Author response: Olfactory receptor accessory proteins play crucial roles in receptor function and gene choice (2017)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Each of the olfactory sensory neurons (OSNs) chooses to express a single G protein-coupled olfactory receptor (OR) from a pool of hundreds. Here, we show the receptor transporting protein (RTP) family members play a dual role in both normal OR trafficking and determining OR gene choice probabilities. Rtp1 and Rtp2 double knockout mice (RTP1,2DKO) show OR trafficking defects and decreased OSN activation. Surprisingly, we discovered a small subset of the ORs are expressed in larger numbers of OSNs despite the presence of fewer total OSNs in RTP1,2DKO. Unlike typical ORs, some overrepresented ORs show robust cell surface expression in heterologous cells without the co-expression of RTPs. We present a model in which developing OSNs exhibit unstable OR expression until they choose to express an OR that exits the ER or undergo cell death. Our study sheds light on the new link between OR protein trafficking and OR transcriptional regulation. https://doi.org/10.7554/eLife.21895.001 eLife digest Olfaction, or the sense of smell, is perhaps the most complicated and least understood of the five basic senses. Olfactory neurons in the nose can detect and distinguish between tens of thousands of different odor producing substances. They do so by using hundreds of unique sensors called olfactory receptors, each of which responds to a specific type of odor. During development, each olfactory neuron “chooses” to produce only one type of olfactory receptor. Once the neuron recognizes that the functional receptor is being generated and transported to the cell surface, it will stop making all the other olfactory receptors. Chaperone proteins are responsible for transporting many olfactory receptors to the cell surface. To investigate how the loss of these chaperones affects how the olfactory system develops, Sharma et al. studied mice that were unable to produce the olfactory chaperone proteins. In these mice, developing neurons that chose to produce a type of olfactory receptor that depends on chaperone protein transport could not fully shut off other olfactory receptor genes. This led either to the neuron attempting to produce another type of receptor, or the death of the neuron. As a result, more neurons than usual produced receptors that do not require chaperone proteins to transport them to the cell surface. The olfactory neurons therefore produced only a fraction of all possible olfactory receptors, which decreased the ability of the mice to respond to odors. In the future, it will be important to understand what determines whether an olfactory receptor can be transported to the cell membrane in the absence of chaperone proteins. Olfactory receptors are G protein-coupled receptors (GPCRs), which are the largest molecular class of drug targets for cancer and diseases that affect the brain and heart. Thus, results presented by Sharma et al. will also be relevant to researchers who study how GPCR malfunction causes diseases. https://doi.org/10.7554/eLife.21895.002 Introduction Seven transmembrane G-protein coupled receptors (GPCRs), are diverse and the largest superfamily of receptors. Their roles are well established in sensing various stimuli including odorants, tastants, light, hormones, neurotransmitters and proteins. Some GPCRs require the presence of specific accessory proteins such as chaperones, vesicular targeting molecules and co-receptors for their cell surface expression (Lu et al., 2003; Salahpour et al., 2004; Dey and Matsunami, 2011; Wu et al., 2003). Mammalian olfactory receptors (ORs), which are GPCRs (Buck and Axel, 1991), are retained in the ER when expressed in non-olfactory cells. RTP1 (Receptor Transporting Protein 1) and RTP2 (Receptor Transporting Protein 2), both single transmembrane proteins strongly and exclusively expressed in the peripheral olfactory organs (Lu et al., 2003; Saito et al., 2004; Zhuang and Matsunami, 2008; Gimelbrant et al., 1999), greatly enhance the trafficking of ORs to the cell surface of heterologous cells. However, the role played by the RTPs in vivo remains unclear. The mouse genome encodes over one thousand intact OR genes (Niimura et al., 2014), which are