Deconstructing Olfactory Stem Cell Trajectories at Single-Cell Resolution

doi:10.1016/j.stem.2017.04.003

. 2017 Jun 1;20(6):817-830.e8.

doi: 10.1016/j.stem.2017.04.003. Epub 2017 May 11.

Deconstructing Olfactory Stem Cell Trajectories at Single-Cell Resolution

Russell B Fletcher¹, Diya Das¹, Levi Gadye², Kelly N Street³, Ariane Baudhuin¹, Allon Wagner⁴, Michael B Cole⁵, Quetzal Flores¹, Yoon Gi Choi⁶, Nir Yosef⁴, Elizabeth Purdom⁷, Sandrine Dudoit⁸, Davide Risso⁹, John Ngai¹⁰

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
² Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA.
³ Division of Biostatistics, University of California, Berkeley, CA 94720, USA; Center for Computational Biology, University of California, Berkeley, CA 94720, USA.
⁴ Department of Electrical Engineering and Computer Science, University of California, Berkeley, CA 94720, USA; Center for Computational Biology, University of California, Berkeley, CA 94720, USA.
⁵ Department of Physics, University of California, Berkeley, CA 94720, USA; Center for Computational Biology, University of California, Berkeley, CA 94720, USA.
⁶ QB3 Functional Genomics Laboratory, University of California, Berkeley, CA 94720, USA.
⁷ Department of Statistics, University of California, Berkeley, CA 94720, USA; Center for Computational Biology, University of California, Berkeley, CA 94720, USA.
⁸ Division of Biostatistics, University of California, Berkeley, CA 94720, USA; Department of Statistics, University of California, Berkeley, CA 94720, USA; Center for Computational Biology, University of California, Berkeley, CA 94720, USA.
⁹ Division of Biostatistics, University of California, Berkeley, CA 94720, USA.
¹⁰ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA; QB3 Functional Genomics Laboratory, University of California, Berkeley, CA 94720, USA. Electronic address: jngai@berkeley.edu.

PMID: 28506465
PMCID: PMC5484588
DOI: 10.1016/j.stem.2017.04.003

Deconstructing Olfactory Stem Cell Trajectories at Single-Cell Resolution

Russell B Fletcher et al. Cell Stem Cell. 2017.

. 2017 Jun 1;20(6):817-830.e8.

doi: 10.1016/j.stem.2017.04.003. Epub 2017 May 11.

Authors

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
² Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA.
³ Division of Biostatistics, University of California, Berkeley, CA 94720, USA; Center for Computational Biology, University of California, Berkeley, CA 94720, USA.
⁴ Department of Electrical Engineering and Computer Science, University of California, Berkeley, CA 94720, USA; Center for Computational Biology, University of California, Berkeley, CA 94720, USA.
⁵ Department of Physics, University of California, Berkeley, CA 94720, USA; Center for Computational Biology, University of California, Berkeley, CA 94720, USA.
⁶ QB3 Functional Genomics Laboratory, University of California, Berkeley, CA 94720, USA.
⁷ Department of Statistics, University of California, Berkeley, CA 94720, USA; Center for Computational Biology, University of California, Berkeley, CA 94720, USA.
⁸ Division of Biostatistics, University of California, Berkeley, CA 94720, USA; Department of Statistics, University of California, Berkeley, CA 94720, USA; Center for Computational Biology, University of California, Berkeley, CA 94720, USA.
⁹ Division of Biostatistics, University of California, Berkeley, CA 94720, USA.
¹⁰ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA; QB3 Functional Genomics Laboratory, University of California, Berkeley, CA 94720, USA. Electronic address: jngai@berkeley.edu.

PMID: 28506465
PMCID: PMC5484588
DOI: 10.1016/j.stem.2017.04.003

Abstract

A detailed understanding of the paths that stem cells traverse to generate mature progeny is vital for elucidating the mechanisms governing cell fate decisions and tissue homeostasis. Adult stem cells maintain and regenerate multiple mature cell lineages in the olfactory epithelium. Here we integrate single-cell RNA sequencing and robust statistical analyses with in vivo lineage tracing to define a detailed map of the postnatal olfactory epithelium, revealing cell fate potentials and branchpoints in olfactory stem cell lineage trajectories. Olfactory stem cells produce support cells via direct fate conversion in the absence of cell division, and their multipotency at the population level reflects collective unipotent cell fate decisions by single stem cells. We further demonstrate that Wnt signaling regulates stem cell fate by promoting neuronal fate choices. This integrated approach reveals the mechanisms guiding olfactory lineage trajectories and provides a model for deconstructing similar hierarchies in other stem cell niches.

Keywords: HBC; Wnt; branching lineage; horizontal basal cell; lineage trajectory; olfactory epithelium; scRNA-seq; stem cells.

