An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism.

BACKGROUND: Simple epithelia encase developing embryos and organs. Although these epithelia consist of only one or two layers of cells, they must provide tight barriers for the tissues that they envelop. Apoptosis occurring within these simple epithelia could compromise this barrier. How, then, does an epithelium remove apoptotic cells without disrupting its function as a barrier? RESULTS: We show that apoptotic cells are extruded from a simple epithelium by the concerted contraction of their neighbors. A ring of actin and myosin forms both within the apoptotic cell and in the cells surrounding it, and contraction of the ring formed in the live neighbors is required for apoptotic cell extrusion, as injection of a Rho GTPase inhibitor into these cells completely blocks extrusion. Addition of apoptotic MDCK cells to an intact monolayer induces the formation of actin cables in the cells contacted, suggesting that the signal to form the cable comes from the dying cell. The signal is produced very early in the apoptotic process, before procaspase activation, cell shrinkage, or phosphatidylserine exposure. Remarkably, electrical resistance studies show that epithelial barrier function is maintained, even when large numbers of dying cells are being extruded. CONCLUSIONS: We propose that apoptotic cell extrusion is important for the preservation of epithelial barrier function during cell death. Our results suggest that an early signal from the dying cell activates Rho in live neighbors to extrude the apoptotic cell out of the epithelium.

Extracellular control of cell size.

Both cell growth (cell mass increase) and progression through the cell division cycle are required for sustained cell proliferation. Proliferating cells in culture tend to double in mass before each division, but it is not known how growth and division rates are co-ordinated to ensure that cell size is maintained. The prevailing view is that coordination is achieved because cell growth is rate-limiting for cell-cycle progression. Here, we challenge this view. We have investigated the relationship between cell growth and cell-cycle progression in purified rat Schwann cells, using two extracellular signal proteins that are known to influence these cells. We find that glial growth factor (GGF) can stimulate cell-cycle progression without promoting cell growth. We have used this restricted action of GGF to show that, for cultured Schwann cells, cell growth rate alone does not determine the rate of cell-cycle progression and that cell size at division is variable and depends on the concentrations of extracellular signal proteins that stimulate cell-cycle progression, cell growth, or both.

Two molecularly distinct intracellular pathways to oligodendrocyte differentiation: role of a p53 family protein.

Both thyroid hormone (TH) and retinoic acid (RA) induce purified rat oligodendrocyte precursor cells in culture to stop division and differentiate. We show that these responses are blocked by the expression of a dominant-negative form of p53. Moreover, both TH and RA cause a transient, immediate early increase in the same 8 out of 13 mRNAs encoding intracellular cell cycle regulators and gene regulatory proteins, but only if protein synthesis is inhibited. Platelet-derived growth factor (PDGF) withdrawal also induces these cells to differentiate, but we show that the intracellular mechanisms involved are different from those involved in the hormone responses: the changes in cell cycle regulators differ, and the differentiation induced by PDGF withdrawal (or that which occurs spontaneously in the presence of PDGF) is not blocked by the dominant-negative p53. These results suggest that TH and RA activate the same intracellular pathway leading to oligodendrocyte differentiation, and that this pathway depends on a p53 family protein. Differentiation that occurs independently of TH and RA apparently involves a different pathway. It is likely that both pathways operate in vivo.

Lack of replicative senescence in cultured rat oligodendrocyte precursor cells.

Most mammalian somatic cells are thought to have a limited proliferative capacity because they permanently stop dividing after a finite number of divisions in culture, a state termed replicative cell senescence. Here we show that most oligodendrocyte precursor cells purified from postnatal rat optic nerve can proliferate indefinitely in serum-free culture if prevented from differentiating; various cell cycle-inhibitory proteins increase, but the cells do not stop dividing. The cells maintain high telomerase activity and p53- and Rb-dependent cell cycle checkpoint responses, and serum or genotoxic drugs induce them to acquire a senescence-like phenotype. Our findings suggest that some normal rodent precursor cells have an unlimited proliferative capacity if cultured in conditions that avoid both differentiation and the activation of checkpoint responses that arrest the cell cycle.

Evidence that axon-derived neuregulin promotes oligodendrocyte survival in the developing rat optic nerve.

