Conditional Math1 over expression in mouse embryonic stem cells specifies the cerebellar granule neuron identity

Abstract

Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, I have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. In the first part of my study, Math1 inducible mouse ESCs lines were constructed based on the Tetracycline-On transactivator system. In the second part of study the appropriate window for the Math1 over-expression was investigated. The transgene over-expression at various stages of ESCs differentiation for two days showed that the yield of granule progenitors was considerably increased when this proneural gene was induced during embryoid body formation stage. Such short term transgene induction however failed to show an increase in the terminally differentiated granule neurons. Based on these results, Math1 over-expression studies during seven days of embryoid body formation stage were followed in the next part. The transgene induction triggered prolonged expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and immediate strong expression of early cerebellar territory markers Engrailed1 and NeuroD1. Three weeks after induction, a decrease in the number of glial cells and an increase in that of neurons albeit still immature was observed. Also many granule neuron markers were found to be increased. In the next part of study it was found that combining conditional Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1 and GABAand#945;6R characteristic of mature granule neurons, formed T-shaped axons typical of granule neurons and generated synaptic contacts and action potentials in vitro.