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. Author manuscript; available in PMC: 2022 May 27.
Published in final edited form as: Cell. 2021 Apr 30;184(11):3056–3074.e21. doi: 10.1016/j.cell.2021.04.003

Figure 2. Epithelial differentiation trajectory reveals progenitor and ciliogenesis programs of ChP epithelial cells during development.

Figure 2.

(A) Embryonic neuronal and glia-like cell subsets. T-SNE of embryonic neuronal and glia-like single cell profiles (scRNA-seq, 955 cells). (B) Diffusion map embedding (components 1 and 3) of neuronal, glia-like and epithelial cell profiles (dots) from 3V ChP, colored by log2(TP10K+1) expression of marker genes of neurons (Tubb3), progenitors (Pax6, Rspo2), cycling (Mki67), ciliogenesis (Ccdc67), and mature epithelial (Krt18) cells. Right: Suggested differentiation trajectories. (C) Schematic of embryonic hindbrain; upper rhombic lip (URL), lower rhombic lip (LRL). Dotted box: area shown in panels (D-K). (D) Proliferating (KI67) progenitors (PAX6) in URL. (E) SmFISH markers of progenitors (Rspo2, Rspo3) and Wnt1. (F) SmFISH of Wnt1 and FoxJ1. (G) High magnification image of (F). (H) Confetti-labeling schematic. (I) E16.5 hindbrain region from Wnt1-CRE2 crossed with Confetti mouse shows distribution of Wnt1-derived cells. (J) Wnt1-derived cRFP or mCFP epithelial cells adjacent to URL. (K) Wnt1-derived cRFP and TUBB3 neurons in URL with DAPI. Inset: split cRFP (top) and TUBB3 (bottom). (L) Confetti labeled cells in 3V ChP. (M) Proliferating cells enriched at LV ChP base. Left: BrdU labeling; Middle: ISH of Ccdc67; Right: AQP1. (N) Left: Scanning EM of multi-ciliated epithelial cell. Right: Median expression level in expressing cells (color) and proportion of expressing cells (circle size) of markers of ciliogenesis (columns) in ciliogenic (developing) and mature epithelial cells (rows). (O) Left: LV ChP immunostained for Ac-Tubulin and CCDC67/DEUP1. Middle and right: Ac-Tubulin with CCDC67 (middle) or SHISA8 (right). (P) Schematic: LV ChP maturation regions. (Q-R) Spatial mapping of maturation domains in 3V and 4V ChP marked by progenitors (Rspo2), ciliogenesis (Ccdc67), differentiated epithelial cells (Krt18) (Genepaint). Schematic: ChP regions with maturing epithelial cells. See also Figure S2.