Retinal ganglion cell axons drive the proliferation of astrocytes in the developing rodent optic nerve.

We show that the proliferation of astrocytes in the developing rodent optic nerve absolutely depends on axons and that this axonal influence depends on axonal transport but not on axonal electrical activity. We also show that purified retinal ganglion cells stimulate DNA synthesis in optic nerve astrocytes in culture and that the effect can be mimicked by fibroblast growth factor but not by neuregulins or several other growth factors. Taken together with previous findings, our present results indicate that axons promote glial cell proliferation and survival in the developing optic nerve by at least three distinct mechanisms.

Glial cells are increased proportionally in transgenic optic nerves with increased numbers of axons.

To study how an increase in axon number influences the number of glial cells in the mammalian optic nerve, we have analyzed a previously described transgenic mouse that expresses the human bcl-2 gene from a neuron-specific enolase promoter. In these mice, the normal postnatal loss of retinal ganglion cell axons is greatly decreased and, as a consequence, the number of axons in the optic nerve is increased by approximately 80% compared with wild-type mice. Remarkably, the numbers of oligodendrocytes, astrocytes, and microglial cells are all increased proportionally in the transgenic optic nerve. The increase in oligodendrocytes apparently results from both a decrease in normal oligodendrocyte death and an increase in oligodendrocyte precursor cell proliferation, whereas the increase in astrocytes apparently results from an increase in the proliferation of astrocyte lineage cells. Unexpectedly, the transgene is expressed in oligodendrocytes and astrocytes, but this does not seem to be responsible for the increased numbers of these cells. These findings indicate that developing neurons and glial cells can interact to adjust glial cell numbers appropriately when neuronal numbers are increased. We also show that the expression of the bcl-2 transgene in retinal ganglion cells protects the cell body from programmed cell death when the axon is cut, but it does not protect the isolated axon from Wallerian degeneration, even though the transgene-encoded protein is present in the axon.

Ciliary neurotrophic factor enhances the rate of oligodendrocyte generation.

Although ciliary neurotrophic factor (CNTF) is a potent survival factor for many types of neurons and glial cells in vitro, there is currently no evidence that it participates in normal development. Here we show that CNTF greatly enhances the rate of oligodendrocyte generation. Proliferation of oligodendrocyte precursor cells purified from rodent optic nerves and cultured in platelet-derived growth factor-containing medium is significantly increased by CNTF. Similarly, the number of proliferating oligodendrocyte precursor cells in developing optic nerves of transgenic mice lacking CNTF is decreased by up to threefold and the number of oligodendrocytes is transiently decreased; proliferation is restored to normal by the delivery of exogenous CNTF into the developing optic nerve. Both oligodendrocyte number and myelination ultimately attain wild-type values in CNTF-deficient adult mice, indicating that CNTF is not necessary for either oligodendrocyte differentiation or myelination, although it normally accelerates oligodendrocyte development by enhancing the proliferation of oligodendrocyte precursor cells.

Quantification of normal cell death in the rat retina: implications for clone composition in cell lineage analysis.

Naturally occurring cell death complicates the analysis of cell lineage studies by making the surviving members of a clone appear more closely related than they actually are. Here we ask how much normal cell death occurs during rat retinal development, and whether that amount of death is sufficient to confuse the analysis of cell lineage relationships. We measure total cell death in the retina by combining relative counts of dead cells with absolute measurements of total cell loss. For most cell types, but not rods, we find that half of the cells generated die during normal retinal development. We use a computer model to quantify the effects of different amounts of cell death in a simulated lineage study. The simulation indicates that 50% cell death means that clonal variability analysed after the cell death period is not necessarily a good indicator of how much variability actually occurs in the underlying lineage.

Evidence for large-scale astrocyte death in the developing cerebellum.

There is increasing evidence that some glial cells die during normal vertebrate development, but the extent of the death and the types of glial cells that die remain uncertain. We have analyzed pyknotic cells in the developing postnatal rat cerebellum. During the first postnatal week, the majority of pyknotic cells are in the developing white matter where their number peaks at about postnatal day 7 (P7) and then declines sharply. Pyknotic cells in the internal granule cell layer peak at P10, while those in the molecular and external granule cell layers peak later. Both electron microscopy and in situ end labeling of DNA catalyzed by terminal deoxynucleotidyl transferase confirm that the pyknotic cells are undergoing apoptosis. Immunohistochemical staining suggests that 50-70% of the pyknotic cells in the white matter and internal granule cell layer are astrocytes. We estimate that at P7, as many as 50% of the white matter cells die and, of these, more than half appear to be astrocytes.

