Smith JC (1987), A mesoderm-inducing factor is produced by Xenop...

XB-ART-28306

Development 1987 Jan 01;991:3-14.

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A mesoderm-inducing factor is produced by Xenopus cell line.

Smith JC .

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Inductive interactions play a major role in the diversification of cell types during vertebrate development. These interactions have been extensively studied in amphibian embryos (usually Xenopus laevis) where the earliest is mesoderm induction, in which an equatorial mesodermal rudiment is induced from the animal hemisphere under the influence of signal from the vegetal hemisphere. The molecular basis of mesoderm induction is unknown, although Tiedemann has isolated a protein form 9- to 13-day chick embryos that has the properties one would expect of a mesoderm-inducing factor. However, the relevance of this molecule to the events of early amphibian development is unclear, and it is a matter of some importance to discover a Xenopus mesoderm-inducing factor. In this paper I show that the Xenopus XTC cell line secretes mesoderm-inducing activity into the culture medium. Isolated animal pole regions cultured in XTC-conditioned medium differentiate into muscle and notochord, while controls form 'atypical epidermis'. Three different cell lines -XL, XL177 and KR- secrete no such activity indicates that the active principle is heat stable, trypsin sensitive, nondialysable, and has an apparent relative molecular mass of about 16,000. Work is in progress to characterize the activity further and to discover whether the mesoderm-inducing factor is also present in normal embryos.

