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During the gastrula stage of Xenopus laevis, mesodermal cells migrate on the blastocoel roof (BCR) toward the animal pole. In this process, mesodermal cells directly adhere to the BCR via adhesion molecules, such as cadherins, which in turn trigger a repulsive reaction through factors such as Eph/ephrin. Therefore, the mesoderm and BCR repeatedly adhere to and detach from each other, and the frequency of this adhesion is thought to control mesoderm migration. Although knockdown of cadherin or Eph/ephrin causes severe gastrulation defects, these molecules have been reported to contribute not only to boundary formation but also to the internal function of each tissue. Therefore, it is possible that the defect caused by knockdown occurs due to tissue function abnormalities. To address this problem, we developed a method to specifically induce adhesion between different tissues using rapalog (an analog of rapamycin). When adhesion between the BCR and mesoderm was specifically enhanced by rapalog, mesoderm migration was strongly suppressed. Furthermore, we confirmed that rapalog significantly increased the frequency of adhesion between the two tissues. These results support the idea that the adhesion frequency controls mesoderm migration, and demonstrate that our method effectively enhances adhesion between specific tissues in vivo.
Figure 1. riCAM; rapalog-induced cell adhesion molecule. (a) Schematic of rapalog-induced cell adhesion. Rapalog-induced riCAM1/2 heterodimerization causes cell-cell adhesion (top). Structure of riCAM1 and riCAM2 (bottom). SP, signal peptide. mCherry, monomeric Cherry. sfGFP, superfolder GFP (a valine codon was inserted after the start codon). TM, transmembrane domain. V5, V5-tag. (b-g) Dissociation and reaggregation assay. riCAM1 (150 pg) or riCAM2 (150 pg) mRNA was injected into the animal side of blastomeres at the 4-cell stage. Dissociated cells were cultured in calcium magnesium-free medium (CMFM) (n = 3) (b, e), CMFM containing 4mM CaCl2 (n = 3) (c, f), and CMFM containing 0.5 μM rapalog (n = 3) (d, g)
Figure 2. Checkerboard-like cell aggregate formation by rapalog. (a-e) Difference in adhesion patterns induced by rapalog or Ca2+. Embryos were co-injected with riCAM1 mRNA (150 pg) and Alexa Fluor 647-dextran (2.5 ng), or with riCAM2 mRNA (150 pg) and Alexa Fluor 488-dextran (2.5 ng), at the 4-cell stage. Cells dissociated in CMFM (a) were reaggregated using Ca2+ (b) or rapalog (c-e) (n = 3). Scale bar = 100μm. Arrowheads, adjacent cells expressing the same riCAM. (f, g) Representative confocal images used to analyze the adhesion patterns in (h). Arrowheads, heterotypic cell adhesion (adhesion between cells expressing different riCAM). Arrows, homotypic cell adhesion (adhesion between cells expressing the same riCAM). Scale bar = 30μm. (h) Quantification of adhesion patterns. Adhesion sites in Ca2+-induced aggregates (n = 21, 296 adhesion sites). Adhesion sites in rapalog-induced aggregates (n = 61, 273 adhesion sites). (i) Schematic of adhesion patterns induced by rapalog or Ca2+
Figure 3. Accumulation of riCAM1/2 in cellâcell contact sites. (a-e) Representative confocal images of cell aggregates induced by rapalog or Ca2+ (n = 3). Embryos were injected with various concentrations of riCAM1 or riCAM2 mRNA together with mTurquoise2-CAAX mRNA (150 pg) at the 4-cell stage. (c) Arrowheads indicate the adhesion sites in which riCAM is accumulated. Dots represent cells where mTurquoise2-CAAX expression is relatively weak. (d, e) Arrowheads indicate accumulation of riCAM in the adhesion sites. Dots represent cells in which riCAM is distributed throughout the cell membrane. (f) Model showing accumulation of riCAM with low expression levels in the adhesion sites
