Skn-1a/Pou2f3 is required for the generation of Trpm5-expressing microvillous cells in the mouse main olfactory epithelium
- Tatsuya Yamaguchi†1,
- Junpei Yamashita†1,
- Makoto Ohmoto†2,
- Imad Aoudé3,
- Tatsuya Ogura3,
- Wangmei Luo3,
- Alexander A Bachmanov2,
- Weihong Lin3,
- Ichiro Matsumoto2Email author and
- Junji Hirota1, 4Email author
© Yamaguchi et al.; licensee BioMed Central Ltd. 2014
Received: 19 November 2013
Accepted: 14 January 2014
Published: 16 January 2014
The main olfactory epithelium (MOE) in mammals is a specialized organ to detect odorous molecules in the external environment. The MOE consists of four types of cells: olfactory sensory neurons, supporting cells, basal cells, and microvillous cells. Among these, development and function of microvillous cells remain largely unknown. Recent studies have shown that a population of microvillous cells expresses the monovalent cation channel Trpm5 (transient receptor potential channel M5). To examine functional differentiation of Trpm5-expressing microvillous cells in the MOE, we investigated the expression and function of Skn-1a, a POU (Pit-Oct-Unc) transcription factor required for functional differentiation of Trpm5-expressing sweet, umami, and bitter taste bud cells in oropharyngeal epithelium and solitary chemosensory cells in nasal respiratory epithelium.
Skn-1a is expressed in a subset of basal cells and apical non-neuronal cells in the MOE of embryonic and adult mice. Two-color in situ hybridization revealed that a small population of Skn-1a-expressing cells was co-labeled with Mash1/Ascl1 and that most Skn-1a-expressing cells coexpress Trpm5. To investigate whether Skn-1a has an irreplaceable role in the MOE, we analyzed Skn-1a-deficient mice. In the absence of Skn-1a, olfactory sensory neurons differentiate normally except for a limited defect in terminal differentiation in ectoturbinate 2 of some of MOEs examined. In contrast, the impact of Skn-1a deficiency on Trpm5-expressing microvillous cells is much more striking: Trpm5, villin, and choline acetyltransferase, cell markers previously shown to identify Trpm5-expressing microvillous cells, were no longer detectable in Skn-1a-deficient mice. In addition, quantitative analysis demonstrated that the density of superficial microvillous cells was significantly decreased in Skn-1a-deficient mice.
Skn-1a is expressed in a minority of Mash1-positive olfactory progenitor cells and a majority of Trpm5-expressing microvillous cells in the main olfactory epithelium. Loss-of-function mutation of Skn-1a resulted in complete loss of Trpm5-expressing microvillous cells, whereas most of olfactory sensory neurons differentiated normally. Thus, Skn-1a is a critical regulator for the generation of Trpm5-expressing microvillous cells in the main olfactory epithelium in mice.
A sense of smell is essential for the survival of both individuals and species. The main olfactory epithelium (MOE) is considered to be responsible for detecting a vast number of airborne odorous chemicals. The MOE consists of four major types of cells: olfactory sensory neurons (OSNs), supporting cells, basal cells, and microvillous cells . The OSNs are ciliated bipolar neurons specialized in detecting odorants and send their information to the axonal target in the main olfactory bulb. The cell bodies of the terminally differentiated OSNs are located in the intermediate position of the MOE. The supporting cells, also called sustentacular cells, protect and support OSNs, much like glial cells in the central nervous system. The supporting cells span the entire basal to apical extent of the MOE, and their somata are located in the apical/superficial layer of the MOE. The basal cells, which are globose and horizontal cells, are considered to function as stem cells that give rise to OSNs and supporting cells.
Although the properties of OSNs, supporting cells, and basal cells have been well studied and characterized in terms of both development and function, those of the microvillous cells remain largely unknown in the MOE. Microvillous cells are less abundant than are OSNs and supporting cells and are scattered in the superficial layer of the MOE [2–5]. Morphologically, at least three different types of microvillous cells have been described . Two of them express the monovalent cation channel transient receptor potential channel M5 (Trpm5). Because Trpm5 plays a critical role in chemical sensing in sweet, umami, and bitter taste cells (so-called type II taste cells) and in solitary chemosensory cells (SCCs) [6–10], and because the chemosensory activities of these taste cells are Trpm5-dependent and thermosensitive , Trpm5-expressing microvillous cells (Trpm5-microvillous cells) in the MOE are considered to be chemo- and/or thermosensitive. Indeed, Trpm5-microvillous cells were shown to express choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter, to respond to chemical or thermal stimuli, and to release acetylcholine to modulate activities of neighboring supporting cells and OSNs . However, molecular mechanisms underlying the generation and differentiation of these cells are not well understood.
