Na+/H+ Exchangers Are Required for the Development and Function of Vertebrate Mucociliary Epithelia

Abstract

Na+/H+ exchangers (NHEs) represent a highly conserved family of ion transporters that regulate pH homeostasis. NHEs as well as other proton transporters were previously linked to the regulation of the Wnt signaling pathway, cell polarity signaling, and mucociliary function. Furthermore, mutations in the gene SLC9A3 (encoding NHE3) were detected as additional risk factors for airway infections in cystic fibrosis patients. Here, we used the Xenopus embryonic mucociliary epidermis as well as human airway epithelial cells (HAECs) as models to investigate the functional roles of NHEs in mucociliary development and regeneration. In Xenopus embryos, NHEs 1-3 were expressed during epidermal development, and loss of NHE function impaired mucociliary clearance in tadpoles. Clearance defects were caused by reduced cilia formation, disrupted alignment of basal bodies in multiciliated cells (MCCs), and dysregulated mucociliary gene expression. These data also suggested that NHEs may contribute to the activation of Wnt signaling in mucociliary epithelia. In HAECs, pharmacological inhibition of NHE function also caused defective ciliation and regeneration in airway MCCs. Collectively, our data revealed a requirement for NHEs in vertebrate mucociliary epithelia and linked NHE activity to cilia formation and function in differentiating MCCs. Our results provide an entry point for the understanding of the contribution of NHEs to signaling, development, and pathogenesis in the human respiratory tract.

Keywords: Airway; Cilia; Na+/H+ exchangers; Slc9a1; Slc9a2; Slc9a3; Xenopus.

Figure 1:. Dynamic slc9a1, 2 and 3 expression during Xenopus development

In situ hybridization on Xenopus laevis embryos from stages (st.) 3 – 30 revealed dynamic expression of slc9a transcripts during early development. At the four-cell stage (st. 3) strong maternal transcript deposition was detected for slc9a1, but slc9a3 was only lowly expressed and no signals were observed for slc9a2. During gastrulation (st. 11), slc9a1 was markedly expressed throughout the prospective ectoderm and mesoderm, while slc9a3 was found at the blastpore (bp), the endoderm as well as involuting mesodermal cells and no specific expression was found for slc9a2. In neurula stage embryos (st. 16 and 20), slc9a1 and 2 transcripts were detected in the epidermal ectoderm (ep) with stronger expression of slc9a1, and slc9a3 was expressed in the endoderm (ed). During late neurulation (st. 20), slc9a2 and 3 were also expressed in the presomitic mesoderm. In tailbud stages (st. 24, 30), slc9a1 and 2 transcripts were detected in the otic vesicle (ov) and the cement gland (eg), but not slc9a3. Additionally, slc9a1 was expressed in the somites (s), and slc9a2 was expressed in the brain (b). St. 3 embryos are depicted in animal view. St. 11 embryos are depicted in vegetal view, dorsal up. St. 16 and 20 embryos are depicted in dorsal view, anterior left. St. 24 and 30 embryos are depicted in lateral view, anterior left. Sections depicting the epidermis of st. 11 and st. 16 embryos are shown in yellow boxes below the whole mount image. Section planes are indicated by yellow lines.

Figure 2:. NHE1-3 Ioss-of-function impairs fluid flow, MCC cilia formation and basal body alignment

