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We have developed a whole-mount immunocytochemical method for Xenopus and used it to map the expression of the intermediate filament protein vimentin during early embryogenesis. We used two monoclonal antibodies, 14h7 and RV202. Both label vimentin filaments in Xenopus A6 cells, RV202 reacts specifically with vimentin (Mr, 55 x 10(3] on Western blots of A6 cells and embryos. 14h7 reacts with vimentin and a second, insoluble polypeptide of 57 x 10(3) Mr found in A6 cells. The 57 x 10(3) Mr polypeptide appears to be an intermediate filament protein immunochemically related to vimentin. In the whole-mount embryo, we first found vimentin at the time of neural tube closure (stage 19) in cells located at the lateral margins of the neural tube. By stage 26, these cells, which are presumably radial glia, are present along the entire length of the neural tube and in the tail bud. Cells in the optic vesicles express vimentin by stage 24. Vimentin-expressing mesenchymal cells appear on the surface of the somites at stage 22/23; these cells appear first on anteriorsomites and on progressively more posteriorsomites as development continues. Beginning at stage 24, vimentin appears in mesenchymal cells located ventral to the somites and associated with the pronephric ducts; these ventral cells first appear below the anteriorsomites and later appear below more posteriorsomites. The dorsal finmesenchyme expresses vimentin at stage 26. In the head, both mesodermally-derived and neural-crest-derived mesenchymal tissues express vimentin by stage 26. These include the mesenchyme of the branchial arches, the mandibular arch, the corneal epithelium, the eye, the meninges and mesenchyme surrounding the otic vesicle. By stage 33, vimentin-expressing mesenchymal cells are present in the pericardial cavity and line the vitelline veins. Vimentin expression appears to be a marker for the differentiation of a subset of central nervous system cells and of head and body mesenchyme in the early Xenopus embryo.
Fig. 4. Expression of vimentin in stage-19, -22 and -24 embryos. (Note: the diagram in this and Figs 5, 6, 7 and 8 indicates the optical section planes presented.) In an anterior-dorsal view of a stage-19 embryo stained with RV202 (A) the neural tube (nt) runs vertically down the centre of the embryo and ends with two bulbs that are the nascent optic vesicles (ov). Vimentin-expressing cells line the sides of the neural tube (nv). Individual round or wedge-shaped cells can be identified through the cleared ectoderm and neural tube. There does not appear to be any staining of the optic vesicles themselves. In an anterior view of a stage-22 embryo stained with RV202 (B), the outer lateral margin of the neural tube contains many cells that are darkly stained for vimentin (nv). There is no staining for vimentin in the optic vesicles. Staining of the cement gland (c) is artifactual. Vimentin-positive cells (nv) can be seen in a lateral view of the optic vesicle of a stage-24 embryo stained with 14h7 (C). Dorsal (dor) is left and ventral (ven) is right in C. Dorsal is at the top and ventral is at the bottom in B. Bars in A and B, 100^m; in C, 250um
Fig. 5. Vimentin expression in stage 20 and 22 embryos. In a lateral optic section of a stage-22 embryo stained with RV202 (A), vimentin-expressing cells of the anterior neural tube (nv) are more elongate. Somites (s) are beginning to form beneath the anterior neural tube; vimentin staining can be seen on their surfaces in different focal planes. In a transverse physical section through the trunk of a stage-20 embryo stained with 14h7 (B), the cells at the outer margin of the neural tube stain intensely and those on the ventricular surface stain weakly (nv). The stained cells on the outer margin of the neural tube are restricted to the lateral neural tube and are absent from both dorsal and ventral regions, c, cement gland; n, notochord; nt, neural tube, ov, optic vesicle. Bars in A, 150/im; in B, 100 fim.
Fig. 6. Vimentin expression in the stage-26 embryo. A lateral optic section of a stage-26 embryo stained with RV202 (A) reveals that vimentin-expressing cells (nv) are present in the neural tube, from the head to the tailbud. Vimentin-expressing somitic mesenchyme (sm) is present on the eight anteriormost trunksomites. Vimentin-expressing mesenchymal cells are found in the head where the branchial arches are forming (hm) as well as below the anteriortrunksomites where the pronephros are forming (pm). An oblique frontal/lateral physical section through posteriortrunk region of the neural tube and somites of a stage-26 embryo stained with RV202 (B) reveals that the vimentin-expressing cells of the neural tube (nv) become gradually less elongated toward the tail, ant, anterior; pos, posterior; c, cement gland; nt, neural tube; ov, optic vesicle; s, somite. Bars, 350 fim in A and 150/xm in B.
Fig. 7. Vimentin expression in the head region of the stage-26 embryo. In a transverse physical section through the head of a stage-26 embryo stained with 14h7 (A), vimentin-expressing headmesenchyme (hm) appears adjacent to the brain and in the region of the mandibular arch (ma). Vimentin-containing processes of cells of the mesencephalon (me) extend in toward the ventricle (nv). Staining in the cement gland is an artifact. In a transverse physical section through an anteriorsomite of the trunk stained with 14h7 (B), the mesenchyme at the surface of the somites (sm) is expressing vimentin. Vimentin- expressing cells (nv) are present at the margin of the neural tube and have small processes that extend midway to the ventricular surface. There are no vimentin-expressing cells in the ventral region of the neural tube. There appear to be vimentin-expressing mesenchymal cells dorsal to the neural tube (unmarked arrow), c, cement gland; ma, mandibular arch; me, mesencephalon; n, notochord; nt, neural tube; ov, optic vesicle; s, somites. Bars, 50 jxm in both parts.
