|
Fig. 1. LEM migrates faster than AM. (A) Scheme of the in vitro migration assay. The BCR, LEM, and AM were dissected from St. 10+ embryos. The BCR was used for substrate conditioning. Green lines on the BCR and culture dish indicate the extracellular matrix. (B) RT-PCR confirmation of the dissected animal cap (AC), LEM, and AM. Epidermal keratin I (epi. keratin I), epidermal marker; Cerberus, LEM marker; Xbra, AM marker; Ornithine decarboxylase (ODC), internal control. WE, whole embryos; –RT, control experiment without reverse transcriptase. (C) Still images from a time-lapse movie of LEM and AM (Movie S1) on a normal BCR-coated dish. Green filled circles indicate the centroid of the explant. Green lines are traced lines. The animal pole on the reproduced substrate is up. Scale bars: 500 μm. (D) Tracings of LEM (left) and AM (right) centroids migrating on a BCR-coated dish. Black lines show individual traces for 5 h. The intersections of the red lines indicate the initial point. In both experiments, wild type-BCR explants were used for the BCR coating.
|
|
Fig. 2. Force measurement with a glass needle. (A) The size of the micro-glass needle and set-up of the experiment are shown at the left and right, respectively. Red filled circle represents the LEM explant. Green lines on the dish indicate the conditioned substrate. (B) Schematic of the experimental strategy for force measurement with a micro-glass needle. The generated force was calculated as described in Materials and methods section. (C) Still images from a time-lapse movie of the force-measurement experiment (Movie S2). Black dotted line indicates the initial position of the micro-glass needle. Anterior of the BCR-substrate is up. Scale bar: 500 μm. (D) An example of the relationship between generated force and migration speed. Red line shows the moving average of migration speed. Blue line indicates generated force. Gray line is the original migration-speed data. Black dotted line indicates the maximum generated force. (E) Schematic of prepared explants and measured maximum force obtained from LEM (n=14, 41.1±11.5 nN, mean±s.d.). Single LEM explants were cut into pieces of about 500×500 μm. (F) Quantification of the maximum force obtained with LEM explants of different sizes (n=27). Circles indicate individual samples. The best-fit line is shown (R2=0.57).
|
|
Fig. 3. AM receives tension from LEM. (A) Schematic of the laser ablation experiment. Explants in which the connections between the LEM, AM, and ectoderm were maintained were cut from a St. 10+ embryo and flattened on a piece of a BCR-coated dish. The explant, held by glass-plates, was then turned upside-down on a culture dish. Brown line indicates the epidermis layer. White dashed line indicates BCR-coating. (B) Bright-field images of explants after a 2 h incubation. Two types of explants were prepared: one included migratory LEM (+LEM) and the other did not (–LEM). (C) and (D′) Laser ablation experiment in the presence (C) and absence (D) of LEM. AM was ablated along the mediolateral axis (red lines). Fluorescent images of memGFP-injected explants were taken just before (magenta) and immediately after (δt=4 s, green) ablation. White boxed region in C and D is magnified in C′ and D′, respectively. Scale bar: 50 μm. (E) and (F) Deformation map generated by PIV analysis showing the magnitude of anteroposteriorly (A–P) directed displacement. Lower displacements are indicated with the color range of purple to blue; regions of high displacements are in the color ranges of yellow to red. White-boxed regions indicate the ROIs for quantification. a, anterior. p, posterior. (G) Mean displacements calculated from the white-boxed regions in E and F. Positive and negative values of vertical axis indicate anteriorly deformation and posteriorly deformation, respectively. +LEM explants generated greater recoils on the anterior and posterior side (n=26, 7 batches, 5.11±1.73 μm (anterior), 3.96±1.73 μm (posterior), mean±s.d.) of the ablation line compared with the –LEM explants (n=27, 7 batches, 4.00±1.79 μm (anterior), 2.98±1.67 μm (posterior), mean±s.d.). *P<0.05.
|
|
Fig. 4. BCR-targeted xFN-MO injection only affects the BCR region. (A) Schematic of BCR-targeted MO injection. At the beginning of the 4-cell stage, 10 nl of MOs was injected into the ventro-animal pole of both blastomeres (green) at 0.35 mM. (B) Immunostaining of flag-β-globin (tracer), fibronectin (FN), and Phalloidin staining in BCR-targeted MO-injected embryos. White boxed regions in the FN-stained images are magnified at the bottom. Dorsal is to the right. (C)–(E) Scheme of experiments and FN immunostaining images in dorsal-lip explants. C, Std.–MO targeted to BCR; D, xFN-MO targeted to BCR; E, xFN-MO targeted to DMZ. Green indicates the MO-injection site and red dotted lines indicate explanted region. (F) Quantification of the mean intensity of FN in dorsal lip explants. Results for Std.–MO injection targeted to the BCR (Cont-BCR), at the left (n=6); xFN-MO targeted to BCR (MO-BCR), in the middle (n=4); and xFN-MO targeted to the DMZ (MO-DMZ) at the right (n=3). Error bars indicate s.d. *P<0.05.
