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Mucus secretion and ciliary motility are hallmarks for muco-ciliary epithelia (MCE). Both, mammalian airways as well as the less complex epidermis of Xenopus embryos show cilia-driven mucus flow to protect the organism against harmful effects by exogenous pathogens or pollutants. Four cell types set up the epidermal MCE in Xenopus. Multi-ciliated cells (MCCs) generate an anterior to posterior flow of mucus. Ion secreting cells (ISCs) are characterized by the expression of ion transporters, presumably to maintain a favorable homeostasis. The largest cell type is represented by goblet cells, which cover most of the epidermis and exhibit secretory properties. Additionally, small secretory cells (SSCs) release mucus, antibiotic compounds, and the monoamine serotonin (5-hydroxytryptamine; 5-HT). We have recently shown that serotonin regulates flow velocity by acting on ciliary beat frequency. Here, we describe the identification and functional characterization of Xenopus polka-dots (Xpod). No homologous genes or proteins were found in other vertebrates, including Xenopus tropicalis. We demonstrate that Xpod serves as an SSC-specific marker, starting to be expressed shortly after SSC specification at neurula stages. Overexpression of a tagged Xpod protein resulted in the localization of secretory granules. Notch signaling induced SSC cell fate, in contrast to its repressing effect on MCC and ISC specification. Xpod loss-of-function revealed that mucus and 5-HT release by SSCs was severely diminished, which impaired the ciliary beating of MCCs. In summary, Xpod specifically marked SSCs and was required for muco-ciliary secretion in Xenopus laevis.
Figure 1. Xpod is expressed in and secreted from SSCs. (a) Xpod amino acid sequence. Translation start sites identified by Yoshii et al. (2011) and in this work marked in red. Signal peptide and cleavage site are indicated in green. (b) Xpod mRNA expression at Stage (st.) 32. (b′) Immunofluorescence for SSC marker PNA co‐localized with Xpod positive cells. (c, d) Myc‐tagged Xpod (mycXpod) is secreted via PNA (c) and serotonin (d) loaded granules. Orthogonal views are shown in insets (c′) and (d′, d″)
Figure 2. Notch‐dependent SSC fate determination. Notch activation (middle) repressed SSC fates, while Notch repression (bottom) induced ectopic SSCs, as compared to wild type controls (top). mRNAs of dominant active intracellular domain of Notch (Nicd) or dominant‐negative suppressor of hairless (Su(H)‐DBM) were injected into the epidermal lineage at the 4–8 cell stage. At Stages 32–33, expression of marker genes for SSCs (Xpod), MCCs (foxj1), and ISCs (atp6ve1) were analyzed. In addition, MCCs and SSCs were identified by IF using anti‐acetylated tubulin (blue) and anti‐serotonin (red) antibodies, respectively. Actin staining by phalloidin (green) marks cell border
Figure 3. Xpod is required for MucXS and serotonin release. Xpod function was analyzed by the XpodMO‐mediated gene knockdown. (a–c) MucXS and serotonin loaded granules were detected by immunofluorescence using PNA (red) and anti‐serotonin antibody (green), respectively. Cell borders were visualized by actin staining (phalloidin, blue). (a–c) Controls (co.; a) showed MucXS and serotonin positive granules, which were reduced in Xpod morphants (b). Co‐injecting XpodMO and rescue‐Xpod mRNA restored secretion (c). (d–f) Statistical evaluation of frequency of PNA (d) and serotonin (e) positive granules per SSC. The number of SSCs per area is shown in (f). Statistical significance was calculated by Wilcoxon test. See Table S1 for p values
Figure 4. Impaired ciliary beating in Xpod morphants. (a–c) Still pictures of representative control (co.; a), XpodMO (b) and rescue mix (c) injected MCCs, which were subjected to time‐lapse videography. Kymographs (a′–c′) depicting ciliary motions over time (1 s), which were generated by the optical orthogonal section at the level indicated by the red line in (a–c). Untreated MCC (a′) showed a fast motion of multiple cilia. Xpod morphant MCC (b′) showed reduced ciliary motion, which can be deduced from cilia keeping their position over time. Co‐injecting XpodMO and rescXpod mRNA restored ciliary beating (c′). Statistical analyses of analyzed MCCs (d) demonstrate concentration‐dependent XpodMO effects (2 pmol vs. 3/4 pmol/embryo) and MO‐specificity, as shown by co‐injecting rescXpod mRNA. Note, exogenous serotonin administration restored ciliary motion in Xpod morphants. See Table S1 for p values
Fig. S1 Xpod mRNA expression during early SSC development. Xpod whole mount in situ hybridization at neurula (A) and early tadpole stages (B‐D). Histological sections (A’‐D’) revealed that SSCs expressed Xpod prior to intercalation into the superficial layer (arrowheads in close ups A”, B′’ or dashed line in C′’). At Stage 24, most SSCs were intercalated (arrowhead in D”).
Fig. S2 Notch signaling regulates SSC fate.
Embryo number and statistics of epidermal cell fates in embryos where Notch signaling was activated (NICD) or repressed (SU[H]DBM). At least three independent experiments for each condition and cell identity was performed. To address the statistical significance of our observations a Chi2 test was conducted. The p values in each case depicts a very highly significant difference to untreated controls (***). See also Table S1 for p values.
Fig. S3 No effect of Xpod knockdown on ISC, SSC and MCC specification as well as MCC ciliogenesis. Marker gene analysis by WMISH of untreated controls (co., A, D, G), Xpod morphants (B, E, H) and specimens co‐injected with MO and rescXpod mRNA (rescue; C, F, I). SSC, ISC and MCC identity were analyzed by tph1 (A‐C), atp6v1e1 (D‐F) and foxJ1 (G‐H) expression, respectively. (J, K) IF using PNA (red) and anti‐ac. tubulin (green) antibody on control (J) and Xpod morphant specimens (K) did not reveal any differences in ciliogenesis. Note that PNA positive granules (red arrowheads) were present in controls but greatly reduced in Xpod morphants. (L‐O) Release of mucus (PNA; red) and serotonin (green) was unaltered by gscMO (3–4 pmol/embryo) injections. (L and N). Statistical acquisition of data by Wilcoxon test. See also Table S1 for p values.
