Godsave SF et al. (1986), The appearance and distribution of intermediate...

XB-ART-28565

J Embryol Exp Morphol 1986 Sep 01;97:201-23.

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The appearance and distribution of intermediate filament proteins during differentiation of the central nervous system, skin and notochord of Xenopus laevis.

Godsave SF , Anderton BH , Wylie CC .

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Antibodies against various intermediate filament proteins have been used to follow cell differentiation in the early Xenopus embryo. Three new monoclonal antibodies against Xenopus cytokeratins raised against Triton-insoluble material from tadpoles (RD35/2a, RD35/3a and D3/3a), two antibodies against mammalian cytokeratins (LE65 and LP3K), monoclonal anti-(rat 200 K neurofilament protein), rabbit anti-(rat glial filament acidic protein), and rabbit antibodies to hamster and calf vimentin were used. We show that cytokeratins are present in the early central nervous system (CNS) and persist in the ependymal cells of the adult CNS. We also show that the notochord contains cytokeratin. The ontogeny of intermediate filament protein appearance in the CNS, skin and notochord between neural fold stage and swimming tadpole stage are described. These results are discussed in particular with regard to the use of the antibodies as differentiation markers.

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Species referenced: Xenopus laevis
Genes referenced: dio3 krt12.4 vim
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	Figs 1-9. Nitrocellulose blotting of extracts of Xenopus and mammalian tissues with anti-intermediate filament protein antibodies. All values are Mr x 10~3. Figs 1, 2, 8, 9 and 10 show autoradiographs of 125I-labelled immunoblots. In these cases the molecular weights marked are those of rat brain and HT29 cell intermediate filament proteins known to react with anti-intermediate filament antigen (IFA) monoclonal antibody (Pruss et al. 1981). For Figs 3-7, immunoperoxidase labelling was used and photographs of the nitrocellulose are shown. The Mr markers shown are: carbonic anhydrase, 29K; egg albumen, 45 K; bovine serum albumen, 66K; phosphorylase b, 97-4K; )3-galactosidase, 116K. Abbreviations: NF, neurofilament; V, vimentin; GFAP, glial filament acidic protein. Fig. 1. Autoradiograph showing anti-IFA staining of Triton X-100 extracts of rat brain (lanes a and e), adult Xenopus spinal cord (lanes b and f), Xenopus swimming tadpoles (lane c), HT29 cells (lane d). Fig. 2. Autoradiograph showing Triton-insoluble extracts of HT29 cells stained with RD35/2a (track a), RD35/3a (track b), D3/3a (track c). Fig. 3. Anti-IFA staining of tadpole Triton-insoluble material (lane a) and whole tadpole protein (lane b). Fig. 4. RD35/2a staining of tadpole Triton-insoluble material (lane a) and whole tadpole protein (lane b). Fig. 5. RD35/3a staining of tadpole Triton-insoluble material (lane a) and whole tadpole protein (lane b). Fig. 6. D3/3a staining of tadpole Triton-insoluble material (lane a) and whole tadpole protein (lane b). Fig. 7. Rabbit anti-vimentin IgG staining of extracts of rat brain (lane a), adult Xenopus spinal cord (lane b), 3T3 cells (lane c). Fig. 8. Rabbit anti-GFAP serum staining of extracts of rat brain (lane a), Xenopus spinal cord (laneb). Fig. 9. Mouse monoclonal anti-neurofilament antibodies RT97 (lanes a-c) and 147 (lanes d and e): reaction with extracts of rat brain (lane a), Xenopus swimming tadpole (lanes b and d), adult Xenopus spinal cord (lanes c and e).
	Figs 10-12. Indirect immunofluorescence labelling of sections of adult Xenopus skin and gut with anti-cytokeratin monoclonal antibodies. Abbreviations: e, epidermis; g, gland; ge, gut epithelium. Bars, 50\|um. Fig. 10. RD35/2a staining of (A) skin and (B) gut. Fig. 11. RD35/3a staining of skin. Fig. 12. D3/3a staining of (A) skin and (B) gut. Fig. 13. Indirect immunofluorescence labelling of adult Xenopus skin with (A) LP3K cytokeratin antibody; (B) LE65 cytokeratin antibody; (C,D) rabbit anti-vimentin IgG. B,D demonstrate the presence of cytokeratin and vimentin in blood vessels of the dermis. Arrows mark densely staining bodies which are also present in controls. Abbreviations: e, epidermis; g, gland. Bars, 50 fim.
	Fig. 14. (A,B) Indirect immunofluorescence labelling of neural folds of stage 17/18 embryos with cytokeratin antibodies: (A) LP3K; (B) RD35/3a. (C) Phase contrast micrograph of stage 17/18 neural folds. Abbreviations: e, epidermis; nf, neural fold; Bars, 20/an. (D-G) Indirect immunofluorescence labelling of the neural tube and notochord of stage 25/26 embryos with (D) LE65 cytokeratin antibody; (E) control culture medium; (F) rabbit antibodies to vimentin; (G) RD35/3a cytokeratin antibody. The fluorescence seen in the epidermis of the LE65 stained section (D) is thought to be non-specific since it was also observed in controls (E). Epidermis-staining anti-cytokeratins give much brighter staining (G). (H) Phase contrast micrograph of stage 25/26 neural tube. Abbreviations: nt, neural tube; s, somites; n, notochord; e, epidermis. Bars, 20/an.
	Fig. 15. Indirect immunofluorescence labelling of the stage 33/34 spinal cord and notochord by (A) RD35/2a; (B) rabbit anti-vimentin IgG; (C) rabbit anti-GFAP serum; (D) D3/3a. (E) Phase contrast micrograph of stage 33/34 spinal cord and notochord. Abbreviations: nt, neural tube; n, notochord; s, somites; e, epidermis. Bars, 20/rni.
	Fig. 16. Indirect immunofluorescence labelling of the spinal cord and notochord of stage 48 embryos with (A) RD35/2a; (B) LP3K; (C) rabbit anti-vimentin serum; (D) rabbit anti-GFAP; (E) monoclonal antibody to neurofilament 200 K protein (147). Abbreviations: n, notochord; sc, spinal cord; m, meninges. Bars, 50jum.
	Fig. 17. Indirect immunofluorescence labelling of adult Xenopus spinal cord with (A) RD35/2a; (B) LE65; (C) LP3K; (D,E) rabbit anti-vimentin serum; (F) rabbit anti-GFAP serum; (G) 147, anti-neurofilament 200K protein monoclonal antibody. Abbreviations: w, white matter; g, grey matter; m, meninges; bv, blood vessels; c, central canal. Bars, 50jum.
	Fig. 18. Electron micrographs showing (A) desmosomes in ependymal cells in the spinal cord of a swimming tadpole; (B) high power view of desmosomes shown in A. Intermediate-sized filaments can be seen in association with the desmosome. (C) Desmosomes in cells of the notochord. Abbreviations: d, desmosome; /, intermediate- sized filaments. Bars,1um