key: cord-0328908-u77jqpwv
authors: Kiyokawa, Hirofumi; Yamaoka, Akira; Matsuoka, Chisa; Tokuhara, Tomoko; Abe, Takaya; Morimoto, Mitsuru
title: Airway tissue stem cells reutilize the embryonic proliferation regulator, Tgfß-Id2 axis, for tissue regeneration
date: 2020-11-24
journal: bioRxiv
DOI: 10.1101/2020.11.23.394908
sha: 552ae0b163c918dd1a92058b13e70f89d9acac83
doc_id: 328908
cord_uid: u77jqpwv

During development, quiescent basal stem cells are derived from proliferative primordial progenitors through the cell cycle slowdown. In contrast, quiescent basal cells contribute to tissue repair during adult tissue regeneration by shifting from slow-cycling to proliferating and subsequently back to slow-cycling. Although sustained basal cell proliferation results in tumorigenesis, the molecular mechanisms regulating these transitions remain unknown. Using temporal single-cell transcriptomics of developing murine airway progenitors and in vivo genetic validation experiments, we found that Tgfß signaling slowed down cell cycle by inhibiting Id2 expression in airway progenitors and contributed to the specification of slow-cycling basal cell population during development. In adult tissue regeneration, reduced Tgfß signaling restored Id2 expression and initiated epithelial regeneration. Id2 overexpression and Tgfbr2 knockout enhanced epithelial proliferation; however, persistent Id2 expression in basal cells drove hyperplasia at a rate that resembled a precancerous state. Together, the Tgfß-Id2 axis commonly regulates the proliferation transitions in airway basal cells during development and regeneration, and its fine-tuning is critical for normal regeneration while avoiding basal cell hyperplasia.

Adult airway epithelium shows a low cellular turnover rate, usually more than 4 months, 58 in rodents (Rock, et al., 2009; Blenkinsopp, 1967) . It possesses a substantial ability to 

In the present study, to unveil the molecular mechanism that establishes slow-cycling In summary, we demonstrate that the Tgfß-Id2 axis is a shared, critical regulator of the 103 transition between the active proliferation and slow-cycling mode in airway stem cells 104 during development and adult tissue regeneration. 

