Frequency and zonal restriction of tTa-driven tet_o-modified P2 alleles in the olfactory epithelium.

<p>(A) Diagram of the olfactory epithelium showing zones of OR expression. The shaded region is the II/III zone of P2 expression. Areas in black boxes depict regions shown in (F–N). (B) Coronal section through the olfactory epithelium of a P2-IRES-GFP control animal reveals expression of the P2 allele at P14. Sections were subject to anti-GFP IHC (green), and nuclei were counterstained with Toto-3 (blue). (C) Coronal section through the olfactory epithelium of a OMP-IRES-tTa/tet-P2-IRES-GFP animal reveals expression of the tet-P2 allele driven by tTa at P14. Sections were subject to anti-GFP IHC (green), and nuclei were counterstained with Toto-3 (blue). (D) High-power image of the boxed region in (B). (E) High-power image of the boxed region in (C). (F–H) Coronal sections through the olfactory epithelium of a CaMKII-tTa/tet-P2-IRES-GFP animal reveal the zonal restriction of expression of the tet-P2 allele driven by tTa at P75 in zone I/II (F), zone II/III (G), and zone IV (H). Sections were subject to anti-GFP IHC (green), and nuclei were counterstained with Toto-3 (blue). (I–K) Coronal sections through the olfactory epithelium of a CaMKII-tTa/tet-P2Δ-IRES-GFP animal reveal the zonal restriction of expression of the tet-P2 allele driven by tTa at P75 in zone I/II (I), zone II/III (J), and zone IV (K). Sections were subject to anti-GFP IHC (green), and nuclei were counterstained with Toto-3 (blue). (L–N) Coronal sections through the olfactory epithelium of a CaMKII-tTa/M71-Tg animal show pervasive expression of tet-linked M71 transgene driven by tTa at P60 in zone I/II (L), zone II/III (M), and zone IV (N). Sections were subject to anti-lacZ IHC (green), and nuclei were counterstained with Toto-3 (blue). (O–T) Zonal restriction of the tet-P2 allele driven by OMP-IRES-tTa examined by two-color RNA in situ hybridization. Coronal sections through olfactory epithelia of P90 OMP-IRES-tTa/tet-P2 animals were hybridized with RNA probes directed against GFP (green) (O and R), and against OMP (red) (P and S), in zonal region II/III (O–Q) and zonal region IV (R–T). Red and green channels are shown merged (Q and T). Nuclei were counterstained with Toto-3 (blue). (U–Z) Increase in frequency of expression of the tet-P2 allele over time. Coronal sections corresponding to zone II/III of the olfactory epithelia of OMP-IRES-tTa/tet-P2 animals subject to IHC with immunoserum directed against GFP (green) at P14 (U), P18 (V), P30 (W), P60 (X), P120 (Y), and P360 (Z). Nuclei counterstained with Toto-3 (blue).</p

Pre-activation of tet-P2 leads to persistent expression independent of tTa.

<p>(A) Diagram of pre-activation strategy of tet-P2 with tTa by administration of doxycycline (dox). The tet-P2 locus is subject to activation by tTa until P60 by CaMKII-tTa. Doxycycline is administered, to ablate tTa binding, for 48 h prior to expression analysis by RNA in situ hybridization. (B) Diagram of the tet-P2 allele showing the location of RNA probes used to differentiate between wild-type (red “+1”) and tet_o (black “+1”) start sites of transcription. The RNA probe shown in red is derived from tet_o sequences and detects message initiated by the endogenous P2 promoter, while the probe shown in green is derived from GFP sequences and hybridizes to messages initiated from either endogenous P2 or tet_o promoters. (C–E) Control experiments demonstrate expression of the tet-P2 gene initiated from the wild-type P2 promoter. Coronal sections of a tet-P2 animal subject to RNA in situ hybridization with probe directed against the tet_o sequences (red) (C), with probe directed against GFP sequences (green) (D), and with red and green channels merged (E). Nuclei were counterstained with Toto-3 (blue). (F–H) Expression of the tet-P2 allele driven by CaMKII-tTa without doxycycline treatment at P60. Coronal sections of a tet-P2 CaMKII-tTa animal subject to RNA in situ hybridization with probe directed against the tet_o sequences (red) (F), with probe directed against GFP sequences (green) (G), and with red and green channels merged (H). Nuclei were counterstained by Toto-3 (blue). (I–K) Continuation of expression of the tet-P2 allele driven by CaMKII-tTa after 48 h of doxycycline treatment at P60. Coronal sections of a tet-P2 CaMKII-tTa animal subject to RNA in situ hybridization with probe directed against the tet_o sequences (red) (I), with probe directed against GFP sequences (green) (J), and with red and green channels merged (K). Nuclei were counterstained with Toto-3 (blue).</p

Suppression of tet_o-modified P2 alleles by the pervasive expression of an OR transgene.

