Diverse homeostatic and immunomodulatory roles of immune cells in the developing mouse lung at single cell resolution
- Division of Critical Care Medicine, Department of Pediatrics, Stanford University School of Medicine, United States
- Center for Excellence in Pulmonary Biology, Stanford University School of Medicine, United States
- Department of Bioengineering, Stanford University, United States
- Prince of Wales Clinical School, Lowy Cancer Research Centre, University of New South Wales, Australia
- Division of Pulmonary, Asthma and Sleep Medicine, Department of Pediatrics, Stanford University School of Medicine, United States
- Department of Chemical and Systems Biology, Stanford University, United States
- Chan Zuckerberg Biohub, United States
- Department of Applied Physics, Stanford University, United States
Figures
Figure 1 with 2 supplements
Diversity of the lung immune landscape increases dramatically after birth.
(A) Overview of the experimental design including the four timepoints (E18.5, P1, P7, P21) corresponding to key stages in late lung development. Lungs were isolated, perfused, and digested and …
Figure 1—figure supplement 1
Optimization of lung tissue digestion.
(A–C) E18.5, P1, P7 and P21 murine lungs were processed for flow cytometry and the frequency of (B) total and (C) CD45+ (immune) dead cells was assessed. (D–E) Frequency of dead cells of P7 murine …
Figure 1—figure supplement 2
Determination of variation between mice.
To quantify whether the different mice contributed spurious variation to the data, a distribution level approach was chosen. For each cell type and time point, 100 pairs of cells from either the …
Figure 2 with 2 supplements
Macrophage populations present before and after birth broadly separate into two populations based on expression of Dab2 and Plac8.
(A) t-SNE plot depicting Cd68 expression in the five macrophage populations. (B) Separate embeddings for prenatal versus postnatal macrophages, identifying developmental timepoint of cell origin …
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Figure 2—source data 1
Source files for quantification of Dab2+ and Plac8+ Cd68+ cells.
This zip archive contains all the fluorescent micrographs used for the quantitative analysis shown in Fig. blank. The individual files are named with the timepoint (for figures containing more than one timepoint), the gene detected by FISH, followed by the color of the label for the gene with ‘G’ for green, ‘R’ for red, ‘W’ for white, and ‘Y’ for yellow.
- https://cdn.elifesciences.org/articles/56890/elife-56890-fig2-data1-v2.zip
Figure 2—figure supplement 1
Multiplex in situ hybridization to detect Dab2 and Plac8 expressing macrophages.
In situ hybridization at E18.5 and P7 to detect Cd68 (white), Dab2 (red), Plac8 (green), and a merged image. Calibration bar = 20 μm.
Figure 2—figure supplement 2
Lineage-defining genes are diffusely expressed across macrophage populations.
t-SNE plots of Adgre1, Lgals3, Mertk, Ly6c2, Ly6c1 and Itgam expression across all macrophage clusters between E18.5 and P21. The color scale is a logarithmic scale with a pseudocount of 0.1 counts …
Figure 3 with 1 supplement
Embryonic macrophages encircle developing blood vessels prior to birth.
(A) t-SNE plots depicting expression of Mki67 and Mcm5 in the macrophage clusters with low expression in purple and high expression in yellow, with quantification of proliferating macrophages at …
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Figure 3—source data 1
Source files for quantification of perivascular and parenchymal Cd68+ cells at E18.5.
This zip archive contains all the fluorescent micrographs used for the quantitative analysis shown in Fig. blank. The individual files are named with the timepoint (for figures containing more than one timepoint), the gene detected by FISH, followed by the color of the label for the gene with ‘G’ for green, ‘R’ for red, ‘W’ for white, and ‘Y’ for yellow.
- https://cdn.elifesciences.org/articles/56890/elife-56890-fig3-data1-v2.zip
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Figure 3—source data 2
Source files for quantification of Mki67+ Cd68+ cells at E18.5.
This zip archive contains all the fluorescent micrographs used for the quantitative analysis shown in Fig. blank. The individual files are named with the timepoint (for figures containing more than one timepoint), the gene detected by FISH, followed by the color of the label for the gene with ‘G’ for green, ‘R’ for red, ‘W’ for white, and ‘Y’ for yellow.
- https://cdn.elifesciences.org/articles/56890/elife-56890-fig3-data2-v2.zip
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Figure 3—source data 3
Source files for quantification of Gal+ and C1qa+ perivascular Cd68+ cells at E18.5.
This zip archive contains all the fluorescent micrographs used for the quantitative analysis shown in Fig. blank. The individual files are named with the timepoint (for figures containing more than one timepoint), the gene detected by FISH, followed by the color of the label for the gene with ‘G’ for green, ‘R’ for red, ‘W’ for white, and ‘Y’ for yellow.
- https://cdn.elifesciences.org/articles/56890/elife-56890-fig3-data3-v2.zip
Figure 3—figure supplement 1
Distribution of proliferating cells among macrophage/monocyte clusters.
Graph of percentage of proliferating macrophages in each cluster at E18.5.
Figure 4 with 4 supplements
Distinct transcriptional profiles and spatial distribution suggest specific physiologic functions for discrete macrophage populations.
