Figure 4. Characterization of neonatal cilia-secretory hybrid cells.

(A) Violin plots showing the expression levels of ciliated cell markers ​Foxj1​, ​Prr18,​ ​and Cdhr3​, as well as secretory cell markers ​Creb3l1,​ ​Spdef,​ and ​Gp2​ in three cell clusters: ciliated cells, secretory cells, and cilia-secretory hybrids. Gene expression has been normalized and log-transformed. (B) RNA FISH analysis of Foxj1​ (magenta), ​Gp2​ (blue), and ​Prr18​ (yellow) mRNA in P3 wild-type trachea. Hybrid cells expressing all three markers are indicated by dashed circles. Foxj1⁺ ​ ​ Prr18⁺ ​ciliated cells are indicated by arrowheads. Gp2⁺ secretory cells are indicated by arrows. Nuclei are marked by DAPI (grey). Scale bar indicates 20 ​μ​m. (C) Fluorescent immunostaining of FOXJ1 (red) and SCGB1A1 (red) in P3 wild-type trachea. Hybrid cells expressing both markers are indicated by dashed circles. FOXJ1⁺ ​ciliated cells are indicated by arrowheads. SCGB1A1⁺ ​secretory cells are indicated by arrows. Cell membranes are marked by E-cadherin (grey). Scale bar indicates 20 ​μ​m. (D) Transmission electron microscopy (TEM) images of P0 tracheal epithelial cells. Intracellular vesicles are indicated by arrows in yellow. Motile cilia are indicated by arrowheads in blue. Scale bar corresponds to 2 ​μ​m. (E) scGPS of disease-associated genes curated from the Online Mendelian Inheritance in Man (OMIM) database. Each dot represents a cell. Colors indicate cell clusters. Full lists of genes used are in Figure 4—source data 1.

Figure 4—figure supplement 1. ​ Characterization of cilia-secretory hybrid cells.

(A) Upper panel: Immunostaining of acetylated-​α ​tubulin (red), which labels motile cilia of ciliated cells, shows abundant ciliated cells with motile cilia in Pofut^-/- mutants compared to littermate controls. Cell membrane is marked by E-cad (green). Lower panel: RNA FISH analysis shows mRNAs of ​Foxj1​ (red) and ​Gp2 (green)​ double positive cells, highlighted by dashed circle, were present in both genotypes. Note that Pofut^-/- mutants lack cells that are only ​Gp2⁺ ​. Scale bar indicates 20 μm. (B) Foxj1Cre^ERT2-GFP mice were crossed to Rosa26mT/mG mice for lineage tracing of Foxj1⁺ cells. Tamoxifen was injected into pregnant females at E14 to E15 to induce Foxj1-CRE expression. Trachea samples from Foxj1-CRE positive pups with mTomato expression and mGFP were examined at P1. (C) Cytoplasmic expression of SCGB1A1 (magenta) labels secretory cells, and mGFP (green) labels Foxj1⁺ lineage. Because mTomato fluorescence signal was quenched after PFA and methanol fixation, we use E-cadherin to label cell membrane in the red channel and was pseudo-colored in grey. Hybrid cells expressing both markers are indicated by dashed circles. mGFP⁺ green ​ciliated cells are indicated by arrowheads. SCGB1A1⁺​ ​secretory cells are indicated by arrows. Scale bar indicates 20 μ​​m. A quantification of mGFP⁺ green ciliated cells and mGFP⁺; SCGB1A1⁺ double positive yellow hybrid cells in P1 trachea (n = 3) was included. Error bars represent S.D. (D) Dot plot depicting expression patterns of genes implicated in the disease severity of COPD and CF. The size of the dot encodes the percentage of cells expressing the gene, while the color encodes the mean of expression level which has been normalized, log-transformed, and z-score transformed.