Extracellular vesicle-localized miR-203 mediates neural crest-placode communication required for trigeminal ganglia formation

Abstract

While interactions between neural crest and placode cells are critical for the proper formation of the trigeminal ganglion, the mechanisms underlying this process remain largely uncharacterized. Here, we show that the microRNA-(miR)203, whose epigenetic repression is required for neural crest migration, is reactivated in coalescing and condensing trigeminal ganglion cells. Overexpression of miR-203 induces ectopic coalescence of neural crest cells and increases ganglion size. Reciprocally, loss of miR-203 function in placode, but not neural crest, cells perturbs trigeminal ganglion condensation. Demonstrating intercellular communication, overexpression of miR-203 in the neural crest in vitro or in vivo represses a miR-responsive sensor in placode cells. Moreover, neural crest-secreted extracellular vesicles (EVs), visualized using pHluorin-CD63 vector, become incorporated into the cytoplasm of placode cells. Finally, RT-PCR analysis shows that small EVs isolated from condensing trigeminal ganglia are selectively loaded with miR-203. Together, our findings reveal a critical role in vivo for neural crest-placode communication mediated by sEVs and their selective microRNA cargo for proper trigeminal ganglion formation.

Keywords: Neural crest; condensation; extracellular vesicles; miR-203; trigeminal ganglion.

Figure 2:. Overexpression of miR-203 promotes ectopic and premature NC condensation and enhanced aggregation into the trigeminal ganglion.

(A) In situ hybridization for Sox10 in HH15–16 embryos unilaterally electroporated with miR-203 (miR-203 OE) or empty control (Control OE) overexpression vectors. (A’) Cross section of miR-203 OE embryo at the level shown in A (dotted line) reveals an ectopic Sox10+ group of cells (white arrowhead) and a denser TG (black arrowhead) on the treated side. (B) Quantification of embryos showing a phenotype (normal versus affected condensation having ectopic condensation and/or denser TG) on Control and miR-203 OE. Numbers in the graph represent the numbers of analyzed embryos. **P=0.001 by contingency table followed by a χ² test.

Figure 3:. miR-203 loss of function in placode cells but not in the NC causes abnormal trigeminal ganglion condensation.

(A-B) Electroporation scheme for region-specific knockdown of miR-203. Embryos at HH9 were unilaterally electroporated in the neural tube (A) or ectodermal placodes (B) with miR-203 or scrambled sponge plasmids and TG condensation was analyzed by ISH for Sox10 at HH17–18. Bar graphs represent the quantification of embryos showing a phenotype (normal versus affected condensation having a less aggregated ganglion) on the miR-203 and Scrambled sponge treated embryos. Numbers in the graphs represent the numbers of analyzed embryos. **P=0.0015 by contingency table followed by a χ² test.

Figure 4:. Extracellular vesicles are actively released by NC cells in the vicinity of placode cells during trigeminal ganglion condensation.

Transmission electron microscopy from dissected trigeminal ganglia at HH16–17 shows that cells have many direct cell-to-cell contacts to each other (A), the presence of multivesicular bodies (MVB) releasing exosomes-like structures into the extracellular space (B), and a shedding vesicle protruding from plasma membrane (C). Bars: 2 μm (A), 200 nm (B and C). (D) Diagram of pHluo-CD63-mScarlet functional assay. (E) Embryos electroporated in the neural tube with pHluo-CD63-mScarlet at HH9- and visualized at stage HH16 exhibited GFP+/mScarlet+ fluorescence in the condensing trigeminal ganglion area (dotted line); in contrast, cells located in the midbrain are enrich in mScarlet+ (white arrowheads). TG, trigeminal ganglion; E, eye. (F) Migratory neural crest cells electroporated with pHluo-CD63-mScarlet exhibit intraluminal MVB (pHluo-/mScarlet+), extracellular/releasing EVs (pHluo+/mScarlet+) and deposit trails behind the cells. (G) Transverse section through the TG showing NC cells electroporated with pHluo-CD63-mScarlet and placode cells immunostained for Tuj1 (magenta). (G’) Zoom of box in G shows a placode cells (Tuj1+) with pHluo puncta incorporated into their cytoplasm (white arrowheads).

Figure 5:. Extracellular vesicles produced by NC cells are engulfed by placode cells in explant co-cultures.