It was previously shown that newly formed oligodendrocytes depend on axons for their survival, but the nature of the axon-derived survival signal(s) remained unknown. We show here that neuregulin (NRG) supports the survival of purified oligodendrocytes and aged oligodendrocyte precursor cells (OPCs) but not of young OPCs. We demonstrate that axons promote the survival of purified oligodendrocytes and that this effect is inhibited if NRG is neutralized. In the developing rat optic nerve, we provide evidence that delivery of NRG decreases both normal oligodendrocyte death and the extra oligodendrocyte death induced by nerve transection, whereas neutralization of endogenous NRG increases the normal death. These results suggest that NRG is an axon-associated survival signal for developing oligodendrocytes.

Differences between the clearance of apoptotic cells by professional and non-professional phagocytes.

Both professional and non-professional phagocytes [1] participate in clearing the massive numbers of cells that undergo apoptosis during animal development [2], but it is not known how they divide this task. Using time-lapse recordings of cells in culture, we show that professional phagocytes (brain macrophages or microglia) are highly motile, ingest apoptotic cells immediately, and digest them quickly. Non-professionals such as BHK and lens epithelial cells are sessile, often recognize apoptotic cells as soon as they die by showing characteristic palpating movements, but delay ingestion until several hours later. By pre-ageing apoptotic cells, we show that this delay is because the apoptotic cells must undergo further changes before non-professionals can ingest them. The difference was also apparent in vivo, using immunofluorescence and electron microscopy of the developing central nervous system. This arrangement favours prompt clearance by professionals if present in adequate numbers; if they are scarce, however, non-professional bystanders will reluctantly clear the apoptotic cells.

Long-term culture of purified postnatal oligodendrocyte precursor cells. Evidence for an intrinsic maturation program that plays out over months.

Oligodendrocytes myelinate axons in the vertebrate central nervous system (CNS). They develop from precursor cells (OPCs), some of which persist in the adult CNS. Adult OPCs differ in many of their properties from OPCs in the developing CNS. In this study we have purified OPCs from postnatal rat optic nerve and cultured them in serum-free medium containing platelet-derived growth factor (PDGF), the main mitogen for OPCs, but in the absence of thyroid hormone in order to inhibit their differentiation into oligodendrocytes. We find that many of the cells continue to proliferate for more than a year and progressively acquire a number of the characteristics of OPCs isolated from adult optic nerve. These findings suggest that OPCs have an intrinsic maturation program that progressively changes the cell's phenotype over many months. When we culture the postnatal OPCs in the same conditions but with the addition of basic fibroblast growth factor (bFGF), the cells acquire these mature characteristics much more slowly, suggesting that the combination of bFGF and PDGF, previously shown to inhibit OPC differentiation, also inhibits OPC maturation. The challenge now is to determine the molecular basis of such a protracted maturation program and how the program is restrained by bFGF.

Evidence that Wallerian degeneration and localized axon degeneration induced by local neurotrophin deprivation do not involve caspases.

The selective degeneration of an axon, without the death of the parent neuron, can occur in response to injury, in a variety of metabolic, toxic, and inflammatory disorders, and during normal development. Recent evidence suggests that some forms of axon degeneration involve an active and regulated program of self-destruction rather than a passive "wasting away" and in this respect and others resemble apoptosis. Here we investigate whether selective axon degeneration depends on some of the molecular machinery that mediates apoptosis, namely, the caspase family of cysteine proteases. We focus on two models of selective axon degeneration: Wallerian degeneration of transected axons and localized axon degeneration induced by local deprivation of neurotrophin. We show that caspase-3 is not activated in the axon during either form of degeneration, although it is activated in the dying cell body of the same neurons. Moreover, caspase inhibitors do not inhibit or retard either form of axon degeneration, although they inhibit apoptosis of the same neurons. Finally, we cannot detect cleaved substrates of caspase-3 and its close relatives immunocytochemically or caspase activity biochemically in axons undergoing Wallerian degeneration. Our results suggest that a neuron contains at least two molecularly distinct self-destruction programs, one for caspase-dependent apoptosis and another for selective axon degeneration.

An analysis of the early events when oligodendrocyte precursor cells are triggered to differentiate by thyroid hormone, retinoic acid, or PDGF withdrawal.

