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Gastrulation is the most dynamic cell movement and initiates the body plan in amphibian development. In contrast to numerous molecular studies on mesodermal induction, the driving force of gastrulation is as yet poorly understood. A novel transmembrane protein, Xoom, was previously reported, which is required for Xenopus gastrulation. In the present study, the role of Xoom during Xenopus gastrulation was further examined in detail. Overexpression and misexpression of Xoom induced overproduction of Xoom protein, but not a changed phenotype. However, Xoom antisense ribonucleic acid (RNA) injection reduced the Xoom protein and caused gastrulation defects without any influence on the involution and translation levels of mesodermal marker genes. Normal migrating activity of dorsal mesodermal cells was recognized in the antisense RNA-injected explant. Morphological examination using artificial exogastrulation showed that convergent extension of mesodermal cells occurred normally, but the ectodermal cell layer significantly shrank in the antisense RNA-injected embryo. Comparison of cell shape among various experimental conditions showed that inhibition of cell spreading occurs specifically in the outer ectodermal layer of the antisense RNA-injected embryo. Cytochemical examination indicated disorganization of F-actin in the ectodermal cells of the antisense RNA-injected embryo. These results suggest that Xoom plays an important role in the epibolic movement of ectodermal cells through some regulation of actin filament organization.
Fig. 1. Effect of antisense ribonucleic acid (RNA) injection on
Xoom transcripts and protein. (A) Xoom and the predicted
protein structure. Arrow indicates the cDNA region that was
used for synthesizing antisense RNA. SP, signal peptide; Cysrich,
cysteine-rich region; N-glycosylation, N-glycosylation site;
Ser/Thr-rich, serine/threonine-rich region; TM, transmembrane
region; UTR, untranslated region. (B) Comparison of Xoom
transcripts. Contents of Xoom transcripts were examined among
the embryos injected with b-gal RNA (gal), Xoom antisense RNA
(anti), Xoom sense RNA (Xoom) or none (cont), using quantitative
reverse transcriptionâpolymerase chain reaction (RT-PCR).
Histone H4 is an internal marker. Polymerase chain reaction
(PCR) without reverse transcriptase (â RT) was performed with
the non-injected sample. (C) Comparison of Xoom protein.
Membrane fraction proteins from aliquots of embryos in (B) were
examined by western blot analysis using anti-Xoom antibody.
Fig. 2. Gastrulation defect caused by Xoom antisense ribonucleic
acid (RNA). Embryos were injected with 5 ng of Xoom antisense
RNA into the animal pole of both blastomeres at the 2-cell
stage. Vegetal view of embryos injected with b-gal RNA (A,C) and
Xoom antisense RNA (B,D). There was no discernible difference
until stage 11 (A,B). Inhibitory phenotype became apparent at
stage 11.5 (C,D). Embryos injected with b-gal RNA (E,G) and
Xoom antisense RNA (F,H) were histologically examined at stage
12. Photographs (G,H) show highly magnified dorsal lip of (E,F)
respectively. Asterisk indicates mesodermal cell mass stacking
around the dorsal lip. Embryos are positioned with the animal pole
at the top and the dorsal side to the right. bc, blastocoele; ac,
archenteron; yp, yolk plug; dl, dorsal lip; vl, vegetal lip. Arrowhead
indicates dorsal blastoporal groove. Bar, 50 μm.
Fig. 3. Gene expression in the embryo injected with Xoom antisense
ribonucleic acid (RNA). Embryos injected with b-gal RNA
(gal), Xoom antisense RNA (anti) or none (cont), were cultured,
and gene expression was examined at stage 10, 11.5 (A) and
24 (B) by reverse transcriptionâpolymerase chain reaction
(RT-PCR) with a reference of histone H4 as an internal marker.
Fig. 4. Morphogenetic movement of mesodermal cells. From
embryos injected with b-gal ribonucleic acid (RNA; gal), Xoom
antisense RNA (anti) or none (cont), dorsal mesoderm was
dissected and cultured on fibronectin-coated dishes (A). Actively
migrating cells with filopodia were observed 4 h after explanting
(BâD). Average distance of individual cells from the center of
explants was analyzed on time-lapse video recording (n = 16;
(E)). Whole embryos were cultured in 1.03modified Barthâs solution
to produce exogastrula after injecting with b-gal RNA (G),
Xoom antisense RNA (H) or none (F). Phenotype was examined
at stage 16. The convergent extension occurred normally in every
case, but ectodermal cell elongation was specifically inhibited
by the antisense RNA injection (arrowheads in (H)). Bar, 5 μm
(BâD).
Fig. 5. Effect of Xoom antisense ribonucleic acid (RNA) on epibolic
movement of the ectoderm. After injecting with synthesized
RNA into the animal pole of both blastomeres at the 2-cell stage,
the animal half dissected at stage 10 was sandwiched with the
non-injected one (A). Normal epibolic expansion was observed
in the b-gal RNA-injected explant (B), but not in the antisense
RNA-injected explant at stage 20 (C). The injected side of
the sandwich was determined by green fluorescent protein
fluorescence (D,E). cont, none; gal, b-gal RNA; anti, Xoom antisense
RNA-injected side.
Fig. 6. Effect of Xoom antisense ribonucleic acid (RNA) on epidermal
cell extension. Animal halves were isolated from embryos
injected with b-gal RNA (A,C) or Xoom antisense RNA (B,D) and
cultured in the medium containing activin (C,D) or none (A,B).
Phenotype was examined at stage 24. Histologic section was
prepared from b-gal RNA-injected (E) and antisense RNAinjected
explant at stage 16 (F). Arrowheads show the boundary
between the outer and inner layer of explants. Cell shape of
the ectodermal layer was examined based on the width/thickness
ratio (G).
Fig. 7. Actin filaments in animal epidermal cells during
gastrulation. Embryos injected with b-gal ribonucleic acid (RNA;
(A,C)) or Xoom antisense RNA (B,D) into the animal pole of both
blastomeres at the 2-cell stage were fixed at stage 11.5 and
stained with rhodamine phalloidin. Samples were mounted
between slide glasses and stained F-actin was visualized as a
red signal in a dark field (C,D). The intensity of rhodamine
fluorescence was measured in each sample (E). au, arbitrary
units and n = 7. Yolk-free cell extract was loaded on sodium
dodecylsulfateâpolyacrylamide gel electrophoresis (SDS-PAGE)
and 43 kDa actin bands were compared with each other (F).
