 29 through hatching there was a gradual decline in the labeling index. After hatching a burst of proliferative activity occurred but the appositional pattern of growth was not seen. The proliferative activity of the littoral cells remained relatively constant when compared to that of the hepatic parenchymal cells. This suggests that the control of their proliferative activity was somewhat different from that of the hepatic parenchymal cells. The relationship of cell proliferation and cytodifferentiation in the liver could not be established. There was a progressive increase in the proliferative index of the heart ventricular myoblasts from stage 12 through stage 20–23. This was followed by a gradual decline in proliferation as embryonic age increased. An appositional growth of the heart was also demonstrated. Cytodifferentiation of heart myoblasts is known to occur as early as stage 10. Almost all of the myoblasts were labeled at stages 32 and 33 of the group exposed to the isotope for 48 h. This indicates that, in the heart, cytodifferentiation and cell proliferation are not mutually exclusive processes.

In vitro analysis of the hormonal basis for the sexual dimorphism in the embryonic development of the mouse mammary gland

Abstract: Factors underlying the sexual dimorphism in the embryonic development of mouse mammary glands were analysed in vitro and the following results were obtained: 1. Mammary gland rudiments of 13-day male embryos, explanted immediately before the onset of their regression, were perfectly capable of developing into female-type glands in vitro . Even some of the glands of 14-day male embryos, where the regression process had already begun, recovered after explantation and underwent female-type morphogenesis. 2. Combined explantation of 13-day testes with mammary rudiments of female embryos of 12–14 days gestation resulted in male-type regression of the glands. 3. The addition of testosterone to the culture medium caused a similar regression of explanted (female) mammary-gland rudiments. The minimal effective concentration of the hormone was 10−9m, or 0·00029 μg/ml. 4. Cultured mammary rudiments of 15-day female embryos were no longer responsive to the presence of testis explants. They failed to undergo regression and continued their development in vitro . From these results the following conclusions were drawn: ( a ) The sexual dimorphism in the embryonic development of mouse mammary glands is caused by their suppression in males and not by their stimulation in female embryos. ( b ) The androgenic hormones in male foetuses are solely responsible for the regression of the mammary rudiments. They exert their effect directly on the gland without the need for involvement of other endocrine organs. ( c ) The genetic sex of the gland itself has no influence on its developmental capacities as: (i) glands of male embryos are able to develop in the absence of androgens, and (ii) glands of female embryos undergo typical male-type regression in vitro when exposed to the presence of foetal testes or of testosterone.

The relationship between cleavage and blastocoel formation in Xenopus laevis. II. Electron microscopic observations.

Abstract: Blastocoel formation in Xenopus laevis was investigated by light microscopy using serial sections of epoxy-embedded, staged embryos. The earliest manifestation of the blastocoel in the embryo appeared during the first cleavage as a modification in the animal pole furrow tip. This modification consisted of an expansion of a localized area of the furrow. As the blastocoel became a distinct entity, it remained stationary, while the furrow tip continued to advance inwardly. In contrast, no such furrow cavity was observed in the vegetal pole furrow during its formation. During subsequent cleavages, up to the late morula stage, furrows on opposite sides of any given blastomere had different morphologies. As further divisions occurred, the mode of furrow formation became identical regardless of location in the embryo. It is suggested that the cytokinetic pattern in early amphibian embryos is modified to allow for the formation of the blastocoel. After the blastocoel has formed, the cytokinetic pattern changes to one which is concerned solely with cell division.

Cell death in the "opaque patch" in the central mesenchyme of the developing chick limb: a cytological, cytochemical and electron microscopic analysis.

