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Development of the Xenopus embryo has provided an adaptable framework for the rapid evaluation of molecular factors that guide patterning and morphogenesis. We present and characterize a novel explant preparation that is useful for such studies. This preparation consists of 180 degrees of the marginal zone of the early Xenopus gastrula cultured on a fibronectin-coated substrate. In addition to a thorough description of its preparation, we analyze gene expression patterns at three stages of development. The stereotypic morphogenesis of this explant can be understood in the context of the intact embryo through a catalog of gene expression patterns providing definitive identities for epidermis, anterior and posterior neural, notochord, somitic mesoderm, and mesendoderm.
Fig. 1. Morphogenesis of the marginal zone explant. (A) An embryo at stage 10 þ is dissected in a series steps that separate the marginal zone from the rest of the embryo. Sub-blastoporal endoderm (tissues vegetal of the contracted bottle cells; dotted region) are included with this preparation. (B) Four frames from a timelapse sequence of an explant cultured for 19 h on fibronectin. The last frame shows the cement gland (arrow) forming at the animal end (an) of the anterior neural anlagen (ana). The dorsal ridge (dr) can be seen after 6 h. All views are en face with the same orientation: animal up (an), vegetal down (vg), and lateral on either side of the midline (dotted line).
Fig. 2. Gene expression in the explant. (A) Mix2, xnr3, cerberus, xwnt8, and sox17a gene expression in explants immediately after isolation from the early gastrulaembryo. (B) Xk81, otx2, neural-specific tubulin, xslit, chordin, and xwnt8 gene expression patterns in explants cultured to the equivalent of the mid-late gastrula stage (approximately stages 12–14). (C) Xrx, xslit, chordin, slug, neural-specific tubulin, sonic hedgehog, xmsr gene expression at the equivalent tadpole stage (stages 25 – 28). Immunolocalization of tor70 (marking differentiated notochord) is shown in red accompanying slug, neural-specific tubulin, and sonic hedgehog. Immunolocalization of 12/101 (marking differentiated somitic mesoderm) is shown in red accompanying the xmsr gene expression pattern. All views are en face with the same orientation of animal up, vegetal down, and lateral on either side of the midline.
Fig. 3. Architecture of the marginal zone explant. (A) Schematic showing the three stages of explant morphogenesis. From left to right: shortly after microsurgery and plating on fibronectin (line shows blastopore), 4 h after isolation (line shows dorsal ridge), and 24 h after isolation (line shows neural anlagen boundary and dorsal ridge). Dashed line insets correspond to the location of XY-sections. Dotted line insets correspond to the location YZ-reconstructed cross- sections through the dorsal ridge. (B) A single en face XY-section through the early explant shows the small cell mesoderm (scm) animal of the blastopore (bp) and large cell mesendoderm (lcm) vegetal of the blastopore. (B0) A cross-section reconstructed from serial XY sections shows the mesendoderm is approximately 2 cells thick while the mesoderm is 4 – 5 cells in thickness. Mesoderm and mesendoderm are separated by the blastopore (arrowhead). (C) An en face XY-section through an explant cultured for 4 h shows mediolaterally elongated mesoderm cells within the dorsal ridge. (C0 ) A cross-section reconstructed from serial XY sections shows the dorsal ridge in cross-section where mesendoderm and neural tissues (me and ne, respectively) are approximately 2–3 cells thick while the dorsal ridge is 6–8 cells thick. (D) An en face stereoscope view of somites (stained with mAb 12/101) in an explant cultured for 24 h (approximate stage 25). Boundaries of the explant (not seen) are indicated by the dotted line. (D0) Higher resolution view of the somitic mesoderm (dashed line inset in D). (E) A single confocal section (75 mm from the coverslip) through the same region as in D0 showing a notochord running through the center of the somitic mesoderm. (E0) A cross-section at the location of blue line in E showing the notochord (arrow) cradled by the somitic mesoderm above the fibronectin coverslip (black line along the left). (E) A single confocal section showing the medial aspect (arrow) of the notochord (no) adjacent to the coverslip (20 mm deep). The notochord separates from the fibronectin substrate laterally (arrowhead) where it is underlaid by somitic mesoderm (so). All panels are en face views with the exception of B0, C0 and E0 and all panels have the same orientation of animal (an) up and vegetal (vg) down.
Fig. 4. Cell movement: from the marginal zone into the dorsal ridge. (A) Frames from a timelapse sequence of a single explant expressing a nuclear localizing GFP. Each frame is a montage of four images collected simultaneously from four quadrants of the explant. (B) A digitally enhanced closeup of the marginal zone showing the location of individual nuclei. (C) Frames from a timelapse sequence shown in the inset (dashed line in B) at 2 h intervals. Thirty-minute trajectories of cell nuclei in explants are overlaid on an image of nuclei positions. Green indicates the current position of the nuclei, yellow indicates positions 6 and 12 min earlier, and red indicated positions 18, 24, and 30 min earlier. (C0 ) The same frames as shown in C showing only the 30 min trajectories of nuclei. All panels are en face views with animal up and vegetal down.