Oligodendrocyte precursor cells withdraw from the cell cycle and terminally differentiate after a limited number of cell divisions. The timing of cell-cycle withdrawal and differentiation is controlled by an intrinsic timer, which consists of a timing component that measures elapsed time and an effector component that arrests the cell cycle and initiates differentiation. The effector component can be triggered by either thyroid hormone (TH) or retinoic acid (RA). In this study we investigate how TH and RA act to trigger differentiation. We show the following: (1) Synthetic retinoids that can inhibit AP-1 transcription factors but do not activate gene transcription cannot trigger the effector mechanism, suggesting that TH and RA do not act only by inhibiting AP-1 activity as previously suggested. (2) Both TH and RA induce a transcriptionally dependent antigenic change in purified precursor cells within 2-4 h. (3) Unexpectedly, even before they differentiate, the precursor cells express ceramide galactosyltransferase (CGT), the enzyme that catalyzes the final step in the synthesis of galactocerebroside, an early marker of oligodendrocyte differentiation. (4) Neither TH nor RA directly activates the transcription of the CGT gene, a number of immediate early genes, or the genes that encode any of the known cyclin-dependent kinase inhibitors. (5) The withdrawal of the mitogen platelet-derived growth factor (PDGF), but not TH or RA treatment, causes a rapid decrease in c-fos, NGFI-A/Krox-24, and cyclin D2 mRNA, even though all three treatments trigger cell-cycle arrest and differentiation. (6) PDGF withdrawal and TH treatment, but not RA treatment, induce an increase in cyclin D3 mRNA within 4 h. Thus, we have not found any early changes in gene expression that occur with all three treatments that trigger oligodendrocyte differentiation.

A role for Sonic hedgehog in axon-to-astrocyte signalling in the rodent optic nerve.

Retinal ganglion cell (RGC) axons have been shown to stimulate the proliferation of astrocytes in the developing rodent optic nerve, but the signals that mediate this effect have not been identified. The following findings suggest that Sonic hedgehog (Shh) is one of the signals. (1) RGCs express both Shh mRNA and protein, whereas the optic nerve contains the protein but not the mRNA. (2) Astrocytes and their precursors in the developing optic nerve express the Hedgehog (Hh) receptor gene Patched (Ptc), suggesting that they are being signalled by an Hh protein. (3) Ptc expression in the nerve is greatly decreased by either nerve transection or by treatment with neutralizing anti-Shh antibodies, suggesting that it depends on axon-derived Shh. (4) Astrocyte proliferation in the developing nerve is reduced by treatment with anti-Shh antibodies, suggesting that Shh normally helps stimulate this proliferation.

Caspase activation in the terminal differentiation of human epidermal keratinocytes.

The epidermis is a multilayered squamous epithelium in which dividing basal cells withdraw from the cell cycle and progressively differentiate as they are displaced toward the skin surface. Eventually, the cells lose their nucleus and other organelles to become flattened squames, which are finally shed from the surface as bags of cross-linked keratin filaments enclosed in a cornified envelope [1]. Although keratinocytes can undergo apoptosis when stimulated by a variety of agents [2], it is not known whether their normal differentiation programme uses any components of the apoptotic biochemical machinery to produce the cornified cell. Differentiating keratinocytes have been reported to share some features with apoptotic cells, such as DNA fragmentation, but these features have not been seen consistently [3]. Apoptosis involves an intracellular proteolytic cascade, mainly mediated by members of the caspase family of cysteine proteases, which cleave one another and various key intracellular target proteins to kill the cell neatly and quickly [4]. Here, we show for the first time that caspases are activated during normal human keratinocyte differentiation and that this activation is apparently required for the normal loss of the nucleus.

An intrinsic timer that controls cell-cycle withdrawal in cultured cardiac myocytes.

