Polygenic transcription of OR genes in olfactory progenitors follows a zonal expression pattern.

(A) Schematic illustrating OR zones along the dorsoventral axis in whole mount views of the MOE (left) and coronal sections (middle). Zone1 (red) is the dorsal-most zone and zone 5 (blue) is the ventral-most zone. Zoomed in view of the MOE (right) shows cell populations at different stages of OSN differentiation organized in a pseudostratified fashion from the basal (least differentiated) to apical (most differentiated) layers: HBC, horizontal basal cell; GBC, globose basal cell; INP, immediate neuronal precursor; iOSN, immature olfactory sensory neuron; mOSN, mature olfactory sensory neuron. MOE: main olfactory epithelium, OB: olfactory bulb.

(B) t-SNE (t-distributed Stochastic Neighborhood Embedding) dimensionality reduction used to visualize clustering of single cells from FAC-sorted MOE cell populations with Seurat, based on expression of the most variable genes. Plot (left panel) shows the separation of single cells into 6 populations, to which we assigned cell identities based on expression of known MOE markers [31] (See also Supplementary Figure S1). Olfactory receptor expression is first detected in INP3 cells (right panel).

(C, D, left panel) t-SNE plot (as in B) of cell populations isolated from either dorsal (zones 1-2) in (C) or ventral (zones 3-5) in (D) MOE microdissections. Cells are colored according to the zonal identity of the most highly expressed OR. (C, D, right panel) Plots depicting zonal identities of all the OR genes detected in individual INP3 cells from dorsal (C) or ventral (D) MOE. Y axis shows OR expression in normalized counts of unique transcripts (UMIs) for different OR genes (separated by black lines). On the X axis each point is a different INP3 cell. ORs are colored according to their zonal identity. Note that while class I OR genes are expressed within zone 1 of the MOE, they may be regulated through a different mechanism and are thus displayed separately.

(E) Expression of OR genes of different zonal identities in olfactory progenitor INP cells (top) and mOSNs (bottom), determined with bulk RNA-seq, in cells isolated from dorsal-most (zone 1) (left), dorsomedial (zones 2-3) (middle), and ventral-most (zones 4-5) MOE microdissections (right). Note that INP and mOSN cells were FAC-sorted from the same exact dissection, thus the mOSN OR expression patterns confirm the accuracy of the dissection.

Heterochromatin deposition silences OR genes from lower zones.

(A) Signal tracks of H3K9me3 and H3K79me3 native ChIP-seq from the whole MOE show heterochromatin deposition over two representative OR gene clusters. These clusters were selected because they harbor OR genes with both dorsal (zone 1) and ventral (zone 5) identities. OR genes are colored according to their zonal identity: zone 1 ORs in red, zone 2 ORs in yellow, zone 5 ORs in blue and ORs with unknown zonal identity in gray. Purple triangle marks the “H” OR gene enhancer that is present within that OR gene cluster.

(B) H3K9me3 (left) and H3K79me3 (right) native ChIP-seq in the whole MOE. Box plots of read density over OR gene bodies, separated by their zonal identity, depict a pattern of deposition that is high on dorsal-most (zone 1) OR genes, progressively decreases with more ventral zonal OR identities, and is absent on class I ORs.

(C) H3K79me3 native ChIP seq in GBC, INP, iOSN and mOSN populations shows onset of H3K79me3 deposition as cells transition from INPs to iOSNs. Each row of the heatmaps shows coverage over an OR gene body (separated into categories by their zonal identity). (See also Supplementary Figure S2A for H3K9me3 heatmap).

(D) H3K79me3 native ChIP-seq in mOSNs from zonally dissected MOE. Colored schematics above each heatmap depict the zone of dissection. (See also Supplementary Figure S2B for H3K9me3 heatmap).

(A-D) Pooled data from two biological replicates is shown for all ChIP experiments.

Zonal OR compartmentalization permits OR genes from more ventral zones to be recruited into the OR compartment.

