Small intensely fluorescent cells in culture: Role of glucocorticoids and growth factors in their development and interconversions with other neural crest derivatives

Abstract

The neural crest gives rise to a number of adrenergic derivatives, including sympathetic neurons and adrenal chromaffin cells, which contain catecholamines (CAs) but differ in other morphological and functional characteristics. Small intensely fluorescent (SIF) cells, which exist primarily as a minority cell population in autonomic ganglia, are a third cell type in the sympathoadrenal branch of the neural crest lineage. In some respects these cells appear intermediate in phenotype between sympathetic neurons and adrenal chromaffin cells. We established pure dissociated cell cultures of SIF cells from rat superior cervical ganglia (SCG) and used these to study the role of environmental factors in SIF cell development and the relationship of these cells to the other cell types of the sympathoadrenal lineage. When cells from neonatal rat SCG were grown for 3 weeks in the presence of glucocorticoid and in the absence of nerve growth factor (NGF), pure cultures of SIF cells developed. The properties of the cells included (i) small cell size and the occasional presence of short neurites, (ii) intense CA histofluorescence and immunoreactivity for CA synthetic enzymes, (iii) synthesis and storage of CA from radioactive precursors, and (iv) characteristic ultrastructure. The concentration of the glucocorticoid and the presence or absence of non-neuronal cell factors influenced which types of SIF cells developed. In micromolar glucocorticoid most of the cells resembled adrenal chromaffin or type II SIF cells: they displayed immunohistochemically detectable phenylethanol-amine-N-methyltransferase (PNMT), synthesized and stored epinephrine, and contained large granular vesicles (100 to 300 nm). When SCG cells were grown in 10-8 M hormone, many fewer SIF cells developed and a higher percentage of these lacked PNMT immunoreactivity, had neurites, and contained vesicles of smaller mean diameter (70 to 130 nm), similar to those of type I SIF cells in vivo. In the presence of conditioned medium (medium conditioned by non-neuronal cells) as well as glucocorticoid, virtually all of the cells morphologically resembled type I SIF cells. In the absence of glucocorticoid, no SIF cells were ever observed after 3 weeks in culture. By following the development of CA histofluorescence and SIF cell ultrastructure in the cultures over time, we demonstrated that SIF cells were not present in large numbers in these cultures immediately after plating, but were induced from an apparently undifferentiated precursor by the hormonal environment, whereas most of the principal neurons died. When corticosteroid was subsequently withdrawn from mature SIF cell cultures and replaced by NGF, the SIF cells responded with neurite outgrowth and conversion into neurons. They increased in size, lost their intense cell body CA histofluorescence and large granular vesicles, and acquired characteristic neuronal ultrastructure including small clear vesicles and morphologically specialized synapses. Thus, there are cells in the SCG with the potential to become either SIF cells or neurons, and NGF and glucocorticoids influence this choice. As described in the preceding paper for adrenal chromaffin cells in NGF (Doupe, A.J., S.C. Landis, and P.H. Patterson (1985) J. Neurosci. 5:2119-2142), type II SIF cells transiently acquired vesicles of SIF type I size during the conversion to neurons. Furthermore, when both NGF and corticosteroid were added to mature SIF cell cultures, many of the cells also assumed an intermediate phenotype resembling type I SIF cells. The studies in this and the preceding paper provide evidence for a role of environmental factors in neural crest differentiation. Similar factors could act during normal development and may explain the characteristic localization and heterogeneity of these phenotypes in vivo. Moreover, the central position of type I SIF cells in the interconversions demonstrated in vitro and the morphological similarity of these cells to early sympathetic precursors suggest that SIF cells may normally be precursors to the entire autonomic lineage of the neural crest in vivo.