Distinct roles for SOX2 and SOX21 in differentiation, distribution and maturation of pulmonary neuroendocrine cells

Abstract

Pulmonary neuroendocrine (NE) cells represent a small population in the airway epithelium, but despite this, hyperplasia of NE cells is associated with several lung diseases, such as congenital diaphragmatic hernia and bronchopulmonary dysplasia. The molecular mechanisms causing the development of NE cell hyperplasia remains poorly understood. Previously, we showed that the SOX21 modulates the SOX2-initiated differentiation of epithelial cells in the airways. Here, we show that precursor NE cells start to develop in the SOX2 + SOX21 + airway region and that SOX21 suppresses the differentiation of airway progenitors to precursor NE cells. During development, clusters of NE cells start to form and NE cells mature by expressing neuropeptide proteins, such as CGRP. Deficiency in SOX2 resulted in decreased clustering, while deficiency in SOX21 increased both the numbers of NE ASCL1 + precursor cells early in development, and the number of mature cell clusters at E18.5. In addition, at the end of gestation (E18.5), a number of NE cells in Sox2 heterozygous mice, did not yet express CGRP suggesting a delay in maturation. In conclusion, SOX2 and SOX21 function in the initiation, migration and maturation of NE cells.

Keywords: Lung development; Neuroendocrine cells; SOX2; SOX21.

Fig. 1

Differentiation to NE cells of SOX2 + airway progenitor cells occurs in SOX21 + airway region. A Immunofluorescence staining of ASCL1 (red) and DAPI (blue) at gestational ages E13.5 and E14.5. C = cluster, S = solitary. Scale bar = 50 µm. B Immunofluorescence staining of ASCL1 (red), SOX21 (green) and SOX2 (blue) at gestational age E13.5 and E14.5. Cells positive for ASCL1 are encircled. Scale bar = 25 µm. C Immunofluorescence staining of ASCL1 (red), SOX21 (green) and SOX2 (blue) at gestational age E14.5. In the encircled cells the MFI of ASCL1, SOX21 and SOX2 is shown at that specific region. Scale bar = 25 µm. The graph shows the MFI of ASCL1, SOX21 and SOX2 in ASCL- and ASCL + airway epithelium. Data are represented as mean ± SD. Two-way ANOVA (n = 3, *** p < 0.001). D Immunofluorescence staining of TRP63 (violet), ASCL1 (red), SOX21 (green) and DAPI (blue) at gestational age E14.5. Cells positive for TRP63 are encircled. Scale bar = 25 µm. The graph shows the MFI of TRP63, SOX21 and SOX2 in TRP63- and TRP63 + airway epithelium. Data are represented as mean ± SD. Two-way ANOVA (n = 3, *** p < 0.001)

Fig. 2

Loss of SOX21 increases differentiation of SOX2 + airway progenitor cell to NE precursor cells. A Immunofluorescence staining of ASCL1 (red) on the first bifurcation of the main bronchus at gestational age E14.5 of wild-type (WT), Sox2^+/−, Sox21^+/− and Sox21^−/− mice. B Quantification of the percentage of NE precursor cells in a box of 400 µm² around the first bifurcation of the main bronchus. Data are represented as mean ± SEM. One-way ANOVA (n WT = 5, n Sox2^+/−  = 4, Sox21^+/−  = 3, Sox21^−/− = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 3

Different abundancy of SOX2 and SOX21 during the maturation of NEBs. A Immunofluorescence staining of SV2 (violet), ASCL1 (red), CGRP (green) and DAPI (blue) at gestational ages E15.5, E16.5 and E18.5. Scale bar = 50 µm. B Immunofluorescence staining of ASCL1 (red), SOX21 (green) and SOX2 (blue) at gestational age E15.5, E16.5 and E18.5. The proximal airway epithelium is characterized by abundant expression of SOX21. Cells positive for ASCL1 are encircled. Scale bar = 25 µm. C The graph shows the MFI of ASCL1, SOX21 and SOX2 in ASCL- and ASCL + airway epithelium in proximal airway epithelium at E15.5, E16.5 and E18.5. Data are represented as mean ± SD. Two-way ANOVA (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001). D Immunofluorescence staining of ASCL1 (red), SOX21 (green) and SOX2 (blue) at gestational age E15.5, E16.5 and E18.5 in the distal SOX2 + SOX21- airway epithelium. Cells positive for ASCL1 are encircled. Scale bar = 25 µm. E Schematic representation of SOX2 and SOX21 protein levels in neuroendocrine bodies (NEBs) at different gestational ages. SOX21 shows a dip and SOX2 a slight decrease in expression at E16.5, when CGRP expression starts and NEBs become innervated (SV2)

Fig. 4

Loss of SOX2 decreases and of SOX21 increases the number NE cells at E18.5. A Immunofluorescence staining of CGRP (green), SOX2 (RED) and DAPI (blue) on lung sections at gestational age E18.5 of wild-type (WT), Sox2^+/−, Sox21^+/− and Sox21^−/− mice. Scale bar = 200 µm. B Quantification of the number of NE cells on E18.5 lung sections, normalized to the area of SOX2 + area measured. Data are represented as mean ± SEM. One-way ANOVA on ASCL1 + cells (left) (n WT = 4, n Sox2^+/−  = 4, Sox21^+/−   = 4, Sox21^−/− = 3), One-way ANOVA on CGRP + cells (right) (n WT = 7, n Sox2^+/−  = 7, Sox21^+/−  = 7, Sox21^−/− = 6) (*p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001). C Immunofluorescence staining of ASCL1 (red), CGRP (green) and SOX2 (blue) on lung sections at gestational age E18.5 of wild-type (WT), Sox2^+/−, Sox21^+/− and Sox21^−/− mice. Scale bar = 50 µm

Fig. 5

Loss of SOX2 decreases and of SOX21 increases the number large NE clusters. A Immunofluorescence staining of CGRP (green) and SV2 (RED) on lung sections at gestational age E18.5 of wild-type (WT), Sox2^+/−, Sox21^+/− and Sox21^−/− mice. Scale bar = 15 µm. B The percentage of CGRP + NE cells that are present as single NE cells, small clusters (2–5 cells) or large clusters (> 5). Data are represented as mean ± SEM. Two-way ANOVA (n WT = 7, n Sox2^+/−  = 7, Sox21^+/−  = 7, Sox21^−/− = 6, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). C Representation of the average number of CGRP + NE cells in clusters > 5. Data are represented as mean ± SEM. One- way ANOVA (n WT = 7, n Sox2^+/−  = 3 (4 did not show any clusters > 5), Sox21^+/−  = 7, Sox21^−/− = 6)