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Single-cell transcriptomics reveals receptor transformations during olfactory neurogenesis

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Science  04 Dec 2015:
Vol. 350, Issue 6265, pp. 1251-1255
DOI: 10.1126/science.aad2456
  • Fig. 1 Olfactory neurons exhibit large-scale shifts in gene expression during development.

    (A) Unsupervised analysis of single-cell gene expression profiles with Monocle revealed a linear trajectory (black line) along which cells develop in pseudotime. Coloring of cells based on the expression of developmental markers shows that the trajectory corresponds to a stepwise development from olfactory progenitors to precursors to immature OSNs to mature OSNs. (B) Global analysis of gene expression kinetics along the trajectory identified 3830 genes that vary significantly over developmental pseudotime (false discovery rate < 5%, determined by a Tobit-valued generalized linear model likelihood ratio test; supplementary materials). Hierarchical clustering of these genes via Ward’s method recovered 11 nonredundant groups that covary over the trajectory. Cluster analysis indicates that multiple large shifts in gene expression occur as neurons progress through development. The bar on top shows the locations of individual cells, colored by stage of development, along this developmental trajectory. The Expression Z score indicates changes in a gene relative to its dynamic range over pseudotime. (C) Kinetic diagrams show the expression of known markers of different developmental stages over the developmental progression. Numbers in parentheses indicate the groups in which genes are found in (B). Dots indicate individual cells and are colored according to developmental stage. Black lines indicate local polynomial regression smoothing (span, 0.75; degree, 2) of log-transformed FPKM values over developmental pseudotime.

  • Fig. 2 Immature neurons can express multiple Olfrs.

    (A) Neurons assigned to different developmental stages were arranged by developmental progress, as measured in pseudotime. Different developmental stages are indicated by differently colored ticks. Different Olfrs are represented by different colors in the bars. The total number of Olfr transcripts per cell shows a steady, though variable, increase during development. (B) Multiple different Olfr transcripts were detected in 12 of 25 early immature, 6 of 13 late immature, and 6 of 25 mature OSNs with Olfr transcripts. (C) The number of different Olfr transcripts per cell was highest in early immature OSNs and then declined over development. Early immature OSNs tended to express similar levels of different Olfrs. In contrast, the majority of mature OSNs expressed only one Olfr or high levels of one Olfr and low levels of one or two additional Olfrs. Each color in a bar represents a single Olfr, except gray, which represents >1 Olfr. (D) Olfrs stimulate neuronal activity via mechanisms involving sensory transduction molecules encoded by Gnal (or possible Gnas in immature OSNs), Adcy3, Cnga2, and Cnga4. Six immature and six mature neurons with >1 Olfr expressed all four genes, suggesting that neuronal activity downstream of odorant receptors is not what reduces the number of Olfrs expressed per neuron. Omp, which is highly expressed in mature OSNs, was absent from six early immature OSNs with >1 Olfr, arguing against contamination from mature OSNs. Gapdh and Actb are housekeeping genes.

  • Fig. 3 Olfrs expressed in the same neuron belong to a regional gene set.

    (A) Dual RNA-FISH of P3 tissue sections using a conventional method showed a small percentage of OSNs co-labeled with an Olfr1507 probe and a mix of probes for other Olfrs expressed in the same zone (zone 4). Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (blue). Two co-labeled cells are shown, one on the left half and the other on the right half of the panel. Scale bar, 5 μm. (B) Dual RNA-FISH of P3 tissue sections using a highly sensitive method showed a small percentage of OSNs coexpressing Olfr1507 and Olfr286. Two co-labeled cells are shown [as in (A)], and a cell labeled with only one probe (red only) is also shown on the right. Scale bar, 5 μm. (C) Dual RNA-FISH shows that Olfrs coexpressed in single immature OSNs (neuron D200 or D243) are singly expressed in neurons in the same or partially overlapping zones in adult olfactory epithelium sections. This correspondence suggests that Olfr expression in the immature OSN is restricted to a spatially determined set of Olfr genes. In the upper row, colored dots indicate the locations of labeled neurons. Boxed areas in the upper row are shown at higher magnification in the lower row. Scale bars, 500 μm (upper row) and 250 μm (lower row).

  • Fig. 4 Immature neurons coexpress Olfrs from multiple chromosomal loci.

    Diagrams show the chromosomal locations of Olfrs expressed in single OSNs of different stages. Each mouse chromosome is indicated by a vertical bar with its number below. The names of neurons, parenthesized number of Olfrs per neuron, and dots indicating the chromosomal locations of those Olfrs are shown in different colors for different neurons.

Supplementary Materials

  • Single-cell transcriptomics reveals receptor transformations during olfactory neurogenesis

    Naresh K. Hanchate, Kunio Kondoh, Zhonghua Lu, Donghui Kuang, Xiaolan Ye, Xiaojie Qiu, Lior Pachter, Cole Trapnell, Linda B. Buck

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S5
    • Tables S1 to S7
    • Full Reference List

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