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.

Early cortical progenitor cells of the ventricular zone (VZ) differ from later progenitor cells of the subventricular zone (SVZ) in cell-type generation and their level of epidermal growth factor receptors (EGFRs). To determine whether differences in their behavior are causally related to EGFR number/density, we introduced extra EGFRs into VZ cells with a retrovirus in vivo and in vitro. This results in premature expression of traits characteristic of late SVZ progenitor cells, including migration patterns, differentiation into astrocytes, and proliferation of multipotential cells to form spheres. The choice between proliferation and differentiation depends on ligand concentration and progenitor cell age and may reflect different thresholds of stimulation. The level of EGFRs expressed by progenitor cells in the cortex may therefore contribute to the timing of their maturation and choice of response to pleiotropic environmental signals.

Abstract. Ciliary neurotrophic factor (CNTF) is a potent survival molecule for a variety of embryonic neurons in culture. The developmental expression of CNTF occurs clearly after the time period of the physiological cell death of CNTF-responsive neurons . This, together with the sites of expression, excludes CNTF as a target-derived neuronal survival factor, at least in rodents . However, CNTF also participates in the induction of type 2 astrocyte differentiation in vitro. Here we demonstrate that the time course of the expression of CNTF-mRNA and protein:in the rat optic nerve (as evaluated by quantitative Northern blot analysis and biological activity,, respectively) is compatible with such a glial differentiation function of CNTF

We have been studying a population of bipotential glial progenitor cells in the perinatal rat optic nerve and brain in an attempt to understand how cells choose between alternative fates in the developing mammalian central nervous system (CNS). This cell population gives rise initially to oligodendrocytes and then to type-2 astrocytes\ both of which apparently collaborate in sheathing axons in the CNS 2 .3. In vitro studies suggest that oligodendrocyte differentiation is the constitutive pathway of development for the oligodendrocyte-type-2-astrocyte (O-2A) progenitor ce1l 4 ,s, whereas type-2 astrocyte differentiation depends on a specific inducing protein 6 . This protein is present in the developing optic nerve when type-2 astrocytes are differentiating and can induce 0-2A progenitor cells in vitro to express glial fibrillary acidic protein (GFAP)6, a marker of astrocyte differentiation'. Here we show that the type-2-astrocyte-inducing protein is similar or identical to ciliary neutrotrophic factor (CNTF)8,9, which promotes the survival of some types of peripheral neurons in vitro 8 , including ciliary ganglion neurons8,IO. This suggests that CNTF, in addition to its effect on neurons, may be responsible for triggering type-2 astrocyte differentiation in the developing CNS.CNTF was purified from rat sciatic nerve using a modification of the method used previousll. When it was added to cultures of postnatal-day-O (PO) rat optic nerve cells it induced GFAP expression in about 20% of 0-2A progenitor cells (Fig. la). The same result was obtained with an extract of 3-4-week-old rat optic nerve (Fig. Ib), as described previously6. CNTF and optic nerve extract had the same effect when added simultaneously as when either was added alone (Fig. lb). Two other neurotrophic factors, nerve growth factor l1 ,12 and brain-derived growth factor 13 , had no effect on GFAP expression in this assay (not shown).CNTF and the type-2-astrocyte-inducing protein share several properties. They are both acidic (ref. 9 and unpublished observations), with a relative molecular mass (Mr) of about 25,000 (25K) (refs 6 and 9), and present at relatively high concentrations in sciatic nerve 6 ,l4 and kidney (ref. 9 and unpublished observations), compared with liver, retina or brain (refs 6, 14 and unpublished observations). If the type-2-astrocyte-inducing activity in optic nerve extracts is CNTF, then these extracts should support ciliary ganglion neuron survival in vitro, which they did (Fig. Id). The responses of both ciliary ganglion neurons and 0-2A progenitor cells were elicited within a similar range of concentrations (Fig. 1); the specific activity of optic nerve extract was about 1,300-fold less than that of CNTF in both assays.When tested on optic nerve cells CNTF had many of the functional properties previously described for optic nerve extract 6 • First, CNTF had no appreciable effect on GFAP expression in type-l astrocytes or their precursors in cultures of embryonic day 17 (E17) optic nerve cells, even though it induced many of ...

The differentiation of multipotential progenitor cells in the vertebrate retina into photoreceptors, neurons and glial cells is regulated in part by cell-cell signalling. Transforming growth factor (TGF)-alpha is one of the extracellular signals implicated in the control of several aspects of retinal development, including proliferation and cell fate. The way cells interpret pleiotropic signals such as TGF-alpha is influenced by the level of expression of epidermal growth factor receptor (EGF-R) in some cell lines. To address the influence of receptor level on responses of retinal progenitor cells to TGF-alpha, additional copies of EGF-Rs were introduced in vitro and in vivo with a retrovirus. Normally in vitro, low concentrations of TGF-alpha stimulated proliferation whereas high concentrations biased choice of cell fate, inhibiting differentiation into rod photoreceptors while promoting differentiation into Müller glial cells. We report here that introduction of extra EGF-Rs into progenitor cells in vitro reduced the concentration of TGF-alpha required for changes in rod and Müller cell differentiation but did not enhance proliferation. Introduction of extra EGF-Rs in vivo increased the proportion of clones that contained Müller glial cells, suggesting that receptor level is normally limiting. These findings demonstrate that responsiveness to extracellular signals during development can be modulated by the introduction of additional receptors, and suggest that the level of expression of receptors for these signals contributes to the regulation of cell fate.