Martin C. Raff

GALACTOCEREBROSIDE IS A SPECIFIC CELL-SURFACE ANTIGENIC MARKER FOR OLIGODENDROCYTES IN CULTURE

A TEMPERATURE-SENSITIVE (ts) mutant of the highly oncogenic group A (ref. 1) human adenovirus type 12 (H12), H12ts401 (ref. 2) is unable to establish stable transformation of cells in restrictive conditions3. Cells transformed by ts401 at the permissive temperature and shifted to nonpermissive temperatures show a reversion to a normal phenotype; wild-type H12-transformed cells, in contrast, exhibit a transformed phenotype when grown in either restrictive or permissive conditions4. The temperature sensitivity of the transformed phenotype of the ts401 mutant-transformed cells suggests that the continued expression of gene 401 is required for maintenance of the transformed cell phenotype. This study was initiated to detect the H12 transformation-specific protein(s) in H12-transformed cell lines. We identify here a 60,000 molecular weight transformation-specific antigen in H12-transformed rat 3Y1 cells5,6 by immunoprecipitation of 35S-methionine labelled polypeptides with serum from H12 tumour-bearing hamsters. Furthermore, the expression of this antigen is temperature dependent in 3Y1 cells transformed by ts401. Further characterisation of the 60,000 MW antigen may lead to an understanding of the molecular mechanism of adenovirus cell transformation.

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.

PROLIFERATION OF OLIGODENDROCYTE PRECURSOR CELLS DEPENDS ON ELECTRICAL-ACTIVITY IN AXONS

OLIGODENDROCYTES myelinate axons in the vertebrate central nervous system. It would, therefore, make sense if axons played a part in controlling the number of oligodendrocytes that develop in a myelinated tract. Although oligodendrocytes themselves normally do not divide, the precursor cells that give rise to them do. Here we show that the proliferation of oligodendrocyte precursor cells in the developing rat optic nerve depends on electrical activity in neighbouring axons, and that this activity-dependence can be circumvented by experimentally increasing the concentration of platelet-derived growth factor, which is present in the optic nerve and stimulates these cells to proliferate in culture. These findings suggest that axonal electrical activity normally controls the production and/or release of the growth factors that are responsible for proliferation of oligodendrocyte precursor cells and thereby helps to control the number of oligodendrocytes that develop in the region.

Does oligodendrocyte survival depend on axons

BACKGROUNDnWe have shown previously that oligodendrocytes and their precursors require signals from other cells in order to survive in culture. In addition, we have shown that about 50% of the oligodendrocytes produced in the developing rat optic nerve normally die, apparently in a competition for the limiting amounts of survival factors. We have hypothesized that axons may control the levels of such oligodendrocyte survival factors and that the competition-dependent death of oligodendrocytes serves to match their numbers to the number of axons that they myelinate. Here we test one prediction of this hypothesis - that the survival of developing oligodendrocytes depends on axons.nnnRESULTSnWe show that oligodendrocyte death occurs selectively in transected nerves in which the axons degenerate. This cell death is prevented by the delivery of exogenous ciliary neurotrophic factor (CNTF) or insulin-like growth factor I (IGF-1), both of which have been shown to promote oligodendrocyte survival in vitro. We also show that purified neurons promote the survival of purified oligodendrocytes in vitro.nnnCONCLUSIONnThese results strongly suggest that oligodendrocyte survival depends upon the presence of axons; they also support the hypothesis that a competition for axon-dependent survival signals normally helps adjust the number of oligodendrocytes to the number of axons that require myelination. The identities of these signals remain to be determined.

Schwann cell growth factors

Purified rat Schwann cells were found to proliferate very slowly in normal growth medium containing 10% fetal calf serum (FCS). Crude extracts of bovine pituitary or brain markedly enhanced Schwann cell growth, while similar extracts of nerve roots, liver and kidney did not. Pituitary extracts were more potent than brain extracts, and extracts from both anterior and posterior pituitary were active. The mitogenic activity of pituitary extracts was reduced by treatment with trypsin, and abolished by pronase and by boiling. A variety of known anterior and posterior pituitary hormones, as well as fibroblast, epidermal and nerve growth factors, were not mitogenic. FCS (greater than 1%) was required for Schwann cell proliferation, but even high concentrations of FCS did not substitute for pituitary or brain extracts, and serum from various other species did not support Schwann cell growth. Although various agents that increase cyclic AMP levels (such as cholera toxin) had been shown to be Schwann cell mitogens, extracts of pituitary or brain did not increase cyclic AMP levels. Extracts of various bovine tissues, including pituitary, brain, liver and kidney, acted synergistically with cholera toxin in stimulating Schwann cell proliferation, although the increase in cyclic AMP induced by the mixture was not greater than that seen with cholera toxin alone. We conclude that there are at least two separate pathways for stimulating Schwann cell division, only one of which involves an increase in intracellular cyclic AMP.

