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Differentiation
2007 Jan 01;751:84-92. doi: 10.1111/j.1432-0436.2006.00114.x.
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In vivo magnetic resonance microscopy of differentiation in Xenopus laevis embryos from the first cleavage onwards.
Lee SC
,
Mietchen D
,
Cho JH
,
Kim YS
,
Kim C
,
Hong KS
,
Lee C
,
Kang D
,
Lee W
,
Cheong C
.
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Differentiation inside a developing embryo can be observed by a variety of optical methods but hardly so in opaque organisms. Embryos of the frog Xenopus laevis--a popular model system--belong to the latter category and, for this reason, are predominantly being investigated by means of physical sectioning. Magnetic resonance imaging (MRI) is a noninvasive method independent of the optical opaqueness of the object. Starting out from clinical diagnostics, the technique has now developed into a branch of microscopy--MR microscopy--that provides spatial resolutions of tens of microns for small biological objects. Nondestructive three-dimensional images of various embryos have been obtained using this technique. They were, however, usually acquired by long scans of fixed embryos. Previously reported in vivo studies did not cover the very early embryonic stages, mainly for sensitivity reasons. Here, by applying high field MR microscopy to the X. laevis system, we achieved the temporal and spatial resolution required for observing subcellular dynamics during early cell divisions in vivo. We present image series of dividing cells and nuclei and of the whole embryonic development from the zygote onto the hatching of the tadpole. Additionally, biomechanical analyses from successive MR images are introduced. These results demonstrate that MR microscopy can provide unique contributions to investigations of differentiating cells and tissues in vivo.
Fig. 1 Magnetic resonance (MR) images
of cytoplasmic rearrangement during initial
cell divisions. Nine in vivo MR images
of a developing embryo recorded during
1.5–2.8 hpf at 9 min intervals. The slice
was positioned perpendicular to the first
cleavage furrow and parallel to the animal–
vegetal axis. Animal and vegetal cytoplasmic
rearrangements accompanying
the progression of cell cycle are readily
visible: In-plane resolution (pixel size)5
2346 mm2, slice thickness5200 mm,
imaging time52.2 min, temperature5
(15 1)1C. Field of view (FOV)5
1.6mm1.6mm (from originally 3mm
3 mm).
Fig. 2 Temporal series of the early stages
of Xenopus laevis embryonic development.
(A) Image from a slice parallel to the animal–
vegetal axis before the first cleavage.
The positions of two slices acquired perpendicular
to this image plane are indicated.
(B) Images from the two slices
indicated in (A), showing an individual
frog developing from the zygote to the
blastula stage. AH, animal hemisphere;
VH, vegetal hemisphere. The cells boxed
in blue (animal half) and light blue (vegetal
half), respectively, in the images taken
at 3.5 and 4 hpf are dividing at right angles
to one another—producing, respectively,
two superficial cells (detailed in C)
or one superficial and one deep cell (detailed
in D). (C) Close-up of cell divisions
in the green-boxed cell during 2.8–3.4 hpf,
recorded with 4.4 min intervals. (D) Cell
divisions in the light blue-boxed cell during
3.5–4.1 hpf. In-plane resolution5
2323 mm2, imaging time54.4 min, temperature5(
18 1)1C. Scale bar51 mm.
Fig. 3 Temporal series covering the complete
embryonic development of Xenopus
laevis. (A) Images from the initial cleavages
to the mid-neurula stage. Arrows
indicate: magenta, blastocoel; dark red,
ectoderm; light blue, mesoderm; blue,
archenteron; light green, endorderm;
dark green, brain ventricle. r, rostral; c,
caudal; d, dorsal; v, ventral. In-plane
resolution52323 mm2, temperature5
(20 1)1C. Imaging time512 min for
images up to 4.5 hpf, and 24 min for the
images thereafter, in accordance with the
slowdown of the cell division cycle at the
mid-blastula transition (Gilbert, 2003).
(B) Images from the mid-neurula stage
to the tailbud stage. Axis labels, scale bar
and arrows are as in (A). Blue at 46.1 hpf,
foregut; light blue, somites. In-plane
resolution52323 mm2, imaging time5
12 min, temperature5(18 1)1C. Scale
bars51 mm.
Fig. 4 Detailed time-lapse image sequence
of gastrulation. Consecutive images (part
of the sequence shown in Movie 3) taken
at intervals of 48 min, starting at 8 hpf.
The arrows indicate the moving cell front
of the dorsal blastopore lip, with the
green one in the second row pointing
at the bottle cells. Coordinate axes were
introduced to quantify epiboly biomechanics
(cf. Fig. 5), with y corresponding
to the ventral–dorsal direction, z to
rostral–caudal. The imaging parameters
were identical to those after 4.5 hpf in
Figure 3A.
