1. Cell Biology
  2. Developmental Biology

SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways

  1. Evelien Eenjes
  2. Marjon Buscop-van Kempen
  3. Anne Boerema-de Munck
  4. Gabriela G Edel
  5. Floor Benthem
  6. Lisette de Kreij-de Bruin
  7. Marco Schnater
  8. Dick Tibboel
  9. Jennifer Collins
  10. Robbert J Rottier  Is a corresponding author
  1. Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Netherlands
  2. Department of Cell biology, Erasmus Medical Center, Netherlands
https://doi.org/10.7554/eLife.57325
Share this article
Cite this article
  1. Evelien Eenjes
  2. Marjon Buscop-van Kempen
  3. Anne Boerema-de Munck
  4. Gabriela G Edel
  5. Floor Benthem
  6. Lisette de Kreij-de Bruin
  7. Marco Schnater
  8. Dick Tibboel
  9. Jennifer Collins
  10. Robbert J Rottier
(2021)
SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways
eLife 10:e57325.
https://doi.org/10.7554/eLife.57325
  2. Download BibTeX
  3. Download .RIS

Abstract

SOX2 expression levels are crucial for the balance between maintenance and differentiation of airway progenitor cells during development and regeneration. Here, we describe patterning of the mouse proximal airway epithelium by SOX21, which coincides with high levels of SOX2 during development. Airway progenitor cells in this SOX2+/SOX21+ zone show differentiation to basal cells, specifying cells for the extrapulmonary airways. Loss of SOX21 showed an increased differentiation of SOX2+ progenitor cells to basal and ciliated cells during mouse lung development. We propose a mechanism where SOX21 inhibits differentiation of airway progenitors by antagonizing SOX2-induced expression of specific genes involved in airway differentiation. Additionally, in the adult tracheal epithelium, SOX21 inhibits basal to ciliated cell differentiation. This suppressing function of SOX21 on differentiation contrasts SOX2, which mainly drives differentiation of epithelial cells during development and regeneration after injury. Furthermore, using human fetal lung organoids and adult bronchial epithelial cells, we show that SOX2+/SOX21+ regionalization is conserved. Lastly, we show that the interplay between SOX2 and SOX21 is context and concentration dependent leading to regulation of differentiation of the airway epithelium.

Introduction

The lungs are composed of a highly branched tubular system of airways lined with specialized cell types, which together moisten and warm the air and filter out inhaled substances. The alveoli are located at the distal end of the airways. They consist of a thin layer of epithelium encircled by a network of blood vessels to facilitate an exchange of oxygen and carbon dioxide. During lung development, growth and differentiation along the proximal-distal axis occur simultaneously. A well-regulated balance between progenitor cell maintenance, proliferation, and differentiation is essential to ensure fully functional lungs at birth.

The formation of lung endoderm starts from the ventral anterior foregut by the development of two lung buds, forming the left and right lung. As the growing lung buds expand, the future trachea is separated from the esophagus proximally of the lung buds. Through a repetitive process of branching of the growing tips and outgrowth of the newly formed branches, a complex bronchial tree develops (Metzger et al., 2008). Regionalization of the branching structures occurs along the proximal-distal axis. Distal progenitors expressing the SRY-box protein SOX9 and the HLH protein ID2 generate new branches and will ultimately give rise to alveolar cells (Rawlins et al., 2009a). While the buds grow and expand, SOX9+ progenitor cells gradually become more distant from the branch-inducing FGF10 signal secreted by the distal mesenchymal cells, resulting in loss of SOX9 expression and initiation of SOX2 expression (Park et al., 1998; Weaver et al., 1999). These SOX2+ progenitor cells mark the non-branching epithelium and give rise to the airway lineages.

SOX2 is a critical transcription factor in the development of the airway and epithelial lineages. SOX2 deficiency results in aberrant tracheobronchial epithelium due to the loss of basal cells (Rock et al., 2009; Tompkins et al., 2009; Wang et al., 2013). In contrast, SOX2 overexpression results in increased basal cell numbers and perturbed branching morphogenesis (Gontan et al., 2008; Ochieng et al., 2014). The necessity of proper SOX2 levels in airway development is therefore well documented, but it remains unclear how the balance between SOX2+ progenitor maintenance and early cell fate determination is regulated.

Previously, we showed that increased SOX2 expression during mouse lung development leads to increased SOX21 expression in airway epithelium (Gontan et al., 2008). While the function of SOX21 in lung development is not known, SOX21 was found to be enriched in SOX2+ stalk (differentiating bronchiole) versus tip progenitor cells of the human fetal lung (Nikolić et al., 2017). Concomitant expression of SOX2 and SOX21 results in either a synergistic or antagonistic function depending on the tissue or environmental stimuli (Mallanna et al., 2010; Freeman and Daudet, 2012; Whittington et al., 2015; Goolam et al., 2016).

Here, we hypothesized that SOX21 is an important regulator of SOX2+ progenitor maintenance during development and regeneration of airway epithelium. In this study, we demonstrate that during lung development SOX21 marks a region in the proximal-distal patterning of the airway tree. SOX21 defines the epithelium of the airways where SOX2+ progenitor cells differentiate into basal cells. Using SOX2 and SOX21 mutant mice, we show that SOX2 and SOX21 have opposite functions, as SOX21 suppresses and SOX2 promotes the differentiation of airway progenitor cells during lung development. In contrast, transgenic mice with high ectopic expression of either SOX2 or SOX21 during lung development presented both airway branching defects and increased basal cell numbers. In mouse adult lungs, co-expression of SOX2 and SOX21 is maintained in the epithelium of the extrapulmonary airways (trachea and main bronchi), the region where basal cells reside. Adult mouse basal cells with reduced SOX21 levels are more prone to differentiate to ciliated cells, while reduced SOX2 levels decreased differentiation towards ciliated cells. This opposing effect on differentiation is similar to what we observed in airway progenitor cells during lung development. Finally, SOX2 and SOX21 have a conserved expression pattern and function in human airway epithelial cells. In human air-liquid interface (ALI) cultures, both the reduction of SOX21 or SOX2 levels resulted in an increased differentiation to ciliated cells. Taken together, our results demonstrate that the balance of SOX2 and SOX21 expression regulates airway progenitor cell maintenance and differentiation during lung development and regeneration.

Results

SOX2 and SOX21 regionalize the extrapulmonary airways in a proximal–distal pattern during branching morphogenesis

To gain more insight into the interplay between SOX2 and SOX21, we first examined the spatial and temporal distribution of SOX21 in mouse lung epithelium with respect to the SOX2–SOX9 proximal–distal patterning at different stages of lung development.

The earliest expression of SOX21 during lung development was found at gestational age E11.5. A few SOX21-expressing cells were found in the most proximal region of the SOX2+ epithelium (Figure 1A). SOX2 expression, which can be detected at E9.5, therefore precedes SOX21 (Que et al., 2007; Gontan et al., 2008). At E11.5, abundant SOX21 expression was seen in cells of the thymus and esophagus, which also co-expressed SOX2. At E12.5, SOX21 expression became more apparent, but stayed restricted to the proximal region of the SOX2+ epithelium, the trachea, and main bronchi. From E13.5 onwards, SOX21 was expressed throughout the trachea and main bronchi, but was absent in the smaller SOX2+ airways. Labeling from proximal to distal, four zones could be distinguished in the developing lung epithelium (Figure 1A): zone 1, the most proximal zone, comprised the developing trachea and main bronchi, which contain SOX2+SOX21+ airway epithelial cells; zone 2, which contains only SOX2+ airway epithelial cells; zone 3, a transition zone, in which distal SOX9+ progenitors transition into SOX2+ airway progenitors (Mahoney et al., 2014); and zone 4, the most distal part of the lung epithelium containing SOX9+ progenitors (Rawlins et al., 2009a; Figure 1A). SOX21 was never observed in the distal buds, but always in cells that also express SOX2. SOX21 shows different expression levels among cells during the early developmental stages (E12.5–E14.5), in stark contrast to the more homogeneous distribution of SOX2 (Figure 1A). At E18.5, SOX21 is homogeneously expressed in the trachea, heterogeneously expressed in the main bronchi and absent in the intrapulmonary airways (Figure 1A). In summary, SOX21 is expressed specifically in the extrapulmonary airways (trachea and main bronchi) during the formation of the airway tree, which is the most proximal part of the SOX2+ epithelium.

Figure 1 with 1 supplement see all
Download asset Open asset
SOX21 is expressed in the proximal region of the SOX2+ non-branching zone of the airway epithelium.

(A) Co-staining of SOX9 (green) for distal buds, SOX2 (blue) for proximal epithelium, and SOX21 (red) at different stages of lung development. Boxed areas are sown as enlarged inserts. Schematic representations show the distribution of SOX9, SOX2, and SOX21 in the different identified zones in the branching airways during gestation. At E12.5, the asterisks indicate the cells in zone three expressing both SOX2 and SOX9. T = thymus, L = lung, E = esophagus, and C = cartilage. Scale bar = 50 µm. (B) Transversal sections at three different locations of an E14.5 trachea and main bronchi stained with SOX2 (blue), SOX9 (green), SOX21 (red), and smooth muscle actin (SMA) (yellow). Location 1 = trachea, 2 = around the carina, and 3 = immediately distal of the carina. SOX9+ mesenchymal cells surround the ventral side of the trachea and bronchi and SMA+ cells surround the dorsal side. The graphs show the ratio of mean fluorescence intensity (MFI) between ventral and dorsal of SOX2 and SOX21 at the three different locations. Scale bar = 50 µm. Eso = esophagus. (C) Sections of E15.5 lungs of doxycycline induced iSox2SPC-rtTA and iSox21SPC-rtTA. Expression of transgenic Myc-tagged Sox2 or Sox21 was induced by giving doxycycline from E6 onwards. Sections are stained with SOX2 (blue), SOX9 (green), SOX21 (red), and MYC (purple). Scale bar = 50 µm. Db = distal bud.

Figure 1—source data 1

Quantification of the number of cells represented as Dorsal-Ventral ratio of SOX2 and SOX21 positive cells.

https://cdn.elifesciences.org/articles/57325/elife-57325-fig1-data1-v2.xlsx

We also observed a unilateral expression of SOX21 in the main bronchi, starting from the bifurcation of the trachea (the carina) (Figure 1A, Figure 1—figure supplement 1A). SOX21 is abundantly expressed on the medial side of the airway, but almost completely absent on the lateral side (Figure 1—figure supplement 1A). This unilateral expression pattern is present during branching morphogenesis but becomes less apparent after E15.5 (Figure 1—figure supplement 1A). Previously, SOX2 was found to be higher expressed in the dorsal compared to the ventral side of the tracheal epithelium (TE) during development (Que et al., 2009). In the developing trachea, the ventral side is marked by SOX9+ cartilage nodules and the dorsal side by smooth muscle actin+ (SMA+) mesenchymal cells (Hines et al., 2013). We therefore made transverse sections from the trachea up to the main bronchi and observed a more abundant expression of SOX21 on the dorsal side, comparable to the location of high SOX2 levels (Figure 1B). We hypothesize that the high expression of SOX2 is important to set up the expression pattern of SOX21 during lung development.

To validate whether high SOX2 levels can induce Sox21 expression, we ectopically expressed a MYC-tagged SOX2 or -SOX21 in peripheral epithelial lung cells using a doxycycline-inducible transgene and a surfactant protein C (SPC) promoter-driven rtTA (iSox2SPC-rtTA or iSox21SPC-rtTA) (Perl et al., 2002; Gontan et al., 2008; Figure 1—figure supplement 1B). Both MYC-SOX2 and MYC-SOX21 expression caused the appearance of cystic structures, albeit much smaller in the MYC-SOX21 model (Figure 1—figure supplement 1C). Although a similar phenotype was observed, MYC-SOX2-expressing cells in the distal lung buds lost SOX9 while inducing Sox21, but MYC-SOX21-expressing cells in the distal bud retained SOX9+ and did not induce Sox2 expression. In the airway, MYC-SOX21+ cells were SOX2 positive, although some cells still expressed SOX9, suggesting that these cells are transitioning from a distal to a proximal cell fate (Figure 1C). We therefore conclude that SOX2 alone can set up Sox21 expression, but SOX21 alone is not sufficient to induce a SOX2+ airway cell fate in the in the presence of distal mesenchymal signaling.

SOX21 and SOX2 are co-expressed in the zone where progenitor cells differentiate

SOX21 marks a specific proximal region of the airway tree during development, and we therefore asked the question what the role of SOX21 is in this distinct zone 1 versus zone 2, which only contains SOX2+ cells. Early during development, at E11.5, SOX2 and SOX21 were both expressed in the lung, esophagus, and thymus. At this embryonic age, the esophagus and thymus already contained TRP63+ epithelial cell progenitors (Figure 2—figure supplement 1A), while TRP63+ basal cells only start to appear in the lung at E12.5 (Figure 2A). Previously, we showed that SOX2 directly regulates Trp63 expression (Ochieng et al., 2014), and we therefore analyzed whether SOX21 plays a role in the differentiation of airway progenitor cells to basal cells. We found that TRP63+ basal cells appear in zone 1 from E12.5 onward, but not in zone 2 (Figure 2A). At E14.5, we found higher expression of SOX2 and SOX21 as well as more basal cells in the epithelium in close proximity to SMA+ mesenchymal cells (Figure 1, Figure 2—figure supplement 1B,C). This localization of basal cells is consistent with previous findings (Que et al., 2009). The specific localization suggests that mesenchymal–epithelial crosstalk is involved in the combined SOX21 and SOX2 expression and in the progenitor to basal cell differentiation (Figure 2—figure supplement 1D).

Figure 2 with 1 supplement see all
Download asset Open asset
SOX2 and SOX21 are co-expressed in the zone where progenitor cells differentiate to basal cells.

