bluuebunny/crossref_metadata_2025 huggingface dataset:
Maps of strength of positional signalling activity in the developing chick wing bud
Authors: Honig Lawrence S. , Summerbell Dennis
Published: April 2021

Tissue from the posterior margin of the developing limb bud, when grafted to the anterior margin, evokes the formation of a mirror-image limb duplication from the host tissue. We present maps of the spatial and temporal distribution of this signalling activity in the chick wing bud based on a bioassay that provides a quantitative measure of the completeness of the additional structures (the strength of activity index). Activity is first detected prior to the initial appearance of the limb primordium as early as Hamburger & Hamilton stage 14. It reaches a maximum during early outgrowth of the bud at stages 19 to 25. It then declines as the limb starts to differentiate into its final morphological pattern. The design of the experiment provides serendipitous data showing that two operators can consistently perform grafts with high reproducibility between them while variability between embryos is somewhat higher. The maps of activity are of particular practical value in precisely defining for the experimental embryologist and molecular biologist those positions and stages at which peak signalling activity resides.
The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium
Authors: Doetschman Thomas C. , Eistetter Harald , Katz Margot , Schmidt Werner , Kemler Rolf
Published: April 2021

The in vitro developmental potential of mouse blastocyst-derived embryonic stem cell lines has been investigated. From 3 to 8 days of suspension culture the cells form complex embryoid bodies with endoderm, basal lamina, mesoderm and ectoderm. Many are morphologically similar to embryos of the 6- to 8-day egg-cylinder stage. From 8 to 10 days of culture about half of the embryoid bodies expand into large cystic structures containing alphafoetoprotein and transferrin, thus being analagous to the visceral yolk sac of the postimplantation embryo. Approximately one third of the cystic embryoid bodies develop myocardium and when cultured in the presence of human cord serum, 30% develop blood islands, thereby exhibiting a high level of organized development at a very high frequency. Furthermore, most embryonic stem cell lines observed exhibit similar characteristics. The in vitro developmental potential of embryonic stem cell lines and the consistency with which the cells express this potential are presented as aspects which open up new approaches to the investigation of embryogenesis.
Abnormal skin development in pupoid foetus (pf/pf) mutant mice
Authors: Fisher Chris , Kollar Edward J.
Published: April 2021

At 13 days of development the epidermis of mice homozygous for the pupoid foetus (pf/pf) mutation varies in thickness between one and ten cell layers. By 16 days of development cells from the dermis have invaded the epidermis and may be found throughout the epidermis and on its surface. Among these cells are nerve fibres and Schwann cells as well as other unidentified cells. Antibodies directed against fibronectin bind to these abnormal groups of cells in the mutant epidermis and on its surface. A basal lamina, as determined by ultrastructure and by the immuno-fluorescent localization of laminin, was always found at the interface of the mutant epidermis and the invading cell population. By 19 days of development the mutant epidermis is thickened and is permeated by a network of cells including nerve fibres, Schwann cells, blood vessels, and collagen and fibronectin-secreting cells. A basal lamina always separates these groups of invading cells from the epidermal cell population.
Dynamics of tubulin structures in Xenopus laevis oogenesis
Authors: Paleček Jiří , Habrová Věra , Nedvídek Josef , Romanovský Alexej
Published: April 2021

The distribution of tubulin and/or tubulin-containing structures was examined in separate classes of Xenopus laevis oocytes and in germinal vesicles isolated from them. Although a monoclonal antibody has been used, the technique applied on paraffin sections does not allow clear-cut definition of the state of tubulin present (monomeric, dimeric or polymerized form); however, the probable existence of assembled microtubules is indicated by supplementary techniques, i.e. histology and immunoperoxidase staining. Immunofluorescence reveals maximum tubulin concentration in the Balbiani body and in a ring-shaped formation around the nucleus in young oocytes. The Balbiani body disintegrates in the course of vitellogenesis, granules formed from its periphery migrate into the cytoplasm and gradually fill the entire cytoplasm as radial cords. In the ring-shaped formation around the nucleus strongly fluorescent cords and fibres are formed, particularly on the future vegetal-half-facing part of the nucleus. Reorganization of tubulin may be related to the establishment of a structure directing two-way shifts (1) of cytoplasmic organelles from the Balbiani body to the cytoplasm, and (2) of yolk proteins containing endosomes derived from the endocytically active oolemma to the yolk platelets. A distinct fluorescent fibrillar network is found inside the isolated germinal vesicles,’ near the nucleus membrane. Peripheral nucleoli, often present in nuclear membrane protuberances, seem to be surrounded by this material, which is oriented along the surface, and as a basket towards the inside of the nucleus. It is assumed that the structures may participate in the transport of nucleoli from the nucleus to the cytoplasm via the nuclear envelope.
The ontogenesis of cranial neuromeres in the rat embryo : II. A transmission electron microscope study
Authors: Tuckett Fiona , Morriss-Kay Gillian M.
Published: April 2021

The morphogenesis of rhombomeres (neuromeres) caudal to the preotic sulcus during neurulation in rat embryos is described. A model is proposed to explain the development of the characteristic neuromeric sulci and interneuromeric gyri based on the cytoskeletal elements and the kinetic behaviour of the neural epithelium.