Time series single-cell RNA sequencing (scRNA-seq) analyses to delineate a 108 developmental roadmap of airway epithelial cells, including basal cells 109 We aimed to identify a high-fidelity marker of early basal progenitors to elucidate the 110 developmental process of mature basal cells; p63 is a well-known marker for basal cells 111 but is not restricted to basal progenitors at early development (Yang, et al., 2018) . We 112 used a droplet-based scRNA-seq with approximately 3500 epithelial cells at six time 113 points from E12.5 to E18.5 ( Figure 1A ). This approach allowed us to delineate a 114 comprehensive lineage map of airway epithelial cells derived from respiratory endoderm 115 during embryogenesis. We visualized the distinct populations with cluster analysis using 116 the t-SNE algorithm. Uniform progenitors at E12.5 and E13.5 changed transcriptome 117 profiles at E14.5 and acquired cell type-specific gene signatures by E16.5 (Figures 1B 118 and S1B, also see Figure 3I ). During E12.5 to E14.5, proliferative markers are frequently 119 detected and are the major determinants for cell clustering analysis ( Figure 1B and see 120 Figure 3A ). Epithelial progenitors eventually differentiated into three major populations, 121 basal, club, and ciliated cells, and one minor population, NE cells ( Figure 1B ). This 134 We sought a novel maker for basal progenitor more committed to the basal lineage than 135 p63-expressing cells, and Krt17 was selected as a candidate (Supplementary Table 1 Krt17 is an important step for commitment to mature basal cells after the expression of 149 p63 ( Figure 2G ). 150 We further identified Scgb3a2 as a marker gene for Krt17 − progenitors by reanalyzing 151 the scRNA-seq data ( Figures S2A-C ). Scgb3a2 appears from E14.5 on in a pattern 152 mutually exclusive with Krt17 ( Figures 2B and S2B ), which suggests that equivalent 153 airway progenitors make binary cell fate decision around E14.5 to acquire either Krt17 + 154 or Scgb3a2 + (Krt17 − ) status that do or do not differentiate into basal cells, respectively. Numbers of cells in G0 phase (quiescent state) significantly increased between E14.5 and 184 E16.5 (5.9% ± 1.8% vs. 22.5% ± 3.3%, mean ± SD) ( Figure S2D) . A BrdU incorporation 185 assay with mouse embryos also showed a substantial decrease in the ratio of proliferative 186 cells from E14.5 to E16.5 (54.0% ± 2.1% vs. 14.3% ± 3.6%, mean ± SD) ( Figures 3C and   187 3D). Additionally, p63 + cells showed a faster decrease in BrdU incorporation than p63 -188 cells ( Figure S2E ). These results suggest that cell cycle slowdown preferentially occurs 189 in p63 + progenitors before the complete commitment into basal cell lineage and 190 subsequently induces Krt17 expression. Based on these results, we hypothesize that 191 epithelial quiescence occurs before basal cell specification. 192 We tested this hypothesis with ex vivo culture of E12.5 developing trachea, where An increase in Id2 expression was detected at 12 hpi before the increase in Ki67-286 expressing cells ( Figure 6B ). These observations prompted us to hypothesize that 287 recurrent Id2 activation stimulated basal cells to re-enter the active cell cycle. We exposed 288 wild-type and Id2 OE adult mice to SO2 gas and examined their respective number of 289 Ki67-expressing cells to test this hypothesis. Airways from the Id2 OE mice showed a 290 more rapid response to injury than airways from wild-type mice at 12 hpi ( Figure 6C After washing the slides with 0.05% Tween in PBS for 3 times, the sections were 

Zymoresearch, R2060) according to the manufacturer's instructions

Reverse transcription reactions were performed with SuperScript™ III Reverse 191

18080) according to the manufacturer's instructions. qRT-PCR 192 was performed on 7500 Real-Time PCR instrument

Toyobo life science, QPS-201). The mRNA levels 194 of target genes were normalized to the Gapdh mRNA level. Primers used for qPCR are 195

Single cell RNA-seq for sequencing library construction

), the epithelial sheet was peeled off from mesenchymal tissue in 199 developing trachea with a tungsten needle after the incubation with 175U/ml collagenase 200 typeⅠ (Worthington Biochemical Corporation, CLS1) at 37℃ for 6 -60 min

25200056) at 37℃ for 15 min, then loaded onto Chromium Single Cell 203 A Chips (10X Genomics, PN-1000009) for the Chromium Single Cell 3′ Library v2

PN-120233) according to the manufacturer's recommendations (10X

After 207 performing GEM-reverse transcriptions (GEM-RTs), GEMs were harvested and the 208 cDNAs were amplified and cleaned up with SPRIselect Reagent Kit

Indexed sequencing libraries were constructed using Chromium Single Cell 3′

PN-120233) for enzymatic fragmentation, end-repair, A-211 tailing, adaptor ligation, ligation cleanup, sample index PCR, and PCR cleanup

The packages listed below was used for processing raw sequencing data and 216 downstream analysis

First, the cells meeting any of the following criteria were omitted 219 from further analyses for the quality control; <1,000 or >5,000 UMIs, > 7.5% of reads 220 mapping to mitochondria genes, or EpCAM negative cells. For clustering, principal-221 component analysis was performed for dimension reduction

Seurat and passed to t-Distributed Stochastic Neighbor Embedding (tSNE) for 224 clustering visualization. To maintain a standard procedure for clustering

Pronuclear Microinjection during S-Phase Increases 281

The dynamics and regulators of cell fate decisions are revealed by pseudotemporal 309 ordering of single cells