Hasegawa K and Kinoshita T (2000), Xoom is required for epibolic movement of anima...

XB-ART-10393

Dev Growth Differ 2000 Aug 01;424:337-46. doi: 10.1046/j.1440-169x.2000.00516.x.

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Xoom is required for epibolic movement of animal ectodermal cells in Xenopus laevis gastrulation.

Hasegawa K , Kinoshita T .

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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.

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Species referenced: Xenopus laevis
Genes referenced: actl6a adrm1 fn1 h4c4

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	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 transcription–polymerase chain reaction (RT-PCR). Histone H4 is an internal marker. Polymerase chain reaction (PCR) without reverse transcriptase (– 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 transcription–polymerase 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 (B–D). 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 Barth’s 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 (B–D).
	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 dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and 43 kDa actin bands were compared with each other (F).