(A) Heatmap of select differentially expressed genes within enriched pathways illustrated. The color scale is logarithmic with a pseudocount of 0.1 counts per million, normalized to the highest …
Figure 4—figure supplement 1
Pathway analysis of differentially expressed genes in the macrophage populations.
Pathway analysis on the top 100 expressed genes from the 5 Mac clusters was performed for each cluster in a one-vs-rest comparison, and the gene set enrichment evaluated with Metascape. Enriched …
Figure 4—figure supplement 2
Select genes involved in pathways regulating lung progenitor cells are expressed by Mac II and Mac IV.
(A) t-SNE plots of genes expressed by Mac II including Ntn1, Ntn4, Hhip, Tgfb1, and Tgfb2 with plot of cell subtype. (B) t-SNE plots of genes expressed by Mac IV including Fgf10, Bmp4, Wnt2, and WNt5…
Figure 4—figure supplement 3
Expression of select genes in Mac IV cluster.
t-SNE plots of (A) H2-Ab1, (B) H2-Eb1, (C) Cd74, and (D) Mrcl1 demonstrating differential expression in a portion of Mac IV (dotted red line). t-SNE plots of (E) Cx3cr1, (F) Mertk, (G) Cclr2, and …
Figure 4—figure supplement 4
Multiplex in situ hybridization to detect specific macrophage clusters.
(A) In situ hybridization at P1 to detect Cd68 (red), Itgax (white), Car4 (green) and a merged image. (b) In situ hybridization at P1 to detect Cd68 (white), Ifitm6 (red), Ccr2 (green) and a merged …
Figure 5
The Mac V cluster harbors distinct subpopulations with developmentally regulated gene expression patterns.
(A) t-SNE plots demonstrating high expression of Plac8 and Ifitm6 in Mac V cells. (B) t-SNE plots of Ccr1, Ccr2, Ace, and Slc12a2 suggesting the presence of two transcriptionally distinct …
Figure 6
Comparison of Macrophage/Monocyte Subtypes in the Perinatal and Adult Lung.
Data sets were merged, restricted to genes that were overlapping, normalized to counts per million reads, feature selected and subjected to PCA, followed by UMAP. UMAP plots of (A) cell type and (B) …
Figure 7
Expression of Fc receptors across macrophage clusters.
t-SNE plots of the expression of Fc receptors Fcgr1, Fcgr2b, Fcgr3, Fcgr4, Fcer1a, Fcer1g, Fcer2a, Fcgrt. For all tSNE embeddings, the color scale is a logarithmic scale with a pseudocount of 0.1 …
Figure 8 with 1 supplement
Multiple dendritic cell populations and lung granulocytes are primed for rapid pathogen response.
(A) Colored schematic and lung immune cell clustering demonstrating three separate clusters of DCs. (B) t-SNE plots of genes discriminating the three DC subclusters including Itgae (cDC1), Cd209a …
Figure 8—figure supplement 1
Expression of dendritic cell associated genes.
t-SNE plots of (A) pan-DC-associated genes Zbtb46 and Flt3; (B) cDC1 specific genes Itgae, Irf8, Xcr1, and Cadm1; (C) cDC2 specific genes Itgam, Sirpa, and Irf4; (D) monocyte-derived specific genes F…
Figure 9 with 1 supplement
Lymphocytes populate the lung at birth but remain naïve during the first three weeks of life (A) Quantification of the abundance of B and T cells at each developmental timepoint expressed on a log (Rodríguez-Castillo et al., 2018) scale as percentage of total immune cells.
(B) Quantification of the percentage of proliferating B cells at each developmental timepoint. (C) B cell heavy and light variable (V) and joining (J) gene identity and their cumulative cell …
Figure 9—figure supplement 1
Transcriptional and flow cytometric profiling of lymphocytes.
t-SNE plots of (A) B cell-associated genes Aicda, Tbx21, Prdm1, and Ebi3. (B) Clonality and t-SNE plots of Mki67 and Syk within the B cell cluster. (C) t-SNE plots of T cell-associated genes Trac …
Tables
Table 1
Top 35 differentially expressed genes in the Mac I cluster.
ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 35 upregulated genes are shown for the Mac I cluser; n = 2 mice.
