Vergara HM et al. (2018), miR-206 is required for changes in cell adhesio...

XB-ART-54743

Dev Biol 2018 Jun 15;4382:94-110. doi: 10.1016/j.ydbio.2018.03.021.

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miR-206 is required for changes in cell adhesion that drive muscle cell morphogenesis in Xenopus laevis.

Vergara HM, Ramirez J, Rosing T, Nave C, Blandino R, Saw D, Saraf P, Piexoto G, Coombes C, Adams M, Domingo CR.

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MicroRNAs (miRNAs) are highly conserved small non-coding RNA molecules that post-transcriptionally regulate gene expression in multicellular organisms. Within the set of muscle-specific miRNAs, miR-206 expression is largely restricted to skeletal muscle and is found exclusively within the bony fish lineage. Although many studies have implicated miR-206 in muscle maintenance and disease, its role in skeletal muscle development remains largely unknown. Here, we examine the role of miR-206 during Xenopus laevis somitogenesis. In Xenopus laevis, miR-206 expression coincides with the onset of somitogenesis. We show that both knockdown and over-expression of miR-206 result in abnormal somite formation affecting muscle cell rotation, attachment, and elongation. In particular, our data suggests that miR-206 regulates changes in cell adhesion that affect the ability of newly formed somites to adhere to the notochord as well as to the intersomitic boundaries. Additionally, we show that β-dystroglycan and F-actin expression levels are significantly reduced, suggesting that knockdown of miR-206 levels affects cellular mechanics necessary for cell shape changes and attachments that are required for proper muscle formation.

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Species referenced: Xenopus laevis
Genes referenced: dag1 fn1 gap43 mtor utrn