(A) In situ Hi-C contact matrices of a 90Mb region of chromosome 2 that contains 3 large OR gene clusters, depicted with boxes under the contact matrices. Hi-C libraries were prepared from mOSNs FAC-sorted from dorsal-most (zone1) and ventral-most (zone 4-5) MOE microdissections, as well as a pure population of Olfr17 (a zone 2 OR) expressing dorsomedial mOSNs. For each zonal contact matrix, magnified views show long-range cis Hi-C contacts between the large OR gene cluster in the middle that contains ORs of every zonal identity with the OR cluster on the left that contains mostly zone 1-2 identity ORs (red box) and the OR cluster on the right that contains mostly zone 4-5 identity ORs (blue box). Cis contacts between OR genes increase from dorsal to ventral mOSNs, but the zone 4-5 identity OR cluster associates with the other ORs only in the ventral-most OSNs (as seen when comparing Hi-C contacts in the blue boxes).

(B) Heatmaps of average interchromosomal Hi-C contacts between OR genes annotated by their zonal identity at 50Kb resolution show increased trans contacts in mOSNs from more ventral zones. OR genes have a similar, intermediate frequency of contacts in the mOSN population where they are expressed, marked with an asterisk. Class I OR genes (which are also expressed in zone 1) make few interchromosomal interactions in all zones (data not shown) and were thus excluded from this analysis.

(C) Dip-C in mOSNs from dorsal and ventral dissected MOE was used to generate haplotype resolved single cell contact matrices and 3D genome structures, as previously described [40].

(D) Analysis of Dip-C contact densities of interchromosomal contacts between ORs genes confirms that ventral mOSNs have increased OR compartment interactions (Wilcoxon rank sum p-value = 9.164e-11).

(E) Single cell heatmaps of pairwise distances between OR genes generated from 3D genome structures in two ventral mOSNs show OR genes from different chromosomes intermingle in a different pattern in the two cells (top). For each cell, heatmaps are sorted by chromosome order and show all OR interactions within 10 particle radii (approximately ∼600nm). Representative 3D structures show the different positioning of three chromosomes (chr19, chr9 and chr2) in the two cells, resulting in a different pattern of OR cluster contacts (bottom). See also Supplementary Figure S3 for heatmaps of Dip-C distances in each of the 48 dorsal and ventral mOSNs.

NFI paralogue gradients regulate zonal OR expression

(A) Heatmaps showing differentially expressed transcription factors in immediate neuronal precursor (INP) and immature olfactory sensory neuron (iOSN) cells isolated from either dorsal, dorsomedial or ventral olfactory epithelium. The shown transcription factors are significantly differentially expressed between dorsal and ventral cells with an adjusted p-value of <0.05, at least a three-fold change in expression, and an expression level of at least 15 TPM. A broader list of zonal transcription factors is included in Supplementary Table S1. The heatmaps are sorted based on expression in ventral cells and the color bar above each heatmap shows the log2 fold change in ventral cells relative to dorsal cells.

(B) Expression levels of NFIA, NFIB, and NFIX at four stages of OSN development in dorsal cells (red), dorsomedial cells (green) and ventral cells (blue).

(C, D) Comparison of OR gene expression in NFI ABX triple knockout and control cells from the whole MOE. NFI transcription factors are deleted either in olfactory progenitors (C) using the Krt5-CreER driver or in mOSNs (D) using the OMP-IRES-Cre driver (as illustrated in Supplementary Figure S4). At the right of each panel, scissors indicate the differentiation stage of NFI ABX deletion, and a red box marks the cell type that was FAC-sorted for RNA-seq analysis. Asterisk: Wilcoxon rank sum test p-value < 0.001.

(E) OR expression in NFI ABX triple knockout, NFI AB double knockout, NFIX knockout, NFI ABX triple heterozygous and control mOSNs from ventrally dissected MOE. Knockout was induced in progenitors with the Krt5-CreER driver. Plots show a different pattern of OR gene transcription in the different genotypes. Quantification of differentially expressed ORs for the three knockout genotypes is shown in Supplementary Figure S4.

