An understanding of cardiac development has influenced the study of cardiovascular disease in children and adults. Defects in cardiac development lead to a wide variety of pathology, including congenital heart defects, cardiomyopathies, and valvular diseases. Sphingosine-1-phosphate (S1P) has been found to play an important role in vertebrate development. S1P is a product of cell membrane metabolism and found ubiquitously throughout the body. Once believed to simply be an end product of cel membrane breakdown, S1P is now recognized as an important regulator of cell proliferation, migration, and death. The effects of S1P are mediated by a family of five G protein-coupled receptors, sphingosine-1-phosphate receptors 1-5 (S1P1-5). They are expressed in a tissue specific manner and confer the diversity of responses to S1P. This project proposes to study the role of the S1P receptor S1P1 in cardiac development. Synthetic oligonucleotide inhibition of S1P1 expression in zebrafish leads to impaired cardiac sarcomere development. These abnormalities appear to be specific to cardiac myocytes.
In Aim 1, the timing of S1P1 function in zebrafish cardiac development will be examined. Light-activated oligonucleotides will be injected into zebrafish embryos and ultraviolet light will be used to initiate inhibition of S1P expression at specific time points. This will allow control over the location and timing of S1P1 inhibition. The effects of S1P1 expression inhibition will be assayed by evaluating cardiac contractility and immunohistochemistry of sarcomere components.
In Aim 2, the cellular changes associated with failed sarcomere assembly will be evaluated in zebrafish subjected to synthetic oligonucleotide inhibition of S1P1 expression. Actin will be visualized using a method of fluorescence tagging. This method does not interfere with actin function and will allow real-time assessment of the sarcomere in vivo. Immunohistochemistry of other important sarcomere components will be performed. The signaling pathways that lead to impaired sarcomere development in this model will also be investigated.
In Aim 3, conservation of this phenotype will be investigated by inhibiting S1P1 expression in mice. To accomplish this, mice that have the S1P1 gene disrupted in the atrial myocardium, the ventricular myocardium, or both the myocardium and endocardium will be generated. The effects of this disruption will then be assayed by echocardiography, immunohistochemistry, and electron microscopy.
An understanding of cardiac development has increasingly influenced the study of cardiovascular diseases in children and adults. Defects in cardiac development lead to congenital heart defects, cardiomyopathies, and valvular diseases. Congenital heart disease affects approximately 800,000 adults in the United States. This work will provide insight into a novel regulator of cardiac development.
| Wu, Roland S; Lam, Ian I; Clay, Hilary et al. (2018) A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish. Dev Cell 46:112-125.e4 |
