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SOX9 maintains human foetal lung tip progenitor state by enhancing WNT and RTK signalling

Abstract The balance between self‐renewal and differentiation in human foetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self‐renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self‐renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID, we place SOX9 at the centre of a transcriptional network, which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof‐of‐principle CRISPRi screen and Targeted DamID tools establish a new workflow for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease.

A B D C Figure EV2. Validation of the CRISPRi screen results.
A qRT-PCR results showing the targeted genes (IRF6, MYBL2 and ZBTB7B) were efficiently knocked down by the inducible CRISPRi system using the gRNAs selected from the CRISPRi gRNA library. B Representative EdU staining images of non-targeting gRNA control and IRF6 or MYBL2 knock-down experiments. C Quantification of the percentage of EdU + cells in each of three parental organoid lines used with non-targeting control, IRF6 knock-down and MYBL2 knock-down. n = 1,649, 1,705, 3,548 cells were scored for NT controls. n = 2,517, 950, and 1,313 cells were scored for IRF6 gRNAs. n = 1,098 and 1,306 cells were scored for MYBL2 gRNAs. D qRT-PCR results showing ARID5B was not knocked down by the inducible CRISPRi system using the gRNAs selected from the CRISPRi gRNA library.
Data information: Error bars: mean AE SEM. Statistical analysis was using the two-tailed paired t-test. P-values are reported as follows: *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. non-significant. N = 3 organoid lines (biological replicates) used for each panel. Source data are available online for this figure.
A Unsupervised hierarchical clustering of non-targeting control, SOX2 knock-down and SOX9 knock-down RNA-Seq results. B Venn diagram showing minimal overlap of differentially expressed genes after SOX2 knock-down in two different parental organoid lines. Overlapping DE genes were labelled in boxes. C qPCR of selected DE genes from SOX9 RNA-seq data following SOX9 knock-down in a further 2 independent organoid lines. Cells harvested 5 days after knock-down.
Error bars: mean AE SEM. Statistical analysis was using the two-tailed paired t-test. P-values are reported as follows: **P < 0.01, ***P < 0.001. N = 3 bio-replicates (Organoid line BRC2174 with two different NT gRNAs and two different SOX9 gRNAs, and Organoid line BRC2136 with 1 NT gRNA and 1 SOX9 gRNA) were used. D Sashimi plot to visualise splicing junction of NT control and SOX9 KD. Upper panel: Sashimi plot was used to visualise splicing junction information in non-targeting gRNA control and SOX9 knock-down groups. Junctional reads between intron #1 and exon #2 were only observed in SOX9 knock-down groups and not in nontargeting gRNA control groups.   Figure EV4. SOX9 directly activates tip cell genes and represses secretory cell genes.
A Summary of enriched TF binding motifs in SOX9 TaDa peaks. The SOX motif was enriched, indicating the SOX9 TaDa faithfully identified SOX9 binding sites across the genome. B SOX9 direct transcriptional target enrichment in human foetal lung scRNA-seq data. All SOX9 direct transcriptional targets were used for scoring. Similar to Fig 2H, SOX9 directly activated targets were enriched in tip progenitor cells (left panel), whereas SOX9 directly repressed targets were enriched in secretory cell lineages (right panel). C SHH was co-expressed with SOX9 in human foetal lung tip progenitor cells. SOX9 in yellow and SHH in red. No-probe controls are shown in the right panel. D LEF1 and WIF1 were co-expressed with SOX9 in human foetal lung tip progenitor cells. SOX9 in red and LEF1 (left panel) and WIF1 (right panel) in yellow. E Lentiviral construct design for overexpressing SOX9 in human foetal lung progenitor cells. F Representative images showing organoid morphology does not change after 3 days of SOX9 overexpression. SOX9 overexpressed organoid indicated with arrow. G qRT-PCR results showing that 3 days of SOX9 overexpression led to ETV5 and MYCN transcription being significantly upregulated, however, ETV4 and CFTR were not changed. N = 4 organoid lines (bio-replicates) were used. Error bars: mean AE SEM. Two-tailed Student's t-tests were performed. P-values are reported as follows: *P < 0.05; ***P < 0.001.
Data information: Scale bars denote 50 lm (C, D) and 100 lm (F). Source data are available online for this figure.
A qRT-PCR showing SOX9 transcription 6 day after FGF10 supplementation (500 ng/ml), or FGF10 supplementation and removal of WNT activators. N = 4 different organoid lines. Error bars: mean AE SEM. Statistical analysis was using the two-tailed paired t-test. P-values are reported as follows: **P < 0.01; ***P < 0.001; n.s. nonsignificant. B Pearson correlation of SOX9, ETV4 and ETV5 TaDa. ETV4 and ETV5 TaDa exhibited great consistency. C Motifs enriched in ETV5 TaDa peaks. The ETS binding motif was highly enriched. D Genomic occupancy annotated features for SOX9 and ETV5 peaks. E Venn diagram showing overlap of differentially expressed genes after ETV4 and ETV5 double knock-down in two different parental organoid lines. Overlapping DE genes related to cell division by GO analysis are labelled in the box. F Heatmap showing expression level of all 42 DE genes after ETV4; ETV5 double knock-down across different organoid lines. Directly regulated genes are marked by asterisks.
Source data are available online for this figure. ◀