Picking (apart) the Nose: Characterization of Nrg1 and ErbB expression in the olfactory epithelium and function in a 3-D model of olfactory regeneration.
ability of the mesenchyme to produce soluble factors that influence epithelial growth
and differentiation is a concept that has been well-studied in multiple tissue systems.
This cross-talk between different cellular compartments is a fundamental requirement of
development, and one that is often exploited in disease, such as cancer. The olfactory
epithelium (OE) is a neuroepithe... read morelial tissue that is appealing to study both because of
the ease of tissue accessibility and the persistent replenishment, and thus active stem
cell niche, of all cell types throughout adult life. As with most tissues, the OE
contains an underlying mesenchymal compartment, the lamina propria (LP), which, together
with the OE, comprise the full olfactory mucosa (OM). Although some effort has been
directed at understanding the role of the LP in the repair of both olfactory sensory
neurons (OSNs) and support cells of the OE after injury, the secretome of this
mesenchyme and its effect on the OE are largely unknown. A goal of this project was to
use an in vitro, 3-D sphere model to study the effects of LP-derived soluble factors on
OE progenitor differentiation. We focused on Neuregulin1 (Nrg1), a protein that exists
in multiple isoforms and that has been studied in many other tissues. Nrg1 is a ligand
that functions by binding to and activating ErbB receptors, initiating signaling
cascades that are crucial for development. Although Nrg1 has been identified as a
component of the LP, its function remains elusive. To better understand the effect Nrg1
exerts on the OE, we addressed 2 major questions-1) What is the expression pattern of
Nrg1 isoforms and ErbB receptors in the OM? and 2) What are the functional consequences
of Nrg1 during OE regeneration? Using a combination of RT-qPCR, FACS, and
immunohistochemistry (IHC), we meticulously identified both the repertoire of Nrg1
isoforms present in the OM, as well as their cell type-specific expression patterns.
These analyses confirmed the presence of 8-9 Nrg1 isoforms-with structure variability
that support both juxtacrine and paracrine signaling mechanisms. Additionally, we
confirmed the presence of 3 ErbB receptors in the OE and provided IHC evidence that ErbB
receptors are in a position to respond to a soluble isoform of Nrg1 during OE
regeneration. We looked at the functional consequence of Nrg1 signaling by utilizing a
3-D in vitro sphere model developed in our lab to assay the differentiation of OE
progenitor cells upon growth factor(s) stimulation. We found that stimulation by a
recombinant form of soluble Nrg1 (rNrg1) alone is capable of inducing sphere growth.
Using RT-qPCR and IHC, we further showed that OE progenitor cells stimulated with rNrg1
develop into spheres that are highly enriched for duct/gland (D/G) cells, a support cell
of the OE. These data suggest that Nrg1 is capable of directing OE progenitor cells
towards a specific, D/G cell lineage. Ultimately, this project thoroughly interrogates
the expression pattern of Nrg1 isoforms and ErbB receptors, defining specific cell
populations that express these proteins and providing a framework to further study the
biochemical significance of Nrg1/ErbB signaling in the OE. Moreover, these data
highlight the ability of a single factor to influence stem cell fate. By confirming the
validity of our 3-D culture system as a method to effectively model the in vivo
environment, these results support a method to study the secretome of the LP and the
effect of other growth factors on OE progenitors. Finally, these data highlight a
previously unappreciated function of D/G cells in OE regeneration by uncovering a role
for Nrg1 in D/G cell expansion.
Thesis (Ph.D.)--Tufts University, 2014.
Submitted to the Dept. of Genetics.
Advisor: James Schwob.
Committee: Phil Hinds, Grace Gill, and Christiane Dammann.
Keywords: Genetics, and Cellular biology.read less