Delineating the early transcriptional specification of the mammalian trachea and esophagus
- Program in Craniofacial Biology, University of California San Francisco, United States
- Department of Cell and Tissue Biology, University of California San Francisco, United States
- Institute for Human Genetics, University of California San Francisco, United States
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, United States
- Developmental and Stem Cell Biology Graduate Program, University of California San Francisco, United States
- Department of Biology, San Francisco State University, United States
- MRC London Institute of Medical Sciences (LMS), United Kingdom
- Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, United Kingdom
Figures
Figure 1 with 5 supplements
Single-cell transcriptomics of the developing foregut epithelium identifies distinct dorsoventral populations.
Dissected, FACS-purified foregut epithelial cells were subjected to droplet-based single-cell RNA sequencing at E10.5 and E11.5. (a) UMAP representation of 6,407 cells identified at E10.5 and (b) …
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Figure 1—source data 1
Top 20 markers of foregut cell populations identified by scRNA-seq of E10.5 and E11.5 foreguts.
- https://cdn.elifesciences.org/articles/55526/elife-55526-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Quality control metrics and characterization of single-cell RNA sequencing experiments.
(a) Representative gating strategy for FACS of mouse embryonic foreguts. Top row shows gating for single cells, and bottom row shows gating for sytox-negative live cells (left) and epCAM-positive …
Figure 1—figure supplement 2
Additional cell type-specific markers of dorsoventral populations identified by scRNA-seq at E10.5.
(a-m) RNA localization of additional marker genes Wnt7b, Pcdh10, Ly6h, and Klf5 identified from differential expression analysis of cell populations in scRNA-seq data at E10.5. First column shows …
Figure 1—figure supplement 3
Cell type-specific markers of trachea, esophagus, and lung populations identified by scRNA-seq at E11.5.
(a) RNA localization of marker genes identified from differential expression analysis of cell populations in scRNA-seq data at E11.5 with immunofluorescent staining of NKX2-1. First three columns …
Figure 1—figure supplement 4
Newly identified esophageal marker genes are more dorsally restricted than SOX2.
RNA localization of esophageal markers genes Klf5, Krt19, and Dcn and protein localization of LRIG1 (green) with immunofluorescent staining of NKX2-1 (magenta) and SOX2 (cyan) in the unseparated …
Figure 1—figure supplement 5
Validation of pharynx and UBB cluster identities.
(a) RNA localization of pharynx marker gene Foxe1 identified from differential expression analysis of cell populations in scRNA-seq data at E10.5 and E11.5. Top row: RNA-localization of Foxe1 …
Figure 2 with 1 supplement
Transcriptomic analysis of Nkx2-1-/- foreguts reveals NKX2-1 regulated tracheal and esophageal transcriptional programs.
(a) Dissected, FACS-purified epithelium from E11.5 Nkx2-1-/- and WT foreguts was sequenced and analyzed for differential gene expression. 109 genes were increased and 148 genes were decreased in Nkx2…
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Figure 2—source data 1
Differentially expressed genes between E11.5 Nkx2-1-/- and WT foregut epithelium identified with bulk RNA-seq.
- https://cdn.elifesciences.org/articles/55526/elife-55526-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
NKX2-1-dependent gene expression in E11.5 scRNA-seq.
(a-b) Combined expression of all genes that increase (a) or decrease (b) in Nkx2-1-/- foreguts as a percentage of all reads in E11.5 scRNA-seq data, projected onto the E11.5 UMAP.
Figure 3 with 1 supplement
Identification of an NKX2-1-independent tracheal and esophageal program.
(a-l) RNA localization of NKX2-1-independent tracheal (Irx2, Ly6h) and esophageal (Dcn, Ackr3) makers identified by scRNA-seq and Nkx2-1-/- mutant RNA-seq data at E11.5. First column shows …
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Figure 3—source data 1
Differentially expressed genes between E11.5 WT trachea and esophagus epithelium identified with bulk RNA-seq.
- https://cdn.elifesciences.org/articles/55526/elife-55526-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Nkx2.5-cre recombination, additional validation of NKX2-1-independent genes.
(a) Lineage trace of Nkx2.5-cre recombination pattern in E10.5 (top) and E11.5 (bottom) foreguts using the ROSA26mTmG reporter (red/green) and NKX2-1 immunofluorescent staining (magenta). (b) RNA …
Figure 4
NKX2-1 binds directly to tracheoesophageal genes in the developing foregut.
(a) ChIP-seq for NKX2-1 in E11.5 foreguts identified 15,861 NKX2-1-bound genomic regions (peaks) shared between replicates (FDR < 0.00001). n = 2 biological replicates with 175 pooled …
Figure 5
NKX2-1 regulates target genes in a SOX2-dependent and independent manner.
