Ventrella R et al. (2023), Bidirectional multiciliated cell extrusion is c...

XB-ART-60220

Development 2023 Sep 01;15017:. doi: 10.1242/dev.201612.

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Bidirectional multiciliated cell extrusion is controlled by Notch-driven basal extrusion and Piezo1-driven apical extrusion.

Ventrella R , Kim SK , Sheridan J , Grata A , Bresteau E , Hassan OA , Suva EE , Walentek P , Mitchell BJ .

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Xenopus embryos are covered with a complex epithelium containing numerous multiciliated cells (MCCs). During late-stage development, there is a dramatic remodeling of the epithelium that involves the complete loss of MCCs. Cell extrusion is a well-characterized process for driving cell loss while maintaining epithelial barrier function. Normal cell extrusion is typically unidirectional, whereas bidirectional extrusion is often associated with disease (e.g. cancer). We describe two distinct mechanisms for MCC extrusion, a basal extrusion driven by Notch signaling and an apical extrusion driven by Piezo1. Early in the process there is a strong bias towards basal extrusion, but as development continues there is a shift towards apical extrusion. Importantly, response to the Notch signal is age dependent and governed by the maintenance of the MCC transcriptional program such that extension of this program is protective against cell loss. In contrast, later apical extrusion is regulated by Piezo1, such that premature activation of Piezo1 leads to early extrusion while blocking Piezo1 leads to MCC maintenance. Distinct mechanisms for MCC loss underlie the importance of their removal during epithelial remodeling.

