Spatial patterns of homeobox gene expression in the developing mammalian CNS.
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Biomedical subjects
C D Stern
Publications and source records attributed to C D Stern.
Mechanisms of vertebrate segmentation.
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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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Changes in the expression of the carbohydrate epitope HNK-1 associated with mesoderm induction in the chick embryo.
We report that a monoclonal antibody, HNK-1, identifies specific regions and cell types during primitive streak formation in the chick blastoderm. Immunohistochemical studies show that the cells of the forming hypoblast are HNK-1 positive from the earliest time at which they can be identified. Some cells of the margin of the blastoderm are also positive. The mesoderm cells of the primitive streak stain strongly with the antibody from the time of their initial appearance. In the epiblast, some cells are positive and some negative at pre-primitive-streak stages, but as the primitive streak develops a gradient of staining intensity is seen within the upper layer, increasing towards the primitive streak. At later stages of development, the notochord and the mesenchyme of the headfold are positive, while the rest of the mesoderm (lateral plate) no longer expresses HNK-1 immunoreactivity. This antibody therefore reveals changes associated with mesodermal induction: before induction, it recognizes the 'inducing' tissue (the hypoblast) and reveals a mosaic pattern in the responding tissue (the epiblast); after primitive streak formation, the mesoderm of the primitive streak that results from the inductive interactions expresses the epitope strongly. Affinity purification of HNK-1-related proteins in various tissues was carried out, followed by SDS-PAGE to identify them. The hypoblast, mesoderm and epiblast of gastrulating chick embryos have some HNK-1-related proteins in common, while others are unique to specific tissues. Attempts have been made to identify these proteins using Western blots and antibodies known to recognize HNK-1-related molecules, but none of the antibodies used identify the bands unique to any of the tissues studied. We conclude that these proteins may be novel members of the HNK-1/L2 family, and that they may have a role in cell interactions during early development.
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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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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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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.
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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.
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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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Do ionic currents play a role in the control of development?
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A historical perspective on the discovery of the accessory duct of the pancreas, the ampulla 'of Vater' and pancreas divisum.
The discovery of the accessory duct of the pancreas is usually ascribed to Giovanni Domenico Santorini (1681-1737), after whom this structure is named. The papilla duodeni (ampulla 'of Vater', or papilla 'of Santorini') is named after Abraham Vater (1684-1751) or after GD Santorini. Pancreas divisum, a persistence through non-fusion of the embryonic dorsal and ventral pancreas is a relatively common clinical condition, the discovery of which is usually ascribed to Joseph Hyrtl (1810-1894). In this review I report that pancreas divisum, the accessory duct and the papilla duodeni (ampulla 'of Vater') had all been observed and the observations published during the 17th century by at least seven anatomists before Santorini, Vater, and Hyrtl. I further suggest, in the light of frequent anatomical misattributions in common usage, that anatomical structures be referred to only by their proper anatomical names.
Ampulla of Vater↗
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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Fluid transport across the epiblast of the early chick embryo.
A simple method is described which allows quantitation of the rate of fluid transport across the isolated epiblast of the early chick embryo. This method consists of allowing the tissue to form spheres, which then spontaneously undergo a large volume increase. The rate of fluid uptake into the spheres can be estimated by measuring the dimensions of the spheres. Pharmacological and electrophysiological studies were performed on the spheres to determine the mechanisms of fluid transport. It was found that fluid is driven into the interior of the spheres by the osmotic gradient generated by unidirectional sodium transport and to a lesser extent by another mechanism, as yet unknown. We discuss possible candidates for this mechanism, and consider the significance of these findings to early development.
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A simple model for early morphogenesis.
Despite the large amount of knowledge which continues to accumulate about early developmental events, very little is known about the processes which control them. Part of the problem may lie in that workers applying different approaches and techniques have different points of view and appear to be reluctant to read each others' literature. My aim in this paper is not to give a generative, formal model for early development, but rather to suggest several connecting strands between the physiological, biochemical, cell biological and experimental embryological approaches which may stimulate new research in fields between those already exploited.
Amphibians↗
Mitotic activity during somite segmentation in the early chick embryo.
The mitotic activity of the somites, segmental plate and posterior mesoderm were investigated in colchicine-treated and untreated chick embryos at st. 7-14. The mitotic figures in the somites are restricted to the proximity of the lumen and have their spindles orientated predominantly tangentially to the cavity. In the segmental plate there is no pattern in terms of the position or orientation of the mitotic spindles, but there is a single region, often found close to the cranial end of the segmental plate, with an elevated mitotic index. This may indicate a certain degree of synchrony among groups of segmental plate cells. These results are discussed in relation to the process of somite segmentation.
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Mini-review: hyaluronidases in early embryonic development.
The foregoing discussion indicates that hyaluronidases probably play an important part in the control of development. In morphogenesis, they may be involved in epithelial-mesenchymal inductive interactions, in non-malignant invasion when one tissue displaces another in normal development, in controlling cell movements, in modulating changes of shape of cells and sheets of cells, in controlling the permeability of tissues and regulating the ionic environment within the embryo. There is also evidence indicating that hyaluronidases are involved in the initiation of cytodifferentiation pathways, perhaps via direct or indirect effects upon the cell division cycle and histone-DNA interactions. The evidence presented indicates that hyaluronidases are important repeatedly at different stages of embryonic development and differentiation, where periods of high activity follow others of reduced activity in localized regions of the embryo. Some new results were also presented, showing the presence of different hyaluronidase activities at early stages of chick embryo development. The highest levels of hyaluronidase activity were found in the primitive streak and mesoderm.
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The roles of node regression and elongation of the area pellucida in the formation of somites in avian embryos.
Experiments have been carried out on explanted chick embryos to test certain widely accepted concepts about the role of Hensen's node in somite formation. The relationship between elongation of the area pellucida and regression of Hensen's node has also been investigated. We conclude from these experiments that: (a) The timing of somite formation is not controlled by the regression of Hensen's node, nor by the shearing of the mesoderm into right and left halves. (b) Somite size and shape are probably controlled by local conditions in the chick embryo. (c) Elongation and regression are two different events. (d) The position of the probably depends on mechanical tensions in the area pellucida. (e) The notochord is not required for the stability of somites in vivo.
Agar↗
Sodium transport and the control of epiblast polarity in the early chick embryo.
The sodium transport properties of chick epiblast during gastrulation were studied by various techniques. It was found that the epiblast is capable of unidirectional apical to basal sodium transport, in towards the underlying intraembryonic space. The Na-K-ATPase was localized by [3H]ouabain binding and autoradiography near the basal surfaces of the cells, and the number of pump sites was quantified. The transport rate of sodium was determined with 22Na. Electrophysiological studies on embryos at primitive streak stages showed a transepithelial potential of about +16 +/- 5 mV (basal side positive) which was sensitive to strophanthidin. Applying similar voltages but of reverse polarity to isolated sheets of epiblast caused a rapid reversal of some of their morphological polarity markers as well as some of their physiological functions. The relevance of these results to development is discussed.
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