bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatially restricted patterns of expression of the homeobox-containing gene Hox 2.1. during mouse embryogenesis
Authors: Holland Peter W. H. , Hogan Brigid L. M.
Published: April 2021

The mouse Hox 2.1 gene contains a homeobox sequence and is therefore a candidate for a vertebrate gene involved in the control of embryonic patterning or positional specification. To investigate this possibility, we have used in situ hybridization to determine the pattern of Hox 2.1 expression during mouse embryogenesis. At 8·5 days post coitum, Hox 2.1 is expressed at a low level in the posterior neuroectoderm and mesoderm, and in the neuroectoderm of the presumptive hindbrain. At 12·5 days p.c., Hox 2.1 is expressed in an anteroposterior restricted domain extending from the hindbrain throughout the length of the spinal cord, predominantly in the dorsal region. Between 12·5 and 13·5 days p.c. the domain becomes localized to the occipital and cervical regions. We also detect Hox 2.1 RNA in the embryonic lung, stomach, mesonephros and metanephros, as well as in myenteric plexus, dorsal root ganglia and the nodose ganglion, and in mature granulocytes. The embryonic expression of Hox 2.1 in neural tissue is compared with that of Hox 3.1, which also shows anteroposterior restricted domains of gene expression. These patterns of expression are not clearly consistent with Hox 2.1 or Hox 3.1 having roles in segmental patterning. However, the data are consistent with these genes having regulatory roles in anteroposterior positional specification in the neuroectoderm and mesoderm, and suggest that Hox 2.1 may also have functions during organogenesis.
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach
Authors: Robertson E. J. , Beddington Rosa
Published: April 2021

Thirty years ago teratocarcinomas were little more than pathological oddities, of passing interest to some oncologists but quite unknown to most biologists. Their emergence from obscurity was largely due to the work of Roy Stevens and Barry Pierce who, between them, gradually impressed on their audiences the special features of these obscure tumours. At a gross level, there was something immediately fascinating in the monstrous form of some teratocarcinomas, the jumble of hair and teeth, skin and brain tissue, intestine and skeletal elements all chaotically cohabiting in a single tumour. But it was the ‘life cycle’ of teratocarcinomas that was especially intriguing, particularly in the context of the major advances in manipulating early mammalian embryos which occurred in the 1960s.
Oncogenes and Growth Control
Authors: Kahn P. , Graf T. , Heath John
Published: April 2021

Circumstantial evidence is accumulating, bit by bit, to convict growth factors and their signalling systems as accomplices in developmental processes. For example, growth factors from cows’ brains turn out to have striking effect on the differentiation of frog embryos and certain developmental mutants in Drosophila turn out to be lesions in genes whose products look a lot like growth factors (dodecaplegic) or their receptors (sevenless). This recently discovered ability to cross taxonomic barriers (often considered the hallmark of ‘important’ molecules) inspires confidence in the general significance of these agents which, for the last twenty years, have principally been denizens of the plastic dish and the sole preserve of mammals.
The restrictive effect of early exposure to lithium upon body pattern in Xenopus development, studied by quantitative anatomy and immunofluorescence
Authors: Cooke Jonathan , Smith Emma J.
Published: April 2021

We have carried out an anatomical study of Xenopus larval and gastrula stages resulting from treatment of synchronous early blastulae for brief periods with Li+. We confirm the proposal that such treatment causes a particular transformation, and partial elimination, of the normal body pattern. Coordinated restriction of pattern, without appreciable loss of cell number, is seen in all three germ layers. The distortion has been investigated by quantitative study of mesoderms at a standard stage, in relation to the normal fate map for mesoderm, and with the help of immunofluorescence on sections for somitic muscle and for blood. In the extreme syndrome, mesoderm arises from all around the blastula as usual, but is symmetrical and corresponds to that arising near the dorsal/anterior meridian of the normally specified egg or embryo with a large posterior subset of the normal pattern values thus missing. The effect is independent of any inhibition of archenteron formation or mesoderm migration (i.e. the cell mechanics of gastrulation) incurred by the treatment. It is also quite separate from a syndrome caused by more prolonged exposure to Li+ during gastrulation. A small, but distinctive, anterior pattern region is also not expressed and, anomalously in relation to their general nature, these forms differentiate considerable blood tissue. We consider the implications of some details of the pattern restriction for our understanding of interaction in the normal development and propose that the Li+ embryo is likely to be useful as a specific ‘differential screen’, in relation to the normal, during the search for those gene products that mediate initial regionalization of the body.
The cadherins: cell-cell adhesion molecules controlling animal morphogenesis
Authors: Takeichi Masatoshi
Published: April 2021

Cadherins are a family of glycoproteins involved in the Ca2+-dependent cell-cell adhesion mechanism which is detected in most kinds of tissues. Inhibition of the cadherin activity with antibodies induces dissociation of cell layers, indicating a fundamental importance of these molecules in maintaining the multicellular structure. Cadherins are divided into subclasses, including E-, N- and P-cadherins. While all subclasses are similar in molecular weight, Ca2+- and proteasesensitivity, each subclass is characterized by a unique tissue distribution pattern and immunological specificity. Analysis of amino acid sequences deduced from cDNA encoding these molecules showed that they are integral membrane proteins of 723–748 amino acids long and share common sequences; similarity in the sequences between subclasses is in a range of 50–60 % when compared within a single animal species.