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Species referenced: Xenopus laevis
Genes referenced: ccdc34 kif22 myh3 prss1
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	Fig. 1. The experimental design. (A) Mesoderm induction is demonstrated by combining animal pole tissue with vegetal pole tissue. (B) A pellet of XTC or XL cells is pressed against the blastocoel surface of an isolated animal pole region. (C) Conditioned medium from XTC cells is used as the culture medium for isolated animal pole regions.
	Fig. 2. Mesoderm induction by a pellet of XTC cells but not by a pellet of XL cells. Isolated Xenopus blastula animal pole regions were pressed against a pellet of XTC or XL cells and allowed to develop for 65 h at 18-22 °C in modified L15 medium containing 10 % foetal calf serum. After fixation and sectioning, samples were analysed by indirect immunofluorescence using an antibody raised against Xenopus myosin heavy chain. (A,B) A combination between XL cells and Xenopus animal pole tissue stained both with 4',6-diamidino-2-phenylindole-dihydrochloride (DAPI), to show nuclei, and with anti-myosiji heavy chain. (A) DAPI staining. The animal pole component, with fewer cells, is to the left. (B) The section is negative for myosin heavy chain. (C,D) A combination between XTC cells and animal pole tissue stained with DAPI and anti-myosin heavy chain. (C) DAPI staining. The animal pole component is to the left. (D) The animal pole cells have formed muscle. Scale bar in (D) is 200 jan and also applies to (A), (B) and (C).
	Fig. 3. Mesoderm induction by XTC-, but not XL-, conditioned medium. Midblastula-stage animal pole regions were cultured in heated conditioned media for about 16 h before being transferred to half-strength NAM for about 48 h. They were then fixed and analysed by indirect immunofluorescence. (A-C) Explants cultured in XL-conditioned medium. (A) This explant has formed a wrinkled ciliated sphere. (B) A section (of a different explant) stained with DAPI to show the nuclei. (C) The same section stained with 12/101; no muscle is formed. (D-F) Explants cultured in XTCconditioned medium. (D) This explant has become elongated and acquired some internal structure. (E) A section (of a different explant) stained with DAPI. (F) The same section stained with 12/101; large amounts of muscle have been formed. Scale bar in (D) is 500 fim and also applies to (A). Scale bar in (F) is 200/an and also applies to (B), (C) and (E).
	Fig. 4. Histological cell types formed in response to heated XTC-conditioned medium. (A) An explant showing notochord (not), muscle (mus), kidney (kid), mesenchyme (mes) and mesothelium (meso). The epidermis (epi) shows good histological differentiation. (B) An explant demonstrating large amounts of notochord (not) with neuroepithelium (new) and melanocytes (met). The epidermis (epi) is well differentiated. (C) An explant cultured in half-strength NAM forms 'atypical epidermis'. Scale bar in (C) is 200/mi and also applies to (A) and (B).
	Fig. 5. Titration of XTC- and XL-conditioned media by Western blotting. Groups of three animal pole explants were exposed for 16h to serial dilutions of XL-conditioned medium, heated or unheated, or XTC-conditioned medium, heated or unheated. They were then transferred to half-strength NAM and cultured for 48 h until controls reached stage 40. Samples were solubilized in gel sample buffer, boiled and run on a linear 5-15 % polyacrylamide gradient gel. The separated proteins were transferred electrophoretically to nitrocellulose and probed simultaneously with an antibody recognizing Xenopus myosin heavy chain (Mr = 205 000) and an antibody against Xenopus keratin-like protein II (which recognizes at least four bands between Mt = 50000 and MT = 60000). Tracks 1-5: XL-conditioned medium (unheated) at protein concentrations of 41-4, 13-8, 4-1, 1-4 and 0-4/igmP1 respectively. Tracks 6-10: XL-conditioned medium (heated to 95°C for 5min) at 34-2, 11-4, 3-4, 1-1 and 0-3/igmr1 respectively. Tracks 11-15: XTC-conditioned medium (unheated) at 20, 6-7, 20, 0-7 and 0-2/igmP1 respectively. Tracks 16-20: XTC-conditioned medium (heated to 95°C for 5 min) at 20, 6-7, 20, 0-7 and 0-2[igm\~l respectively. Note that XL-conditioned medium is only slightly active even after heating, and that the mesoderm-inducing activity of XTC-conditioned medium is enhanced by a factor of 10 as a result of heating.
	Fig. 6. The mesoderm-inducing activity of XTC-conditioned medium has an apparent relative molecular mass of about 16000. 120 ml of serum-free XTC-conditioned medium was prepared from ten confluent 150mm tissue culture plates. One plate contained serum-free L15 with 10% of the normal methionine concentration and 20 ^Ci of [^SJmethionine. The conditioned medium was centrifuged to remove cell debris, heated to 95CC for 5min, and centrifuged at 10 000 g for 20min. This medium was concentrated with an Amicon YM2 membrane, ammonium sulphate precipitated and dissolved in 5ml of 0-lM-NaCl, lmM-EDTA, 20mM-tris pH80, 0 1 % sodium deoxycholate. 4ml of this sample was applied to an AcA 54 gel filtration column equilibrated in the same buffer, and 4 ml fractions were collected. The fractions were assayed at a 1/20 dilution. The figure shows protein absorbance at 280nm (•) and 35S-radioactivity (O). Mesoderm-inducing activity was assayed by Western blotting and the region of the blot containing the myosin heavy chain is shown above the graph. Only the odd-numbered fractions are presented here. Fractions 31 and 33 induced substantial quantities of myosin heavy chain while a very weak band is visible at fraction 35. Arrows indicate relative molecular mass markers: BSA (MT = 66000), ovalbumin (OV; Mr = 45000), soybean trypsin inhibitor (SBTI; MT = 20100) and cytochrome C (CYT C; M, = 12300).
	Fig. 7. Protein synthesis by XTC and XL cells. Confluent cultures of XTC or XL cells were rinsed three times with serum-free modified L15 medium and then incubated for 24 h in the same medium but with the methionine concentration at 10 % of the normal level, and with 25/iCiml"1 [35S]methionine. Samples of labelled conditioned medium, either heated or unheated, were prepared for gel electrophoresis by acetone precipitation and solubilization in gel sample buffer. The labelled XTC and XL cells were rinsed in 70 % PBS-A and solubilized directly into gel sample buffer. After boiling, equal counts were run on a linear 5-15 % polyacrylamide gradient gel, which was fluorographed and exposed to Xray film. Lane 1, proteins associated with XTC cells; lane 2, proteins associated with XL cells. Lane 3, XTCconditioned medium; lane 4, heated XTC-conditioned medium; lane 5, XL-conditioned medium; lane 6, heated XL-conditioned medium. Relative molecular mass markers are shown (xlO~3).