Project Summary
In the past decade, restoring the intrinsic axon growth ability of mature neurons has received promising results
in promoting axon regeneration in the central nervous system (CNS). However, to date, axon regeneration that
leads to successful functional recovery in the CNS is still practically impossible, primarily due to the inadequate
distance of regeneration and the low number of regenerating axons. Previous studies and my preliminary data
have shown that many genes mediating the intrinsic axon growth ability are differentially expressed at different
developmental stages in neurons, indicating the altered gene expression level during neuronal maturation is an
important factor underlying the diminished intrinsic axon growth capacity. However, how the altered gene
expression program is regulated remains largely unknown. Transcription factors (TFs) play important roles during
neuronal development, shaping the spatiotemporal gene expression landscape to control cellular activities
including axon elongation. Thus, understanding the intricate transcriptional regulatory network orchestrating
axon growth during development is critical for solving the challenge of mammalian CNS axon regeneration. In
this proposed study, I will perform parallel RNA-seq and ATAC-seq of purified retinal ganglion cells (RGCs) at
multiple developmental time points, and use advanced integrative bioinformatics analysis to obtain a
comprehensive view of the transcriptional regulatory network controlling the axon elongation function during
RGC development, and identify key TFs that function as core regulators of axon growth. The identified TFs will
be functionally tested in mouse optic nerve regeneration model to verify if they play important roles in RGC axon
regeneration and cell survival. RGCs are comprised of more than forty molecular distinct subtypes. Different
RGC subtypes vary in vulnerability to axonal injury and have distinct responses toward gene modulations. I will
conduct single-cell RNA-seq (scRNA-seq) in RGCs 2 weeks after optic nerve crush from control and TF-
manipulated groups to acquire the frequency of each RGC subtype in the final population, and determine what
specific RGC subtypes are protected by the manipulation of a specific TF by comparing the frequencies of RGC
subtypes between control and TF-manipulated groups. TFs whose manipulations are found to improve survival
in distinct RGC subtypes will be combined in the next step to determine if simultaneously manipulating these
TFs could protect a wide variety of RGC subtypes from injury-induced cell death and induce synergistic
promoting effect on RGC axon regeneration. In addition, I will also combine the manipulations of these TFs with
non-muscle myosin IIA/B deletion in RGCs, which produces axon regeneration by modifying cytoskeletal
dynamics in the growth cone of injured axons, to find out if this combinatory approach could lead to
unprecedented long-distance axon regeneration.