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	Fig. 1. Genomic architecture and expression analysis of miR-206 in Xenopus laevis. (A) Evolutionary conservation of miR-206/miR-133b cluster across various species. Top: Genomic sequence conservation landscape plot of miR-206/miR-133b cluster using the Homo sapiens genome as a reference. The immature sequences of both microRNAs are highlighted (grey). From top to bottom, species used to produce this multiple alignment are Mus musculus, Gallus gallus, Xenopus laevis, Xenopus tropicalis, and Danio rerio. On the right, the distance between the two microRNAs are indicated for each species. Bottom. Multiple sequence alignment of the immature miR-206 sequence for the species listed above. The box highlights the conserved mature miR-206 sequence. (B) Whole mount in situ hybridization analysis using a miR-206 probe (Exiqon) in X. laevis embryos at stage 32, lateral view, head to the left. A cross section at the level indicated by “B1” is shown to the right (B1). (C) Control in situ hybridization using DIG-labeled “scramble” probe (Exiqon). Magnification: 10 × . (D) Box plot showing the relative expression analysis of miR-206 and miR-133b across developmental stages using quantitative RT-PCR in uninjected embryos.
	Fig. 2. miR-206 morpholino specifically knocks down endogenous miR-206 levels. (A) miR-206 whole mount in situ hybridization in stage 28 embryo injected with 72 ng of miR-206 morpholino at the one-cell stage. (B) Control, miR-206 whole mount in situ hybridization in stage 28 uninjected embryo. (C) Box plot showing the relative expression levels of miR-206 calculated by quantitative RT-PCR in stage 28 embryos injected at the one-cell stage with variable amounts of either standard or miR-206 morpholinos.
	Fig. 3. Temporal analysis of miR-206 morphant embryos. Images of live uninjected embryos (A-E), embryos injected at the one-cell stage with standard MO (F-J) or miR-206 MO (K-O), and embryos injected at the two-cell stage in only one blastomere with standard MO (P-T) or miR-206 MO (U-Y). The first two columns show a blastoporal view, the third and fourth column show a dorsal view (head up), and the fifth column shows a lateral view (head up, ventral left). Magnification: 10 × .
	Fig. 4. Knockdown of miR-206 disrupts somite morphogenesis in X. laevis. Composite dorsal confocal images of stage 28 miR-206 (A) and standard (B) half-morphants showing the overall morphology of the paraxial mesoderm and surrounding tissues. A composite image shows the merged expression pattern of the lissamine-tagged MO (red), the muscle specific marker 12/101 (green), and the membrane-bound expression of GAP43 (white) in miR-206 (A1) and standard (B1) morphants The subsequent panels show each channel independently. The lissamine-tagged morpholino is restricted to the left side of the miR-206 (A2) and standard (B2) morphants. The muscle-specific marker, 12/101, shows muscle formation in both the miR-206 (A3) and standard (B3) morphant embryos. GAP43-GFP images were inverted and then pseudo-colored. The single asterisk represents the anterior-most somite undergoing rotation whereas the double asterisk represents the anterior-most region of the PSM. Magnification: 20 × . (C) Quantification of the miR-206 morphant phenotype. Morphant phenotypes were evaluated using a scaling rubric that spans from “Normal” to “Severe” phenotypes. The criteria for each score is described under each category and is based on various features including 12/101 expression levels, somite rotation, intersomitic boundaries and extracellular space between the notochord and somites. Embryos were first injected at the one cell stage with GAP43-GFP and then injected at the two-cell stage with miR-206 or standard MO. Between stages 26–28, embryos were stained with the muscle specific marker 12/101 and then scored using the described rubric. (D) A graph shows the percent of embryos that reflect phenotypes ranging from “Normal” to “Severe” for miR-206 MO-injected embryos and half embryos in comparison to standard MO-injected embryos.
	Fig. 5. Knockdown of miR-206 disrupts the adhesive interaction between the somites and the notochord. (A) Single-plane confocal image of standard-half morphant embryo stained for muscle (12/101) and notochord (Tor 70) illustrating the close juxtaposition between the notochord and the somites. (B) miR-206 half morphant embryo is detached from the notochord on the left side and Tor 70 expression is found in the intersomitic boundaries of the detached somites. (C) miR-206 MO injected at the one cell stage showing detachment of somites on both sides of the notochord. (D) Close-up of a miR-206 morphant somite showing the fine notochord sheath filaments (asterisks) indicating that adhesion to the notochord occurs throughout the medial edge of the somite with the highest concentration at the medial edge of the intersomitic boundary. (E) Graph showing the surface area (µm2) between the somites and notochord in miR-206 half morphants, standard half morphants, and the uninjected side of embryos between stages 24–26. An example of the surface area used in these measurements is highlighted by yellow dots around the notochord and somites shown in B. Significance is calculated using the Two-Sample T-test analysis, p < 0.005 for both comparisons.
	Fig. 6. Scanning electron microscopy shows that knockdown of miR-206 levels disrupts the overall somite architecture. Dorsal view of scanning electron micrograph (SEM) images of uninjected (A), standard (B) and miR-206 half morphant (C) embryos at stage 26. (A’-C’) Black box shows a close up of the mid-trunk region of the paraxial mesoderm. (A”-C”) Blue box provides a magnification of the PSM region. Asterisks in C” show the increasing separation of cells from the notochord as they begin to undergo rotation. (D-F) Dorso-lateral surface of embryos showing the neural tube and dorsal surface of the paraxial mesoderm of stage 24 embryos. (D’-F’) Black box shows a close-up view of the dorsal surface of somites.
	Fig. 7. Ectopic expression of miR-206 specifically disrupts somite morphogenesis. Composite image shows the merged expression pattern of stage 28 embryos injected at the one cell stage with GAP43 mRNA (white), and at the two-cell stage with 12 ng of either control (A) or miR-206 (B) duplex along with rhodamine dextran amine (RDA; red), a cell lineage tracer. Embryos were stained for muscle using 12/101 (green). Subsequent panels show each channel independently. RDA shows that the mimic is restricted to the left side in control (A’) and miR-206 (B’) mimic-injected embryos. Muscle-specific marker, 12/101, shows muscle formation in both control (A”) and miR-206 (B”) mimic-injected embryos. Cell shapes are highlighted by the expression of GAP43 GFP and images were inverted and pseudo-colored. (BIV) Asterisks highlight the irregularly-shaped cells at the lateral edge of many somites. Magnification: 20 × .
	Fig. 8. Impact of miR-206 levels on the distribution of β-integrin, fibronectin and collagen II during somite morphogenesis. Distribution of β-integrin, fibronectin and collagen II in standard MO (A, C, E), miR-206 MO (B, D, F), mimic control (G, I, K), and miR-206 mimic (H, J, L) embryos injected at the two-cell stage. The injected side is always on the left side of the embryo. 12/101 expression is shown in green. Sample size is indicated. Magnification: 25 × .
	Fig. 9. Changes in miR-206 levels significantly affect the expression levels of β-dystrogylcan, laminin and utrophin. Distribution of β-dystrogylcan, laminin and utrophin levels in standard MO (A, C, E), miR-206 MO (B, D, F) mimic control (G, I, K), and miR-206 mimic (H, J, L) embryos injected at the two-cell stage. The injected side is always on the left side of the embryo.12/101 expression shown in green. Sample size is indicated. Magnification: 25X.
	Fig. 10. Changes in miR-206 levels decrease β-dystroglycan protein levels. (A) Ethidium stained agarose gels of RT-PCR reactions. RT-PCR was performed on RNA extracted from three different samples consisting of uninjected and MO or mimic injected stage 28 embryos. mRNA levels of β-dystroglycan and Ornithine decarboxylase (ODC) were examined from three different experiments. No differences were detected between experimental and control samples. (B) Western blot analysis of β-dystroglycan from stage 28 embryos that were uninjected or injected with standard MO, control mimic, miR-206 MO, or miR-206 mimic. β-tubulin was used as a loading control. A significant decrease in β-dystroglycan protein levels is observed in both miR-206 morphant and mimic embryos in comparison to controls.
	Fig. 11. Knockdown of miR-206 disrupts actin filament formation. Maximum projections of cross sections through the trunk region of miR-026 (A) and standard (B) morphant embryos stained with phalloidin. F-actin staining is significantly reduced on the miR-206 MO side (Bâ€™) in comparison to the contralateral side and to the standard MO embryo (Aâ€™). Magnification: 25X.
	Fig. 12. miR-206 mimic specifically rescues miR-206 morphant phenotype. (A-C) Comparison between embryos co-injected with miR-206 MO and mimic (A), control mimic (B) and miR-206 MO only (C). Composite images represent the MO-lissamine (red) and the muscle marker 12/101 (green). (D) Bar plot showing the average length (µm) of 12/101 expression of each side of the embryo (injected or non-injected sides). (E-G) Comparison between embryos co-injected with miR-206 MO and mimic (E), control mimic (F) and miR-206 MO only (G). Composite image represents the MO-lissamine (red) and β-integrin (white) to highlight cell shapes. (H) Histogram summarizes the resultant phenotypes using the scoring rubric established earlier (see Fig. 4).