Molecular milestones that signal axonal maturation and the commitment of human spinal cord precursor cells to the neuronal or glial phenotype in development

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Abstract
Insights into the programmatic induction of neuronal and glial genes during human embryogenesis have depended largely on extrapolations of data derived from experimental mammals. However, the assumptions upon which these extrapolations are based have not been rigorously tested. Indeed, practically no information is available even on the human counterparts of the relatively small subset of well‐characterized, developmental regulated neuron and glial specific genes of the mammalian CNS. Thus, the developmental programs upon which human neural embryogenesis are based remain largely undeciphered. We have addressed this problem in immunohistochemical studies conducted on 22 human fetal spinal cords with gestational ages (GAs) that ranged from 6 to 40 weeks by using monoclonal antibodies to several classes of neuron or glial specific polypeptides. These polypeptides included: representatives of four different types (Types I–IV) of intermediate filament proteins, i.e., vimentin filament protein (VFP), glial fibrillary acidic protein (GFAP), different phospho‐isoforms of the high (NF‐H), middle (NF‐M), and low (NF‐L) molecular weight (Mr) neurofilament (NF) subunits, both acidic and basic cytokeratin (CK) proteins; three different micro tubule associated proteins (MAPs), i.e., MAP2, MAP5, and tau; two different synaptic or coated vesicle proteins, i.e., synaptophysin (SYP) and clathrin light chain B (LCb); an oligodendroglial specific protein, i.e., myelin basic protein (MBP); and a receptor for a CNS trophic factor, i.e., the nerve growth factor receptor (NGFR). The major findings derived from these studies may be summarized as follows: (1) the most primitive neuroepithelial cells only expressed VFP and MAP5; (2) postmitotic, postmigratory neurons transiently expressed NGFR in the earliest developmental stages, while NF‐H, NF‐M, NF‐L, MAP2, MAP5, clathrin LCb, and SYP were expressed throughout development although the time of initial onset of each of these proteins differed; for example, NF‐M isoforms generally appeared before NF‐L and NF‐H isoforms, and the most highly phosphorylated NF‐H variants emerged much later than NF‐M; moreover, the induction of SYP in anterior horn cells followed the induction of proteins that are thought to determine neuronal polarity (e.g., NF‐L, NF‐M, NF‐H, MAP2, tau); (3) GFAP positive astrocytes became evident after the appearance of many neuron specific proteins although radial glia transiently expressed VFP earlier in development; (4) MBP appeared in the cell bodies of glial cells contemporaneously with GFAP, and in the myelin sheaths of white matter well before axons acquired a fully mature complement of cytoskeletal proteins; and (5) although programmed neuron death undoubtedly occurred during the GAs examined here, this process was not associated with the presence of debris containing any of the developmentally regulated polypeptides examined in this stud. We conclude that human neurogenesis and gliogenesis in the developing spinal cord is a highly orchestrated process in which neuron specific and specific genes are induced manner quite similar to that described in previous studies of other experimental animals. However, unlike some reports on neurogenesis in rodents and birds, the acquisition of the molecular neuronal phenotype in the human spinal cord was exclusively a postmitotic, postmigratory series of events. Nevertheless, as in these other species, the induction of neuron specific gene products in human spinal cord neurons occurred in a step‐wise, asynchronous manner. Finally, the absence of cellular debris containing any of the developmentally regulated antigens we studied here, at a time during which massive neuron death is likely to be in progress, suggests that the induction of these neuron specific genes may identify subsets of neurons destined to survive into maturit.