(A) Immunofluorescence of SOX2 (blue), SOX21 (red), and TRP63 (green) on lung sections of E12.5, E13.5, and E14.5 showing a spatial co-localization of the three proteins. Scale bar = 100 µm. (B) Schematic representation of the iSox2CC10-rtTA and iSox21CC10-rtTA mouse models. Doxycycline was given to the dams from E6 till E18.5. The percentage of secretory, ciliated, and basal cells was analyzed at the medial and lateral side in a 400 µm area around the carina. (C) Quantification of the percentage of SCGB1A1+ secretory cells at E18.5 in wild-type (WT; open bars), iSox2CC10-rtTA, and iSox21CC10-rtTA mice (closed bars) represented as percentage of medial (blue), lateral (red), and total (grey) cells. Two-way ANOVA (n = 3; *p<0.05, **p<0.01, ***p<0.001). Scale bar = 50 µm. (D) Immunofluorescence of SCGB1A1 (red) and FOXJ1 (green) in wild-type (WT), iSox2CC10-rtTA, and iSox21CC10-rtTA lung sections at E18.5. Quantification of the percentage of FOXJ1+-ciliated cells at E18.5 in wild-type (WT; open bars), iSox2CC10-rtTA, and iSox21CC10-rtTA mice (closed bars) represented as percentage of medial (blue), lateral (red), and total (grey) cells. Two-way ANOVA (n = 3; *p<0.05). Scale bar = 50 µm. (E) Immunofluorescence of TRP63 (red), KRT5 (grey) basal cells, and KRT8 (red) luminal cells in wild-type (WT), iSox2CC10-rtTA, and iSox21CC10-rtTA lung sections at E18.5. Quantification of the percentage of TRP63+ basal cells at E18.5 in wild-type (WT; open bars), iSox2CC10-rtTA, and iSox21CC10-rtTA (closed bars) mice represented as percentage of medial (blue), lateral (red), and total (grey) cells. Two-way ANOVA (n = 3; *p<0.05, **p<0.01, ***p<0.001). Scale bar = 50 µm. (F) In normal lung development, secretory cells can renew or give rise to ciliated cells. Upon induction of MYC-SOX2 or MYC-SOX21, the secretory cell identity is rapidly lost as shown by the loss of SCGB1A1 expression. Concomitantly, the SCGB1A1/CC10-driven MYC transgene is no longer expressed and cells adopt a TRP63+ basal cell fate.

Figure 2—source data 1

Quantification of the number of SCGB1A1, FOXJ1 and TRP63 positive cells represented in the plots.

https://cdn.elifesciences.org/articles/57325/elife-57325-fig2-data1-v2.xlsx

In our iSox2SPC-rtTA and iSox21SPC-rtTA mouse models, zone 1 is extended to the distal buds and the airways lack zone 2, where only SOX2 is expressed (Figure 2—figure supplement 1E). Within the extended SOX2+SOX21+ zone 1, TPR63+ basal cells were also present, showing that induction of both SOX2 and SOX21 is sufficient for the differentiation of airway progenitors to TPR63+ basal cells and can be independent on anatomical localization (Figure 2—figure supplement 1E). Previously, conditional deletion of β-catenin in SPC+ cells during lung development caused the appearance of cystic structures, just like after SOX2 and SOX21 induction. Upon β-catenin deletion, an increased number of TRP63+ basal cells was found, of which a subset co-expressed SOX9 (Ustiyan et al., 2016). Similarly, we found SOX9+ basal cells in the iSox2SPC-rtTA and iSox21SPC-rtTA mouse lungs, while only sporadic SOX9+ basal cells were detected in the control (Figure 2—figure supplement 1E).

To further investigate the effect of SOX2 and SOX21 in proximal differentiated airway epithelium during development, we induced expression of MYC-tagged Sox2 or Sox21 transgenes in secretory cells using a CC10/SCGB1A1-driven rtTA-inducible model (iSox2CC10-rtTA and iSox21CC10-rtTA) (Tichelaar et al., 2000). Doxycycline was giving from E6 onwards to target as much secretory cells as possible during airway development (Figure 2B). The number of secretory, basal, and ciliated cells were counted on the medial and lateral side of the main bronchi, immediate distal of the carina (Figure 2B). In both models, we found a decreased number of secretory cells (Figure 2C), a slight decrease of total number of ciliated cells (Figure 2D, grey bar) and an increase in the total number of basal cells (Figure 2E, grey bar). Reminiscent of the unilateral expression of SOX21 during development, TRP63+ basal cell numbers increased mostly on the lateral side of the airway, while a decrease in secretory cells was observed on both sides. This suggests that a difference in medial and lateral mesenchymal-epithelial signaling directs the induced differentiation of MYC-SOX2 or MYC-SOX21 expressing cells. Focusing on the MYC expressing cells, we observed that these cells were mostly negative for basal cell marker TRP63, secretory cell marker SCGB1A1, and ciliated cell marker FOXJ1 (Figure 2—figure supplement 1F). This suggests that upon the induction of MYC-SOX2 or MYC-SOX21, the secretory cell identity is rapidly lost as shown by the loss of SCGB1A1 expression. Concomitantly, the SCGB1A1/CC10-driven MYC transgene is no longer expressed and cells adopt a TRP63+ basal cell fate (Figure 2F).

SOX21 and SOX2 regulate maintenance and differentiation of the airway progenitor state

Next, we studied how reduced levels of SOX21 affect the differentiation of progenitor cells during development in Sox21 heterozygous (Sox21+/−) and homozygous (Sox21−/−) knockout mice. Sox21−/− mice do not show respiratory distress at birth (Kiso et al., 2009). Their lungs are smaller compared to wild-type littermate controls but have no apparent branching defect (data not shown). To analyze how reduced levels of SOX21 affect epithelial differentiation, we evaluated airway specific cells numbers at E14.5 and E18.5 distal to the carina, at the medial side where SOX2 and SOX21 levels are highest (Figure 1—figure supplement 1A). At E14.5, we found an increased number of basal cells in Sox21+/− mice, which was even more pronounced in Sox21-/- mice (Figure 3A). In contrast, a decrease in the number of basal cells is observed in lungs of Sox2+/− at E14.5 (Figure 3—figure supplement 1A), corresponding with the dose-dependent role described for SOX2 in airway differentiation (Que et al., 2007). Hence, reduced levels of SOX2 and SOX21 appear to oppositely affect airway progenitor cell differentiation. SOX21 maintains the SOX2 progenitor state, while SOX2 expression is needed to initiate progenitor to basal cell differentiation.

Figure 3 with 1 supplement see all
Download asset Open asset
SOX21 counter balances SOX2+ progenitor differentiation to airway-specific cell types.

(A–D) Immunofluorescence and quantification of the number of TRP63+ basal cells (A), TRP73+ cells (B), FOXJ1+-ciliated cells (C), and KI67+-dividing cells (D) at E14.5 in wild-type (WT), Sox21+/−, and Sox21−/− mice. The number of cells were counted within the first 400 µm immediately distal of the carina at the medial side of the airway. One-way ANOVA (n = 3; *p<0.05). Scale bar = 50 µm. (E) Immunofluorescence and quantification of the number of TRP63+ (green) basal cells, FOXJ1+ (red) ciliated cells, and SCGB1A1+ (yellow) secretory cells at E18.5 in wild-type (WT), Sox21+/−, and Sox21−/− mice. The number of cells were counted within the first 400 µm immediately distal of the carina at the medial side of the airway. One-way ANOVA (n = 3; *p<0.05, **p<0.01, ***p<0.001). Scale bar = 50 µm. (F) Luciferase assay to test the transcriptional activity of the Sox2 promotor region from −2002 till −128 andTrp63 promotor region from −1090 till +1180 (+1 is considered the transcriptional start site). Blue squares showing SOX21 binding motifs and the orange bar shows the region bound by RNA polymerase II. The graph shows luciferase activity induced after transfection of FLAG-SOX2 and/or increasing amounts of MYC-SOX21. One-way ANOVA (n = 3; **p<0.01, ***p<0.001, ****p<0.0001, when not indicated otherwise, stars show significance compared to control). (G) Schematic overview of epithelial progenitor maintenance and differentiation during murine lung development. From proximal to distal a transition of SOX protein levels takes place. The most proximal part of the lung, the trachea, and the two main bronchial branches, show high expression levels of SOX2 and co-expression of SOX21. SOX21 balances the maintenance of the SOX2 progenitor state in a zone where progenitor cells are prone to differentiate to airway-specific cell types.

Figure 3—source data 1

Quantification of the number of positive cells represented in the plots.

https://cdn.elifesciences.org/articles/57325/elife-57325-fig3-data1-v2.xlsx

To determine the role of SOX2 and SOX21 for differentiation to airway-specific cell types, we investigated the differentiation to ciliated cells. Ciliated cells started to appear at E14.5 and can be quantified using TRP73, an early marker for differentiation, and FOXJ1, a more mature marker (Marshall et al., 2016). At E14.5, there was a small increase of TRP73+- and FOXJ1+-ciliated cells in Sox21+/− airways and an even larger increase in Sox21−/− airways (Figure 3B,C), reaffirming that SOX21 levels are important for maintenance of the progenitor state and suppression of differentiation. In the Sox2+/− mice, TRP73+- and FOXJ1+-ciliated cell numbers did not differ from wild-type controls (Figure 3—figure supplement 1B,C), suggesting that decreasing SOX2 levels do not influence the initiation of ciliated cell differentiation. Moreover, overall proliferation was unaltered during development in Sox2+/− airways and only slightly increased in Sox21−/− airways (Figure 3D, Figure 3—figure supplement 1D), suggesting that SOX2 and SOX21 act mainly on differentiation and not proliferation.

At E18.5, the increase in basal cell numbers in Sox21−/− observed at E14.5 was no longer detectable, although increased numbers of ciliated cells were still present. Additionally, we also observed a decrease in secretory cells at E18.5 (Figure 3E). Thus, it seems that in the absence of SOX21 the increase in ciliated cells is at the expense of secretory cells, in agreement with the reported finding that the secretory cell lineage gives rise to ciliated cells (Rawlins et al., 2009b). In Sox2+/− lungs, only a minor decrease in secretory cells was observed at E18.5 (Figure 3—figure supplement 1E).

To better understand the effect of SOX21 on SOX2-induced gene expression, we analyzed promotor regions that are activated upon expression of SOX2 by luciferase assays. We found that SOX2 activates its own promoter, as well as the promoter regions of the Trp63 and Trp73 genes (Figure 3F, Figure 3—figure supplement 1F). SOX21 binding motifs have been characterized through chromatin immunoprecipitation (Matsuda et al., 2012), and we subsequently identified putative binding sites for SOX21 in the promoter regions of Sox2, Trp63, and Trp73 (Figure 3F, Figure 3—figure supplement 1F). When increasing amounts of SOX21 were titrated in with SOX2, luciferase activity with the Trp63 promotor significantly decreased (Figure 3F, Figure 3—figure supplement 1F). This shows that SOX21 can suppress promotor regions of SOX2 target genes.

Taken together, we propose that high levels of SOX2 initiate SOX21 expression, marking a zone of the proximal airways where progenitor cells start to differentiate. Within this zone, SOX21 and SOX2 in progenitor cells have a regulatory function, by maintaining a balance between progenitor maintenance and differentiation (Figure 3G).

SOX2 drives and SOX21 represses basal cell differentiation to ciliated cells

At E18.5, SOX2 and SOX21 remain expressed in the extrapulmonary airways, the region where basal cells are maintained. Basal cells are important for regeneration after injury, as they can proliferate and differentiate to secretory and ciliated cells (Rock et al., 2009). To better understand how SOX2 and SOX21 regulate the differentiation of basal cells, we used an in vitro differentiated ALI culture method of mouse tracheal epithelial cells (MTECs; Eenjes et al., 2018). ALI cultures provide standardized conditions to study basal cell differentiation at different time points. Using this model, we show that Sox2 and Sox21 were both expressed at baseline levels at the start of ALI and that their levels gradually increased in the initial days of ALI differentiation (Figure 4A). Genes reflecting the differentiation status of ciliated and secretory cells were increasingly expressed in a similar fashion (Figure 4—figure supplement 1A). Immunofluorescence analysis on ALI day 10 showed expression of SOX2 and SOX21 in basal and apical cells, although both proteins were heterogeneously distributed among the two cell layers (Figure 4B). To determine whether SOX2 and SOX21 levels correlated, we measured the fluorescence intensity of both proteins (Figure 4—figure supplement 1B). Cells highly expressing SOX2 were frequently high in SOX21 expression and vice versa (Figure 4—figure supplement 1C). Basal cells were mainly Sox2lowSox21low (80.5%), while ciliated cells were either SOX2highSOX21high (77.7%) or SOX2highSOX21low (14.5%; Figure 4C). In the adult mouse TE, SOX2 and SOX21 levels were similar between cell types in vivo (Figure 5A), suggesting that these are stable for the maintenance of TE and only increase when differentiation is initiated (Figure 4—figure supplement 1D).

Figure 4 with 1 supplement see all
Download asset Open asset
SOX2 and SOX21 are inversely correlated with basal cell differentiation to ciliated cells.

(A) Schematic overview of mouse tracheal epithelial cell (MTEC) culture. QPCR analysis of Sox2 and Sox21 expression during differentiation of MTECs on air–liquid interface (ALI). One-way ANOVA (n = 5; *p<0.05, ** p<0.01, ***p<0.001). (B) Immunofluorescence staining on MTEC 10 days after ALI of SOX2 (purple), SOX21 (green), and TRP63 (blue, left images) or FOXJ1 (blue, right images). Dotted lines show representation of measured nuclei for fluorescence intensity. Scale bar = 25 µm. (C) Dot-plot of the MFI of SOX2 (x-axis) and SOX21 (y-axis). The green filled circles are TRP63+ basal cells and the number in each quadrant represents the percentage of basal cells in each quadrant. Bar graph is the quantification of basal cells that are either high (MFI > 1) or low (MFI < 1) in expression of SOX2 and SOX21. The red filled circles are FOXJ1+-ciliated cells and the number in each square represents the percentage of ciliated cells in each quadrant. Bar graph is the quantification of ciliated cells that are either high (MFI > 1) or low (MFI < 1) in expression of SOX2 and SOX21. One-way ANOVA (n = 3; **p<0.01, ***p<0.001). (D) Analysis of MTEC cultures of tracheal cells derived from wild-type (WT), Sox2+/-, or Sox21+/- animals after 10 days of ALI culture. Immunofluorescence staining of ciliated cells (FOXJ1, red) or secretory cells (SCGB3A2, green). Scale bar = 50 µm. Quantification of the number of ciliated cells per 775 µm2 field. One-way ANOVA (n = 3; **p<0.01). (E) Schematic representation of the iSox2CC10-rtTA and iSox21CC10-rtTA mouse models. Doxycycline was given to adult mice for 6 weeks. Immunofluorescence and quantification of the number of SCGB1A1+ (yellow) secretory cells, FOXJ1+ (red) ciliated cells and TRP63+ (green) basal cells in wild-type (WT) compared to iSox2CC10-rtTA and iSox21CC10-rtTA mice. One-way ANOVA (n = 3; *p<0.05, **p<0.01). Scale bar = 50 µm. (F) Upon homeostasis, tracheal epithelium has a low turnover. The induction of MYC-SOX2 or MYC-SOX21 results in a loss of secretory cell phenotype. The induction of MYC-SOX2 drives the appearance of basal cells. The induction of MYC-SOX21 appears insufficient to drive differentiation toward a basal or ciliated cell type.

