Exosomes Mediate Epithelium-Mesenchyme Crosstalk in Organ Development

Abstract

Organ development requires complex signaling by cells in different tissues. Epithelium and mesenchyme interactions are crucial for the development of skin, hair follicles, kidney, lungs, prostate, major glands, and teeth. Despite myriad literature on cell-cell interactions and ligand-receptor binding, the roles of extracellular vesicles in epithelium-mesenchyme interactions during organogenesis are poorly understood. Here, we discovered that ∼100 nm exosomes were secreted by the epithelium and mesenchyme of a developing tooth organ and diffused through the basement membrane. Exosomes were entocytosed by epithelium or mesenchyme cells with preference by reciprocal cells rather than self-uptake. Exosomes reciprocally evoked cell differentiation and matrix synthesis: epithelium exosomes induce mesenchyme cells to produce dentin sialoprotein and undergo mineralization, whereas mesenchyme exosomes induce epithelium cells to produce basement membrane components, ameloblastin and amelogenenin. Attenuated exosomal secretion by Rab27a/b knockdown or GW4869 disrupted the basement membrane and reduced enamel and dentin production in organ culture and reduced matrix synthesis and the size of the cervical loop, which harbors epithelium stem cells, in Rab27a^ash/ash mutant mice. We then profiled exosomal constituents including miRNAs and peptides and further crossed all epithelium exosomal miRNAs with literature-known miRNA Wnt regulators. Epithelium exosome-derived miR135a activated Wnt/β-catenin signaling and escalated mesenchymal production of dentin matrix proteins, partially reversible by Antago-miR135a attenuation. Our results suggest that exosomes may mediate epithelium-mesenchyme crosstalk in organ development, suggesting that these vesicles and/or the molecular contents they are transporting may be interventional targets for treatment of diseases or regeneration of tissues.

Keywords: Wnt; development; epithelium; exosomes; mesenchyme; miR135a; miRNA.

Figure 1

Exosome characterization in epithelium, mesenchyme, and basement membrane. (A) Dental epithelium with cervical loop (arrowhead) was dissected from dental mesenchyme (D). (B, E) Isolated dental epithelial cells and mesenchymal cells. Transmission electron microscopy showing extracellular vesicles with ~100 nm diameters isolated from epithelium (C) and mesenchyme (F). (G, H) Size analysis revealed epithelium vesicles with diameters of 100.1 ± 2.2 nm and mesenchyme vesicles of 116.0 ± 2.3 nm. (I) Total proteins extracted from nanometer vesicles and parent cells probed by anti-CD63 and anti-GM-130 antibodies. (J, J′) CD63 (red); (K, K′) DAPI (blue); (L, L′) overlay. e: epithelium, m: mesenchyme, bm: basement membrane (arrowheads).

Figure 2

Preferential exosome uptake reciprocally by epithelium and mesenchyme cells. (A) Cy3-labeled siRNA electroporated mesenchyme-derived exosome (M/exo) incubated with epithelium (Epi) cells for 12 h and PBS control. (B) Mesenchyme (Mes) cells incubated with epithelium-derived exosomes (E/exo) for 12 h and PBS control. (C, D) Preferential endocytosis reciprocally by epithelium or mesenchyme cells upon exosome delivery (5.0 or 10.0 μg/mL exosomal protein content) (mean ± SD; six to 10 independent experiments). *P < 0.05, **P < 0.01 (one-way ANOVA and LSD tests).

Figure 3

Mesenchyme-derived exosomes induced epithelial cell differentiation and matrix synthesis. (A, B) Mesenchyme exosomes stimulated epithelium cells to produce ameloblastin (Ambn) and amelogenin (Amelx) mRNAs and proteins. (C, D) Collagen IV (Col IV) and Laminin (Lam) production by epithelium cells upon stimulation by mesenchyme exosomes at mRNA and protein level (mean ± SD; three to five independent experiments). *P < 0.05 (one-way ANOVA and LSD test).

Figure 4

Epithelium-derived exosomes induced mesenchymal cell differentiation and mineralization. (A) Epithelial exosomes promoted alkaline phosphatase (ALP) with higher magnification, quantified in B. (C) Alizarin Red (AR)-positive mineral nodule formation was increased with different doses of epithelium exosomes, with higher magnification and quantification (D). (E, F) Epithelium exosomes stimulated mesenchyme cells to produce Dsp at mRNA and protein (mean ± SD; five independent experiments). *P < 0.05 (one-way ANOVA and LSD test).

Figure 5

Attenuated exosome secretion evokes epithelium–mesenchyme dysmorphogenesis. (A) Rab27a and Rab27b expression in mesenchyme (Mes) and epithelium (Epi) cells following silencing of Rab27a and/or Rab27b. (B, C) Total exosomal proteins from stably transfected Rab27a and/or Rab27b per million Mes and Epi cells (mean ± SD; four independent experiments). *P < 0.05 (one-way ANOVA and LSD test). (D, E, G, H) Epithelium and mesenchyme in E16.5 tooth germs were microdissected and reconstituted/cultured for 4 days with scramble (NC) or Rab27a/b knockdown. (F, I) Fluorescence of F-actin and collagen IV (Col IV) in basement membrane (arrows). (J, M) Reconstituted tooth germs cultured for 10 days with scramble (NC) or Rab27a/b knockdown. Dentin formation in control (K, L) but not in Rab27a/b knockdown group (N, O).

Figure 6

Development deficiency in Rab27aash/ash mutation mouse. (A, C) HE staining of postnatal day 0 (P0) incisor of wild-type (WT) and Rab27a^ash/ash mice. Amelogenin (Amgn) (red) and Dsp (green) expression in WT incisor (B) but with reduced expression in Rab27a^ash/ash incisor (D), with enamel and dentin area quantified (I). HE staining of the cervical loop of E18.5 incisor tooth organ in WT (E) and Rab27a^ash/ash (G) mice, with E-cadherin (E-cad) (red) expression in WT (F) and Rab27a^ash/ash (H) mice, and quantified cervical loop area (J) (mean ± SD; five independent experiments, *P < 0.05, **P < 0.01, independent t test).

Figure 7

Profiling of exosomal constituents. (A) Heat map of microRNA profiles differentially expressed by epithelium cells and exosomes (exo). (B) A total of 35 miRNAs were identified by crossing all 390 miRNAs of our microarray with known miRNAs that regulate Wnt signaling (68). (C) Relative luciferase activity of Topflash reporter with PBS (Ctrl), Wnt3a (50 ng/mL), or 10.0 μg/mL epithelium exosomes (E/exo) (mean ± SD; five independent experiments). (D) miR135a expression in epithelium cells (Epi), epithelium exosomes (E/exo), and mesenchyme cells (Mes). (E) β-Catenin staining (green) of mesenchyme cells transfected with miRNA mimic, miR135a, or Antago-miR135a. Arrowheads indicate β-catenin transnucleation. (F) Relative luciferase activity of the Topflash reporter of mesenchymal cells transfected with miRNA mimic, miR135a, or Antago-miR135a (mean ± SD; seven independent experiments). (G, H) mRNA expression of Apc, Axin2, Dsp, Bglap, and Runx2 of mesenchymal cells incubated with epithelium exosomes (E/exo) that are transfected with miRNA mimic, miR135a, or Antago-miR135a (mean ± SD; four independent experiments). *P < 0.05, **P < 0.01 (one-way ANOVA and LSD test).