Sonic hedgehog Promotes G₁ Cyclin Expression and Sustained Cell Cycle Progression in Mammalian Neuronal Precursors

Abstract

Sonic hedgehog (Shh) signal transduction via the G-protein-coupled receptor, Smoothened, is required for proliferation of cerebellar granule neuron precursors (CGNPs) during development. Activating mutations in the Hedgehog pathway are also implicated in basal cell carcinoma and medulloblastoma, a tumor of the cerebellum in humans. However, Shh signaling interactions with cell cycle regulatory components in neural precursors are poorly understood, in part because appropriate immortalized cell lines are not available. We have utilized primary cultures from neonatal mouse cerebella in order to determine (i) whether Shh initiates or maintains cell cycle progression in CGNPs, (ii) if G₁ regulation by Shh resembles that of classical mitogens, and (iii) whether individual D-type cyclins are essential components of Shh proliferative signaling in CGNPs. Our results indicate that Shh can drive continued cycling in immature, proliferating CGNPs. Shh treatment resulted in sustained activity of the G₁ cyclin-Rb axis by regulating levels of cyclinD1, cyclinD2, and cyclinEmRNA transcripts and proteins. Analysis of CGNPs from cyclinD1^−/− orcyclinD2^−/− mice demonstrates that the Shh proliferative pathway does not require unique functions of cyclinD1 or cyclinD2 and that D-type cyclins overlap functionally in this regard. In contrast to many known mitogenic pathways, we show that Shh proliferative signaling is mitogen-activated protein kinase independent. Furthermore, protein synthesis is required for early effects on cyclin gene expression. Together, our results suggest that Shh proliferative signaling promotes synthesis of regulatory factor intermediates that upregulate or maintain cyclin gene expression and activity of the G₁cyclin-Rb axis in proliferating granule neuron precursors.

ACKNOWLEDGMENTS

We are especially grateful to Charles Stiles, Peter Sicinski, Heide Ford, and Rosalind Segal for criticism and helpful comments on the manuscript. We also thank Kevin Williams (Biogen, Inc.) for providing Sonic hedgehog; Jane Johnson (University of Texas Southwestern Medical Center) and Rosalind Segal (Dana-Farber Cancer Institute) for generously providing antisera against Math-1 and Zic, respectively; and Peter Sicinski for the cyclinD1 andcyclinD2 knockout mice. Sovann Som, Dongin Yuk, and the staff of the Jimmy Fund FACS facility provided much appreciated technical assistance.

A.M.K. is supported by the Justin Porter/American Brain Tumor Association Fellowship. These studies were funded by grants from the Charles Hood Foundation, the NIH (HD01182), and the Edward Mallinkrodt Jr. Foundation. D.H.R. is a Claudia Adams Barr Investigator and a recipient of a Basil O'Connor Starter Scholar Award from the March of Dimes Foundation.