Evidence obtained from a study of control, cytochalasin D-treated and colchicine-treated embryos, at the electron microscopic level, supports the proposed model. The longitudinally expanding cranial neural epithelium bulges between microtubule blocks present within the interneuromeric gyri, causing a bulge to develop along the line of least resistance, away from the microfilament-rich luminal border of the neuromeric sulcus region.
Autoradiographic analysis of RNA synthesis in the oocyte—nurse cell complex of the polychaete Ophryotrocha labronica
Authors: Emanuelsson Hadar
Published: April 2021

Females of the polychaete Ophryotrocha labronica have been pulse labelled with [5-3H] uridine, and the incorporation of label into the RNA of the oocyte-nurse cell complex was followed by light and electron microscope autoradiography. Up to its regression the polyploid nurse cell displays an intense synthesis of rRNA and mRNA, which sustains an extensive production of electron-dense protein granules in it. Concomitantly rRNA and mRNA are synthesized also in the oocyte. Short-term treatment (7 h) of polychaete females with α-amanitin provokes serious disturbances of oogenesis and subsequent embryonic development, irrespective of when it is applied during oogenesis. In contrast actinomycin gives such effects only when it is applied at the onset of oogenesis.

A previous investigation has demonstrated that nurse cell granules are gradually exported through an intercellular canal to the oocyte, where a fraction of them is incorporated into the typical yolk granules. The present labelling experiments indicate that nurse cell RNA is associated with the exported yolk precursor material. From inhibition experiments with α-amanitin and from in situ hybridizations with a poly(U)probe it appears that mRNA is particularly involved.

At the final collapse of the nurse cell practically all its contents are transferred to the oocyte. From that time the ooplasm is found to contain nuage-like RNA aggregates, which in contrast to other [5-3H]uridine-labelled ooplasmic structures (yolk granules, and minor granules and aggregates) have a non-uniform distribution. The possible origin and function of these aggregates is discussed. The investigation indicates that the nurse cell has a significant export of RNA essentially similar to that from insect nurse cells.
Accumulation of pigment granules around nuclei in early embryos of Anura (Amphibia)
Authors: Kobayakawa Yoshitaka
Published: April 2021

A dark spot was found to appear in each blastomere of the vegetal surface of blastulae of Rana rugosa, Hyla arborea. The spot divided into two before division of the blastomere, so that one new spot was allotted to each daughter cell. These dark spots were formed at early blastula stage, and persisted until the end of yolk plug stage.

Cytological observations showed that each dark spot corresponds to a mass of accumulated pigment granules around the nucleus of a blastomere. The accumulation increases with development during the cleavage period more rapidly in blastomeres of the vegetal hemisphere than in those of the animal hemisphere.

This accumulation of pigment granules around nuclei during development indicates that the granules are transported toward the nuclei during the cleavage period, suggesting some sort of directional flow of cytoplasm in blastomeres of early amphibian embryos.
Absence of a sex vesicle in meiotic foetal germ cells is consistent with an XY sex chromosome constitution
Authors: Hogg Heather , Mclaren Anne
Published: April 2021

Sex vesicles were not seen in meiotic germ cells isolated from male and female foetal adrenals, although they were readily identified in adult male meiotic germ cells prepared by the same airdrying method. It is suggested that the failure of the XY germ cells from the male adrenals to develop a sex vesicle is due to their embarking on oogenesis rather than spermatogenesis, and that the absence of a sex vesicle does not necessarily indicate lack of a Y chromosome.
Dynamics of the control of body pattern in the development of Xenopus laevis: I. Timing and pattern in the development of dorsoanterior and posterior blastomere pairs, isolated at the 4-cell stage
Authors: Cooke Jonathan , Webber John A.
Published: April 2021

Xenopus embryos have been selected in which the second cleavage is occuring in a frontal plane, i.e one tending to lie at right angles to the prospective plane of bilateral symmetry for the body pattern. Some of these have been used to deduce a map of the disposition of materials for the normal mesodermal pattern (the normal ‘fate map’) by injecting blastomeres to found fluorescently marked clones from 4-to 32-cell stages. Other such 4-cell embryos have been separated into two isolates across this second cleavage; in fate-map terms, prospective dorsoanterior and posterior isolates. These have been allowed to develop to control axial larval stages, with examination of the time schedule of their gastrulation movements in relation to cofertilized whole controls. The patterns of mesoderm produced have been examined and interpreted in the light of quantitative knowledge about the normal pattern, and our current understanding of the map.