L cells, with very little endogenous cadherin activity, transfected with the cadherin cDNA acquired high cadherin-mediated aggregating activity. Their colony morphology was altered by the ectopic expression of cadherins from the dispersed type to the compact type, providing direct evidence for a key role of cadherins in cell-cell adhesion. It has been suggested that cadherins bind cells by their homophilic interactions at the extracellular domain and are associated with actin bundles at the cytoplasmic domain.

It appears that each cadherin subclass has binding specificity and this molecular family is involved in selective cell-cell adhesion. In development, the expression of each cadherin subclass is spatiotemporally regulated and associated with a variety of morphogenetic events; e.g. the termination or initiation of expression of a cadherin subclass in a given cell collective is correlated with its segregation from or connection with other cell collectives. Antibodies to cadherins were shown to perturb the morphogenesis of some embryonic organs in vitro. These observations suggest that cadherins play a crucial role in construction of tissues and the whole animal body.
Retinoic acid coordinately proximalizes regenerate pattern and blastema differential affinity in axolotl limbs
Authors: Crawford Karen , Stocum David L.
Published: April 2021

An assay that detects position-related differences in affinity of axolotl regeneration blastema cells in vivo was used to test whether retinoic acid, which proximalizes regenerate pattern, simultaneously proximalizes blastema cell affinity. The assay involved autografting or homografting late bud forelimb blastemas derived from the wrist, elbow or midupper arm levels to the dorsal surface of the blastema-stump junction of an ipsilateral, medium-bud-stage hindlimb regenerating from the midthigh level. The grafted blastemas consistently displaced to their corresponding levels on the proximodistal axis of the host regenerate, indicating the existence of level-specific differences in blastema cell affinity. Retinoic acid proximalized the pattern of donor forelimb regenerates to the level of the girdle and abolished their displacement behaviour on untreated host hindlimbs. Conversely, untreated forelimb donor blastemas displaced distally to their corresponding levels on host ankle regenerates, that had been proximalized to the level of the girdle by retinoic acid. These results indicate that positional memory in regenerating limbs is directly related to blastema cell affinity, and that very similar or identical sets of level-specific affinity properties are shared by forelimb and hindlimb cells.
Carbohydrate-binding properties of boar sperm proacrosin and assessment of its role in sperm-egg recognition and adhesion during fertilization
Authors: Jones R. , Brown C. R. , Lancaster R. T.
Published: April 2021

Recent evidence indicates that a carbohydrate recognition mechanism is involved in the early stages of sperm-egg interaction in mammals. In this communication, we describe a potential zona-ligand molecule on boar spermatozoa that has the capacity to recognize and bind to carbohydrate moieties of zona pellucida glycoproteins as well as neoglycoproteins, BSA-fucose and BSA-mannose. The molecule has broad specificity for carbohydrate binding and there is a requirement for a polysaccharide structure or for ‘clustering’ of saccharides on a protein backbone. The molecule has been identified as proacrosin, the zymogen form of the acrosomal protease acrosin. Strong similarities exist between proacrosin and ‘bindin’, the lectin-like protein that is thought to mediate sperm-egg adhesion in echinoderms. An hypothesis is proposed for spermegg interaction in mammals in which proacrosin, released during the early stages of the acrosome reaction, mediates secondary or consolidated binding of spermatozoa to the zona pellucida by virtue of its carbohydrate-binding capacity. The localized proteolytic action of active acrosin on the zona enhances this interaction in a manner analogous to the requirement for trypsinization of erythrocytes before agglutination by certain lectins. This hypothesis, which is supported by evidence from in vitro fertilization experiments, is discussed in relation to current concepts on sperm-egg recognition.
The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro
Authors: Papaioannou V. E. , Ebert K. M.
Published: April 2021

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12 –16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched.

Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.
Influence of chromosomal determinants on development of androgenetic and parthenogenetic cells
Authors: Surani M. Azim , Barton Sheila C. , Howlett Sarah K. , Norris Michael L.
Published: April 2021

We have examined the role of germline-specific chromosomal determinants of development in the mouse. Studies were carried out using aggregation chimaeras between androgenetic ↔ fertilized embryos and compared with similar parthenogenetic ↔ fertilized chimaeras. Several adult chimaeras were found with parthenogenetic cells but none were found with androgenetic cells. Analysis of chimaeras at mid-gestation showed that parthenogenetic cells were detected in the embryo and yolk sac but that androgenetic cells were found only in the trophoblast and yolk sac and not in the embryo. The contribution of parthenogenetic cells to the embryo and yolk sac was increased by aggregating 2-cell parthenogenetic and 4-cell fertilized embryos but the contribution of parthenogenetic cells in extraembryonic tissues remained negligible even after aggregation of 4-cell parthenogenetic and 2-cell fertilized embryos. Furthermore, parthenogenetic cells were primarily found in the yolk sac mesoderm and not in the yolk sac endoderm.