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Species referenced: Xenopus laevis
Genes referenced: ccl20 mcc mcidas notch1 piezo1 tub
GO keywords: Notch signaling pathway [+]
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	Fig. 1. The loss of cilia-driven fluid flow accompanies the loss of multiciliated cells. (A) Quantification of cilia-driven fluid flow, as measured by fluorescent bead displacement across the surface of the epithelium showing that flow peaks at 4 dpf and is essentially lost by 9 dpf, n=8 animals per time point (data are mean±s.d.). (B) Quantification of MCC number using both the transgenic line TgTub-memRFP driving MCC-specific expression of membrane RFP and antibody staining of acetylated tubulin, n>3 embryos per time point. Box and whiskers plot represents 25th-75th percentiles (boxes), minimum and maximum values (whiskers), with the line representing the median. (C) Representative images of the progression of MCC loss in embryos between ST38 and ST48, showing acetylated Tub (white), TgTub-memRFP (magenta), phalloidin (purple) and DAPI (blue). Scale bars: 500 µm.
	Fig. 2. Lineage tracing of MCC fate. (A) Experimental design showing lineage-tracing constructs of the CMV DsRed loxP GFP cassette together with the Tub-driven CRE. (B) Lineage-tracing experiment showing the conversion of DsRed to GFP in ST42 MCCs upon expression of CRE specifically in MCCs under control of the Tub promoter, and the loss of MCC-specific CMV expression of GFP despite the maintenance of broad CMV-driven DsRed expression at ST48. (C) Developmental quantification of embryos containing GFP-positive cells in lineage-tracing experiments, n=102 transgenic animals over five independent experiments (data are mean±s.d.). Scale bars: 20 µm.
	Fig. 3. MCCs are extruded both basally and apically. (A) Representative frames from a time-lapse movie of TgTub-memRFP embryos injected with LifeACT-GFP showing a MCC undergoing basal extrusion with the RFP-positive cell remnants remaining visible for at least 400 min. Lower panels represents a side projection (see Movie 2). (B) Representative frames from a time-lapse movie showing a MCC undergoing apical extrusion. Lower panel represents a side projection (see Movie 3). (C) Representative example of RFP-positive MCC remnants in fixed tissue stained with phalloidin. Lower panel represents a side projection of boxed area. Scale bars: 10 µm. (D) Quantification of the rate of apical and basal extrusion events using long-term light-sheet imaging between ST42 and ST46 (P=0.04), and between ST46 and ST48 (n.s.), n=132 extrusion events from five ST42-ST46 embryos, and 69 events from six embryos post ST46-ST48. (E) Percentage of extrusion events that are basal between ST42 and ST46, and ST46 and ST48 (P=0.01). Box and whiskers plots represent 25th-75th percentiles (boxes), minimum and maximum values (whiskers), with the line representing the median (see Movie 4).
	Fig. 4. Skin transplants display age-dependent loss of MCCs. (A) Experimental design showing the transplantation of ST11 donor skin of Xla.Tg(CMV:eGFP-OMP25)Wtnbe onto ST28 host skin of a CMV:memb-RFP-injected embryo. (B) Representative image of an area containing both host tissue (red, ST48) and donor tissue (green, ST47) stained for acetylated tubulin (white). Scale bar: 50 µm. (C) Quantification of MCC number in host versus donor tissue, n=7 transplants, P=0.02. Box and whiskers plot represents 25th-75th percentiles (boxes), minimum and maximum values (whiskers), with the line representing the median.
	Fig. 5. Deciliation of MCCs reboots the transcriptional program and extends MCC lifespan. (A-C) Representative whole-tail images of TgTub-memRFP (magenta) embryos stained with acetylated tubulin (white) and phalloidin (purple), showing MCCs in ST48 control embryo (A), as well as ST48 embryos deciliated at ST28 (B) and ST46 (C). (A′-C′) Zoom in of outlined areas in A-C showing acetylated tubulin (top; white) and TgTub-memRFP (bottom; white). Scale bars: 500 μm (A-C); 100 μm (A′-C′). (D) Quantification of MCC number at ST48 in control (n≥10 embryos) and embryos that were deciliated at ST28 (n≥10 embryos, P=0.014) or ST46 (n=6 embryos, P=0.001). Box and whiskers plot represents 25th-75th percentiles (boxes), minimum and maximum values (whiskers), with the line representing the median (see Fig. S4).
	Fig. 6. Maintenance of MCC fate protects against MCC loss. (A,B) Whole-tail images of control and Tub-MCIDAS transgenic embryos with TgTub-memRFP (red), acetylated tubulin (white) and phalloidin (purple). Scale bars: 500 µm. (C) Quantification of MCC number in control and Tub-MCIDAS embryos at ST48, n≥12 embryos, P=0.007. Box and whiskers plot represents 25th-75th percentiles (boxes), minimum and maximum values (whiskers), with the line representing the median (see Fig. S5).
	Fig. 7. Piezo1 mechanosensation regulates MCC apical extrusion. (A-B) Representative images of DMSO-treated (A) and 100 µM Yoda-treated (B) embryos with TgTub-mem-RFP (magenta), acetylated tub (white) and phalloidin (purple). Scale bars: 500 µm. (C) Quantification of MCCs (scored with acetylated Tub) in embryos treated with DMSO and 100 µM Yoda, n≥7 embryos, P=4.2E-5. (D) Quantification of RFP-positive clusters reflecting apoptotic MCCs in embryos treated with DMSO and 100 µM Yoda, n≥7 embryos, P=0.68. (E,F) Representative images of DMSO-treated (E) and 2 µM GsMTX-treated (F) embryos with TgTub-memRFP (magenta), acetylated tub (white) and phalloidin (purple). Scale bars: 500 µm. (G) Quantification of MCCs (scored with acetylated Tub) in embryos treated with DMSO and 2 µM GsMTX, n≥5 embryos, P=0.02. (H) Quantification of RFP-positive vesicles reflecting apoptotic MCCs in embryos treated with DMSO and 2 µM GsMTX, n≥5 embryos, P=0.05. Box and whiskers plots represent 25th-75th percentiles (boxes), minimum and maximum values (whiskers), with the line representing the median.
	Fig. S1. Basal extrusion of apoptotic cells. (A-D) ST47 TgTub-memRFP embryo showing MCCs and RFP + MCC remnants (magenta (A), white (C)) stained with anti- acetylated tubulin (green (A), white (B)) to mark cilia and anti- Cleaved Caspase 3 (cyan (A), white (D)) to mark apoptotic cells. Scale is 50 m.
	Fig. S2. MCC apical and basal extrusion and RFP vesicle maintenance. (A-C) Representative examples from long-term light-sheet movies showing maintenance of RFP posit ve vesicles (arrows) after basal extrusion for 13.5 Hrs (A), 16.6 Hrs (B) and 18.3 Hrs (C.also see Movie 52) when the cells/embryo eventually moved out of frame (out of frame depicted via dotted line). (D) Light-sheet imaging showing MCC disappearance during apical extrusion (arrow) and RFP cluster formation during basal extrusion (arrowhead;see Movie 54). Scale is 50µm.
	Fig. S3. Mesodermal Notchis required for MCCloss. (A) Day 15 ST50 animal cap that still maintains a large number of MCCs. (B) Day 30 ST55 animal cap from a TgTub-memRFP embryo that shows the presence of multiple MCCs but also the loss of cilia and MCC maintenance in some RFP positive cells. (C) Comparison of MCCs in caps from WT embryos and embryos injected with Tub-NICO.(D) Quantification of MCCs in caps with and without TubNICO (n > 3 caps; p = 4.SE-8). Scale bar is 20µm.
	Fig. S4. Multiciliated cell deciliation. (A-E) Representative images of a embryos stained with phalloidin (magenta) and acetylated tubulin (green). (A-B) Representative images of a con- trol embryo at ST38 (A) and a ST38 embryo directly after deciliation (B). (C-E) Representative images of a controlembryo at ST42 (C), a ST42 embryo that was deciliated at ST38 (D), and an embryo that was deciliated at ST38 but that was also treated with cycloheximide (E). Scale is 20µm.
	Fig. S5. Tub promoter activity is downstream of MCIDAS. (A-B) ST36 embryo mosaically injected with the MCC-inducing factor MCIDAS, showing broad expression of the TgTub-memRFP (magenta) in ectopic MCCs labeled with acetylated tubulin (green) with phalloidin staining (purple) (A) and the same image showing only the ectopic TgTub driven mem-RFP (B). Scale is 50µm.

References [+] :

Andrade, Apoptotic regulation of epithelial cellular extrusion. 2011, Pubmed