Enomoto T , Nishida H , Iwata T , Fujita A , Nakayama K , Kashiwagi T , Hatanaka Y , Kondo H , Kajitani R , Itoh T , Ohmoto M , Matsumoto I , Hirota J .

	Fig. 1. Loss-of-function mutation of Bcl11b biases the OR class choice of OSNs. a Distributions of class I (blue) and class II OSNs (magenta) and ISH with RNA probes for Bcl11b and a dorsal marker, Acsm4, in consecutive coronal sections of the MOE at P30. b Combination of IHC for Bcl11b (green) and ISH for OR genes (magenta). Arrowheads and arrow indicate co-labeled and not co-labeled OSNs, respectively. c Bar graphs showing the percentages of Bcl11b-positive cells that are labeled with each OR probe (n = 3 animals. The quantification data are summarized in Supplementary Data 1). d Microarray analysis of the expression of OR genes in the wild type and Bcl11b−/− MOE. A heat-map representation was obtained by hierarchical clustering using 36 OR gene probe sets (blue: class I genes; magenta: class II genes). Each row refers to independent preparations (n = 5 control mice, 6 Bcl11b−/− mice). Color scale indicates the log2 value of the signal intensity of OR gene normalized to the internal control, GAPDH (Supplementary Data 2). e ISH with mixed RNA probes for the eight class I and the eight class II genes in coronal sections of the wild type and Bcl11b−/− MOE at P0. f Quantification of the number of OSNs expressing class I or class II genes per section (control: black circles, Bcl11b−/−: open circles). Bar represents the mean values ± s.e.m. The quantification data and number of animals analyzed are summarized in Supplementary Data 1. g Bar graphs showing the percentage of change in the number of cells expressing each OR gene in Bcl11b−/− versus wild type mice (serial sections throughout the MOE at 100 μm interval were analyzed). The quantification data and number of animals analyzed are summarized in Supplementary Data 1. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 (two-tailed t-test, n = at least 3, as the correction for multiple testing, p-values were adjusted based on false-discovery rate using the Benjamini–Hochberg (BH) method). D, dorsal; V, ventral; M, medial; L, lateral. Scale bars, 100 μm in (a) and (e); 10 μm in (b)
	Fig. 2. Abnormal axonal projections and glomerular domain organization in Bcl11b−/− mice. a–d Axonal projections of class I-OSNs and class II-OSNs were visualized using Olfr545-IRES-tauGFP mice (a, c) and Olfr151-IRES-taulacZ mice (b, d), respectively with control and Bcl11b−/− backgrounds. a Olfr545-expressing class I-OSNs project to ectopic and multiple glomeruli in the Bcl11b−/− OB (arrows). b Olfr151-expressing class II OSNs send their axon to a few glomeruli in the OB of control mice (arrowheads), while those of Bcl11b−/− mice are barely detected. c Axonal termini of Olfr545-expressing class I-OSNs were immunostained using anti-GFP (green) and anti-protocadherin (Pcdh) 21 (red) antibodies and counterstained with DAPI (blue). Numbers represent the section numbers of Olfr545-positive axonal termini observed in the consecutive coronal sections of OB. Arrowheads indicate Olfr545-positive glomeruli. d Axonal termini of Olfr151-expressing class II OSNs were immunostained using anti-β-gal (green) and anti-Pcdh21 (red) antibodies and counterstained with DAPI (blue). The section number of Olfr151-positive axonal termini observed were shown as in (d). Arrowheads indicate Olfr151-positive glomeruli. Arrow indicates Olfr151-positive axonal terminus that did not innervate into the glomerular layer. e Axonal projection domains of class I OSNs were visualized using ΔOlfr545-YFP mice with control and Bcl11b−/− backgrounds. Dotted lines indicate the OB outline. Coronal sections of the OB were immunostained as in (c). Dotted boxes are magnified. Arrowheads indicate ΔOlfr545-positive axonal termini in the ventral OB. f Axonal projection domains of class II OSNs in P-lacZ Tg mice with control and Bcl11b−/− backgrounds. Coronal sections of the OB were immunostained as in (d). Dotted boxes are magnified. D, dorsal; V, ventral; R, rostral; C, caudal; A, anterior; P, posterior. Scale bars, 100 μm