These results suggest that maternal chromosomes in parthenogenetic cells permit their participation in the primitive ectoderm lineage but these cells are presumably eliminated by selective pressure or autonomous cell lethality from the primitive endoderm and troph-ectoderm lineages. Conversely paternal chromosomes in androgenetic cells confer opposite properties since the embryonic cells can be detected in the trophoblast and the yolk sac but not in the embryos, presumably because they are eliminated from the primitive ectoderm lineage. The spatial distribution of cells with different parental chromosomes may occur partly because of differential expression of some genes, such as proto-oncogenes, and partly due to their ability to respond to a variety of diffusible growth factors.
Evidence that the nerve controls molecular identity of progenitor cells for limb regeneration
Authors: Fekete Donna M. , Brockes Jeremy P.
Published: April 2021

Adult urodele amphibians can regenerate their limbs after amputation by a process that requires the presence of axons at the amputation plane. Paradoxically, if the limb develops in the near absence of nerves (the ‘aneurogenic’ limb) it can subsequently regenerate in a nerve-independent fashion. The growth zone (blastema) of regenerating limbs normally contains progenitor cells whose division is nerve-dependent. A monoclonal antibody that marks these nerve-dependent cells in the normal blastema does not stain the mesenchymal cells of developing limb buds and only stains the amputated limb bud when axons have reached the plane of amputation. This report shows that the blastemal cells of the regenerating aneurogenic limb also fail to react with the antibody in situ. These data suggest that the blastemal cells arising during normal regeneration have been altered by the nerve. This regulation may occur either at the time of amputation (when the antigen is expressed) or during development (when the limb is first innervated).
Inductive effects of fibroblast growth factor and lithium ion on Xenopus blastula ectoderm
Authors: Slack J. M. W. , Isaacs H. V. , Darlington B. G.
Published: April 2021

We have studied the response of Xenopus blastula ectoderm to fibroblast growth factor and to lithium ion. The properties of acidic and basic FGF are very similar showing a 50 % induction level at 1–2 ng ml−1 and a progressive increase of muscle formation up to concentrations of 100–200 ng ml The elongation of explants also shows a dose-response relationship. The minimum contact requirement for induction of ectoderm explants is about 90 min and the stage range of ectodermal competence extends from midblastula to early gastrula, both these figures resembling those obtained in embryological experiments with vegetal tissue as the inducer. Lithium chloride concentrations which produce anteriorization of whole embryos have no effect on isolated ectoderms unless accompanied by FGF. Simultaneous treatment with FGF and Li lead to a marked enhancement of both elongation and muscle formation over that produced by FGF alone. By contrast, ventral marginal explants show increased elongation and muscle formation if treated with lithium alone suggesting that they have already received a low-dose FGF treatment within the embryo. It is concluded that endogenous FGF may be solely responsible for inducing the ventral mesoderm and that dorsalization of ventral mesoderm to the level of somitic muscle might be achieved either by a very high local concentration of FGF in the dorsal region, or by the action of a second, synergistic, agent in the dorsal region.
Purification, partial characterization and biological effects of the XTC mesoderm-inducing factor
Authors: Smith J. C. , Yaqoob M. , Symes K.
Published: April 2021

The mesoderm of Xenopus laevis is formed through an inductive interaction in which a signal from the vegetal hemisphere of the blastula acts on overlying animal pole cells. We have recently reported that the Xenopus XTC cell line secretes a mesoderm-inducing factor (MIF) which may resemble the natural signal. In this paper, we describe the purification and biological effects of XTC-MIF. XTC-MIF is a hydrophobic protein with an isoelectric point of 7·8 and an apparent relative molecular mass (Mr) of 23500. On reduction, XTC-MIF loses its biological activity and the protein dissociates into two inactive subunits with apparent Mr of about 15 000. These properties closely resemble those of transforming growth factor type β (TGF-β2), and it is interesting that TGF-)32 has recently been shown to have mesoderm-inducing activity.

The biological response to XTC-MIF is graded. After exposure to 0·2 – 1·0 ng ml−1 XTC-MIF, stage-8 animal pole explants form mesenchyme and mesothelium. At higher concentrations, up to about 5 ng ml−1, muscle is formed, occasionally with neural tissue. In response to concentrations of XTC-MIF greater than 5·10 ng ml−1, notochord and neural tissue are usually formed. The formation of notochord and neural tissue in response to XTC-MIF represents a qualitative difference between this inducing factor and the other known group of MIFs, the heparin-binding growth factors.
Cyclic AMP–phosphodiesterase induces dedifferentiation of prespore cells in Dictyostelium discoideum slugs: evidence that cyclic AMP is the morphogenetic signal for prespore differentiation
Authors: Wang Mei , Driel Roel Van , Schaap Pauline
Published: April 2021