(A) Extracellular fluid flow was quantified by addition of fluorescent beads to the medium and high-speed fluorescent microscopy for at least 10s at 50 – 100 frames per second. Uninjected embryos were used as controls (n = 15). Morphants were injected with the indicated MO (6 pmol). slc9a1MO, n = 13; slc9a2MO, n = 16; slc9a3MO, n = 14. Flow velocities were calculated relative to median values of uninjected controls. Boxes depict 50% of values, the median is depicted by the horizontal line, the mean is depicted by the cross. Whiskers indicate the upper and lower quartiles, outliers are depicted as circles. *** = P<0.001, Mann-Whitney test. (B) Embryos were injected with centrin4-cfp (blue) and clamp-rfp (red) mRNAs (control) to visualize cell borders and to identify targeted cells. Morphants were co-injected with 6 pmol of the indicted MO. Cilia were visualized by immunofluorescence staining against acetylated-α-tubulin (Ac.-α-Tub., green). Dashed boxes in micrographs on the left indicate magnified areas shown in B’-B’”. Non-targeted MCCs are indicated by asterisks. (C) Embryos were injected with centrin4-cfp (grey) and clamp-rfp (red) mRNAs (control) to visualize basal bodies and rootlets, respectively. Morphants were co-injected with 6 pmol of the indicted MO. F-actin was visualized by fluorescence staining with phalloidin (green). Boxes in micrographs on the left indicate magnified areas shown in the right panels. Direction of basal bodies is indicated by arrows. Scale bars indicate magnification.

Figure 3:. NHE1-3 loss-of-function affects neural tube closure as well as subapical actin formation and basal body positioning in MCCs

(A) slc9a1. 2 or 3 were knocked down unilaterally (visualized by co-injection of RFP encoding mRNA; individual knockdown at 4pmol, combined knockdown at 1.33pmol of each MO) and neural tube closure was analyzed at stage 19-20. In contrast to control-injected embryos and the non-injected control side of individual embryos, slc9a morphants displayed various degrees of neural tube closure defects (quantified in Fig. S2A). Representative examples are shown in anterior-dorsal view and the outline of the injected neural folds is marked by dashed blue line. (B) Embryos were injected with clamp-rfp (red) mRNAs and indicated MOs (same set of embryos as depiced in A). Immunofluorescence staining against acetylated-α-tubulin (Ac.-α-Tub., blue) revealed cilia and phalloidin stained the actin cytoskeleton (green). Loss of NHE function induced cilia defects as well as loss of subapical actin links between basal bodies in all treatments. Lateral projections further revealed severe basal body apical transport defects in slc9a1 and 2 morphants, but only mild or no defects after combined knockdown or after slc9a3MO injections. Scale bars indicate magnification.

Figure 4:. NHE1-3 Ioss-of-function affects gene expression in MCCs and secretory cells of the mucociliary epidermis

In situ hybridization for cell type markers in the embryonic mucociliary epidermis. Embryos were injected unilaterally on the right side (inj.) and the uninjected left side (uninj.) served as internal control. Unmanipulated specimens were used as additional controls (control). (A) St. 17 embryos are shown in dorsal view, anterior up. foxj1 marks MCCs, foxi1e marks ion secreting cells, foxa1 marks small secretory cells and otogelin is a marker of mucus secreting goblet cells. Downregulation is indicated by red arrowheads, upregulation is indicated by yellow arrowheads. (B) Quantification of results. * = P<0.05, *** = P<0.001, chi-squared test.

Figure 5:. Pharmacological inhibition of NHE function impairs MCC cilia formation and regeneration in human airway epithelial cells

Human airway epithelial cells (HAECs) were grown in ALI cultures and treated either with vehicle (DMSO controls) or 25μM of the NHE inhibitor El PA for seven days. (A) Top view on cultured cells at ALI day 28 after seven days of treatment during week 4 of regeneration. Immunofluorescence staining for MCC cilia (Ac.-α-Tub., magenta), F-actin (green) and nuclei (DAPI, blue) revealed defective MCC ciliation in EIPA-treated specimens. Scale bars indicate magnification. (B) Immunofluorescence staining for MCC cilia (Ac.-α-Tub., green), Keratin 5-positive basal cells (KRT5, magenta) and nuclei (DAPI, blue) revealed ciliation defects in EIPA-treated specimens, but no loss of basal cells. Scale bars indicate magnification. Morphology of control (DMSO) and El PA treated HAECs at ALI day 28, after treatment during the indicated week of regeneration revealed successful epithelialization of HAECs in controls and EIPA-treated specimens. Fixation directly after El PA treatment (week 4) further revealed a thinning of the epithelium. Apical surface up. Scale bars indicate magnification.