(a-c) Schematic of Nkx2-1; Sox2 compound mutant analysis phenotypes and resulting NKX2-1 and SOX2 expression patterns in E11.5 control embryos (a), Nkx2-1-TrKO mutants (b), Nkx2-1-TrKO; Sox2-TrKO …
Figure 6 with 1 supplement
NKX2-1 regulates mesenchymal specification via Wnt7b and Shh-mediated epithelial-mesenchymal crosstalk.
(a) Alcian blue staining of tracheal cartilage in E18.5 control and Nkx2-1-/- embryos. 3/3 embryos examined exhibited similar cartilage phenotype. (b) ChIP-seq of direct NKX2-1 binding near Wnt7b …
Figure 6—figure supplement 1
NKX2-1 regulation of epithelial-mesenchymal signaling.
(a) RNA localization of Wnt7b (red) and Axin2 (yellow), and (b) Shh (red) and Ptch1 (yellow) in WT and Nkx2-1-/- E11.5 foreguts. (c) Schematic of rostral-caudal position of images in (d-g). (d-g) …
Figure 7
Proposed model of NKX2-1-dependent and -independent tracheoesophageal specification.
(a) Schematic of NKX2-1 and SOX2 regulation of gene expression in WT trachea and esophagus, and epithelial-mesenchymal signaling downstream of NKX2-1. NKX2-1 negatively regulates esophageal genes Shh…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (mouse) | CD1 | Harlan/Envigo | Cat#: 030 | |
| Genetic reagent (mouse) | Nkx2-1fl/fl | Kusakabe et al., 2006 | MGI: 3653645 | |
| Genetic reagent (mouse) | Sox2fl/fl | Shaham et al., 2009 | MGI: 4366453 | |
| Genetic reagent (mouse) | Tmem163Tg(Actincre) | Lewandoski et al., 1997 | MGI: 2176050 | |
| Genetic reagent (mouse) | Nkx2.5cre | Stanley et al., 2004 | MGI: 2448972 | |
| Antibody | α-NKX2-1 (rabbit polyconal) | Millipore | Cat#: 07601 | 1:200 for IF, 5ug for ChIP |
| Antibody | α-SOX2 (goat polyclonal) | Neuromics | Cat#: GT15098 | 1:250 for IF |
| Antibody | α-SOX9 (Rabbit polyclonal) | Santa Cruz | Cat#: sc-20095 | 1:250 for IF |
| Antibody | α-PITX1 (Rabbit polyclonal) | NovusBio | Cat: NBP188644 | 1:200 for IF |
| Antibody | α-LRIG1 (Goat polyclonal) | R&D Systems | Cat#: AF3688 | 1:250 for IF |
| Antibody | α-SMA (Rabbit polyclonal) | Abcam | AB5694 | 1:300 for IF |
| Antibody | α-SMA-cy3 (mouse monoclonal) | Sigma | C6198 | 1:300 for IF |
| Antibody | α-EpCAM (rat polyclonal) | BioLegend | Cat#: 118219 | 1:200 for FACS |
| Sequence-based reagent | RNAscope probe mm-Irx2 | Advanced Cell Diagnostics | Cat#: 519901 | |
| Sequence-based reagent | RNAscope probe mm-Wnt7b | Advanced Cell Diagnostics | Cat#: 401131 | |
| Sequence-based reagent | RNAscope probe mm-Pcdh10 | Advanced Cell Diagnostics | Cat#: 477781-C3 | |
| Sequence-based reagent | RNAscope probe mm-Tppp3 | Advanced Cell Diagnostics | Cat#: 586631 | |
| Sequence-based reagent | RNAscope probe mm-Ly6h | Advanced Cell Diagnostics | Cat#: 587811 | |
| Sequence-based reagent | RNAscope probe mm-Krt19 | Advanced Cell Diagnostics | Cat#: 402941 | |
| Sequence-based reagent | RNAscope probe mm-Klf5 | Advanced Cell Diagnostics | Cat#: 444081 | |
| Sequence-based reagent | RNAscope probe mm-Foxe1 | Advanced Cell Diagnostics | Cat#: 509641 | |
| Sequence-based reagent | RNAscope probe mm-Crabp1 | Advanced Cell Diagnostics | Cat#: 474711-C3 | |
| Sequence-based reagent | RNAscope probe mm-Bmp4 | Advanced Cell Diagnostics | Cat#: 401301-C2 | |
