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R J Keynes

Publications and source records attributed to R J Keynes.

At least 37 records · Page 2Linked to original sources
PubMed1990

Segmental and developmental regulation of a presumptive T-cell oncogene in the central nervous system.

Although most proto-oncogenes such as c-myc are involved in cell proliferation, being expressed in a wide range of tissues as well as in progenitors of transformed cells, others may normally function in cellular differentiation. We now report on a gene on human chromosome 11, at the junction of the T-cell tumour-associated chromosomal translocation t(11; 14) (p15; q11) and known as the 11p15 gene or Ttg, which is believed to be involved in the pathogenesis of the tumour. It has two transcriptional promoters (both retained by the translocated allele) and is expressed in tumour cells with neuro-endocrine properties, suggesting that normal expression may occur in nerve cells. Using fusion constructs of one 11p15 promoter and lacZ in transgenic mice, we found that the gene is expressed in a segment-specific manner in rhombomeres of the developing mouse hind-brain. During subsequent development, the gene is more widely expressed, again in precisely defined regional patterns, but in post-mitotic neurons confined to the central nervous system. Thus, this presumptive T-cell oncogene is both developmentally regulated and segmentally restricted in a tissue different from that in which the original tumour arose.

Animals↗
PubMed1990

Isolation from chick somites of a glycoprotein fraction that causes collapse of dorsal root ganglion growth cones.

The segmented pattern of peripheral spinal nerves in higher vertebrates is generated by interactions between nerve cells and somites. Neural crest cells, motor axons, and sensory axons grow exclusively through anterior-half sclerotome. In chick embryos, posterior cells bind the lectins peanut agglutinin (PNA) and Jacalin. When liposomes containing somite extracts are applied to cultures of chick sensory neurons, growth cones collapse abruptly, recovering within 4 hr of liposome removal. Collapse activity is eliminated by immobilized PNA, and SDS-PAGE demonstrates two major components (48K and 55K), which are absent from anterior-half sclerotome. Rabbit polyclonal antibodies against these components recognize only posterior cells and may also be used to eliminate collapse activity. We suggest that spinal nerve segmentation is produced by inhibitory interactions between these components and growth cones.

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PubMed1989

Periodic segmental anomalies induced by heat shock in the chick embryo are associated with the cell cycle.

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 heat-shock 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.

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PubMed1989

Molecular differences between the rostral and caudal halves of the sclerotome in the chick embryo.

It is known that both neural crest cell migration and motor axon outgrowth in most vertebrate embryos are segmented because of restrictions imposed upon their distribution by the neighbouring sclerotomes, each of which is divided into a rostral and a caudal half. The caudal half does not allow crest migration or axon outgrowth, while the rostral half does. In this paper, we investigate the expression of proteins and glycoproteins in the two halves of the sclerotome of the chick embryo at stages between 20 and 32 pairs of somites by two-dimensional SDS-polyacrylamide gel electrophoresis. We find that the patterns of expression are complex, and that polypeptides and glycoproteins vary both spatially and temporally: of those that are expressed differentially by the sclerotome, some differ quantitatively and others qualitatively. Some macromolecules change their spatial distribution with developmental age, and some appear or disappear as the embryos become older.

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PubMed1989

J1/tenascin-related molecules are not responsible for the segmented pattern of neural crest cells or motor axons in the chick embryo.

It has been suggested that substrate adhesion molecules of the tenascin family may be responsible for the segmented outgrowth of motor axons and neural crest cells during formation of the peripheral nervous system. We have used two monoclonal antibodies (M1B4 and 578) and an antiserum [KAF9(1)] to study the expression of J1/tenascin-related molecules within the somites of the chick embryo. Neural crest cells were identified with monoclonal antibodies HNK-1 and 20B4. Young somites are surrounded by J1/tenascin immunoreactive material, while old sclerotomes are immunoreactive predominantly in their rostral halves, as described by other authors (Tan et al. 1987--Proc. natn. Acad. Sci. U.S.A. 84, 7977; Mackie et al. 1988--Development 102, 237). At intermediate stages of development, however, immunoreactivity is found mainly in the caudal half of each sclerotome. After ablation of the neural crest, the pattern of immunoreactivity is no longer localised to the rostral halves of the older, neural-crest-free sclerotomes. SDS-polyacrylamide gel electrophoresis of affinity-purified somite tissue, extracted using M1B4 antibody, shows a characteristic set of bands, including one of about 230 x 10(3), as described for cytotactin, J1-200/220 and the monomeric form of tenascin. Affinity-purified somite material obtained from neural-crest-ablated somites reveals some of the bands seen in older control embryos, but the high molecular weight components (120-230 x 10(3] are missing. Young epithelial somites also lack the higher molecular mass components. The neural crest may therefore participate in the expression of J1/tenascin-related molecules in the chick embryo. These results suggest that these molecules are not directly responsible for the segmented outgrowth of precursors of the peripheral nervous system.