We investigated whether cyclic AMP is an essential extracellular stimulus for the differentiation of prespore cells in slugs of D. discoideum. A local reduction of the extracellular cAMP level inside the slug was induced by implantation of cAMP-phosphodiesterase (cAMP-PDE)-coated spheres in intact slugs. This treatment caused the disappearance of prespore antigen in the vicinity of the sphere. A general reduction of extracellular cAMP levels in slugs, induced by submerging slugs in 0·25i.u.ml−1 cAMP-PDE, reduced the proportion of prespore cells from 66 % to 15 %, without affecting slug morphology. The cAMP-PDE-induced dedifferentiation of prespore cells was counteracted by cAMP and was not due to the production of the hydrolysis product 5′AMP, but to the reduction of extracellular cAMP levels. We conclude that extra-cellular cAMP is the major morphogenetic signal for the differentiation of prespore cells in the multicellular stages of D. discoideum development and we present a working hypothesis for the generation of the prestalk/ prespore pattern during multicellular development.
Reinitiation of chondroitin sulphate proteoglycan synthesis in regenerating skeletal muscle
Authors: Carrino David A. , Oron Uri , Pechak David G. , Caplan Arnold I.
Published: April 2021

Previous work from this laboratory involved the characterization of a large chondroitin sulphate proteoglycan unique to chick skeletal muscle. This proteoglycan is synthesized by embryonic skeletal muscle both in ovo and in culture but is not synthesized by adult muscle in vivo and myotubes in advanced cultures. Because regenerating skeletal muscle has been found to recapitulate synthesis of embryonic muscle protein isoforms, an analysis was performed to assess whether synthesis of chondroitin sulphate proteoglycans is reinitiated during muscle regeneration. Adult chicken pectoral and leg (gastrocnemius) muscle was injured by excision of a small piece of tissue or by cold injury; in the latter, the basement membrane has been reported to remain intact. At various times after injury, whole animals were exposed to [35S]sulphate and the proteoglycans were isolated by ion-exchange chromatography and analysed. Synthesis of only small proteoglycans, typical of normal adult skeletal muscle, is observed in the contralateral, uninjured muscle. In the regenerating muscle 4 days after injury, there is increased sulphate incorporation and abundant synthesis of chondroitin sulphate proteoglycans. This is observed in both pectoral and leg muscle irrespective of the type of injury, which suggests that the presence of basement membrane does not affect reinitiation of chondroitin sulphate proteoglycan synthesis. By 25 days after injury, synthesis of chondroitin sulphate proteoglycans is still detected but is significantly diminished. These data are consistent with the notion that skeletal muscle regeneration involves a recapitulation of embryonic events and give further credence to the hypothesis that skeletal muscle chondroitin sulphate proteoglycans play a role in some early aspect of myogenesis.
The distribution of fibronectin and tenascin along migratory pathways of the neural crest in the trunk of amphibian embryos
Authors: Epperlein Hans-Henning , Halfter Willi , Tucker Richard P.
Published: April 2021

It is generally assumed that in amphibian embryos neural crest cells migrate dorsally, where they form the mesenchyme of the dorsal fin, laterally (between somites and epidermis), where they give rise to pigment cells, and ventromedially (between somites and neural tube), where they form the elements of the peripheral nervous system. While there is agreement about the crest migratory routes in the axolotl (Ambystoma mexicanum), different opinions exist about the lateral pathway in Xenopus. We investigated neural crest cell migration in Xenopus (stages 23, 32, 35/36 and 41) using the X. laevis-X. borealis nuclear marker system and could not find evidence for cells migrating laterally. We have also used immunohistochemistry to study the distribution of the extracellular matrix (ECM) glycoproteins fibronectin (FN) and tenascin (TN), which have been implicated in directing neural crest cells during their migrations in avian and mammalian embryos, in the neural crest migratory pathways of Xenopus and the axolotl. In premigratory stages of the crest, both in Xenopus (stage 22) and the axolotl (stage 25), FN was found subepidermally and in extracellular spaces around the neural tube, notochord and somites. The staining was particularly intense in the dorsal part of the embryo, but it was also present along the visceral and parietal layers of the lateral plate mesoderm. TN, in contrast, was found only in the anterior trunk mesoderm in Xenopus; in the axolotl, it was absent. During neural crest cell migration in Xenopus (stages 25–33) and the axolotl (stages 28–35), anti-FN stained the ECM throughout the embryo, whereas anti-TN staining was limited to dorsal regions. There it was particularly intense medially, i.e. in the dorsal fin, around the neural tube, notochord, dorsal aorta and at the medial surface of the somites (stage 35 in both species). During post migratory stages in Xenopus (stage 40), anti-FN staining was less intense than anti-TN staining. In culture, axolotl neural crest cells spread differently on FN- and TN-coated substrata. On TN, the onset of cellular outgrowth was delayed for about 1 day, but after 3 days the extent of outgrowth was indistinguishable from cultures grown on FN. However, neural crest cells in 3-day-old cultures were much more flattened on FN than on TN. We conclude that both FN and TN are present in the ECM that lines the neural crest migratory pathways of amphibian embryos at the time when the neural crest cells are actively migrating. FN is present in the embryonic ECM before the onset of neural crest migration. In contrast, the appearance of TN is correlated with the initiation of migration. Since amphibian neural crest cells find FN, but not TN, to be adhesive migratory substrata in vitro, our results suggest that an interaction between these ECM components is important in regulating the onset and pathways of neural crest cell migration in the amphibian embryo.
Developmental control of N-CAM sialylation state by Golgi sialyltransferase isoforms
Authors: Breen Kieran C. , Regan Ciaran M.
Published: April 2021

