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We have isolated the Xenopus homologue of Muscle LIM protein (MLP, CRP3) and examined its expression during early embryonic development. MLP is only expressed in the differentiated heart during early development and is expressed in a subset of other striated muscles during later stages. There is no MLP expression during primary myogenesis in the somites, although it is found in adult skeletal muscle.
Fig. 1.Sequence analysis of Xenopus MLP. (A) A comparison of the Xenopus, rat, and human MLP amino acid sequences (GenBank accession numbers AAF78241, X81193 and AAA92571 respectively). Differences in the Xenopus MLP sequence, when compared to the mammalian sequences, are underlined. The cysteine/histidine residues that form the two zinc fingers are in bold and are conserved across all three sequences. (B) A dendrogram showing the relationship of XMLP to other members of the CRP family showing that the Xenopus sequence clusters with other vertebrate CRP3 sequences rather than CRP1 or CRP2.
Fig. 2. Expression of MLP during Xenopus embryogenesis. (A) MLP expression is first detected at stage 27 (staged according to Nieuwkoop and Faber, 1994) in the heart (h) by whole mount in situ hybridization. (B) A stage 36 embryo showing that MLP expression (seen in blue) is still restricted to the heart at this stage. (C) At stage 40/41, MLP expression can be found in both the heart (h) and intermandibularis (i) muscle. In addition, expression was observed in the lymph heart (l) near the anteriorsomites. (D) An enlarged view of a stage 40/41 embryo showing the regions of expression in the head and clearly showing the lack of expression in the developing somites. (E) Ventral view of a Xenopus embryo showing the two patches of intermandibularis muscle spanning the lower jaw and expressing MLP. (F) A frontal section through the heart of a stage 38 embryo showing expression of MLP in the ventricle (v), atria (a) and outflow tracts (o) but not the endocardium (e). (G) Northern blot of adult intestine (1), kidney (2), liver (3), skeletal muscle (4), and heart (5) RNA probed with MLP. There was approximately equal loading in each lane based on rRNA levels. MLP expression is restricted to the heart and skeletal muscle. As in mammals, a higher level of expression was observed in the heart, in which a second, larger transcript was also observed (Arber et al., 1994).
Fig. 3. MLP expression is disrupted by retinoic acid. Expression of MLP in (A) a normal heart (h, arrow) and (B) an embryo treated with 1 μM retinoic acid. Treatment with retinoic acid results in a complete loss of MLP expression in the heart region.
csrp3 (cysteine and glycine-rich protein 3 (cardiac LIM protein)) gene expression in a Xenopus laevis embryo, NF stage 40, as assayed by in situ hybridization. Lateral view: anteriorleft, dorsal up.