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PubMed1988

Heat shock causes repeated segmental anomalies in the chick embryo.

A single heat shock, given to 2-day-old chick embryos, can generate multiple but discrete somite and skeletal anomalies. Each of these anomalies is restricted to one, or at the most two, consecutive segments. The anomalies are separated from each other by a distance of 6-7 somites or vertebrae, or a multiple of this distance. These results argue against the 'clock and wavefront' model; while they support the idea of a cellular clock, they are not consistent with a single propagating wave gating cells destined to form each segment. Heat shock also alters the size and number of segments, as well as the rostrocaudal proportions of the sclerotome. The results are consistent with the rostrocaudal fate of sclerotome cells being determined during segmentation. From our observations, we speculate on the implications for regionalization of the vertebral column.

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PubMed1988

A cell lineage analysis of segmentation in the chick embryo.

We have studied the lineage history of the progenitors of the somite mesoderm and of the neural tube in the chick embryo by injecting single cells with the fluorescent tracer, rhodamine-lysine-dextran. We find that, although single cells within the segmental plate give rise to discrete clones in the somites to which they contribute, neither the somites nor their component parts (sclerotome, dermatome, myotome or their rostral and caudal halves) are 'compartments' in the sense defined in insects. Cells in the rostral two thirds or so of the segmental plate contribute only to somite tissue and divide about every 10 h, while those in the caudal portions of this structure contribute both to the somites and to intermediate and lateral plate mesoderm derivatives. In the neural tube, the descendants of individual prospective ventral horn cells remain together within the horn, with a cycle time of 10 h. We have also investigated the role of the cell division cycle in the formation and subsequent development of somites. A single treatment of 2-day chick embryos with heat shock or a variety of drugs that affect the cell cycle all produce repeated anomalies in the pattern of somites and vertebrae that develop subsequent to the treatment. The interval between anomalies is 6-7 somites (or a multiple of this distance), which corresponds to 10 h. This interval is identical to that measured for the cell division cycle. Given that cell division synchrony is seen in the presomitic mesoderm, we suggest that the cell division cycle plays a role in somite formation. Finally, we consider the mechanisms responsible for regionalization of derivatives of the somite, and conclude that it is likely that both cell interactions and cell lineage history are important in the determination of cell fates.

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PubMed1987

The differing effects of occipital and trunk somites on neural development in the chick embryo.

In all higher vertebrate embryos the sensory ganglia of the trunk develop adjacent to the neural tube, in the cranial halves of the somite-derived sclerotomes. It has been known for many years that ganglia do not develop in the most cranial (occipital) sclerotomes, caudal to the first somite. Here we have investigated whether this is due to craniocaudal variation in the neural tube or crest, or to an unusual property of the sclerotomes at occipital levels. Using the monoclonal antibody HNK-1 as a marker for neural crest cells in the chick embryo, we find that the crest does enter the cranial halves of the occipital sclerotomes. Furthermore, staining with zinc iodide/osmium tetroxide shows that some of these crest-derived cells sprout axons within these sclerotomes. By stage 23, however, no dorsal root ganglia are present within the five occipital sclerotomes, as assessed both by haematoxylin/eosin and zinc iodide/osmium tetroxide staining. Moreover, despite this loss of sensory cells, motor axons grow out in these segments, many of them later fasciculating to form the hypoglossal nerve. The sclerotomes remain visible until stages 27/28, when they dissociate to form the base of the skull and the atlas and axis vertebrae. After grafting occipital neural tube from quail donor embryos in place of trunk neural tube in host chick embryos, quail-derived ganglia do develop in the trunk sclerotomes. This shows that the failure of occipital ganglion development is not the result of some fixed local property of the neural crest or neural tube at occipital levels. We therefore suggest that in the chick embryo the cranial halves of the five occipital sclerotomes lack factors essential for normal sensory ganglion development, and that these factors are correspondingly present in all the more caudal sclerotomes.

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PubMed1987

The neural tube origin of ventral root sheath cells in the chick embryo.

