CONFOCAL MICROSCOPY OF A NEWLY IDENTIFIED PROTEIN ASSOCIATED WITH HEART DEVELOPMENT IN THE MEXICAN AXOLOTL

Recessive mutant gene c for ''cardiac nonfunction'' in the mexican axo lotl, Ambystoma mexicanum, results in a failure of affected embryos to develop contracting hearts. Mutant embryos survive approximately 4 we eks after fertilization, but eventually die from a lack of circulation . Morphological studies show that mutant hearts lack organized sarcome ric myofibrils. This abnormality can be corrected by co-culturing earl y mutant hearts with normal anterior endoderm/mesoderm tissues, by cul turing them in a medium ''conditioned'' by this normal tissue, or by R NA isolated from normal endoderm/mesoderm. Additionally, RNA isolated from normal anterior endoderm/mesoderm conditioned medium corrects the mutant hearts in a dose-dependent manner. A cDNA library is construct ed using this RNA. On the basis of sequence analyses on this cDNA libr ary, it was estimated that 56% of the total RNA present in the conditi oned medium is rRNA, while 44% is nonribosomal RNA. One of the nonribo somal RNAs that showed no significant homology with other known sequen ces in the Genebank was examined further. An RT-PCR analysis showed th at this RNA (designated ''N1'') is expressed in juvenile skeletal musc le, brain, and heart in significant amounts, less in the lung and not at all in the liver tissue. Affinity-purified polyclonal antipeptide a ntibodies were produced against the most antigenic portion of the poly peptide which was deduced from this RNA. Western blot analyses of adul t heart homogenates, using these antibodies, showed a specific doublet staining at 67 kDa and 65 kDa. These doublets were purified and analy zed for their amino acid composition which showed that both bands most likely belong to the same protein. The N1-protein was further investi gated to determine its localization in normal isolated hearts at embry onic stages 35, 38, and 41 and on cross-sections through the heart reg ions of whole normal embryos at stages 16, 33-34, 37-38, and 41-42 usi ng immunohistochemical techniques and confocal microscopy. In addition , mutant embryos at stage 37-38 were studied for the presence and dist ribution of the N1-protein on cross-sections through their heart regio ns. The N1-protein staining was significantly reduced in mutant hearts when compared to normal.