Fig. 8.C Transverse sections of stage-35 embryos stained for vimentin. In the area of the embryoventral to the region illustrated in B (C) are found the lateral body wall and the yolky endodermventral to the pronephros. The pronephric mesoderm is at the top (pm) and below it, between the mesoderm and the endoderm are the vitelline veins (vv) which contain vimentin-expressing mesenchyme. vv, vitelline veins. pm, pronephric ducts. Bar 25 um in C.
Fig. 9. Vimentin staining in the stage-35 embryo. In a lateral optic section of stage-35 embryo stained with RV202 (A), vimentin can be found in the neural tube, in the dorsal fin (df) and on the somites (the somitic mesenchyme is mostly out of focus in this picture). Vimentin is expressed in the pronephric mesenchyme (pm) below the first six somites with progressively less mesenchyme below the more posteriorsomites. The reticulated pattern of the vitelline veins (vv) can be seen throughout the belly around the yolky endoderm. Vimentin-expressing mesenchyme is present on the branchial arches (ba), around the otic vesicle (oi), covering the eye (e) and on the surface of the brain (b). A close-up optical section of the dorsal fin (B) reveals that the morphology of individual dorsal fin mesenchymal cells (dm) can be distinguished by the vimentin staining. In a lateral optical section midway through the embryo (C), vimentin-expressing mesenchyme surrounds the heart in the pericardial cavity (cm). A close lateral optical section of the anteriorsomites (D) reveals individual, vimentin-expressing mesenchymal (sm) cells. Individual pronephric mesenchymal cells (pin) are visible below the somites. A close lateral view of the region ventral to the anteriorsomites is shown in D. (E) reveals that the pronephric mesenchyme (pm) expresses vimentin, as do cells of the vitelline veins (vv). b, brain; ba, branchial arch; c, cement gland; df, dorsal fin; e, eye; nt, neural tube; ot, otic vesicle; s, somite; vv, vitelline veins. Bars, 250um in A, 50um in B, 20um in C, 100um in D and 20um in E.
Fig. 1. 14h7 and RV202 staining of A6 cells. Methanol-fixed A6 cells were stained with anti- tubulin (A,B), 14h7 (C,D) or RV202 (E,F). In control cells (A,C,E), both 14h7 and RV202 stain an extended filamentous system, distinct from the microtubule system. Cells treated with nocodazole have depolymerized microtubules (B) and collapsed filament systems stained by 14h7 and RV202 (D,F). Cytokeratin filaments remain extended in nocodazole-treated cells (not shown). Bar in A marks 15/zm.
Fig. 2. Western immunoblot analysis of A6 cells and embryo residues. Whole cell and detergent-insoluble residues of A6 cells were separated by SDS-polyacrylamide gel electrophoresis and either stained with Coomassie brilliant blue (A) (we, whole cell; cr, cell residue) or were electrophoretically transferred to nitrocellulose paper and probed with the monoclonal antibody RV202 (B) or 14h7 (C). RV202 labelled a single band of 55xlO in both whole cell (we) and cell residue (cr) lanes. Occasionally a band below the 55 x 103 polypeptide was labelled (asterisk), this is presumably a proteolytic degradation product of the 55 x 103 polypeptide. Typically 14h7 appears to react weakly with a 55x10 polypeptide in whole cell, and with polypeptides of 33
55xlO (large arrow) and 57xlO (small arrow) in cell
residue (C). To illustrate that RV202 and 14h7 react with 3
the same 55xlO polypeptide, a single lane of A6 cell residue was cut down the middle (D) and probed with 14h7
3
(left side) or RV202 (right side); the labelled 55xlO polypeptides align perfectly. To define the specificity of 14h7 and RV202 in embryos, insoluble residues from stage- 35 tadpoles were separated by SDS-polyacrylamide gel electrophoresis and either stained with Coomassie brilliant blue (E) or transferred to nitrocellulose and probed with
either RV202 (F) or 14h7 (G). RV202 reacted with poly- 3
peptide of 55xlO , 14h7 reacted with this same polypeptide
(large arrow), and very weakly with a polypeptide of 3
57xlO . A minor band that presumably represents a proteolytic degradation product of the 55 x 103 polypeptide was also labelled by both antibodies (asterisk in F and G).
Dash marks on the left side of A and E mark the position 3
of the molecular weight markers phosphorylase (97xlO ), 33
bovine serum albumin (66X10 ), ovalbumin (45xlO ) and 3
carbonic anhydrase (29x10 3)
Fig. 3. The 57x 10 3 polypeptide carries the IFA-epitope. Western blots of A6 cell residues were probed with 14h7 (B), IFA (C), RV202 (D) or a rabbit anti-vimentin antiserum (Rb-V; E); the Coomassie-stained gel is shown in A. Both the 55x10 3 vimentin polypeptide and the 57x10 3 anti-vimentin antiserum reacts preferentially with the 57x10 3 polypeptide. Asterisk marks weakly stained polypeptide that is likely to be a proteolytic degradation product of vimentin.