| Mac I cluster top 35 differentially expressed genes | |||
|---|---|---|---|
| Gene name | Statistic | P value | Log2 fold change |
| Gal | 0.6133 | 5.42E-53 | 3.6207 |
| Crispld2 | 0.5 | 1.89E-34 | 3.6018 |
| Ncapd2 | 0.4567 | 1.40E-28 | 3.3349 |
| Pclaf | 0.49 | 4.88E-33 | 3.1388 |
| Mcm5 | 0.5067 | 2.07E-35 | 2.9792 |
| Cdca8 | 0.44 | 1.74E-26 | 2.8548 |
| Lig1 | 0.45 | 9.89E-28 | 2.7655 |
| AI506816 | 0.5233 | 7.05E-38 | 2.7046 |
| Asf1b | 0.4433 | 6.73E-27 | 2.5712 |
| Smc2 | 0.4367 | 4.44E-26 | 2.4042 |
| Fkbp5 | 0.5533 | 1.42E-42 | 2.3039 |
| Mcm7 | 0.44 | 1.74E-26 | 2.1166 |
| Cks1b | 0.45 | 9.89E-28 | 2.0842 |
| A930007I19Rik | 0.46 | 5.23E-29 | 2.0187 |
| Psat1 | 0.52 | 2.24E-37 | 2.0158 |
| Pfkl | 0.4467 | 2.59E-27 | 1.9417 |
| Ezh2 | 0.4433 | 6.73E-27 | 1.8453 |
| Dkc1 | 0.4733 | 9.21E-31 | 1.8027 |
| Cluh | 0.44 | 1.74E-26 | 1.7959 |
| Scd2 | 0.44 | 1.74E-26 | 1.7455 |
| Ruvbl2 | 0.4433 | 6.73E-27 | 1.7334 |
| Mif | 0.4933 | 1.67E-33 | 1.6941 |
| Tmem273 | 0.44 | 1.74E-26 | 1.628 |
| Stmn1 | 0.4433 | 6.73E-27 | 1.5905 |
| Spint1 | 0.5267 | 2.20E-38 | 1.5781 |
| Tpi1 | 0.5033 | 6.29E-35 | 1.562 |
| C230062I16Rik | 0.45 | 9.89E-28 | 1.5462 |
| Nt5dc2 | 0.4467 | 2.59E-27 | 1.5376 |
| Ran | 0.4567 | 1.40E-28 | 1.5078 |
| Trf | 0.5933 | 2.32E-49 | 1.4397 |
| Gcnt1 | 0.44 | 1.74E-26 | 1.4095 |
| Abcd2 | 0.46 | 5.23E-29 | 1.3537 |
| Anp32b | 0.47 | 2.56E-30 | 1.3418 |
| Dbi | 0.5167 | 7.04E-37 | 1.2018 |
| Atp5g3 | 0.52 | 2.24E-37 | 1.1808 |
Table 2
Top 35 differentially expressed genes in the Mac II cluster.
ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 35 upregulated genes are shown for the Mac II cluster; n = 2 mice.
| Mac II cluster top 35 differentially expressed genes | |||
|---|---|---|---|
| Gene name | Statistic | P value | Log2 fold change |
| Adgrl3 | 0.5713 | 8.85E-42 | 3.8593 |
| Adarb1 | 0.4216 | 5.92E-23 | 2.6952 |
| Ms4a8a | 0.4528 | 2.11E-26 | 2.4485 |
| Nav2 | 0.4751 | 4.97E-29 | 2.1319 |
| Tmcc3 | 0.4289 | 9.85E-24 | 2.0579 |
| Ldhb | 0.3809 | 8.26E-19 | 2.0235 |
| Acp5 | 0.3837 | 4.45E-19 | 2.0198 |
| Dapk1 | 0.3658 | 2.20E-17 | 1.8704 |
| Itgax | 0.5825 | 2.02E-43 | 1.8680 |
| Csf2rb2 | 0.3459 | 1.11E-16 | 1.7791 |
| H2-DMa | 0.5022 | 2.22E-32 | 1.7642 |
| Trerf1 | 0.3850 | 3.31E-19 | 1.7402 |
| Dmxl2 | 0.4117 | 6.59E-22 | 1.7264 |
| Tgm2 | 0.3663 | 1.99E-17 | 1.6906 |
| Ear2 | 0.5159 | 3.79E-34 | 1.6602 |
| Dok2 | 0.3695 | 1.00E-17 | 1.6373 |
| Flt1 | 0.3655 | 2.34E-17 | 1.5457 |
| Axl | 0.5068 | 5.74E-33 | 1.4698 |
| AU020206 | 0.3723 | 5.40E-18 | 1.4207 |
| Ece1 | 0.3962 | 2.54E-20 | 1.4075 |
| Ear10 | 0.3938 | 4.41E-20 | 1.4069 |
| Il18 | 0.4084 | 1.46E-21 | 1.4021 |
| Clec7a | 0.4959 | 1.37E-31 | 1.3413 |
| Spint1 | 0.3724 | 5.29E-18 | 1.3407 |
| Mpeg1 | 0.5454 | 4.05E-38 | 1.3269 |
| Il1rn | 0.3463 | 1.11E-16 | 1.2922 |
| Clec4n | 0.4454 | 1.45E-25 | 1.2765 |
| Fn1 | 0.4088 | 1.34E-21 | 1.2497 |
| Il1b | 0.4002 | 1.02E-20 | 1.2281 |
| Dst | 0.4484 | 6.72E-26 | 1.1994 |
| F11r | 0.4129 | 4.97E-22 | 1.1982 |
| Nceh1 | 0.3548 | 2.24E-16 | 1.1299 |
| Neurl3 | 0.3721 | 5.74E-18 | 1.1091 |
| Tnfaip2 | 0.3785 | 1.40E-18 | 1.0880 |
| Plet1 | 0.4461 | 1.23E-25 | 1.0845 |
Table 3
Top 35 differentially expressed genes in the Mac III cluster.
ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 35 upregulated genes are shown for Mac III cluster; n = 2 mice.
| Mac III cluster top 35 differentially expressed genes | |||
|---|---|---|---|
| Gene name | Statistic | P value | Log2 fold change |
| Slc39a2 | 0.6600 | 3.98E-62 | 9.2983 |
| Atp6v0d2 | 0.9533 | 1.14E-151 | 7.1784 |
| Krt19 | 0.7533 | 1.56E-83 | 6.8066 |
| Cd200r4 | 0.7533 | 1.56E-83 | 5.9139 |
| Gpnmb | 0.6967 | 5.32E-70 | 5.0666 |
| Ear1 | 0.6267 | 1.66E-55 | 4.9379 |
| Car4 | 0.7500 | 1.10E-82 | 4.6965 |
| Ly75 | 0.7867 | 2.26E-92 | 4.4964 |
| Bhlhe41 | 0.7800 | 1.51E-90 | 4.4452 |
| Slc7a2 | 0.7933 | 3.16E-94 | 4.1798 |
| Lrp12 | 0.6533 | 9.21E-61 | 3.7640 |
| Ccl6 | 0.8700 | 4.58E-118 | 3.7161 |
| Spp1 | 0.6267 | 1.66E-55 | 3.4907 |
| Serpinb1a | 0.6600 | 3.98E-62 | 3.4778 |
| Mgll | 0.8067 | 4.96E-98 | 3.4554 |
| Il1rn | 0.6567 | 1.93E-61 | 3.2640 |
| Kcnn3 | 0.7600 | 3.03E-85 | 3.2372 |
| Ralgds | 0.7000 | 9.49E-71 | 3.1251 |
| Dst | 0.7633 | 4.13E-86 | 3.1042 |
| Atp10a | 0.6600 | 3.98E-62 | 3.0453 |
| Ccnd2 | 0.7233 | 3.77E-76 | 2.9813 |
| Fabp5 | 0.6233 | 7.18E-55 | 2.9667 |
| Cd36 | 0.7600 | 3.03E-85 | 2.9174 |
| Vat1 | 0.7467 | 7.57E-82 | 2.8682 |
| Myof | 0.7600 | 3.03E-85 | 2.8483 |
| Plet1 | 0.7567 | 2.19E-84 | 2.7934 |
| Hvcn1 | 0.6967 | 5.32E-70 | 2.7689 |
| Fth-ps3 | 0.6933 | 2.94E-69 | 2.7627 |
| Lima1 | 0.6533 | 9.21E-61 | 2.7359 |
| Itgax | 0.7567 | 2.19E-84 | 2.7210 |
| Card11 | 0.6367 | 1.93E-57 | 2.6520 |
| Mir692-2 | 0.7567 | 2.19E-84 | 2.5974 |
| Ctsd | 0.8167 | 5.70E-101 | 2.5402 |
| Klhdc4 | 0.6500 | 4.35E-60 | 2.5367 |
| Mir692-1 | 0.6500 | 4.35E-60 | 2.4553 |
Table 4
Top 35 differentially expressed genes in the Mac IV cluster.
ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 35 upregulated genes are shown for the Mac IV cluster; n = 2 mice.
| Mac IV cluster top 35 differentially expressed genes | |||
|---|---|---|---|
| Gene name | Statistic | P value | Log2 fold change |
| Fcrls | 0.7367 | 2.28E-79 | 12.2139 |
| Pf4 | 0.7900 | 2.70E-93 | 11.4117 |
| Gas6 | 0.6433 | 9.38E-59 | 9.6283 |
| C1qc | 0.8733 | 3.18E-119 | 9.1247 |
| Ccl12 | 0.6400 | 4.28E-58 | 8.7381 |
| C1qa | 0.8700 | 4.58E-118 | 8.3676 |
| Stab1 | 0.8033 | 4.55E-97 | 7.6076 |
| C1qb | 0.8600 | 1.17E-114 | 7.0450 |
| Sdc4 | 0.7200 | 2.32E-75 | 6.3860 |
| Igfbp4 | 0.7433 | 5.15E-81 | 6.2878 |
| Ms4a7 | 0.7200 | 2.32E-75 | 6.0618 |
| Slc40a1 | 0.6033 | 3.72E-51 | 6.0358 |
| Tmem176b | 0.8600 | 1.17E-114 | 5.5402 |
| Maf | 0.7500 | 1.10E-82 | 5.4450 |
| S1pr1 | 0.6367 | 1.93E-57 | 5.3458 |
| Igf1 | 0.6700 | 3.28E-64 | 4.8862 |
| Tmem37 | 0.5900 | 9.02E-49 | 4.8077 |
| Ophn1 | 0.5933 | 2.32E-49 | 4.6672 |
| Tmem176a | 0.7167 | 1.41E-74 | 4.6367 |
| C3ar1 | 0.7100 | 4.98E-73 | 4.5725 |
| Smagp | 0.6700 | 3.28E-64 | 4.5052 |
| Cxcl16 | 0.5800 | 4.98E-47 | 4.2439 |
| Sesn1 | 0.5633 | 3.27E-44 | 4.0183 |
| Apoe | 0.7600 | 3.03E-85 | 4.0149 |
| Hpgds | 0.6333 | 8.62E-57 | 3.9920 |
| Lgmn | 0.7267 | 6.04E-77 | 3.9551 |
| Ms4a4a | 0.5667 | 9.10E-45 | 3.5567 |
| Selenop | 0.6200 | 3.07E-54 | 3.4452 |
| Cx3cr1 | 0.6000 | 1.49E-50 | 3.3845 |
| Abca1 | 0.5433 | 5.69E-41 | 3.2834 |
| Tcf4 | 0.5433 | 5.69E-41 | 3.1404 |
| Blvrb | 0.5533 | 1.42E-42 | 3.1103 |
| Aif1 | 0.6933 | 2.94E-69 | 3.0826 |
| Abca9 | 0.5833 | 1.32E-47 | 3.0325 |
| Pea15a | 0.5400 | 1.91E-40 | 3.0198 |
Table 5
Top 35 differentially expressed genes in the Mac V cluster.
ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 35 upregulated genes are shown for the Mac V cluster; n = 2 mice.
| Mac V cluster top 35 differentially expressed genes | |||
|---|---|---|---|
| Gene name | Statistic | P value | Log2 fold change |
| I830127L07Rik | 0.4867 | 1.42E-32 | 7.0634 |
| Ifitm6 | 0.8800 | 1.41E-121 | 6.9362 |
| Pglyrp1 | 0.6867 | 8.67E-68 | 6.2150 |
| Serpinb10 | 0.5333 | 2.09E-39 | 6.1537 |
| Plac8 | 0.9600 | 6.02E-155 | 5.9647 |
| Adgre4 | 0.6033 | 3.72E-51 | 5.8672 |
| Nxpe4 | 0.5000 | 1.89E-34 | 4.8441 |
| Ifitm3 | 0.9267 | 1.17E-139 | 4.6727 |
| Plbd1 | 0.7267 | 6.04E-77 | 3.9525 |
| Susd3 | 0.5033 | 6.29E-35 | 3.9210 |
| Hp | 0.6333 | 8.62E-57 | 3.8550 |
| Pmaip1 | 0.4767 | 3.29E-31 | 3.7016 |
| Adgre5 | 0.7500 | 1.10E-82 | 3.5335 |
| Ifi27l2a | 0.5667 | 9.10E-45 | 3.3416 |
| Atp1a3 | 0.4900 | 4.88E-33 | 3.3206 |
| Ifitm2 | 0.7600 | 3.03E-85 | 3.2359 |
| Xdh | 0.4833 | 4.07E-32 | 3.2003 |
| Pou2f2 | 0.4733 | 9.21E-31 | 3.1216 |
| Rnase6 | 0.5067 | 2.07E-35 | 3.0629 |
| Pla2g7 | 0.6800 | 2.43E-66 | 3.0556 |
| Thbs1 | 0.5767 | 1.86E-46 | 3.0133 |
| Itga4 | 0.6600 | 3.98E-62 | 2.9055 |
| Itgam | 0.5500 | 4.91E-42 | 2.7702 |
| Napsa | 0.6000 | 1.49E-50 | 2.6112 |
| Sorl1 | 0.5033 | 6.29E-35 | 2.5328 |
| Wfdc17 | 0.4967 | 5.64E-34 | 2.5299 |
| Clec4a1 | 0.5300 | 6.81E-39 | 2.4922 |
| Slc11a1 | 0.5267 | 2.20E-38 | 2.4649 |
| Fyb | 0.6400 | 4.28E-58 | 2.4454 |
| Srgn | 0.6633 | 8.14E-63 | 2.2151 |
| Cnn2 | 0.4933 | 1.67E-33 | 2.1880 |
| Samhd1 | 0.6167 | 1.30E-53 | 2.1715 |
| Lst1 | 0.6667 | 1.65E-63 | 2.1340 |
| Cd300a | 0.5100 | 6.78E-36 | 2.1007 |
| Nr4a1 | 0.6100 | 2.24E-52 | 2.0001 |
Table 6
Top 20 differentially expressed genes in the Mig-DC vs cDC2 cluster.
ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 20 differentially expressed genes are shown for the migDC vs cDC2 cluster; n = 2 mice.
| Mig-DC versus cDC2 cluster top 20 differentially expressed genes | |||
|---|---|---|---|
| Gene name | Statistic | P. value | Log2 fold change |
| Ccl5 | 1.0000 | 3.45E-13 | 16.9849 |
| Il12b | 0.7857 | 6.97E-07 | 15.3906 |
| Fscn1 | 1.0000 | 3.45E-13 | 14.5028 |
| Nudt17 | 0.9286 | 2.48E-10 | 13.7990 |
| Zmynd15 | 0.9286 | 2.48E-10 | 13.5523 |
| Il15ra | 0.9286 | 2.48E-10 | 13.5174 |
| Cacnb3 | 1.0000 | 3.45E-13 | 13.4891 |
| Cd63-ps | 1.0000 | 3.45E-13 | 13.4587 |
| H2-M2 | 0.8571 | 2.06E-08 | 13.4381 |
| Tnfrsf4 | 0.7143 | 1.31E-05 | 13.4073 |
| Serpinb6b | 0.9286 | 2.48E-10 | 13.2991 |
| AW112010 | 0.8571 | 2.06E-08 | 13.1876 |
| Laptm4b | 0.8571 | 2.06E-08 | 12.9752 |
| Lad1 | 0.7143 | 1.31E-05 | 12.8948 |
| Cd63 | 1.0000 | 3.45E-13 | 12.8680 |
| Dnase1l3 | 0.7143 | 1.31E-05 | 12.7389 |
| Apol7c | 0.5000 | 7.75E-03 | 12.7302 |
| Tm4sf5 | 0.5000 | 7.75E-03 | 12.6086 |
| Spint2 | 0.9286 | 2.48E-10 | 12.2644 |
| Zfp872 | 0.6429 | 1.57E-04 | 11.4802 |
Table 7
Top 20 differentially expressed genes in the Mig-DC vs cDC1 cluster.
ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 20 differentially expressed genes are shown for the mig-DC vs cDC1 cluster; n = 2 mice.
| Mig-DC versus cDC1 cluster top 20 differentially expressed genes | |||
|---|---|---|---|
| Gene name | Statistic | P. value | Log2 fold change |
| H2-M2 | 0.8571 | 2.34E-09 | 14.0574 |
| Tnfrsf4 | 0.7143 | 3.71E-06 | 13.6282 |
| Zfp872 | 0.6429 | 5.96E-05 | 11.348 |
| Arc | 0.7143 | 3.71E-06 | 10.9369 |
| Gbp8 | 0.6429 | 5.96E-05 | 10.6692 |
| Ramp3 | 0.5 | 4.51E-03 | 10.6263 |
| Insl6 | 0.6429 | 5.96E-05 | 10.5787 |
| Gbp4 | 0.9286 | 1.19E-11 | 10.4833 |
| Adcy6 | 0.8201 | 2.13E-08 | 10.4176 |
| Atxn1 | 0.7143 | 3.71E-06 | 10.3352 |
| Ankrd33b | 0.9286 | 1.19E-11 | 10.1242 |
| Dscaml1 | 0.6429 | 5.96E-05 | 9.8945 |
| Src | 0.5 | 4.51E-03 | 9.7618 |
| Tmem150c | 0.6429 | 5.96E-05 | 9.566 |
| Slc4a8 | 0.7116 | 4.01E-06 | 9.5518 |
| Nudt17 | 0.9101 | 6.10E-11 | 9.5137 |
| Il21r | 0.6958 | 7.81E-06 | 9.3355 |
| Fas | 0.6243 | 1.12E-04 | 9.3023 |
| Clec2i | 0.5 | 4.51E-03 | 9.2653 |
| Cacnb3 | 0.9815 | 2.44E-13 | 8.6751 |
Table 8
Top 20 differentially expressed genes in the cDC2 vs cDC1 cluster.
ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 20 differentially expressed genes are shown for the cDC2 vs cDC1 cluster; n = 2 mice.
| cDC2 versus cDC1 cluster top 20 differentially expressed genes | |||
|---|---|---|---|
| Gene name | Statistic | P. value | Log2 fold change |
| Mgl2 | 0.7857 | 5.55E-15 | 12.5236 |
| Cd209d | 0.5476 | 4.76E-07 | 12.1889 |
| Cd209b | 0.3333 | 7.77E-03 | 12.1445 |
| Ms4a6d | 0.5714 | 1.14E-07 | 11.7772 |
| Ms4a4c | 0.5476 | 4.76E-07 | 11.2462 |
| Plaur | 0.3810 | 1.42E-03 | 11.0707 |
| Casp4 | 0.3810 | 1.42E-03 | 10.0788 |
| Il1rl1 | 0.6190 | 5.17E-09 | 10.0357 |
| Cd300lg | 0.3333 | 7.77E-03 | 9.9463 |
| Cd33 | 0.4762 | 2.21E-05 | 9.7976 |
| Emilin2 | 0.4048 | 5.52E-04 | 9.5336 |
| Il1rl2 | 0.3810 | 1.42E-03 | 9.5332 |
| Creb5 | 0.3915 | 9.39E-04 | 9.2747 |
| Cd209c | 0.4762 | 2.21E-05 | 9.0161 |
| Cd209a | 0.9577 | 3.33E-16 | 9.0160 |
| Ccl9 | 0.6772 | 7.43E-11 | 9.0010 |
| Fcrls | 0.3810 | 1.42E-03 | 8.9226 |
| Il18rap | 0.3915 | 9.39E-04 | 8.7118 |
| Wfdc17 | 0.7249 | 1.46E-12 | 8.5219 |
| Tent5a | 0.6534 | 4.52E-10 | 8.2999 |
Table 9
Top 25 differentially expressed genes in the mast cell cluster.
ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 25 upregulated genes are shown for the mast cell cluster; n = 2 mice.
| Mast cell top 25 differentially expressed genes | |||
|---|---|---|---|
| Gene name | Statistic | P. value | Log2 fold change |
| Tpsb2 | 1.0000 | 8.9E-16 | 18.7534 |
| Mcpt4 | 0.8750 | 2.4E-07 | 17.4253 |
| Tpsab1 | 0.8750 | 2.4E-07 | 15.5899 |
| Cma1 | 1.0000 | 8.9E-16 | 15.0710 |
| Slc45a3 | 0.7500 | 5.2E-05 | 11.7029 |
| Rprm | 0.8750 | 2.4E-07 | 11.6969 |
| Hs6st2 | 0.7500 | 5.2E-05 | 11.1665 |
| Maob | 0.8683 | 3.4E-07 | 10.7834 |
| Cpa3 | 0.9900 | 1.8E-13 | 10.6394 |
| Ednra | 0.7500 | 5.2E-05 | 10.2131 |
| Gata1 | 0.7500 | 5.2E-05 | 10.0639 |
| Kit | 1.0000 | 8.9E-16 | 9.2771 |
| Mlph | 0.7467 | 5.9E-05 | 8.5668 |
| Cma2 | 0.7500 | 5.2E-05 | 8.5455 |
| Cyp11a1 | 0.9933 | 4.9E-14 | 8.4308 |
| Tph1 | 0.8717 | 2.8E-07 | 8.3091 |
| Rab27b | 0.8717 | 2.8E-07 | 8.1436 |
| Il1rl1 | 0.9733 | 1.4E-11 | 7.7399 |
| Itga2b | 0.8350 | 1.8E-06 | 7.6658 |
| Gata2 | 0.9933 | 4.9E-14 | 7.5597 |
| Poln | 0.7367 | 8.2E-05 | 7.2024 |
| Slc18a2 | 0.9600 | 1.4E-10 | 6.8570 |
| Stard13 | 0.7467 | 5.9E-05 | 6.7772 |
| Adora3 | 0.7033 | 2.3E-04 | 6.4180 |
| Kcne3 | 0.8050 | 6.6E-06 | 6.4033 |
Table 10
Top 25 differentially expressed genes in the basophil cluster.
ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 25 upregulated genes are shown for the basophil cluster; n = 2 mice.
| Basophil top 25 differentially expressed genes | |||
|---|---|---|---|
| Gene name | Statistic | P. value | Log2 fold change |
| Mcpt8 | 0.7347 | 2.22E-16 | 15.6663 |
| Ms4a2 | 0.8676 | 2.22E-16 | 12.3460 |
| Gata2 | 0.9084 | 2.22E-16 | 9.7841 |
| Il4 | 0.6872 | 2.22E-16 | 8.7980 |
| Cd200r3 | 0.9525 | 2.22E-16 | 8.6767 |
| Il6 | 0.8309 | 2.22E-16 | 8.5795 |
| Ccl4 | 0.9021 | 2.22E-16 | 7.0748 |
| Ifitm1 | 0.9363 | 2.22E-16 | 6.8128 |
| Cyp11a1 | 0.9729 | 2.22E-16 | 6.6271 |
| Ccl3 | 0.9225 | 2.22E-16 | 5.9632 |
| Hgf | 0.7326 | 2.22E-16 | 5.7653 |
| Csf1 | 0.8921 | 2.22E-16 | 5.4838 |
| Ccl9 | 0.8825 | 2.22E-16 | 5.3108 |
| Ifitm7 | 0.7559 | 2.22E-16 | 5.1284 |
| Aqp9 | 0.7563 | 2.22E-16 | 5.0857 |
| Rab44 | 0.6534 | 2.22E-16 | 5.0335 |
| Hdc | 0.9133 | 2.22E-16 | 4.9976 |
| Cd69 | 0.7454 | 2.22E-16 | 4.9828 |
| Cdh1 | 0.7197 | 2.22E-16 | 4.9643 |
| Il18r1 | 0.6539 | 2.22E-16 | 4.5920 |
| Csf2rb2 | 0.6930 | 2.22E-16 | 4.3472 |
| Lilr4b | 0.8788 | 2.22E-16 | 4.1383 |
| Rgs1 | 0.7001 | 2.22E-16 | 4.0712 |
| Il18rap | 0.7571 | 2.22E-16 | 4.0030 |
| Osm | 0.6559 | 2.22E-16 | 3.6933 |
Table 11
Top 25 differentially expressed genes in the neutrophil cluster.
ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 25 upregulated genes are shown for the neutrophil cluster; n = 2 mice.