Developing cardiac myocytes divide a limited number of times before they stop and terminally differentiate, but the mechanism that stops their division is unknown. To help study the stopping mechanism, we defined conditions under which embryonic rat cardiac myocytes cultured in serum-free medium proliferate and exit the cell cycle on a schedule that closely resembles that seen in vivo. The culture medium contains FGF-1 and FGF-2, which stimulate cell proliferation, and thyroid hormone, which seems to be necessary for stable cell-cycle exit. Time-lapse video recording shows that the cells within a clone tend to divide a similar number of times before they stop, whereas cells in different clones divide a variable number of times before they stop. Cells cultured at 33 degrees C divide more slowly but stop dividing at around the same time as cells cultured at 37 degrees C, having undergone fewer divisions. Together, these findings suggest that an intrinsic timer helps control when cardiac myocytes withdraw from the cell cycle and that the timer does not operate by simply counting cell divisions. We provide evidence that the cyclin-dependent kinase inhibitors p18 and p27 may be part of the timer and that thyroid hormone may help developing cardiac myocytes stably withdraw from the cell cycle.

Are caspases involved in the death of cells with a transcriptionally inactive nucleus? Sperm and chicken erythrocytes.

We show that mouse sperm die spontaneously within 1-2 days in culture and that treatment with either staurosporine (STS) and cycloheximide (CHX) or a peptide caspase inhibitor does not accelerate or delay the cell death. Chicken erythrocytes, by contrast, are induced to die by either serum deprivation or treatment with STS and CHX, and embryonic erythrocytes are more sensitive than adult erythrocytes to both treatments. Although these erythrocyte deaths display a number of features that are characteristic of apoptosis, they are not blocked, or even delayed, by peptide caspase inhibitors, and most of the cells die without apparently activating caspases. A small proportion of the dying erythrocytes do activate caspase-3, but even these cells, which seem to be the least mature erythrocytes, die just as quickly in the presence of caspase inhibitors. Our findings raise the possibility that both mouse sperm and chicken erythrocytes have a death programme that may not depend on caspases and that chicken erythrocytes lose caspases as they mature. Chicken erythrocytes may provide a useful 'stripped down' cell system to try to identify the protein components of such a death programme, which may serve to back-up the conventional caspase-dependent suicide mechanism in many cell types.

Cell number control and timing in animal development: the oligodendrocyte cell lineage.

Our studies of oligodendrocyte development in the rodent optic nerve provide clues as to how cell numbers and the timing of differentiation may be controlled during mammalian development. Both cell number and the timing of differentiation depend on intracellular programs and extracellular signals, which together control cell survival and cell division. As the cells seem to compete for limiting amounts of both survival signals and mitogens, the levels of these extracellular signals must be tightly regulated, but it is not known how this is achieved. The timing of cell-cycle exit, and therefore the onset of differentiation, seems to depend in part on the progressive accumulation of the intracellular Cdk inhibitor p27/Kip1, but it is still unclear how the level of this protein is controlled over time in the dividing cells. The timing of cell-cycle exit is also regulated by thyroid hormone, which, along with other hormones, seems to coordinate the timing of development in various organs, much as the timing of the multiple changes in metamorphosis in both vertebrates and invertebrates is coordinated by hormones. In this sense, one might think of mammalian development as a prolonged metamorphosis.

p27Kip1 alters the response of cells to mitogen and is part of a cell-intrinsic timer that arrests the cell cycle and initiates differentiation.

BACKGROUND: In many vertebrate cell lineages, precursor cells divide a limited number of times before they arrest and terminally differentiate into postmitotic cells. It is not known what causes them to stop dividing. We have been studying the 'stopping' mechanism in the proliferating precursor cells that give rise to oligodendrocytes, the cells that make myelin in the central nervous system. We showed previously that the cyclin-dependent kinase inhibitor p27Kip1 (p27) progressively accumulates in cultured precursor cells as they proliferate and that the time course of the increase is consistent with the possibility that p27 accumulation is part of a cell-intrinsic timer that arrests the cell cycle and initiates differentiation at the appropriate time. RESULTS: We now provide direct evidence that p27 is part of the intrinsic timer. We show that although p27-/- precursor cells stop dividing and differentiate almost as fast as wild-type cells when deprived of mitogen, when stimulated by saturating amounts of mitogen they have a normal cell-cycle time but tend to go through one or two more divisions than wild-type cells before they stop and differentiate. Cells that are p27+/- behave in an intermediate way, going through at most one extra division, indicating that the levels of p27 matter in the way the timer works. We also show that p27-/- precursor cells are more sensitive than wild-type cells to the mitogenic effect of platelet-derived growth factor. CONCLUSIONS: These findings demonstrate that p27 is part of the normal timer that determines when oligodendrocyte precursor cells stop dividing and differentiate, at least in vitro. It seems likely that p27 plays a similar role in many other cell lineages, which could explain the phenotypes of the p27-/- and p27+/- mice.

