Investigation of Common Bases of Sympathetic Nervous System and Neuroblastoma Development

by Shi, Huilin

Sympathetic ganglia are primarily composed of noradrenergic neurons and satellite glial cells. Although both cell types originate from neural crest cells, the identities of the progenitor populations at intermediate stages of the differentiation process remain to be established. Here we report the identification in vivo of glial and neuronal progenitor cells in postnatal sympathetic ganglia, using mouse superior cervical ganglia as a model system. There are significant levels of cellular proliferation in mouse superior cervical ganglia during the first 18 days after birth. A majority of the proliferating cells express both nestin and brain lipid-binding protein (BLBP). BrdU fate-tracing experiments demonstrate that these nestin and BLBP double positive cells represent a population of glial progenitors for sympathetic satellite cells. The glial differentiation process is characterized by a marked downregulation of nestin and upregulation of S100, with no significant changes in the levels of BLBP expression. We also identify a small number of proliferating cells that express nestin and tyrosine hydroxylase, a key enzyme of catecholamine biosynthesis that defines sympathetic noradrenergic neurons. Together, these results establish nestin as a common marker for sympathetic neuronal and glial progenitor cells and delineate the cellular basis for the generation and maturation of sympathetic satellite cells. This research in the normal postnatal sympathetic development provides the basis for the neuroblastoma study. Neuroblastoma is the most common childhood malignant tumor which originates from sympathetic nervous system. 90% of children with this disease are diagnosed before 6 years old. Therefore, the tumorigenesis of neuroblastoma may represent an abnormal embryonic or postnatal sympathetic development. Transformed neural crest stem cells or malignant sympathetic precursor cells that obtain stem cell abilities are recognized as neuroblastoma stem cells that might be the origin of neuroblastoma. BE(2)-C cells, a human neuroblastoma cell line enriched with tumorigenic stem cells, can be induced to undergo either neuronal or glial differentiation. We use BE(2)-C cells as a system to identify the genes that regulate neuroblastoma stem cell activities, such as self-renewal and differentiation. One of these genes is GATA3, a zinc-finger transcription factor with an essential role in sympathetic development. Downregulation of GATA3 by siRNA promotes BE(2)- C cell proliferation and overexpression of GATA3 decreases the proliferation of BE(2)-C cells. GATA3 regulates cell proliferation through distinct pathways involving Cyclin D1 and E2F1, as evidenced by an increased expression of Cyclin D1 and a decreased expression of E2F1 in GATA3 knockdown cells. In addition, GATA3 knockdown induces BE(2)-C cells to undergo glial differentiation, as indicated by an increase in the expression of GFAP, a glial cell marker, and a decrease in the expression of neuronal marker SNAP25. GATA3 overexpression promotes neuronal differentiation of BE(2)-C cells, as indicated by a reduction of GFAP expression and an upregulation of SNAP25 expression. GATA3 knockdown also downregulates Phox2b and GATA3 overexpression upregulates Mash1, two transcription factors that are expressed in neuronal progenitor cells and are essential for sympathetic development. Together, these findings suggest that GATA3, Phox2b and Mash1 may function in a regulatory network in the control of neuroblastoma cells in a stem/progenitor cell state.