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Effects of antisense misexpression of CFC on downstream flectin protein expression during heart looping
Kersti K Linask1, Ming-Da Han, Kaari L Linask
1Department of Cell Biology, University of Medicine and Dentistry of New Jersey-SOM, Stratford, New Jersey, USA. klinask@hsc.usf.edu
Abstract:
Dextral looping of the heart is regulated on multiple levels. In humans, mutations of the genes CFC and Pitx2/RIEG result in laterality-associated cardiac anomalies. In animal models, a common read-out after the misexpression of laterality genes is heart looping direction. Missing in these studies is how laterality genes impact on downstream morphogenetic processes to coordinate heart looping. Previously, we showed that Pitx2 indirectly regulates flectin protein by regulating the timing of flectin expression in one heart field versus the other (Linask et al. [2002] Dev. Biol. 246:407-417). To address this question further we used a reported loss-of-function approach to interfere with chick CFC expression (Schlange et al. [2001] Dev. Biol. 234:376-389) and assaying for flectin expression during looping. Antisense CFC treatment results in abnormal heart looping or no looping. Our results show that regardless of the sidedness of downstream Pitx2 expression, it is the sidedness of predominant flectin protein expression in the extracellular matrix of the dorsal mesocardial folds and splanchnic mesoderm apposed to the foregut wall that is associated directly with looping direction. Thus, Pitx2 can be experimentally uncoupled from heart looping. The flectin asymmetry continues to be maintained in the secondary heart field during looping.
Insights
Heart looping direction is determined by flectin protein expression asymmetry, not solely by Pitx2 gene activity. This finding decouples Pitx2 from direct regulation of cardiac looping direction.
Area of Science:
- Developmental biology
- Molecular genetics
- Cardiovascular research
Background:
- Heart looping is crucial for cardiac development and is regulated by laterality genes like CFC and Pitx2.
- Mutations in these genes can lead to cardiac anomalies.
- The precise downstream mechanisms by which laterality genes control heart looping remain unclear.
Purpose of the Study:
- To investigate how CFC gene expression influences heart looping direction.
- To determine the role of flectin protein expression in coordinating heart looping.
- To examine the relationship between Pitx2 expression and heart looping direction.
Main Methods:
- Utilized a loss-of-function approach by interfering with chick CFC gene expression using antisense treatment.
- Assayed for flectin protein expression during heart looping.
- Analyzed the correlation between flectin expression asymmetry and heart looping direction.
Main Results:
- Antisense CFC treatment led to abnormal or absent heart looping.
- Predominant flectin protein expression asymmetry in specific embryonic tissues directly correlated with heart looping direction.
- This correlation held true irrespective of the sidedness of downstream Pitx2 expression.
Conclusions:
- Flectin protein expression asymmetry is the direct determinant of heart looping direction.
- Pitx2's role in heart looping can be experimentally uncoupled, suggesting it acts upstream or indirectly.
- Flectin asymmetry is maintained in the secondary heart field during cardiac looping.
