Homozygosity for the lethal cardiac mutant allele in Mexican axolotls results in failure to develop organized myofibrils and contracting hearts. This is corrected by coculturing mutant hearts with normal anterior endoderm, culturing them in conditioned medium from normal endoderm cultures, or culturing them in RNA isolated from endodermal conditioned medium. A cDNA library constructed from this RNA and a clone (pN1) with a unique partial nucleotide sequence was identified and its deduced 88 amino acid sequence was given the name, N1. Polyclonal antibody was raised against a 14mer peptide of this presumptive N1 protein and western blot analysis of adult axolotl heart homogenates suggest that the protein was present in anterior endoderm (a potent heart inductor tissue) and in stage 16 endoderm (heart induction stage) localized adjacent to the presumptive cardiogenic mesoderm. The N1 protein staining was significantly reduced in mutant hearts when compared to normal hearts, suggesting the N1 is a novel protein that may play a significant role in cardiogenic induction of mesoderm by the ventral anterior endoderm. The hypothesis being tested is that expression of N1 is essential for normal heart development and the alteration of N1 gene expression results in specific abnormalities during heart development leading to a failure in myofibril formation and a corresponding lack of contractile function.
Specific aims are to: (1) isolate and sequence the full length N1 cDNA and subject this sequence to computer assisted analysis to identify potential homologies and domain/motif structures of the deduced amino acid sequence; (2) determine the temporal and spatial patterns of expression for the N1 mRNA and protein in normal and mutant embryos beginning at the early developmental stages using the mutant (c/c) fertilized eggs obtained by spawning chimeric c/c axolotls; (3) determine the effect of N1 on cardiogenesis by microinjecting N1 expression vector constructs into fertilized axolotl eggs of known genotype, in effect, creating transgenic axolotls and; (4) isolate and sequence the N1 genomic DNA and analyze and characterize potential cis elements.
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