EsrrG Plays a Role in Suppressing Motor Neurons and Inducing V2b Interneurons

Abstract

One of the major challenges in neuroscience is understanding how diverse neuronal subtypes are generated from a relatively uniform neural precursor. Elucidation of molecular mechanism regarding neuronal development will be of much value to our society as it would provide crucial basis for developing new drugs or cell therapy for treating neurodegenerative diseases. The goal of my research project was to gain further insight into genes that are involved in neuronal development based on the ChIP-seq and RNA-seq data on Motor neuron hexamer by Professor Seunghee Lee (MN-hexamer). Among the 500 genes identified as potential targets of motor neuron hexamer (MN-hexamer), 52 also possessed DNA binding response elements of Estrogen Related Receptor (ERRE). The research began with hypothesis that Estrogen Related Receptor could, independently or in cooperation with the MN-hexamer, direct motor neuron development. To see if the putative genes were expressed at motor neuron domain in the spinal cord, in situ hybridization (ISH) was performed with the mouse and chick spinal cord tissue transverse sections using the probes that I cloned. After screening for mRNA localization patterns of numerous genes, a few of them were chosen for further study. Interestingly, Estrogen Related Receptor Gamma (EsrrG) displayed migratory expression pattern depending on developmental stages. Luciferase reporter assay and GFP reporter assay were performed with the constructs that I cloned. Reporter assays confirmed that the target gene promoters were indeed activated by EsrrG. This was further validated by Chromatin Immunoprecipitation (ChIP) which showed that EsrrG physically bound to ERRE located upstream of Notch4. In order to gain insight into the function of EsrrG during spinal cord development, I performed the function analysis: For the gain of function analysis, chick embryo spinal cords were electroporated with the expression plasmids, either full length construct or the construct bearing transcription activating domain VP16. For the loss of function analysis, both chick and mice were analyzed. Chick embryos had the spinal cords electroporated with shRNA knockdown constructs or constructs bearing engrailed repressor domain, which acts as a dominant-negative form. Mutant mice embryos were harvested at various developmental stages by breeding EsrrG-floxed mice with Nestin-Cre mice, and obtaining knockout embryos which have EsrrG deleted at early neural progenitor stage. Surprisingly, I was able to conclude that EsrrG favored V2b inhibitory interneuron and played a certain role in suppressing motor neuron formation. I obtained consistent results during embryonic stem cell differentiation to motor neuron using the doxycycline inducible EsrrG stem cell line (iEsrrG) that I established. During differentiation of iEsrrG-ESC to motor neurons by treatment with retinoic acid and Sonic hedgehog agonist, doxycycline treatment led to very significant decreases in motor neuron markers Hb9 and Isl1 both in RT-PCR and IHC. My research has identified, for the first time, a novel role EsrrG plays in spinal cord neural development by establishing V2b interneuron and motor neuron cell fate decision. Further molecular mechanistic study and mutant mice study are in progress and closer examination of EsrrG at various embryonic stages and elucidation of its downstream signaling network will be the subjects for immediate future studies.