Figure 4—source data 1

Quantification of the number of positive cells represented in the plots.

https://cdn.elifesciences.org/articles/57325/elife-57325-fig4-data1-v2.xlsx
Figure 5 with 1 supplement see all
Download asset Open asset
Regeneration is delayed in SOX2 deficient tracheal epithelium.

(A) Immunofluorescence staining on tracheal sections of adult wild-type mice for SOX2 (red), SOX21 (green), and KRT5 (grey, top row) or smooth muscle actin (grey, SMA, bottom row). TE = tracheal epithelium. SMG = submucosal gland. Closed arrowheads (u) indicate single SOX2+ cells. Scale bar = 100 µm. (B) Schematic overview of the experimental set up of the Naphthalene injury and recovery in wild-type (WT), Sox2+/− (S2+/−), and Sox21+/− (S21+/−) mice. (C) Immunofluorescence and quantification of the number of SOX9+ cells, 5 days post injury (DPI), in the upper TE from Cartilage (C) ring 0 till C1 in WT, Sox2+/− and Sox21+/− mice. Scale bar = 100 µm. One-way ANOVA (WT n = 8, Sox2+/− n = 8, Sox21+/− n = 5, *p<0.05). (D) Immunofluorescence of FOXJ+ (red) cells and SCGB1A1+ (yellow) cells in the TE, of WT, Sox2+/− and Sox21+/− mice at 20 DPI. Scale bar = 100 µm. Quantification of the number of FOXJ1+-ciliated cells and SCGB1A1+ secretory cells from C0 till C6 between genotypes. One-way ANOVA (WT n = 6, Sox2+/− n = 7, Sox21+/− n = 6, *p<0.05, ***p<0.001). Quantification of the number of ciliated cells, 20 DPI compared to the corn oil (CO) exposed mice of each WT, Sox2+/− and Sox21+/− mice. Unpaired T-test (WT: CO n = 5 and 20DPI n = 6, Sox2+/−: CO n = 3 and 20DPI n = 7, Sox21+/−: CO n = 5 and 20DPI n = 6, *p<0.05). (E) Immunofluorescence with TRP63 (red) and KI67 (green) to mark dividing basal cells in the TE, 20 DPI, in WT, Sox2+/−, and Sox21+/− mice. Scale bar = 100 µm. Quantification of the number of dividing basal cells from C0 till C6 between genotypes. One-way ANOVA (WT n = 6, Sox2+/− n = 7, Sox21+/− n = 6, ***p<0.001). Quantification of dividing basal cells, 20 DPI compared to CO mice of each wild-type (WT), Sox2+/−, and Sox21+/−. Unpaired T-test (WT: CO n = 5 and 20 DPI n = 6, Sox2+/−: CO n = 3 and 20 DPI n = 7, Sox21+/−: CO n = 5 and 20 DPI n = 6, *p<0.05).

Figure 5—source data 1

Quantification of the number of FOXJ1, SCGB1A1, TRP63 and KI67 positive cells represented in the plots.

https://cdn.elifesciences.org/articles/57325/elife-57325-fig5-data1-v2.xlsx

To test the importance of SOX2 and SOX21 levels in basal cell differentiation, we analyzed ALI day 10 cultures of WT, Sox2+/−, and Sox21+/− mice. After 10 days, there were comparable numbers of basal cells in Sox2+/−, Sox21+/−, and WT ALI cultures (Figure 4—figure supplement 1C). In Sox21+/− ALI cultures, more basal cells differentiated to FOXJ1+ ciliated cells compared to WT (Figure 4D). TRP73 is one of the earliest markers expressed by basal cells upon initiation of ciliated cell differentiation, followed by the loss of TRP63 expression (Marshall et al., 2016). While there was an increase in FOXJ1+ ciliated cells, TRP73+/TRP63+ or single TRP73+ cell numbers remained stable (Figure 4—figure supplement 1C). In Sox2+/− ALI cultures, fewer basal cells differentiated to ciliated cells compared to WT, as was shown by a significant decrease of both single TRP73+ and FOXJ1+ cells (Figure 4D; Figure 4—figure supplement 1C). The differentiation capacity towards secretory cells was not affected in either Sox2+/− or Sox21+/− ALI cultures compared to WT (Figure 4D). Therefore, SOX2 and SOX21 levels are mainly involved in balancing the differentiation of basal cells to ciliated cells, with SOX2 stimulating differentiation and SOX21 inhibiting differentiation (Figure 4—figure supplement 1D).

To further understand the role of SOX2 and SOX21 in differentiated airway epithelium, we used iSox2CC10-rtTA and iSox21CC10-rtTA adult mice to induce expression of Sox2 or Sox21 in secretory cells for 6 weeks (Figure 4E). Similar to the CC10-rtTA induction of MYC-SOX2 or MYC-SOX21 during development, the MYC transgene expressing cells were mostly negative for other cell-specific markers (Figure 4—figure supplement 1E). Induction of either MYC-SOX2 or MYC-SOX21 resulted in a decrease in secretory cells, while MYC-SOX2 also increased TRP63+ basal cell numbers (Figure 4E). Thus, MYC-SOX2 induction in secretory cells drives the appearance of basal cells. In addition, the induction of MYC-SOX21 in secretory cells of adult airway epithelium resulted in a loss of secretory phenotype but appears insufficient to drive differentiation toward a basal or ciliated cell type (Figure 4F).

Deficiency of SOX2 decreases airway epithelial repair after naphthalene-induced injury while SOX21 stimulates it

Because fine-tuning of SOX2 and SOX21 levels is important in basal cell differentiation in vitro, we investigated whether SOX21 is also important for basal cell differentiation after injury in vivo. SOX21 remains expressed throughout the tracheal epithelium (TE) in both basal and non-basal cells in adult mice (Figure 5A). In addition, we observed SOX21-expressing cells in the submucosal glands (SMG), which also contribute to TE regeneration after injury (Lynch et al., 2018; Tata et al., 2018; Figure 5A). To test whether SOX21 and SOX2 levels are important to control adult stem cell differentiation, we exposed wild-type (WT), Sox2+/–, and Sox21+/– mice to corn oil (CO) or naphthalene to induce transient epithelial injury. We examined the immediate response after 2 days and the recovery after 5 and 20 days post-injury (DPI) (Figure 5B). Due to the fragility of Sox21–/– mice, we were unable to study adult Sox21–/– TE after naphthalene injury (Kiso et al., 2009). Neither Sox2+/– nor Sox21+/– TE showed significant differences in basal, dividing basal, or ciliated cell numbers compared to WT after CO exposure (Figure 5—figure supplement 1D).

SOX9+ SMG cells surface at the TE after administering a high naphthalene dose and thereby contribute to repair of the TE (Lynch et al., 2018; Tata et al., 2018). The high concentration of naphthalene administered to the mice causes severe injury by removing most luminal epithelial cells and leaving few basal cells (Figure 5—figure supplement 1A), as reported (Cole et al., 2010; Tata et al., 2018). Previously, it was shown that SOX2 expression was largely extinguished from SOX9+ SMG cells at 1 DPI, suggesting that changes in SOX2 expression are important in the early contribution of SMG cells to repair (Lynch et al., 2018). However, we found that SOX2 or SOX21 expression was unaltered at 2 DPI compared to CO exposed mice, suggesting that SOX2 downregulation at 1 DPI is temporary and a consequence of naphthalene administration (Figure 5A, Figure 5—figure supplement 1B). After 5 days, we observed a decrease of SOX9+ cells in Sox2+/− TE, and a small increase of SOX9+ cells in Sox21+/− TE compared to WT and to each other (Figure 5C). This shows that SOX2 and SOX21 levels are important for TE regeneration by SOX9+ SMG cells.

To determine whether SOX2 or SOX21 deficiency affects regeneration after naphthalene injury, we quantified the percentage of secretory, ciliated, non-dividing, and dividing basal cells. The number of ciliated and dividing basal cells was similar between WT, Sox21+/−, and Sox2+/− at 5 DPI (Figure 5—figure supplement 1C). At 20 DPI, fewer FOXJ1+ ciliated cells, but more dividing and non-dividing basal cells were observed in Sox2+/− TE compared to WT, Sox21+/−, or to Sox2+/− CO exposed mice (Figure 5D–E, Figure 5—figure supplement 1D). Both WT and Sox21+/− TE had a similar number of ciliated cells, non-dividing basal cells, and dividing basal cells at 20 DPI compared to CO exposed mice (Figure 4D, Figure 5—figure supplement 1D). Thus, recovery of injury in the WT and Sox21+/− TE appears the same, while Sox2+/− TE show a delayed recovery (Figure 5E, Figure 5—figure supplement 1D), indicating an important role for SOX2 in TE regeneration.

SOX2 and SOX21 expression is conserved in human airway epithelial cells

The data above show that SOX21 is an important factor to maintain the balance between progenitor maintenance and differentiation in both mouse lung development and adult basal cell differentiation. Next, we asked whether this role is conserved in human airway epithelial development. SOX21 is enriched in SOX2+ stalk (differentiating bronchiole) versus tip (distal) progenitor cells of the human fetal lung (Nikolić et al., 2017). We observed sporadic SOX21-expressing cells in lung sections at post-conceptional week (PCW) 17. The SOX21-expressing cells were mainly found in larger airways, where basal cells were also present (Figure 6A). Additionally, we cultured human fetal lung tip (PCW 17) organoids as previously described (Figure 7—figure supplement 1A; Nikolić et al., 2017). All cells within the organoids were positive for SOX2 and co-expressed either SOX9, SOX21, or both, unlike human PCW 17 lung sections (Figure 6A,B). Some SOX21-expressing cells in the fetal lung organoids were positive for the basal cell marker TP63, just like in human fetal lung sections (Figure 6A,B, white arrowheads). To determine whether SOX21 increases upon further lung development, we differentiated human fetal lung tip organoids into a more mature airway organoid. To induce maturation, we replaced fetal lung organoid medium with airway organoid medium as recently described (see Materials and Methods, Table 1; Sachs et al., 2019). This caused the lung organoids to take on a differentiated airway epithelium phenotype, with an increase in basal cells and the appearance of ciliated cells (Figure 6C). In these airway organoids, SOX21 expression was observed in most cells (Figure 6C), and we confirmed expression of SOX21 in adult airway epithelial cells using human bronchial sections (Figure 6D).

Figure 6
Download asset Open asset
Evolutionary conserved expression of SOX21 in human airway epithelium.

(A) Immunofluorescence on human fetal lung sections post-conceptional week (PCW) 13, 16.5, and 17. Proximal–distal patterning of human developing airways is analyzed with SOX9 (red) and SOX2 (blue) (left). In the small developing airways (left), no expression of SOX21 (green) was observed. On the right, co-staining of SOX2 (blue), SOX21 (green), and TP63 (red). The closed white arrowheads show cells co-expressing TP63 and SOX21. Scale bar = 50 µm. (B) Top and middle row: Immunofluorescence analysis of fetal lung organoids shows SOX2 (grey), SOX9 (red), and SOX21 (green) positive cells. White closed triangles (►) show cells positive for SOX2 and SOX9, open triangles (w) show cells positive for SOX2, SOX21, and SOX9. Arrows (g) show cells positive for SOX21 and SOX2. Bottom row: Immunofluorescence analysis of SOX2 (grey), TP63 (red), and SOX21 (green) in fetal lung organoids. White closed arrowheads (►) show cells positive for SOX2, TP63, and SOX21. Scale bar = 50 µm. (C) Fetal lung organoids differentiated to airway epithelium, as shown by the presence of ciliated cells (TUBIV; left) or basal cells (TP63, right), have an abundant expression of SOX21 (green). Scale bar = 50 µm. (D) Immunofluorescence analysis of sections of human adult bronchi shows co-localization of SOX2 (red) and SOX21 (green) throughout the epithelium (1, top row). SOX21 (green) is expressed in luminal (KRT8; grey) and basal (KRT5; red) cells (2, middle row). SOX21 is expressed in basal (TP63; red) and ciliated (FOXJ1; grey) and is high expressed in cells absent of TP63 and FOXJ1(g) (3, bottom row). Scale bar = 25 µm.

