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Zaraisky AG
,
Ecochard V
,
Kazanskaya OV
,
Lukyanov SA
,
Fesenko IV
,
Duprat AM
.
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At the beginning of gastrulation the homeobox-containing gene, XANF-1, is expressed at a low level throughout the animal hemisphere of Xenopus laevis embryos, with a local maximum of expression in the region of the dorsal blastopore lip. By the end of gastrulation expression ceases everywhere except in the most anterior part of the neurectoderm. We have investigated the functions of this gene by microinjecting XANF-1 mRNA in the blastomeres of the 32-cell stageembryo and have observed the following effects. First, microinjections of the mRNA in the animal blastomeres and the blastomeres of the marginal zone elicited massive migration of cells to the interior of the embryo at the early gastrula stage. Second, overexpression of XANF-1 in the ventral marginal zone (VMZ) resulted in the appearance of an additional centre of gastrulation movements and the formation of a secondary axis. In addition we showed that synthetic XANF-1 mRNA was able to cause dorsal-type differentiation in VMZ explants extirpated from the microinjected embryos at the beginning of gastrulation. These results suggest that XANF-1 may control the main functions of cells of the Spemann organizer.
Fig. 1. Whole-mount in situ hybridization with XANF-1 anti-sense RNA labelled with
digoxigenin. (A) At the beginning of gastrulation (stage 10.25) the strongest expression of
XANF-1 is localized on the dorsal side of the embryo, in the Spemann organizer region (arrow).
Vegetal view, the dorsal side is to the top. (B) The same embryo, as in A, but cut in half. Within
the Spemann organizer region, XANF-1 transcripts are present predominantly in the deep zone
cells (dc), corresponding to the prospective headmesoderm and endoderm, and are much less
abundant in the surface cells (sc). Lateral view, dorsal side is to the right, animal pole is to the
top. Arrow indicates the position of the dorsal blastopore lip. The blastocoel roof (animal cap)
was destroyed during the whole-mount procedure. (C) At stage 10.5, XANF-1 begins to be
expressed intensely in the presumptive neurectoderm (pn), located just above the anteriormesoderm (am), which still expresses this gene. Lateral view, dorsal side is to the right, animal
pole is to the top. Arrow indicates the position of the dorsal blastopore lip. The blastocoel roof
(br) was flattened during the whole-mount procedure. (D) By the middle-late gastrula stage
(stage 12), the expression of XANF-1 has increased and is restricted toward the anterior region of
the presumptive neurectoderm (pn). The embryo is shown from the anterior, dorsal side up.
(E) At the middle-late gastrula stage, the expression of XANF-1 is localised exclusively in cells
of both neurectodermal (ne) layers, but is absent from cells of the underlying anteriormesoderm
(am). The embryo, cut in half, is shown from the left side. The dorsal side is to the top, anterior
end is to the left. (F) At the mid-neurula (stage 15), the hybridization signal is horse-shoeshaped,
localised on the surface of the anterior neural ridge, with two stripes of higher intensity
bordering the medialanteriorridge from the anterior and posterior sides. Orientation of the
embryo is the same as in D. (G) At the late neurula stage (stage 17), the hybridization signal
gradually recedes into the posterior region of the initial horse-shoe-shaped zone (Fig.
1G).Orientation of the embryo is the same as in D. (H) At the end of neurulation (stage 20), the
only labelled spot is seen in a region corresponding to the location of the prospective anterior
pituitary. Orientation of the embryo is the same as in D. Scale bar, 200 mm.
Fig. 2. XANF-1 overexpression in the
ventral marginal zone elicits formation
of a secondary axis. (A) A diagram of a
32-cell embryo with blastomeres
designated according to Dale and Slack
(1987). The microinjected blastomere,
C4, is labelled. (B) In the control
embryos at the beginning of
gastrulation, only one centre of
invagination appears in the dorsal
marginal zone (upper row, arrows), but
the embryos injected in the ventral
blastomere with XANF-1 mRNA display
additional centres of invagination in the
ventral marginal zone (lower row,
triangles). Arrows indicate normal
centres of invagination. (C) The
embryos injected with XANF-1 mRNA
and displaying an additional centre of
invagination, develop into tadpoles with
a partial secondary axis (sa).
