XB-ART-48635Development April 1, 2014; 141 (7): 1526-33.
A novel serotonin-secreting cell type regulates ciliary motility in the mucociliary epidermis of Xenopus tadpoles.
The embryonic skin of Xenopus tadpoles serves as an experimental model system for mucociliary epithelia (MCE) such as the human airway epithelium. MCEs are characterized by the presence of mucus-secreting goblet and multiciliated cells (MCCs). A third cell type, ion-secreting cells (ISCs), is present in the larval skin as well. Synchronized beating of MCC cilia is required for directional transport of mucus. Here we describe a novel cell type in the Xenopus laevis larval epidermis, characterized by serotonin synthesis and secretion. It is termed small secretory cell (SSC). SSCs are detectable at early tadpole stages, unlike MCCs and ISCs, which are specified at early neurulation. Subcellularly, serotonin was found in large, apically localized vesicle-like structures, which were entirely shed into the surrounding medium. Pharmacological inhibition of serotonin synthesis decreased the velocity of cilia-driven fluid flow across the skin epithelium. This effect was mediated by serotonin type 3 receptor (Htr3), which was expressed in ciliated cells. Knockdown of Htr3 compromised flow velocity by reducing the ciliary motility of MCCs. SSCs thus represent a distinct and novel entity of the frog tadpole MCE, required for ciliary beating and mucus transport across the larval skin. The identification and characterization of SSCs consolidates the value of the Xenopus embryonic skin as a model system for human MCEs, which have been known for serotonin-dependent regulation of ciliary beat frequency.
PubMed ID: 24598162
Article link: Development
Species referenced: Xenopus
Genes referenced: actl6a ddc foxa1 foxj1 foxj1.2 htr1a htr1e htr1f htr2a htr2b htr2c htr3a htr4 htr5a htr6 htr7 mcc nfs1 slc18a1 slc18a2 slc6a4l tph1 tph2 tuba4b
Antibodies: Fluro-phalloidin Ab2 Peanut Agglutinin Seratonin Ab2 Tuba4b Ab17
Morpholinos: htr3a MO1 htr3a MO2
Article Images: [+] show captions
|Fig. 1. Small secretory cells (SSCs) in the Xenopus tadpole epidermis. (A) Stage 33 tadpole stained for serotonin (red) and cilia (green) using anti-serotonin and anti-acetylated-α-tubulin antibodies. (A′,A′) Higher magnification reveals vesicular nature of serotonin staining. Please note a serotonin-positive vesicle attached to cilia (white arrowhead in A′). (B) Localization of serotonin-containing vesicles in SSCs. Cell boundaries marked by actin staining using phalloidin. Higher magnifications (left panels in B′-B′′) and orthogonal projection of z-stack slices (white dashed lines; right panels in B′-B′′) demonstrate serotonin-positive vesicles beneath, at, or above the apical cell membrane (B′-B′′, green arrowheads). (C) SEM of stage 32 tadpole displaying MCCs and SSCs. (C′) Higher magnification of two SSCs. Red arrowheads highlight detaching vesicles. Scale bars: 50 μm in A′; 10 μm in A′; 5 μm in B-B′′; 20 μm in C; 5 μm in C′.|
|Fig. 2. SSCs express tph1. (A) Expression pattern of tph1 mRNA at stage 31. Signals were detected in the brain and in a punctate pattern in the epidermis. A sagittal histological section (A′) demonstrated tph1expression in the epiphysis (A′) in SSCs and in a subset of neuronal cells (n) in the floor plate of the neural tube (A′′). (B) SSCs express tph1 (blue arrowhead), as demonstrated by whole-mount in situ hybridization followed by IF for serotonin (green, green arrowhead) and cilia (acetylated-α-tubulin, red, red arrowhead). (C) Punctate expression pattern of aromatic-L-amino-acid decarboxylase (DOPA-decarboxylase; ddc) in the embryonic skin. Inset shows close-up. Embryos are shown in lateral views. a, anterior; d, dorsal; epi, epiphysis; l, left; r, right.|
|Fig. 3. Inhibition of serotonin synthesis impairs cilia-driven flow. (A) Experimental setup. Embryos were incubated with TPH inhibitor p-chloro-phenylalanin (PCPA) from stage 25-32, followed by assessment of flow (B), serotonin localization (C) and tph1 expression (D). (B) Reduction of flow velocity in PCPA-treated specimen was partially rescued by co-culture with exogenously applied serotonin. Flow was analyzed by data processing from time-lapse movies following the addition of fluorescent beads to the medium. The mean velocity in control embryo was set to 100%. (C) Absence of serotonin staining (red arrowheads) upon PCPA incubation (C′). (D) Unaltered tph1 expression following PCPA treatment (D′).|