|
|
Fig. 5. BCR-targeted xFN-MO injection affected the migratory activity of LEM and resulting force. (A) and (B) Still images from a time-lapse movie of LEM on BCR coating from Std.-MO-injected embryos (A) or xFN-MO-injected embryos (B). Green filled circles indicate the centroid of the explants. Green lines trace the movements of the centroid. The anterior of the reproduced substrate is up. The right graphs shows traces of LEM centroid migratory path on the BCR coating from Std.-MO-injected embryos (upper, n=6) or xFN-MO-injected embryos (bottom, n=7). Black lines show individual traces obtained for 5 h. The intersection of the red lines indicates the initial point. The animal pole of the reproduced substrate is up. In both experiments, wild-type LEM explants were used. (C) and (D) Scheme of experiments and deformation map generated by PIV analysis showing the magnitude of A–P directed displacement. Explants were prepared as shows in Fig. 3. C, Explants were placed on the Std.–MO BCR. D, Explants placed on the xFN-MO BCR. Red lines indicate ablation lines. White boxed regions indicate the ROIs for quantification. a, anterior. p, posterior. (E) The mean displacements along A–P direction calculated from the white boxed regions in C and D. Explants on the control BCR-coating showed significantly greater recoils (n=50, 12 batches, 4.67±2.59 μm (anterior), 4.56±2.34 μm (posterior), mean±s.d.) than explants on the xFN-MO BCR-coating (n=38, 9 batches, 3.11±1.87 μm (anterior), 3.16±1.66 μm (posterior), mean±s.d.). **P<0.01. (F) Scheme of the DMZ shrinkage assay. See Materials and methods section for details. (G) Bright-field views of the DMZ in the shrinkage assay. White dotted lines indicate the initial position of the edge. Scale bars: 200 μm. (H) Relative length of the AM during shrinking. Blue indicates control (n=13), red indicates BCR-FN morphants (n=15). Error bars indicate s.d. **P<0.01.
|
|
Fig. 6. Migrating LEM is necessary for normal gastrulation movement and elongation of AM. (A) and (B) Dorsal views of BCR-injected embryos at St. 12.5. The white brackets indicate the diameter of the blastopore. (C) and (D) Expression of Xnot, an AM marker, in a Std.-MO (control) (C) or xFN-MO (D) -injected embryo. The black and gray brackets in (D) indicate the widened and shortened notochord. (E) Quantification of embryos showing gastrulation defects (G.D.). Almost all control embryos were normal (n=39), whereas the BCR-FN morphants (n=30) showed a higher frequency of G.D. If the size of the yolk plug was bigger than a third of the diameter of the embryo, we categorized the sample as severely defective. (F) Quantification of the length and width of Xnot staining. Compared with controls (n=8), the BCR-FN morphants (n=7) showed a widened and shortened notochord. Error bars indicate s.d. **P<0.01. (G) Morphants at a late stage (St. 31). Scale bar: 1 mm. (H) Quantification of the A–P length of the late morphants. The dorsal axis extension was moderately reduced in the BCR-FN morphants (n=31), compared with the Std.-MO-injected embryos (n=21). Error bars indicate s.d. **P<0.01.
|
|
Fig. 7. Cell orientation and elongation in the AM were disrupted by the reduction of LEM migratory activity. (A) Scheme of notochord imaging. Gray square shows the plane of section. Membrane-localized RFP and GFP (memRFP/memGFP) were each injected into one side of the embryo. (B)–(D′) Confocal image and analysis of control embryos (B)–(D) and BCR-FN morphants (B′)–(D′) at St. 12.5. (B) and (B′) AM cells expressing memRFP/memGFP within a confocal section. Yellow dotted lines indicate the notochord-somite boundaries. (C) and (C′) Cell aspect ratio (AR) analysis. Yellow and orange indicate high AR cells; blue and purple indicate low AR cells. The mean AR in the controls (C) was 2.39±1.06 (n=843 cells from 3 embryos, mean±s.d.); in the morphants (C′), the mean AR was 1.93±0.78 (n=1042 cells from 3 embryos, mean±s.d.). Dotted line indicates the mean AR of controls. ***P<0.005. (D) and (D′) Analysis of cell long-axis angle. Sample numbers were the same as in C and C′, respectively. Rose diagrams show the frequency distribution of the cells′ angles. Dots along the outer periphery indicate individual cell angles. an, anterior; ml, mediolateral; p, posterior. Anterior of the embryos is up. (E) Scheme of tissue dissection for western blot analysis. Dorsal regions which did not contain MO-injected BCR regions were isolated at St. 12.5. Red line indicates cut plane. (F) The dorsal activities of RhoA and Rac1 were not changed in BCR-FN morphants. DMZ explants and ventral marginal zone (VMZ) explants were dissected from St. 10.5 embryos and used for control; RhoA and Rac1 were highly activated in DMZ, but not in VMZ.
|
|
Fig. 8. Simultaneous knockdown of LEM′s anterior migration and the Wnt/PCP pathway causes severe defects in gastrulation movements. (A) Scheme of combined knockdown experiment. xFN-MO (0.35 mM) was targeted to the BCR by injection at the 2-cell stage, and Xdd1 mRNA (200 pg) was targeted to the DMZ by injection at the 4-cell stage. (B) Quantification of embryos that showed gastrulation defects. Almost all control embryos were normal (n=120). The combined knockdown (n=117) caused severer defects than the single injection of xFN-MO (n=118) or Xdd1 mRNA (n=115). The notochord structure was confirmed by in situ hybridization of Xnot.
|
|
Fig. S4.
No effect of xFN-MO injection into the BCR on patterning of the dorsal region. In situ hybridization analysis of control and BCR-FN morphants. Cerberus is a dorsoanterior mesendoderm marker. Xbra is a pan-mesoderm marker. Xnot is a notochord marker. MyoD is a somite maker. Although the notochord structure was shortened, the dorsal patterning was not affected by the BCR-targeted xFN-MO injection.
|