expressed in a singular and monoallelic fashion in each olfactory sensory neuron (OSN) (Shykind et al., 2004; Chess et al., 1994; Serizawa et al., 2000; Malnic et al., 1999). Each OR is not chosen at random; rather, OSNs express different ORs with dramatically varying probabilities (Khan et al., 2011; Ibarra-Soria et al., 2014). OSNs in the olfactory epithelium (OE) are organized in overlapping zones defined by the expression of each OR (Ibarra-Soria et al., 2014; Ressler et al., 1993; Vassar et al., 1993; Miyamichi et al., 2005; Kanageswaran et al., 2015; Saraiva et al., 2015) as well as in a pseudostratified manner with progenitor cells forming the basal layer and mature neurons forming the upper layers. Mature OSN dendrites project into the nasal cavity forming a dendritic knob at the surface of the OE where they express ORs to interact with odorant molecules. Mature OSN axons expressing the same OR project to the olfactory bulb (OB) to converge onto specific glomeruli (Mombaerts et al., 1996; Ressler et al., 1994; Vassar et al., 1994; Hayar et al., 2004; Aungst et al., 2003; Gire et al., 2012). When the β2 Adrenergic Receptor (β2AR) is expressed instead of an OR, the β2AR –expressing OSNs target their axons to the OB and form glomeruli (Feinstein et al., 2004a, 2004b; Omura et al., 2014; Nakashima et al., 2013). Hence, the development of the peripheral olfactory system is dependent on functional GPCRs. The mechanisms by which an OSN makes an OR choice have not been fully elucidated. Locus control-region like enhancers scattered on the genome and relative location of ORs from these elements have important roles in determining the probabilities of OR gene choice (Khan et al., 2011; Serizawa et al., 2003; Markenscoff-Papadimitriou et al., 2014). Various epigenetic mechanisms, for example histone modification by lysine demethylase (LSD1), allow the escape of an OR gene from repression (Magklara et al., 2011; Lyons et al., 2013, 2014; Armelin-Correa et al., 2014; Kilinc et al., 2016). OR expression is unstable until one OR is functionally expressed which then represses the expression of other OR alleles via negative feedback signaling through the unfolded protein response (UPR) and G proteins (Serizawa et al., 2003; Dalton et al., 2013; Wang et al., 2012; Li and Matsunami, 2013; Lewcock and Reed, 2004; Ferreira et al., 2014; Nguyen et al., 2007; Abdus-Saboor et al., 2016). Here, we generated Rtp1 and Rtp2 double knockout mice (RTP1,2DKO) to investigate their role in the functioning and development of the olfactory system in vivo. We show that the RTP1,2DKO have OR trafficking defects, a substantial reduction in the number of mature OSNs, and an overall diminished olfactory capacity. Unexpectedly, we found that some ORs are overrepresented (referred to as oORs) while others are underrepresented (referred to as uORs) in RTP1,2DKO. Cells expressing a uOR lack stable gene choice in the mutant compared to wild-types while cells expressing an oOR do not show this instability, a result that links OR protein trafficking and OR transcriptional regulation. Results Generation of RTP1,2DKO mice In order to study the role played by RTP1 and RTP2 in regulating OR expression and trafficking in vivo, we consecutively knocked out these genes while the intervening ~500 kb genomic region was not disrupted in ES cells (Figure 1A). Following chimeric mice production and germline transmission, we established mouse lines with Rtp1 and Rtp2 double knock out alleles. We found no phenotypic difference between Rtp1(+/+);Rtp2(+/+) (wild-type) and Rtp1(+/−);Rtp2(+/−) (het) mice. The Rtp1(−/−);Rtp2(−/−) homozygous mutants (RTP1,2DKO) showed no gross defects outside the olfactory system. Heterozygous crosses gave rise to wild-type, heterozygous and homozygous adults in roughly 1:2:1 ratio (Rtp1(+/+);Rtp2(+/+) 19, Rtp1(+/−);Rtp2(+/−) 34, Rtp1(−/−);Rtp2(−/−) 15, n = 10 mating pairs), suggesting no embryonic or postnatal lethality. We validated the absence of RTP1 and RTP2 transcripts in RTP1,2DKO by performing RNA in situ hybridization (Figure 1B). Figure 1 Download asset Open asset Deletion of RTP1 and RTP2 causes defects in the OE. (A) Strategy for knocking out RTP1 and RTP2 in series. (B) RNA in situ hybridization with probes specific to RTP1 and RTP2 in both wild-type and RTP1,2DKO mice showing