Esmond J. Sanders

Programmed Cell Death in Development

Although cell death has long been recognized to be a significant element in the process of embryonic morphogenesis, its relationships to differentiation and its mechanisms are only now becoming apparent. This new appreciation has come about not only through advances in the understanding of cell death in parallel immunological and pathological situations, but also through progress in developmental genetics which has revealed the roles played by death in the cell lineages of invertebrate embryos. In this review, we discuss programmed cell death as it is understood in developmental situations, and its relationship to apoptosis. We describe the morphological and biochemical features of apoptosis, and some methods for its detection in tissues. The occurrence of programmed cell death during invertebrate development is reviewed, as well as selected examples in vertebrate development. In particular, we discuss cell death in the early vertebrate embryo, in limb development, and in the nervous system.

The vertebrate tail bud: three germ layers from one tissue.

SummaryThe tail bud of amniote embryos comprises a mass of apparently undifferentiated mesenchymal cells located at the caudal limit of the embryo, representing the remains of Hensens node and the primitive streak. These cells have the potential to give rise to a variety of different tissues including the posterior or ‘secondary’ neural tube, the tail gut, and somites and their derivatives. This seemingly homogeneous accumulation of cells therefore has the capacity to differentiate into tissues which in more cranial regions of the embryo are derived from cells of different germ layers. In this review, the tissue contributions of the tail bud in various vertebrate classes are discussed, with particular attention to the mesenchymal-to-epithelial transformation that characterizes the process of secondary neurulation, and which distinguishes it from the epithelial folding that occurs during primary neurulation in more cranial regions. Recent studies suggest that the transformation is accompanied by extensive changes in the cell surface oligosaccharide complement of the differentiating cells, and that the sialyted form of N-CAM is expressed both temporally and spatially in a manner that suggests a role for it in the process. The pluripotential nature of the tail bud mesenchyme may be revealed experimentally by grafting the tissue ectopically, or by culturing it on different substrata. In the latter case, the mesenchyme can be demonstrated to give rise to myocytes, chondrocytes, neuroepithelium and neural crest derivatives such as melanocytes, depending on the nature of the culture substratum. It is concluded that the tail bud mesenchyme represents a developing system which is readily amenable to experimentation and should provide insights into the general mechanisms of cell differentiation and transformation.

Chicken oocyte growth: receptor-mediated yolk deposition

During the rapid final stage of growth, chicken oocytes take up massive amounts of plasma components and convert them to yolk. The oocyte expresses a receptor that binds both major yolk lipoprotein precursors, vitellogenin (VTG) and very low density lipoprotein (VLDL). In the present study, in vivo transport tracing methodology, isolation of coated vesicles, ligand- and immuno-blotting, and ultrastructural immunocytochemistry were used for the analysis of receptor-mediated yolk formation. The VTG/VLDL receptor was identified in coated profiles in the oocyte periphery, in isolated coated vesicles, and within vesicular compartments both outside and inside membrane-bounded yolk storage organelles (yolk spheres). VLDL particles colocalized with the receptor, as demonstrated by ultrastructural visualization of VLDL-gold following intravenous administration, as well as by immunocytochemical analysis with antibodies to VLDL. Lipoprotein particles were shown to reach the oocyte surface by passage across the basement membrane, which possibly plays an active and selective role in yolk precursor accessibility to the oocyte surface, and through gaps between the follicular granulosa cells. Following delivery of ligands from the plasma membrane into yolk spheres, proteolytic processing of VTG and VLDL by cathepsin D appears to correlate with segregation of receptors and ligands which enter disparate sub-compartments within the yolk spheres. In small, quiescent oocytes, the VTG/VLDL receptor was localized to the central portion of the cell. At onset of the rapid growth phase, it appears that this pre-existing pool of receptors redistributes to the peripheral region, thereby initiating yolk formation. Such a redistribution mechanism would obliterate the need for de novo synthesis of receptors when the oocytes energy expenditure is to be utilized for plasma membrane synthesis, establishment and maintenance of intracellular topography and yolk formation, and preparation for ovulation.

