Ascl3 transcription factor marks a distinct progenitor lineage for non-neuronal support cells in the olfactory epithelium

www.nature.com/scientificreports OPEN received: 12 August 2016 accepted: 04 November 2016 Published: 02 December 2016 Ascl3 transcription factor marks a distinct progenitor lineage for non-neuronal support cells in the olfactory epithelium Pei-Lun Weng1, Mridula Vinjamuri2 & Catherine E. Ovitt2 The olfactory epithelium (OE) is composed of olfactory sensory neurons (OSNs), sustentacular supporting cells, and several types of non-neuronal cells. Stem and progenitor cells are located basally, and are the source of all cell types needed to maintain OE homeostasis. Here, we report that Ascl3, a basic helix-loop-helix transcription factor, is expressed in the developing OE. Lineage tracing experiments demonstrate that the non-neuronal microvillar cells and Bowman’s glands are exclusively derived from Ascl3+ progenitor cells in the OE during development. Following chemically-induced injury, Ascl3 expression is activated in a subset of horizontal basal cells (HBCs), which repopulate all microvillar cells and Bowman’s glands during OE regeneration. After ablation of Ascl3-expressing cells, the OE can regenerate, but lacks the non-neuronal microvillar and Bowman’s gland support cells. These results demonstrate that Ascl3 marks progenitors that are lineage-committed strictly to microvillar cells and Bowman’s glands, and highlight the requirement for these cell types to support OE homeostasis. The mammalian olfactory epithelium (OE) is a pseudostratified epithelium composed predominantly of olfactory sensory neurons (OSNs), which are generated in the basal region and extend apically to the nasal cavity. They are supported by an apical layer of glial-like sustentacular cells1,2. Scattered throughout the OE are the non-neuronal microvillar cells and Bowman’s glands. Bowman’s glands consist of clustered acinar cells located under the OE in the lamina propria, linked to ducts that span the epithelium to transport mucus to the apical surface3. At least three types of microvillar cells have been described in the OE4. Two types, distinguished by different morphologies, express the transient receptor potential channel M5 (Trpm5)5. The third type is characterized by expression of phospholipase C β2 (PLC β2), and type 3 IP3 receptor (IP3R3), both involved in calcium-mediated signal transduction, and of CD736,7. The latter microvillar cell type has been identified as the primary source of neuropeptide Y (NPY) in the OE, which binds specific receptors to stimulate proliferation of basal progenitor cells and neurogenesis8,9. Knockout of NPY, or its receptor, results in reduced stem cell proliferation and decreased production of OSNs9,10. Numerous lines of evidence have indicated that the microvillar cells play an important role in OE homeostasis9,11–13. The OE undergoes constant turnover, which is fueled by basally located proliferative progenitors, and quiescent stem cells14–16. Under normal conditions, a heterogeneous population of active progenitors, known as globose basal cells (GBCs), expressing markers such as Lgr5, Ascl1, c-Kit or SEC8 generates the cell types to maintain the integrity of the OE17–23. In contrast, the multipotent horizontal basal cells (HBCs) are relatively quiescent, and are activated only after extensive lesioning of the OE, which removes both sustentacular cells and GBCs14. Re-activated HBCs can regenerate all cell types in the OE14,24. Ascl genes, members of the achaete scute-like complex family, are basic helix-loop-helix transcription factors (bHLH), which are expressed in progenitor cells of various tissues at the time of cell type specification. In the OE, Ascl1 is found in a subset of GBCs, which give rise to OSNs and sustentacular cells22. A second family member, Ascl2, is a critical regulator of intestinal stem cell fate and follicular T-helper cell specification25,26. Ascl3, the least 1 Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, USA. 2Center for Oral Biology and Department of Biomedical Genetics, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, USA. Correspondence and requests for materials should be addressed to C.E.O. (email: ) Scientific Reports | 6:38199 | DOI: 10.1038/srep38199 1 www.nature.com/scientificreports/ Figure 1. Ascl3 is expressed in the OE during embryonic development. (A) The Ascl3 gene locus includes 2 exons. In Ascl3EGFP-Cre/+ mice, the non-coding exon1 was maintained, and a fusion cassette encoding EGFPCre replaced the entire Ascl3 coding sequence in exon 2, through homologous recombination. (B) OE was isolated from Ascl3EGFP-Cre/+ mice at E12.5, E14.5, E16.5 and E18.5 of development. Immunohistochemistry was performed using antibodies to EGFP and TuJ1. Ascl3-EGFP+ cells (arrowheads) remain apically localized and are not colocalized with the marker for immature neurons, TuJ1, during embryonic development. Dotted line indicates basal lamina. Nuclei are stained by DAPI (blue). Scale bars: 25 μm. characterized member of the family, is a marker of progenitor cells in the salivary glands, and Ascl3-expressing precursor cells generate both duct and acinar cells in vitro27–29. Here we demonstrate that Ascl3 is also expressed in the OE, and marks precursors of the non-neuronal microvillar cells and Bowman’s glands during development and regeneration. We report that Ascl3 expression is activated in progenitors during development, and in a subset of HBCs immediately after injury, which generate all microvillar cells and the Bowman’s glands. Using cell-specific ablation and injury-induced regeneration, we show that in the absence of these cell types, neurogenesis of OSNs is reduced but not blocked, which may be due to the decreased number of GBCs and increase in apoptotic cells. Our data provide new insight into the lineage of non-neuronal support cells, and their requirement for OE homeostasis. Results Precursors of non-neuronal support cells express Ascl3 during OE development. To investigate the expression profile of Ascl3 during embryonic development of the OE, we used a previously generated mouse strain carrying a fusion cassette of EGFP and Cre recombinase knocked into the Ascl3 gene locus, which replaced the entire Ascl3 coding sequence (Fig. 1A)29. In this strain, EGFP expression is driven by the endogenous Ascl3 promoter. We observed EGFP as early as embryonic day 12.5 (E12.5) in the developing OE (Fig. 1B). EGFPpositive cells were detectable throughout embryonic development, at E14.5, E16.5 and E18.5, in cells localized at the apical region of the developing OE (Fig. 1B). There was no overlap observed between the EGFP-labeled cells and OSNs labeled with antibody to TuJ1. To follow the fate of the Ascl3-expressing cells found in the embryonic OE, we traced the lineage of these cells, using the Ascl3EGFP-Cre strain crossed with the R26tdTomato reporter. In Ascl3EGFP-Cre/+/R26tdTomato/+ mice, Ascl3-ex (...truncated)