CLONING AND CHARACTERIZATION OF MYOSIN-RELATED CDNAS FROM XENOPUS LAEVIS

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In order to understand the role of the Xenopus forkhead protein (Xfkh3) in the differentiation of presomitic mesoderm, I have tried to overexpress and misexpress this protein by injecting synthetic mRNA in the animal pole region of both blastomeres of 2 cell stage embryos. 0.25, 0.5 and 1 ng of mRNA was injected. At stage 8, animal caps were dissected and cultured until sibling embryos reached tailbud stage in the presence or absence of activin. Although misexpression of the message by itself does not induce any muscle in the animal caps, still, muscle induction by activin is not inhibited. Further analysis of these results is currently under progress. By whole mount in situ hybridization, I have localized the distribution of Xenopus nonmuscle myosin heavy chain B (Bhatia-Dey et al., Proc. Natl. Acad. Sci. USA 90, 2856, 1993) using a probe that comprises part of the rod and the entire 3' UTR. The transcript is expressed ubiquitously in blastula and gastrula stage embryos, however, at stage 21, the transcripts begin to localize in the anterior part of the embryo, especially in the developing eye and fully differentiated anterior somites. At early tailbud stage, the transcripts become more concentrated in differentiated somites and in the eye. No expression is detected in the presomitic mesoderm. In swimming tadpoles, the transcripts are localized in differentiated somites, the eye and the branchial arches. Using a probe from a similar region of Xenopus nonmuscle myosin heavy chain A, no localized transcripts were detected throughout embryogenesis. However, Northern analysis, using the same probe, detected 2 transcripts during development. A 7.5 kb transcript is detected throughout embryogenesis from unfertilized eggs to swimming tadpoles and in a number of adult tissues. Another 8.3 kb transcript is first detected around stage 15/16, peaks between stage 25 to 30, gradually decreases in swimming tadpoles and is no longer detected in stage 47 embryos. Preliminary analysis suggests that the larger transcript arises due to differential polyadenylation.