Identification, regulation and role of tissue inhibitor of metalloproteinases-4 (TIMP-4) in human platelets

Matrix metalloproteinase‐2 (MMP‐2) released during activation of human platelets by aggregating agents and cancer cells is known to stimulate platelet aggregation. The expression, activity and role of tissue inhibitors of metalloproteinases (TIMPs), natural inhibitors of MMPs, in isolated human platelets were investigated. Western blot, reverse zymography, immunogold electron microscopy, aggregometry (collagen‐, thrombin and HT‐1080 human fibrosarcoma cells‐induced aggregation), flow cytometry and the release of 14C‐serotonin from labelled platelets recruited to the aggregate were used to characterize the presence and function of platelet TIMPs. TIMP‐4 (23 kDa) has been identified as the major MMP inhibitor (12–16 ng per 108 platelets) in human platelets. Platelets expressed lower (<1 ng per 108 platelets) amounts of TIMP‐1. No other TIMPs were detected using Western blot analysis. TIMP‐4 co‐localized with MMP‐2 in resting platelets and was released upon platelet aggregation induced by collagen and thrombin. Collagen resulted also in the release of higher molecular weight (60 kDa) complexes of TIMP‐4. The release of TIMP‐4 was reduced by prostacyclin and S‐nitroso‐glutathione (GSNO), an NO donor. Human recombinant TIMP‐4 (rTIMP‐4), but not human rTIMP‐1, inhibited partially both platelet aggregation and recruitment. The recombinant TIMP‐4 potentiated the recruitment inhibitor effects of GSNO. TIMP‐4 was not released during platelet aggregation induced by HT‐1080 cells. Human rTIMP‐4 exerted a biphasic effect on HT‐1080 cells‐induced aggregation. Thus, TIMP‐4 is the major intraplatelet MMP inhibitor and it is involved in regulation of platelet aggregation and recruitment.

Development of the basal lamina and extracellular materials in the early chick embryo.

SummaryChick embryos at developmental stages up to primitive streak formation were fixed in a mixture of tannic acid and glutaraldehyde. A basal lamina was present in the unincubated embryo and consisted of a lucent lamina interna and a lamina densa. At the primitive streak stage the lamina densa showed a periodicity of stained elements. Densely stained materials were present on the cell surfaces lining the cavity between the epiblast and endoblast, and on the mesoderm cells within this cavity. Considerable amounts of extracellular material were observed in the cavity. Hyaluronidase treatment removed the cell surface and extracellular material, indicating that hyaluronic acid is a major component. This enzyme disrupted the basal lamina, leaving a fibrillar remnant with no periodic structure. It is therefore suggested that the dense periodicities consist of glycosaminoglycan built on an enzyme-resistant framework which is probably collagen. Enzyme-resistant fibrils, presumably collagen precursors, are present elsewhere within the tissue spaces.

Growth hormone as an early embryonic growth and differentiation factor

In this review we consider the evidence that growth hormone (GH) acts in the embryo as a local growth, differentiation, and cell survival factor. Because both GH and its receptors are present in the early embryo before the functional differentiation of pituitary somatotrophs and before the establishment of a functioning circulatory system, the conditions are such that GH may be a member of the large battery of autocrine/paracrine growth factors that control embryonic development. It has been clearly established that GH is able to exert direct effects, independent of insulin-like growth factor-I (IGF-I), on the differentiation, proliferation, and survival of cells in a wide variety of tissues in the embryo, fetus, and adult. The signaling pathways behind these effects of GH are now beginning to be determined, establishing early extrapituitary GH as a bona fide developmental growth factor.

Ultrastructural identification of apoptotic nuclei using the TUNEL technique

SummaryWe describe an ultrastructural adaptation of the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) for the identification of DNA fragmentation. Thin sections of tissue embedded in hydrophilic resin were nick end labelled with biotinylated dUTP which was subsequently labelled with avidin conjugated to gold particles. The technique was validated by labelling the nuclei of L929-8 cells treated with tumour necrosis factor α. These cells are known to respond to treatment with the factor by undergoing apoptosis. The method was then used on tissue from the chick embryo which is known to be undergoing programmed cell death. This tissue was from the neural tube and the posterior necrotic zone of the limb bud, where cells can be identified as undergoing apoptosis based on the morphology of their nuclei. The method specifically labelled heterochromatin adjacent to the nuclear envelope as well as the associated with the nucleolus of cells from regions of the embryo where programmed cell death was expected. In addition to labelling the nuclei of cells that were clearly undergoing apoptosis, the method also identified nuclei of apparently normal cells. This method, used in conjunction with corroborating techniques, provides a means for the early detection of cells undergoing DNA fragmentation, before the onset of gross apoptotic morphology, and in cells that do not show classical apoptotic characteristics.