| Sequence-based reagent | RNAscope probe mm-Nrp2 | Advanced Cell Diagnostics | Cat#: 500661 | |
| Sequence-based reagent | RNAscope probe mm-Dcn | Advanced Cell Diagnostics | Cat#: 413281-C3 | |
| Sequence-based reagent | RNAscope probe mm-Has2 | Advanced Cell Diagnostics | Cat#: 465171-C2 | |
| Sequence-based reagent | RNAscope probe mm-Meis2 | Advanced Cell Diagnostics | Cat#: 436371-C3 | |
| Sequence-based reagent | RNAscope probe mm-Ackr3 | Advanced Cell Diagnostics | Cat#: 482561-C2 | |
| Sequence-based reagent | RNAscope probe mm-Axin2 | Advanced Cell Diagnostics | Cat#: 400331-C3 | |
| Sequence-based reagent | RNAscope probe mm-Shh | Advanced Cell Diagnostics | Cat#: 314361 | |
| Sequence-based reagent | RNAscope probe mm-Ptch1 | Advanced Cell Diagnostics | Cat#: 402811-C2 | |
| Sequence-based reagent | Nkx2-1-geno1 | This paper | PCR primer | 5'-CTA-GGG-AGG-CTA -GGA-ACT-CGG-3' |
| Sequence-based reagent | Nkx2-1-geno2 | This paper | PCR primer | 5'-CCG-ACC-CAC-GTA-GAG-CC-3' |
| Sequence-based reagent | Nkx2-1-geno3 | This paper | PCR primer | 5'-CTC-TTA-TCT-GGG-ATC-GCC-TGA-G-3' |
| Sequence-based reagent | Sox2-flox-geno1 | Steevens et al., 2019 | PCR primer | 5'-TGG-AAT-CAG-GCT-GCC-GAG-AAT-CC-3' |
| Sequence-based reagent | Sox2-flox-geno2 | Steevens et al., 2019 | PCR primer | 5'-TCG-TTG-TGG-CAA-CAA-GTG-CTA-AAG-C-3' |
| Sequence-based reagent | Sox2-flox-geno3 | Steevens et al., 2019 | PCR primer | 5'-CTG-CCA-TAG-CCA-CTC-GAG-AAG-3' |
| Sequence-based reagent | Cre-geno1 | Liang et al., 2005; Vauclair et al., 2005 | PCR primer | 5'-GTT-CGC-AAG-AAC-CTG-ATG-GAC-A-3' |
| Sequence-based reagent | Cre-geno2 | Liang et al., 2005; Vauclair et al., 2005 | PCR primer | 5'-CTA-GAG-CCT-GTT-TTG-CAC-GTT-C-3' |
| Commercial assay or kit | MicroChIP Diapure columns | Diagenode | Cat#: C03040001 | DNA extraction (ChIP) |
| Commercial assay or kit | Microplex Library Preparation Kit v2 | Diagenode | Cat#: C05010012 | ChIP library prep |
| Commercial assay or kit | Rneasy Micro kit | Qiagen | Cat# 74004 | RNA extraction (bulk) |
| Commercial assay or kit | SMARTer Stranded Total RNAseq kit V2 | Takara | Cat#: 634411 | Bulk RNAseq library prep |
| Commercial assay or kit | RNA 6000 Pico Bioanalyzer kit | Agilent | Cat# 5067-1513 | Bioanalyzer (RNA) |
| Commercial assay or kit | High Sensitivity DNA Bioanalyzer kit | Agilent | Cat#: 5067-4626 | Bioanalyzer (DNA) |
| Commercial assay or kit | QuBit dsDNA HS Assay kit | Invitrogen | Cat#: Q32854 | DNA quantification |
| Commercial assay or kit | Chromium Single Cell ' Reagent Kit V2 | 10X Genomics | Cat#: PN-120237, PN-120236, PN-120262 | Single cell RNA-seq |
| Other | Sytox Blue nuclei acid stain | Thermo | Cat: S11348 | Live/dead stain for FACS, use at 1uM |
| Other | TSAplus Fluoresceine | Akoya Biosciences | NEL741001KT | RNAscope, 1:1500 |
| Other | TSAplus Cy3 | Akoya Biosciences | NEL744001KT | RNAscope, 1:1500 |
| Other | TSAplus Cy5 | Akoya Biosciences | NEL745001KT | RNAscope, 1:1500 |
| Other | DynaBeads, protein G | Invitrogen | Cat#: 10003D | ChIP |
| Other | AMPure XP beads | Beckman Coulter | Cat#: A63881 | Library cleanup |
| Other | TrypLE Express | Thermo | Cat# 12604013 | Tissue dissociation |