that the knock out mice do not express either of these proteins. Scale bar = 50 μm. (C) Schematic depiction of M71-IRES-tau GFP. (D) Antibody against M71 (red) stains the dendrite in the wild-type OE (top) but not the RTP1,2DKO OE. On the other hand, the antibody against GFP shown in green stains the entire neuron from RTP1,2DKO;M71-IRES-tauGFP mice, which shows that M71 positive OSNs have dendrites. https://doi.org/10.7554/eLife.21895.003 RTP1,2DKO OSNs show defects in OR trafficking It has been previously shown that RTP1 and RTP2 promote cell surface expression of ORs in the heterologous expression assays (Saito et al., 2004; Zhuang and Matsunami, 2007). Therefore, we used M71-IRES-tauGFP mice in which Olfr151 (also known as M71 and MOR171-2) expressing OSNs co-express tauGFP to examine the OSNs for OR trafficking defects (Feinstein et al., 2004a) (Figure 1C). In the RTP1,2DKO;M71-IRES-tauGFP OE, GFP staining was observed in the dendrites of Olfr151 positive OSNs (Figure 1D), indicating that the morphology of their OSNs remains unchanged. In contrast, immunostaining against Olfr151 (Barnea et al., 2004) was restricted to the cell body, indicating these OSNs are unable to traffic the OR to the dendrite (Figure 1D). Altogether, the data suggest that RTP1 and RTP2 are essential for OR trafficking. RTP1,2DKO mice have fewer mature sensory neurons Upon examination of the OE, we found that its thickness was significantly reduced in RTP1,2DKO mice. (p=0.02 paired student t test) (Figure 2A). We therefore examined the expression of various OSN developmental markers and signaling molecules in the OE to evaluate areas occupied by mature and immature OSNs in RTP1,2DKO. We compared OMP and adenylate cyclase 3 (ACIII), markers for mature neurons (Carter et al., 2004; Rogers et al., 1987), in 21 day old RTP1,2DKO mice and their littermates (Figure 2B, Figure 2—figure supplement 1). We measured the area occupied by RNA in situ hybridization signal against OMP and found that mice showed an average of 22% reduction in RTP1,2DKO when compared to the wild-type (p<0.0001, paired student t test, wild-type mean area 71% ± 5 (SD), RTP1, 2DKO mean area 49% ± 4 (SD)) (Figure 2C, See methods for details). Comparison of the OMP positive layer from wild-type and RTP1,2DKO OE collected at 1-day-old, 21-day-old and 6-month-old mice showed a significant reduction in OMP expression at 1 day and 21 days (p=0.0003, Mann Whitney U test, p=0.0003, Mann Whitney U test) but not at 6 months (Figure 2D). Immunohistochemical analysis of expression of adenylate cyclase 3 (ACIII), a signaling molecule expressed in mature OSNs (Wei et al., 1998; Wong et al., 2000; Col et al., 2007) showed a 17% decrease in the area occupied by the staining in 21 day old RTP1,2DKO OE (p=0.0001, Mann Whitney U test, wild-type mean area 44% ± 9 (SD), RTP1,2DKO mean area 27%, ± 3 (SD)) (Figure 2B). Consistent with OMP expression, we observed a significant difference in ACIII expression at 1 day old (p=0.0079, Mann Whitney U test) but not at 6 months (Figure 2E). Figure 2 with 1 supplement see all Download asset Open asset RTP1,2DKO mice have fewer mature sensory neurons. (A) Paired comparison of the thickness of the OE measured at five matched positions (see methods) between RTP1,2DKO and their wild-type littermate. (p=0.02, paired student t test). (B) RNA in situ hybridization against OMP (top), GAP43 (bottom) and IHC against ACIII (middle) at 1 day, 21 days and 6 months old. Scale bar = 25 μm. (C) Quantification of the percent area occupied by OMP RNA in situ hybridization signal from matched positions in the OE. Pair wise student t test shows a significant reduction in the area occupied by OMP staining in RTP1,2DKO. Error bars indicate SEM, p<0.0001, Paired student t test. (D) Comparison of percent area occupied by OMP between RTP1,2DKO and their het/wild-type littermates at different ages showing that RTP1,2DKO has fewer mature OSNs at 1 day (p=0.0003 Mann Whitney U test) and 21 day (p=0.0003 Mann Whitney U test) but there is no difference at 6 months (p=0.7, Mann Whitney U test). (E) Quantification of the area occupied by ACIII staining between RTP1,2DKO and their control genotype (hetetrozygous or wild-type) littermates at different ages showing that RTP1,2DKO has fewer mature OSNs at 1 day (p=0.0079 Mann Whitney