Spatial transcriptomics shows dorsalization and homogenization of the MOE upon NFI A, B, and X deletion.

(A) Schematic depicting our analysis pipeline: Spatial transcriptomics was performed on sections of wt control and NFI ABX cKO MOE. Dimensionality reduction was performed, and spatial spots were clustered based on normalized expression of OR genes.

(B) Heatmaps showing scaled, normalized expression levels of the top 20 highest expressed OR genes per zone in the control dataset. Unbiased neighborhood analysis and clustering grouped spatial spots into 5 clusters for both control and cKO MOE (depicted in distinct colors on the top of the heatmaps). Clustering of spatial spots in the control sample reproduces anatomical zones, as spots within each cluster express OR genes with the corresponding zonal identity (left heatmap). The same clusters were generated for NFI cKO sample (right heatmap). Although cluster 1 expresses exclusively zone 1 ORs, like in control MOEs, clusters 2-5 exhibit homogenous OR expression, with ventral expansion of zone 2/3 ORs, and reduced representation of zone 4/5 ORs.

(C) Average normalized per-spot expression of the 20 highest expressed OR genes from zone 1, zone 2, and zone 5 is overlaid against H&E histological image of control (top) and NFI cKO (bottom) MOE sections. Expression of zone 1 OR genes is confined to the same anatomical region for both control and NFI cKO sections. Zone 2 OR gene expression is spread to more ventral regions in the NFI cKO compared to control sections, while expression of zone 5 OR genes is almost completely absent in the NFI cKO sample.

(D) Spatial spots are colored according to their zonal assignment, which was determined based on the highest summed normalized expression of OR genes per zonal identity within that spot. Zonal spot assignment of the control sample visually reproduces known anatomical zones. In the NFI cKO sample, spots in the dorsal region have the highest expression of class I and zone 1 OR genes, similar to the control sample. However, in the rest of the NFI cKO MOE, most spots have a zone 2 OR identity. Spots assigned the identity ‘none’ did not contain any OR transcripts and were excluded from cluster analysis.

NFI A, B and X regulate chromatin state and OR compartment formation

(A) Native ChIP-seq for H3K9me3 (top) and H3K79me3 (bottom) in NFI ABX knockout mOSNs from ventral MOE. Heatmaps show ChIP signal over OR gene bodies, scaled to 6kb with 2kb flanking on either side. There is a decrease of both histone marks on zone 3-5 identity OR genes in NFI ABX knockout compared to control. Triple NFI deletion was induced with the Krt5-CreER driver (before OSN differentiation).

(B) Hi-C in in NFI ABX knockout and control mOSNs from ventral MOE. Left: In situ Hi-C contact matrices of a 90Mb region of chromosome 2 from control (top) and NFI ABX triple knockout (bottom) ventral mOSNs, as described in Figure 3A. The contact matrix shows long-range cis interactions between 3 large OR gene clusters: one enriched for dorsal, zone 1-2, identity ORs (left), one containing ORs of every zonal identity (middle), and one enriched for ventral, zone 4-5, identity ORs. Note that long range cis contacts between the zone 4-5 identity enriched cluster and the mixed identity cluster dissipate in the triple NFI cKO (bottom, blue box), whereas the contacts of the mixed identity cluster with the zone 1-2 identity enriched cluster are preserved (bottom, red box). Right: Heatmaps of average interchromosomal Hi-C contacts between OR genes annotated by their zonal identity (as described in Figure 3B) in control (top) and triple NFI cKO (bottom) mOSNs from ventral MOE. Trans contacts between zone 4-5 ORs dissipate, whereas trans contacts between zone 2-3 ORs reach intermediate levels typically detected between OR genes with the “correct” zonal identity for a given MOE segment (see Figure 3).