Abstract: Cell death in the ‘opaque patch’ of central mesenchyme of the developing chick forelimb was investigated by a variety of light and electron-microscope cytological and cytochemical techniques. Cell death appears first at stage 23/4 (4 days) and reaches its maximum extent at stages 24 and 25 (4½ and 5 days), at which it separates the ulnar and radial mesenchymal condensations. It then decreases in size to a small area separating the proximal parts of radius and ulna and disappears at stage 28. Cytological studies show the presence of a few isolated dead cells, of mesenchymal cells containing 1–3 ingested dead cells and of macrophages containing up to 18 dead cells in various stages of digestion. These findings are interpreted as showing that isolated dead cells are ingested by neighbouring mesenchymal cells which thus become transformed into macrophages, first ingesting and then digesting further dead cells. Histochemical studies show that isolated dead cells and recently ingested dead cells contain no more acid phosphatase activity, either discrete or diffuse, than either neighbouring living mesenchymal cells, or mesenchymal cells which have ingested 1–3 dead cells. Increased acid phosphatase activity is found within the macrophages, where activity is localized within the digestive vacuoles (‘secondary lysosomes’) containing the dead cells, and within the Golgi apparatus and Golgi vesicles (‘primary lysosomes’) of macrophage cytoplasm. Loss of staining capacity by the dead cell is correlated with high acid phosphatase activity: this is interpreted as indicating the digestion of dead cells within the macrophage by acid hydrolases. There is circumstantial evidence that viable mesenchyme cells in the ‘opaque patch’ area autophagocytose part of their own cytoplasm in secondary lysosomes (1·2–2µm). The role of the ‘opaque patch’ in relation to the pattern of limb chondrogenesis is discussed. It is suggested that cell death may play a role in separation of radius and ulna, and that autophagocytosis may indicate a change in the pathway of differentiation of the mesenchyme cells lying between radius and ulna.

Hop-sterile, a mutant gene affecting sperm tail development in the mouse

Abstract: SUMMARY Hop-sterile (hop) is a new mutation in the mouse, the effects of which include complete male sterility, a 'hopping' gait and preaxial polydactyly of all feet. Ultrastructural studies of spermiogenesis show that sperm tails are absent or highly modified. Second meiotic division is frequently abnormal or incomplete, often with four centrioles per cell. These centrioles usually fail to form fiagella and sperm tail development is arrested.

Abnormal spermiogenesis in two pink-eyed sterile mutants in the mouse.

Abstract: Abnormal spermiogenesis is described in two neutron-irradiation induced alleles at the pink-eye locus in the mouse. Testicular hypotrophy and abnormal spermateliosis are present, resulting in the formation of giant sperm with abnormal heads and multiple tails. Abnormal sperm-head shape results from abnormal proacrosome formation and multiplication of tail number from a failure in cytokinesis. The pleiotropic effects of the locus on coat colour, fertility, behaviour and growth are reviewed in conjunction with similar mutants in other rodents: it is suggested that pituitary dysfunction could explain all aspects of the ‘pink-eye’ syndrome.

Early limb development of Xenopus laevis.

Abstract: This investigation has used histological techniques and the scanning electron microscope to establish the presence of an apical ectodermal ridge in the developing limbs of Xenopus laevis The ridge appeared at stage 50, reached its maximal size at stage 51, and subsequently disappeared by stage 53 The course of the ridge was consistently related to a marginal sinus in the underlying mesenchyme The other features of limb morphogenesis, such as the formation of a paddle and the sequence of condensation of skeletal rudiments in the mesenchyme, corresponded closely to those seen in other vertebrates It remains to be seen whether the ridge we have demonstrated in Xenopus exercises a similar function to that claimed for its counterpart in the chick

The retinotectal projections after uncrossing the optic chiasma in Xenopus with one compound eye.

