XB-ART-24185J Cell Sci January 1, 1992; 101 ( Pt 1) 151-60.
Distinct distribution of vimentin and cytokeratin in Xenopus oocytes and early embryos.
We report the identity of a major component of Triton-insoluble extracts from Xenopus oocytes and early embryos. In a previous paper we showed that an antibody, Z9, cross-reacts with two polypeptides from such extracts (Mr 56,000 and 57,000) as well as Xenopus vimentin. Direct microsequencing of the Mr 57,000 protein shows near identity of three tryptic fragments with regions of the predicted amino acid sequence of XCK1(8), a basic cytokeratin whose mRNA is known to be expressed in Xenopus oocytes. We have raised an antibody, CK7, against a fusion protein generated from this cDNA. The specificity of this antibody has been tested using 1- and 2-dimensional immunoblotting, which show that it is specific for the Mr 56,000 and 57,000 proteins, suggesting that these two proteins may be the products of two non-allelic XCK1(8) genes. The antibody does not cross-react with vimentin. We have used CK7 to follow the distribution of XCK1(8) throughout development by immunoblotting and immunocytochemistry. In larval stages, strong staining is seen in the notocord, the apical epithelia of the gut, the mesentery, and a few cells in the spinal cord. In oocytes and early embryos, two distinct intermediate filament (IF) networks can be distinguished: a cortical cytokeratin network, and a deeper vimentin one. In addition, the oocyte germ plasm stains with Z9 but not CK7. We propose that such distinct distributions of each IF protein reflect functional differences during early development.
PubMed ID: 1373733
Article link: J Cell Sci
Genes referenced: ckb krt12.4 krt8.1 vim
Antibodies: Vim Ab3
Article Images: [+] show captions
|Fig. 1. Sequences of three tryptic fragments obtained from the Mr 57,000 protein, arbitarily named PI, P2 and P3. A comparison of these with XCK1(8) is shown. Amino acid positions within the XCK1(8) clone are given above each comparison, mismatches are in bold type.|
|Fig. 2. 2-D immunoblots of Triton-insoluble extracts from stage 5 (A), stage 17 (B), and stage 38 (C) Xenopus embryos, using affinity-purified CK7 antibody (2/ig/ml). MT 56,000 and 57,000 proteins can be seen in each case (indicated in A). Arrows in C mark three smaller, more acidic cross-reacting proteins. D shows a Coomassie blue stain of stage 38 Tritoninsoluble proteins. CK7 immunoreactive proteins are arrowed, and the position of vimentin shown (v).' Loadings: A-C, 5 embryos; D, 20 embryos.|
|Fig. 3. (A and B) show immunoblots of Triton-insoluble proteins from 10 defolliculated oocytes, either uninjected (-) or injected with 5ng of synthetic vimentin mRNA 20 hours prior to preparation of the extract (+). A is blotted with affinity-purified CK7 antibody (2^g/ml), and B with affinity-purified Z9 (5/^g/ml). Arrows show MT 55, 56 and 57,000 proteins. Overexpressed vimentin is detected by Z9 (B; lane 1), but not by CK7 (A; lane 1). C shows Triton extracts of 5 stage 5, 10, 17, 23 embryos, and 2 stage 38 embryos blotted with CK7 (2ug/ml)|
|Fig. 4. RNase protection assay using the CK22 probe (see Materials and methods) demonstrating the levels of XCK1(8) during development (CK). RNA recovery is controlled for using a 5 S RNA probe (5 S). Size markers are 220 and 150 nucleotide fragments of ^P-labelled Hinfldigested pBR322.|
|Fig. 5. Comparison of CK7 (A, D) and Z9 (B, E) staining of larval tissues. (A, B and C) show the neural tube (nt), notocord (no), and somites (s) stained with 4fytg/ml CK7 (A), 80/ig/ml Z9 (B). (C) is the same section as in B viewed under phase contrast. (D, E and F) show the dorsal body wall and aorta (a), somites (s), and mesentry (m) stained with CK7 (D), Z9 (E). (F) is the same section as in E viewed under phase contrast showing the aorta (a), somite (s), kidney (k) and mesentry (m). Vimentin staining is seen only around the somite, in cells within the developing kidney, and the wall of the aorta. All these tissues are negative with CK7 (D).xl60.|
|Fig. 6. Further analysis of CK7 and Z9 in larval tissues. High power view of three different sections of a loop of gut: (A) under phase contrast, (B) stained with 40/ig/ml CK7, and (C) stained with 80^g/ml Z9. Lu, lumen of gut. x600.|
|Fig. 8. (A, B and C) Grazing sections through the animal pole of a fully grown oocyte stained with CK7 (A; f, follicle cell layers) and with Z9 (B). (C) is the same section as in A viewed under phase contrast. (D) shows CK7 staining in the surface of one vegetal blastomere of a stage 7 embryo stained as a whole mount. x200.|