Figure 1. Clonal Analysis of HBC-Derived Cells During Regeneration.

(A) Schematic of olfactory cell types produced by HBCs during regeneration. HBC (green), horizontal basal cell; GBC (cyan), globose basal cell; Sus (magenta), sustentacular cell; olfactory sensory neuron (OSN, orange). (B) Maximum projection of a 40μm tissue section of regenerating olfactory epithelium at 14 DPI from a Krt5-CreER; Rosa26^Confetti animal in which Cre was sparsely activated. Reporter localization detected using a GFP antibody coupled with antibodies to SOX2 (expressed by HBCs, GBCs, and sustentacular cells; magenta) and P63 (expressed by HBCs alone; white) were used along with cellular morphology to discriminate cell types and clonal relationships in YFP- and CFP-positive cells. Scale bar, 50 μm. (C) Distributions of ratios of HBCs, OSNs, and sustentacular cells with respect to total cells counted across all clones, by animal and by experimental time-point (7 DPI and 14 DPI); the mean, across animals, is indicated by a dash in each condition. P-values were calculated using a negative binomial regression model, and the Benjamini-Hochberg method was used to adjust for multiple testing (STAR Methods). (D) Distributions of neurons (top) and sustentacular cells (bottom) per clone in clones possessing at least one of each cell type, respectively, at 14 DPI. (E) Ratios of clones scored by lineage representation at 7 DPI and 14 DPI. Clones were binned on the basis of containing at least one cell of one of the three major lineages (HBC, green; neuron, magenta; sustentacular cell, purple). Multi-lineage clones are indicated by different colored rectangles from left to right, while uni-lineage clones are represented by a single colored rectangle. (F–H) HBCs were lineage-traced in Krt5-CreER; Rosa26^Confetti animals; tissue was collected at 48 HPI to assess the fates of HBC-derived doublets. (F) Representative maximum projections of 40μm confocal z-stacks show the three most commonly observed doublet compositions (top: symmetric renewing; middle: symmetric differentiating; bottom: asymmetric) based on expression of marker genes. Basement membrane, dotted line; scale bar, 10 μm. (G) Percent of doublet cells that are renewed HBCs, differentiating HBCs, and unknown cells at 48 HPI. (H) Doublet compositions at 48 HPI. See Figure S1.