Neurons are largely non-proliferative in mammals after injury leading to intense interest aimed at
reprogramming the neighboring glial cells into neurons1. In the retina of many fish and amphibians
regeneration of neurons from Müller glia occurs naturally after injury2,3. This regenerative capacity relies on the
highly conserved transcription factor Ascl14. Intriguingly, engineered overexpression of Ascl1 in mice leads to
the production of some neuronal cell types, such as bipolar cells, but not photoreceptors or ganglion cells5–7. I
aim to develop and use single-cell technologies to push Müller glia towards more diverse cell types and to
define which cis-regulatory elements drive those changes in cell fate. Production of photoreceptors from glia
would mark exceptional progress towards the development of regenerative treatments for blindness.
The single-cell sequencing revolution now makes it possible, within a single experiment, to recognize
the individual transcriptional responses of cell populations to up to thousands of treatment conditions8–10.
Combined with the growing movement towards assaying multiple regulatory steps simultaneously in a single
cell11–24, there has never been a better time to apply and develop single-cell technologies aimed at decoding
the regulation of fate changes in heterogeneous cell populations, such as those undergoing reprogramming.
In Aim 1, I will set out to uncover conditions which drive the conversion of Müller glia to novel
reprogramed fates. A recent study indicated that the efficiency of Müller glia to neuronal transition is vastly
improved by the treatment of cells with a histone deacetylase inhibitor (HDACi)6. I hypothesize that chromatin
modifications, not disrupted by HDACi treatment, prevent a more diverse rewiring of cell types upon the
induction of Ascl1. By harnessing sci-Plex, a technique recently developed in the Trapnell lab, I will culture
cells under hundreds of different small molecule treatments, each targeting aspects of the epigenome, and
then readout single-cell transcriptomes10. Through the systematic perturbation of chromatin biology, I hope to
expand the reprogramming potential of Müller glia and gain a better understanding of retinal cell specification.
In Aim 2, I propose engineering an assay able to read out both the transcriptome and the genomic
localization of histone modifications in a single cell. Multimodal single-cell technologies are powerful methods
to assess how aspects of the gene regulatory process interact with each other. The output of an assay such as
what I aim to develop could, through the use of regression analysis, allow the construction of maps linking
regulatory sites to genes. This can be used to determine the cis-regulatory elements most responsible for each
Müller glia reprogramming trajectory. If successful, similar techniques could be used to improve countless other
differentiation protocols and to better understand the regulatory landscape driving differentiation.