Distinct retinoic acid receptor (RAR) isotypes control differentiation of embryonal carcinoma cells to dopaminergic or striatopallidal medium spiny neurons

Anna Podleśny-Drabiniok,Wojciech Krężel,Krystyna Gołembiowska,Angel R De Lera,Małgorzata Cebrat,Katarzyna Kamińska,Joanna Sobska,Pascal Dollé

doi:10.1038/s41598-017-13826-x

Abstract

Embryonal carcinoma (EC) cells are pluripotent stem cells extensively used for studies of cell differentiation. Although retinoic acid (RA) is a powerful inducer of neurogenesis in EC cells, it is not clear what specific neuronal subtypes are generated and whether different RAR isotypes may contribute to such neuronal diversification. Here we show that RA treatment during EC embryoid body formation is a highly robust protocol for generation of striatal-like GABAergic neurons which display molecular characteristics of striatopallidal medium spiny neurons (MSNs), including expression of functional dopamine D2 receptor. By using RARα, β and γ selective agonists we show that RARγ is the functionally dominant RAR in mediating RA control of early molecular determinants of MSNs leading to formation of striatopallidal-like neurons. In contrast, activation of RARα is less efficient in generation of this class of neurons, but is essential for differentiation of functional dopaminergic neurons, which may correspond to a subpopulation of inhibitory dopaminergic neurons expressing glutamic acid decarboxylase in vivo.

Highlights

Retinoic acid (RA), the bioactive form of vitamin A, is essential for embryonic development by virtue of controlling proliferation, differentiation and homeostasis of diverse cell types, contributing to organogenesis and formation of different types of tissues including nervous tissue
Immunocytochemistry analyses revealed that following ATRA treatment, 88.5 ± 2.5% cells expressed a pan-neuronal marker, beta III-tubulin (TUJ1 antibody), indicating that neurons are the major cell type obtained after ATRA-induced differentiation (Fig. 1B)
We found that a vast majority of neurons (81.0 ± 1.3% of all cells and 90% of TUJ1+ cells) were inhibitory and expressed glutamate decarboxylase 65/67 (GAD65/67), whereas analyses of glutamate receptor 2/3 (GluR2/3) expression showed no evidence of excitatory glutamatergic neurons (Fig. 1C)

Summary

Introduction

Retinoic acid (RA), the bioactive form of vitamin A, is essential for embryonic development by virtue of controlling proliferation, differentiation and homeostasis of diverse cell types, contributing to organogenesis and formation of different types of tissues including nervous tissue. RARα and RARβ are the only two RARs which could mediate such RA activity (RARγ is not expressed in the LGE)[17,18,19], genetic ablation of RARβ in mice affected development of only striatonigral projection neurons, a subpopulation of MSNs expressing dopamine receptor Drd[1], and did not affect generation of striatopallidal MSNs expressing Drd[219,20] These two populations of inhibitory, GABAergic neurons define two main output pathways of the striatum, and their unbalanced signaling is at the origin of physiopathology of several disorders of basal ganglia[21]. Neither the function nor the developmental origin of these neurons is known

Methods

Results

Conclusion