The Generation of Peripheral Neuron Diversity from Mammalian Progenitor Cells In Vitro

The vertebrate peripheral nervous system is comprised of many different kinds of neurons that develop from a common progenitor pool, the neural crest. Factors that control the specification of most neuronal subtypes are not well understood. The studies presented in this thesis describe how and when sensory precursors become different from those of the autonomic lineage, and how subtype diversity is generated within the sensory neuron class. The development of peripheral neurons was assayed in vitro using rat neural tube explant cultures as a source of neural crest, and antibody staining for lineage-specific transcription factors to identify neuronal subtypes. A chemically defined medium supported the differentiation of sensory but not autonomic neurons in these explants. When cultures were challenged with the extrinsic factor BMP2 to induce autonomic neurons, the division and differentiation of sensory precursors was unperturbed. Therefore, the neural crest contains a population of di vi ding precursors that are operationally determined to the sensory neuron fate. The majority of sensory neurons that differentiated in defined medium displayed characteristics of the muscle afferent subtype including dependence on the neurotrophins BDNF and NT-3, expression of the marker ER81, and lack of expression of trkA. In contrast, the addition of serum or BMP2 induced the development of many neurons that expressed the cutaneous afferent marker, trkA. Neither serum nor BMP2 is sufficient to induce trkA in post-migratory, differentiated neurons; instead, these factors induce the neural tube to produce additional sensory precursors, some of which differentiate into cutaneous afferents. We also observed that the neurotrophins BDNF and NT-3, which could directly regulate ER81 expression in differentiated neurons, did not prevent the development of trkA expressing neurons. This implies that serum/BMP2 have early affects on the specification of sensory subtypes while neurotrophins regulate acquisition of particular subtype characteristics. In addition to their ability to promote the development of the cutaneous afferent sensory subtype, serum!BMP2 induced patterning in neural tube explants. In the presence of these factors, the location of sensory and autonomic neurons in the neural crest outgrowth of the explant resembled the arrangement of these neuronal subtypes along the dorsoventral axis in vivo.