Figure 4. Accumulation of riCAM1/2 by rapalog in vivo. (a, b) No obvious differences were observed between rapalog-injected and control embryos at stage 40. Control (n = 48, 98%). Rapalog-injected embryo (n = 48, 90%). Control, embryos with no injection into the blastocoel. +Rapalog, embryos injected with rapalog into the blastocoel. (c-f) Accumulation of riCAM in the adhesion sites. Representative confocal images of animal cap cells expressing riCAM1 and riCAM2 separately at stage 11. Various concentrations of riCAM1 and riCAM2 mRNA were separately injected into the animal side of a blastomere at the 4-cell stage. Rapalog (100 μM/12 nL) was injected into the blastocoel at stage 9. Arrowheads indicate accumulation of riCAM. n = 3 (c); n = 3 (d); n = 7 (e); n = 6 (f). Control, embryos with no injection into the blastocoel. +Rapalog, embryos injected with rapalog into the blastocoel
Figure 5. Inhibiton of mesoderm migration by rapalog. (a) Experimental procedure for regulation of adhesion between the mesoderm and BCR using rapalog. At the 8-cell stage, riCAM1 mRNA (150 pg) and Alexa Fluor 647-dextran (2.5 ng) were co-injected into the animal blastomeres, while riCAM2 mRNA (150 pg) and Alexa Fluor 488-dextran (5 ng) were co-injected into the vegetal blastomeres. At stage 9, rapalog (100 μM/6 nL) was directly injected into the blastocoel. (b-f) Fluorescent dyes injected by the method in (a) efficiently label the BCR and mesoderm separately. The dorsal (D; arrowheads in green-labeled tissue) and ventral (V; arrowheads in green-labeled tissue) mesoderm migrate on the BCR (magenta) toward the animal pole during the early to mid gastrula stage (b-e). At the late gastrula stage, the dorsal and ventralmesoderm make contact in the anterior region of the embryo (dotted circle in f). (g-j) Rapalog strongly suppresses mesoderm migration in riCAM1/2-expressing embryos. riCAM1/2-expressing embryos (g); riCAM1/2-expressing embryos injected with rapalog (h); riCAM1-expressing embryos injected with rapalog (i); riCAM2-expressing embryos injected with rapalog (j). (k) Arc length between the dorsal and the ventralmesoderm tips was normalized by the diameter of the embryo. Kruskal-Wallist test with Dunn's multiple comparisons test; ****P < 0.0001. (-), embryos with no injection into the blastocoel. (+Rap), embryos injected with rapalog into the blastocoel
Figure 6. Increased frequency of intertissue adhesion by rapalog. (a-c) Representative confocal images of the Brachet's cleft using BABB-cleared embryos at stage 11. Embryos were injected with riCAM1 (150 pg) and riCAM2 (150 pg) mRNA together with Alexa Fluor 647-dextran (2.5 ng) at the 8-cell stage. Rapalog (100 μM/6 nL) was injected into the blastocoel at stage 9. Embryo injected with fluorescent dye (n = 11) (a); embryo injected with riCAM1/2 mRNA and fluorescent dye (n = 17) (b); embryo injected with riCAM1/2 mRNA, fluorescent dye, and rapalog (n = 22) (c). Arrowheads represent the boundary between the mesoderm and BCR. (d) Schematic of tissue separation assay. An aggregate of mesoderm or inner-layer ectoderm excised from embryos is placed on the BCR fragment, and the adhesion dynamics between the two tissues are analyzed by live imaging. (e-h) Snapshot of merged images from representative time-lapse imaging of attachment-detachment dynamics between two tissues: BCR expressing mCherry-CAAX, and ectoderm expressing mNeonGreen-CAAX (e); BCR expressing mCherry-CAAX and mesoderm expressing mNeonGreen-CAAX (f); BCR expressing riCAM1 and mesoderm expressing riCAM2 with (h) or without (g) rapalog. Detachment and reattachment between the mesoderm and the BCR were observed in (f) and (g). Arrowheads represent retraction fibers in the gap. (e’, h’) Single color images of (e) and (h). Arrowheads indicate riCAM, particularly riCAM2, accumulated in the adhesion sites of the BCR and mesoderm. No obvious accumulation of CAAX was observed. (i) Measurement of tissue separation. The number of times that BCR cells detached from ectoderm or mesoderm cells over one hour was recorded. n = 50 (e); n = 32 (f); n = 31 (g); n = 38 (h). (-), embryos with no injection into the blastocoel. (+Rap), embryos injected with rapalog into the blastocoel
