Transcription factor Runx1 inhibits proliferation and promotes developmental maturation in a selected population of inner olfactory nerve layer olfactory ensheathing cells
Introduction
Olfactory ensheathing cells (OECs) are a unique class of glial cells found exclusively in the olfactory system, one of the few neural tissues with the capacity to support lifelong neuronal regeneration (Franssen et al., 2007, Su and He, 2010). Residing in both the olfactory epithelium (OE) peripherally and the olfactory bulb (OB) centrally, OECs are remarkable in their ability to perform a vast array of functions in the context of olfactory system development and regeneration. They accompany and ensheath the axons of olfactory sensory neurons (OSNs) in the OE as they project to the olfactory bulb (OB). They also produce growth factors, cell adhesion molecules and extracellular matrix proteins that promote the growth of OSN axons and their correct targeting to the appropriate glomeruli in the OB. OECs are also endowed with phagocytic activity and engulf apoptotic olfactory nerve debris (Babiarz et al., 2011, Sasaki et al., 2011, Schwarting et al., 2004, Su and He, 2010, Su et al., 2013, Woodhall et al., 2001).
Because of their important biological functions in the context of olfactory system development and homeostasis, their ability to cross the boundary between the peripheral and central (CNS) nervous systems, and the demonstration that they can promote axonal regrowth and remyelination under experimental conditions, OECs are considered as attractive candidates for cell-based therapies to repair the injured CNS. Also supporting this possibility is the demonstration that OECs can associate with astrocytes without inducing astrocytosis and a glial scar, one of the key barriers to the repair of the injured CNS (Fitch and Silver, 2008, King-Robson, 2011, Radtke and Kocsis, 2012, Ramón-Cueto and Muñoz-Quiles, 2011, Tetzlaff et al., 2011). The potential application of OECs to neural repair in the CNS has been examined extensively; however, results have been mixed. Some groups reported that transplantation of OECs into the spinal cords of experimentally injured rats led to increase in tissue sparing, nerve fiber regeneration and axon outgrowth, and robust myelination, as well as increased impulse conduction and functional improvement. However, other groups observed little evidence of ascending or descending sensory fiber regrowth, no reduction in cavity formation and little to no functional recovery after OEC transplantation (Deumens et al., 2006, Imaizumi et al., 2000, Li et al., 2011, Li et al., 2012, Richter et al., 2005, Steward et al., 2006, Toft et al., 2007).
One of the reasons believed to underlie the inconsistent results of OEC-based strategies to promote neural repair is the lack of a detailed understanding of OEC biology. OECs represent a heterogeneous population of cells that are distinguishable from one another based on their spatial distribution within the olfactory nerve layer (ONL) in the OB, as well as their morphology, gap junction connectivity, and expression of specific sets of proteins in varying combinations (Franssen et al., 2007, Honoré et al., 2012, Kaplinovsky and Cunningham, 2011, Rela et al., 2010, Su and He, 2010, Thyssen et al., 2013, Ubink and Hökfelt, 2000). Different classes of OECs are believed to perform distinct subtype-specific functions. OECs in the outer region of the ONL are thought to be involved in OSN axonal outgrowth and guidance toward the OB, in part by selectively displaying adhesive or repulsive behaviors toward outgrowing axons via cell surface receptors that include Ret and p75 neurotrophin receptor (p75) (Honoré et al., 2012, Kaplinovsky and Cunningham, 2011, Su and He, 2010, Thyssen et al., 2013). OECs in the inner ONL are believed to be involved in axon sorting, refasciculation and targeting to appropriate glomeruli, partially through the distinct spatiotemporal expression of molecules such as neuropeptide Y (NPY) and Semaphorin 3A (Schwarting et al., 2004, Su and He, 2010, Ubink and Hökfelt, 2000). These observations point to a need for a better understanding of the mechanisms that mediate the acquisition of the characteristic molecular profiles, settling positions within the ONL, and specific cellular functions of individual OEC subpopulations.
The aim of the present study was to characterize new factors involved in OEC development and subtype identity acquisition. It is shown here that the Runt-related transcription factor Runx1, which is important for OSN neurogenesis in the OE (Theriault et al., 2005), is expressed in a defined OEC subpopulation located in the inner portion of the ONL, as well as in their precursors en route to the OB. We also provide evidence suggesting that Runx1 is important for the developmental maturation of the specific OEC subtype in which it is expressed by inhibiting proliferation and promoting the acquisition of a more developmentally mature phenotype. These findings identify Runx1 as a useful new marker of a selected OEC subpopulation postulated to play a role in the precise and appropriate innervation of the OB and reveal a previously unknown function for Runx1 during the development of specific OECs in the inner ONL.
Section snippets
Animals and tissue preparation
Runx1LacZ/+ mice were genotyped as previously described (Theriault et al., 2005). The recombined locus of Runx1LacZ/+ mice encodes a fusion protein of the N-terminal 242 amino acids of Runx1, containing a nuclear localization sequence, and bacterial β-galactosidase (βGal). As a result, the βGal-containing fusion protein expressed in Runx1LacZ/+ mice is localized to the nucleus. The expression of βGal faithfully recapitulates that of Runx1 (Stifani et al., 2008, Zagami et al., 2009, Zusso et
Runx1 is expressed in the developing olfactory bulb
Previous studies have shown that Runx1 gene expression marks OSN progenitor cells located on the basal side of the OE (Theriault et al., 2005). To determine whether Runx1 might also be expressed in other regions of the olfactory system, we took advantage of previously described Runx1LacZ/+ knock-in mice in which the expression of a βGal protein that localizes to the nucleus faithfully recapitulates Runx1 protein expression (Stifani et al., 2008, Zagami et al., 2009, Zusso et al., 2012). Runx1
Discussion
OECs are a molecularly and functionally heterogeneous class of glial cells playing multiple key roles to support the development and lifelong regeneration of the olfactory system. Little is presently known about the mechanisms controlling the development and functional specification of specific OEC subtypes. In this study, we have obtained evidence that the transcription factor Runx1 is a new useful marker of a particular subset of OECs located in the inner region of the ONL. Moreover, we have
Conflict of interest
The authors declare that they have no competing interests.
Acknowledgments
We thank Dr. Phil Barker for his generous gift of the p75 antibody and Rita Lo for invaluable assistance with primary OEC cultures. This work was supported by the Canadian Institutes of Health Research Operating Grant MOP-84577 to S.S., who is a James McGill Professor of McGill University.
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Equal contributions.