Abstract: The nature of the retinotectal projection from a compound (NN or TT) eye in Xenopus raises certain problems concerning the mode of formation of connexions between the eye and the tectum. Each half of the compound eye appears to spread its connexions across the entire extent of the (apparently normal) contralateral tectum. This could indicate a certain plasticity in the way in which optic fibres can connect with the tectum. Alternatively, it is conceivable that each (similar) half of the compound eye is only able to innervate its corresponding half-tectum; in which case the uninnervated half-tectum could remain undeveloped and the innervated half-tectum could overgrow to resemble a normal tectum. This mechanism would preserve the idea of a rigidly fixed cell-to-cell specificity between retina and tectum. In an attempt to distinguish between these two mechanisms (spreading or overgrown half-tectum) we have given each of a series of Xenopus embryos at stage 32 one compound eye (NN or TT). Then, shortly after metamorphosis, we uncrossed the optic chiasma and 6 months later recorded the retinotectal projections from each eye to the tecta. Thus by connecting up the normal eye to the suspect tectum, and the compound eye to the normal tectum, we used the normal side in each case to provide an indication of the degree of abnormality with which the other side was connected. The results showed that a compound eye (NN or TT), connected to a normal tectum, gave a typical reduplicated map across the entire tectum, whereas the normal eye, when connected to the tectum which was previously innervated by the compound eye, gave an approximately normal projection across the whole of that tectum. These results lead us to conclude that, in the Xenopus visual system, no strict cell-to-cell type specificity exists; rather, what is preserved throughout these experimental manoeuvres is the polarity and extent of the projection.

The control of cell adhesion in a morphogenetic system

Abstract: A new general type of morphogenetic process has been revealed by experiments on the phenomenon of non-coalescence between different strain types ( alpha and delta ) in the sponge Ephydatia fluviatilis . The question investigated was whether any process of cell adhesion was responsible for the phenomenon. Preliminary results suggested that the cells might show specific adhesion but further results indicated that a more complex system existed. Each strain produces a soluble factor that increases the adhesiveness of homologous cells but decreases that of cells of heterologous strains. The adhesion of cells, even in the presence of these factors, is non-specific but the factors specifically control adhesion and determine its quantitative value. The adsorption of the factors to the cells was tested for with inconclusive results. Heterologous factors may irreversibly alter a cell9s adhesiveness. It is shown that this system, particularly by reason of its negative effect on adhesiveness, ( a ) accounts adequately for the phenomenon of non-coalescence, ( b ) provides a model system for many forms of morphogenesis and ( c ) allows many apparently contradictory results obtained by other workers to be reconciled.

Behaviour of mouse primordial germ cells in the chick embryo

Abstract: Hind guts of 9½-day mouse embryos were transplanted into the posterior part of the coelomic cavity of 2½-day chick embryos. The hosts were sacrificed after 1-7 days and the mouse primordial germ cells (PGCs) in the graft and in the surrounding host tissues were searched for by means of the histochemical technique for alkaline phosphatase. Altogether 94 grafts were examined. During the first 3 days of intracoelomic development of the graft accumulations of mouse PGCs close to the mesonephros, the mesentery or the gonad of a chick embryo were observed in 26 out of 51 cases. In 12 grafts single PGCs crossed the boundary between the host and the graft and settled in host tissues such as the mesonephros, the mesentery or the gonad. After 3 days mouse PGCs are no longer visible in the chick tissues. However, the number of PGCs in the grafts also gradually decreases and from the 4th day onwards many of the grafts contain no PGCs. The ability of mouse PGCs to survive extragonadally, even in the embryonic hind gut, is thus limited. In some of the 4- to 7-day-old grafts PGCs occur on the periphery of the graft in the form of single aggregations. From the 6th day the only PGCs which survive are those in aggregations. The experiments indicate that the gonads, together with adjacent tissues (mesonephros, mesentery) of a chick embryo are attractive to mouse primordial germ cells and that the hypothetical attractive substance is not species specific.