The embryonic origin of peripheral nerve Schwann/sheath cells is still uncertain. Although the neural crest is known to be an important source, it is not clear whether the ventral neural tube also contributes a progenitor population for motor axons. We have used the techniques of immunohistochemistry, electron microscopy and quail-chick grafting to examine this problem. Immunohistochemistry with monoclonal antibody HNK-1 identified a cluster of immunoreactive cells in the sclerotome, at the site of the future ventral root. With the electron microscope, nucleated cells could not be seen breaching the basal lamina of the neural tube, exclusively in the region of the ventral root and preceding axon outgrowth. After grafting a length of crest-ablated quail neural tube in place of host chick neural tube, a population of quail cells was found localized to the ventral root exit zone, associated with the ventral root axons. Taken together, these observations support the possibility of a neural tube origin for ventral root sheath cells, although we found no evidence for a more extensive migration of these cells. The ventral root cells share certain phenotypic traits, such as HNK-1 immunoreactivity, with neural-crest-derived Schwann cells, but are not necessarily identical to them. We argue that while they may help motor axons to exit the neural tube at the correct position, they are unlikely to guide axons beyond the immediate vicinity of the neural tube.

Animals↗
PubMed1987

Interactions between somite cells: the formation and maintenance of segment boundaries in the chick embryo.

We have investigated the interactions between the cells of the rostral and caudal halves of the chick somite by carrying out grafting experiments. The rostral half-sclerotome was identified by its ability to support axon outgrowth and neural crest cell migration, and the caudal half by the binding of peanut agglutinin and the absence of motor axons and neural crest cells. Using the chick-quail chimaera technique we also studied the fate of each half-somite. It was found that when half-somites are placed adjacent to one another, their interactions obey a precise rule: sclerotome cells from like halves mix with each other, while those from unlike halves do not; when cells from unlike halves are adjacent to one another, a border is formed. Grafting quail half-somites into chicks showed that the fates of the rostral and caudal sclerotome halves are similar: both give rise to bone and cartilage of the vertebral column, as well as to intervertebral connective tissue. We suggest that the rostrocaudal subdivision serves to maintain the segmental arrangement when the mesenchymal sclerotome dissociates, so that the nervous system, vasculature and possibly vertebrae are patterned correctly.

Animals↗
PubMed1987

The specificity of motor innervation of the chick wing does not depend upon the segmental origin of muscles.

In vertebrate embryos, motor axons originating from a particular craniocaudal position in the neural tube innervate limb muscles derived from myoblasts of the same segmental level. We have investigated whether this relationship is important for the formation of specific nerve-muscle connections, by altering the segmental origin of muscles and examining their resulting innervation. First, by grafting quail wing somites to a new craniocaudal position opposite the chick wing, we established that the segmental origin of a muscle can be altered: presumptive muscle cells migrated according to their new, rather than their original, somitic level, colonizing a different subset of muscles. However, after reversal of a length of brachial somitic mesoderm along the craniocaudal axis, or exchange or shift of brachial somites, the craniocaudal position of wing muscle motoneurone pools within the spinal cord was undisturbed, despite the new segmental origin of the muscles themselves. While not excluding the possibility that muscles and their motor nerves are labelled segmentally, we conclude that specific motor axon guidance in the wing does not depend upon the existence of such labels.

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PubMed1986

Muscle basal lamina: a new graft material for peripheral nerve repair.

The suitability of muscle basal lamina as a graft material for the repair of peripheral nerves was investigated. Grafts were prepared by evacuating the myoplasm from muscles excised from rats and rabbits. This produced a material consisting mainly of basal lamina and connective tissue, with the basal lamina arranged as parallel tubes. Rat- and rabbit-derived graft material in 0.5-cm lengths was sutured into rat sciatic nerves, and 4-cm lengths of rabbit-derived graft material were interposed into rabbit sciatic nerves. For controls, 0.5-cm nerve autografts were grafted into rats and 4-cm autografts into rabbits. After 2 to 3 months, the success of the grafts was assessed functionally, electrophysiologically, and anatomically. By all these criteria the basal lamina grafts were as successful as nerve autografts; essentially the same number of axons of the same size grew through both graft types, animals recovered their limb function equally well, and the nerve conduction velocities and relative refractory periods were the same in both groups of animals. In rats, following both basal lamina and nerve autografts, the number of axons distal to the grafts was approximately the same as that proximal to them, but axon diameter and speed of conduction were significantly less than normal. The authors conclude that muscle basal lamina grafts are as effective as nerve autografts for repairing severed rat or rabbit peripheral nerves, and suggest that grafts prepared in this way may prove to be useful for nerve repair in humans.

