Notch signaling in the adult olfactory epithelium: roles in stem cell quiescence, lineage specification, and neuronal maturation
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). Both ... read morepopulations 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
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