(D) Colloidal gold (CG) labelling
reveals a massive accumulation of cells
containing XANF-1 mRNA in the
anterior part of the secondary axis
(arrow). Some of the labelled cells are
also seen in more posterior regions.
(E) In the control embryo co-injected
with CG and truncated XANF-1 mRNA
labelled cells do not demonstrate a
preferentially anterior location.
Fig. 3. XANF-1 not only promotes formation of an additional centre of
gastrulation movements, but elicits differentiation of more dorsal cell types.
(A) A diagram of the experiment. Explants from the ventral marginal zone were
removed at the early gastrula (stage 10.25) from embryos injected into the
ventral blastomeres with full-length XANF-1 mRNA or control truncated XANF-
1 mRNA, and incubated in saline for 2 days. (B) Differentiated explants from
embryos injected with full-length XANF-1 mRNA have an elongated shape and
contain numerous melanocytes. (C) Histological 7 mm section of one of the
explants, shown in B, revealing a row of well developed myotomes (mt)
surrounded by an ectodermal sac. (D) Analogous explants from embryos injected
with control truncated XANF-1 mRNA are round and have no melanocytes.
(E) Only mesenchymal (mes) and blood (b) cells can be seen in the explants of
embryos injected with control truncated XANF-1 mRNA. Scale bars: B,D 100
mm; C,E 30 mm.
Fig. 4. XANF-1 overexpression in the DMZ and animal cap leads to enhanced cell migration to the interior of embryo and, in the case of the
migration from the DMZ, to the movement of these cells to an anterior position. (A) The embryo was injected with XANF-1 mRNA, at the 32-
cell stage, into blastomere B1. At the beginning of gastrulation, cells that have plunged to the interior of the embryo form a furrow
(arrowheads) on the surface of the presumptive neurectoderm. Dorsal view. Animal pole is to the top, natural invagination of the blastopore is
to the bottom, beyond the field of view. (B) Cells starting to migrate from the blastocoel roof (animal cap) into the blastocoel (bl) of the embryo
injected with XANF-1 mRNA into A1-A4 blastomeres have a bottle-shaped form (bc). Their neighbours are elongated along the blastocoel
roof, which suggests that the bottle cells generate mechanical tensions by active contraction of their apices. At the same time, cells that have
already left the blastocoel roof are spherical in shape and fail to form tight contacts either with each other or with other cells. Semithin 2 mm
sagittal section of embryos fixed at the early gastrula stage (stage 10.5). Animal pole is to the top. (C) The embryo was injected into blastomere
B1 with colloidal gold (CG) and synthetic XANF-1 mRNA (left). By the late gastrula stage, the majority of cells containing this mixture have
migrated into the interior of embryo. In the control embryo injected with CG mixed with the control truncated XANF-1 mRNA, many of the
labelled cells remain on the surface, in the presumptive neurectoderm (right). Both embryos are shown from the dorsal side, anterior end is to
the top, posterior to the bottom. Position of blastopore (bp) is indicated in each embryo. (D) Histological 7 mm medial sagittal section of the
left-hand embryo shown in C reveals that the majority of cells containing CG and synthetic XANF-1 mRNA have become incorporated into the
axial mesoderm (axm) and have migrated within it toward the prospective head mesoderm and endoderm. At the same time the presumptive
neurectoderm (pne) is almost totally lacking labelled cells. Dorsal is to the top, anterior to the left. The dorsal blastopore lip (dbl) and ventral
blastopore lip (vbl) are indicated. (E) An analogous section of the control embryo (C, right) demonstrates that the majority of cells containing
both CG and control truncated XANF-1 mRNA remain in the presumptive neurectoderm. Only a minor proportion of the labelled cells involutes
through the dorsal blastopore lip with the posterior mesoderm. Scale bars: A, 20 mm; B, 10 mm; C-F, 200 mm.