A role for caspases in lens fiber differentiation.

There is increasing evidence that programmed cell death (PCD) depends on a novel family of intracellular cysteine proteases, called caspases, that includes the Ced-3 protease in the nematode Caenorhabditis elegans and the interleukin-1beta-converting enzyme (ICE)-like proteases in mammals. Some developing cells, including lens epithelial cells, erythroblasts, and keratinocytes, lose their nucleus and other organelles when they terminally differentiate, but it is not known whether the enzymatic machinery of PCD is involved in any of these normal differentiation events. We show here that at least one CPP32 (caspase-3)-like member of the caspase family becomes activated when rodent lens epithelial cells terminally differentiate into anucleate lens fibers in vivo, and that a peptide inhibitor of these proteases blocks the denucleation process in an in vitro model of lens fiber differentiation. These findings suggest that at least part of the machinery of PCD is involved in lens fiber differentiation.

Continuous observation of multipotential retinal progenitor cells in clonal density culture.

All neural cell types in the vertebrate retina, except astrocytes, have been shown to develop from multipotential progenitor cells. It is not known, however, to what extent the progenitor cells are heterogeneous in their developmental potential or to what extent cell-cell interactions versus cell-autonomous factors influence the types of cells they become. To address these issues we developed a clonal-density cell culture system where mouse retinal progenitor cells can survive, divide, and differentiate. We followed the development of clones both by continuous time-lapse video microscopy and by daily microscopic observation. We show that even when cultured at clonal density in a homogeneous general environment, where they cannot contact cells outside their own clone, the retinal progenitor cells vary in proliferative capacity, cell cycle time, and in the cell types that they generate. In addition, we show that under these conditions single progenitor cells can generate both neurons and glia, in which case the neurons almost always develop before glial cells, as is the case in vivo.

Müller-cell-derived leukaemia inhibitory factor arrests rod photoreceptor differentiation at a postmitotic pre-rod stage of development.

In the present study, we examine rod photoreceptor development in dissociated-cell cultures of neonatal mouse retina. We show that, although very few rhodopsin+ rods develop in the presence of 10% foetal calf serum (FCS), large numbers develop in the absence of serum, but only if the cell density in the cultures is high. The rods all develop from nondividing rhodopsin- cells, and new rods continue to develop from rhodopsin- cells for at least 6-8 days, indicating that there can be a long delay between when a precursor cell withdraws from the cell cycle and when it becomes a rhodopsin+ rod. We show that FCS arrests rod development in these cultures at a postmitotic, rhodopsin-, pre-rod stage. We present evidence that FCS acts indirectly by stimulating the proliferation of Müller cells, which arrest rod differentiation by releasing leukaemia inhibitory factor (LIF). These findings identify an inhibitory cell-cell interaction, which may help to explain the long delay that can occur both in vitro and in vivo between cell-cycle withdrawal and rhodopsin expression during rod development.

Is programmed cell death required for neural tube closure?

Programmed cell death (PCD) plays an important part in animal development. It is responsible for eliminating the cells between developing digits, for example, and is involved in hollowing out solid structures to create cavities (reviewed in [1] [2]). There are many cases, however, where PCD occurs in developing tissues but its function is unknown. Important examples are seen during the folding, pinching off, and fusion of epithelial sheets during vertebrate morphogenesis, as in the formation of the neural tube and lens vesicle [2]; PCD is an invariable accompaniment to these processes, but it is unclear whether it is required for the processes to occur or is just an unavoidable consequence of them. There is increasing evidence that PCD in animals is mediated by a family of cysteine proteases, known as caspases, which are thought to act in a proteolytic cascade, cleaving one another and key intracellular proteins to kill the cell in a controlled way [3] [4]. Inhibitors of caspases are, therefore, potential tools for studying the roles of PCD during animal development [5] [6]. Here, we show that peptide caspase inhibitors block neural tube closure in explanted chick embryos, suggesting that PCD is required for this crucial developmental process.