Skn-1a (also known as Pou2f3), a POU (Pit-Oct-Unc) transcription factor, is expressed in Trpm5-expressing chemosensory cells: type II taste cells in taste buds of oropharyngeal epithelium and SCCs in nasal respiratory epithelium. Its loss-of-function mutation resulted in defective generation and/or functional differentiation of type II taste cells and SCCs [13, 14]. Thus, Skn-1a functions as a determinant for the generation and functional differentiation of these cells. Here we show that Skn-1a is expressed in the MOE, where neither taste cells nor SCCs have been observed. We characterized Skn-1a-expressing cells and investigated the function of Skn-1a in the MOE using Skn-1a-deficient mice. We demonstrate that Skn-1a is necessary for the generation of Trpm5-microvillous cells.
The expression of Skn-1ain the main olfactory epithelium
To our knowledge, neither SCCs nor taste cells have been found in the MOE. Both cell types share expression of Tas1r3, Tas2r family genes, Gnat3 (gustducin), Plcb2, and Trpm5. We examined the mRNA expression of these genes in the MOE and detected only Trpm5 (Figure 1D). The Trpm5 mRNA signals were observed in the superficial layer, where Skn-1a mRNA expression was also observed, indicating a possible role of Skn-1a in generation of cells of the MOE.
To analyze the population of Skn-1a-expressing cells, we performed two-color in situ hybridization for Skn-1a in combination with either Mash1 or Trpm5 and counted the number of single- and double-positive cells at postnatal day 30 (Figure 2D). Because of total number of cells counted differed between sections due to the scattered expression of Skn-1a and Trpm5, we represent the population of Skn-1a-expressing cells in percentage. Quantitative analyses revealed that 8.34 ± 2.82% (mean ± SD) of the Skn-1a-expressing cells were Mash1 positive (n = 3, see Additional file 1: Table S1) and 77.7 ± 5.95% were Trpm5 positive (n = 3, see Additional file 1: Table S1). Thus, a large population of Skn-1a-expressing cells is Trpm5 positive, and Mash1-positive cells are a minor population. In the OSN lineage, Mash1-positive olfactory progenitors rarely expressed Skn-1a (1.41 ± 0.564%, n = 3, see Additional file 1: Table S1; Figure 2E), whereas 36.9 ± 15.0% of Trpm5-expressing cells coexpress Skn-1a (n = 3, see Additional file 1: Table S1). These results suggest involvement of Skn-1a in the Trpm5-microvillous cell lineage rather than in the OSN lineage.
Impact of Skn-1adeficiency on olfactory sensory neuronal lineage
Impact of Skn-1adeficiency on Trpm5-positive microvillous cells
Quantitative comparison of ChAT/Trpm5-expressing microvillous cell density in Skn-1a-/- and ChAT-eGFPmice
We found that Skn-1a is expressed in both Mash1-positive olfactory progenitors and Trpm5-microvillous cells in the MOE. Although Skn-1a is expressed in both cell lineages, the loss-of-function mutation of Skn-1a had differential impacts: grossly normal differentiation of OSNs and complete loss of Trpm5-microvillous cells.
In the absence of Skn-1a, OSNs differentiated normally except for a partial defective differentiation in a limited region of the MOE: ectoturbinate 2. Because only a small population of Skn-1a-expressing cells coexpressed Mash1, and most of Mash1-expressing progenitors did not coexpress Skn-1a, Skn-1a could not be a determining factor for OSN differentiation. Considering the partial penetrance of this minor phenotype (defective differentiation in ectoturbinate2), it could be due to a secondary effect of loss of Skn-1a function. However, we could not exclude the possibility that Skn-1a somehow interacts with Mash1 genetic pathways and might cause this phenotype in Skn-1a-/- mice.