A rat brain Golgi sialyltransferase activity capable of the differentiation-dependent control of N-CAM sialylation state is described. The specific activity of Golgi sialyltransferase was found to be developmentally regulated with respect to both endogenous and exogenous protein acceptors, with a particular elevation on postnatal days 10-12 when the heavily sialylated or ‘embryonic’ form of N-CAM is re-expressed. The subsequent developmental decrease in activity was associated with a significant decrease in apparent Km for the CMP-NeuNAc substrate, but not for the asialofetuin exogenous acceptor, which could not be attributed to the temporal expression of an endogenous competitive inhibitor. The apparent Vmax remained constant for CMP-NeuNAc but was significantly reduced for asialofetuin. Sialyltransferase activity, which was optimal at pH 7·0 – 7·5, was also modulated by various cations. Zinc abolished enzyme function, in contrast to ferric ions which stimulated activity fourfold-sevenfold. The marked activation of the adult form of the enzyme by potassium and magnesium ions, together with the alterations in kinetic constants, suggested this activity to be distinct from that derived from postnatal day-12 tissue. The kinetics of [l4C]sialic acid incorporation into immunoprecipitated N-CAM demonstrated the individual polypeptides to be sialylated, possibly by addition of polysialosyl units, in a developmental sequence. The presence of four distinct sialyltransferase activities was demonstrated by non-denaturing gel electrophoresis followed by solid-phase enzyme assay. These isoforms were temporally expressed during development, two being correlated with the postnatal reexpression of the ‘embryonic’ form of N-CAM.
Determination of somite cells: independence of cell differentiation and morphogenesis
Authors: Aoyama Hirohiko , Asamoto Ken
Published: April 2021

Somites are mesodermal structures which appear transiently in vertebrates in the course of their development. Cells situated ventromedially in a somite differentiate into the sclerotome, which gives rise to cartilage, while the other part of the somite differentiates into dermomyotome which gives rise to muscle and dermis. The sclerotome is further divided into a rostral half, where neural crest cells settle and motor nerves grow, and a caudal half. To find out when these axes are determined and how they rule later development, especially the morphogenesis of cartilage derived from the somites, we transplanted the newly formed three caudal somites of 2·5-day-old quail embryos into chick embryos of about the same age, with reversal of some axes. The results were summarized as follows. (1) When transplantation reversed only the dorsoventral axis, one day after the operation the two caudal somites gave rise to normal dermomyotomes and sclerotomes, while the most rostral somite gave rise to a sclerotome abnormally situated just beneath ectoderm. These results suggest that the dorsoventral axis was not determined when the somites were formed, but began to be determined about three hours after their formation. (2) When the transplantation reversed only the rostrocaudal axis, two days after the operation the rudiments of dorsal root ganglia were formed at the caudal (originally rostral) halves of the transplanted sclerotomes. The rostrocaudal axis of the somites had therefore been determined when the somites were formed. (3) When the transplantation reversed both the dorsoventral and the rostrocaudal axes, two days after the operation, sclerotomes derived from the prospective dermomyotomal region of the somites were shown to keep their original rostrocaudal axis, judging from the position of the rudiments of ganglia. Combined with results 1 and 2, this suggested that the fate of the sclerotomal cells along the rostrocaudal axis was determined previously and independently of the determination of somite cell differentiation into dermomyotome and sclerotome. (4) In the 9·5-day-old chimeric embryos with rostrocaudally reversed somites, the morphology of vertebrae and ribs derived from the explanted somites were reversed along the rostrocaudal axis. The morphology of cartilage derived from the somites was shown to be determined intrinsically in the somites by the time these were formed from the segmental plate. The rostrocaudal pattern of the vertebral column is therefore controlled by factors intrinsic to the somitic mesoderm, and not by interactions between this mesoderm and the notochord and/or neural tube, arising after segmentation.
Mechanism of erythropoietin action on the erythroid progenitor cells induced from murine erythroleukemia cells (TSA8)
Authors: Matsui Hiroaki Fukumoto Yasuhisa , Obinata Masuo
Published: April 2021

Erythropoietin is a well-known erythroid differentiation and growth factor, but the mechanism of its action is not well understood. In this work, we have examined its mechanism of action on the erythropoietin-responsive murine erythroleukemia cells (TSA8). TSA8 cells become responsive to erythropoietin after induction with DMSO. Stimulatory effects on erythropoietin response are observed with the addition of compounds affecting the cAMP level such as forskolin, phosphodiesterase inhibitor and cholera toxin only in the presence of erythropoietin. cAMP analogues themselves show no stimulatory effect on TSA8 cells, nor does erythropoietin increase cAMP level in the cells. Thus, it is suggested that cAMP does not act as a direct second messenger for signal transduction through erythropoietin receptors, but as a stimulator of the erythropoietin receptor pathway and/or as a second messenger in combination with the receptor pathway. The mechanism for acquisition of responsiveness to growth and differentiation factors of progenitor cells is discussed.
Periodic segmental anomalies induced by heat shock in the chick embryo are associated with the cell cycle
Authors: Primmett D. R. N. , Norris W. E. , Carlson G. J. , Keynes R. J. , Stern C. D.
Published: April 2021