A meaningful fate map exists for the egg material even at this early, essentially acellular stage, and it differs appreciably from what might have been expected in view of that traditionally shown for early gastrula stages. The patterns developed in the isolates show that at least in many eggs, widespread information that positively specifies material as to its body position is available from at most 1 h after the events that give rise to bilateral symmetry upon fertilization. This information usually leads to a mosaic development of the appropriate mesodermal part-pattern in dorsoanterior isolates, and frequently allows development that approximates to this in the reciprocal posterior part. Regulation, i.e. the replacement of removed information to specify a development more complete than the normal contribution in isolates, is not observed. The results suggest a revision of former claims for regulative ability in at least this amphibian embryo. They also imply that systems for ascribing position value (positional information) to early embryonic tissue can be diverse in dynamics, even among embryos whose body plans are obviously homologous as are those of vertebrates.
Expression of the Tcp-1 locus of the mouse during early embryogenesis
Authors: Sánchez Edwin R. , Erickson Robert P.
Published: April 2021

The synthesis of proteins by preimplantation mouse embryos was studied by two-dimensional (2D) gel electrophoresis of proteins labelled with [35S]methionine. The product of the Tcp-1 locus of the mouse, p63/6.9, is synthesized by morula and blastocyst stage embryos, but is not detectable in 2-cell embryos. These results indicate that maternal expression (oocyte-specific) of this gene is not occurring and that this gene may be developmentally regulated. Blastocysts which are allowed to implant in vitro have rates of protein synthesis that permit fluorographs of single embryos to be performed. These embryos can be unambiguously identified for their t-complex genotype. Quantitation of the amounts of the Tcp-la and Tcp-lb allelic products in morula embryos suggested delayed expression of the paternal allele.
Amino acid transport in mouse blastocyst compartments
Authors: Miller J. G. O. , Schultz G. A.
Published: April 2021

Properties of uptake and exchange of L-methionine associated with the compartments of the blastocyst were examined in the mouse embryo. The inner cell mass (ICM) was derived from the 102 h blastocyst by immunosurgical procedures and studied in isolation. The ICM possessed strong exchange and efflux transport character. The Vmax of transport was about one-fifth that of the intact blastocyst, but the Km (660 μM) was about 30 times as high as that of the intact blastocyst. Consequently, at a concentration of about 100 μM-methionine, the velocity of uptake into the ICM is about one-thirtieth of that into the intact blastocyst. Uptake of amino acid into the intact blastocyst was resolved into cellular and cavity components by mechanically collapsing the blastocyst following uptake of radiolabelled methionine. By this method, it was found that about 70 % of the label accumulated by the blastocyst was in the cavity.
Developmental potencies of area opaca and marginal zone areas of early chick blastoderms
Authors: Khaner Oded , Mitrani Eduardo , Eyal-Giladi Hefzibah
Published: April 2021

The marginal zone, in pregastrulating chick blastoderms, has been defined as the intermediate ring between the epiblast proper and the most external region, the area opaca (Spratt & Haas, 1960). Azar & Eyal-Giladi (1979) have shown that the marginal zone of a stage XIII blastoderm has the capacity of regenerating an inductive layer which when in contact with a competent stage XIII epiblast can cause the formation of axial structures.

The present work demonstrates that at stage×(Eyal-Giladi & Kochav, 1976) the marginal zone can both induce and respond to its own inductive stimulus by forming an embryonic axis. By stage XIII the marginal zone seems to have lost its competence to respond to an inductive stimulus and cannot form an embryonic axis. It is further shown that at stages X-XIII the area opaca is neither competent to develop any sort of axial structures nor has the capacity to generate an inductive layer.
Epidermal development in Xenopus laevis: the definition of a monoclonal antibody to an epidermal marker
Authors: Jones Elizabeth A.
Published: April 2021

A monoclonal antibody 2F7.C7 which identifies an epidermal antigen common to both anuían and urodele amphibia is described. This antigen has a high molecular weight and is expressed at stage first inside and then on the surface of epidermal cells. It is not expressed on presumptive nervous tissue. It is expressed on all cells of the larval epidermis including ciliated and non-ciliated cells and sucker. The relationship of the epidermal antigen to ‘epimucin’ is investigated by blocking experiments using Peanut agglutinin. These studies suggest that either the molecule recognized by 2F7.C7 is not epimucin, or that its epitope is far away from the lectin-binding site. Studies using this antibody in grafting and sandwich experiments are discussed and an overall scheme of epidermal development in Xenopus is presented.
Distribution of nuclear proteins during maturation of the Xenopus oocyte
Authors: Hausen Peter , Wang Ya Hui , Dreyer Christine , Stick Reimer , Müller Ursula , Riebesell Metta
Published: April 2021

