Mitogenic and anti‐proliferative signals for neural crest cells and the neurogenic action of TGF‐β1

The influence of pertinent growth factors on proliferation and differentiation of quail neural crest cell was assessed by in vitro colony assay in a serum-free (0.5% chick embryo-extract supplemented) culture medium. The factors tested included basic fibroblast growth factor (bFGF; FGF-2), neurotrophins, and transforming growth factor-beta-1 (TGF-beta). Both bFGF and neurotrophins are implicated in the development of the peripheral nervous system, whereas TGF-beta can affect cell differentiation and modulate the action of other growth factors. Bromodeoxyuridine (BrdU) incorporation indicated that bFGF is mitogenic to pluripotent neural crest cells (and/or their immediate progeny) and to committed melanogenic cells. However, this was not reflected in an increase in colony size. In contrast, colony size did increase when nerve growth factor (NGF) 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 for survival. Sequential addition of the factors showed that exposure to bFGF was required prior to the presence of a neurotrophin, thus favoring the latter possibility. All three neurotrophins tested, NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), were capable of supporting survival of pluripotent neural crest cells (or their closely related progeny) in the presence of bFGF. In the absence of bFGF, neurotrophins did not affect colony size. Although the BrdU data indicated that bFGF is also a mitogen for committed melanogenic cells, the size of pigmented colonies did not change in the presence of bFGF alone or of bFGF plus a neurotrophin. This suggested that another, yet to be determined, factor is required for the survival of proliferating melanogenic cells. Colony assays were also performed in the presence 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 melanogenic cells, causing a decrease in colony size. When TGF-beta was added 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 when 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 and sensory neuron precursors increased in TGF-beta-treated neuroblast-positive colonies. The following new insights were derived from this study: 1) basic FGF is a mitogen for pluripotent neural crest cells (and/or their immediate derivatives); 2) pluripotent and committed melanogenic neural crest cells that have been exposed to bFGF become dependent 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 melanogenic 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 previous reports by other investigators, who showed that bFGF promotes and TGF-beta inhibits proliferation of pigment cells.