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Identification of vimentin and novel vimentin-related proteins in Xenopus oocytes and early embryos.
Torpey NP
,
Heasman J
,
Wylie CC
.
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We have made antibodies against fusion proteins of Xenopus vimentin. We show for the first time the distribution of vimentin in larval stages, where it is found in cells of mesenchymal origin, and in radial glial cells. In sections of Xenopus oocytes and early embryos, immunocytochemistry reveals the presence of an extensive cytoplasmic network, distributed in an animal-vegetal gradient. Germ plasm stains particularly strongly. The form of the IF proteins in this network is unusual. In immunoblot experiments the anti-vimentin antibodies detect a number of distinct proteins. We have identified those that are the products of the two known vimentin genes, by injection of synthetic mRNA transcribed from cloned vimentin cDNAs into oocytes, followed by two-dimensional Western blotting. This has demonstrated unambiguously that one Xenopus vimentin, Vim1, is present in oocytes and early embryos. However, two other immunoreactive proteins detected in Triton extracts of oocytes and early embryos are not the products of Vim1, since depletion of vimentin mRNA by antisense oligonucleotide injection has no effect on the synthesis of these proteins. These results suggest that novel IF-like proteins are expressed in Xenopus oocytes and early embryos.
Fig. 1. Cross reaction of affinity-purified Z9 antibody with Xenopus tissue sections. (A) Spinal cord; (B) dorsal structures
including spinal cord and somite; (C) mesonephric region (m- mesentry of gut, a- dorsal aorta); (D) high power view of
the somite showing positive staining in the connective tissue, but not muscle; (E) two adjacent gut loops showing staining
only in the connective tissue layer (c), not the epithelia (e); (F) previtellogenic oocyte from adult ovary, showing staining
in the follicle cells and mitochondrial cloud (m) of the oocyte. A-E are taken from stage 48 larvae, F from adult ovary.
Magnifications are x215 (A,D,E) and xllO (B,C,F).
Fig. 2. (A) Triton extracts from stage 5, 17 and 38
embryos Western blotted with Z9 serum. Three bands in
the A/r range 55-57000 are visible at each stage (arrowed).
The level of the 57k band declines relative to the two
lower Mr bands as development procedes. The cross
reacting proteins at a lower Mr at stage 38 are degradation
products of the major bands odserved. 1 and p are the two
yolk proteins lipovitellin and phosvitin. (B) Western blots
of Triton extracts of uninjected oocytes, oocytes injected
with viml mRNA and oocytes injected with vim4 mRNA.
The antibody used was affinity-purified Z9, which does not
react with yolk proteins. (C) Triton extract of 15 oocytes
blotted with affinity-purified Z9, showing again that there
is no cross reaction of the affinity-purified antibody with
yolk proteins.
Fig. 3. Two-dimensional Western blots of Triton extracts of stage 5 (A), stage 17 (B) and stage 38 (C) embryos using Z9
serum (A) or affinity purified antibody (B-D). Proteins x, y and z are labelled (see text for details). D is a Triton extract
from oocytes injected with both viml and vim4 mRNA. The overexpressed proteins are labelled vl and v4 respectively.
Fig. 4. A and B are fluorograghs of Triton extracts from
oocytes injected with XIF3 (control) andl242 (anti-sense
vimentin) oligos, respectively. The basic proteins v and z
that cross react with antibody Z9 are arrowed. C is a
Northern blot of RNA from these oocytes probed with a
Viml cDNA probe. Lanes 1 and 2 were loaded with RNA
from oocytes injected with XIF3 and 1242 after 2 h
incubation, and lanes 3 and 4 loaded with RNA from
oocytes after incubation for 16h. D shows the same blot
reprobed with an XCK1(8) probe.
Fig. 5. Immunostaining of a section of a fully grown Xenopus oocyte using affinity-purified Z9 antibody at lOO.ugml '.
shows the pattern of staining in the whole oocyte. B and C show high power views of the animal and vegetal poles,
respectively. Magnification of A x!45; B,C x550.
Fig. 6. Immunostaining of pre-midblastula transition embryos (stage 5) using affinity-purified Z9 antibody. A and B are
fluorescence and phase-contrast images of the same section of an animal blastomere. C and D are fluorsescence images of
a vegetal blastomere showing germ plasm and the perinuclear area, respectively. Magnifications are x550.