To functionally assess the role of SOX21 in human adult airway epithelium, we isolated airway epithelial cells from human bronchial tissue. Both SOX2 and SOX21 are expressed throughout the bronchial epithelium (Figure 6D). ALI cultures showed that SOX2 and SOX21 were also expressed in vitro, as with murine cells. Prolonged culturing resulted in a significant increase of SOX2 and a slight increase of SOX21 (Figure 7A, Figure 7—figure supplement 1B). Both SOX2 and SOX21 were higher expressed in the apical layer compared to the basal layer (Figure 7B). Most TP63+ basal cells were low in SOX2 and SOX21 expression (88.3%) (Figure 7—figure supplement 1D). Next, we determined whether SOX2high and/or SOX21high cells in the apical layer were FOXJ1+-ciliated cells. Comparing FOXJ1+ and FOXJ1− nuclei showed that ciliated cells were high in SOX2 expression, while SOX21 was highest in non-ciliated cells (Figure 7B, Figure 7—figure supplement 1C). Fluorescence intensity of SOX2 and SOX21 also showed that most ciliated cells were SOX2highSOX21low (75%; Figure 7—figure supplement 1D). Furthermore, the intensities of either SOX2 or SOX21 for ciliated (FOXJ1+), basal (TP63+), and FOXJ1− TP63− cells, showed that SOX21 was mainly high in double-negative cells (Figure 7C). We think that these double-negative cells are intermediate cells transitioning from basal cells to a ciliated or sercretory cell fate, the para-basal cell. In accordance with this observation, sections of human airway epithelium show SOX21high cells in between the basal and luminal layer, which also express both basal cell marker KRT5 and luminal cell marker KRT8 (Figure 6D 1–3, arrows). Comparison of our expression results to published single-cell RNA data on human primary bronchial epithelial ALI culture (Plasschaert et al., 2018) confirmed that high levels of SOX21 mRNA denote an intermediate cell type. Furthermore, in line with our observation, high levels of SOX2 were observed both in the intermediate state and in differentiated ciliated cells (Figure 7—figure supplement 1E).

Figure 7 with 1 supplement see all
Download asset Open asset
SOX2 and SOX21 in human basal cell differentiation.

(A) Schematic overview of human primary bronchial epithelial cell (HPBEC) culture. QPCR analysis of SOX2 and SOX21 expression during differentiation of HPBECs on air–liquid Interface (ALI). One-way ANOVA (n = 4 (ALI cultures of four different donors); ***p<0.001). (B) Immunofluorescence analysis of HPBEC 16 days after ALI of SOX2 (red), SOX21 (green), and TP63 (blue; left) or FOXJ1 (blue; right). XZ plane shows basal cells (TP63+) at the basal membrane and ciliated cells (FOXJ1+) at the apical side. Scale bar = 25 µm. The grey bar graphs the MFI of SOX2 or SOX21 in basal and apical cells. The red bar graphs show the MFI of SOX2 or SOX21 in FOXJ+ and FOXJ− apical cells. The circled FOXJ1+ nuclei show high expression of SOX2 with sporadic high expression of SOX21(t) as well. Paired T-test (n = 3 [ALI cultures of three different donors]; *p<0.05, ***p<0.001). (C) Dot-plot of the MFI of SOX2 (y-axis: left graph) or SOX21 (y-axis: right graph). Each dot represents a cell, and ALI cultures of two different donors are included. The red filled circles are FOXJ1+-ciliated cells, green filled circles are TP63+ basal cells and non-filled are FOXJ1− TP63− cells. (D) Knockdown of SOX2 or SOX21 after lentiviral transduction of one of two different shRNAs directed against different regions of the SOX2 or SOX21 gene. Scr are HPBECs transduced with lentivirus and expressing a scrambled shRNA. The HPBEC ALI culture was transduced 1 day before exposure to air. The number of FOXJ1+ nuclei were analyzed after 14 days of ALI. Immunofluorescence of FOXJ1+ (green) ciliated cells and mCherry+ (red) transduced cells. Scale bar = 25 µm. The graphs show quantification of the average number of FOXJ1+ cells per area of 375.5 µm2 (Top) and the percentage of FOXJ1+mCherry+ cells of total mCherry+ cells (bottom). Paired T-test (n = 4 [four different donors]; *p<0.05, ***p<0.001). (E) Immunofluorescence of HPBECs transduced with lentiviruses to knockdown SOX2 (shSOX2-B) or SOX21 (right; shSOX21-B) stained with TUBIV+ (green) to visualize differentiated ciliated cells, MUC5B+ (grey) for goblet cells and mCherry+ (red) denotes transduced cells. Scr are HPBECs transduced with lentivirus and expressing a scrambled shRNA. Scale bar = 25 µm. The area of MUC5B+ was measured after 14 days of ALI. The graphs show quantification of the average MUC5B+ per area of 375.5 µm2. n = 3 (three different donors), each treated with two different shRNA constructs.

Figure 7—source data 1

Quantification of the number of positive cells represented in the plots.

https://cdn.elifesciences.org/articles/57325/elife-57325-fig7-data1-v2.xlsx

Finally, we assessed the effects of reduced SOX2 or SOX21 levels on human basal cell differentiation by transducing ALI cultures with lentiviruses expressing short-hairpin RNAs (shRNA) to knockdown SOX2 or SOX21, and a mCherry to mark transduced cells. Cells were transduced at D0 of ALI, and the differentiation to ciliated and MUC5B+ secretory goblet cells was assessed at D14. SCGB1A1+ secretory cells were rarely observed in our cultures and therefore not analyzed. We compared the fluorescence intensity of SOX2 or SOX21 in mCherry+ transduced cells with mCherry− non-transduced cells and observed an average lower intensity in two of three shRNAs used (Figure 7—figure supplement 1F). We observed an overall increase of FOXJ1+ cells upon knockdown of SOX2 or SOX21 in several independent donor ALI cultures (Figure 7D). This increase in FOXJ1+ cells was also observed when analyzing the mCherry+ cells only (Figure 7D). Thus, knockdown of SOX2 or SOX21 in the human airway cells favor a FOXJ1+ cell type, partly resembling the mouse ALI experiments (Figure 3C, Figure 3—figure supplement 1C). Despite the increase in FOXJ1+ nuclei, we did not observe an increase in fully differentiated ciliated cells as determined by the formation of TubulinIV+ cilia (Figure 7—figure supplement 1G). However, the increase in FOXJ1+ nuclei is seemingly combined with an impaired ability to differentiate into MUC5B+ goblet cells (Figure 7E). In conclusion, our data suggest that both SOX2 and SOX21 suppress basal to ciliated cell differentiation, while both seem to positively affect goblet cell differentiation in the human airways.

Discussion

During lung development, a proximal-to-distal epithelial gradient is observed by the separation of proximal SOX2- and distal SOX9-expressing cells. Here we show a further evolutionary conserved regionalization of the proximal epithelium by marking a SOX2+/SOX21+ proximal zone. Within this zone, progenitor cells differentiate into basal cells. With the use of human embryonic lung sections and fetal lung organoids, we confirmed SOX21 expression in SOX2+ progenitor stalk cells (Nikolić et al., 2017). In addition, SOX21 becomes more widely expressed when human fetal lung organoids are differentiated into airway organoids. Based on our findings, we propose that (1) SOX2+SOX21+ cells in murine and human fetal lung organoids are progenitor cells in an environment destined for differentiation and (2) that SOX21 is important in balancing maintenance and differentiation of SOX2+ airway progenitor cells.

SOX2 is a key regulator in regulating proliferation and differentiation in many different stem cells populations; however; the chromatin regions targeted by SOX2 are cell-type specific. The regulation of stem cells by SOX2 is dependent on both its co-factors and expression levels (Brafman et al., 2013; Hagey et al., 2018). We observed a correlation between high SOX2 expression and appearance of SOX21 on the dorsal side of the proximal airways and trachea. Additionally, ectopic induction of SOX2 in distal cells resulted in an upregulation of SOX21. We suggest that SOX2 requires a certain threshold level of expression to induce SOX21. Interestingly, SOX21 expression was first observed during the embryonic stage (E10.5–11.5), at a time when cells adopt a proximally restricted fate for the extrapulmonary airways (Yang et al., 2018). We suggest that SOX21 is a downstream effector of SOX2 during formation of the extrapulmonary airways, separating extrapulmonary airway progenitors from intrapulmonary airway which are only SOX2 positive. A similar induction of SOX21 by SOX2 has been described in the four-cell-stage embryo, in embryonic stem cells and in neuronal progenitor cells. The function of SOX21 and its synergy or antagonism with SOX2 activity is highly context dependent (Kopp et al., 2008; Mallanna et al., 2010; Chakravarthy et al., 2011; Goolam et al., 2016; Graham et al., 2003; Ohba et al., 2004; Sandberg et al., 2005; Matsuda et al., 2012). As an example of the context-dependent function of SOX21, we showed that upon ectopic expression of SOX21, SOX9+ distal progenitor cells were maintained, with no differentiation to SOX2+ progenitor cells. Thus, SOX21 itself is not capable of driving differentiation to SOX2+ progenitor cells in the presence of distal mesenchymal signaling. However, when co-expressed with SOX2, SOX21 is present in the region where SOX2 progenitor cells differentiate into basal cells. SOX2 and SOX21 can physically interact (Mallanna et al., 2010), and preliminary data of a genome-wide chromatin immunoprecipitation of the human airway ALI cultures showed that SOX2 and SOX21 not only have unique binding sites, but also co-occupy the same genomic regions (Figure 7—figure supplement 1H). This supports our findings that SOX2 and SOX21 are involved in the same cellular differentiation processes.

Using Sox21+/− and Sox21−/− mice, we showed that the presence of SOX21 is important to suppress the differentiation of SOX2+ progenitor cells into basal and ciliated cells. Surprisingly, basal cell numbers increased upon deletion of SOX21, but also after ectopic expression of a doxycycline-inducible SOX21 using an SPC-promotor- or SCGB1A1-promotor-driven rtTA during development. This seemingly contradictory result may be caused by the difference in timing between the mouse models. The Sox21−/− mice lack expression of Sox21 from the fertilization of the oocyte, whereas the transgenic overexpressing mice only start to express SOX21 when the SPC or SCGB1A1 promoters become active. Thus, the Sox21−/− mice lack Sox21 expression from the onset of lung development, which may trigger an expansion of basal cells, whereas the overexpression of SOX21 is induced in differentiated epithelial cells. The SPC or SCGB1A1 promoter-driven expression of SOX21 may cause a reversal of differentiated cells to basal cells. Alternatively, high levels of SOX21 may force cells to lose their identity followed by redifferentiation according to downstream cues received from the surrounding mesenchyme. For example, SPC-promoter-driven induction of SOX21 initiates the reversal of SOX9+ cells to a proximal phenotype, but this only becomes apparent once these SOX9+ cells are sufficiently distanced from distal mesenchymal FGF10 signaling. The latter causes the cells to express SOX2, leading to a more pronounced induction of the proximal fate and an increase in TRP63+ basal cells. The initiation of a proximal cell fate by ectopic expression of SOX21, followed by SOX2 expression, may explain why the cysts in SOX21-expressing lungs are smaller than the cysts observed upon ectopic expression of SOX2, the major proximal cell fate inducer.

SOX21 and SOX2 co-expression continues in adult TE and SMGs, which are both regions where progenitor cells reside. We used naphthalene injury and in vitro analysis to study whether SOX21 and SOX2 function in adult progenitors is similar to function during development. We showed that reduced SOX2 levels inhibit the contribution of SOX9+ SMG cells to repair of TE injury, while reduced SOX21 levels promote their contribution. Furthermore, reduced SOX2 levels decreased differentiation of basal to ciliated cells in vitro and in vivo, while reduced SOX21 levels only increased basal cell differentiation in vitro but not in vivo. The latter might be explained by the fact that differentiation of progenitor cells in vivo is regulated by an epithelial–mesenchymal interaction, which might inhibit further differentiation to ciliated cells when regeneration is complete (Volckaert et al., 2017). In summary, both during development and regeneration of the airway epithelium, endogenous SOX21 acts as a suppressor of SOX2+ progenitor cell differentiation, while SOX2 levels are important for stimulating differentiation. Using a luciferase assay, we specifically show that SOX21 can antagonize SOX2 function on certain promotor regions. However, to fully understand the function of SOX21 within SOX2+ airway progenitors, signaling/environmental cues, additional co-factors, and expression levels are likely to play a role.

SOX2 and SOX21 showed a similar but not identical expression pattern in human airway epithelium. As opposed to mouse tracheal epithelium which has a basal and luminal cell layer, the human proximal airway epithelium contains an additional layer of intermediate cells or para-basal cells (Mercer et al., 1994; Boers et al., 1998). Using human primary bronchial epithelial ALI cultures, we showed high SOX21 levels in TP63− FOXJ1− cells and suggest that these cells represent the intermediate layer of para-basal cells transitioning to a luminal cell fate. Besides the different expression pattern compared to mice, SOX2 and SOX21 also act differently on basal cell differentiation, as reduced expression levels of either protein resulted in more ciliated cells and fewer goblet cells. In mouse ALI cultures, reduced SOX2 expression resulted in a decrease in ciliated cells, whereas reduced SOX21 expression resulted in an increase in ciliated cells without affecting secretory cell numbers. Recently, human basal cells were shown to comprise at least four basal cell types, whereas mice only have one basal cell population (Travaglini et al., 2020). For future studies, it is important the understand which genes are regulated by SOX2 and SOX21 in human and mouse, and how they subsequently affect airway epithelial homeostasis and regeneration.

Our data provide a new understanding of proximal–distal patterning of the airways and the regulation of SOX2 progenitor cells within development and regeneration of airway epithelium. De-regulation of SOX2 and SOX21 expression levels can alter branching morphogenesis and differentiation of airway epithelium. We show that SOX21 can act as a suppressor of differentiation when SOX2 expression levels are high and when progenitor cells are prone to differentiate.

