Walk This Way: Stepping Back along the Lineage Highway with Progenitor Cells of the Olfactory Epithelium
Lin, Brian.
2017
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Abstract: Neurally
regenerative adult tissues in mammalian model systems are difficult to pin down: the few
known centers of adult neurogenesis are subject to caveats about how active and
functional they truly are. There is one tissue, however, which undergoes constant
neuronal turnover while maintaining a functional neural epithelium throughout adult
life—the olfactory epithelium (OE). In ... read morethis thesis, I use the OE to study the
effects of injury on lineage commitment and the regeneration of olfactory sensory
neurons. In Chapters 2 and 3, I provide a basis for key experimental techniques and
optimize them to the OE for their use later on in the thesis. First, I explore the use
of single cell mRNAseq in identifying cell types of the OE de novo without applying any
a priori knowledge. I conclude that either large numbers of cells or targeted capture of
a specific heterogeneous cell population is required to make it viable and later apply
it to identify heterogeneously regulated pathways in Chapter 4. Next, I use Gibson
Assembly and Taguchi Methods to generate a novel tri-cistronic doublet fluorescent
reporter construct, which is then knocked into the endogenous Ascl1 gene locus in vivo
at high efficiency using CRISPR/Cas9. This mouse is later used in Chapter 4 for FACS
isolation of the Ascl1+ neuronal progenitor population for transplantation. In Chapter
4, I demonstrate that neuronal lineage commitment is more a guideline than a rule, as
neuronally committed Ascl1+ progenitors or Neurog1+ immediate neuronal precursors are
capable of generating significant numbers of non-neuronal cells after injury. I show
that this induced multipotency is partially cell-autonomous using transplantation
assays. Single cell mRNAseq revealed significant heterogeneous cell populations within
the tissue, as well as several enriched pathways. I validated that the epigenetic
regulator Ezh2 plays a significant role in modulating this induced plasticity, while
canonical Notch signaling does not—in stark contrast to other tissues.
Furthermore, I demonstrate that this induced plasticity is a form of reprogramming,
mediated by some of the Yamanaka factors (Sox2, KLF4, and MYC targets), involving
stepping backwards developmentally. Finally, I show that this dedifferentiation
initially requires Sox2 for its induction, but may not be necessary for maintaining the
multipotent state afterwards. In Chapter 5, I show that while canonical Notch signaling
plays no role in dedifferentiation to multipotency, it does play a role in neuronal
maturation. Heterozygous knockout of RBPJ is sufficient to result in a severe neuronal
maturation phenotype where cells fail to properly target the olfactory bulb and
innervate glomeruli. As in Chapter 4, injury induces significant differences in this
population: knockout neurons are capable of targeting and innervating the bulb,
expressing mature markers and olfactory receptors. Interestingly, after olfactory
bulbectomy, the loss of trophic support does not affect these neurons as they are
eminently able to mature and survive for months past their wildtype brethren. These data
support a hypothesis where RBPJ plays a role in regulating trophic dependence, and the
possible existence of an injury-related neuronal maturation program distinct from
uninjured conditions. Taken together, this thesis provides the tools for future deep
molecular interrogation of cell fate commitment. It characterizes a novel method of
tissue regeneration in the OE and identifies part of the mechanism mediating this
dedifferentiation. Finally, it identifies a Notch-independent role of RBPJ in neuronal
maturation and suggests the existence of an injury-specific neuronal maturation
program.
Thesis (Ph.D.)--Tufts University, 2017.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisor: James Schwob.
Committee: Charlotte Kuperwasser, Grace Gill, Janis Lem, and Connie Cepko.
Keywords: Cellular biology, and Neurosciences.read less - ID:
- th83m8901
- Component ID:
- tufts:20682
- To Cite:
- TARC Citation Guide EndNote