	Fig. 3. Specific depletion of Bcl11b in postmitotic OSNs switches the OR class from class II to class I. a Genetic strategy to generate Bcl11b cKO mice. b Schematic illustration of the genetic manipulation of Bcl11b expression during OSN differentiation. GBC, globose basal cell; INP, immediate neuronal precursor; iOSN, immature OSN; mOSN, mature OSN. IHC analyses of the expression of Cre recombinase during the OSN-differentiation in the MOE of Goofy-Cre-IRES-Venus Tg mice are presented in Supplementary Fig. 4. c Combination of IHC against Bcl11b (green) and ISH with either Neurod1 or Gap43 probes (magenta) on coronal sections of the MOE of control, Bcl11b−/−, and Bcl11b cKO mice. Bcl11b-immunoreactivity is detected in Neurod1-expressing cells but not detected in Gap43-expressing cells in Bcl11b cKO, indicating specific depletion of Bcl11b in postmitotic OSNs. d ISH with mixed RNA probes for the eight class I and class II genes in coronal sections of the control, Bcl11b−/−, and Bcl11b cKO mice. Quantification data of coexpression analyses is summarized in Supplementary Fig 4d and Supplementary Data 1. Scale bars, 50 μm in (c); 100 μm in (d)
	Fig. 4. Bcl11b negatively regulates the expression of class I OR genes. a Genetic strategy of the gain-of-Bcl11b-function analysis in OSNs. Robust expression of Bcl11b throughout the MOE of the gain-of-function mutant mice was confirmed by IHC (Supplementary Fig. 5a). b ISH with mixed RNA probes for the four class I and dorsal class II genes in coronal sections of the MOE of the control and gain-of-function mutant mice. Scale bar, 100 μm. c Quantification of the number of OSNs expressing either class I or class II genes per section. Bars represent the mean values ± s.e.m. of the control (gray) and gain-of-function mutant mice (class I in blue, class II in magenta), respectively: Class I genes: 33.8 ± 1.60 in control and 3.78 ± 1.82 in mutant mice, p = 0.000243, two-tailed t-test, n = 3 independent experiments; Class II genes: 25.6 ± 1.57 in control and 26.4 ± 3.15 in mutant mice, p = 0.838, two-tailed t-test, n = 3 independent experiments. ****p < 0.001, NS, not significant. Quantification data and statistical details are summarized in Supplementary Data 1. d The log2-fold change values of OR gene expression analyzed by RNA-seq are arranged according to their relative positions along the chromosomes. Class I genes (blue), atypical class I genes (purple), and class II genes (magenta). Increased- and decreased OR genes (p < 0.05) are represented by filled circles. FPKMs in the control and gain-of-function mutant MOEs are summarized in Supplementary Data 3. e Merged representation of the bee-swarm and box-plots of RNA-seq FPKM values for class I (n = 128) and class II genes (n = 968) of the control (gray) and gain-of-function mutant (class I genes in blue; class II genes in magenta) mice
	Fig. 5. Bcl11b suppresses the enhancer activity of the J element. a Class I and class II enhancer activities were visualized using J-Venus and P-LacZ Tg mice in the presence or absence of Bcl11b by IHC against Venus and β-gal in coronal sections of the MOE of J-Venus and P-LacZ Tg mice, respectively. b Genetic strategy to analyze the functional relationship between Bcl11b and the J element. If there is no functional relationship between the induced Bcl11b and the J element, the OSN should be colored yellow by Venus (green) and mRFP (magenta). Alternatively, if the induced Bcl11b negatively regulates the J element, the expression of gapVenus will be turned off and OSNs will be colored magenta by mRFP. IHC analyses of class I OSN-specific expression of Venus in J-Cre-IRES-Venus Tg mice were shown in Supplementary Fig. 6. c, IHC against Venus (green) and RFP (magenta) in the MOE of J-Cre-IRES-Venus Tg mice without and with the CAGp-LSL-Bcl11b-IRES-mRFP transgene. Green and magenta arrows indicate Venus- and mRFP-single positive cells, respectively. Yellow arrowheads indicate Venus/mRFP-double positive cells. d Merged representation of the bee-swarm and box-plots for the relative position of OSNs along the basal-apical axis (0–1.0) of the MOE. Venus-positive cells (433 cells in control, 200 cells in double Tg mutant) and/or RFP-positive cells (251 cells in double Tg mutant) collected from three animals of each mutant strain. Quantification data are summarized in Supplementary Data 1. Scale bars, 100 μm in (a); 50 μm in (c)
	Fig. 6. OSN-specific depletion and overexpression of Bcl11b alters physiological responses to aversive odorants. a–d Odor-responsive glomeruli in the MOB of control and Bcl11b cKO mice stimulated with 2MBA (a, c) and TMT (b, d). IHC against protein of the immediate early gene Egr1 in the dorsal-medial (DM) and dorsal-lateral (DL) regions of the MOB is shown in (a, b). The dotted lines indicate the boundary of the glomerular layer (GL) and the external plexiform layer (EPL). Unrolled odor maps for the expression of Egr1 in the GL are shown in (c, d). The magenta and yellow lines indicate the outline of maps and the boundary of Ocam-positive and negative, respectively. The color code indicates