Materials and methods

Key resources table
Reagent type (species)
or resource		DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (M. musculus)Sox2-CreERTPMID:21982232MGI:5295990Sox2tm1(cre/ERT2)Hoch
The Jackson Laboratories Stock No: 017593
Genetic reagent (M. musculus)Sox21-KOPMID:19470461MGI:2654070Dr. Stavros Malas
Genetic reagent (M. musculus)iSox2PMID:18374910MGI:98364Dr. Robbert Rottier
Genetic reagent (M. musculus)iSox21-MGI:2654070Dr. Robbert Rottier
Genetic reagent (M. musculus)SPC-rtTAPMID:11874100MGI:109517Prof. Jeffrey Whitsett
Genetic reagent (M. musculus)CC10-rtTAPMID:10766812MGI:98919Prof. Jeffrey Whitsett
AntibodyMouse anti-β-TUBULIN IVBioGenexMU178-UC1:100
AntibodyMouse anti-FOXJ1eBioscience14–99651:200
AntibodyRabbit anti-KRT5BiolegendPoly190551:500
AntibodyRat anti-KRT8DSHBTROMA-I1:100
AntibodyRabbit anti-MYC (Immunofluorescence)AbcamAB91061:500
AntibodyRabbit anti-SCGB1A1AbcamAB408731:200
AntibodyMouse anti-SCGB3A1R and D systemsAF17901:200
AntibodyGoat anti-SCGB3A2R and D systemsAF34651:500
AntibodyMouse anti-SMANeomarkersMS-113-P11:500
AntibodyRabbit anti-SOX2Seven-HillsWRAB-SOX21:500
AntibodyGoat anti-SOX2Immune systemsGT150981:500
AntibodyGoat anti-SOX21R and D systemsAF35381:100 (TSA: 9 min)
AntibodyRabbit anti-SOX9AbcamAB1852301:500
AntibodyMouse anti-TRP63AbcamAB7351:100
AntibodyRabbit anti-TRP73AbcamAB406581:100
AntibodyRabbit anti-FLAGSigmaF7425Western blot, 1:1000
AntibodyRabbit anti-MYCAbcamAB91061:1000
AntibodyRabbit anti-MUC5BSigmaHPA0082461:500
AntibodyMouse anti-β -TUBULINSigmaT83281:2000
AntibodyAlexa Fluor 405, 488, 594 Donkey anti Goat IgGJackson ImmunoResearch705-475-147,
705-545-147,
705-585-147		1:500
AntibodyAlexa Fluor 488, 594, 647 Donkey anti Mouse IgGJackson ImmunoResearch715-545-151,
715-585-151,
711-605-151		1:500
AntibodyAlexa Fluor 488, 594, 647 Donkey anti Rabbit IgGJackson ImmunoResearch711-545-152,
711-585-151,
711-605-152		1:500
AntibodyAlexa Fluor 488, 594 Donkey anti Rat IgGJackson ImmunoResearch712-545-150
712-585-153		1:500
AntibodyDonkey anti-Goat HRP conjugatedJackson ImmunoResearch705-035-1471:500

Mice

All animal experimental protocols were approved by the animal welfare committee of the veterinary authorities of the Erasmus Medical Center. Mice were kept under standard conditions. Mouse strains SPC-rtTA and CC10-rtTA (gifts of Jeffrey Whitsett), Sox2-CreERT (Jackson Labs, stock number 017593), SOX21-KO (gift of Stavros Malas), and pTT::myc-SOX2 (iSox2) have been described (Gontan et al., 2008; Kiso et al., 2009; Arnold et al., 2011). The iSox21 transgenic mouse line was developed by cloning a murine Sox21 cDNA with a N-terminal myc epitope in a modified pTRE-Tight (Clontech) vector (pTT::myc-sox21). Pronuclear microinjection of a linearized fragment was performed, and three independent lines were tested. To induce expression of Sox2 or Sox21 during lung development, the pTT::myc-Sox2 or pTT::mycSox21 mice were crossed with SPC-rtTA or CC10-rtTA mice, and doxycycline was administered to dams in the drinking water (2 mg/ml doxycycline, 5% sucrose) from gestational day 6.5 onwards during development or for 6 weeks to 2–3 months old mice. Wild-type animals were non-transgenic littermates.

Naphthalene injury

Request a detailed protocol

Adult mice (~8–12 weeks) were injured with a single intraperitoneal injection of 300 mg/kg naphthalene. Naphthalene (Sigma; 184500) was freshly prepared and dissolved in corn oil (Sigma; C8267). Corn oil injection served as baseline control. Groups of mice were sacrificed 2, 5, and 20 days post-injury (DPI) (number of mice per group is indicated in each figure).

Mouse tracheal epithelial cell culture

Request a detailed protocol

Mouse tracheal epithelial cell (MTEC) culture was performed as previously described (Eenjes et al., 2018). Briefly, MTECs were isolated from mice adult trachea and cultured in KSFM expansion medium (Table 1) on collagen-coated plastic (50 µg/cm2 of rat tail collagen Type IV [SERVA, 47256.01] in 0.02N acetic acid [Sigma; 537020]). After expansion, 8 × 104 MTECs were plated per collagen-coated 12-well insert (Corning Inc, Corning, USA) in proliferation medium (Table 1) for ALI culture. When confluent, MTECs were exposed to air by removing proliferation medium and adding MTEC differentiation medium (Table 1) to the lower chamber. MTECs were cultured in standard conditions, at 37°C in a humidified incubator with 5% CO2.

Table 1
Medium.
KSFM-hPBEC medium
ReagentCompanyCat. no.Final concentration
KSFMGibco17005034n/a
Penicillin/streptomycinLonzaDE17-602e100 U/ml 100 µg/ml
Bovine pituitary extractGibco130280140.03 mg/ml
Human EGFPeprotech315–0925 ng/ml
IsoproterenolSigmaI-65041 µM
KSFM-MTEC expansion medium
ReagentCompanyCat. no.Final Concentration
KSFMGibco17005034n/a
Penicillin/streptomycinLonzaDE17-602e100 U/ml 100 µg/ml
Bovine pituitary extractGibco130280140.03 mg/ml
Mouse EGFPeprotech315–0925 ng/ml
IsoproterenolSigmaI-65041 µM
Rock Inhibitor (Y27632)Axon MedChem168310 µM
DAPTAxon MedChem14845 µM
MTEC proliferation medium
ReagentCompanyCat. no.Final concentration
DMEM:F12Gibco1133032n/a
Penicillin/streptomycinLonzaDE17-602e100 U/ml 100 µg/ml
NaHCO3Gibco250800940.03% (w/v)
Fetal calf serumHyCloneSH30071.035%
L-GlutamineGibco250300811.5 mM
Insulin-transferin-seleniumGibco414000451×
Cholera toxinSigmaC80520.1 µg/ml
Bovine pituitary extractGibco130280140.03 mg/ml
Mouse EGFPeprotech315–0925 ng/ml
Rock inhibitor (Y27632)Axon MedChem168310 µM
Retinoic acidSigmaR26250.05 µM
MTEC differentiation medium
ReagentCompanyCat. no.Final concentration
DMEM:F12Gibco1133032n/a
Penicillin/streptomycinLonzaDE17-602e100 U/ml 100 µg/ml
NaHCO3Gibco250800940.03% (w/v)
Bovine serum albuminGibco152600370.1% (w/v)
L-GlutamineGibco250300811.5 mM
Insulin-transferin-seleniumGibco414000451×
Cholera toxinSigmaC80520.025 µg/ml
Bovine pituitary extractGibco130280140.03 mg/ml
Mouse EGFPeprotech315–095 ng/ml
Retinoic acidSigmaR26250.05 µM
Human fetal organoid medium
ReagentCompanyCat. no.Final concentration
Advanced DMEM:F12Invitrogen12634–034n/a
R-SpondinPeprotech120–38500 ng/ml
NogginPeprotech120–10C100 ng/ml
Fgf10Peprotech100–26100 ng/ml
Fgf7Peprotech100–19100 ng/ml
EGFPeprotechAF-100–1550 ng/ml
CHIR99021Stem Cell Techn.720523 µM
SB431542Tocris161410 µM
B27 supplement (- VitA)ThermoFisher12587–0101×
N-AcetylcysteineSigmaA91651.25 mM
Glutamax 100×Invitrogen12634–0341×
N2ThermoFisher17502–0481×
HepesGibco15630–5610 mM
Penicillin/streptomycinLonzaDE17-602e100 U/ml 100 µg/ml
PrimocinInvivogenAnt-pm-150 µg/ml
Human airway organoid medium
ReagentCompanyCat. no.Final concentration
Advanced DMEM:F12Invitrogen12634–034n/a
R-SpondinPeprotech120–38500 ng/ml
NogginPeprotech120–10C100 ng/ml
Fgf10Peprotech100–26100 ng/ml
Fgf7Peprotech100–1925 ng/ml
SB202190SigmaS7067500 nM
A83-01Tocris2939500 nM
Y-27632Axon MedChem16835 µM
B27 supplementGibco17504–441×
N-AcetylcysteineSigmaA91651.25 mM
NicotinamideSigmaN06365 mM
Glutamax 100×Invitrogen12634–0341×
HepesGibco15630–5610 mM
Penicillin/streptomycinLonzaDE17-602e100 U/ml 100 µg/ml
PrimocinInvivogenAnt-pm-150 µg/ml

Human primary epithelial cell culture

Request a detailed protocol

Human primary airway epithelial cell (HPBEC) culture was performed as previously described (Amatngalim et al., 2018). Lung tissue was obtained from residual, tumor-free material obtained at lung resection surgery for lung cancer. The Medical Ethical Committee of the Erasmus MC Rotterdam granted permission for this study (METC 2012–512).

Briefly, cells were isolated from healthy bronchial tissue by incubation in 0.15% Protease XIV (Sigma; P5147) for 2 hr at 37°C. The inside of the bronchi was scraped in cold PBS (Sigma; D8537), and the obtained airway cells were centrifuged and resuspended in KSFM-HPBEC medium for expansion (Table 1). Culture plates were coated with 10 μg/ml human fibronectin (Millipore; FC010), 30 μg/ml BSA (Roche; 1073508600), and 10 μg/ml PureCol (Advanced Biomatrix; 5005-B) for 2 hr at 37°C. Upon confluency, HPBECs were frozen (4 × 105 cells/vial) and stored for later use.

When used for ALI culture, cells were thawed and seeded in a coated 10 cm dish, grown until confluent in KSFM-HPBEC medium, trypsinized, and 8 × 104 of HPBECs were plated per 12-transwell insert (Corning Inc, Corning, USA). On inserts, the HPBECs were cultured in bronchial epithelial growth medium (ScienCell Research Laboratories, Carlsbad, USA; 3211). Basal bronchial epithelial growth medium was first diluted 1:1 with DMEM (Gibco; 41966). Next, 1× supplement and 1× pen/strep (Lonza; DE17-602e) were added to 500 ml BEGM. Retinoic acid (1 nM RA) was freshly added. When cells reached full confluency, the BEGM medium was removed from the apical chamber and only supplied to the basal chamber and freshly supplemented with 50 nM RA. The medium was changed every other day, and the apical chamber was rinsed with PBS. HPBECs were cultured in standard conditions, at 37°C in a humidified incubator with 5% CO2.

Human fetal organoid culture

Request a detailed protocol

The culture of human fetal lung (17 weeks of gestation) and adult airway organoids was performed as previously described (Nikolić et al., 2017; Miller et al., 2018).

Lung lobes were dissociated using dispase (Corning; 354235) on ice for 30 min. Tissue pieces were then incubated in 100% FBS on ice for 15 min, after which they were transferred to a 10% FBS solution in Advanced DMEM with Glutamax, pen/strep, and HEPES. Lung bud tips were separated from mesenchymal cells through repeated pipetting. Pieces of tissue were resuspended in 30 μl of basal membrane extract (BME type 2, Trevigon; 3533-010-02), transferred to a 48-well plate, and incubated at 37°C to solidify the BME. After 5 min, 300 μl of self-renewing fetal lung organoid medium (Table 1) was added (Nikolić et al., 2017). Medium was refreshed every 3–4 days. Every 2 weeks, organoids were split 1:3 to 1:6. The medium was aspirated and cold PBS was added to the well to re-solidify the BME. Organoids were disrupted using a 1000 μl tip with on top a 2 μl tip. The disrupted organoids were centrifuged at 300 g for 5 min, resuspended in BME, and re-plated. To differentiate fetal lung organoids to organoids containing solely airway epithelium, fetal lung organoids were split, resuspended in BME, and re-plated with human airway organoid medium (Table 1; Sachs et al., 2019). Organoids were grown under standard culture conditions (37°C, 5% CO2).

Immunofluorescence

Tissue

Request a detailed protocol

Mouse embryonic lungs and human bronchial tissue were fixed overnight in 4% PFA (Sigma; 441244) at 4°C. Post-fixation, samples were washed with PBS, de-hydrated to 100% ethanol, transferred to xylene, and processed to paraffin wax for embedding.

Organoids were retrieved from the BME by adding cold PBS to the 48 wells. Organoids were centrifuged at 150 g for 5 min and fixed overnight in 4% PFA at 4°C. Post-fixation, organoids sink to the bottom of the tube. Centrifuging was avoided after fixation to keep the organoids intact. The organoids were embedded in 4% low-melting agarose. The organoids were washed in PBS for 30 min and manually de-hydrated by 50 min incubation steps in 50%, 70%, 85%, 95%, and two times 100% ethanol. Organoids were further processed by three times xylene for 20–30 min and washed three times for 20–30 min in 60°C warm paraffin to remove all traces of xylene. The organoids were placed in a mold and embedded in paraffin. Paraffin blocks were sectioned at 5 µm and dried overnight at 37°C.

Sections were deparaffinized by three times 3 min xylene washes, followed by rehydration in distilled water. Antigen retrieval was performed by boiling the slides in Tris–EDTA (10M Tris, 1M EDTA) buffer pH = 9.0 for 15 min. Slides were cooled down for 30 min and transferred to PBS. For SOX21 staining, the tyramide signal amplification (TSA) kit was used (Invitrogen, B40922, according to manufacturer’s protocol). When using the TSA kit, a hydrogen peroxide (35%) blocking step was performed after boiling. Sections were blocked for 1 hr at room temperature (RT) in 3% BSA (Roche; 10735086001), 0.05% Tween (Sigma; P1379) in PBS. Primary antibodies (Key resources table) were diluted in blocking buffer and incubated with the sections overnight at 4°C. The next day, sections were washed three times for 5 min at RT in PBS with 0.05% Tween. Secondary antibodies (Key resources table) were added in blocking buffer and incubated for 2 hr at RT. DAPI (4′,6-diamidino-2-phenylindole) solution (BD Pharmingen, 564907, 1:4000) was added to the secondary antibodies for nuclear staining. After incubation, three times 5 min washes in PBS-0.05% Tween and one wash in PBS were performed, and sections were mounted using Mowiol reagent (for 100 ml: 2,4% m/v Mowiol (Sigma; 81381), 4,75% m/v glycerol, 12 % v/v Tris 0.2M pH = 8.5 in dH2O till 100 ml). All sections were imaged on a Leica SP5 confocal microscope.