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Figures

**Figure 1. Experimental Strategy for Olfactory Stem Cell Lineage Analysis with Single-Cell RNA-Seq**
**(A)** Schematic of the olfactory epithelium describing the constituent cells: horizontal basal cell (HBC, green), globose basal cell (GBC, blue), sustentacular cell (Sus, pink), olfactory sensory neuron (OSN, purple), microvillous cell (MV, dark blue), Bowman’s gland (yellow). **(B)** Immunohistochemistry for the HBC lineage tracer YFP (green) and SOX2 (magenta) shows basal resting HBCs in the wild type (WT) background (left panel) and asynchronous differentiation following *Trp63* conditional knockout (cKO) (center, right). **(C)** YFP(+) cells were collected by FACS at the indicated times following tamoxifen administration from mice carrying the *Krt5-CreER; Rosa26^eYFP* transgenes and either the *Trp63^+/+* (WT) or *Trp63^lox/lox* (cKO) alleles. **(D)** Sox2-eGFP(+)/ICAM1(−)/SCARB1(−)/F3(−) cells were collected by FACS; this enriched for the GBC, INP, and MV fates over Sus cells. **(E)** Data from both experimental designs were combined, filtered, normalized, clustered, and used in downstream analyses. – Scale bars, 50 microns. See Figure S1.

**Figure 2. Statistical Analysis of Single-Cell RNA-Seq Data Predicts Distinct Cell States and Branch Points in the Olfactory Stem Cell Trajectory**
**(A, B)** t-SNE plot (perplexity = 10) based on the 500 most variable genes shows the separation of the cells into discrete groups congruent with the clustering. Cluster medoids are displayed as larger circles with initial assignments of cluster identity based on the expression of a small number of marker genes (B). **(C)** t-SNEs as in (A,B), colored by experimental condition. Differentiation is asynchronous, but cells from the later lineage tracing time-points and the Sox2-eGFP+ cells contribute to more differentiated cell types. **(D, E)** Three-dimensional representation of single cell gene expression profiles based on principal component analysis (D); cells are colored by cluster. *Slingshot* predicts an early bifurcation in the lineage trajectories of the neuronal (orange) and sustentacular cell (magenta) lineages whereas the MV lineage (blue) is predicted to branch later off of the neuronal lineage from the GBCs (E). See Figure S2.

**Figure 3. Patterns of Coordinated Gene Regulation in the Neuronal and Sustentacular Cell Lineages Reveal Different Strategies for Differentiation**
**(A, C)** Developmental distance of cells within each lineage as inferred by Slingshot. Mean +/− standard deviation of developmental distance is indicated for each cluster. **(B, D)** Heatmaps display the average scaled expression profile for each gene cluster (numbered at the left of each row) with cells (columns) ordered according to their developmental positions within the neuronal (B) and sustentacular cell (D) lineages. There are numerous step-like transitions in the neuronal lineage but fewer, wave-like changes in the sustentacular cell lineage. The lower row in each heatmap represents a set of 40 cell cycle (CC) genes. See Figures S3 and S4.

**Figure 4. Clonal Lineage Tracing In Vivo Validates Branching Lineage Assignment Predictions**
**(A–D)** Example images of clones from *Krt5-CreER; Trp63^lox/lox; Rosa26^Confetti* transgenic animals analyzed 14 days following tamoxifen induction of the CreER driver: neuronal only (A), neuronal and sustentacular (arrow) (B), sustentacular cell only (C), and neuronal and microvillous cell (arrowhead) (D). **(E)** Most clones represent unipotent differentiation events, supporting the prediction that the lineages bifurcate early: the majority of clones contain only cells in the GBC/neuronal lineage, and just over half of all sustentacular cell-containing clones are composed of only sustentacular cells. Almost all microvillous cells are found together with neurons. One out of 99 clones contained a labeled Bowman’s gland together with neurons. **(F, G, H)** Clone size distribution. The number of neurons in neuron-containing clones (F) is larger, validating that the neuronal lineage contains amplification stages whereas the sustentacular cells are usually present as single cells (G). Microvillous cells are usually found as singlets and typically are found in clones together with neurons (H). **(I)** Percentage of *Krt5-CreER; Trp63^lox/lox; Rosa^eYFP* lineage-traced cells co-labeled with activated CASPASE3 at 24, 48 and 96 hours and 7 days following tamoxifen injection. The low frequency of apoptosis (0–0.6 %) in the HBC lineage is inconsistent with cell death as a mechanism to generate clones containing single cells. **(J, K, L)** *Ascl1-CreER; Rosa26^eYFP* lineage tracing at 21 days following tamoxifen induction shows that although *Ascl1*-positive GBCs usually form neurons (~ 98%) (J), they occasionally form microvillous cells (K). The percentage (mean +/− SD) of each cell type formed from lineage-traced cells (1072 cells from three animals) is summarized in (L). Scale bars, 25 microns. See Figure S5.

