International Mammalian Genome Society

The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

G28 NFATC1 Activates and Suppresses Multiple Genes During Cardiac Development

Bin Zhou, Bingruo Wu, Justin Morabito, Craig Mickanin and H. Scott Baldwin. Children's Hospital of Philadelphia and the University of Pennsylvania School of Medicine

NFATc1 null mutation die in late gestation (E13.5-15.5) due to cardiac failure resulting from absent semilunar formation with no qualitative defects in AV valve morphogenesis. Since NFATc1 expression is transient and restricted to the endocardium of developing heart, we hypothesized that this burst of endocardial-specific NFATc1 expression is required for transcription activation of genes which are critical for semilunar valve morphogenesis. To test this hypothesis, we compared expressions of the known targets (IL1b, 2, 3, 4, 5, 6, 10, 13, TNFa, GMCSF, IFNg) of NFATc1 in the outflow tract of E12.5 wild type to that of mutant hearts by multiple RNase protection assays. These studies document that most of the known factors are either undetectable at this stage of development or, as in the case of TGFß1-3, were not altered in the hearts of NFATc1-/- embryos as compared to normal embryos. We then utilized a PCR-based cDNA subtractive hybridization approach in an attempt to identify novel "down stream" targets of NFATc1 activation that were either diminished or accentuated in the null mutant embryonic hearts. A total of 89 subtracted clones of NFATc1+/+ minus NFATc1-/- were obtained and sequenced. Of these, 57 unique clones were identified and used for sequential reverse northern analysis with cDNA probes transcribed from mRNA of both E12.5 NFATc1+/+ and NFTAc1-/- embryonic hearts. Six of these clones were clearly downregulated or absent in NFTAc1-/- embryonic hearts. In a reversed subtracted hybridization (NFTAc1-/- minus NFATc1+/+ hearts), a total 97 clones were sequenced and among these 72 unique clones were isolated. Nine of these clones were clearly upregulated or exclusively expressed in the NFATc1-/- hearts. Interestingly, one third of the clones in both subtracted pools were unknown in Genebank database searching. Furthermore, developmentally regulated as well as tissue-specific, including heart, vascular endothlium, and liver, expression of certain genes were revealed by northern blot and in situ hybridization. These data suggest that NFATc1 may be involved in both activation and suppression of expression of multiple genes during semilunar valve ontogeny.

Supported by NIH ARO1 HL56583, P50 HL61014, and AHA 95002720.


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