Wake Up and Smell the Media: Developing a Culture Model of the Olfactory Epithelium
Peterson, Jesse.
2017
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Abstract: The
olfactory neuroepithelium (OE) is capable of regeneration throughout adult life to an
extent unparalleled within the mammalian nervous system. The OE accomplishes this
regeneration through the concerted effort of at least two resident stem cell
populations: the flattened, horizontal basal cells (HBCs) and the globose basal cells
(GBCs) that reside just apical to the HBCs and ... read morebelow the olfactory sensory neurons
(OSNs). HBCs exhibit the behavior of a classic reserve stem cell: they are quiescent and
slowly dividing until activated by severe tissue loss. In contrast, GBCs are highly
proliferative and reactive, responding to daily cell turnover by frequent
differentiation. Recently, the transcription factor p63 was identified as the master
regulator of HBC quiescence, providing a unique opportunity to investigate HBC
activation and to further explore stem cell heterogeneity. In this thesis I develop a
set of additional tools to study p63 in vitro and explore the relationship between p63
and the major developmental pathway Wnt. In Chapter 3 I demonstrate an OE basal cell
primary culture that may be utilized to further investigate p63 regulation. I establish
a relatively pure basal epithelial culture based on the classic bronchial epithelial
growth media with modifications to accommodate the metabolic demands of neurobasal stem
cells. The histological profile of these cells is compared with the basal progenitors
and mature cell types of the OE and found to be identical to p63+ HBCs in vivo. I
confirm that these cells remain undifferentiated in culture until treated with either
retinoic acid or by genetic ablation of p63 which results in differentiation into
neuronal and non-neuronal cell types. By utilizing genetic lineage traces, I can
identify the cells of origin for these HBC-like cells and determine they come from
Ascl1+ GBCs as well as HBCs. Interestingly, immediate neural precursor Neurog1+ GBCs can
only give rise to basal cultures following olfactotoxic lesion. The plasticity of
olfactory cultures is further demonstrated by directing them toward a respiratory cell
fate. Finally I develop a 3D air-liquid interface based system for generating complex
neuronal and non-neuronal organoids. Chapter 4 is broad characterization of Wnt
signaling in HBCs under normal physiological conditions and during recovery from injury.
I also explore the consequences of aberrant Wnt expression in HBCs. Utilizing a Wnt
reporter model I identify its peak activity in the basal compartment after olfactotoxic
lesion and through immunohistochemistry, quantitative PCR, and transgenic animal models
I begin to characterize the previously unexplored relationship between Wnt, p63 and HBC
proliferation. My data suggest that Wnt may be dispensible for HBC self-renewal, and
utilizing mutant models I find that HBCs appear relatively resistant to moderate Wnt
manipulation in contrast to recent findings in GBCs. However, in the presence of a
stabilized β-catenin, HBCs exhibit dysplastic growth. I identify the non-canonical
Wnt receptor Ror2 in HBCs, which to our knowledge represents the first histological
marker that distinguishes between these cells and the basal cells of the neighboring
respiratory epithelium. I proceed to explore Ror2 expression changes relative to
canonical Wnt levels after injury, although the significance of this relationship
remains to be determined. Taken together, this thesis introduces a number of new tools
for investigating the role of p63 in HBC quiescence/activation and clarifies our
understanding of the relationship between Wnt and
p63.
Thesis (Ph.D.)--Tufts University, 2017.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisor: Grace Gill.
Committee: Amy Yee, Li Zeng, and Bradley Goldstein.
Keyword: Cellular biology.read less - ID:
- 08613096n
- Component ID:
- tufts:20683
- To Cite:
- TARC Citation Guide EndNote