Timing of the phases of the cell cycle during the period of asynchronous division up to the 49-cell stage in Lymnaea

Abstract: The duration of the phases of the cell cycle (M-G 1 –S-G 2 ) has been determined from the 8-up to the 49-cell stage in eggs of Lymnaea , using autoradiography and cytophotometry of Feulgen-stained nuclei. Division asynchrony of corresponding cells in different quadrants is primarily caused by unequal lengthening of the G 2 phases. In general it appeared that in the vegetative cells lengthening of the cell cycles is chiefly due to an extension of the G 2 phases, whereas in the cells of the animal half the duration of both the S and the G 2 phases are extended. DNA synthesis is not blocked in cells which stop dividing and start to differentiate. A conspicuous lengthening of the cell cycles is observed in the 16- and 24-cell embryo; this is accompanied with the reappearance of distinct nucleoli. Supporting evidence has been obtained for the assumption that bilateral symmetry at the animal pole of the embryo is induced by cells from the vegetative hemisphere, presumably by the macromere 3D , during the 24-cell stage.

The mechanism of axial rotation in the rat embryo: an experimental study in vitro.

Abstract: Axial rotation has been studied in 9- to 11-day rat embryos grown in culture by New9s watch-glass technique. Unlike the mouse, the rat embryo rotates towards its right side and rotation starts with the head end only. The twist then passes caudaiwards until the whole axis has reversed its dorsoventral orientation and curvature. Contractions in cervical and cardiac regions appear to initiate the rotation. Posterior parts of 9- and 10-day embryos, isolated by transections at mid-trunk or cervical levels, show much less ability to rotate than unoperated controls: the frequencies of fully turned, partially turned and unturned embryos have been compared between control and experimental groups and show significant differences. There is more marked inhibition of rotation when the operation is performed at 9 days than at 10 days, and more with cervical than with mid-trunk transections. In all, 67 % of embryos transected at the mid-trunk level and 98 % transected at the cervical level were unable to rotate the posterior parts. Extrusion of embryos from the amniotic cavity also resulted in abnormal or incomplete axial rotation. The role of the membranes in facilitating rotation is discussed briefly.

Transmission and spread of embryonic induction: I. Temporal relationships in transfilter induction of kidney tubules in vitro

Abstract: The question of whether inductive tissue interactions involve factors transmitted by long-range diffusion was investigated. In transfilter experiments with spinal cord as the inductor and metanephric mesenchyme as the responding tissue it was established that interposition of a second TA Millipore filter (pore size 0·8 µm, thickness 25 µm) prolongs the induction time by about 12 h. The prolongation of the induction time was concluded to be due to the time taken by the inductor to pass through the second filter. It was also demonstrated that metanephric mesenchyme partially lost its competence if precultivation lasted more than 12 h. This explains why 100% induction never was achieved in the double-filter experiments. In order to rule out the possibility that the filters, owing to their inhomogeneous structure and negative surface charge, seriously restricted diffusion, the diffusion of several substances through the filter was measured under conditions as closely similar as possible to those in the transfilter experiments. Although there was some restriction of diffusion, the calculated diffusion constants were of the same order of magnitude as those reported in the literature, indicating that the filter is no major obstacle. Calculations based on four different hypotheses that the inductor is transmitted by diffusion, indicate that diffusion can hardly explain the long transmission time.

Further evidence for the sorting out of cells in the differentiation of the cellular slime mold Dictyostelium discoideum.

Abstract: SUMMARY The observation of Takeuchi's that the denser cells of Dictyostelium discoideum tend to sort out towards the anterior end of the migrating slug (and the lighter cells towards the posterior end) has been confirmed using spore size as a method of identifying cells populations. A fraction of the anterior and posterior ends of a slug are isolated and allowed to fruit; their spores are then measured. The same is done for preaggregation cells which have been separated into heavy and light fractions, using Takeuchi's technique of centrifugation of the cells in a dextrin solution equal to the mean specific gravity of the cells. Invariably, in three experiments with different strains of D. discoideum, the spores derived from dense cells corresponded perfectly with spores derived from the anterior cells of the slug, and a similar correspondence was found between spores derived from light cells and posterior slug cells. Contrary to a previous view (Bonner, 1959), cell size did not always correlate with position; in one strain the anterior cells were larger, in the other two they were smaller.