Animals↗
PubMed1986

Interactions between neurites and somite cells: inhibition and stimulation of nerve growth in the chick embryo.

After neural processes emerge from the neural tube in the chick embryo, their growth is restricted to the cranial halves of the neighbouring somites. In this study we have developed an in vitro system to model the interactions between these tissue types. Pioneer neurites display a hierarchy of preferences in terms of the substrates they can grow on. As expected, tissue culture plastic does not support neural outgrowth, but this can be overcome by coating the plastic substrate with either collagen or poly-L-lysine. Neural crest, cranial half somite, and a number of other tissues support growth well, while caudal half somite and tail bud mesenchyme do so to a much smaller extent. The binding pattern of a variety of lectins was assessed in cryostat sections of embryos and in cultured cells of the above tissues. It was found that peanut agglutinin can discriminate between cranial and caudal sclerotome both in vitro and in the embryo, since it binds preferentially to caudal sclerotome in both cases. This difference is expressed as soon as the sclerotome forms. The significance of these findings is twofold: first, they show that the interactions that take place during peripheral neural segmentation can be modelled in vitro; second, they represent the first instance of a molecular difference between the cranial and caudal halves of the sclerotome, detectable both in culture and in the embryo.

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PubMed1985

Postnatal growth of motor nerve terminals in muscles of the mouse.

A reduced silver stain was used to examine the development of complexity of motor nerve terminals in the postnatal period. Terminals in three histochemically different muscles were examined in mice aged 12 days to 3 years. The total number and total length of intraterminal axon branches increases with age, but only until animals are 3 months old. Terminals become longest and most branched in the histochemically glycolytic tensor fascia latae (TFL) muscle, shortest and least branched in the oxidative diaphragm, and intermediate in the histochemically mixed gluteus muscle. In addition, myelinated terminal branches develop in TFL and to a lesser extent in the gluteus between 1 and 3 months of age. These myelinated branches appear to be produced by nodal sprouting from the penultimate node of Ranvier of the terminal axon, and also by myelination of pre-existing terminal branches. The diameter of muscle fibres also increases until animals are 3 months old, and there is a good correlation between mean fibre diameter and either mean terminal length or mean number of terminal branches when all muscles at all ages are compared. This suggests that terminal growth could be determined by muscle fibre growth; however, within any given muscle there is little or no correlation between the diameter of a muscle fibre and either the length or number of branches of its nerve terminal, suggesting that terminal morphology is not controlled solely by muscle fibre growth. The presence of a myelinated branch in a nerve terminal is also unrelated to fibre diameter within a given muscle, but again when means are compared there are good, but significantly different correlations for the three different muscles. Thus some kind of muscle or nerve type-specific property additional to a general effect of muscle fibre size influences the development of myelinated terminal branches. Between 3 and 12 months of age terminal complexity remains constant or may decrease slightly. At 19 months or older, when mice are becoming senile, a large proportion of synapses have terminal sprouts and muscle fibres become innervated by two or more distinct axons. These changes can be attributed to the death of some motor neurons and sprouting of the remaining axons.

Aging↗
PubMed1985

The migration of neural crest cells and the growth of motor axons through the rostral half of the chick somite.

We have studied the pathway of migration of neural crest cells through the somites of the developing chick embryo, using the monoclonal antibodies NC-1 and HNK-1 to stain them. Crest cells, as they migrate ventrally from the dorsal aspect of the neural tube, pass through the lateral part of the sclerotome, but only through that part of the sclerotome which lies in the rostral half of each somite. This migration pathway is almost identical to the path which presumptive motor axons take when they grow out from the neural tube shortly after the onset of neural crest migration. In order to see whether the ventral root axons are guided along this pathway by neural crest cells, we surgically excised the neural crest from a series of embryos, and examined the pattern of axon outgrowth approximately 24 h later. In somites which contained no neural crest cells, ventral root axons were still found only in the rostral half of the somite, although axonal growth was slightly delayed. These axons were surrounded by sheath cells, which had presumably migrated out of the neural tube, to a point about 50 micron proximal to the growth cones. With appropriate antibodies we found that the extracellular matrix components fibronectin and laminin are evenly distributed between the rostral and caudal halves of the somite. Neither of these molecules therefore plays a critical role in determining the specific pathway of neural crest cells or motor axons through the rostral half of the somite.

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