**Figure 5. Coordinated Transcription Factor Networks Associated with Lineage Progression**
**(A, B)** Heatmaps of the top DE genes for the neuronal (A) and sustentacular cell (B) lineages. **(C, D)** Connectivity graphs of the most correlated DE transcription factors for the neuronal (C) and sustentacular cell (D) lineages colored by the cluster in which expression is highest; the size of each node indicates magnitude of expression. **(E)** Connectivity graph for the neuronal lineage colored by rank across the clusters within the lineage. For each transcription factor, we computed the average expression in each cluster and color-coded the corresponding node to indicate the cluster with the highest average expression. See Figure S6 and Table S3.

**Figure 6. Wnt Signaling Is Necessary and Sufficient for HBC Activation to Form Neurons**
**(A)** Wnt signaling pathway gene expression in the resting HBC cluster, color-coded according to log2-fold-change (log2FC) between the resting HBC cluster and the other neuronal lineage clusters (“one vs. all”, see STAR Methods). **(B–K)** The indicated alleles were on a *Krt5-CreER; Rosa26^eYFP* transgenic background. **(B–E, J)** Conditionally activating Wnt signaling via removal of exon 3 of *β-catenin* (C) results in HBCs that change shape and begin to differentiate (compare with cells from heterozygous *Trp63^lx/+* controls, shown in panel B). Coupled with the removal of one allele of *Trp63* (D), activation of Wnt signaling in HBCs induces proliferation and differentiation into neurons, similar to the *Trp63^lx/lx* phenotype (E). **(J)** Quantitation of the effect of activating *β-catenin* in the *Trp63^lx/+* background, n = 4 (*Trp63^lx/+*), n = 3 (*Trp63^lx/+*; *β-catenin*^lxex3/+) at 14–21-days post tamoxifen (DPT); mean is shown in red; p-value = 0.01. **(F–I, K)** Quiescent HBCs (F) differentiate into neurons (assessed by suprabasal position, cell shape, and lack of SOX2 expression) and support cells after conditional knockout of *Trp63* (G). Inhibition of Wnt signaling by conditional knockout of *β-catenin* in the *Trp63^lx/lx* background decreases the capacity of HBCs to form neurons (H, I, K). **(K)** There are fewer neurons (assessed by suprabasal position, cell shape, and lack of SOX2 expression) in the double knockout (14 DPT), n = 3 for each genotype, p-value = 0.04. See Figure S6.

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References

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[1] Ali S, Mohs A, Thomas M, Klare J, Ross R, Schmitz ML, Martin MU. The Dual Function Cytokine IL-33 Interacts with the Transcription Factor NF- B To Dampen NF- B-Stimulated Gene Transcription. The Journal of Immunology. 2011;187:1609–1616. - PubMed

[2] Ali S, Mohs A, Thomas M, Klare J, Ross R, Schmitz ML, Martin MU. The Dual Function Cytokine IL-33 Interacts with the Transcription Factor NF- B To Dampen NF- B-Stimulated Gene Transcription. The Journal of Immunology. 2011;187:1609–1616. - PubMed

[3] Arnold K, Sarkar A, Yram MA, Polo JM, Bronson R, Sengupta S, Seandel M, Geijsen N, Hochedlinger K. Adult Stem and Progenitor Cells Are Important for Tissue Regeneration and Survival of Mice. Stem Cell. 2011;9:317–329. - PMC - PubMed

[4] Arnold K, Sarkar A, Yram MA, Polo JM, Bronson R, Sengupta S, Seandel M, Geijsen N, Hochedlinger K. Adult Stem and Progenitor Cells Are Important for Tissue Regeneration and Survival of Mice. Stem Cell. 2011;9:317–329. - PMC - PubMed

[5] Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. - PMC - PubMed

[6] Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. - PMC - PubMed

[7] Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003;19:185–193. - PubMed

[8] Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003;19:185–193. - PubMed

[9] Brault V, Moore R, Kutsch S, Ishibashi M, Rowitch DH, McMahon AP, Sommer L, Boussadia O, Kemler R. Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development. 2001;128:1253–1264. - PubMed

[10] Brault V, Moore R, Kutsch S, Ishibashi M, Rowitch DH, McMahon AP, Sommer L, Boussadia O, Kemler R. Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development. 2001;128:1253–1264. - PubMed

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Deconstructing Olfactory Stem Cell Trajectories at Single-Cell Resolution

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Deconstructing Olfactory Stem Cell Trajectories at Single-Cell Resolution

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