Programmed cell death and the control of cell survival.

We draw the following tentative conclusions from our studies on programmed cell death (PCD): (i) the amount of normal cell death in mammalian development is still underestimated; (ii) most mammalian cells constitutively express the proteins required to undergo PCD; (iii) the death programme operates by default when a mammalian cell is deprived of signals from other cells; (iv) many normal cell deaths may occur because cells fail to obtain the extracellular signals they need to suppress the death programme; and (v) neither the nucleus nor mitochondrial respiration is required for PCD (or Bcl-2 protection from PCD), raising the possibility that the death programme, like mitosis, is orchestrated by a cytosolic regulator that acts on multiple organelles in parallel.

Autocrine signals enable chondrocytes to survive in culture.

We recently proposed that most mammalian cells other than blastomeres may be programmed to kill themselves unless continuously signaled by other cells not to. Many observations indicate that some mammalian cells are programmed in this way, but is it the case for most mammalian cells? As it is impractical to test all of the hundreds of types of mammalian cells, we have focused on two tissues--lens and cartilage--which each contain only a single cell type: if there are cells that do not require signals from other cells to avoid programmed cell death (PCD), lens epithelial cells and cartilage cells (chondrocytes) might be expected to be among them. We have previously shown that rat lens epithelial cells can survive in serum-free culture without signals from other cell types but seem to require signals from other lens epithelial cells to survive: without such signals they undergo PCD. We show here that the same is true for rat (and chick) chondrocytes. They can survive for weeks in culture at high cell density in the absence of other cell types, serum, or exogenous proteins or signaling molecules, but they die with the morphological features of apoptosis in these conditions at low cell density. Medium from high density cultures, FCS, or a combination of known growth factors, all support prolonged chondrocyte survival in low density cultures, as long as antioxidants are also present. Moreover, medium from high density chondrocyte cultures promotes the survival of lens epithelial cells in low density cultures and vice versa. Chondrocytes isolated from adult rats behave similarly to those isolated from developing rats. These findings support the hypothesis that most mammalian cells require signals from other cells to avoid PCD, although the signals can sometimes be provided by cells of the same type, at least in tissues that contain only one cell type.

Programmed cell death and Bcl-2 protection in the absence of a nucleus.

The molecular basis of programmed cell death (PCD) is unknown. An important clue is provided by the Bcl-2 protein, which can protect many cell types from PCD, although it is not known where or how it acts. Nuclear condensation, DNA fragmentation and a requirement for new RNA and protein synthesis are often considered hallmarks of PCD. We show here, however, that anucleate cytoplasts can undergo PCD and that Bcl-2 and extracellular survival signals can protect them, indicating that, in some cases at least, the nucleus is not required for PCD or for Bcl-2 or survival factor protection. We propose that PCD, like the cell cycle, is orchestrated by a cytoplasmic regulator that has multiple intracellular targets.

Programmed cell death and the control of cell survival: lessons from the nervous system.

During the development of the vertebrate nervous system, up to 50 percent or more of many types of neurons normally die soon after they form synaptic connections with their target cells. This massive cell death is thought to reflect the failure of these neurons to obtain adequate amounts of specific neurotrophic factors that are produced by the target cells and that are required for the neurons to survive. This neurotrophic strategy for the regulation of neuronal numbers may be only one example of a general mechanism that helps to regulate the numbers of many other vertebrate cell types, which also require signals from other cells to survive. These survival signals seem to act by suppressing an intrinsic cell suicide program, the protein components of which are apparently expressed constitutively in most cell types.

Large-scale normal cell death in the developing rat kidney and its reduction by epidermal growth factor.

Although normal cell death is known to occur in many developing vertebrate organs, it has not been thought to play an important part in the development of the mammalian kidney. We show here that normal cell death is found in both the nephrogenic region and medullary papilla of the developing rat kidney and, in each of these areas, it follows a distinct developmental time course. As many as 3% of the cells in these areas have a typical apoptotic morphology and the dead cells seem to be cleared rapidly (within 1-2 hours) by phagocytosis by neighbouring parenchymal cells. These values are similar to those in vertebrate neural tissues where 50% or more of the cells die during normal development, suggesting that large-scale death is a normal feature of kidney development. We also show that in vivo treatment with epidermal growth factor inhibits cell death in the developing kidney, consistent with the possibility that the cells normally die because they lack sufficient survival factors. Our findings suggest that the extent of normal cell death in developing animals is still greatly underestimated and they raise the possibility that many of these cell deaths may reflect limiting amounts of survival factors.

