Notch signaling in the adult olfactory epithelium: roles in stem cell quiescence, lineage specification, and neuronal maturation
Herrick, Daniel.
2018
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Abstract: The adult
olfactory epithelium (OE) has the remarkable capacity to regenerate fully both
neurosensory and non-neuronal cell types after severe epithelial injury. Life-long
persistence of two stem cell populations brings about OE regeneration when damaged: the
horizontal basal cells (HBCs), which are quiescent and held in reserve, and mitotically
active globose basal cells (GBCs). Bo... read moreth populations regenerate all cell types of the OE
after injury, but the mechanisms underlying HBC activation, neuronal vs. non-neuronal
lineage commitment after recruitment of the stem cell pools, and the role of Notch in
neuronal maturation all remain unknown. In the first set of experiments (Chapter 3), I
first provide a thorough immunohistochemical characterization of Notch in the adult OE
and a thorough analysis of the differential Notch gene regulation in the setting of
various lesion types. I first performed cell-population specific ablations using either
surgical olfactory bulbectomy (OBX) to selectively deplete the mature neuron layer or a
transgenic mouse to selectively deplete Sustentacular support cells using Sus-cell
specific expression of diphtheria toxin subunit A. We show for the first time that Sus
cell ablation is sufficient for HBC activation to multipotency. Through genetic
manipulation of Notch receptors, the data also suggest that Notch signaling contributes
to maintenance of HBC quiescence through a positive regulation of p63, the master
regulator of HBC quiescence. Additionally, Notch1 plays a critical role in maintaining
quiescence in the presence of severe neural injury or increased neurogenesis.
Importantly, this study reveals that Notch1 and Notch2 do not play redundant roles in
HBCs and the impact of Notch-ON on maintaining p63 levels is not Notch receptor
dose-dependent. In Chapter 4, I show that once HBCs are activated, Notch signaling
determines progenitor cell fate. We utilized both retroviral transduction and mouse
lines that permit conditional cell-specific genetic manipulation as well as the tracing
of progeny to study the role of canonical vs. non-canonical Notch signaling in the
determination of neuronal vs. non-neuronal lineages in the regenerating adult OE.
Excision of either Notch1 or Notch2 genes alone in HBCs did not alter progenitor fate
during recovery from epithelial injury, while conditional knockout of both Notch1 and
Notch2 together, retroviral transduction of progenitors with a dominant-negative form of
MAML, or excision of the downstream cofactor RBPJ caused progeny to adopt a neuronal
fate exclusively. Conversely, we show that overexpressing the Notch1-intracellular
domain (N1ICD) either genetically or by transduction blocks neuronal differentiation
completely. However, N1ICD overexpression requires both alleles of the canonical
cofactor RBPJ to specify downstream lineage. Taken together, our results suggest that
canonical RBPJ-dependent Notch signaling through redundant Notch1 and Notch2 receptors
is both necessary and sufficient for determining neuronal vs. non-neuronal
differentiation in the regenerating adult OE. Finally, in Chapter 5, using drivers
specific for neuronally committed progenitors (Ascl1 and Neurog1), I determined the role
of Notch signaling in neural progenitors after lineage had been determined. In the
uninjured epithelium, a low level of neurogenesis occurs throughout adult life. In
neuronally committed progenitors, RBPJ is required for neuronal maturation. In fact,
RBPJ haploinsufficiency is sufficient to prevent neuronal maturation. However, upon loss
of mature neurons by OBX or loss of all differentiated cells by chemical lesion, RBPJ is
no longer required for neuronal maturation.
Thesis (Ph.D.)--Tufts University, 2018.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisor: James Schwob.
Committee: Charlotte Kuperwasser, Tom Gridley, and John Castellot.
Keyword: Cellular biology.read less - ID:
- bz60d775g
- Component ID:
- tufts:25423
- To Cite:
- DCA Citation Guide EndNote
