CLONING AND CHARACTERIZATION OF MYOSIN-RELATED CDNAS FROM XENOPUS LAEVIS

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In collaboration with Dr. Milan Jamrich, we have cloned a cDNA (Xfkh3) for a novel class of forkhead genes from Xenopus using degenerate primer PCR. Xfkh3 encodes for a 483 amino acid protein with a highly conserved putative DNA binding domain of 77 amino acids. RNA blot analysis detected a 2.3 kb transcript first at stage 10.5 and at a constant level throughout embryogenesis up to swimming tadpoles, suggesting that the transcription of this gene is solely zygotic. Whole mount in situ hybridization revealed that at stage 10.25, the transcript is expressed throughout the mesoderm. As gastrulation proceeds, the transcript becomes more restricted. At stage 11.5-12.5, a strong signal is detected in the presomitic mesoderm and no expression is detected in the notochord and in the ventral mesoderm. During neurulation, a strong signal is detected in the presomitic mesoderm whereas newly differentiated somites show only weak expression, suggesting the requirement of this protein prior to somite differentiation. In a cross section of the tailbud embryo through a differentiated somite, the signal is detected in sclerotomes and not in dermatomes and myotomes. In swimming tadpoles, a strong signal is detected in the branchial arches, in the region around the eye, and in the presomitic mesoderm at the tip of the tail. With the animal cap induction assays, Xfkh3 RNA is detected only in animal caps treated with activin, but not with bFGF or retinoic acid. Earlier, we have shown that mRNA for Xenopus nonmuscle myosin heavy chain-B is expressed throughout development, starting from unfertilized eggs to swimming tadpoles (Bhatia-Dey et al., Proc. Natl. Acad. Sci. USA 90: 2856, 1993). In situ hybridization reveals that the transcript is expressed ubiquitously in blastula and gastrula stage embryos, but begins to localize in the anterior part of the embryo in late neurula. In swimming tadpoles, the strong signal is detected in differentiated somites, the eye and the branchial arches. Currently, I am investigating the role of this myosin isoform in somitogenesis.