Conditional Induction of Math1 Specifies Embryonic Stem Cells to Cerebellar Granule Neuron Lineage and Promotes Differentiation into Mature Granule Neurons

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, we 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. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1.Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAa6R characteristic of mature granule neurons, formed ''T-shaped'' axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage. STEM CELLS 2013;31:652-665 Disclosure of potential conflicts of interest is found at the end of this article.

Proneural Transcription Factor Atoh1 Drives Highly Efficient Differentiation of Human Pluripotent Stem Cells Into Dopaminergic Neurons

“…These results suggest that Atoh1-induced neurons respond to extrinsic factors for generating lineage-specific neurons. A recent report shows that in embryonic bodies derived from mouse ESCs, Atoh1 induction in combination with extrinsic factors promotes the generation of cerebellar granule neurons [40]. Atoh1 induction has also been found to induce inner ear hair cell-like cells from mouse ESC-derived embryonic bodies [41].…”

Control of hair cell development by molecular pathways involving Atoh1, Hes1 and Hes5

“…Therefore, in embryonic stem cells, after being upregulated by transient activation of Atoh1, Barhl1 and Barhl2 expressions continue independently of ATOH1 (Srivastava et al, 2013).…”

“…1). On the other hand, induction of Atoh1 expression in embryonic stem cells increased the number of neurons that express Zic1 (Srivastava et al, 2013) (Table 1), suggesting that Atoh1 can in turn be an upstream gene of Zic1.…”

“…Furthermore, during the differentiation of embryonic stem cells, Barhl1 and Barhl2 are directly involved in the formation of granule neuron precursors and induce a strong expression of En1 and NeuroD1, both early cerebellar territory markers (Srivastava et al, 2013).…”

Endogenous cerebellar neurogenesis in adult mice with progressive ataxia

“…Following injection, Alzet pumps (DURECT, Cupertino, CA, USA) filled with ciclosporin (10 mg/kg per day) were transplanted subcutaneously and replaced monthly. Procedures for sample preparation and immunolabeling were performed as described 5. All antibodies and concentrations used are listed in Table2.…”