The internal structure of the Xenopus oocyte is reorganized during the hormone-induced egg maturation. A cytological survey of the intracellular movements and changes is described. The behaviour of the nuclear lamina protein and of three nucleoplasmic proteins during these processes was studied by immunocytology. The proteins are finally deposited in the egg in different patterns brought about by their differential behaviour during the process of maturation.
The role of gap junctions in amphibian development
Authors: Warner Anne E.
Published: April 2021

The possibility that communication through gap junctions may be important during embryonic development has often been raised since gap junctions were first described between early embryonic cells. It is now known that this direct cell-to-cell communication pathway disappears between groups of embryonic cells with different developmental fates as the embryo progresses through development, suggesting that transfer through the gap junctional pathway may play some part in controlling events during development. Supportive evidence for a role for gap junctions comes from experiments demonstrating that the properties of gap junctions differ at the border separating each segment in insect epidermis. Recently it has been shown that the ability to exchange small dyes between cells in the amphibian embryo depends on the position of each cell with respect to the grey crescent. When communication through gap junctions is prevented, by injecting antibodies to gap junctions protein, pattern formation is severely disturbed in the non-communicating region. The paper describes experiments on the pattern of junctional communication at early stages of development of the amphibian embryo and illustrates how anti-gap junction antibodies are being used to determine when and where communication through gap junctions may play an important role during development.
The system specifying body position in the early development ofXenopus, and its response to early perturbations
Authors: Cooke Jonathan
Published: April 2021

Evidence is presented that the system setting up preliminary specifications for contributions to the axial body plan, across vegetal regions of the Xenopus embryo, acts in a widespread way at early stages. Mechanisms that regulate the spatial profile of this primary positional variable, and thus ensure the constancy and harmony of the body plans normally achieved, have lost this integrative ability by the 4-cell stage one hour after the plasm shifts that precede first cleavage and symmetrize the egg. Abnormal, partial or distorted profiles of the positional system across whole eggs or isolates, recorded by these times, are retained to give correspondingly partial or imbalanced mes/endodermal pattern at tailbud larval stages. There is evidence that subsequent ‘back-up’ positional interactions, which can heal gross positional discontinuities in isolated presumptive lateral half-eggs and so restore bilateral symmetry, also do this at the price of loss of complete pattern specification. This is probably because of an asymmetrical principle whereby relatively activated (dorsoanterior specified) material can raise the level of originally posterior material on contact, whereas the reverse interaction cannot occur. The observations are discussed in relation to apparently different behaviour in certain other amphibian embryos, and to our knowledge of other positional interactions, normal and also experimentally provoked, such as those that set up the germ layers.
The Importance of Mesenchymal Factors in the Differentiation of Chick Epidermis
Authors: McLoughlin C. B.
Published: April 2021

Experiments were undertaken to investigate the degree to which the underlying mesenchyme influences the differentiation of an epithelium. Isolated epidermis of the 5-day chick embryo was explanted in vitro in contact with three different types of mesenchyme from chick embryos of the same age. These mesenchymes were derived from the gizzard, proventriculus, and heart. In a control series epidermis was re-implanted on limb mesenchyme. In each combination the epidermis behaved in a different and characteristic way. The cellular processes by which an isolated epithelium becomes established on mesenchyme in vitro are described. In the control series, epidermis re-implanted on limb mesenchyme keratinized normally, but in contact with cartilage it degenerated. On gizzard mesenchyme epidermis is prevented from keratinizing and is induced to secrete mucus and sometimes to become ciliated. On proventriculus mesenchyme epidermis is initially prevented from keratinizing and secretes mucus, but after about 7 days it reverts to its normal differentiation and keratinizes. Expiants of heart mesenchyme become subdivided into two regions: a central myoblastic area surrounded by a zone of fibroblastic outgrowth. On the central region of myoblasts the epidermis is prevented from keratinizing and spreads to a single-layered squamous epithelium. On heart fibroblasts, however, the epidermis keratinizes more densely than in the controls. The f our types of mesenchyme differ in the nature of the intercellular material that they contain, and in the amount of epithelial spreading they permit. The various effects of the mesenchyme are probably exerted specifically by the fibroblasts, which arrange themselves in contact with the epithelium in most types of culture. These epithelio-mesenchymal interactions are discussed in relation to the initiation and maintenance of embryonic and adult differentiation. It is suggested that the intercellular material of the mesenchyme may participate in the mechanisms underlying these reactions.
Further Investigations on the Cytotoxic and Morphogenetic Effects of Some Nitrogen Mustard Derivatives
Authors: Jurand A.
Published: April 2021