ALI culture

Request a detailed protocol

Human or mouse ALI cultures were washed with PBS and fixed on inserts in 4% PFA at RT for 15 min. Inserts were then washed 3 times for 5 min in 0.3% TritonX (Simga; T8787) in PBS and blocked for 1 hr at RT in 5% normal donkey serum (NDS, Millipore; S30), 1% BSA 0.3% TritonX in PBS. Primary antibodies (Key resources table) were diluted in blocking buffer and incubated overnight at 4°C. The next day, inserts were three times rinsed with 0.03% TritonX in PBS followed by three washes for 10 min at RT in PBS with 0.03% TritonX. Secondary antibodies (Key resources table) were added in blocking buffer and incubated for 2 hr at RT. DAPI solution (BD Pharmingen, 564907, 1:2000) was added to the secondary antibodies. After incubation, inserts were three times rinsed with 0.03% TritonX in PBS followed by three washes for 10 min at RT. Inserts were covered by a coverslip using Miowol reagent. Images were collated on a Leica SP5 confocal microscope.

Image analysis

Fluorescence intensity measurements

Request a detailed protocol

Intensity measurements in MTEC and HPBEC cultures were performed on three separate isolations from wild-type mice or donors and measured using ImageJ. Of each n, more that 500 nuclei were manually selected on the DAPI staining. In each nucleus, the intensity of SOX2 and SOX21 was measured. The MFI for each n and each intensity measurement was calculated by dividing it by the average intensity of that measurement in the same n.

For the knockdown ALI HPBEC culture, SOX2 and SOX21 intensity was measured in each experiment of each shRNA in 20 mCherry+ and 20 mCherry− nuclei.

Counting

Request a detailed protocol

To standardize counting between animals, basal and ciliated cells were counted during lung development in a square of 400 µm around the first branch at the medial side of the bronchi. If counted on the lateral side, this is mentioned in the figure. In this manner, we could determine a position in the SOX21−/− animal where in the wildtype SOX21 is highest expressed. Of each genotype and each n, three sections were counted and the percentage of ciliated and basal cells were calculated based on the total number of airway epithelial (SOX2+) cells.

Five days after Naphthalene injury, the number of basal (TRP63+), ciliated (FOXJ1+), dividing (KI67+), secretory cells (SCGB1A1+), and SOX9+ cells were counted in the tracheal epithelium from cartilage ring (C) 0 till C1 for 5 dpi and from C0 till C6 for 20 dpi. Of each animal, three sections were counted throughout the trachea.

For the adult iSox2CC10-rtTA or iSox21CC10-rtTA experiments, the number of ciliated (FOXJ1+), secretory (SCGB1A1+), and basal (TRP63+) were counted in the trachea around cartilage ring (C) 1, C6, and C10. Of each animal, one section was counted.

The number of FOXJ1+ nuclei in the MTEC cultures were counted per 775 µm2. For determining, the differentiation to secretory cells, the percentage of SCGB3A2+ area per 775 µm2 was measured. The number of TRP63+ basal and TRP73+ cells were counted, respectively, to the number of nuclei present in each field. Each n are separate isolations of different animal, and per n, 5 fields of 775 µm2 were counted.

ALI HPBEC cultures of four different donors were transduced with viruses to express shRNAs against Sox2 or Sox21. Of each transduced culture, the number of FOXJ1+ nuclei and Cherry+ nuclei on three fields of 375.5 µm2 was counted. The percentage of positive area MUC5B, TUBIV, and Cherry were measured on three fields of 375.5 µm2 per insert.

RNA isolation, cDNA synthesis, and qRT-PCR analysis

Request a detailed protocol

Human or mouse airway cells were removed from the insert by scraping them off the insert into cold PBS. Cells were collected in an Eppendorf tube and centrifuged at 800 g for 5 min at 4°C. PBS was aspirated, and the cell pellet was snap frozen in liquid nitrogen and stored at −80°C till RNA isolation.

To isolate RNA, 500 µl of TRI Reagent (Sigma, T9424) was added to the cell pellet. RNA extraction was performed according to the TRI Reagent protocol. RNA concentrations were measured using NanoDrop (ThermoFisher Scientific). First strand cDNA was synthesized using 2 µg RNA, MLV Reverse transcriptase (Sigma, M1302), and Oligo-dT primers (self-designed: 23xT+A, 23xT+C, and 23xT+G). For one qRT–PCR, 0.5 µl of cDNA was used with Platinum Taq polymerase (Invitrogen, 18038042) and SybrGreen (Sigma, S9430). The primer combinations for the qRT-PCR are listed in Table 2. Normalized gene expression was calculated using the ddCT method relative to GAPDH (mouse) or B-ACTIN (human) control.

Table 2
RT-PCR primers.
RT-primers
GeneForward (5’→3’)Reverse (5’→3’)Species
Foxj1CAGACCCCACCTGGCAGAATTCAAAGGCAGGGTGGATGTGGACTMouse
Gapdh (housekeeping)CCTGCCAAGTATGATGACATGTCCTCAGTGTAGCCCAAGMouse
Krt5TACCAGACCAAGTATGAGGAGTGGATCATTCGGTTCATCTCAGMouse
Scgb1a1GCAGCTCAGCTTCTTCGGACATCCTGGTCTCTTGTGGGAGGGMouse
Scgb3a2GTGGTTATTCTGCCACTGCCCTTTCGTCCACACACTTCTTCAGTCCMouse
Sox2AACATGGCAATCAAATGTCTTGCCAGTACTTGCTCTCATMouse
Sox21TTGAAAGATGCCTCTCACCAAATAAGCTAAATGGGAAGGGAGMouse
Trp63GGAAAACAATGCCCAGACTCGATGGAGAGAGGGCATCAAAMouse
Actin (housekeeping)ATTGGCAATGAGCGGTTCGGATGCCACAGGACTCCATHuman
Foxj1CCCACCTGGCAGAATTCAATCCGCAGTCGCCGCTTCTTGAAAGCHuman
Scgb1a1GCTCCGCTTCTGCAGAGATCTGGCTTTTGGGGGAGGGTGTCCAHuman
Scgb3a1TGCTGGGGGCCCTGACAACGTTTATTGAGAGGGGCCGGHuman
Sox2AATGCCTTCATGGTGTGGTCTTGCTGATCTCCGAGTTGTGHuman
Sox21CCACTCGCTTGGATTTCTGACACATCGACTCAAACTTAGGGCAACGAHuman
Trp63CCACCTGGACGTATTCCACTGTCGAATCAAATGACTAGGAGGGGHuman

Luciferase assay

Cloning

Request a detailed protocol

Promotor regions were PCR amplified (primers listed in Table 3) from mouse genomic DNA. To each primer, a restriction site was added, to clone the promotor region into the pGL4.10 [luc2] construct (Promega; E6651). The promotor sequence included the sequence of a transcriptional active area, which was identified with a RNA polymerase II ChIP in MTECs (Marshall et al., 2016). We used in silico analysis to predict SOX21 binding sites (MatInsepctor, GenomatixSuite v3.10 and http://jaspar2016.genereg.net/).

Table 3
Primers used for pGL4.10[luc2] cloning.
Primers used for pGL4.10[luc2] cloning
NamePrimers (5’→3’) cut siteCut site
Trp63Forward: CAGGGTACCGGGCACATTCCATCTTTCCTKpnI
Reverse: CAGCTCGAGAGACTGGTCAAGGCTGCTCTXho
Sox2Forward: CAGGGTACCCGCGAGAGTATTGCAGGGAAKpnI
Reverse: CAGGCTAGCCGGAGATCTGGCGGAGAATANheI
Trp73Forward: CAGGGTACCGGACACGCATCTGTTGTGGAKpnI
Reverse: CAGCTCGAGTCTGCACACGCTGAGGAGCTXho

Luciferase assay

Request a detailed protocol

HELA cells were plated in a 12-well plate in DMEM + 10% FCS and transiently transfected using Lipofectamine3000 (Thermofisher, L3000001). Each transfection consisted of 250 ng expression plasmid pcDNA3-control (Addgene; n/a), pcDNA3-Sox2FLAG (homemade), pcDNA3-Sox21MYC (homemade), 250 ng pGL4.10[luc2] reporter plasmid, and 2.5 ng TK-Renilla plasmid (Promega; E2241) (transfection control). Luciferase activity was measured 48 hr after transfection using the Dual-Luciferase Reporter Assay System (Promega, E1910). Plate reader VICTOR X4 was used to measure Firefly and Renilla luminescence. Firefly luminescence of each sample was calculated by dividing the Firefly luminescence by the Renilla luminescence. The increase or decrease of luciferase activity was then normalized to the pGL4.10[luc2] reporter plasmid transfected with the pcDNA3-control expression plasmid.

Western blot

Request a detailed protocol

Samples were prepared from cell lysates used for luciferase measurements. Cells were lysed in the lysis buffer that was included in the Dual-Luciferase Reporter Assay System (Promega, E1910). To the cell lysate, 8M urea (to denature the DNA), 50 mM 1,4-dithiothreitol (DTT, Sigma), and 1× SDS sample buffer was added. Samples were boiled and loaded on a 12% SDS-polyacrylamide gel and blotted onto a PVDF membrane (Immobilon-P transfer membrane, Millipore). The blots were blocked for 1 hr in PBS containing 0.05% Tween-20% and 3% BSA at RT, and probed overnight with primary antibodies at 4°C (Key resources table). Next day, membranes were washed three times with PBS containing 0.05% Tween-20 and incubated for 1 hr with horseradish peroxidase (HRP)-conjugated secondary antibodies (DAKO) at a dilution of 1:10,000. Signal was detected with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). Blots were developed using the Amersham Imager 600GE (GE Healthcare).

Knockdown of SOX2 and SOX21 in HPBEC ALI cultures

Lentiviral production

Request a detailed protocol

Lentiviral constructs containing an U6 promoter to express shRNAs targeted at SOX2 or SOX21 were ordered at Genecopoeia (HSH110118-LVRU6MP-a to c, HSH091508-LVRU6MP-a to c). Three constructs targeting different parts of SOX2 or SOX21 were used, and a non-targeting scrambled shRNA construct was used as negative control (CSHCTR001-1-LVRU6MP; referred to as shScrm). To visualize efficient transduction into HPBECs, all plasmids contained a mCherry expression cassette to visualize transduced cells.

Lentivirus was generated by transfection of HEK293T cells. A 10 cm dish of HEK293T cells was transfected using 100 µg PEI with 20 µg shRNA plasmid, 15 µg Pax2 (packaging vector), and 5 µg VSV-G (envelope vector). After 4 hr, medium was replaced with 8 ml DMEM + 10% FCS + 1× pen/strep. Medium was collected and refreshed after 24 hr, 48 hr, and 72 hr. The collected medium was centrifuged at 1500 rpm for 5 min, and supernatant was filtered through a 0.45 µm filter to discard all remaining cells. To collect lentiviral particles, the supernatant was centrifuged at 20,000 rpm, at 4°C for 135 min. Virus pellets were resuspended in 100 µl BEGM medium.

Knockdown of SOX2 or SOX21 in HPBEC ALI cultures

Request a detailed protocol

1 × 105 HPBECs were seeded onto 12 mm inserts, 0.4 µm pore (Corning Inc, Corning, USA), and grown submerged until confluent. The HPBECs were transduced with 10–20 µl lentiviral particles in 500 µl BEGM + 1 nM EC23 (retinoic acid analog, Sigma; SML2404) + 2 µg/ml Polybrene in the apical compartment of the insert. After 24 hr, the apical compartment was washed once with PBS and the PBECs were put to ALI. Medium was changed in the lower chamber to BEGM + 50 nM EC23. Transduced HPBECs were cultured on ALI for 14 days, after which they were fixed and processed for immunofluorescence staining.

Statistics

Statistical analysis was performed using Prism5 (GraphPad). For all measurement, three or more biological replicates were used. Data are represented as means ± standard error of mean (SEM) with the data points present in each graph. Statistical differences between samples were assed with Student’s unpaired or paired t-test, one-way ANOVA (post-test: Tukey), or two-way ANOVA (post-test: Bonferroni). p-values below 0.05 are considered significant. The number of replicates and statistical tests used are indicated in the figure legends.

Single-cell RNA sequencing of HPBEC

Request a detailed protocol

Expression levels of SOX2 and SOX21 in ALI cultures of HPBEC were analyzed using the dataset previously published (Plasschaert et al., 2018). The dataset is available at the Klein Lab SPRING viewer.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1 to 5, and 7.

References

Decision letter

  1. Melanie Königshoff
    Reviewing Editor; University of Pittsburgh, United States
  2. Edward E Morrisey
    Senior Editor; University of Pennsylvania, United States
  3. Stijn De Langhe
    Reviewer; University of Alabama at Birmingham, United States
  4. Finn Hawkins
    Reviewer; Boston University and Boston Medical Center, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This study aims at advancing our understanding about the molecular mechanisms that control airway stem cell maintenance and differentiation. The analysis focuses on Sox21 in lung development, homeostasis, and injury, using wide array of methods including mouse human cell-based ALI cultures and organoids. The authors describe a new role for Sox21 downstream of the key airway development transcription factor Sox2. They suggest opposing roles for these genes whereby Sox21 prevents and Sox2 promotes differentiation of immature airway progenitors into basal cells. This study thus describes a role of Sox21 in the (patho)biology of airway progenitors and the balance of cell types in the airways.

Decision letter after peer review:

Thank you for submitting your article "SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Edward Morrisey as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Stijn De Langhe (Reviewer #2); Finn Hawkins (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

This is a comprehensive analysis of Sox21 in lung development, homeostasis, and injury, using wide array of methods including mouse human cell-based ALI cultures and organoids. The authors describe a new role for Sox21 downstream of the key airway development transcription factor Sox2. They suggest opposing roles for these genes based on analysis of Sox2 and Sox21 heterozygous knock-out mice whereby Sox21 prevents and Sox2 promotes differentiation of immature airway progenitors into basal cells. This raises interesting questions regarding the role of Sox21 in the biology of airway progenitors and the balance of cell types in the airways. In adult mice no obvious differences are detected in Sox21+/- mice in terms of regenerative capacity. In in vitro assays of adult mouse basal cell differentiation (ALI culture) failed to identify obvious differences in basal cell number of differentiation into specific lineages in Sox21+/- cells. In human ALI cultures the authors identify a similar possible role for Sox21 in differentiating basal cells in humans. Overall, the molecular mechanisms that control airway stem cell maintenance and differentiation are of great interest to the field. However, in its current form the studies remain primarily descriptive and several data inconsistencies remain, which makes it difficult to draw impactful conclusions at the current stage. The potential suitability for eLife would significantly increase with additional mechanistic experiments and data.