Potential roles for tumour necrosis factorα during embryonic development

This paper reviews the evidence indicating possible roles for tumour necrosis factor-alpha (TNFα) in development. It is proposed that TNFα may have essentially three major roles during embryonic development, which may be analogous to its roles in the immune system and during inflammation: a role in programmed cell death; a role as a cellular growth and differentiation factor; and also a role in the remodelling of extracellular matrix, and the regulation of cell adhesion molecules and integrins. The concept of the existence of a cytokine array during embryogenesis, analogous to that occurring in inflammation, is discussed, as well as potential roles for TNFα in the induction of ubiquitin; protective mechanisms embryonic cells may employ against TNFα-mediated cytotoxicity; and a consideration of the role TNFα may play in a “free radical theory of development”.

The effects of axotomy on bullfrog sympathetic neurones.

1. The effects of axotomy on the electrical properties of B cells in paravertebral sympathetic ganglia were studied using standard intracellular recording techniques. The effects were apparent after 1 week and persisted throughout the 47 days of study. 2. Action potential duration (spike width) and amplitude (spike height) were significantly increased in axotomized neurones. 3. The duration of the after‐hyperpolarization which followed the action potential showed considerable scatter in control neurones (mean +/‐ S.E. of mean, 159.0 +/‐ 5.8 ms for 100 cells). Following axotomy, the duration was significantly reduced (50.9 +/‐ 2.3 ms for 97 cells). The amplitude of the after‐hyperpolarization was also significantly smaller in axotomized neurones. 4. Changes in the characteristics of the action potential and the after‐hyperpolarization in axotomized neurones were not due to alteration in resting membrane potential or input resistance which were unchanged after axotomy. Rheobase current was significantly increased. 5. There was neither a significant depression of the rate of rise or the amplitude of orthodromically evoked nicotinic e.p.s.p.s nor any obvious ultrastructural alteration following axotomy. 6. Despite the decrease in the duration of the after‐hyperpolarization, the rate of discharge in response to constant current injection was little changed in axotomized neurones. 7. Although axotomy produces significant changes in several measurable electrophysiological parameters in bullfrog sympathetic ganglion cells, the present results imply that mature neurones are able to maintain relatively normal electrical activity despite injury.

The mechanosensory apparatus of the femoral tactile spine of the cockroach, Periplaneta americana.

SummaryTactile spines are large cuticular sense organs that appear to provide insects with a sense of touch which is spatially coarse but of great sensitivity. Cockroach legs have a number of these spines on each leg and a particularly prominent spine on the end of each femur, the femoral tactile spine. The ease of recording afferent activity from this spine during mechanical stimulation has made it one of the most thoroughly studied insect mechanoreceptors and yet it has never been examined by electron microscopy.We report here the results of an examination of the femoral tactile spine by both scanning and transmission electron microscopy, as well as by light microscopy. The spine is shown to be innervated by a single sensory bipolar neuron with its soma located in the base of the spine. A canal through the wall of the spine leads to the outside and emerges just above the junction between the base of the spine and its articulating socket membrane. The sensory dendrite of the neuron passes from the soma through this canal and forms a modified ciliary sensory ending with the typical dendritic sheath and dense tubular body that is characteristic of insect mechanosensory cuticular sensilla. The tubular body is embedded in a cuticular terminal plug which closes the exterior end of the canal but appears to be fastened to the spine by a very flexible ring of cuticle. This plug is connected to the socket membrane by a specialized socket attachment which presumably serves to move the plug relative to the wall of the spine during movement of the spine within the socket. The morphology of this sensillum is discussed in terms of the possible ways in which it is stimulated by movements of the spine and also in light of the dynamic behaviour of the receptor which is now very well described.