Davis RL and Kirschner MW (2000), The fate of cells in the tailbud of Xenopus lae...

XB-ART-11773

Development 2000 Jan 01;1272:255-67. doi: 10.1242/dev.127.2.255.

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The fate of cells in the tailbud of Xenopus laevis.

Davis RL , Kirschner MW .

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The vertebrate tailbud and trunk form very similar tissues. It has been a controversial question for decades whether cell determination in the developing tail proceeds as part of early axial development or whether it proceeds by a different mechanism. To examine this question more closely, we have used photoactivation of fluorescence to mark small neighborhoods of cells in the developing tailbud of Xenopus laevis. We show that, in one region of the tailbud, very small groups of adjacent cells can contribute progeny to the neural tube, notochord and somitic muscle, as well as other identified cell types within a single embryo. Groups averaging three adjacent cells at a later stage can contribute progeny with a similar distribution. Our data suggest that the tailbud contains multipotent cells that make very late germ-layer decisions.

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Species referenced: Xenopus laevis
Genes referenced: actc1 actl6a dnai1 fgf4 ncam1 not tbx2

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	Fig. 1. (A) Schematic of procedure. Embryos are injected at the 2-cell stage with caged fluorescein dextran conjugate (CFD). Photoactivation is performed at stage 31 (stage 36 for small aperture activations). Embryos are imaged immediately to record photoactivation marks and returned to the dark, or are imaged by confocal microscopy to observe the number of cells labeled. Embryos returned to the dark are then imaged again at stage 45-46 (47-48 for small aperture photoactivations) to record the fate of progeny cells. (B) Stage 31 tailbud embryo following in situ hybridization for somites (cardiac actin, magenta; Stutz and Spohr, 1986), neural tube (NCAM, navy blue; Krieg et al., 1989), and chordoneural hinge (Xnot, originally navy blue, but pseudocolored green in this image; von Dassow et al., 1993). This embryo has also been stained with a monoclonal antibody to keratan sulfate (MZ15, cyan; Smith and Watt, 1985), labeling the outer sheath of the notochord. (C) Drawing of tailbud region of stage 31 embryo based on several focal plane images of embryo in B. The posterior epidermis has been peeled away to reveal the underlying axial and paraxial structures.
	Fig. 2. Regions of tailbud photoactivation. (A) Tailbud of CFD injected, but not photoactivated, stage 31 embryo. The range of dorsal, ventral, and posterior photoactivation regions is outlined in black. The mean ± 1 s.d. of these regions is outlined in white. (B) Same embryo as in A at stage 45. (C) Tailbud region of a stage 31 embryo, stained for eFGF expression by whole-mount in situ hybridization (medium blue; Isaacs et al., 1992; Isaacs et al., 1995). Photoactivation regions are outlined. (D) Same as C, with anatomical structures noted. Scale bars, 100 mm.
	Fig. 3. Example of regional photoactivation. (A) Photoactivation spot in stage 31 embryo tailbud (TB12p7, dorsal region, Table 1). Anterior is to the left; dorsal is top. (B) Magnified image of C, showing separate fluorescence in notochord (No) and neural tube (NT). (C) Posterior tail region of same embryo at stage 45, with fluorescence signal in notochord (No) and neural tube (NT). (D) More anterior image of same embryo, with fluorescence signal in several myocytes (Dorsal Myo). This signal is the same as the out-of-focus signal at the far left (anterior) of C. (E) Fluorescence image in C superimposed on DIC image. (F) Fluorescence image in D superimposed on DIC image. Fluoresence is pseudocolored green in E and F. Scale bars, 100 mm.
	Fig. 4. Confocal imaging of photoactivation marks. (A) Wide-field digital camera image of a photoactivation mark in the dorsal region of a stage 31 embryo. (B) Single confocal plane of tailbud region of stage 31 embryo previously injected with RNAs encoding membrane-localized and nuclear-localized GFP proteins (see Materials and Methods). (C) Single confocal plane of photoactivation mark of embryo in A. (D) Summed image of confocal z-series of photoactivation mark in same embryo after horizontal and vertical edge filters to enhance cell boundaries. 25 images are summed in D. Nine fluorescent cells are labeled. Note that the single confocal plane in C does not accurately summarize the entire z series, when compared to D. Scale bars, 100 mm.
	Fig. 5. Examples of regional photoactivation. (A,B) Merged fluorescence and DIC images of the same dorsal region photoactivated embryo (TB25-9, Table 1). Anterior is to the left; dorsal is top. Note label in myocytes (Dorsal Myo), notochord (No) and neural tube (NT). (C) Merged fluorescence and DIC image of a dorsal region marked embryo (TB18-1, Table 1) showing label in dorsal fin mesenchymal cells. (D,E) Merged fluorescence and DIC images of the same ventral region photoactivated embryo (TB23-6, Table 2). Anterior is to the right; dorsal is top. Note, E is a higher magnification image of the field of view in D. (F,G) Merged fluorescence and DIC images of the same posterior region photoactivated embryo (TB24-7, Table 3). Anterior is to the left; dorsal is top. Note, G is a higher magnification image of the field of view in F. For ventral and posterior regions, only labeled myocytes (Dorsal Myo and Ventral Myo) are seen (other cells are labeled more posteriorly, but not shown; however, no signal is found in notochord or neural tube in these embryos). Fluorescence is pseudocolored green. Scale bars, 100 mm.
	Fig. 6. Confocal imaging of small photoactivation marks. (A) Widefield digital camera image of a photoactivation mark (using 30 mm pinhole aperture) in stage 36 embryo. (B) Single confocal plane of photoactivation mark of embryo in A. (C) Summed image of confocal z-series of photoactivation mark in same embryo after horizontal and vertical edge filters to highlight cell boundaries. 25 images are summed in C. Three fluorescent cells are labeled. Compare cell size and shape with Fig. 4B. Scale bars, 100 mm.
	Fig. 7. Example of small aperture photoactivation. (A,B) Fluorescent images of embryo at stage 48 after photoactivation at stage 36 (TB33-12, Table 4). Anterior is to the right; dorsal is top. Myocytes (Dorsal Myo) are labeled in A. Two of four labeled myocytes are significantly brighter. Two weakly fluorescent epidermal cells are also present. Several notochord (No) cells and neural tube (NT) cells are labeled in B, which is more posterior than the image in A. (C,D) Merged images of A and B, respectively, with DIC images. Fluorescence is pseudocolored green in C and D. Scale bars, 100 mm.