CYTOKINES REGULATE THE CELLULAR PHENOTYPE OF DEVELOPING NEURAL LINEAGE SPECIES

The patterns and mechanisms of action of inductive signals that orches trate neural lineage commitment and differentiation in the mammalian b rain are incompletely understood. To examine these I developmental iss ues, we have utilized several culture systems including conditionally immortalized cell lines, subventricular zone progenitor cells and prim ary neuronal cultures. A neural stem and progenitor cell line (MK31) w as established from murine embryonic hippocampus by retroviral transdu ction of temperature-sensitive alleles of the simian virus 40 large tu mor antigen. At the non-permissive temperature for antigen expression (39 degrees C) in serum-free media, the neural stem cells give rise to a series of increasingly mature neuronal progenitor and differentiate d cellular forms under the influence of a subset of hematolymphopoieti c cytokines (interleukins 5, 7, 9 and 11), when individually co-applie d with transforming growth factor alpha, after pretreatment with basic fibroblast growth factor. These cellular forms elaborated a series of progressively more mature neurofilament proteins, a sequential patter n of ligand-gated channels, and inward currents and generation of acti on potentials with mature physiological properties. Because the factor s regulating the development of central nervous system astrocytes have been so difficult to define, we have chosen to focus, in this manuscr ipt, on the elaboration of this cell type. At 39 degrees C, applicatio n of a subfamily of bone morphogenetic proteins of the transforming gr owth factor beta superfamily of growth factors sanctioned the selectiv e expression of astrocytic progenitor cells and mature astrocytes, as defined by sequential elaboration of the Yb subunit of glutathione-S-t ransferase and glial fibrillary acidic protein. These lineage specific cytokine inductive relationships were verified using subventricular z one neural progenitor cells generated by the application of epidermal growth factor, alone or in combination with basic fibroblast growth fa ctor, to dissociated cellular cultures derived from early embryonic mu rine brain, a normal non-transformed developmental population. Finally , application of a different series of cytokines from five distinct fa ctor classes (basic fibroblast growth factor, platelet-derived growth factor-AA, insulin-like growth factor 1, neurotrophin 3 and representa tive gp130 receptor subunit-related ligands) caused the elaboration of oligodendroglial progenitor species and post-mitotic oligodendrocytes , defined by progressive morphological maturation and the expression o f increasingly advanced oligodendroglial and oligodendrocyte lineage m arkers. In addition, seven different gp130-associated neuropoietic (ci liary neurotrophic factor, leukemia inhibitory factor, oncostatin-M) a nd hematopoietic (interleukins 6, 11, 12, granulocyte-colony stimulati ng factor) cytokines exhibited differential trophic effects on oligode ndroglial lineage maturation and factor class interactions. Examinatio n of the expression of hematolymphopoietic cytokines and their recepto rs in brain and neural cultures has confirmed that these epigenetic si gnals are present at the appropriate developmental times to mediate th eir neurotrophic actions. These cytokines signal through alternate rec eptor subunit motifs distinct from those of the traditional neurotroph ins. The bone morphogenetic protein ligands, in particular, exhibit a complex spatiotemporal pattern of transcript expression that suggests a broad spectrum of developmental roles for these transforming growth factor beta subclass factors. To examine the cellular action of the bo ne morphogenetic proteins on astroglial lineage elaboration in greater detail, we utilized several complementary developmental systems. When primary neuronal cultures from multiple brain regions of mid-gestatio nal (embryonic day 15) fetuses in serum-free media were exposed to the same combination of bone morphogenetic proteins that sanctioned astro glial lineage elaboration from neural stem and progenitor cells, they exhibited significant suppression of neuronal viability. By contrast, application of the same factors to late embryonic day 17-18 neuronal c ultures resulted in a regional and factor-specific potentiation of cel lular survival and differentiation. The neurotrophic effects of the bo ne morphogenetic proteins appear to be indirect and mediated by stimul ation of non-neuronal cells. Further, application of the bone morphoge netic proteins to purified O-2A progenitor cells, derived from early p ostnatal brain and from a colonal progenitor cell line resulted in the selective induction of type II astrocytes, suggesting that these tran sforming growth factor beta subclass factors are acting directly on th ese bipotent astrocytic/oligodendroglial progenitor cells. These diver se experimental observations suggest that a single central nervous sys tem neural stem cell can give rise to all three major cellular element s of the mammalian brain. Cytokines from the three major growth factor superfamilies (neurotrophins, hemopoietins and transforming growth fa ctor beta-related factors) exhibit a differential pattern of neurotrop hic actions on distinct central nervous system lineage species during sequential developmental stages. These observations suggest that a com plex hierarchy of interacting epigenetic signals is required for centr al nervous system neurogenesis.