|Fig. 4. The serotonin receptor Htr3 controls cilia motility in the tadpole epidermis. (A,B) Punctate serotonin receptor Htr3 mRNA expression in stage 31 epidermis (A) colocalizes to MCCs, demonstrated by double-staining with anti-acetylated tubulin antibody (B). (C) Reduction of flow velocity in Htr3 morphants. Knockdown of Htr3 by MO1 and MO2 reduced cilia-driven flow velocity compared with control specimens (co). Velocity was partially rescued by co-injection of a mutated Htr3 mRNA not targeted by MO1. Flow was analyzed by adding fluorescent beads to the medium and data processing of time-lapse movies. The mean velocity in control embryo was set to 100%. (D,E) Cilia motility in the epidermis of co-MO and Htr3-MO injected specimens. Cilia were visualized by co-injection of mRFP mRNA. (D) Maximum z-projections (stacks of 450 frames) from time-lapse movies (5-second duration). Blurring indicates motion, acuity lack thereof. (E) Quantification of results. Note rescue of ciliary motility by co-injection of mutated Htr3 mRNA or DNA construct, which is not targeted by Htr3-MO. a, anterior; d, dorsal; l, left; r, right.|
|Fig. 5. Regulation and function of cell types at the Xenopus tadpole epidermis: a model. SSCs constitute a novel cell type in the mucociliary epithelium of the Xenopus laevis tadpole skin. Serotonin is released in vesicles (green circles) and transported to MCCs by flow. Following release, serotonin modulates ciliary beat frequency via serotonin receptor 5-HT3.|
|Fig. S1. Cilia association of serotonin-containing vesicles and vesicle maturation. (A, B) Co-staining of serotonin (green) and cilia (red; anti acetylated--tubulin) at stage 32 reveals occasional co-localization in the epidermis (white arrowheads). (B) Higher magnification of two MCCs in side view. Note the vesicular nature of serotonin staining in all cases. (C) Vesicle maturation in SSCs. Z-stack analysis of a single SSC stained for serotonin (red) and actin (phalloidin, green). Apical to basal horizontal optical sections (I-V) were used to reconstruct an orthogonal hypothetical model of vesicle maturation (lower panel in C). Vesicles localized basally were small (0.2 μm diameter), with a continuous increase in diameter to a maximum of 1 μm towards the apical pole of the cell. (D, D') Vesicular co-localization of serotonin (green) and peanut agglutinin (PNA; red) in the epidermis of Xenopus laevis tadpoles at stage 39. Scale bars in (D) and (D') represent 50 and 5μm, respectively.|
|Fig. S2. Embryonic expression of serotonin pathway components. (A, B) Epidermal expression of monoamine transporter slc18a1 mRNA in stage 14 neurula (A) and stage 35/36 tadpole (B) embryos. Transverse sections (A', B') reveal uniform superficial staining in the epidermis. Note lack of expression in the neural plate at stage 14. (C) tph2 expression was restricted to the hindbrain at stage 30. (C') Transverse histological section. Embryos are shown in lateral views except for panel (A), which shows a dorsal perspective. (D, D') tph1 mRNA expressing SCCs (blue arrowheads) secrete PNA positive vesicles (red arrowheads). (D) Close-up bright field image of a transverse section of a tph1 stained tadpole at stage 36 displaying three SSCs. (D') Fluorescent PNA signals (red) co-localize with tph1 mRNA.|
|Fig. S3. MO-specificity, foxj1 expression and ciliogenesis in Htr3 morphants. (A-C) MO specificity. MO1 and MO2 specificity was tested using an eGFP-reporter construct. A 60 bp fragment of Htr3 including the translational start site as well as bindings sites for both MO1 and MO2 (cf. Fig. 1C) was cloned in frame with eGFP (cf. Gessert et al., 2010). Injection into animal blastomeres of 4-cell embryos were performed using rhodamine-B dextran as lineage tracer. (A) Co-injection of co-MO did not affect green fluorescence of reporter construct in targeted regions at stage 9. (B, C) Absence of green fluorescence upon co-injection of MO1 (B) or MO2 (C) demonstrated MO-specificity. (D, E) Unaltered foxj1 mRNA expression in the frog epidermis of Htr3 morphant. Embryos shown in lateral view, anterior to the left. (F, G) Wildtype and morphant epidermis at stage 34 stained for cilia (red) and actin (green) using anti-acetylated-α-tubulin antibodies and phalloidin. MCC number and ciliation were unaffected in morphants (F', G', F'', G''). Increasing magnifications. Scale bars represent 100μm (F, G), 50μm (F', G') and 10μm (F'', G'').|
|tph2 (tryptophan hydroxylase 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.|
|slc18a1 (solute carrier family 18 (vesicular monoamine transporter), member 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35 & 36, lateral view, anterior left, dorsal up.|
|slc18a1 (solute carrier family 18 (vesicular monoamine transporter), member 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view, anterior left.|
|ddc (DOPA-decarboxylase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34 & 35, lateral view, anterior left, dorsal up.|