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Fig. 1. Cell number and mitotic incidence in synchronous
normal and UV siblings during midblastula transition.
Nuclear number, on the ordinate, refers to the sum of
nuclei actually transected in every fifth 7/an section, in
that portion of the blastula shown in the inset diagram
(ap, animal pole). Estimates of total cells in the whole
blastulae, derived by including the animal cap and using
the appropriate Abercrombie correction (1946), lie in the
expected range at 5000-8400. Each time point is
represented by three control (â¢) and three u.v.-irradiated
(O) blastulae, and the first time point is at the projected
onset of the 13th round of cleavage extrapolated from the
pre-MBT cleavage intervals. Thus it is seen that in the
population as a whole, somewhat over two thirds of the
blastomeres initially present divide over the hour spanned
by the observations, whereas pre-MBT cycles were 27 min
long at 20°C in the egg batch concerned. The overall lag
in cell number in the UV samples is barely statistically
significant (0-05 <P<O-1), though a similar marginal
deficit was seen in the other MBT experiment. Control
sample mitotic incidences are given above each set of cell
number points, UV, below, but no difference at any time
point was significant. A photograph of the cellular texture
of the marginal region of midblastular material is
included, but the delicate Feulgen staining of nuclear
figures at such early stages is not well revealed. Bar,
100 Um.
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Fig. 2. Histological structure of UV5 embryos and controls at control stage 13. (A-C) Anterior, middle and posterior
levels of transverse section through a normal stage 13, early neurula. A includes only prechordal mesoderm, B includes
notochord and dorsally converging somitogenic tissue and C is in the thick circumblastoporal zone of mesodenn
recruitment. (D-F) Three equivalent levels of section through a UV5 sibling at an equivalent stage of blastopore
closure, 20min older than the control. Little evidence of deviation from radial symmetry of architecture is seen. Bar,
100 um.
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Fig. 3. The mesoderm at stage 13. In A-F, tracings of the sections of Fig. 2 are laid out as in that figure, with the
mesodenn profile shaded to demarcate it from the inner endoderm mass (which may exhibit an archenteric cavity), and
from the enclosing ectoderm. Below this are diagrammatic midsagittal sections of the normal (G) and UV5 (H)
development. These show, respectively, the dorsal-ventral and axial character of mesoderm and endoderm, and its
absence in the experimental development, pc, prechordal, nc, notochord, r, recruitment zone, bl, blastocoel remnant.
Bars: A-F, 100um; G,H, 300um.
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Fig. 4. Histological structure of UV5 embryos and controls at control stage 32. (A-C) Head, middle (pronephric) and
rx)sterior levels of transverse section through a normal axial larva. (D-F) Three positions of transverse section through
a UV5 of the same age, containing early-, middle-, and late-recruited mesoderm. nc, notochord, 5, somite,
pn, pronephros, Ipl, lateral plate character of mesoderm, br, brain parts including eye. Bar, 100 um.
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Fig. 5. Muscle myosin and larval globin synthesis in
normal and UV5 development at control larval stage 38.
(A) Western blot of total larval protein, probed with
anti-muscle myosin antiserum. Two sets of four tracks
bearing serial twofold dilutions are shown. On the left,
the sample was prepared from a set of ten normal larvae.
On the right, preparation was from ten synchronous UV5
examples. The arrow marks the band revealing myosin,
and the first (right-hand) track of each series,
representing one embryo's worth of protein, is indicated
above.
(B) Lanes from a Western blot probed with anti-
Xenopus larval globins. The left-hand lane represents one
embryo's worth of protein from a normal larval sample as
in (A). The right-hand lane represents one embryo's
worth of protein from a UV5 sample. The arrow marks
the band revealing globin concentration.
Larvae were solubilized in Laemmli sample buffer and
loaded onto linear 5-15 % polyacrylamide gradient gels
1-5 mm thick. The separated proteins were transferred
electrophoretically to nitrocellulose. The membrane was
'blocked' overnight at room temperature in 10 % normal
goat serum and 4% bovine serum albumin in PBS-A. It
was incubated for 2 h in a 1/200 dilution of anti-globin
serum in 1 % BSA in PBS-A. After five washes in PBS-A
over 1 h, it was incubated in a 1/500 dilution of HRPconjugated
affinity-purified goat-anti-rabbit IgG (Miles)
for 2h, then re washed. The nitrocellulose was probed for
peroxidase activity using DAB.
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Fig. 6. Distribution of larval globin production in normal and UV5 development at stage 34. 10/an wax sections
transverse to the long axis, treated by indirect immunofluorescence with rabbit anti-Xenopus globin and FITC-conjugated
goat anti-rabbit IgG and counterstained with aqueous eriochrome black.
(A) The normal heart level, showing absence of red blood cells in the circulatory spaces at this stage.
(B) Level midway between heart and blastopore in normal larva, showing the restricted and compact midventral sector
from which blood originates. The sector is never wider than shown and diminishes towards the blastopore. The tissue is
not structurally distinguishable from adjacent lateral plate at these stages.
(C-F) Successive sectional levels through typical UV5 body, from anterior grazing section of mesoderm and covering
ectoderm to a level halfway back to the terminal blastopore. Distribution of globin is ubiquitous and apparently solid in
mesoderm of first two levels, but the appearance at levels around E shows that there is a rather sharp though irregular
posterior boundary to the globin-producing region, again unrevealed by any structural distinction in ordinary light
microscopy although the tissue tends more towards a multilayered and open texture anteriorly. Globin is never seen from
level F back. The fluorescence associated with globin production, while perfectly distinct, tends to be slightly less in the
UV5 case at an identical time after fertilization, as might be expected in view of the nonspecific delay of 3-4 h in
developmental stage shown by partial axial UV forms at these stages, h, heart; end, yolky endoderm; Ipl, mesoderm of
lateral plate character; ect, ectoderm; Hb, fluorescent appearance marking haemoglobin production. Bar, 100um.
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Fig. 7. Suggested events delimiting the area of larval
blood production in normal and in radialized UV
development. Longitudinal sections of normal (A) and
UV5 (B) embryos are shown at the close of gastrulation.
The mesoderm in section is shown stippled and, in (A),
this corresponds to the sagittal plane of left-right
symmetry. The anterior mesoderm border, corresponding
to the earliest involution, is shown at the left by a heavy
dashed line in each case, while arrows more posteriorly
indicate the direction and vigour of migration (and dorsal
convergence in the normal case) of the later-involuting
tissue. The final area of production of haemoglobincontaining
cells, extending back from the anterior
mesodermaJ border, is delimited by a thin black line. In
the normal case, the dorsal and posterior zone devoted to
somite tissue is delimited by a single dashed line, and this
incorporates the later-involuting mesoderm from almost
all around the blastopore. Normally, 'dorsal' pattern
elements probably act to limit by suppression the area in
which 'ventral' developments such as blood occur,
whereas such signals are absent in the UV5 embryo. But
the mechanism whereby blood is specified only in earlyrecruited
or 'anterior' mesoderm remains. In the normal
neurula, mesoderm initially behind this is swept upwards
past the blastopore to join the somite axis in the
dorsalization process, thus giving the definitive blood
domain a posterior border that abuts on the proctodaeum
or blastopore remnant in the later larva.
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