In contrast, none of the markers for Trpm5-microvillous cells were detectable in the Skn-1a-/- MOE, and a drastic loss of microvillous cells was clearly demonstrated. Therefore, Skn-1a is not simply required for the expression of marker genes, but is necessary for generation of Trpm5-microvillous cells. Previous studies have shown that Skn-1a is essential for the generation and/or functional differentiation of chemosensory cells, such as sweet, umami, and bitter taste cells in taste buds and SCCs in nasal epithelium [13, 14]. Both types of chemosensory cells share molecular characteristics of chemoreceptors, intracellular signaling molecules, and physiological functions to detect noxious substances [7, 9, 13, 15]. Although they do not express taste-cell-like signaling molecules except for Trpm5 and ChAT [5, 7, 12], Trpm5-microvillous cells function as chemo- and thermo-sensitive cells by responding to certain chemical or thermal stimuli, and they release acetylcholine to modulate activities of neighboring supporting cells and OSNs . It is intriguing that Skn-1a is commonly critical to generate these chemosensory cells.
There are at least three types of microvillous cell in the MOE : two are Trpm5-microvillous cells, and one is a Trpm5-negative microvillous cell. Our quantitative analyses of the density of superficial cells showed that the reduction in the density of Trpm5-microvillous cells in Skn-1a-/- mice is comparable to the percentage of ChAT/Trpm5-expressing microvillous cells, and that there are residual superficial cells, presumably non-Trpm5-microvillous cells. Currently, the identity of non-Trpm5-microvillous cells is unknown. The MOE, however, has a population of non-Trpm5-microvillous cells, called IP3 receptor type 3-expressing microvillous cells (IP3R3-microvillous cells) that express distinct cell markers, such as TRPC6, IP3R3, and PLC-β2 [2–5]. Double-label immunostaining against IP3R3 showed that immuno-signal of IP3R3 remained in Skn-1a-/- mice, whereas that of Trpm5 was abolished, suggesting that one of remaining superficial microvillous cells in Skn-1a-/- mice would be IP3R3-microvillous cells. (see Additional file 2: Figure S1). These indicate that Skn-1a is involved in the generation of Trpm5-microvillous cells but that its deficiency would not cause loss or expansion of non-Trpm5-microvillous cells; this differs from the case in taste buds, where Skn-1a regulates the fates of type II (sweet, umami, and bitter) and type III (sour) taste cells. In the microvillous cell lineages in the MOE, Skn-1a would not function to determine the lineage between Trpm5- and non-Trpm5-microvillous cells but would promote functional differentiation of Trpm5-microvillous cells. Further analysis of the function of Skn-1a in the olfactory epithelial cell lineages would provide us better understanding on the olfactory epithelial cell lineages.
Here we show that in the MOE, Skn-1a is expressed mainly in Trpm5-expressing microvillous cells and is required for their generation in the MOE. Combined with previous observations, this study shows that Skn-1a is a critical transcription factor for generation and/or functional differentiation of several types of chemosensory cells, that is, sweet, umami, and bitter taste cells, SCCs, and Trpm5-microvillous cells in the nasal and oropharyngeal epithelium. It is possible that Skn-1a could be involved in generation of chemosensory cells in other epithelial tissues, such as brush cells in trachea and intestine. The expression and function of Skn-1a in those cell types will be investigated in future studies to reveal common molecular mechanisms of Skn-1a function in generation of closely related chemosensory cells.
Skn-1a/Pou2f3-deficient mice (Skn-1a-/-) and Mash1-deficient mice (Mash1-/-) were generated as described elsewhere [13, 16]. The ChAT (BAC) -eGFP transgenic (ChAT-eGFP) mice were kindly provided by Dr. M. I. Kotlikoff . All mice used in this study were C57BL/6 background, and mutant and wild-type mice/embryos of either sex were used. For embryo staging, midday of the day of the vaginal plug was designated as embryonic day 0.5. The day of birth was designated postnatal day 0. All mouse studies were approved by the institutional animal experiment committees of University of Maryland, Baltimore County, of Monell Chemical Senses Center, and of Tokyo Institute of Technology and were performed in accordance with institutional and governmental guidelines.
