Spinal Cord Neuronal Precursors Generate Multiple Neuronal Phenotypes in Culture

Abstract

Neuronal restricted precursors (NRPs) () can generate multiple neurotransmitter phenotypes during maturation in culture. Undifferentiated E-NCAM+ (embryonic neural cell adhesion molecule) immunoreactive NRPs are mitotically active and electrically immature, and they express only a subset of neuronal markers. Fully mature cells are postmitotic, process-bearing cells that are neurofilament-M and synaptophysin immunoreactive, and they synthesize and respond to different subsets of neurotransmitter molecules. Mature neurons that synthesize and respond to glycine, glutamate, GABA, dopamine, and acetylcholine can be identified by immunocytochemistry, RT-PCR, and calcium imaging in mass cultures. Individual NRPs also generate heterogeneous progeny as assessed by neurotransmitter response and synthesis, demonstrating the multipotent nature of the precursor cells. Differentiation can be modulated by sonic hedgehog (Shh) and bone morphogenetic protein (BMP)-2/4 molecules. Shh acts as a mitogen and inhibits differentiation (including cholinergic differentiation). BMP-2 and BMP-4, in contrast, inhibit cell division and promote differentiation (including cholinergic differentiation). Thus, a single neuronal precursor cell can differentiate into multiple classes of neurons, and this differentiation can be modulated by environmental signals.

Sonic hedgehogRegulates Proliferation and Inhibits Differentiation of CNS Precursor Cells

“…However, the expansion and patterning abnormalities clearly require Shh, and the induction of ventral cell types (e.g., Nkx-2.2) is most consistent with direct Shh signaling. Moreover, our observations are entirely consistent with a number of in vitro studies of Shh proliferative effects on CNS precursors (Jensen and Wallace, 1997;Kalyani et al, 1998;Wechsler-Reya and Scott, 1999) and the finding that Patched mutations in mice result in highly proliferative cerebellar tumors (Goodrich et al, 1997). An obvious question remains whether Shh has a role in regulating proliferation during normal spinal cord development, as has been reported recently for cerebellar granule cells (Wechsler-Reya and Scott, 1999).…”

“…Although inactivating mutations of PATCHED can result in medulloblastoma, a tumor of cerebellar granule cells, the spinal cord is not affected in such patients. Nevertheless, Shh causes proliferation in spinal cord precursor cells in vitro (Kalyani et al, 1998) and in vivo (present study). Our results suggest that severe temporal restrictions on cellular competence could limit a putative "tumorigenic window" to only a few days of embryogenesis in the spinal cord.…”

“…Thus, it is unlikely that the failure to form Isl-1ϩ neurons is related to the dosage of Shh in Shh-Tg mice. A second possibility was that Shh actually inhibited the differentiation of Isl-1 motor neurons, as suggested from in vitro studies (Kalyani et al, 1998). We therefore analyzed Shh-Tg dorsal spinal cord cells in dispersed explant cultures for generation of Isl-1 neurons.…”

“…Several studies using primary CNS precursor cell cultures have demonstrated proliferative effects of the biologically active N-Shh protein after treatment for 36 -48 hr (Jensen and Wallace, 1997;Kalyani et al, 1998). Whether Shh functions as a direct mitogen in vitro, however, has not been established (Jensen and Wallace, 1997).…”

“…Upregulation of HES-1, in particular, suggests that such a mechanism may be f unctioning in Shh-Tg mice. Interestingly, Kalyani et al (1998) and Wechsler-Reya and Scott (1999) recently reported that Shh can directly inhibit differentiation of neuronally restricted precursor cells in vitro.…”