Saint-Jeannet JP et al. (1992), Ventrolateral regionalization of Xenopus laevis...

XB-ART-23498

Development 1992 Aug 01;1154:1165-73.

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Ventrolateral regionalization of Xenopus laevis mesoderm is characterized by the expression of alpha-smooth muscle actin.

Saint-Jeannet JP , Levi G , Girault JM , Koteliansky V , Thiery JP .

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Mesodermal patterning in the amphibian embryo has been extensively studied in its dorsal aspects, whereas little is known regarding its ventrolateral regionalization due to a lack of specific molecular markers for derivatives of this type of mesoderm. Since smooth muscles (SM) are thought to arise from lateral plate mesoderm, we have analyzed the expression of an alpha-actin isoform specific for SM with regard to mesoderm patterning. Using an antibody directed against alpha-SM actin that recognized specifically this actin isoform in Xenopus, we have found that the expression of alpha-SM actin is restricted to visceral and vascular SM with a transient expression in the heart. The overall expression of the alpha-SM actin appears restricted to the ventral aspects of the differentiating embryo. alpha-SM actin expression appears to be activated following mesoderm induction in animal cap derivatives. Moreover, at the gastrula stage, SM precursor cells are regionalized since they will only differentiate from ventrolateral marginal zone explants. Using the animal cap assay, we have found that alpha-SM actin expression is specifically induced in treated animal cap with bFGF or a low concentration of XTC-MIF, which induce ventral structures, but not with a high concentration of XTC-MIF, which induces dorsal structures. Altogether, these results establish that alpha-SM actin is a reliable marker for ventrolateral mesoderm. We discuss the importance of this novel marker in studying mesoderm regionalization.

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Species referenced: Xenopus laevis
Genes referenced: acta2 acta4 actc1 actl6a amh fgf2 mif
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	Fig 1. Western blot analysis of extracts from Xenopus adult tissues. Skeletal muscles of the legs (lane 1) and of the back (lane 2), brain (lane 5), and heart (lane 6) do not present any reactivity to the anti-a-SM actin antibody. Lung (lane 3), aorta (lane 4), skin (lane 9), and liver to a lesser extent (lane 7), display a single band with an apparent relative molecular mass of 45 ´ 103. Intestine (lane 8) and bladder (lane 10) present also a major band at 45´103 and additional bands with lower relative molecular masses, which are probably degradation products. Relative molecular masses of markers are respectively, 106, 80, 49.5 and 32.5 ´103.
	Fig. 2. Developmental expression of a-SM actin expression. (A) Schematic representation of the developmental distribution of a-SM actin. From stage 37/38 to stage 46, a-SM actin is detected in the heart. From stage 40 and on, a-SM actin expression is predominently expressed in vascular and visceral SM cells. (B) At stage 23, no labeling is detected in the lateral plate (arrowheads). (C) By stage 37/38, a faint labeling is observed in the heart anlage (h), just beneath the pharyngial cavity (ph). (D) Stage-41 embryo displaying an a-SM actin immunoreactivity confined to the outer layer of the endoderm, where the first intestinal loop starts to differentiate. Two sections in the anterior (E) and posterior (F) regions of an embryo at stage 43 present a staining delineating the differentiating endoderm. (G) At the same stage heart is also labeled (arrowhead). (H) Enlargement of the heart area shown in (G); staining is restricted to the atrium (a) whereas ventricule (v) remains negative. (I,J) Stage-47 embryo where labeling is detected in the outer coat of the gut loops in anterior (I) and more posterior (J) regions. (K) Detail of a stage-47 embryo stained with the a-SM actin antibody, aorta (ao), lung (lu) and intestine (in) present a strong immunoreactivity whereas liver (li), notochord (n) and somites (s) do not. Magnification: B, D-G and I-J ´40; C, H and K ´80.
	Fig. 3. Expression of a-SM actin in stage-8 animal cap, vegetal pole and conjugates. (A) Blastula-isolated animal caps do not present any immunoreactivity with the a-SM actin antibody. (B) Corresponding phase contrast. (C) Vegetal pole cells are also negative for this antibody. (D) Corresponding phase contrast. (E,F) When both tissues are combined as conjugates, an a-SM actin immunoreactivity can be observed in the animal cap derivatives (arrowheads). Magnification: A-I, ´50.
	Fig. 4. Immunodetection of a-SM actin in explants isolated at stage 11 and cultured for 5 days. Animal caps (A) and endodermal tissues (C) do not present any immunoreactivity to the anti-a-SM actin antibody; (B,D) corresponding phase contrast. (E) Explants derived from the dorsal marginal zone, in which notochord (n) differentiate, are also negative to this antibody; (F) corresponding phase contrast. (G) Ventral marginal zone-derived explants express a-SM actin in patches of cells in the centre of the explant; (H) corresponding phase contrast. (I-L) Lateral marginal zone-derived explants present also an immunoreactivity to the a-SM actin antibody, either as a cluster of cells within the explant (I,J) or as a layer of cells associated with the differentiated epidermis (L). (K) Phase contrast of (J). Magnification: A-K, ´ 50; L, ´ 100.
	Fig. 5. Immunodetection of a-SM actin in stage-8 animal caps treated with peptide growth factors. (A) Untreated animal caps are negative to the anti-a-SM actin antibody; (B) corresponding phase contrast. (C) Animal caps treated with undiluted XTC-GTX-11-conditioned medium display induction of axial structures such as notochord (n) but do not express a-SM actin; (D) corresponding phase contrast. (E) Animal caps treated with XTCGTX- 11 conditioned medium at a 1:3 dilution present strong a-SM actin immunoreactivity; staining is associated with a layer of cells commonly designated as mesothelium in ventrally induced explants; (F) corresponding phase contrast. (G) Animal caps treated with bFGF at 200 ng ml-1 are intensely labeled with the anti-a- SM actin antibody; (H) corresponding phase contrast. Magnification: A-H, ´ 60.