(A) Scheme of a chicken embryo at HH9⁻ with neural tube electroporated with a pHluo (pseudocolored in yellow) and placode cells electroporated with membrane RFP (pseudocolored in magenta), respectively. The electroporated dorsal neural tube (mostly containing NC cells) and ectodermal trigeminal placode cells were dissected at HH9⁺ and HH10⁻, respectively, and co-cultured to examine their interactions. (B) NC cells (yellow) exhibiting trails (possibly migrasomes or retraction fibers) and cytonemes in very close contact with placode cells (magenta). (B’) Higher magnification of box in B showing the cytonemes from NC cells contacting placode cells. See also Supplementary Movie 1. (C) Placode cells showing vesicular structures like macropinosomes in their membrane (white arrowheads). See also z-stack in Supplementary Movie 2. (D-D’) NC cells expressing pHluo were surrounded by numerous extracellular pHluo+ puncta (possibly EVs deposits) and trails (possibly migrasomes or retraction fibers). Co-cultured placodal cells showing intra-cytoplasmic vesicular structures containing pHluo+ puncta (white arrowheads). See also Supplementary Movie 3 and 4. (E) Placode cell co-cultured with NC cells expressing pHluo produce filopodia that move toward the EVs deposits and engulf them. (E’) Zoom of box in E showing the time-lapse showing placode cells engulfing a pHluo+ puncta (white arrowhead). See also Supplementary Movie 5. (F) 3D reconstruction of placode protrusion observed in (E) demonstrating the internalized pHluo+ puncta (white arrowhead). See also Supplementary Movie 6.

Figure 6:. miR-203 generated in NC cells inhibits a sensor target electroporated in placode cells both in vivo and ex vivo.

(A) Scheme of chick embryo with neural tube electroporated with miR-203 or empty control overexpressing vectors (with cytoplasmic EGFP) and placode cells electroporated with a dual-colored sensor vector (pSdmiR-203) containing two copies of complementary sequences to the mature miR-203. pCAG, Chick β-actin promoter; d4EGFPn nuclear-localized destabilized EGFP with a half-life of 4 h; mRFPn, nuclear-localized monomeric red fluorescent protein. Box of the condensing trigeminal ganglion indicates area of enlarged inset exemplifying the transfer of miR-203 from NC (green) to placode cells (orange because of the expression of both EGFP and RFP), thus affecting the EGFP translation (red cell). (B) Transverse section of HH17 embryos immunostained for Tuj1 (magenta), to identify the trigeminal ganglia with coalescing NC (electroporated with miR-203 OE vector) and placode (electroporated with the dual-colored sensor vector. See white arrowheads) cells. (B’) Zoom of box in B identifying migratory NC cells with cytoplasmic EGFP+ (black arrowhead) and placode cells with nuclear EGFP+/RFP+ (white arrowhead) or only RFP+ (dotted circles). (C) Time-lapse of co-cultured placode ectodermal cells (electroporated with the dual-colored sensor vector) and dorsal neural tube (electroporated with miR-203 OE) explants. Placodal cells (PC1–6) interact with NC cells (NC1–2) where the EGFP channel was pseudocolored (red>white>blue) to visualize the EGFP decay in placode cells over time. See also Supplementary Movie 7. (D) Scatter plot from in vivo experiments analyzing the EGFP/RFP intensity for individual placode cells reaching the condensing area at HH17 from miR-203 OE or Control electroporated embryos (cells counted in 2–5 sections per embryo, n=4 embryos from two independent electroporations). ****P<0.0001 calculated using unpaired Student’s t-test. Error bars indicate standard deviation. (E) Scatter plot from ex vivo co-cultured experiment analyzing the EGFP/RFP normalized intensity for individual placode cells interacting with NC cells from miR-203 OE or Control electroporated embryos (100 cells, n=4 co-cultured explants for each treatment from 2 independent experiments). ****P<0.0001 calculated using unpaired Student’s t-test. Error bars indicate standard deviation.

Figure 7:. Endogenous miR-203 is selectively loaded into sEVs produced during trigeminal ganglion coalescence.

(A) Graphs show particle concentration, size, and mode size using nanoparticle tracking (NTA) from enriched sEVs samples isolated from dissected trigeminal ganglia at HH17. (B) Transmission electron microscopy (TEM) image showing isolated exosomes after sEVs purified from dissected trigeminal ganglia. (C) RT-PCR from two independent whole dissected trigeminal ganglia (TG1 and TG2) and isolated sEVs from condensing trigeminal ganglia (sEV1 and sEV2) raised against the endogenous miR-203 (containing the EXOmotif) and miR-34a-5p (containing the CELLmotif).