Godsave SF and Shiurba RA (1992), Xenopus blastulae show regional differences in ...

XB-ART-23668

Dev Biol 1992 Jun 01;1512:506-15. doi: 10.1016/0012-1606(92)90189-n.

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Xenopus blastulae show regional differences in competence for mesoderm induction: correlation with endogenous basic fibroblast growth factor levels.

Godsave SF , Shiurba RA .

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Single cells from the animal cap and marginal zone (MZ) of mid-blastula stage embryos can undergo mesodermal or ectodermal differentiation as small clones under defined conditions in culture. Here we report that cells treated with Xenopus basic fibroblast growth factor (XbFGF), a mesoderm-inducing factor, usually differentiated into muscle. MZ cells, which normally give rise to most of the mesoderm, responded to lower concentrations of XbFGF than animal pole (AP) presumptive ectoderm cells. This difference in sensitivity correlated with immunocytochemical staining patterns that showed much greater levels of endogenous bFGF within MZ than AP cells in early embryos. At the mid-late blastula stage, nuclei of MZ cells were strongly immunoreactive. Nuclear staining persisted during gastrula and neurula stages, and extracellular bFGF also became apparent. Subsequently in somites, immunoreactivity of nuclei declined while that of the extracellular matrix was retained during tailbud stages. Nuclear localization of bFGF appeared to be temporally correlated with new transcription of muscle-specific genes, and extracellular bFGF was present during morphological differentiation. The results suggest that a cell's competence for mesoderm induction is related to its position in the embryo.

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Species referenced: Xenopus
Genes referenced: diras1 fgf2 fn1 kcnt1
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	FIG. 1. Single cell culture method. Explants were first dissected from stage 8 blastulae as indicated in a and b. (a) Cross section through the animal pole (AP) and vegetal pole (VP) of an embryo. (b) Diagram of an embryo viewed from above the AP. Tissue pieces were excised from around the AP and from parts of the animal hemisphere close to the equator, comprising one-quarter of the circumference of the embryo on the dorsal and ventral sides. Subequatorial MZ explants were cut from beneath the supraequatorial MZ pieces in the vegetal hemisphere. Following dissection, explants were transferred to petri dishes containing Ca2+/MgZt-free NAM medium. (c) Explants were gently dissociated by irrigation with a stream of medium. (d) A single cell was then picked up with a micropipet, and it was placed in a Terasaki plate well. (e) Wells were coated with 2 ~1 of a solution containing laminin and fibronectin (100 @g/ml each). Culture medium NAM/2 contained r-globulins at 2 mg/ml (Godsave and Slack, 1989, 1991) and XbFGF ranging in concentration from 0.02 to 10 rig/ml. (f) Differentiation occurred after 2 to 3 days of culture at 25°C. Then cells were fixed in methanol:acetic acid (3:1), and they were immunocytochemically stained with an anti-muscle monoclonal antibody, MAb 12-101.
	FIG. 2. Induction of single cells by XbFGF.
	FIG. 3. Development of muscle in single cell cultures. Appearance of cultures when control embryos reach (A) stage 11.5 to 12, (B) stage 23, (C) stage 28/29, and (D) stage 33. (E) A clone fixed at the tadpole stage and stained with anti-muscle antibody, MAb 12-101. (A, C, E) Bright field; (B, D) phase contrast. The 100 PM bar applies to all photomicrographs.