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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.
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.