U test) and 21 day (p<0.0001 Mann Whitney U test) but there is no significant difference at 6 months (p=0.1143, Mann Whitney U test). (F) Quantification of the area occupied by GAP43 staining between RTP1,2DKO and their het or wild-type littermates at different ages showing that RTP1,2DKO has more immature neurons at 21 day (p=0.0343 Mann Whitney U test). https://doi.org/10.7554/eLife.21895.004 Figure 2—source data 1 OE thickness and percent area occupied by the OMP layer, ACIII layer and GAP43 layer in the wild-type and RTP1,2DKO. https://doi.org/10.7554/eLife.21895.005 Download elife-21895-fig2-data1-v2.xlsx GAP43 is a marker for immature olfactory neurons in the OE (Meiri et al., 1988; Verhaagen et al., 1990; Treloar et al., 1999) and area occupied by it shows a 7% increase in the area of the OE it occupies in 21-day-old RTP1,2DKO (p=0.03 Mann Whitney U test, wild-type mean area 20%, ± 5 (SD), RTP1,2DKO mean area 27%, ± 6 (SD)) (Figure 2B). No significant difference in the GAP43 positive layer is observed between RTP1,2DKO and their littermates at 1 day nor at 6 months (Figure 2F). Odorant evoked electrophysiological responses in RTP1,2DKO mice are diminished Upon observation of fewer OSNs in RTP1,2DKO mice and lack of OR trafficking to the cilia (Figure 1D), we sought to test the olfactory ability by electroolfactogram (EOG). We tested a diverse set of 7 odorants, in both wild-type and RTP1,2DKO littermates. Wild-type mice show robust EOG responses to all odorants at concentrations as low as 0.01% (Figure 3A). In contrast, RTP1,2DKO mice showed striking deficits in their response. Responses to most odors were identical to the blank stimulus (air only), although some sensitivity was maintained for a subset of odorants (2-heptanone, amyl acetate, isomenthone) compared to the wild-types (Figure 3B–C). Thus, the reduction of mature OSNs and the loss of surface OR expression corresponds to a dramatic loss of odorant sensitivity in RTP1,2DKO. Figure 3 Download asset Open asset Diminished activity in response to odorants in RTP1,2DKO. (A) Electroolfactograms show the response to seven odorants wild-type. The grey line denotes the air only blank averaged over multiple interleaved trials interspersed within the series. (B) RTP1,2DKO responses to the same odors (C) Quantification of the EOG amplitudes for each of the seven odorants showing that only a few of the odors elicit responses from the RTP1,2DKO OE and these responses are lower than the wild-type. Each bar represents the difference between the peak of the odor minus the peak of the air only blank. https://doi.org/10.7554/eLife.21895.007 OR expression is biased in RTP1,2DKO To obtain a comprehensive view of gene expression changes in RTP1,2DKO, we performed an RNA-Seq on isolated whole olfactory mucosa including the OE and surrounding tissues. Differential expression analysis comparing RTP1,2DKO to wild-type littermates revealed that 3.8% of all genes (926/24,661) were differentially expressed between the two genotypes, among which 805 were downregulated and 121 were upregulated in RTP1,2DKO (FDR corrected p<0.05, see Experimental Procedures for details). Canonical signaling molecules known to be expressed in mature OSNs including Gnal (Gαolf), Adcy3 (ACIII), and Cnga2 were less abundant in the RTP1,2DKO consistent with a reduced number of mature OSNs in absence of RTP1 and RTP2. We found no significant difference in the expression levels of housekeeping genes like Gapdh and β actin (Supplementary file 1) (Kouadjo et al., 2007), neither did we see any compensatory increase in other RTP family members Rtp3 or Rtp4. We then asked whether the loss of RTP1 and RTP2 equally affected all ORs. In a comparison between wild-type and RTP1,2DKO we found that 62% of intact ORs (678/1088) were significantly affected by the loss of RTP1 and RTP2 (Figure 4A). Close to half of the annotated intact ORs (562/1088) were downregulated in RTP1,2DKO (FDR corrected p<0.05), consistent with fewer OSNs in the mutant. Unexpectedly however, a small subset of OR transcripts (116/1088) were upregulated in RTP1,2DKO mice (FDR corrected p<0.05) (Figure 4B, Figure 4—figure supplement 1(A-B)). Figure 4 with 1 supplement see all Download asset Open asset Representation of ORs in RTP1,2DKO. (A) Comparison of all transcripts between the wild-type