Control of lens epithelial cell survival.

We have studied the survival requirements of developing lens epithelial cells to test the hypothesis that most cells are programmed to kill themselves unless they are continuously signaled by other cells not to do so. The lens cells survived for weeks in both explant cultures and high-density dissociated cell cultures in the absence of other cells or added serum or protein, suggesting that they do not require signals from other cell types to survive. When cultured at low density, however, they died by apoptosis, suggesting that they depend on other lens epithelial cells for their survival. Lens epithelial cells cultured at high density in agarose gels also survived for weeks, even though they were not in direct contact with one another, suggesting that they can promote one another's survival in the absence of cell-cell contact. Conditioned medium from high density cultures promoted the survival of cells cultured at low density, suggesting that lens epithelial cells support one another's survival by secreting survival factors. We show for the first time that normal cell death occurs within the anterior epithelium in the mature lens, but this death is strictly confined to the region of the anterior suture.

Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA.

When the mammalian proto-oncogene bcl-2 is overexpressed it can protect various types of cells both from normal and from experimentally induced apoptosis, but the molecular mechanisms involved are unknown. Although the Bcl-2 protein is membrane-associated, its subcellular location is controversial: two studies have suggested that it is mainly associated with the nuclear envelope and endoplasmic reticulum, whereas another study has suggested that it is mainly located in the inner mitochondrial membrane. The latter study has suggested that Bcl-2 might protect cells from apoptosis by altering mitochondrial function and that mitochondria may be involved in apoptosis. Here we report that human mutant cell lines that lack mitochondrial DNA (mtDNA), and therefore do not have a functional respiratory chain, can still be induced to die by apoptosis, and that they can be protected from apoptosis by the overexpression of bcl-2, suggesting that neither apoptosis nor the protective effect of bcl-2 depends on mitochondrial respiration. We also show that the Bcl-2 protein in overexpressing cells is associated with the nuclear envelope and endoplasmic reticulum, as well as with mitochondria.

Visualization of O-2A progenitor cells in developing and adult rat optic nerve by quisqualate-stimulated cobalt uptake.

Some macroglial cells of the O-2A lineage express glutamate receptor channels of the quisqualate/kainate type and take up extracellular cobalt when activated by glutamate agonists. These cells can be identified both in vitro and in situ following precipitation and intensification of the intracellular cobalt. We have used this technique to characterize these cells in the developing and adult rat optic nerve. In purified cultures of optic nerve cells, O-2A progenitor cells and type 2 astrocytes took up cobalt in the presence of quisqualate, while oligodendrocytes, type 1 astrocytes, and microglial cells did not. When whole optic nerves of various postnatal ages were exposed to quisqualate and cobalt, a subpopulation of glial cells took up cobalt. Cobalt uptake in vitro and in situ was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. The number, morphology, and spatial distribution of cobalt-filled cells in situ varied with age. In perinatal nerves, 9% of glial cells took up cobalt. These cells had a simple unipolar or bipolar morphology and were two to three times more concentrated at the chiasm end than at the eye end of the nerve. During subsequent development, this gradient disappeared and the cobalt-filled cells became progressively more complex in morphology and increased in number and density, reaching a peak toward the end of the second postnatal week. The number subsequently declined to about 16,000 (7%) in the adult nerve. The processes of some cobalt-filled cells appeared to contact nodes of Ranvier. All cobalt-filled cells in 2 1/2-week-old optic nerves had a similar ultrastructural appearance and did not resemble either mature oligodendrocytes or astrocytes. Our results suggest that the cells stimulated by quisqualate to take up cobalt in the optic nerve are the in vivo counterpart of O-2A progenitor cells. We found no evidence that any of these cells are type 2 astrocytes.

Cell death in the oligodendrocyte lineage.

We have recently found that about 50% of newly formed oligodendrocytes normally die in the developing rat optic nerve. When purified oligodendrocytes or their precursors are cultured in the absence of serum or added signalling molecules, they die rapidly with the characteristics of programmed cell death. This death is prevented either by the addition of medium conditioned by cultures of their normal neighboring cells in the developing optic nerve, or by the addition of platelet-derived growth factor (PDGF) or insulin-like growth factors (IGFs). Increasing PDGF in the developing optic nerve decreases normal oligodendrocyte death by up to 90% and doubles the number of oligodendrocytes, suggesting that this normally occurring glial cell death might result from a competition for limiting amounts of survival signals. These results suggest that competition for limiting amounts of survival factors is not confined to developing neurons, and raise the possibility that a similar mechanism may be responsible for some naturally occurring cell deaths in nonneural tissues.