(C) OR expression by zonal identity in INP cells isolated from ventral NFI ABX knockout and control MOE. Triple NFI deletion was induced with the Krt5-CreER driver (before OSN differentiation) and NFI ABX INP cells were isolated as described in Supplementary Figure S4. Log2 fold change of OR expression in NFI ABX vs control INP cells shows a significant decrease in expression of zone 4-5 ORs (right).

Genetic induction of OR transcription in olfactory progenitors determines OR choice in mOSNs

(A) Genetic strategy for transcriptional induction of OR Olfr17 (a zone 2 identity OR) from its endogenous genomic locus. A genetically modified “tetO-Olfr17” allele contains a tetO promoter immediately downstream of the endogenous Olfr17 promoter and an IRES GFP reporter after the coding sequence[48]. In the presence of tTA a high level of tetO-Olfr17 is induced from the tetO promoter (top), while in the presence of a high amount of doxycycline (DOX) tTA is inhibited and transcription is regulated by the endogenous promoter. See also Supplementary Figure S7A for information on the genomic locus of this Olfr17 allele.

(B) tTA driven by the Gng8 promoter is expressed in INP and iOSN cells in the MOE[57]. When Gng8-tTA drives expression of a tetO-GFP allele, transcription is detected only in progenitor cells located on the basal side of the MOE, where the tTA is expressed (left)[50]. In contrast, when Gng8-tTA drives expression of tetO-Olfr17, expression persists in mature OSNs where tTA is no longer present (right). See also Supplementary Figure S7B,C for the sustained and widespread expression of the tetO-Olfr17 allele after 35 days of high DOX treatment and Supplementary Figure S7D for Gng8 expression during OSN differentiation.

(C) In situ Hi-C in tetO-Olfr17 expressing cells shows enriched contacts with interchromosomal olfactory receptor (“Greek Island”) enhancers over the Olfr17 locus, suggesting tetO-Olfr17+ OSNs are using endogenous mechanisms to sustain Olfr17 expression after Gng8-tTA is no longer present.

(D) tetO-Olfr17 expression in coronal sections of the MOE determined by GFP fluorescence. In the absence of tTA tetO-Olfr17 expression occurs only in zone 2 of the MOE (right); with high tTA induction in progenitor cells tetO-Olfr17 expression occurs throughout all zones of the MOE (left); and with low tTA induction in progenitor cells, due to the addition of a low amount of doxycycline, tetO-Olfr17 expression occurs in zone 2 and spreads dorsally to zone 1 (middle) only. Magnified views show tetO-Olfr17 expression in its native zone 2 (i) and ectopic expression in the most ventral zone 5 (ii). Mice on low DOX treatment were provided doxycycline at 1ug/ml in water throughout gestation and postnatal life.

(E) Quantification of tetO-Olfr17 expression (determined by GFP fluorescence in immunofluorescence images) relative to a normalized zonal position (illustrated on the left) in coronal sections of the MOE from tetO-Olfr17 without tTA driver (bottom), tetO-Olfr17 with Gng8-tTA driver (top), and tetO-Olfr17 with Gng8-tTA driver on low DOX (middle). 6 sections from two replicates were analyzed for tetO-Olfr17 with Gng8-tTA; 9 sections from two replicates were analyzed from tetO-Olfr17 with Gng8-tTA and low DOX; 29 sections from two replicates were analyzed for tetO-Olfr17 without tTA. The plot displays a maximum of 1000 cells randomly selected for each condition.

(F) H3K9me3 native ChIP signal over the Olfr17 locus in mOSNs from dorsal (red), dorsomedial (green) and ventral (blue) MOE shows a higher level of heterochromatin in ventral MOE.

(G) Model of OR choice in each zone of the MOE, regulated by the interplay of low-level polygenic OR transcription in INP cells, which defines the OR repertoire that can be chosen in each zone, and heterochromatic silencing, which prevents ectopic expression of more dorsal ORs. Both polygenic OR transcription in INP cells and heterochromatin deposition are influenced by NFI A, B and X transcription factors, expressed in a dorsal-low ventral-high gradient across the MOE.