Essential revisions:

In particular, the following major points require additional experimental data (details are outlined below):

1) The functional Sox21 role in the adult lung is not clear.

– Mouse studies would benefit from inclusion of overexpression of Sox21 in the airways.

– In human airways and the models used (organoids and ALI cultures) gain and loss of function studies are not performed and would also behelpful to further determine the phenotype of the intermediate para-basal cells in the human airway.

2) Evidence for genetic interaction between Sox21 and Sox2 in the adult lung needs to be expanded.

3) Presence of Sox9 positive basal cells as additional atypical basal cells needs to be further investigated.

4) Quantification the % of club cells in the corn oil and naphthalene treated tracheas of the different mutants as these are the main cells that should be replaced upon naphthalene injury.

Reviewer #2:

This is an interesting but also a bit confusing manuscript by Evelien Eenjes and colleagues.

I was wondering whether the seemingly conflicting effect of Sox21 on basal cell differentiation could be due to an increase or decrease in Sox9 positive basal cells. These atypical basal cells have been described in a number of manuscripts. For example https://www.ncbi.nlm.nih.gov/pubmed/26869074.

It appears from the figures that Sox21 may increase Sox9 expression. Can the authors look at the presence of Sox9 positive basal cells in the trachea of the different mutants?

It would be important to quantify the % of club cells in the corn oil and naphthalene treated tracheas of the different mutants as these are the main cells that should be replaced upon naphthalene injury. I am not sure that people normally look at the number of ciliated cells reappearing after naphthalene injury as ciliated cells are not thought to be killed by naphthalene.

The authors should also look at Sox9 positive basal cells in the tracheas of the corn oil and naphthalene treated ctrl and mutant mice.

Are the intermediate para-basal cells in the human airway differentiation experiment club cells? Why do the authors think these cells are on their way to become ciliated cells? Doesn't Sox21 inhibit ciliated cell differentiation?

Reviewer #3:

In their manuscript titled "SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways" from Eenjes et al. the authors describe a new role for Sox21 in the developing and adult airway epithelium. Building on prior work they observe a unique distribution of Sox21 expression in the developing airways and through careful and elegant immunostaining and over-expression studies they suggest that Sox21 is downstream of a key airway development transcription factor Sox2. They suggest opposing roles for these genes based on analysis of Sox2 and Sox21 heterozygous knock-out mice whereby Sox21 prevents and Sox2 promotes differentiation of immature airway progenitors into basal cells. This raises interesting questions regarding the role of Sox21 in the biology of airway progenitors and the balance of cell types in the airways. In adult mice no obvious differences are detected in Sox21+/- mice in terms of regenerative capacity. In in vitro assays of adult mouse basal cell differentiation (ALI culture) failed to identify obvious differences in basal cell number of differentiation into specific lineages in Sox21+/- cells. The authors report an overall similar pattern of SOX21 expression in a human fetal lung organoid platform. Finally, in human ALI cultures the authors identify SOX21 expression most abundant in paranasal cells suggesting a similar possible role for this genes in differentiating basal cells in humans. Overall, the molecular mechanisms that control airway stem cell maintenance and differentiation are of great interest to the field.

The development data is clear based on the studies presented. The immunostaining and figures are elegant. It does not appear the Sox21 is necessary for airway development. A key question raised by this work is how important and what precisely is the role of Sox21? For example, overexpression of Sox21 in the developing and adult airways might have been more instructive in answering these key questions rather than under the control of the SPC-promoter.

The adult mouse data and human data seem to overall suggest that SOX2 and SOX21 are upregulated in differentiating cells in vitro and are expressed at lower levels in basal cells but real mechanism or phenotype related to SOX21 function is observed in the SOX21 het mouse cells.

The attempt to assess human fetal airways is admirable. However, sections of fetal lungs would be more relevant. Can the authors address how similar fetal lung cultures are to in vivo airways and at what developmental time point ?

Overall the relevance of SOX21 expression in the adult airways is unclear.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Edward Morrisey as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Finn Hawkins (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

The manuscript improved significantly several additional experiments included that have been ask for in the first revision. These experiments further clarified and support the dynamic and context-dependent role for Sox21 in airway cell differentiation. No further experimental data are required, however, some textual changes to clarify the results and enhance readability.

1) The manuscript is still very hard to read, especially given the variety in model systems and different results based on these. Please carefully revise and modify the text accordingly to clarify the results and enhance readability.

2) Adding 1-2 small figures to explain / visualize the experimental setups/ findings would also be helpful.

Reviewer #1:

The manuscript has significantly improved. Additional experiments have been performed including in vivo over expression of Sox21 and Sox 2 (Figure 4) as well as loss of function studies for Sox21 in Figure 7. These data further support a role for Sox21 in inhibiting differentiation of airway progenitors and adult basal cells. Specifically, airway cell transitioning from basal cells to a luminal para-basal cell while cilia cell transition/maturation is suppressed by Sox21. The manuscript is still very hard to read and textual clarification is needed.

Reviewer #2:

This article is very difficult to read. As currently written I do not understand it.

Reviewer #3:

This manuscript is of relevance to research into lung development and the mechanisms that relegate the maintenance and differentiation of progenitor cells in the airway epithelium.

The authors include several new experiments in response to concerns raised in the initial submission. The resubmission is significantly revised.

To address the role of SOX21 in airways in development and in adult mice under homeostatic conditions the authors over-expressed SOX21under the control of the SCGB1A1 promoter in mice using an inducible system. Over-expression during development led to an increase in basal cells. Over-expression of SOX21 in adult secretory cells led to fewer secretory cells not did not alter basal cell numbers. Interestingly, in both experiments the majority of cell over expressing the MYC-tag were not clearly secretory, basal or ciliated cells. While the experiments are helpful in evaluating the functional role of SOX21 in the airways the latter observation is troubling and not really addressed.

Concern in initial submission: The adult mouse data and human data seem to overall suggest that SOX2 and SOX21 are upregulated in differentiating cells in vitro and are expressed at lower levels in basal cells but (no) real mechanism or phenotype related to SOX21 function is observed in the SOX21 het mouse cells. In response the authors performed knockdown of SOX21 and SOX2 in human ALI cultures which suggest a role for both genes in suppressing the differentiation of basal cells to ciliated cells. While an increase in FOXJ1 was detected in these knockdown experiments, the authors did not detect an increase in multiciliated cells.

In addition to assessment of SOX21 in human fetal lung organoids sections of human fetal lung and adult human lung are now included.

Overall, the authors have performed several experiments that have improved the manuscript. It appears that the role of SOX21 is context dependent and despite the authors detailed experiments the biology remains complicated to interpret.

https://doi.org/10.7554/eLife.57325.sa1

Author response

Essential revisions:

In particular, the following major points require additional experimental data (details are outlined below):

1) The functional Sox21 role in the adult lung is not clear.

– Mouse studies would benefit from inclusion of overexpression of Sox21 in the airways.

We added the analysis of the effects of overexpressing SOX21 (and SOX2) on the epithelial differentiation of the airways late in gestation at E18.5 in figure 3E. Moreover, we studied the effect of SOX21 (and SOX2) overexpression during adult homeostasis using additional mouse experiments, and added figure 4E and figure 4—figure supplement 1E to the manuscript to discuss the results.

– In human airways and the models used (organoids and ALI cultures) gain and loss of function studies are not performed and would also behelpful to further determine the phenotype of the intermediate para-basal cells in the human airway.

We have performed SOX2 and SOX21 knock down experiments in our human Air Liquid Interface (ALI) cultures and added the data to figure 7 and figure7-supplement 1.

2) Evidence for genetic interaction between Sox21 and Sox2 in the adult lung needs to be expanded.

For the genetic interaction, we have tried to obtainSox2+/-Sox21-/- mice, but the main difficulty resides in the fact that it is nearly impossible to breed these mice, and we did not obtain (sufficient) mice to perform reasonable experiment. We did analyze Sox2+/-, Sox21+/-, and Sox21-/- E18.5 lungs, which we added to the manuscript (figure 3E, supplemental figure 1E). We obtained only one Sox2+/-Sox21+/- mouse pup at E18.5, of which the lungs were analyzed. Since this is only one sample, we did not include it in the manuscript, but we like to share these preliminary data with the reviewer (see Author response image 1). It appears that the Sox2+/-Sox21+/- lung is comparable to the individual Sox2 and Sox21 heterozygous lungs.

Author response image 1
Download asset Open asset
Bar graphs representing the percentage of FOXJ1+, TRP63+ and SCGB1A1+ cells in E18.

5 lungs of WT, Sox2Sox2+/-, Sox21+/-, Sox21-/- and Sox2Sox2+/-Sox21+/- mice.

Previously, we observed a direct interaction between SOX2 and SOX21, which was already published by others (Mallanna et al., Stem Cells 28, 1715-1727, 2010). Thus, there is a direct link between the two proteins. Moreover, we performed SOX2 and SOX21 ChIP-seq experiments using human ALI cultures. However, we will not report on the full data set (yet), but have included four genome tracks to show single SOX2 or SOX21, and double SOX2-SOX21 occupancy at different genomic loci (Figure 7—figure supplement 1H). We included the supplemental figure, and refer to the data in the discussion line (452-457), to underly the importance of studying the interaction between these proteins further. Of note, the human ALI system is a culture method with a diverse range of cell types and all cell types are in different status of differentiation. It is challenging to study the interaction and function(s) of SOX2 and SOX21 at the cellular level. As described previously and in the discussion (line 446-448), the interaction of both SOX2 and SOX21 are highly context dependent, emphasizing the importance of obtaining a suitable system to study this interaction in the human and mouse airway epithelium.

3) Presence of Sox9 positive basal cells as additional atypical basal cells needs to be further investigated.

We have further investigated the putative presence of SOX9+ basal cells in our experiments, and found that there are some rare SOX9+ basal-like cells. To acknowledge these findings, we have adapted the text (line 178-184) and added supplemental figure2-supplement 2E. As SOX9+ basal cells lack expression of the KRT5 basal marker (Utisyan 2016), we added a KRT5 staining of Sox2+/-, Sox21+/- and Sox21-/- E18.5 lungs to analyze the number of basal cells (Figure 3E and figure 3 supplement 1E).

4) Quantification the % of club cells in the corn oil and naphthalene treated tracheas of the different mutants as these are the main cells that should be replaced upon naphthalene injury.

We have quantified the percentage of Club cells before and after the treatment with naphthalene, and we did not observe significant differences between the different genotypes, or between treated and non-treated animals. We have included these data in figure 5D

Reviewer #2:

This is an interesting but also a bit confusing manuscript by Evelien Eenjes and colleagues.

I was wondering whether the seemingly conflicting effect of Sox21 on basal cell differentiation could be due to an increase or decrease in Sox9 positive basal cells. These atypical basal cells have been described in a number of manuscripts. For example https://www.ncbi.nlm.nih.gov/pubmed/26869074.

It appears from the figures that Sox21 may increase Sox9 expression. Can the authors look at the presence of Sox9 positive basal cells in the trachea of the different mutants?

The reviewer points out an interesting observation. In Figure 1C we indeed find that when Sox21 is induced in the lung bud, SOX9 remains expressed. Furthermore, in the airway we find some iSOX21 cells that are still low in expression of SOX9, but these are scarce and not all iSOX21 expressing cells do.

We did find the presence of TRP63 expressing SOX9+ basal cells in the SPC induced animals. However, we did not elaborate! As described by Ustiyan et al., the observed SOX9+ TRP63+ atypical basal cells are KRT5 negative. We included therefore KRT5 in our staining panel to analyze the number of basal cells in the proximal airways upon induction of Sox2 or Sox21 using the CC10 rTta (Figure 2B-E). All basal cells analyzed were also positive for KRT5. KRT5 was also added to analyze the number of basal cells in Sox2+/-, Sox21+/- and Sox21-/- mice at E18.5 (Figure 3E).

We have included these findings in the text and figures:

“Previously, conditional deletion of βcatenin in SPC+ cells during lung development showed the appearance of cystic structures, similar to the induction of SOX2 and SOX21. […] Similarly we also found SOX9+ basal cells in the iSox2SPC-rtTA and iSox21SPC-rtTA mouse lungs, and sporadic SOX9+ basal cells were also detected in the control (Figure 2—figure supplement 1E).”

It would be important to quantify the % of club cells in the corn oil and naphthalene treated tracheas of the different mutants as these are the main cells that should be replaced upon naphthalene injury. I am not sure that people normally look at the number of ciliated cells reappearing after naphthalene injury as ciliated cells are not thought to be killed by naphthalene.

The reviewer has indeed a good point, and we have quantified the number of Club cells before and after treatment with naphthalene. We have adapted the figure and the text accordingly:

“To determine whether SOX2 or SOX21 deficiency affects regeneration after naphthalene injury, we quantified the percentage of secretory, ciliated, non-dividing and dividing basal cells at 20 DPI. Fewer FOXJ1+ ciliated cells were observed in Sox2+/- TE compared to WT, Sox21+/- , or to Sox2+/- CO exposed mice (Figure. 5D, Figure 5-supplement 1D).”

The authors should also look at Sox9 positive basal cells in the tracheas of the corn oil and naphthalene treated ctrl and mutant mice.

The SOX9+ basal cells is missing in the analysis and unfortunately were not able to add it to the naphthalene experiments, due to lack of material to quantify. However, we did stain TRP63 and SOX9 at 5 days post injury and SOX9+ cells are present at the surface epithelium together with TRP63+ basal cells , we cannot exclude that these basal cells are SOX9+ and whether there is a difference. However, at day 5 post-injury we did not see a difference in the number of basal cells between genotypes but a small difference in SOX9+ cells.