This study provides evidence that cells destined to segment together into somites have a degree of cell division synchrony. We have measured the duration of the cell division cycle in somite and segmental plate cells of the chick embryo as 9·5 h using [3H]thymidine pulse-and-chase. Treatment of embryos with any of a variety of inhibitors known to affect the cell division cycle causes discrete periodic segmental anomalies: these anomalies appear about 6–7 somites after treatment and, in some cases, a second anomaly is observed 6 to 7 somites after the first. Since somites take 1·5 h to form, the 6- to 7-somite interval corresponds to about 9–10 h, which is the duration of the cell cycle as determined in these experiments. The anomalies are similar to those seen after heat shock of 2-day chick embryos. Heat shock and some of the other treatments induce the expression of heatshock proteins (hsp); however, since neither the expression nor the distribution of these proteins relate to the presence or distribution of anomalies seen, we conclude that hsps are not responsible for the pattern of segmental anomalies observed. The production of periodic segmental anomalies appears to be linked to the cell cycle. A simple model is proposed, in which we suggest that the cell division cycle is involved directly in gating cells that will segment together.
Presence of basic fibroblast growth factor in the early Xenopus embryo
Authors: Slack J. M. W. , Isaacs H. V.
Published: April 2021

Mesoderm-inducing activity can be extracted from Xenopus embryos, eggs or whole ovary. It binds to heparin and can be neutralized by heparin or anti-bFGF but not by anti-TGFβ. Two molecular forms can be identified by Western blotting and have molecular weights of about 19 and 14K. The content in embryos is about 7 units g-1 (approximately 7 ng ml-1) which would be sufficient for it to be acting as an endogenous inducer of ventral mesoderm. Attempts to detect TGFβ-like inducing factors in embryos were not successful.
Cell rearrangement and segmentation in Xenopus: direct observation of cultured explants
Authors: Wilson Paul A. , Oster George , Keller Ray
Published: April 2021

We make use of a novel system of explant culture and high resolution video-film recording to analyse for the first time the cell behaviour underlying convergent extension and segmentation in the somitic mesoderm of Xenopus. We find that a sequence of activities sweeps through the somitic mesoderm from anterior to posterior during gastrulation and neurulation, beginning with radial cell intercalation or thinning, continuing with mediolateral intercalation and cell elongation, and culminating in segmentation and somite rotation. Radial intercalation at the posterior tip lengthens the tissue, while mediolateral intercalation farther anterior converges it toward the midline. This extension of the somitic mesoderm helps to elongate the dorsal side of intact neurulae. By separating tissues, we demonstrate that cell rearrangement is independent of the notochord, but radial intercalation - and thus the bulk of extension - requires the presence of an epithelium, either endodermal or ectodermal. Segmentation, on the other hand, can proceed in somitic mesoderm isolated at the end of gastrulation. Finally, we discuss the relationship between cell rearrangement and segmentation.
Embryonic development of Xenopus studied in a cell culture system with tissue-specific monoclonal antibodies
Authors: Mitani Shohei , Okamoto Harumasa
Published: April 2021

An in vitro microculture system of early gastrula cells of Xenopus laevis has been developed; deep layer cells from the lateral marginal zone (prospective somite region) or ventral ectoderm (prospective epidermis region) were fully dissociated, and the desired number of each (1–100) was distributed into a microculture well and cultured under appropriate conditions. When examined with the tissue-specific Mabs (Mul for muscle and E3 for epidermis, respectively), a substantial portion of the deep layer cells from the two regions followed their respective normal embryonic fates. It was found that reproducible cellular differentiation was dependent on the intimate reaggregation of dissociated cells and on the size of the resultant aggregate. About 20 lateral marginal zone cells were found to be sufficient, when put into a culture well, for supporting successful muscle differentiation, whereas about 100 ventral ectoderm cells were necessary for epidermal differentiation.
Xwnt-8, a Xenopus Wnt-1/int-1-related gene responsive to mesoderminducing growth factors, may play a role in ventral mesodermal patterning during embryogenesis
Authors: Christian Jan L. , Mcmahon Jill A. , Mcmahon Andrew P. , Moon Randall T.
Published: April 2021

