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During a process of differential display screening for lithium-responsive mRNA in Xenopus embryos, we found a maternal transcript which is remarkably reduced by lithium. The isolated cDNA, designated Xoom, encoded a novel oocyte membrane protein with a signal sequence in the N-terminus and a single transmembrane domain. The extracellular domain contained a cysteine-rich region and a serine/threonine-rich region, which suggests an extracellular association with some proteins. Expression of Xoom maternally occurred in the whole oocyte at the early stage of oogenesis and the transcript was gradually localized in the animal hemisphere of full-grown oocytes. The zygotic expression was detected at first in the dorsoanterior region of the neural fold stage embryo. Thereafter, localized expression of Xoom was observed in neural crest cells of the neural tube stage embryo and in optic and otic vesicles of tadpole. Xoom has been expressed ubiquitously in adult organs, especially with a high level in the eye, heart, liver and kidney. In examining a relation between Xoom and the dorsoventral patterning, lithium-treatment at 32-cell stageembryo decreased Xoom mRNA level within an hour, but coinjection of lithium with myo-inositol reversed the decreasing Xoom mRNA to normal level. UV-irradiation had no effect on the maternal mRNA level of Xoom. Overexpression of Xoom showed no effect on development, but antisense Xoom RNA causes interference with normal gastrulation movement. These results suggest that maternally expressed and membrane-associated Xoom is closely involved in the gastrulation movement through a lithium-inducible signal pathway.
Fig. 1. Nucleotide and predicted amino acid sequences derived from Xoom cDNA clone.
The putative signal sequence at the N-terminus and the presumed transmembrane domain
are boxed and shadowed, respectively. Cysteine residues are circled. The potential Nglycosylation
site is heavily underlined. The serine/threonine-rich region is shown by a dashed
line and the polyadenylation signal is underlined.
Fig. 2. Northern blot analysis of Xoom. To determine the size of Xoom
transcript, 50 mg total RNA (lane 1) and 4 mg poly A(+) RNA (lane 2) from an
unfertilized egg were electrophoresed, blotted, and hybridized with
digoxigenine-labeled Xoom cDNA. Arrow indicates 1.5 kb size of Xoom
mRNA.
Fig. 3. Comparison of molecular structure between Xoom and human Mr 110,000 antigen. The percentage of sequence homology in each domain
is indicated in the space between the two proteins. Xoom protein consists of 404 amino acids (accession number in DDBJ: AB026995). Human Mr 110,000
antigen consists of 407 amino acids (accession number in DDBJ: D64154).
Fig. 4. Expression profiles of Xoom. Gene expression was examined by
quantitative RT-PCR in developing whole embryos (A) and in adult organs
(B). Total RNAs for RT-PCR were isolated from unfertilized egg (Egg), 32-
cell stage embryo (32c), stage 8 blastula (Bla), stage 10 early gastrula
(eG), stage 11.5 late gastrula (lG), stage 14 early neurula (eN), stage 21
late neurula (lN), stage 27 tailbud (Tb) and stage 38 tadpole (Td) during
normal development, and from ovary (Ov), testis (Tes), brain (Br), eye
(Eye), lung (Lun), heart (Hea), stomach (St), liver (Liv), spleen (Spl) and
kidney (Kid) of 2 years-old female. Histone H4 was used as an internal
marker for standard.
Fig. 5. Spatial expression of Xoom during
oogenesis and embryogenesis. Whole-mount
in situ hybridization with digoxigenin-labeled
Xoom RNA probe was performed in albino oocytes
and embryos. (A) Oocyte stage I to VI (left
to right). Animal pole is up. (B) Lateral view of
early blastula (st. 7). Animal pole is up. (C) Animal
view of gastrula (st. 10). (D) Dorsal view of early neurula (st. 15). Zygotic expression occurs in the dorsoanterior region
(arrow). (E) Dorsal view of late neurula (st. 19). Expression of Xoom is
localized in both sides of the neural tube (arrow) and upper space of the eye
vesicles (arrowheads). (F) Anterior view of late neurula (st. 19). Expression
of Xoom is localized in both sides of neural tube (arrow) and upper space of
eye vesicles (arrowheads). (G) Transverse section at the trunk region of late
neurula (st. 19). nt, neural tube; nc, notochord; so, somite. Xoom specifically
expresses in neural crest cells around the neural tube. (H) Lateral view of
tadpole (st. 35). (I) High magnification of cleared tadpolehead. Intense
expression of Xoom is observed in the optic vesicle (arrow) and the otic
vesicle (arrowhead).
Fig. 6. Effect of lithium and myo-inositol on Xoom transcripts. (A) Depletion of
Xoom transcripts induced by lithium. Normal (cont), lithium-treated (Li) and UVirradiated
embryos (UV) were cultured and total RNAs were extracted at stage 7.
Quantitative RT-PCR analyses were performed with a reference of histone H4 as an
internal marker. (B) Rescue of Xoom transcript depletion by myo-inositol. Quantitative
analyses of Xoom transcripts were performed in embryos injected with 0.2M
LiCl (0.2M Li), 0.2M LiCl and 0.1M myo-inositol (0.2M Li+0.1M I), 0.2M LiCl and 0.2M
myo-inositol (0.2M Li+0.2M I), 0.2M LiCl and 0.5M myo-inositol (0.2M Li+0.5M I)
and 0.5M myo-inositol (0.5M I) or none (Cont). Total 5 nl of the solution was injected
into each of the two ventral blastomeres at 4-cell stage.
Fig.7 Effect of Xoom RNAs on gastrulation movement. Xenopus embryos were injected with b-
galactosidase RNA (A,D), sense Xoom RNA (B,E) or antisense Xoom RNA (C,F). In every case,
normal invagination was observed on the vegetal view of stage 10 embryo (arrowhead of A,B,C).
Gastrulation of the embryo injected with the sense Xoom RNA (E) proceeded normally as control
embryo (D). In contrast, gastrulation movement was inhibited at the initial phase in the embryo
injected with antisense Xoom RNA (F). Arrows of D, E and F shows the boundary of the blastopore
on the vegetal view at st. 11.5.
adrm1 (adhesion regulating molecule 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, oocyte stages I-VI, horizontal view, animal up.
adrm1 (adhesion regulating molecule 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, dorsal view, anteriorleft.
adrm1 (adhesion regulating molecule 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35, lateral view, dorsal up, anteriorleft.