Cell death and control of cell survival in the oligodendrocyte lineage.

Dead cells are observed in many developing animal tissues, but the causes of these normal cell deaths are mostly unknown. We show that about 50% of oligodendrocytes normally die in the developing rat optic nerve, apparently as a result of a competition for limiting amounts of survival signals. Both platelet-derived growth factor and insulin-like growth factors are survival factors for newly formed oligodendrocytes and their precursors in culture. Increasing platelet-derived growth factor in the developing optic nerve decreases normal oligodendrocyte death by up to 90% and doubles the number of oligodendrocytes in 4 days. These results suggest that a requirement for survival signals is more general than previously thought and that some normal cell deaths in nonneural tissues may also reflect competition for survival factors.

Platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture.

The various cell types in a multicellular animal differentiate on a predictable schedule but the mechanisms responsible for timing cell differentiation are largely unknown. We have studied a population of bipotential glial (O-2A) progenitor cells in the developing rat optic nerve that gives rise to oligodendrocytes beginning at birth and to type-2 astrocytes beginning in the second postnatal week. Whereas, in vivo, these O-2A progenitor cells proliferate and give rise to postimitotic oligodendrocytes over several weeks, in serum-free (or low-serum) culture they stop dividing prematurely and differentiate into oligodendrocytes within two or three days. The normal timing of oligodendrocyte development can be restored if embryonic optic-nerve cells are cultured in medium conditioned by type-1 astrocytes, the first glial cells to differentiate in the nerve: in this case the progenitor cells continue to proliferate, the first oligodendrocytes appear on the equivalent of the day of birth, and new oligodendrocytes continue to develop over several weeks, just as in vivo. Here we show that platelet-derived growth factor (PDGF) can replace type-1-astrocyte-conditioned medium in restoring the normal timing of oligodendrocyte differentiation in vitro and that anti-PDGF antibodies inhibit this property of the appropriately conditioned medium. We also show that PDGF is present in the developing optic nerve. These findings suggest that type-1-astrocyte-derived PDGF drives the clock that times oligodendrocyte development.

Evidence that migratory oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells are kept out of the rat retina by a barrier at the eye-end of the optic nerve.

There is evidence that oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells migrate along the developing rat optic nerve from the chiasm toward the eye before differentiating into oligodendrocytes that myelinate the retinal ganglion cell axons in the nerve. Why, then, do these progenitor cells not migrate into the eye, differentiate into oligodendrocytes and myelinate the nerve fibre layer of the retina? Myelination would opacify the neural retina and thereby severely impair vision. Here we provide evidence that there is a barrier at the eye-end of the rat optic nerve that prevents the migration of O-2A progenitor cells into the retina. Our findings in the rat support a previous hypothesis that such a barrier keeps myelin-forming glial cells out of the human retina.

The role of laminin and the laminin/fibronectin receptor complex in the outgrowth of retinal ganglion cell axons.

Chick embryo retinal ganglion cell (RGC) axons grow to the optic tectum along a stereotyped route, as if responding to cues distributed along the pathway. We showed previously that, in culture, RGCs from embryonic Day 6 retina are responsive to the neurite-promoting effects of the extracellular matrix glycoprotein laminin and that this response is lost by RGCs at a later stage of development. Here we report that, before axon outgrowth is initiated in vivo, laminin, is expressed along the optic pathway at nonbasal lamina sites that are accessible to the growth cones of RGC axons. The distribution of laminin within the pathway is consistent with its localization at the end-feet of neuroepithelial cells that line the route, and it continues to be expressed at these marginal sites during the first week of embryonic development. At later stages, concomitant with the loss of response by RGCs in culture, laminin becomes restricted to basal laminae at the retinal inner limiting membrane and pial surface of the optic pathway. Neurofilament-positive RGC axons bind a monoclonal antibody, JG22, which recognizes the laminin/fibronectin receptor complex, and continue to do so throughout embryonic development. We show that, in vitro, the JG22 antigen expressed by RGCs appears to function as a laminin receptor, by demonstrating that JG22 antibody blocks neurite outgrowth on a substrate of laminin. These findings are consistent with the possibility that laminin defines a transient performed pathway specifically recognized by early RGC growth cones as they navigate toward their central target.