| Neutrophil top 25 differentially expressed genes | |||
|---|---|---|---|
| Gene name | Statistic | P. value | Log2 fold change |
| Retnlg | 0.7833 | 1.9E-91 | 16.7841 |
| Stfa2 | 0.9067 | 1.6E-131 | 13.3347 |
| BC100530 | 0.9033 | 3.1E-130 | 11.5407 |
| Mmp9 | 0.8867 | 5.5E-124 | 11.2071 |
| Asprv1 | 0.7500 | 1.1E-82 | 10.6669 |
| Stfa2l1 | 0.9300 | 4.5E-141 | 8.5427 |
| S100a9 | 0.9567 | 2.7E-153 | 8.3546 |
| 2010005H15Rik | 0.8467 | 2.9E-110 | 8.1305 |
| BC117090 | 0.8800 | 1.4E-121 | 7.8635 |
| Slpi | 0.8767 | 2.1E-120 | 7.5069 |
| Stfa1 | 0.8967 | 1.1E-127 | 6.8526 |
| Il1r2 | 0.7133 | 8.4E-74 | 6.6465 |
| S100a8 | 0.9467 | 1.6E-148 | 6.2103 |
| Slc7a11 | 0.6933 | 2.9E-69 | 5.9640 |
| Csf3r | 0.7400 | 3.5E-80 | 5.7411 |
| Cxcr2 | 0.7433 | 5.2E-81 | 5.6599 |
| Wfdc21 | 0.7733 | 9.5E-89 | 5.3370 |
| Pglyrp1 | 0.8367 | 4.4E-107 | 4.9416 |
| Mxd1 | 0.6800 | 2.4E-66 | 4.5560 |
| Hdc | 0.8267 | 5.5E-104 | 4.2867 |
| Grina | 0.7033 | 1.7E-71 | 4.0795 |
| Il1b | 0.6567 | 1.9E-61 | 3.8434 |
| Thbs1 | 0.6733 | 6.5E-65 | 2.7913 |
| S100a11 | 0.7600 | 3.0E-85 | 2.6263 |
| Srgn | 0.6600 | 4.0E-62 | 2.3914 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (M. musculus) | C57BL/6J | Charles Rivers Laboratories | ||
| Antibody | anti-CD3e (Hamster monoclonal) | BD Biosciences | Cat# 553062; RRID:AB_394595 | FACS (1:100) |
| Antibody | anti-CD4 (Rat monoclonal) | BD Biosciences | Cat# 552051; RRID:AB_394331 | FACS (1:100) |
| Antibody | anti- CD8a (Rat monoclonal) | BioLegend | Cat# 100721; RRID:AB_312760 | FACS (1:100) |
| Antibody | anti-Tbet (Mouse monoclonal) | BioLegend | Cat# 644808; RRID:AB_15955479 | FACS (1:100) |
| Antibody | anti-GATA3 (Mouse monoclonal) | BD Biosciences | Cat# 563510; RRID:AB_2738248 | FACS (1:100) |
| Antibody | anti-Foxp3 (Rat monoclonal) | eBioscience | Cat# 12-5773-82; RRID:AB_465936 | FACS (1:100) |
| Antibody | anti-Rorγt (Mouse monoclonal) | BD Biosciences | Cat# 562607; RRID:AB_11153137 | FACS (1:100) |
| Antibody | anti-IFNγ (Rat monoclonal) | BioLegend | Cat# 505829; RRID:AB_10897937 | FACS (1:100) |
| Antibody | anti-IL-4 (Rat monoclonal) | BD Biosciences | Cat# 562045; RRID:AB_10895799 | FACS (1:100) |
| Antibody | IL-10 (Rat monoclonal) | BioLegend | Cat# 505027; RRID:AB_2561522 | FACS (1:100) |
| Antibody | IL-17 (Rat monoclonal) | Miltenyi Biotec | Cat# 130-095-732; RRID:AB_10828821 | FACS (1:100) |
| Commercial assay or kit | FISH probe: anti-mouse Plac8 | Advanced Cell Diagnostics (ADC) | Cat# 532701 | RNAscope: 50 μl per slide |
| Commercial assay or kit | FISH probe: anti-mouse Dab2 | ADC | Cat# 558131-C2 | RNAscope: (1 μl per 50 μl) |
| Commercial assay or kit | FISH probe: anti-mouse Cd68 | ADC | Cat# 316611-C2 | RNAscope: (1 μl per 50 μl) |
| Commercial assay or kit | FISH probe: anti-mouse-Cd68 | ADC | Cat# 316611-C3 | RNAscope: (1 μl per 50 μl) |
| Commercial assay or kit | FISH probe: anti-mouse-Cd68 | ADC | Cat# 316611 | RNAscope: 50 μl per slide |
| Commercial assay or kit | FISH probe: anti-mouse-Cdh5 | ADC | Cat# 312531-C2 | RNAscope: (1 μl per 50 μl) |
| Commercial assay or kit | FISH probe: anti-mouse-Mki67 | ADC | Cat# 416771-C2 | RNAscope: (1 μl per 50 μl) |
| Commercial assay or kit | FISH probe: anti-mouse-Car4 | ADC | Cat# 468421-C2 | RNAscope: (1 μl per 50 μl) |
| Commercial assay or kit | FISH probe: anti-mouse-Gal | ADC | Cat# 400961-C2 | RNAscope: (1 μl per 50 μl) |
| Commercial assay or kit | FISH probe: anti-mouse-C1qa | ADC | Cat# 441221 | RNAscope: 50 μl per slide |
| Commercial assay or kit | FISH probe: anti-mouse-Epcam | ADC | Cat# 418151-C2 | RNAscope: (1 μl per 50 μl) |
| Commercial assay or kit | FISH probe: anti-mouse-Itgax | ADC | Cat# 311501-C3 | RNAscope: (1 μl per 50 μl) |
| Commercial assay or kit | FISH probe: anti-mouse-Ifitm6 | ADC | Cat# 511321-C3 | RNAscope: (1 μl per 50 μl) |
| Commercial assay or kit | FISH probe: anti-mouse-Lair1 | ADC | Cat# 509151 | RNAscope: 50 μl per slide |
| Commercial assay or kit | FISH probe: anti-mouse-Ccr2 | ADC | Cat# 501681 | RNAscope: 50 μl per slide |