Previous experiments with TEM (triethanomelamine) on chick and mouse embryos (Jurand, 1958, 1959) have shown that some embryonic regions are more sensitive than others to its cytotoxic activity. TEM appeared to be specifically active against mesodermal structures, particularly against the somitic mesoderm, being relatively less active against other tissues. In amphibian embryos, however, the neural tube cells were the most sensitive (Waddington, 1958).
A Study of Nuclei and Intercellular Ground Substance during thein situDifferentiation of Somites inTaricha torosa
Authors: Finnegan Cyril V.
Published: April 2021

The differentiation of T. torosa somite mesoderm between stage 25 and 15 mm. has been examined with regard to variation in nuclear morphology, mitosis, and the development of intercellular matrix. Along with conventional techniques a fluorescent Schiff reagent has been used to obtain a fluorescent Feulgen for observation of DNA pattern, and a fluorescent PAS for observation of the appearance of intercellular mucopolysaccharide. The development of three kinds of nuclear morphology (DNA pattern) and of the PAS-positive intercellular material of the differentiating somite is described. The study indicates that nuclear changes and appearance of ground substance spread laterally from the always more differentiated medial portion of the somite and that mitotic activity is frequently observed in those more superficial areas containing cells with nuclei of the first-stage DNA pattern and is not (or is rarely) observed in those more medial areas containing cells With Other DNA patterns and in which the PAS-positive intercellular material is present. It is concluded that in the differentiating somite through stage 40 there exists a superficially located population of mitotically active, presumably undifferentiated, mesoderm cells.
Observations on cell lineage of internal organs of Drosophila
Authors: Lawrence Peter A. , Johnston Paul
Published: April 2021

Adult Drosophila mosaics can be used to study cell lineage and to map relative positions of primordia at the blastoderm stage. This information can define which germ layer an organ comes from and can help build models of genetic regulation of development. Here we use the sdh cell marker to map internal organs in mosaics made by nuclear transplantation. We confirm that oenocytes arise from the same progenitors as the adult epidermis, but that muscles and fat body have a separate (mesodermal) origin and that the precursors of epidermis and central neurones are closely intermingled in the ventral, but not dorsal, epidermis. We find that the malpighian tubules are more closely related to the hindgut than the midgut and are therefore ectodermal in origin. We find that each intersegmental muscle in the thorax arises from one specific parasegment in the embryo, but that very small numbers of myoblasts wander and contribute to muscles of inappropriate segments. We present evidence indicating that the visceral muscles of the midgut have a widely dispersed origin (over much of the embryo) while the somatic mesoderm of the female gonad comes from a small number of abdominal segments. The visceral mesoderm of the hindgut develops from a localized posterior region of the embryo.
The role of cell adhesion in the synchronization and orientation of polarization in 8-cell mouse blastomeres
Authors: Johnson Martin H. , Maro Bernard , Takeichi Masatoshi
Published: April 2021

A detailed investigation into the activity of the homotypic, Ca2+-dependent cell-cell adhesion system (CDS) m the early mouse embryo has revealed its involvement in (i) the synchronizing of the time of polarization of 8-cell blastomeres, and (ii) the orienting of the axis of polarization. Since polarization marks an important and early event in the process of cell diversification in the mouse embryo, it is concluded that the CDS provides an important component of the system by which the temporal and spatial elements of normal development are integrated.
The pituitary adrenocorticotropes originate from neural ridge tissue in Xenopus laevis
Authors: Eagleson Gerald W. , Jenks Bruce G. , van Overbeeke A. P.
Published: April 2021

A series of grafting experiments was conducted to determine pituitary origins prior to brain tube closure in Xenopus laevis. Extirpation experiments indicated that the ventral neural ridge (VNR) tissue of stage-18+ embryos was essential for pituitary development. Bolton-Hunter reagent was used to label stage-18+ VNR tissue with 125I, and this tissue was then returned to the donor and its subsequent ontogenesis followed. Labelled tissue was ultimately found in the ventral hypothalamus, the ventral retina, and the anterior pituitary. Using immunocytochemical techniques with antisera to adrenocorticotropin (ACTH), it was found that some of the VNR-derived cells were corticotropes. A region of the nucleus infundibularis which was radioactive labelled also gave ACTH-positive immunoreaction. This might indicate that some ACTH-containing neurones of the hypothalamus are VNR in origin. We suggest that stage-18+ VNR is the site of attachment of brain and anterior pituitary ectoderm. Part of this adherence point is eventually incorporated into the anterior pituitary and will form corticotropes. It is concluded that the ventral retina, the preoptic region of the hypothalamus, some hypothalamic ACTH-immunoreactive cells, and the most anterior portion of the adenohypophysis are all ventral neural ridge in origin.
Some histochemical and ultrastructural observations on the early foetal pig testis
Authors: van Vorstenbosch C. J. A. H. V. , van Rossum-Kok C. M. J. E. , Colenbrander B. , Wensing C. J. G.
Published: April 2021

Testes of foetal pigs between 26 to 35 days postcoitum (p.c.) were investigated histochemically and ultrastructurally.