and RTP1,2DKO, the green dots represent ORs, higher read counts for ORs are observed in the wild-type compared to RTP1,2DKO. (B) A comparison of the expression levels of ORs between the wild-type (x – axis) and RTP1,2DKO (y-axis). Red indicates uORs and oORs with p<0.01, blue indicates p<0.05. (C) A volcano plot showing the fold change of the expression levels (x-axis) of the ORs between wild-type and RTP1,2DKO using read counts normalized by OR genes. Red dots are ORs with p<0.01, blue: p<0.05, yellow dots signify candidate uORs chosen for validation. (D) Representative images for an in situ analysis with a probe specific to Olfr522 (uOR) where there are fewer positive OSNs in RTP1,2DKO when compared to the wild-type. Scale bar = 25 μm. (E) Quantification of the OSNs expressing uORs shown in (C); all the tested ORs showed smaller fractions of positive OSNs in RTP1,2DKO compared to the wild-type. p<0.05, Mann-Whitney U Test, n = 3 mice. (F) Volcano plot showing oORs with read counts normalized by OR genes. (G) Representative images for an in situ hybridization analysis with a probe specific to Olfr414 (oOR) where there are more positive OSNs in RTP1,2DKO when compared to the wild-type. Scale bar = 25 μm. (H) Quantification of the OSNs expressing oORs shown in (G); all the tested ORs showed greater fractions of positive OSNs in RTP1,2DKO compared to wild-type. p<0.05, Mann-Whitney U Test, n = 3 mice. (I) Plot of the mean abundance where each dot represents a single olfactory receptor classified as an uOR/ oOR/ NS based on normalization by ORs. The horizontal bars denote mean abundance (FPKM). oORs are significantly more abundant than uORs, NS are less abundant than both oORs and uORs (p<0.0001, one-way ANOVA, Tukey’s post hoc test). (J) zoomed in view of the plot showing uOR/oOR and NS abundance, horizontal bars denote mean abundance (FPKM). Figure data 1 positive cell counts for the uORs and oORs in Figure and 4 Download The in the abundance of transcripts for these ORs the of a difference in probabilities of OSNs expressing each OR in RTP1,2DKO. To any possible from we normalized read counts using only on intact ORs and found that were underrepresented and were overrepresented (FDR corrected p<0.05) (Figure Figure 4—figure supplement 1C). To changes in numbers of OSNs expressing ORs in RTP1,2DKO mice, we out RNA in situ hybridization with probes against either underrepresented ORs (Figure or overrepresented ORs (Figure all uORs fewer OSNs were positive in RTP1,2DKO (Figure Mann-Whitney U test). In contrast, the for the tested oORs were greater in RTP1,2DKO Mann-Whitney U test) (Figure results that OR gene choice is biased in RTP1,2DKO a specific subset of receptor we found that oORs as a are more expressed than uORs in the wild-type. The OR genes that were not classified as either underrepresented nor overrepresented not a of changes in expression levels between the wild-type and RTP1,2DKO, but are expressed at significantly lower abundance levels than both oORs and uORs (Figure (p<0.0001 one-way ANOVA, Tukey’s post hoc test). The of OSNs expressing oORs in RTP1,2DKO mice We what to the of OSNs expressing uORs and oORs in RTP1,2DKO mice at different We performed RNA in situ hybridization with a a probe uORs and a probe 25 all expressed in the region of the OE on and 6-month-old OE. In the of the uOR wild-type showed an increase for OSNs expressing the uORs we tested both at 21 days and 6 months (Figure consistent to the of mature OSNs in the OE by larger OMP and ACIII (Figure However, RTP1,2DKO showed no increase in the fraction of cells expressing these ORs with while on the other hand, in RTP1,2DKO the number of neurons expressing oORs showed a dramatic increase both from 1 day old to 21 days and from 21 days to 6 months (p<0.0001 one-way ANOVA, Tukey’s post hoc test) (Figure that the RTP1,2DKO OE is by Figure 5 Download asset Open asset The of OSNs expressing oORs in RTP1,2DKO. (A) Representative images from 1 day, 21 day and 6 OE with a probe against of the uORs expressed in the OE. (B) Quantification of the percent uOR positive cells at different ages in RTP1,2DKO and their het or wild-type littermates. The fraction of cells positive for this probe significantly with only in wild-type (p<0.0001 one-way ANOVA, Tukey’s post hoc test). (C) Representative