In amphibian embryos, formation of the basic body plan depends on positional differences in the mesoderm. Although peptide growth factors involved in mesoderm induction have tentatively been identified, additional signals are required to generate pattern in this tissue. We have isolated a Xenopus cDNA for a Wnt-1 related gene, designated Xwnt-8, which is activated in ‘response to mesoderm-inducing growth factors. Xwnt-8 transcripts are transiently expressed, being most abundant during gastrulation at which time expression is confined primarily to ventral mesodermal cells. Embryos dor-soanteriórized by exposure to lithium exhibit greatly reduced levels of Xwnt-8 mRNA, supporting a correlation between Xwnt-8 expression and a ventral mesodermal cell fate. Surprisingly, ectopic expression of Xwnt-8 in embryos causes a dorsoanterior-enhanced phenotype. These findings suggest that Awni-S may be a secondary signalling agent which is produced in response to mesoderm-inducing factors and is involved in the early steps of mesodermal patterning.
Lineage of neurons and glia in chick dorsal root ganglia: analysis in vivo with a recombinant retrovirus
Authors: Frank Eric , Sanes Joshua R.
Published: April 2021

We used retrovirus-mediated gene transfer to study the lineage of neural crest cells in chick embryos. Individual crest cells were infected before they migrated from the neural tube, and their clonal progeny were subsequently revealed in sensory ganglia and associated structures by a histochemical stain for the viral gene product (lacZ). We found that crest cells were multipotential in several respects. (1) Many clones contained both ventrolateral (VL) and dorsomedial (DM) neurons, which had been suggested to be lineally distinct. (2) Many clones contained both large and small neurons, which are known to innervate distinct targets. (3) Many clones contained multiple glial subtypes, e.g. both Schwann cells, which ensheath axons, and satellite cells, which ensheath neuronal somata. (4) Many clones contained both neurons and glial cells. On the other hand, a sizeable minority of clones was homogenous, e.g. they contained only neurons or only glial cells – suggesting that some progenitors may be, or become, restricted in potential. Finally, this study provides the first opportunity to compare directly the two methods currently available for tracing cell lineage in vertebrate embryos, retroviral infection and tracer injection: our results and those of Bronner-Fraser and Fraser (1989), who used the latter method, provide complementary but consistent views of crest lineage.
Neurothelin: molecular characteristics and developmental regulation in the chick CNS
Authors: Schlosshauer Burkhard
Published: April 2021

Neurothelin has recently been identified as a cell surface protein specific for chick endothelial cells forming the blood-brain barrier. Neurons of the adult brain are essentially devoid of neurothelin. In contrast, neurons of the chick retina, which lack blood vessels and accessory astrocytes, express neurothelin. Here we demonstrate that during chick brain development initially neurothelin is expressed probably in all neuroblasts. With proceeding cytodifferentiation, such as vascularization and gliogenesis, brain neurons become neurothelin negative. Coincidentally the endothelial cells forming the blood-brain barrier start to synthesize neurothelin. In contrast to brain neurons, in retina neurons, neurothelin expression increases by one order of magnitude during the course of histogenesis. Coculturing of chick retinal cells with purified rat astrocytes in vitro results in reduction of neural neurothelin expression as quantified by ELISA. Conversely, disruption of the glia-neuron interactions by culturing brain neurons as individualized cells in vitro leads to a reexpression of neurothelin. This is consistent with the hypothesis that astrocytes inhibit neurothelin expression in neurons.

Biochemical characterization classifies neurothelin as an integral membrane protein. Temperature-induced-detergent phase separation, phospholipase C digestion and sodium carbonate treatment were employed to distinguish between integral membrane proteins, lipid- anchored proteins and peripheral membrane proteins. Two-dimensional gel electrophoresis reveals an isoelectric point of about 6.4 for neurothelin. Polysaccharide analysis by glycosidase digestion and lectin binding indicates that neurothelin is highly glycosylated. The relative molecular mass of glycosylated neurothelin is 41 × 103, whereas the peptide backbone is only 25 × 103.

The very strict spatiotemporal regulation of neurothelin expression in the central nervous system suggests that neurothelin fulfils possibly a crucial function such as transport of low relative molecular mass components that are essential for neuronal metabolism. The proposed biological activity of neurothelin might be specifically affected by some of its distinct biochemical features.
Hepatocyte differentiation initiates during endodermal-mesenchymal interactions prior to liver formation
Authors: Cascio S. , Zaret K.S.
Published: April 2021

Previous studies with embryonic tissue explants showed that cellular interactions with mesenchyme are required for endodermal cells to differentiate into hepatocytes. However, these studies assayed hepatocyte characteristics that were evident after days of culture, leaving open the question of whether the primary inductive interactions initiated hepatocyte differentiation, or whether subsequent steps, such as may occur during cell aggregation to form the liver, were necessary. Using the technique of in situ hybridization, we find that serum albumin mRNA, a liver-specific gene product, is first detected in hepatic precursor cells of the endoderm as early as 9.5 days of mouse embryo development, a full day prior to cell aggregation and liver formation. The endodermal cells express albumin mRNA upon migration into strands of connective tissue matrix within mesenchyme. Thus, the onset of differentiation of the endoderm is coincident with its interaction with mesenchyme. Early albumin transcripts are initiated at the same site of the albumin promoter as in adult hepatocytes, suggesting that at least a subset of the transcription factors that control albumin transcription in the adult may be involved in executing the early steps of hepatic determination. We also observe a sharp increase in albumin mRNA levels shortly after the definitive formation of the liver, apparently reflecting cell interactions that enhance hepatocyte differentiation. Hepatocyte differentiation is therefore similar in several respects to pancreatic exocrine cell development, and may represent a general pattern for gut-derived tissues. For both cell types, early interactions with mesenchyme are coincident with the initial expression of differentiated gene products at a low level in proliferating endoderm, and the initial pattern of expression is amplified upon organ formation.
An evaluation of myelomeres and segmentation of the chick embryo spinal cord
Authors: Lim T.M. , Jaques K.F. , Stern C.D. , Keynes R.J.
Published: April 2021