Diaphorase and Δ5-3β-hydroxysteroid dehydrogenase activities were studied using, respectively, NADH and pregnenolone and dihydroxy androsterone as substrates.

Ultrastructurally, attention was focused on the development of mesenchymal cells and on the sustentacular cells in the primitive sex cords in an attempt to detect the origin of Leydig cells.

Histochemically there is a concentration of activity toward the interstitium with increasing age. Also the reactions increase in intensity. Ultrastructurally no evidence for Leydig cell development from Sertoli cells could be observed. Mesenchymal cells between the sex cords show a development toward Leydig cells. This is absent in mesenchymal cells in the future tunica albuginea.

Before 30 days p.c. no ‘true’ Leydig cells can be observed morphologically. The role of the rough endoplasmic reticulum/mitochondrial complex, which is present in many mesenchymal and sustentacular cells, is discussed.
Sensory nerve routes in chick wing buds deprived of motor innervation
Authors: Swanson Gavin J. , Lewis Julian
Published: April 2021

To what extent do motor and sensory nerve fibres depend on one another for guidance during the development of peripheral nerve patterns? This question has been examined by looking at the paths taken by sensory nerve fibres growing into the embryonic chick wing in the absence of motor axons. The precursors of the motoneurones were destroyed by irradiating the appropriate part of the neural tube with a focused beam of ultraviolet light, before axons had grown out. The limb nerve patterns seen 5 to 7 days later revealed that sensory fibres followed the usual paths of main nerve trunks and formed cutaneous nerve branches in an almost normal way. However, the sensory fibres did not take the paths where muscle nerve branches are normally seen. Apparently, sensory axons for the most part do not depend on motor axons for guidance, except in the case of proprioceptive fibres, which require guidance from motor axons over the final steps of their path into muscle.
Embryonic differentiation of fibre types in normal, paralysed and aneural avian superior oblique muscle
Authors: Sohal G. S. , Sickles D. W.
Published: April 2021

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 acidstable, alkali-labile; type II alkali-stable, acid-labile; and type III both acid- and alkali-stable. These types showed variable mitochondrial α-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 % to 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.
Cinemicrographic study of the cell movement in the primitive-streak-stage mouse embryo
Authors: Nakatsuji N. , Snow M. H. L. , Wylie C. C.
Published: April 2021

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 μm 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.
Histochemical and autoradiographic study of the cultured rat visceral yolk sac
Authors: Sobis H. , Goebels J. , Vandeputte M.
Published: April 2021

The proliferation and differentiation potentiality of the rat visceral yolk sac was investigated both in organ culture and after grafting in vivo. Using alkaline phosphatase as a marker for germ cells, it was shown that these cells are absent in the 12-day-old visceral yolk sac examined before and after organ culture. Therefore, the only cells that proliferate and differentiate must be of endodermal and/or mesodermal origin. By labelling the cells with [3H]thymidine both the endodermal and mesodermal cells were found to proliferate. After 10 days in organ culture or implantation in vivo differentiated tissues (e.g. squamous epidermis, endodermal cysts and giant trophoblast cells) were regularly detected. Several of these differentiated cells contained the radiolabel indicating that they derived from the initial proliferating endodermal and/or mesodermal cells.
Quantification of the transition from oocyte-coded to embryo-coded glucose phosphate isomerase in mouse embryos
Authors: West John D. , Leask Rosemary , Green J. F.
Published: April 2021

A quantitative electrophoretic analysis of glucose phosphate isomerase (GPI-1) allozymes produced by heterozygous Gpi-1sa/Gpi-1sb mouse embryos has enabled us to estimate separately the contributions of GPI-1 enzyme that were (1) oocyte coded, (2) encoded by the embryonic, maternally derived Gpi-1sa allele and (3) encoded by the embryonic, paternally derived Gpi-1sb allele. The oocyte-coded GPI-1 activity is stable until days and then declines and is exhausted by to days post coitum (p.c.). The maternally and paternally derived Gpi-1s alleles are probably usually activated synchronously but several possible exceptions were observed. This activation was first detected in -day embryos. Total GPI-1 activity falls to a minimum around to days, even though embryonic gene expression has already begun. The profile of oocyte-coded GPI-1 activity is consistent with the suggestion (Harper & Monk, 1983) that there is a mechanism for the removal of oocyte-coded gene products at around days p.c. The method of analysis described is applicable to other dimeric enzymes with electrophoretic variants.
Retinoic acid-binding protein in the chick limb bud: identification at developmental stages and binding affinities of various retinoids
Authors: Maden M. , Summerbell D.
Published: April 2021

