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RNA from ventralized Xenopus laevis embryos has been used to generate a subtracted library consisting of genes whose expression is dependent upon dorsoanterior development. Clones were selected on the basis of two criteria: (1) at least 10-fold reduction in ventralized versus control embryo RNA, and (2) absence of hybridization to RNA from adult muscle. One of these clones, designated UVS.2, was characterized further. UVS.2, was found by in situ hybridization to be expressed exclusively in the anterior neural fold of neurula stage embryos. By the tailbud stage, the UVS.2 protein and transcript were localized in specialized cephalic ectoderm, in a region probably corresponding to the hatching gland. UVS.2 expression could be induced in ectodermal explants by contact with dorsal mesodermal tissue. Furthermore, in lithium-treated embryos, which have exaggerated dorsoanterior structures, UVS.2 expression was increased four- to fivefold. A molecular marker such as UVS.2 will be useful in defining and studying the induction and organization of cephalic tissues.
FIG. 1. Developmental expression pattern of UVS.2. Total RNA (7.5
pg/lane) from embryos of the indicated stages was analyzed by RNA
gel blotting, using as probe a 32P-labeled DNA fragment derived from
the UVS.2 cDNA clone. All RNA samples were prepared in parallel
and were equally undegraded as judged from the ethidium bromide
staining pattern (not shown). Exposure time was â7 days at -70°C.
FIG. 2. Localization of UVS.2 transcript at the neurula stage by in situ hybridization. The planes of sections corresponding to A-E are shown
in the top left diagram, a dorsal view of the embryo. The region that hybridized to the UVS.2 35S-labeled antisense RNA probe, depicted in
black, is shown in an anterior view of a stage 17 embryo (top right diagram). The micrographs show the in situ hybridization with dark-field
optics as well as the phase-contrast view of the same sections stained with Giemsa. Hybridized sections were exposed for 10 days at 4°C. Labels
indicate the neural fold (nf) and cement gland (cg).
FIG. 3. Localization of the UVS.2 protein using immunofluorescence.
Whole mounts of stage 36 embryos were immunostained with
an anti-peptide antibody specific for the UVS.2 protein (see Materials
and Methods). The antigen was visualized using a rhodamine-labeled
second antibody. (B, D) Specific staining of putative hatching gland
cells with the anti-UVS.2 peptide antibody. (A, C) Negative controls
in which the antibody reactions were blocked by addition of 25 rg/ml
of the UVS.2 peptide. (E) A section through the head of a stage 36
embryo that has been immunostained with the UVS.2 peptide antibody,
as seen in dark-field optics. The plane of this section is indicated
in A by the dashed line. The cells staining are noted by the arrows,
and the brain (b) and eye (e) are marked.
FIG. 4. Expression of the UVS.2 RNA in ectodermal cap/dorsal
mesoderm recombinants. As shown in the diagram, recombinants
were made using stage 10 dorsal mesoderm sandwiched between two
blastula stage animal caps. Gel blots of total RNA were hybridized
with cDNA probes for either a-actin (left panel, 1 pg RNA per lane) or
UVS.2 (right panel, 2.5 rg RNA per lane). Lane a, ectodermal cap
explants; lane b, dorsal mesoderm; lane c, ectodermal cap/dorsal mesoderm
recombinant; lane d, stage 28 embryo. Note that 1 Kg of total
RNA corresponds to 1.5 ectodermal caps, 2 dorsal mesoderm explants,
0.5 recombinants, and 0.2 whole embryos. Therefore, on a per emho
basis, 4~ more material originating from dorsal mesoderm was
loaded on lane b compared to lane c. Arrows mark cytoskeletal actin
(cyto) and muscle actin (m). The positions of the 28 S and 18 S rRNA
bands are indicated.
FIG. 5. Expression of UVS.2 RNA in lithium-treated embryos. RNA
gel blots were hybridized with the UVS.2 cDNA probe. Lane a, 2.0 Mg
of total RNA from lithium-treated embryos (equivalent of stage 28);
lane b, 2.0 Kg of total RNA from control stage 28 embryos.