<p>(A–C) Coronal sections through the olfactory epithelium of an OMP-IRES-tTa/tet-P2 mouse subject to immunohistochemical detection of lacZ (red) (A) and GFP (green) (B), and with merged signals (C). Nuclei (blue) revealed by Toto-3 counterstaining. (D–F) Coronal sections through the olfactory epithelium of an OMP-IRES-tTa/tet-P2/tet-M71 animal subject to immunohistochemical detection of lacZ (red) (D) and GFP (green) (E), and with merged signals (F). Nuclei (blue) revealed by Toto-3 counterstaining. (G–I) Coronal sections through the olfactory epithelium of an OMP-IRES-tTa/tet-P2Δ mouse subject to immunohistochemical detection of lacZ (red) (G) and GFP (green) (H), and with merged signals (I). Nuclei (blue) revealed by Toto-3 counterstaining. (J–L) Coronal sections through the olfactory epithelium of an OMP-IRES-tTa/tet-P2Δ/tet-M71 mouse subject to immunohistochemical detection of lacZ (red) (J) and GFP (green) (K), and with merged signals (L). Nuclei (blue) revealed by Toto-3 counterstaining.</p

Construction of the tet-P2Z allele and predominant allelic exclusion of the homozygous tet_o-modified P2 alleles.

<p>(A) Modification of the endogenous P2 locus by homologous recombination to generate the tet-P2Z allele. (I) The tet-P2Z targeting construct allows bicistronic expression of the P2 OR protein and the marker protein tau-lacZ, both driven by the tet operator inserted at the start site of transcription of the P2 locus. Flanking P2 promoter regions are preserved in the construct, shifted 5′ of the tet operator. (II) The unmodified genomic P2 locus. (III) Homologous recombination in mouse ES cells followed by self-excision of the ACN selection cassette yields the tet-P2Z allele. (B) Diagram of the genetic strategy used to for biallelic expression of the tet_o-modified P2 alleles in the mouse olfactory epithelium in vivo. The tet-P2 and tet-P2Z alleles have the potential to be transcribed in all olfactory sensory neurons of the olfactory epithelium by the ubiquitous expression of tTa from the CaMKII-tTa transgene. (C and D) Expression of the tet-P2Z allele in the olfactory epithelium (C) and the VNO (D) revealed by IHC in coronal sections with antibody directed against lacZ (red) in a CaMKII-tTa/tet-P2Z animal. Nuclei are revealed by Toto-3 counterstain. (E–G) Expression of the tet-P2 and tet-P2Z alleles in a compound heterozygous animal CaMKII-tTa/tet-P2/tet-P2Z shown by immunohistochemical detection of GFP (green) (E) and lacZ (red) (F), and with merged signals (G). Nuclei are revealed by Toto-3 counterstaining. (E′–G′) High-power magnification of a region of the fields shown in panels (E–G), respectively. An olfactory neuron exhibiting biallelic expression of the tet-P2 alleles is shown by the arrows. (H) Distribution of single (purple) and double (orange) tet-P2+ cells in olfactory epithelia of CaMKII-tTa/tet-P2/tet-P2Z animals. The mean relative position, normalized to the height of the epithelium, of single tet-P2+ cells was 0.501 and of double tet-P2+ cells was 0.424 (<i>n</i> = 100, <i>p</i><0.0068, unpaired <i>t</i>-test, two-tailed).</p

Molecular characterization of projection neuron subtypes in the mouse olfactory bulb.

Projection neurons (PNs) in the mammalian olfactory bulb (OB) receive input from the nose and project to diverse cortical and subcortical areas. Morphological and physiological studies have highlighted functional heterogeneity, yet no molecular markers have been described that delineate PN subtypes. Here, we used viral injections into olfactory cortex and fluorescent nucleus sorting to enrich PNs for high-throughput single nucleus and bulk RNA deep sequencing. Transcriptome analysis and RNA in situ hybridization identified distinct mitral and tufted cell populations with characteristic transcription factor network topology, cell adhesion and excitability-related gene expression. Finally, we describe a new computational approach for integrating bulk and snRNA-seq data, and provide evidence that different mitral cell populations preferentially project to different target regions. Together, we have identified potential molecular and gene regulatory mechanisms underlying PN diversity and provide new molecular entry points into studying the diverse functional roles of mitral and tufted cell subtypes