We have investigated whether the neuromeres of the developing chick spinal cord (myelomeres) are manifestations of intrinsic segmentation of the CNS by studying the patterns of cell proliferation and neuronal differentiation. Treatment of 2-day embryos with colchicine does produce exaggerated myelomeres, in confirmation of Kallen (Z. Anat. Entwickl.-Gesch. 123, 309–319, 1962). However, this does not imply that myelomeres are segmental proliferation centres: the undulations caused by colchicine are irregular alongside the unsegmented mesoderm, and another mitotic inhibitor, bromodeoxyuridine, has no such effects. In contrast to lower vertebrate embryos, there is no evidence for segmental groups of primary motor neurons in the chick: the earliest motor neurons express cholinesterase, and project their axons into the adjacent sclerotome, at random positions in relation to the somite boundaries. The population of motor neurons projecting HRP-labelled axons into a single somite lies out of phase with both myelomere and somite, and is placed symmetrically about the anterior half-sclerotome. The earliest intrinsic spinal cord neurons, as stained with zinc iodide-osmium tetroxide or anti-68 × Mr neurofilament antibody, show no segmental patterns of differentiation. We conclude that, in contrast to the rhombomeres of the developing hindbrain, myelomeres are not matched by segmental groupings of differentiating nerve cells, and result from mechanical moulding of the neuroepithelium by the neighbouring somites.
Segmental lineage restrictions in the chick embryo spinal cord depend on the adjacent somites
Authors: Stern C.D. , Jaques K.F. , Lim T.M. , Fraser S.E. , Keynes R.J.
Published: April 2021

We have investigated whether the developing spinal cord is intrinsically segmented in its rostrocaudal (anteroposterior) axis by mapping the spread of clones derived from single labelled cells within the neural tube of the chick embryo. A single cell in the ventrolateral neural tube of the trunk was marked in situ with the fluorescent tracer lysinated rhodamine dextran (LRD) and its descendants located after two days of further incubation. We find that clones derived from cells labelled before overt segmentation of the adjacent mesoderm do not respect any boundaries within the neural tube. Those derived from cells marked after mesodermal segmentation, however, never cross an invisible boundary aligned with the middle of each somite, and tend to be elongated along the mediolateral axis of the neural tube. When the somite pattern is surgically disturbed, neighbouring clones derived from neuroectodermal cells labelled after somite formation behave like clones derived from younger cells: they no longer respect any boundaries, and are not elongated mediolaterally. These results indicate that periodic lineage restrictions do exist in the developing spinal cord of the chick embryo, but their maintenance requires the presence of the adjacent somite mesoderm.
Mutations in a newly identified Drosophila melanogaster gene, mago nashi, disrupt germ cell formation and result in the formation of mirror-image symmetrical double abdomen embryos
Authors: Boswell Robert E. , Prout Mary E. , Steichen Jessica C.
Published: April 2021

The mago nashi (mago) locus is a newly identified strict maternal effect, grandchildless-like, gene in Drosophila melanogaster. In homozygous mutant mago females reared at 17°C, mago+ function is reduced, the inviable embryos lack abdominal segments and 84–98 % of the embryos die. In contrast, at 25°C, some mago alleles produce a novel gene product capable of inducing the formation of symmetrical double abdomen embryos. Reciprocal temperature-shift experiments indicate that the temperature-sensitive period is during oogenetic stages 7–14. Furthermore, embryos collected from mago1 homozygous females contain no apparent func- tional posterior determinants in the posterior pole. In viable F1 progeny from mago mutant females, regardless of genotype and temperature, polar granules are reduced or absent and germ cells fail to form (the grandchildless-like phentoype). Thus, we propose that the mago+ product is a component of the posterior determinative system, required during oogenesis, both for germ cell determination and delineation of the longitudinal axis of the embryo.
Microtubules mediate the localization of bicoid RNA during Drosophila oogenesis
Authors: Pokrywka Nancy Jo , Stephenson Edwin C.
Published: April 2021

We have examined cytoskeletal requirements for bicoid (bed) RNA localization during Drosophila oogenesis, bed is an anterior morphogen whose proper function relies on the localization of its messenger RNA to the anterior cortex of the egg. Drugs that depolymerize microtubules perturb all aspects of bed RNA localization. During recovery from drug treatment, bed RNA relocalizes to the oocyte cortex, suggesting that the localization machinery is a component of the cortical cytoskeleton.

Taxol, a drug that stabilizes microtubules, also effectively disrupts bed RNA localization, and the effects of taxol treatments on exuperantia and swallow mutants suggest general roles for these gene products in the multi-step bed RNA localization process.