Are the intermediate para-basal cells in the human airway differentiation experiment club cells? Why do the authors think these cells are on their way to become ciliated cells? Doesn't Sox21 inhibit ciliated cell differentiation?

The SOX21 high expressing cells in the ALI culture did not correlate with SCGB1A1 expression (Author response image 2, n=1). We did not continue these experiments, as the single cell RNA seq of human ALI cultures shows a high expression of SOX21 in para-basal cells (Figure 7- figure supplement 1E) and the localization of high expressing SOX21 on human sections are found in the intermediate layer (Figure 6D). As there are some FOXJ1+ nuclei high in expression of SOX21 (Figure 7—figure supplement 1D) we interpreted the data as cells transition to ciliated cells. We observed with the added knock-down experiments that SOX21, similar as in mouse, inhibits ciliated cell differentiation in human ALI cultures. We excluded this possibly premature statement from the manuscript and now state the following:

“We hypothesize that these cells are intermediate cells transitioning from basal cells to a luminal cell fate, the para-basal cell. […] This analysis confirmed that high levels of SOX21 mRNA denote an intermediate cell type.”

Author response image 2
Download asset Open asset
Intensity of SOX2 or SOX21 was measured in nuclei of SCGB3A1+ cells and SCGB3A1- cells.

Circles were put around nuclei that were SCGB3A1+. Staining of SCGB3A1 was analyzed throughout the z-stack.

Reviewer #3:

[…] The development data is clear based on the studies presented. The immunostaining and figures are elegant. It does not appear the Sox21 is necessary for airway development. A key question raised by this work is how important and what precisely is the role of Sox21? For example, overexpression of Sox21 in the developing and adult airways might have been more instructive in answering these key questions rather than under the control of the SPC-promoter.

The reviewer raises a good point, and indeed, the SOX21 knockout mice suggests that SOX21 is not necessary for lung development. However, SOX21 has a clear influence on the distribution of the different cell types, as we report.

Initially, we choose to use the SPC-driven rtTA, as this transgene is expressed early in development in most epithelial cells (e.g. Gontan et al., 2008). These data were included in the original manuscript. We have now expanded this data set using a mouse model to induce SOX2 or SOX21 in differentiating epithelial cells, using the rtTA under the control of the CC10 promoter. This resulted in additional embryonic data, as well as new adult data, which we have included. For the embryonic data, we included the following text and accompanying figures:

“To further investigate the role of SOX2 and SOX21 in differentiation of airway epithelium during development, we induced expression of the MYC-tagged Sox2 or Sox21 transgenes in secretory cells using a CC10/SCGB1A1 driven rtTA inducible model (iSox2CC10-rtTA and iSox21CC10-rtTA) (Tichelaar, Lu et al. 2000). […] So, ectopic expression of SOX2 and SOX21 during development stimulates the appearance of basal cells and decrease the number of ciliated cells.”

And for the adult data set:

“To further understand the role for SOX2 and SOX21 in adult airway epithelium, we used the iSox2CC10-rtTA and iSox21CC10-rtTA mice to induce expression of Sox2 or Sox21 in secretory cells for 6 weeks (Figure 4E). […] Similar as during development, the MYC+ transgene expressing cells were mostly negative for other cell specific markers in both mouse models (Figure 4—figure supplement 1E).”

The adult mouse data and human data seem to overall suggest that SOX2 and SOX21 are upregulated in differentiating cells in vitro and are expressed at lower levels in basal cells but real mechanism or phenotype related to SOX21 function is observed in the SOX21 het mouse cells.

We are not sure what the exact question is of the reviewer. In light of this, we have now expanded the human data with knock down experiments using shRNA constructs in human ALI cultures (figure 7 and figure 7—figure supplement 1). These data show comparable effects of reduced SOX2 or SOX21 as observed in the mouse cells.

The attempt to assess human fetal airways is admirable. However, sections of fetal lungs would be more relevant. Can the authors address how similar fetal lung cultures are to in vivo airways and at what developmental time point ?

We have performed immunofluorescence staining on human sections throughout gestation and included these in figure 6A. We have restructured the figures, so figure 6 now displays human fetal lung sections, organoids (fetal tip organoids and differentiated organoids), and adult bronchi. The ALI cultures we have used for the mechanistic experiments mostly resemble the adult bronchus.

Overall the relevance of SOX21 expression in the adult airways is unclear.

We have performed additional experiments to investigate the role of SOX2 and SOX21 in the differentiating human epithelium by shRNA knockdown experiments, described in the text, figure 7 and supplemental figure 6. We have added the following text:

“Next, we assessed the effects of reduced levels of SOX2 or SOX21 on human basal cell differentiation by transducing ALI cultures with lentiviruses expressing short hairpin RNAs (shRNA) to knockdown SOX2 or SOX21. […] Overall, our data suggest that both SOX2 and SOX21 suppress basal to ciliated cell differentiation, while both seem to have a positive role in the differentiation of goblet cells in the human airways.”

Combined with the mouse data (Sox2+/-, Sox21+/- and Sox21-/-; Naphthalene; CC10rTTA adult), we hope to better indicate the relevance of SOX21 in the adult airways.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Essential revisions:

The manuscript improved significantly several additional experiments included that have been ask for in the first revision. These experiments further clarified and support the dynamic and context-dependent role for Sox21 in airway cell differentiation. No further experimental data are required, however, some textual changes to clarify the results and enhance readability.

1) The manuscript is still very hard to read, especially given the variety in model systems and different results based on these. Please carefully revise and modify the text accordingly to clarify the results and enhance readability.

We agree with the reviewers that the inclusion of new experimental data in the revised version resulted in loss of clarification of the some of our data, which regrettably led to reduced readability. We have carefully revised the new manuscript, and made several textual changes to enhance readability. To do so, we have consulted three independent researchers, including a native speaker, to better foreword our data, conclusions and discussion. We hope the reviewers agree that the manuscript has indeed been improved. Since we have made several changes, we have included a marked-up version to better highlight the changes to the manuscript.

2) Adding 1-2 small figures to explain / visualize the experimental setups/ findings would also be helpful.

We have included two new panels (figure 2F and figure 4F) to summarize and highlight our findings. We believe that these two extra cartoons indeed clarify the data in an appropriate manner, and thank the reviewers for this suggestion. Moreover, throughout the manuscript’s figures and figure supplements, we have several cartoons to explain our experimental setup (e.g. 2B, 4A, 4E, 5B, 7A), as well as three data-summarizing cartoons (Figures 3G, Figure 2-supplement 1D, Figure 4-supplement 1D).

In addition, we have made several (minor) changes to the figures to comply with the changes in the text:

Figure 1

Panel A: Add “Trachea” and “Carina”

Panel C: Add the duration of Dox treatment Figure 1—figure supplement 1:

Panel B: changed text color of DOX to red

Panel C: Line made red to show that the mice received doxycycline from E6 to E18

Figure 2

Panel B: Removed “1st branch” above cartoon, added “Carina”

Panel F: New scheme to illustrate the expression of MYC-SOX2 or MYC-SOX21 in secretory cells during development.

Figure 4

Panel F.: Added a scheme to illustrate the expression of MYC-SOX2 or MYC-SOX21 in SCGB1A1+ secretory cells in adult tracheal epithelium.

Figure 7

Panel B: Changed “luminal” to “apical” Figure 7—figure supplement 1:

Panel C: Changed graph y-ax to apical.

https://doi.org/10.7554/eLife.57325.sa2

Article and author information

Author details

  1. Evelien Eenjes

    Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, Netherlands
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared

  2. Marjon Buscop-van Kempen

    Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, Netherlands
    Contribution
    Software, Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared

  3. Anne Boerema-de Munck

    Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, Netherlands
    Contribution
    Software, Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared

  4. Gabriela G Edel

    Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, Netherlands
    Contribution
    Data curation, Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared

  5. Floor Benthem

    Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, Netherlands
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared

  6. Lisette de Kreij-de Bruin

    Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, Netherlands
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared

  7. Marco Schnater

    Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, Netherlands
    Contribution
    Validation, Writing - review and editing
    Competing interests
    No competing interests declared

  8. Dick Tibboel

    Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, Netherlands
    Contribution
    Funding acquisition, Writing - review and editing
    Competing interests
    No competing interests declared

  9. Jennifer Collins

    Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, Netherlands
    Contribution
    Validation, Writing - review and editing
    Competing interests
    No competing interests declared

  10. Robbert J Rottier

    1. Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, Netherlands
    2. Department of Cell biology, Erasmus Medical Center, Rotterdam, Netherlands
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    r.rottier@erasmusmc.nl
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9291-4971

Funding

Sophia Foundation for Medical Research (S14-12)

  • Evelien Eenjes

Sophia Foundation for Medical Research (S17-20)

  • Gabriela G Edel

Sophia Foundation for Medical Research (S16-17)

  • Jennifer Collins

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank, Mart Lamers and Bart Haagmans (Department of Viroscience, Erasmus MC) for assistance and supplying the human fetal lung organoids, Thomas Koudstaal (department of Pulmonary Medicine, Erasmus MC) for supplying lung resection material for the isolation of human primary bronchial epithelial cells, Rutger Brouwer (Department of Biomics, Erasmus MC) for Figure 7-supplement 6H and Frank Grosveld for critically reading the manuscript (Department of Cell Biology, Erasmus MC). This work was supported by grants from the Sophia Foundation for Medical Research (grant numbers S14-12 EE; S17-20 GGE; S16-17 JJPC).

Ethics

Animal experimentation: All animal experimental protocols were approved the national committee ("Centrale Commissie Dierproeven"; number AVD101002017871) and by the animal welfare committee of the veterinary authorities (prtocol numbers 17-871-01 and 17-871-02) of the Erasmus Medical Center.

Senior Editor

  1. Edward E Morrisey, University of Pennsylvania, United States

Reviewing Editor

  1. Melanie Königshoff, University of Pittsburgh, United States

Reviewers

  1. Stijn De Langhe, University of Alabama at Birmingham, United States
  2. Finn Hawkins, Boston University and Boston Medical Center, United States

Version history

  1. Received: March 27, 2020
  2. Preprint posted: June 4, 2020 (view preprint)
  3. Accepted: July 20, 2021
  4. Accepted Manuscript published: July 21, 2021 (version 1)
  5. Version of Record published: August 3, 2021 (version 2)

Copyright

© 2021, Eenjes et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,139
    Page views
  • 166
    Downloads
  • 8
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Evelien Eenjes
  2. Marjon Buscop-van Kempen
  3. Anne Boerema-de Munck
  4. Gabriela G Edel
  5. Floor Benthem
  6. Lisette de Kreij-de Bruin
  7. Marco Schnater
  8. Dick Tibboel
  9. Jennifer Collins
  10. Robbert J Rottier
(2021)
SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways
eLife 10:e57325.
https://doi.org/10.7554/eLife.57325

Categories and tags

Research organisms

Further reading

    1. Cell Biology

    PM2.5 leads to adverse pregnancy outcomes by inducing trophoblast oxidative stress and mitochondrial apoptosis via KLF9/CYP1A1 transcriptional axis

    Shuxian Li, Lingbing Li ... Xietong Wang
    Research Article

    Epidemiological studies have demonstrated that fine particulate matter (PM2.5) is associated with adverse obstetric and postnatal metabolic health outcomes, but the mechanism remains unclear. This study aimed to investigate the toxicological pathways by which PM2.5 damaged placental trophoblasts in vivo and in vitro. We confirmed that PM2.5 induced adverse gestational outcomes such as increased fetal mortality rates, decreased fetal number and weight, damaged placental structure, and increased apoptosis of trophoblasts. Additionally, PM2.5 induced dysfunction of the trophoblast cell line HTR8/SVneo, including in its proliferation, apoptosis, invasion, migration and angiogenesis. Moreover, we comprehensively analyzed the transcriptional landscape of HTR8/SVneo cells exposed to PM2.5 through RNA-Seq and observed that PM2.5 triggered overexpression of pathways involved in oxidative stress and mitochondrial apoptosis to damage HTR8/SVneo cell biological functions through CYP1A1. Mechanistically, PM2.5 stimulated KLF9, a transcription factor identified as binding to CYP1A1 promoter region, which further modulated the CYP1A1-driven downstream phenotypes. Together, this study demonstrated that the KLF9/CYP1A1 axis played a crucial role in the toxic progression of PM2.5 induced adverse pregnancy outcomes, suggesting adverse effects of environmental pollution on pregnant females and putative targeted therapeutic strategies.

    1. Cell Biology
    2. Microbiology and Infectious Disease

    PerTurboID, A targeted in situ method reveals the impact of kinase deletion on its local protein environment in the cytoadhesion complex of malaria causing parasites

    Heledd Davies, Hugo Belda ... Moritz Treeck
    Tools and Resources

    Reverse genetics is key to understanding protein function, but the mechanistic connection between a gene of interest and the observed phenotype is not always clear. Here we describe the use of proximity labeling using TurboID and site-specific quantification of biotinylated peptides to measure changes to the local protein environment of selected targets upon perturbation. We apply this technique, which we call PerTurboID, to understand how the P. falciparum exported kinase, FIKK4.1, regulates the function of the major virulence factor of the malaria causing parasite, PfEMP1. We generated independent TurboID fusions of 2 proteins that are predicted substrates of FIKK4.1 in a FIKK4.1 conditional KO parasite line. Comparing the abundance of site-specific biotinylated peptides between wildtype and kinase deletion lines reveals the differential accessibility of proteins to biotinylation, indicating changes to localization, protein-protein interactions, or protein structure which are mediated by FIKK4.1 activity. We further show that FIKK4.1 is likely the only FIKK kinase that controls surface levels of PfEMP1, but not other surface antigens, on the infected red blood cell under standard culture conditions. We believe PerTurboID is broadly applicable to study the impact of genetic or environmental perturbation on a selected cellular niche.

    1. Cell Biology

    CRB3 navigates Rab11 trafficking vesicles to promote γTuRC assembly during ciliogenesis

    Bo Wang, Zheyong Liang ... Peijun Liu
    Research Article

    The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. Crb3 knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific Crb3 knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.