the blue to red color with correspond to the number of Egr1-positive cells in a single column. Each graph in c, d shows the histogram for the number of response column (Weak, 5~15; Medium, 15~25; High, more than 25 positive cells in a single column) of Ocam-negative dorsal MOB in Bcl11b cKO (blue for 2MBA-stimulation; magenta for TMT-stimulation) and control (Black). e–h Odor-responding glomeruli in the MOB of control and Bcl11b gain-of-function mutant mice stimulated with 2MBA (e, g) and TMT (f, h). Representation of the panels and graph in (e–h) is same as in (a–d). Bcl11b gain-of-function mutant (cyan for 2MBA-stimulation; orange for TMT-stimulation) and control (gray) are shown in (g) and (h), respectively. Reconstitution of unrolled odor maps from the immunostaining images of Egr1 and Ocam is represented in Supplementary Fig. 8. DM, dorsal-medial; DL, dorsal-lateral; GL, glomerular layer; EPL, external plexiform layer. Scale bar, 100 μm
	Fig. 7. OSN-specific depletion and overexpression of Bcl11b alters behavioral responses to aversive odorants. a A video frame of the behavioral test, in which a male mouse is exposed to a filter paper impregnated with a particular aversive odor. Centre of a filter paper and the opposite side to a filter paper were determined to ‘0’ (black dot) and ‘1’ (dotted line), respectively. The white dot indicates center of the mouse body excluding tail. b, c Raster plots representing occupancy of each animal in two areas (magenta or blue) during the 10-min test period (x-axis) of Bcl11b cKO mice (n = 12 animals for 2MBA, n = 7 for TMT, n = 13 for DW) and control (n = 14 for 2MBA, n = 11 for TMT, n = 13 for DW) (b) and Bcl11b gain-of-function mutant (n = 16 for 2MBA, n = 4 for TMT, n = 8–9 for DW) and control (n = 12 for 2MBA, n = 4 for TMT, n = 9 for DW) (c). The two-color representation corresponds to color discrimination in (a). The graph of time bins is presented in Supplementary Fig. 9. d Representative trajectory plots of mouse positioning during the 10-min test period to 2MBA (black: control; blue: Bcl11b cKO) and TMT (black: control; magenta: Bcl11b cKO). Dotted circles indicate that mice tried to escape from the cage by climbing walls. e, f Aversion index of control (black) and Bcl11b cKO (blue: 2MBA; magenta: TMT; gray: DW) mice during the first 1 min (e) and 10-min (f) of test period. Each bar indicates merged representation of the bee-swarm and box-plots. *p < 0.05 (two-tailed t-test). g Representative trajectory plots of mouse positioning during the 10-min trials to 2 MBA (black for control; cyan for Bcl11b gain-of-function mutant) and TMT (black for control; orange for Bcl11b gain-of-function mutant). g Aversion index of control (black) and Bcl11b gain-of-function mutant (cyan: 2MBA; orange: TMT; gray: DW) mice during the first 1-min (h) and 10-min (i) of test period. Each bar indicates merged representation of the bee-swarm and box-plots. *p < 0.05, ***p < 0.005 (two-tailed t-test). All behavioral analysis data are summarized in Supplementary Data 1
	Fig. 8. Model of the OR class specification of OSNs and terrestrial adaptation. a Schematic representation of the class-specific expression of Bcl11b in the OE of frog and mouse and the molecular mechanism of the OR class specification of OSNs. In tadpoles, the OE called “water-nose” expresses class I genes. During metamorphosis, the OE undergoes remodeling to form two distinct OE, called “water nose” and “air nose”, and Bcl11b starts expressing in the future air nose to allow the expression of class II ORs. In mice, class I and class II OSNs are Bcl11b-negative and -positive, respectively, as in adult frog. In mouse OSNs, the class I OR enhancer J-element is active in the absence of Bcl11b, whereas the presence of Bcl11b suppresses the J-element enhancer activity to permit choosing a functional class II enhancer from the class II enhancer repertoire spread through chromosomes to activate transcription of class II OR gene. b Schematic representation of changes in olfactory behavior caused by the biased OR class choice of OSNs. Behavioral outputs against two distinct aversive odorants, 2MBA (spoiled foods odor) and TMT (predators’ odor) depend on the populations of class I and class II OSNs. 2MBA, which is mainly detected by class I OSNs, induces stronger aversive response in Bcl11b cKO mice (class I-dominant) and less aversive in Bcl11b gain-of-function mutant mice (class II dominant). Class II-responsible odorant, TMT induces weaker aversive response when the population of class II OSNs is decreased

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