ER81 expression in a unilateral naris closed mouse model of odor deprivation. ER81 protein levels were substantially decreased in the OB ispilateral to naris closure.

Immunohistochemical analysis of PEA3 in the forebrain. A, PEA3 is expressed in the subventricular zone (SVZ) and rostral migratory stream (RMS). B and C, higher magnification images of PEA3 immunoreactivity in the SVZ and RMS, respectively. PEA3 is expressed in the nuclei of cells in SVZ and RMS, but no expression was observed in the OB granule cell layer (gcl) or cortex (cx). Bar = 150 μm for A; 75 μm for B and C.

TH expression in the OB of Er81 mutant mice. A and B, immunohistochemistry revealed a drastic reduction in the number of TH labelled cells in the OB of P10 Er81 mutant mice relative to wild-type litter mates. Bar = 40 μm in A and B; 20 μm in A′ and B′.

PAX6 expression in the OB of Er81 mutant mice. A and B, immunohistochemistry revealed that there was no significant change in the number of PAX6 labelled cells in the glomerular layer of Er81 mutant mice relative to wild-type litter mates. Bar = 125 μm in A and B; 50 μm in the insets.

Expression of the dopaminergic gene cassette members in a unilateral naris closed mouse model of odor deprivation. A, qualitative analysis of dopamine gene cassette expression levels in the OB of an adult wild-type mouse. The Th and Dat genes are expressed at the highest levels. Aadc and Vmat2 are substantially lower, and Gtpch is nearly undetectable. B, relative expression levels of dopamine gene cassette members in the OB either ispilateral or contralateral to naris closure as measured by quantitative RT-PCR. For each gene, expression levels ipsilateral to naris closure are shown relative to the levels in the contralateral OB. Gtpch expression was too low for reliable measurement, and was not included in the analysis. Expression levels for both Th and Dat were significantly decreased in the OB ispilateral to naris closure (indicated by asterisk), whereas levels for Aadc and Vmat2 did not significantly differ (n.s.).

Expression of the dopaminergic gene cassette members in the OB of wild-type and Er81 mutant P10 mice as measured by quantitative RT-PCR. For each gene, expression levels are shown relative to the wild-type OB. Gtpch expression was too low for reliable measurement, and was not included in the analysis. Only Th expression was significantly decreased (p<0.01) in the OB of the Er81 mutant mouse (indicated by asterisk), whereas levels for Aadc, Vmat2 and Dat were not significantly changed (n.s.).

Immunofluorescence of ERM and ER81 expression in the OB. In either the glomerular layer (A– D and A′–D′) or the mitral and granule cell layers (E–H and E′–H′), cells containing ERM (arrowhead) did not co-express ER81. These results suggest that ERM and is not functionally redundant to ER81 in the OB. Abbreviations: external plexiform layer (epl), internal plexiform layer (ipl), granule cell layer (gcl), mitral cell layer (m). Bar = 75 μm for A–H, 30 μm for A′–H′.

OMP and c-FOS expression in the OB of Er81 mutant mice. A and B, OMP expression was detected in the glomeruli of both wild-type and Er81 mutant mice, respectively. This finding indicated that the loss of functional ER81 protein did not prevent innervation by the axons of olfactory receptor neurons. C and D, c-FOS expression was detected in the glomerular layer of both wild-type and Er81 mice, respectively. Since c-FOS expression in the OB glomerular layer is activity-dependent, this finding indicated that olfactory receptor neurpon axons were functional and capable of transducing afferent synaptic activity. Bar = 40 μm.

Species-specific ER81 binding to the Th proximal promoter. A, location of consensus ER81 binding sites in the 9kb upstream regions of the rat and mouse Th genes. ER81 binding sites are indicated by “E” and conserved binding sites are indicated by shading between the two upstream regions. B, phylogenetic sequence comparison of equivalent Th proximal promoter regions from rats, mice, humans, cows and dogs. The ER81 consensus sequence (Brown and McKnight, 1992) is shaded in green, and the DA-motif position weight matrix (Flames and Hobert, 2009)is shaded in red. The core ETS DNA binding domain recognition sequence is boxed. Nucleotides that conform to both the DA-motif and ER81 consensus sequences are shaded yellow, whereas sequences that only conform to either the DA-motif or ER81 consensus sequence are shaded in red and green, respectively. Degenerate DNA code: R=A/G, S=C/G, M=A/C, W=A/T, Y=C/T. C, chromatin immunoprecipitation (ChIP) experiments with mouse OB tissue indicated that ER81 bound the mouse Th and Mmp-13 proximal promoters in vivo. By contrast, ChIP experiments with dog OB tissue indicated that ER81 bound only the Mmp-13, but not the Th, proximal promoters in vivo. The previously identified and conserved ER81 binding site in the Mmp-13 (Mmp-1/collagenase) proximal promoter was used as a positive control. D, electromobility shift assays (EMSA) revealed that bacterially expressed murine ER81 bound to the DA-motif/ER81 consensus binding site in the rodent proximal Th promoter, but not the equivalent site in the human Th proximal promoter. The human and mouse ER81 protein amino acid sequences are approximately 99% identical and the DNA binding domains of these proteins are 100% identical. Thus, the human and mouse ER81 proteins almost certainly have the same affinity for the binding sites in either the human or rodent Th promoters.