Fig 2: CXCL13-positive and neuroendocrine cells are unevenly distributed in the trachea. a–d Tracheal whole mount immunohistochemistry, CLSM, with antibodies against CXCL13 (green) and CGRP (red), labeling single neuroendocrine cells and nerve fibers. The density of neuroendocrine (CGRP-positive) cells, CXCL13-immunoreactive cells, and nerve fibers is higher between the cartilage rings (Ca). The density of neuroendocrine cells and CXCL13-immunoreactive cells decreases from cranial (larynx) (b) to caudal (bifurcation) (d); each channel is individually shown in Supplementary Fig. 1. Numerous CGRP-positive nerve fibers ( <) are visible throughout the whole trachea. Maximum intensity projection of z-stack of confocal optical sections. e, f Cell densities (mean ± SEM) quantified on tracheal whole mounts double-labelled for CXCL13 and PGP9.5 (neuroendocrine cell marker). Immunoreactive cells dominate in intercartilage regions (e), and their density continuously declines along the cranio-caudal axis (f). In f, counts include both the area overlying a cartilage and the next intercartilage region; colour coding along the cranio-caudal axis identifies data from the same trachea. g RT-qPCR. Cxcl13 expression is about 3 times higher in tracheal rings 1–3 compared to rings 8–10

Fig 3: In silico analysis of single-cell mRNA sequencing data revealed CXCL13 expression predominantly in neuroendocrine cells of the tracheal epithelium. a–c In silico analysis of published sequencing data (GSE102580) of murine tracheal epithelial cells (Plasschaert et al. 2018). a SPRING plot (Uniform Manifold Approximation and Projection, UMAP) shows eight distinct cell clusters, namely basal, secretory, Krt4/13+, ciliated, solitary cholinergic chemosensory (brush/tuft), cycling basal and solitary neuroendocrine cells, and ionocytes. b SPRING and violin plots showing that Cxcl13-mRNA is predominantly expressed within the neuroendocrine cell cluster. c Heat map shows the most differentially expressed genes (fold change > 1) between CXCL13+ and CXCL13- neuroendocrine cells among the 200 highest expressed genes within the neuroendocrine cell cluster and typical neuroendocrine cell marker genes (e.g., Ascl1, Uchl1, Calca, Chga, Chgb)

Fig 4: CXCL13 is restricted to neuroendocrine cells in the tracheal epithelium. a, b High-resolution of tracheal whole mount immunohistochemistry, labeling CXCL13+ cells in green and CGRP+ cells and nerve fibers in red, revealed CXCL13 and CGRP colocalization within the same cell. Inset in a shows the magnified region of the labeled cell with only limited overlap of immunoreactivites (scale bar 1 µm). Images were acquired using a confocal laser scanning microscope (FLUOVIEW FV3000; Olympus), single confocal optical section. b CXCL13+ cell in contact to a CGRP+ nerve fiber ( <). Maximum intensity projection of z-stack of confocal optical sections. c Immunohistochemistry of a tracheal cryosection from a ChAT-eGFP animal. In the tracheal epithelium, single cells are double-stained with antibodies against PGP9.5 and CXCL13 (arrowhead) or PGP9.5 only (arrow). An eGFP-positive cell (asterisk) is not labeled with antibodies against CXCL13 or PGP9.5. PGP9.5+ nerve fibers are in contact to PGP9.5+ epithelial cells. d Triple-immunofluorescence of tracheal cryosection shows co-labeling of single epithelial cells for CXCL13 (green), CGRP (yellow), and PGP9.5 (red) (arrowheads). A single PGP9.5-labeled cell with neither CXCL13 nor CGRP immunolabeling is also present (asterisk). e–f Transmission electron microscopy. e Ultrastructure of a tracheal neuroendocrine cell (NEC) with a pyramidal or flask-like shape and a small apical part reaching the lumen with microvilli. e' Higher magnification of the basal part, showing the presence of numerous dense core vesicles (DCV). f Ultrastructural immunohistochemistry with antibodies against CXCL13 shows an immunoreactive cell with the diffuse DAB reaction product. f' Higher magnification of the basal part, showing the presence of numerous DCV

Fig 5: Cxcl13- and Calca-mRNAs are present in the murine tracheal epithelium. RT-PCR experiments with cDNA obtained from tracheal epithelium of two C57BL/6RJ animals using primers for Cxcl13 (250 bp), Calca (158 bp), and ß-actin (300 bp). Amplicons of Calca, Cxcl13, and ß-actin are detected in both tracheal epithelium samples (TE). Controls = RNA samples processed without reverse transcriptase (-RT) and water (H2O) without adding cDNA