Two glial cell lineages diverge prenatally in rat optic nerve

Three types of glial cells have been previously described in cultures of neonatal rat optic nerve--oligodendrocytes, type 1 astrocytes, and type 2 astrocytes--which can be distinguished using three different antibodies: antigalactocerebroside antibodies recognize oligodendrocytes; antibodies against glial fibrillary acidic protein recognize both types of astrocytes, while the A2B5 monoclonal antibody distinguishes between the two, binding to type 2 but not type 1 astrocytes. It was subsequently shown that oligodendrocytes and type 2 astrocytes, but not type 1 astrocytes, develop in cultures of 7 day optic nerve from a common, A2B5+ progenitor cell. In the present study, the distribution of rat neural antigen-2 (Ran-2), a cell-surface antigen defined by a monoclonal antibody, has been examined on optic nerve cells. It is demonstrated that, in contrast to A2B5, Ran-2 is present on type 1 but not type 2 astrocytes in optic nerve cultures. More importantly, it is shown that Ran-2 and A2B5 antibodies react with largely nonoverlapping populations of cells in cell suspensions of embryonic Day 17 (E17) and postnatal Day 1 (P1) optic nerve, and that the Ran-2+, A2B5- population contains type 1 astrocytes and their precursors while the A2B5+,Ran-2- population contains the progenitor cells for oligodendrocytes and type 2 astrocytes. These findings provide strong evidence that the glial cells of the rat optic nerve develop as two distinct lineages--one giving rise to type 1 astrocytes and the other to oligodendrocytes and type 2 astrocytes--and that the two lineages diverge as early as E17.

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.

The Id4 HLH protein and the timing of oligodendrocyte differentiation

An intracellular timer is thought to help control the timing of oligodendrocyte differentiation. We show here that the expression of the helix–loop–helix gene Id4 in oligodendrocyte precursor cells decreases in vivo and in vitro with a time course expected if Id4 is part of the timer. We also show that Id4 expression decreases prematurely when the precursor cells are induced to differentiate by mitogen withdrawal. Both Id4 mRNA and protein decrease together under all of these conditions, suggesting that the control of Id4 expression is transcriptional. Finally, we show that enforced expression of Id4 stimulates cell proliferation and blocks differentiation induced by either mitogen withdrawal or treatment with thyroid hormone. These findings suggest that a progressive fall in Id4 transcription is part of the intracellular timer that helps determine when oligodendrocyte precursor cells withdraw from the cell cycle and differentiate.

Differences in the way a mammalian cell and yeast cells coordinate cell growth and cell-cycle progression

Background It is widely believed that cell-size checkpoints help to coordinate cell growth and cell-cycle progression, so that proliferating eukaryotic cells maintain their size. There is strong evidence for such size checkpoints in yeasts, which maintain a constant cell-size distribution as they proliferate, even though large yeast cells grow faster than small yeast cells. Moreover, when yeast cells are shifted to better or worse nutrient conditions, they alter their size threshold within one cell cycle. Populations of mammalian cells can also maintain a constant size distribution as they proliferate, but it is not known whether this depends on cell-size checkpoints. Results We show that proliferating rat Schwann cells do not require a cell-size checkpoint to maintain a constant cell-size distribution, as, unlike yeasts, large and small Schwann cells grow at the same rate, which depends on the concentration of extracellular growth factors. In addition, when shifted from serum-free to serum-containing medium, Schwann cells take many divisions to increase their size to that appropriate to the new condition, suggesting that they do not have cell-size checkpoints similar to those in yeasts. Conclusions Proliferating Schwann cells and yeast cells seem to use different mechanisms to coordinate their growth with cell-cycle progression. Whereas yeast cells use cell-size checkpoints, Schwann cells apparently do not. It seems likely that many mammalian cells resemble Schwann cells in this respect.

Importance of Intrinsic Mechanisms in Cell Fate Decisions in the Developing Rat Retina

Cell diversification in the developing nervous system is thought to involve both cell-intrinsic mechanisms and extracellular signals, but their relative importance in particular cell fate decisions remains uncertain. In the mammalian retina, different cell types develop on a predictable schedule from multipotent retinal neuroepithelial cells (RNECs). A current view is that RNECs pass through a series of competence states, progressively changing their responsiveness to instructive extracellular cues, which also change over time. We show here, however, that embryonic day 16-17 (E16-17) rat RNECs develop similarly in serum-free clonal-density cultures and in serum-containing retinal explants--in the number of times they divide, the cell types they generate, and the order in which they generate these cell types. These surprising results suggest that extracellular signals may be less important than currently believed in determining when RNECs stop dividing and what cell types they generate when they withdraw from the cell cycle, at least from E16-17 onward.