XB-ART-56656Nat Commun January 1, 2020; 11 (1): 665.
Tissue mechanics drives regeneration of a mucociliated epidermis on the surface of Xenopus embryonic aggregates.
Injury, surgery, and disease often disrupt tissues and it is the process of regeneration that aids the restoration of architecture and function. Regeneration can occur through multiple strategies including stem cell expansion, transdifferentiation, or proliferation of differentiated cells. We have identified a case of regeneration in Xenopus embryonic aggregates that restores a mucociliated epithelium from mesenchymal cells. Following disruption of embryonic tissue architecture and assembly of a compact mesenchymal aggregate, regeneration first restores an epithelium, transitioning from mesenchymal cells at the surface of the aggregate. Cells establish apico-basal polarity within 5 hours and a mucociliated epithelium within 24 hours. Regeneration coincides with nuclear translocation of the putative mechanotransducer YAP1 and a sharp increase in aggregate stiffness, and regeneration can be controlled by altering stiffness. We propose that regeneration of a mucociliated epithelium occurs in response to biophysical cues sensed by newly exposed cells on the surface of a disrupted mesenchymal tissue.
PubMed ID: 32005801
PMC ID: PMC6994656
Article link: Nat Commun
Genes referenced: arhgef2 fn1 hpse itln1 krt12.5 myc nhs notch1 prkci rho yap1
GO keywords: regeneration
Article Images: [+] show captions
|Fig. 1: Surface cells of deep ectoderm aggregates undergo epithelial-like phenotypic transition. a) Schematic of the assembly of deep ectoderm cell aggregates from early Xenopus embryo (Stage 10). b) Surface F-actin and fibronectin (FN) from maximum intensity projections at 1.5, 5, and 24 h post aggregation (hpa). Three panels on the right are higher resolution views of the inset region (white box) in the third column. Arrows indicate margin of FN where dense circumapical F-actin suggests epithelial cell phenotype. Scale bar for aggregate images is 100 µm. c) Transverse sectional view through the ectoderm of NHS-Rhodamine surface-labelled embryos. Scale bar, 50 µm. Rhodamine is restricted to the apical surface of outer epithelial cells. d) Deep ectoderm aggregates generated from NHS-Rhodamine surface-labelled embryos. Scale bar, 100 µm. Lack of rhodamine indicates absence of contaminating epithelia. e) Percent of epithelial cell phenotype found on the surface of different-sized deep ectoderm aggregates at 24 hpa. Aggregates assembled with varying amounts of embryo-ectoderm explants (1/2 explant, n = 6; 1 explant, n = 5; 2 explants, n = 5; 4 explants, n = 8). Box plot shows minimum, first quartile, median, third quartile, and maximum values. f) Labeled nuclei shown in deep ectoderm aggregates from 1/2- and 4-embryo-ectoderm explant containing aggregates. Scale bar, 100 µm.|
|Fig. 2: Epithelialization precedes differentiation of mucus-secreting goblet cells. a) Apical polarity protein aPKC localizes on the apical surface of aggregates by 5 hpa. Scale bar, 10 µm. b) Apical localization of epithelial cytoskeleton keratin is restricted to the outer surface of deep ectoderm aggregates. Cross sectional view of aggregates at 5 hpa (Scale bar, 100 µm) and 24 hpa (Scale bar, 50 µm). c) Maximum intensity projection of epithelial tight junctional protein ZO-1 expression in aggregates at 5 hpa (top; immunofluorescence staining) and 24 hpa (bottom; GFP-ZO-1 expression). Epithelialized cells are marked by arrows (inset). Scale bar, 100 µm. d) Representative frames from a time-lapse sequence of an aggregate expressing GFP-ZO-1. Top panel: maximum intensity projection of GFP-ZO-1 shows cells undergo epithelialization (outlined with red on lookup-table-inverted images in lower panel) on the surface of aggregates from 2 to 6 hpa. Scale bar, 100 µm. e) Percentage of cells having undergone epithelialization increases over time (9 aggregates tracked from three clutches). Box plot shows minimum, first quartile, median, third quartile, and maximum values. f) qPCR expression profiling in aggregates made of ectoderm, deep layer, or superficial layer cells. Expression of epithelial (Cdh1, ZO-1, Krt12, and Itln1) and mesenchymal (FN, VimA, and Snai1) genes are analyzed for CT-based fold changes from 3 hpa to 24 hpa. g) At 24 hpa the deep ectoderm aggregate is covered by epithelial cells including differentiated mucus-secreting goblet cells (itln1) and radially intercalated multiciliated cells (acetylated tubulin). Scale bar, 100 μm.|
|Supplementary Figure 1: Epithelialization and goblet cell specification is independent from Notch signaling. a-b, Activation of Notch signaling by over-expression of the intracellular domain of NotchICD inhibits the differentiation of multiciliated cells but does not inhibit regeneration of goblet cells in deep ectoderm aggregates (24 hpa). Inset regions of third columns (white boxes) are shown in fourth column. Scale bars are 100 μm for all image panels except insets.|
|Supplementary Figure 2: F-actin and ZO-1 co-localize on the boundary of epithelial cells. Aggregates expressing ZO-1 RFP (red) and stained for F-actin (green) reveal co-localization of tight junctions and circumapical actin in epithelialized cells (white) in both control and Calyculin A treated aggregates. Inset regions from first and third rows (white boxes) are shown in second and fourth rows, respectively. Scale bars are 100 µm for all panels except insets.|
|Supplementary Figure 3: ΔC-C-cadherin expressing cells are not able to transition to epithelial on the surface of deep ectoderm aggregates. F-actin and myc stained deep ectoderm aggregates at 5 hpa. Cells expressing myc-tagged ΔC-C-cadherin are detected by Anti-myc staining. Non-expressing cells epithelialize (a) while ΔC-C-cadherin expressing cells (yellow) do not (b) within same aggregate. Scale bar for whole aggregate (top row) is 100 µm.|
|Supplementary Figure 4: ROCK inhibition blocks epithelialization driven by constitutive active Rho-GEF. a. Representative maximum projection confocal images of F-actin labeled aggregates. Epithelialization proceeds in control 5 hpa aggregates is enhanced in arhgef2C55R expressing aggregates. Incubation of arhgef2C55R expressing aggregates in ROCK inhibitor Y27362 inhibits epithelialization. Inset regions in first column (white boxes) are shown in second column. Scale bar for all panels except the insets are 100 µm. b. Percent of epithelialization from treatments shown in (a; controls, arhgef2C55R , arhgef2C55R + Y27362, n=6, 5, 6, respectively). (See Statistical Analysis in Methods for detailed description of statistical methods.)|
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