Growth and differentiation of rat egg-cylinders under the kidney capsule

Abstract: Two groups of embryonic shields were transferred under the kidney capsule, on days 7 ½ and 9 of normal pregnancy. The first group was without mesoderm and the second one with well-formed mesoderm. Random-bred and inbred Fischer rats were used. Killed and fixed 15 and 30 days after the operation, the grafts looked like teratomata with well-differentiated tissues. Differentiation was the same in embryos explanted at 7½ and 9 days, and from the two strains of rat. In order to study the growth potential, transfers within and between each strain of rat were carried out. A difference in weight was found 15 days after the operation among some series. This was not observed 30 days after the transfer. Although the grafts in random-bred rats were larger than in the inbred Fischer strain, factors other than the immunological influence were invoked for the explanation of the initial difference in growth. Long-term grafts were studied only in the inbred strain. Two, 4 and 6 months after the operation all well-differentiated tissues were still present. The amount of growth was very variable within each series, but no graft was found totally resorbed. Only in the largest graft, 6 months after the operation, large masses of cells were found which seemed to be undifferentiated.

The effect of early thymectomy on histogenesis of the lymphoid organs in Xenopus laevis.

Abstract: The role of the thymus in the ontogenetic development of the lymphoid system of the clawed toad, Xenopus laevis , was investigated by removing the organ at stage 49 of Nieuwkoop & Faber (1967), a stage when small lymphocytes are present in the thymus but have not yet appeared in the peripheral lymphoid organs. Complete thymectomy, confirmed histologically, was achieved in nine larvae killed at stage 56 and in 23 larvae killed at stage 59. The spleen was smaller in thymectomized larvae than in sham-thymectomized controls in the series killed at stage 56, but the difference was no longer significant at stage 59. In both series, thymectomized larvae showed a fall in the number of extra-follicular lymphocytes and an increase in the reticulo-myeloid elements of the splenic red pulp. The pharyngeal ventral cavity bodies were moderately or severely depleted of lymphocytes. In other areas, histogenesis proceeded normally in the absence of the thymus. In the splenic white pulp, the follicles, which comprised immature cells of the lymphoblast type at the time of thymectomy, developed their normal complement of lymphocytes. All larvae grew and developed at the normal rate.

Accelerating effect of hydrocortisone on the keratinization of chick embryonic skin growing in a chemically defined medium.

Abstract: SUMMARY In an attempt to examine histologically, chemically and biochemically the effect of hydrocortisone in a minimal concentration on keratinization of 13-day chick embryonic shank skin, a simple replicate culture method ('Millipore' filter-roller-tube method) was devised to cultivate rather large pieces of the skin in a chemically defined medium, BGJb supplemented with ascorbate. Hydrocortisone added in a minimal concentration of 0001 /tg/ml produced a heavily cornified eosinophilic layer over the epidermis after 4 days' cultivation, whereas in the absence of the steroid no sign of cornification could be found during culture. Determination of total protein and analysis of amino acid composition of whole protein of the epidermis indicated that hydrocortisone accelerated epidermal cornification as compared with in ovo development. Pregnenolone and progesterone showed no effect on the in vitro keratinization of the epidermis and deoxycorticosterone gave a slight effect: thus the cornification-accelerating effect of hydrocortisone seems to be attributable to its glucocorticoid activity.

Organization of the chick blastoderm edge.