The application of retinoic acid (RA) to the developing chick limb bud causes 6-digit double posterior limbs to form instead of the normal 3-digit limb. As an attempt to begin a molecular analysis of this phenomenon we have identified and characterized a soluble cytoplasmic receptor for RA, namely cytoplasmic retinoic acid-binding protein (CRABP), from the cells of the chick limb bud. It is present from stages 20–35 at similar levels and has an apparent Kd of 140–280 nM. In competition experiments with other retinoids Ro 13-7410 was found to be the most effective at competing for sites on CRABP followed by all-trans-RA, 13-cw-RA, Ro 10-1670 and retinal. Retinol, retinyl palmitate, retinyl acetate, etretinate and arotinoid showed low or no affinity for CRABP. Specificity for binding was thus demonstrated since analogues with an acid end group competed effectively, the aldehyde competed less effectively and the ester or alcohol groups did not compete. At the concentration of RA that needs to be administered to cause duplications in the pattern of the limb bud, we estimate that 4 % of the CRABP present in the limb bud has RA bound. The similarities between steroid receptors in the mediation of steroid hormone action and CRABP in the mediation of RA action is discussed. In this regard we note that while there are 104 steroid receptors per cell in other cell types we estimate that there are about 105 RA receptors per cell in the chick limb bud.
A mesoderm-inducing factor is produced by aXenopuscell line
Authors: Smith J. C.
Published: April 2021

Inductive interactions play a major role in the diversification of cell types during vertebrate development. These interactions have been extensively studied in amphibian embryos (usually Xenopus laevis) where the earliest is mesoderm induction, in which an equatorial mesodermal rudiment is induced from the animal hemisphere under the influence of a signal from the vegetal hemisphere. The molecular basis of mesoderm induction is unknown, although Tiedemann has isolated a protein from 9- to 13-day chick embryos that has the properties one would expect of a mesoderm-inducing factor. However, the relevance of this molecule to the events of early amphibian development is unclear, and it is a matter of some importance to discover a Xenopus mesoderm-inducing factor.

In this paper I show that the Xenopus XTC cell line secretes mesoderm-inducing activity into the culture medium. Isolated animal pole regions cultured in XTC-conditioned medium differentiate into muscle and notochord, while controls form ‘atypical epidermis’. Three different cell lines - XL, XL177 and KR - secrete no such activity. Preliminary characterization of the XTC mesoderm-inducing activity indicates that the active principle is heat stable, trypsin sensitive, nondialysable, and has an apparent relative molecular mass of about 16000. Work is in progress to characterize the activity further and to discover whether the mesoderm-inducing factor is also present in normal embryos.
Studies on the behavior of mitochondrial DNA: Synthesis of mitochondrial dna occurs actively in a specific region just above the quiescent center in the root meristem of Pelargonium zonale
Authors: Kuroiwa Tsuneyoshi , Fujie Makoto , Kuroiwa Haruko
Published: April 2021

The fate of mitochondrial nuclei (known as nucleoids or mt-nuclei), which contain extremely small amounts of DNA, was followed in thin sections of the root meristem of Pelargonium zonale by embedding of samples in Technovit 7100 resin and double staining with 4′-6-diamidino-2-phenylindole (DAPI) and acridine orange, in combination with light-microscopic autoradiography and microphotometry. The synthesis of cell-nuclear DNA and cell division occurs actively in the root meristem, between 150 μm and 700 μm from the tip of the root. For simplicity, cells in S phase in the cortex were selected for main analysis as the model system for examination of cell proliferation. It is estimated, on the basis of the length of the cells in longitudinal median sections, that the cells in the cortex, which are generated in the area just above the quiescent center (QC) about 150 μm from the tip, enter the elongation zone after at least five divisions. In the entire cortex, individual cells in S phase have approximately 230 mitochondria that each contain one mt-nucleus. The observation suggests that individual mitochondria divide once per mitotic cycle in the entire region of the meristem. By contrast, on the basis of incorporation of [3H]thymidine into mt-nuclei, the synthesis of mitochondrial DNA (mtDNA) occurs independently of the mitotic cycle in a restricted region just above the QC. Fluorimetry, using a videointensified microscope photon-counting system (VIM-PICS), revealed that the mtDNA content per mt-nucleus in the cells just above QC, where the synthesis of mtDNA is active, corresponds to approximately 3000 kilobase pairs (kbp) but, in the meristematic cells just below the elongation zone of the root it falls to less than 170 kbp. These findings strongly suggest that the amount of mtDNA per mitochondrion which has been synthesized in the region just above the QC is reduced stepwise as a result of continuous divisions of mitochondria in the absence of the synthesis of mtDNA. This phenomenon would explain why differentiated cells with a large vacuole in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA.
A functional comparison of mutations in integrin β cytoplasmic domains
Authors: Bodeau Amy L. , Berrier Allison L. , Mastrangelo Anthony M. , Martinez Robert , LaFlamme Susan E.
Published: April 2021