images from 1 day, 21 day and 6 OE with a probe against 25 of the oORs expressed in the OE. (D) Quantification of the percent oOR positive cells at different ages between RTP1,2DKO and their het or wild-type littermates. The fraction of cells positive for this probe significantly with in RTP1,2DKO (p<0.0001 one-way ANOVA, Tukey’s post hoc test). Figure data 1 positive cell counts for the uOR and oOR probe at 1 day, 21 day and 6 old OE. Download of an OR in RTP1,2DKO depends the protein and not on the genomic location We whether the OR expression to the of RTP on the elements of an gene or the In we did not an or for the genomic nor did we or uORs or oORs (Figure file In an to the of the we used a mouse expressing from the Olfr151 (Feinstein et al., (Figure and asked whether the numbers of OSNs expressing Olfr151 or β2AR are affected in RTP1,2DKO. We chose Olfr151 as it is a uOR and β2AR it is a of the cell surface without the RTPs in heterologous and can a functional OR in OSNs et al., 2014). We that fewer Olfr151 expressing OSNs were present in RTP1,2DKO Mann-Whitney U as for a more β2AR positive OSNs were present in RTP1,2DKO compared with wild-type Mann-Whitney U test) (Figure suggesting that it is the protein and not the of the OR that determines whether a OR is underrepresented or Figure 6 Download asset Open asset OR protein in RTP1,2DKO. (A) showing uORs in and oORs in (B) Schematic depiction of (C) The percent Olfr151 positive cells is smaller in RTP1,2DKO mouse receptor expressed from the Olfr151 shows more β2AR cells in RTP1,2DKO mouse (D) Quantification of the percent positive Olfr151 and β2AR cells in wild-types RTP1,2DKO Mann-Whitney U test, n = 3 mice. (E) Representative data the number of cells the of staining expressing uORs Each represents an (F) Representative data the number of cells the of staining expressing oORs Each represents an (G) Comparison of the normalized mean of the for all uORs all oORs The are normalized to Mann-Whitney U test, uOR n = oOR n = mean is by the cells. Figure data 1 mean for from Download that β2AR is known to be to the cell surface when expressed in the absence of the we asked whether uORs and oORs show in cell surface trafficking in heterologous cells. We out cell surface staining of cells with either uORs or oORs in the absence of RTP1 and RTP2. In order to the surface we out to the surface OR oORs as a showed more OR surface expression than uORs Mann-Whitney U test) (Figure the ORs that show most robust cell surface expression were all trafficking mechanisms between OSNs and cells are to be data are consistent with the that trafficking of ORs be to of OSNs expressing OSNs expressing oORs mature and Our results suggest that OSNs expressing oORs are to despite the loss of the RTPs. In order to test we examined whether OSNs expressing uORs or oORs co-express OMP (Figure We found that the number of immature OSNs expressing uORs are in RTP1,2DKO and het the number of OSNs expressing uORs show a decrease in RTP1,2DKO. Mann Whitney U test) (Figure Figure 7 Download asset Open asset OSNs expressing oORs from RTP1,2DKO can mature and (A) Representative images showing the of (uOR) and (oOR) with OMP (red) for het (top) and RTP1,2DKO OMP negative OSNs are with (B) Quantification of OMP positive OSNs for uORs and oORs as a uORs there is a significant decrease Mann Whitney U test, het n = RTP1,2DKO n = in the number of OMP positive OSNs in RTP1,2DKO no significant difference is observed for oORs test, het n = RTP1,2DKO n = (C) Quantification of the number of OSNs OMP for (D) Quantification of the number of OSNs OMP for (E) Representative images for staining with either a uOR or an oOR in response to their het shows RTP1,2DKO shows in response to an odor that the oOR but not the one that the positive neurons are by (F) Quantification of the fold change in staining for positive cells in het and RTP1,2DKO mice (red) in response to is a significant increase in the in het (p<0.0001 one-way ANOVA, Tukey’s post hoc test) but not RTP1,2DKO when to the odor. (G) Quantification of the fold change in staining for positive cells in het and RTP1,2DKO mice (red) in response to is a significant increase in the in both het and RTP1,2DKO when to the odor one-way ANOVA, Tukey’s post hoc test). (H) Wild-type and RTP1,2DKO OE with an antibody a