Abstract: A band of cells forming the edge of the chick blastoderm, and attached to the vitelline membrane, causes the expansion of the blastoderm in the first few days of incubation by active migration across the vitelline membrane. The structure and organization of these cells was examined by light microscopy (both on whole mounts and sections) and transmission electron microscopy. The account presented differs markedly from previous descriptions of these cells. The band of cells at the blastoderm edge is an association, between 90 and 130 µm wide, of flattened, non-dividing cells forming a multilayer; some of these cells, and no other cells of the blastoderm, are attached to the vitelline membrane. Each attached cell has a thin flattened lamella, centrifugally oriented and underlapping the next cell distally, except (1) the most distal cell, whose lamella is thick and long, though tapering, and is not overlain by other cells; and (2) the most proximal attached cell which has a short centripetally oriented lamella, as well as a centrifugal underlapping one. The cells of the edge band not attached to the vitelline membrane also have flattened lamellae attached to the cells below; these lamellae are, however, unoriented. The cells of the edge band all have plentiful cortical filaments and cytoplasmic microtubules. Specialized plaques are involved in the attachment of edge band cells to the vitelline membrane. The form of this edge structure is compared with the outgrowth edge of a chick yolk sac epiblast explant cultured on vitelline membrane. It seems likely that the way the blastoderm edge cells are organized may explain their prodigious migratory activity.

Histological features of neural induction in Xenopus laevis

Abstract: It was first established by grafting experiments that neural induction occurs in Xenopus laevis and that it is the mesoderm in the dorsal lip of the blastopore which normally exercises this function. The subsequent histological work provided the following information: At stage 10½ mesodermal invagination was already well under way, in advance of the formation of the archenteric cavity. This confirms the earlier observations of Nieuwkoop & Florschutz(1950). The first evidence of neural induction, thickening of the mid-dorsal ectoderm combined with the development of an inner tier of columnar cells, occurred at stage 11½. By stage 12 there was generalized thickening of the dorsal ectoderm and between stage 12½ and 13 the brain and spinal cord regions of the neural plate became distinguishable. The dorsal mesoderm segregated into notochord rudiment and two lateral masses at stage 13 and the latter further subdivided into paraxial mesoderm and lateral plates by stage 14. The margins of the neural plate were clearly distinguished from presumptive epidermis by stage 15 and the median neural groove was also well marked. In the next two stages the folding of the neural plate in the line of this groove proceeded rapidly. The dorsoventral enlargement of the somites and the relative shrinkage of the notochord were considered to contribute to the mechanism of neurulation. Regionalization of the brain into prosencephalon, mesencephalon and rhombencephalon was in progress at stages 18 and 19. These results indicate that induction consists of an initial activation of dorsal ectoderm (generalized thickening) followed by gradual transformation of the neural plate to form the different parts of the central nervous system (regionalization). Intercellular metachromatic material was noted in various parts of the embryo. This was most plentiful between stage 10½ and stage 13 and thereafter gradually decreased. It was the only feature which persisted long enough to represent a possible inductive agent. At all stages the archenteron was lined with a continuous layer of endoderm. This indicates that the mode of formation of the gastro-intestinal tube in Xenopus is different to that in urodeles. It further implies that the mesoderm is not present on the blastular surface prior to gastrulation but lies in deeper layers.

The origin and movement of the limb-bud epithelium and mesenchyme in the chick embryo as determined by radioautographic mapping.

Abstract: The origin of the limb-bud cells was determined by tracing the movements of [ 3 H]thymidine-labelled grafts excised from late medium-streak to 5-somite stage chick embryos and transplanted to the epiblast, streak, and endoderm-mesoderm of similarly staged recipient embryos. Although exact definition of the prelimb areas was not possible because of the small number of grafts placed at each developmental stage, the study showed in general that at the late medium-streak stage the future limb-bud epithelium is in the epiblast (dorsal) layer near the lateral margin of the area pellucida. It moves medially toward the embryonic axis, just lateral to the premesoderm cells which will be invaginated at the primitive streak. With regression of the streak, the limb-bud epithelium moves relatively anteriorly into a position dorsal to the limb-bud mesoderm, beginning at least as early as the early head-fold stage. At the definitive-streak stage, the future limb-bud mesoderm is in the epiblast layer about halfway from the streak to the lateral margin of the area pellucida, at a level about halfway between the anterior and posterior ends of the streak. From this position the prelimb mesoderm migrates medially to the streak, and is invaginated into the mesoderm layer at a position about halfway between the anterior and posterior ends of the streak; after the head-process stage, it migrates anteriorly and laterally into the somatic layer of the lateral plate, ventral to the limb-bud epithelium. Mesoderm which will form the anterior limb-bud migrates anterior to mesoderm which will form the posterior limb-bud; mesoderm which will form the ventral portion of each limb-bud migrates posterolateral to mesoderm which will form the dorsal portion of each limb-bud.

