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We describe a method of generating three-notochord explants to analyze the cell movements of convergent extension (CE) during Xenopus laevis gastrulation and neurulation. This method uses standard microsurgical techniques under a fluorescence stereomicroscope to combine notochordal sectors of gastrulae, side by side (lateral surfaces apposed) into a single explant. Three-notochord explants cultured on bovine serum albumin (BSA)-coated glass converged mediolaterally and extended in the anterior-posterior direction. The individual notochordal cells showed the mediolaterally oriented, bipolar tractional motility and the resulting mediolaterally oriented cell intercalation characteristic of CE, thereby reproducing both the in vivo tissue and the cell movements in an explant. Image analysis of three-notochord explants reveals the effects of overexpressions or knockdowns of genes, of manipulation of the extracellular matrix, and of exposure to chemical reagents on morphogenesis during gastrulation and neurulation, compared with control explants. Moreover, since three-notochord explants provide two zones of cell intercalation between notochords, individual cell behaviors between notochords of different characteristics and experimental treatments can be observed at the same time.
Figure 1. Making an imaging chamber. (A) The required materials include a Bunsen burner (a), lighter (b), cork borer (c), 60 × 15 mm Petri dish (d), 20 ml plastic syringe (e), silicone grease (f), 100 × 15 mm Petri dish (g), 50 × 70 mm cover glass (h), and forceps (i). (B) Heat the cork borer with a Bunsen burner. (C) Melt through the bottom of the plastic a 60 × 15 mm Petri dish with the hot cork borer. (D) Extract the plug of plastic to make a hole at the bottom of the plastic Petri dish. (E) Inject silicone grease into the plastic syringe. (F) Grease-filled plastic syringe. (G) Apply silicone grease along the outer edge of the hole in the Petri dish. (H) Submerge a 50 × 70 mm cover glass in ethanol in the Petri dish. (I) Grasp the cover glass with forceps, and flame it with a Bunsen burner. (J) Press the silicone grease rimmed dish against the coverslip to seal the imaging chamber
Figure 2. Making a notochord sector. (A–C) Hair tools. (A) Hair loop, upper. Eyebrow knife, lower. (B) Enlarged image of hair loop. (C) Enlarged image of eyebrow knife. (D–E) Alexa 488-dextran (green tracer; 100 pg/blastomere) or Alexa 594-dextran (red tracer; 300 pg/blastomere) was microinjected into the two dorsal blastomeres of the four-cell embryo. The microinjected embryos were observed at stage 10. (D) Bright field microscopic image. (E) Red fluorescence image. (F) Green fluorescence image. (G, H) Gastrula stage embryos after removing the vitelline membrane. Vegetal view. Dorsal side is on the right side of the figure. (G) Bright-field microscopic image. (H) Red fluorescence image. (I, J) The dorsal marginal sector of the gastrula-stage embryo was cut out at an angle of 120° from the center. (I) Bright-field microscopic image. (J) Red fluorescence image. (K–N) Shaving the endodermal/mesendodermal cells off from the dorsal marginal sector’s inner surface. Animal pole side is on the upper side of the figure. (K) Inner side of the dorsal marginal sector before shaving. (L) Shaving large endodermal mass from the dorsal marginal sector. (M) Shaving remained mesendodermal cells from the dorsal marginal sector. (N) The dorsal marginal sector after shaving the endodermal/mesendodermal cells. (O) The outer side view of the dorsal marginal sector of Figure 2N. (P) Cut out an area of notochord, including epithelium, from the dorsal marginal sector. (Q) The notochord area with epithelium. (R) The notochord area, leaving part of the epithelium at the animal pole side
Figure 3. Schematic of making a notochord sector. (A) Cut the dorsal marginal sector of a gastrula-stage embryo (stage 10). (B) Shave off the endodermal cells. (C) Cut out the notochord area with the width of 0.2–0.3 mm, including epithelium. (D) Remove most of the epithelium from the notochord sector
Figure 4. Making a three-notochord explant. Far left columns show bright-field microscopic images. Second columns from left show red fluorescence images. Third columns from left show green fluorescence images. Far right columns show merged images of red and green fluorescence. (A) Three notochord sectors with different fluorescence color signals in an alternating pattern before pressing with small cover glasses. (B) Three notochord sectors that were pressed by three small cover glasses. (C) The three notochord sectors that were pressed horizontally from four directions with cover glasses and covered with another cover glass from above. (D) The three grafted and healed notochord sectors with epithelium. (E) The three-notochord explant after removing epithelium.
Figure 5. Schematic of making a three-notochord explant. (A) Three notochord sectors with different fluorescence color signals arranged in an alternating pattern, pressed from the left, top, and right with small cover glasses. (B) Three covered notochord sectors pressed together with three small cover glasses now covered with a large cover glass. (C) Three notochord sectors pressed from the bottom with another coverslip. (D) Cut off the epithelium of the three grafted notochords. (E) Apply silicone grease at some distance on both sides of the three-notochord explant. (F) Record the cell behaviors of the three-notochord explants using an inverted fluorescence microscope
Figure 6. The shape of three-notochord explants and their individual cell behaviors. The elapsed time is indicated at the bottom. (A) Green tracer (Alexa 488-dextran) or red tracer (Alexa 594-dextran) was co-injected with XCXCLC mRNA (experimental) or without (control) into the two dorsal blastomeres of four-cell embryos. Time-lapse fluorescence images of three-notochord explants were recorded every 2 min for 10 h. The far right columns are high magnifications of the last frames of the three-notochord explants. The enlarged images showing the gray-scale version of green fluorescence provides better contrast than the two-color versions. Arrowheads indicate a protrusion. The dotted line indicates the midline of the explant. (B) The average convergence index for the three-notochord explants are indicated. (C) The cell shape of the three-notochord explants. The length-to-width ratio (left) and orientation (right) of 12 cells of the typical three-notochord explants are shown
