The expression of Xsmα-actin transcripts was assessed by in situ hybridization and transcripts encoding this mRNA, unlike their mammalian and avian homologues (Woodcock-Mitchell et al., 1988; Ruzicka and Schwartz, 1988; Sawtell and Lessard, 1989), were never detected in the somites or skeletal muscle of developing embryos. Xsmα-actin mRNAs were first detected in a bilateral pair of triangular patches that are separated by the ventral midline and lie posterior to the cement gland of the late tail bud embryo (stages 26/27; Fig. 6A,B). The bilateral appearance of the Xsmα-actin mRNA in this heart-forming region of the embryo coincides with the onset of cardiac muscle differentiation (Mohun et al., 2000) and the location of its expression is similar to that observed for transcripts encoding the X. laevis cardiac muscle-specific proteins myosin heavy chain-α, myosin light chain 2a (Mohun et al., 2000), and cardiac troponin I (Drysdale et al., 1994). Interestingly, the expression of this mRNA in the heart-forming region also coincides with the expression of the cardiac (Fig. 6C,D) and skeletal muscle (Mohun et al., 1984; Hemmati-Brivanlou et al., 1990; Logan and Mohun, 1993) α-actin isoforms in this region of the Xenopus embryo. The apparent, coordinated expression of these three actin isoforms in early cardiogenesis is in contrast to the situation in mammals and birds, where the developing heart appears to sequentially expresses mRNAs encoding the vascular smooth muscle α-actin, cardiac muscle α-actin, and skeletal muscle α-actin (Woodcock-Mitchell et al., 1988; Ruzicka and Schwartz, 1988; Sawtell and Lessard, 1989). As development proceeds the expression of Xsmα-actin transcripts extends across the ventral midline and is detectable throughout the heart up to and including the onset of chamber formation (Fig. 6E–G; see Kolker et al., 2000; Mohun et al., 2000). After the heart chambers are formed (stage 46) and before metamorphic climax (stage 61), the expression of Xsmα-actin transcripts becomes confined to the outflow tract of the tadpole heart (Fig. 6H,I). The observation that the expression of this gene is never detected in the visceral tissues and is confined to the vascular system once the heart chambers are formed clearly distinguishes it from a more visceral-like smooth muscle α-actin isoform previously identified and thought to be the only smooth muscle actin isoform in Xenopus (Saint-Jeannet et al., 1992). In fact, based on functional criteria, the subsequent restricted expression of this gene to the vascular system identifies it as one that encodes the Xenopus homologue of the avian and mammalian vascular smooth muscle α-actin.
Figure 6. Expression of Xsmα-actin in developing Xenopus laevis embryos. A–D: Lateral and ventral views of late tail bud embryos (stage 26/27) after whole mount in situ hybridization to detect smooth muscle α-actin (Xsmα-actin; A,B) and cardiac α-actin (Xcα-actin1; C,D) gene expression. Although transcripts from both genes are colocalized in the bilateral domains of the heart-forming (h) region in these embryos, Xsmα-actin mRNA, unlike Xcα-actin1 mRNA, is not present in the somites (s). E: A lateral view of a stage 31 Xenopus embryo showing that Xsmα-actin expression is throughout the developing heart tube. F,G: Lateral views of the anterior portion of Xenopus embryos at stages 34 (F) and 41 (G) showing that Xsmα-actin transcripts are throughout the heart, up to and including the onset of chamber formation. H: In situ hybridization of a cross-section through the heart of a stage 59 tadpole showing that after the heart chambers [atria (a) and ventricle (v)] are formed Xsmα-actin transcripts are confined to the outflow tract (oft) of the heart. I: A higher magnification image of the outflow tract seen in H.
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