MITOGENIC AND ANTIPROLIFERATIVE SIGNALS FOR NEURAL CREST CELLS AND THE NEUROGENIC ACTION OF TGF-BETA-1

The influence of pertinent growth factors on proliferation and differe ntiation of quail neural crest cell was assessed by in vitro colony as say in a serum-free (0.5% chick embryo-extract supplemented) culture m edium. The factors tested included basic fibroblast growth factor (bFG F; FGF-2), neurotrophins, and transforming growth factor-beta-1 (TGF-b eta). Both bFGF and neurotrophins are implicated in the development of the peripheral nervous system, whereas TGF-beta can affect cell diffe rentiation and modulate the action of other growth factors. Bromodeoxy uridine (BrdU) incorporation indicated that bFGF is mitogenic to pluri potent neural crest cells (and/or their immediate progeny) and to comm itted melanogenic cells. However this was not reflected in an increase in colony size. In contrast, colony size did increase when nerve grow th factor (NOF) was present in addition to bFGF. This indicated either that both factors are required to initiate cell proliferation or that at least some bFGF-exposed cells become dependent on neurotrophins fo r survival. Sequential addition of the factors showed that exposure to bFGF was required prior to the presence of a neurotrophin, thus favor ing the latter possibility. All three neurotrophins tested, NGF, brain -derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), were c apable of supporting survival of pluripotent neural crest cells (or th eir closely related progeny) in the presence of bFGF. In the absence o f bFGF, neurotrophins did not affect colony size. Although the BrdU da ta indicated that bFGF is also a mitogen for committed melanogenic cel ls, the size of pigmented colonies did not change in the presence of b FGF alone or of bFGF plus a neurotrophin. This suggested that another, yet to be determined, factor is required for the survival of prolifer ating melanogenic cells. Colony assays were also performed in the pres ence and absence of TGF-beta, both alone and in combination with bFGF plus NGF. TGF-beta inhibited proliferation of both pluripotent neural crest cells (and/or their immediate derivatives) and of committed mela nogenic cells, causing a decrease in colony size. When TGF-beta was ad ded to the culture medium together with the bFGF/NGF combination, this also caused a significant decrease in colony size, similar to the one observed with TGF-beta alone. TGF-beta blocked proliferation even whe n the cells were exposed 24 to 48 hr to the bFGF/NGF combination prior to addition of TGF-beta. Neurogenesis increased significantly in the presence of TGF-beta. The number per colony of both adrenergic cells a nd sensory neuron precursors increased in TGF-beta-treated neuroblast- positive colonies. The following new insights were derived from this s tudy: 1) basic FGF is a mitogen for pluripotent neural crest cells (an d/or their immediate derivatives); 2) pluripotent and committed melano genic neural crest cells that have been exposed to bFGF become depende nt on trophic support; 3) all neurotrophins tested (NGF, BDNF or NT-3) can fulfill the trophic requirement of bFGF-exposed pluripotent cells , but not for melanogenic cells; 4) TGF-beta is an anti-proliferative signal for pluripotent neural crest cells and for committed melanogeni c cells; 5) the TGF-beta-mediated anti-proliferative signal dominates over the bFGF/neurotrophin-mediated mitogenic signal; and 6) TGF-beta enhances sensory and adrenergic neurogenesis, possibly by acting upon a common neurogenic precursor cell. Furthermore, our work confirms pre vious reports by other investigators, who showed that bFGF promotes an d TGF-beta inhibits proliferation of pigment cells. (C) 1997 Wiley-Lis s, Inc.