Timing of the phases of the cell cycle with tritiated thymidine and Feulgen cytophotometry during the period of synchronous division in Lymnaea.

Abstract: The duration of the phases of the cell cycle during the 1-, the 2- and the 4-cell stage of the Lymnaea egg were determined with [3H]thymidine and with Feulgen cytophotometry. The M, S and G2 phases occupy 48, 27 and 25% of the first three cell cycles. A G1 phase cannot be observed. Only from the 4-cell stage was [3H]thymidine readily incorporated into DNA. The theory that an increase in respiration during the S phase of the 4-cell stage is connected with the energy requirements of DNA synthesis is discussed.

Organ-culture studies of achondroplastic rabbit cartilage: evidence for a metabolic defect in glucose utilization.

Abstract: Organ-culture studies were made using cartilage from achondroplastic (dwarf) rabbits ( ac / ac ) and their phenotypically normal litter-mates. A significantly higher incorporation of 14C from glucose and galactose was measured in the dwarf; incorporation of 35S from sulfate and 3H from thymidine was equal in the two types of explant. Also, 14CO2 and [14C]lactate production from glucose by the dwarf cartilage was increased. No difference in the ratio of 14CO2 evolution from [1-14C]- and [6-14C]glucose was found between the two cartilage types. Explants of dwarf cartilage utilized more glucose from the medium than did the controls. Radioautographs of tissue sections from the explants showed an increased number of grains from [14C]glucose overlying the dwarf cartilage, and this difference was particularly great over the central portions of cartilage. The proportion of 14C grains from glucose was greater over the dwarf nuclei, less over the dwarf matrix and equal over the cytoplasm of the two tissues. Grain counts of sulfate and of thymidine did not differ in the two types of cartilage.

Histochemical observations on early implantation in the mouse.

Abstract: Acid phosphatase, polysaccharide complexes and lipids were studied in the uterus and embryo of the mouse from 90 to 120 h post coitum ( p.c. ). The pattern of acid phosphatase-rich vesicles (ERV) in the uterine epithelium and its manner of degeneration are discussed. At 90 h p.c. there are large ERV in the inner cell mass indicating degenerating cells; these are not detectable at 96 h p.c. A border of lysosomes is prominent in regions of the peripheral trophoblastic cytoplasm adjoining the epithelium at 96 h p.c. and this may be involved in membrane changes which facilitate closer contact and interactions between the embryonic and maternal cells. Discrete aggregates of large ERV are present in the epithelium and trophoblast at sites scattered around the abembryonic region, from 96 to 104 h p.c. From 94 to 100 h p.c. macrophage-like cells can be found in the uterine epithelium. In the later stages of implantation, 110–120 h p.c. , the pattern of acid phosphatase-rich vesicles becomes progressively more complicated with the whole of the epithelium around the blastocyst gradually becoming degenerate.