Probes for Skn-1a, Trpm5, Plcb2, Gnat3, Tas1r3, Tas2r105, Tas2r108, Mash1, Ngn1, NeuroD, GAP43, and OMP were prepared as previously described [13, 14, 18]. The MOE was cryosectioned coronally at 10 μm thick. Single- and two-color in situ hybridization was performed according to the method described previously [19, 20]. For two-color in situ hybridization, the tyramide signal amplification-dinitrophenyl system (PerkinElmer) was used. The images were taken on an Olympus BX51 microscope with a DP71 digital CCD camera for bright-field images and a Leica SPE confocal microscope for fluorescent images.
To quantify the number of Skn-1a-, Mash1-, and Trpm5-expressing cells, every 10th coronal section (10 μm thickness) throughout the MOE was collected for in situ hybridization experiments, and the number of positive cells was counted. Experiments were conducted using three mice at P30, and the populations were calculated as the mean value.
Immunohistochemistry was performed according to a previously described method using coronal cryosections of 10 μm thick [12, 21]. The following primary antibodies and dilutions were used: goat anti-villin antibody (1:50; #sc-7672, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Trpm5 antibody (1:500; #ACC-045, Alomone Labs, Jerusalem, Israel), goat anti-ChAT antibody (1:100; #AP144P, Millipore, Billerica, MA), mouse anti-IP3R3 antibody (1:500; #61312, BD Biosciences, San Jose, CA) with the Vector M.O.M. Immunodetection Kit (Vector Laboratories, Burlingame, CA). The following appropriate secondary antibodies were used: Alexa-546-conjugated anti-goat IgG antibody, Alexa-555-conjucated anti-goat IgG antibody (both from Invitrogen, Carlsbad, CA), and biotin-conjugated goat anti-rabbit IgG antibody (Vector Laboratories) with streptoavidin-Alexa-488 fluorescence (Invitrogen). We performed antigen-retrieval pretreatments in Target Retrieval Solution, pH 9.0 (Dako, Glostrup, Denmark) for 20 min at 80°C. The sections were coverslipped with Vectashield mounting medium with DAPI (Vector Laboratories) or Fluomount-G including DAPI for nuclear staining (Southern Biotechnology, Birmingham, AL).
Quantitative analysis of the microvillous cell density
The density of the microvillous cells and nuclei in the most superficial layer of the MOE, which corresponds to the region above the nucleus layer of the supporting cells, was determined. Olfactory epithelial tissues from ChAT-eGFP control mice, in which ChAT/Trpm5-expressing microvillous cells are found throughout the entire MOE , and from Skn-1a-/- mice were prepared as described previously [5, 12], and a pair of noses from ChAT-eGFP and Skn-1a-/- mice, respectively, were embedded side by side in a single block using Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA). Every 20th coronal section (14 μm thickness) throughout the MOE was examined. Images of dorsal recess and septum region of the both sides of MOE in the sections were taken using an epifluorescence microscope (Olympus BX41, 10x objective) equipped with a CCD camera (QImaging Retiga 4000) and reconstituted using the MosaicJ plug-in of NIH Image J software. In the ChAT-eGFP mouse, both GFP-positive (GFP+; ChAT/Trpm5-expressing) microvillous cells and DAPI-stained nuclei in the most superficial layer of the MOE above the nuclei of supporting cells were counted manually. To avoid including supporting cells in the count, we counted only nuclei separated from the supporting cell nucleus layer, regardless the GFP expression. The surface area where the counting was conducted was determined by measuring the epithelial length using NIH Image J multiplied by the thickness of the section (14 μm). The density of both ChAT/Trpm5-expressing GFP+ cells and DAPI-stained nuclei of the MOE superficial layer was calculated using the number of cells or nuclei counted divided by the MOE surface area where the counting was conducted. Similarly, in Skn-1a-/- mouse, DAPI-stained nucleus density of the MOE superficial layer was determined. Data were expressed in number of cells ± SD per mm2, and Student’s t-test was used to determine the statistical significance.
This work was supported in part by grant support from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for Scientific Research (C) (20570208 to JH), the Mishima Kaiun Memorial Foundation and Yamazaki Spice Promotion Foundation to JH, and NIH grants DC009269 and DC012831 to W Lin, DC00882 to AAB, and DC011143 to IM. Parts of the histochemical analyses were performed at the Monell Histology and Cellular Localization Core, which is supported, in part, by funding from NIH Core Grant P30DC011735 (to Robert F. Margolskee, Monell Chemical Senses Center). MO was a Japan Society for the Promotion of Science Postdoctoral Fellow for Research Abroad.
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