Cell adhesion is a multistep process that requires the interaction of integrins with their ligands in cell attachment, the activation of integrin-triggered signals, and cell spreading. Integrin β subunit cytoplasmic domains (β tails) participate in regulating each of these steps; however, it is not known whether the same or different regions within β tails are required. We generated a panel of amino acid substitutions within the β1 and β3 cytoplasmic domains to determine whether distinct regions within β tails regulate different steps in adhesion. We expressed these β cytoplasmic domains in the context of interleukin 2 (IL-2) receptor (tac) chimeras and tested their ability to activate tyrosine phosphorylation, to regulate β1 integrin conformation and to inhibit β1 integrin function in cell attachment and spreading. We found that many of the mutant β1 and β3 chimeras either had no effect on these parameters or dramatically inhibited the function of the β tail in most assays. However, one set of analogous Ala substitutions in the β1 and β3 tails differentially affected the ability of the tac-β1 and tac-β3 chimeras to activate tyrosine phosphorylation. The tac-β1 mutant containing Ala substitutions for the VTT motif did not signal, whereas the analogous tac-β3 mutant was able to activate tyrosine phosphorylation, albeit not to wild-type levels. We also identified a few mutations that inhibited β tail function in only a subset of assays. Ala substitutions for the Val residue in the VTT motif of the β1 tail or for the conserved Asp and Glu residues in the membrane-proximal region of the β3 tail greatly diminished the ability of tac-β1 and tac-β3 to inhibit cell spreading, but had minimal effects in other assays. Ala substitutions for the Trp and Asp residues in the conserved WDT motif in the β1 tail had dramatic effects on the ability of tac-β1 to regulate integrin conformation and function in cell spreading, but had no or intermediate effects in other assays. The identification of mutations in the β1 and β3 tails that specifically abrogated the ability of these β tails to regulate β1 integrin conformation and function in cell spreading suggests that distinct protein interactions with β tails regulate β cytoplasmic domain function in these processes.
Transient expression of phosphatidylserine at cell-cell contact areas is required for myotube formation
Authors: van den Eijnde Stefan M. , van den Hoff Maurice J. B. , Reutelingsperger Chris P. M. , van Heerde Waander L. , Henfling Mieke E. R. , Vermeij-Keers Christl , Schutte Bert , Borgers Marcel , Ramaekers Frans C. S.
Published: April 2021

Cell surface exposure of phosphatidylserine (PS) is shown to be part of normal physiology of skeletal muscle development and to mediate myotube formation. A transient exposure of PS was observed on mouse embryonic myotubes at E13, at a stage of development when primary myotubes are formed. The study of this process in cell cultures of differentiating C2C12 and H9C2 myoblasts also reveals a transient expression of PS at the cell surface. This exposure of PS locates mainly at cell-cell contact areas and takes place at a stage when the structural organization of the sarcomeric protein titin is initiated, prior to actual fusion of individual myoblast into multinucleated myotubes. Myotube formation in vitro can be inhibited by the PS binding protein annexin V, in contrast to its mutant M1234, which lacks the ability to bind to PS. Although apoptotic myoblasts also expose PS, differentiating muscle cells show neither loss of mitochondrial membrane potential nor detectable levels of active caspase-3 protein. Moreover, myotube formation and exposure of PS cannot be blocked by the caspase inhibitor zVAD(OMe)-fmk. Our findings indicate that different mechanisms regulate PS exposure during apoptosis and muscle cell differentiation, and that surface exposed PS plays a crucial role in the process of myotube formation.
Synapsin I is expressed in epithelial cells: localization to a unique trans-Golgi compartment
Authors: Bustos Rodrigo , Kolen E. Robert , Braiterman Lelita , Baines Anthony J. , Gorelick Fred S. , Hubbard Ann L.
Published: April 2021

Synapsin I is abundant in neural tissues. Its phosphorylation is thought to regulate synaptic vesicle exocytosis in the pre-synaptic terminal by mediating vesicle tethering to the cytoskeleton. Using anti-synapsin antibodies, we detected an 85 kDa protein in liver cells and identified it as synapsin I. Like brain synapsin I, non-neuronal synapsin I is phosphorylated in vitro by protein kinase A and yields identical 32P-peptide maps after limited proteolysis. We also detected synapsin I mRNA in liver by northern blot analysis. These results indicate that the expression of synapsin I is more widespread than previously thought. Immunofluorescence analysis of several non-neuronal cell lines localizes synapsin I to a vesicular compartment adjacent to trans-elements of the Golgi complex, which is also labeled with antibodies against myosin II; no sub-plasma membrane synapsin I is evident. We conclude that synapsin I is present in epithelial cells and is associated with a trans-Golgi network-derived compartment; this localization suggests that it plays a role in modulating post-TGN trafficking pathways.