Tentacular and oral-disc regeneration in the sea anemone, Aiptasia diaphana: III. Autoradiographic analysis of patterns of tritiated thymidine uptake

Abstract: Autoradiography with [ 3 H]thymidine and electron microscopy were used to determine ( a ) the patterns of cellular division exhibited by intact anemones, ( b ) if measurable increases in cellular proliferation accompany oral-disc regeneration, ( c ) whether interstitial cells are present in Aiptasia , and ( d ) if these cells could be responsible for the latter proliferative patterns. An oral-aboral gradient in cellular proliferation was exhibited by the epidermis of uncut anemones, with the highest levels in the tentacles. Wound healing did not require cell proliferation and did not immediately stimulatecellular division which was associated with subsequent morphogenetic events. Indices of presumptive oral-disc [ 3 H]thymidine uptake into nuclei increased tenfold with the outgrowth of the new tentacles. This increase occurred in the epidermis, while only small amounts of gastrodermal proliferation were detected. It is hypothesized that the epidermis contributes new cells to the expanding gastrodermis during tentacle budding. Most of the [ 3 H]thymidine-labeled nuclei were localized in the basal portions of the epidermis of intact anemones and 1- to 2-day-old regenerates; very few gastrodermal nuclei accumulated the label. Nests of interstitial cells and transforming interstitial cells were localized in the exact epidermal regions where nuclear labeling took place, suggesting that the proliferative patterns of intact and regenerating Aiptasia are a function of their interstitial cell distribution.

Transient inhibition of DNA synthesis by methotrexate, in the rat embryo and foetus

Abstract: Massive doses of methotrexate, a folic acid inhibitor, followed by folinic acid, the specific antagonist, have been used to produce a period in which the embryo and foetus are exposed to tetrahydrofolate deficiency with subsequent inhibition of DNA synthesis. The effects of this inhibition vary at different stages of gestation, and in late foetal life provide a useful method of inducing a delay in the appearance of vertebral body ossification centres. This defect is rapidly repaired, although there may be permanent sequelae. It is hoped that this technique will be useful in the study of cellular events in ‘catch-up’ growth.

Etude par cytochimie ultrastructurale des corpuscules perinucleaires presents dans les jeunes oocytes de Ilyanassa obsoleta Say (Mollusca gasteropode)

Abstract: An ultrastructural cytochemical study of the perinuclear corpuscles found in young oocytes of Ilyanassa obsoleta Say (molluscan gastropod) Ultrastructural study of the perinuclear region of young oocytes of Ilyanassa obsoleta shows the existence of numerous corpuscles which we have called ‘perinuclear corpuscles’. These are composed of filaments, of variable thickness (15–45 nm) and frequently show contacts with the nuclear envelope. With the development of the oocyte, they scatter in the cytoplasm and then disappear. Treatment of ultrathin sections by pronase or by pepsin provokes the disappearance of the main part of the perinuclear corpuscle. The residual structures of these corpuscles are not digested either by RNase or by DNase. However, if a digestion is carried out with DNase and pronase together, it increases the contrast of the residual structures. On the other hand, the contrast of the perinuclear corpuscles is not altered by specific techniques for polysaccharides. The constitution and the role of the perinuclear corpuscles is discussed.

Spotting genes and internal pigmentation patterns in the mouse.

Abstract: Hereditary white spotting in the mouse may be caused by genes at over a dozen loci. It is thought that some genes achieve their effects by acting through the melanoblasts, and others by acting through the host tissue. The genes mi , Mi wh , W v and s are believed to belong to the former category. But it is not known how the abnormality of the melanoblasts is transformed into the spotting patterns observed. According to one view, the capacity of the melanoblasts to respond to some melanogenesis-promoting factor in the host tissues is impaired, and as all melanoblasts are affected about equally, the pattern depends on normal variations in the distribution of this factor in the host tissue. According to another, a proportion of the melanoblasts are ‘preprogrammed’ to die before differentiation. Both views are largely based on studies on the coat. It was thought that an investigation of the spotting patterns in the choroid, the Harderian gland and the inner ear might throw fresh light on the problem. This was carried out in the genotypes +/ mi , Mi wh /+, W v /+, W v / W v and s / s . The results provide strong support for the view that all melanoblasts are affected and the host tissue plays an important role in determining the pattern of spotting. However, there appear to be some indications that all melanoblasts may not be affected to the same degree.