DEVELOPMENT OF CARDIAC ASYMMETRY IN ZEBRAFISH

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The development of left-right (LR) asymmetry of the heart and abdominal organs during vertebrate embryogenesis is a fundamental, yet poorly understood, process. Errors in the development of normal asymmetry give rise to various forms of situs inversus, including dextrocardia, inversion of the abdominal viscera, and a host of other congenital anomalies. The long-term goal of this project is twofold: to define the inductive role of the developing axial mesoderm in LR patterning and to understand the signals required for proper specification of the axis. We will concentrate on the earliest morphologic sign of asymmetry, namely rightward looping of the heart-tube. The zebrafish, Danio rerio, has been chosen as the model system for this project because it develops externally in a transparent embryo, produces hundreds of eggs daily, and is particularly amenable to genetic and cellular manipulation. One of the best-studied sources of signaling activity for vertebrate organogenesis is the notochord, which organizes structures along the main body axis. Interestingly, zebrafish with mutations of the notochord or adjacent dorsal midline structures have recently been found to have associated abnormalities of normal cardiac looping. By studying this relationship between mutations of axial mesoderm development and errors in cardiac asymmetry, we can begin to understand the regulation of LR specification. We will begin by thoroughly characterizing the mutants phenotypically, to find local relationships that may be responsible for the abnormal cardiac orientation. We will then attempt to rescue normal cardiac situs in the mutants by cell transplantation experiments between wild-type and mutant embryos. Using progenitor cells from the blastula stage of normal embryos, we can repopulate the deficient cell line of the mutant and thereby attempt to rescue normal LR orientation of the heart. These experiments will help to identify structures with important signaling roles in LR organization. We will then examine the effects of experimental ablation of specific groups of cells of the developing axial mesoderm in otherwise normal zebrafish. Disrupting structures needed for LR patterning of the heart will lead to aberrant looping of the heart- tube. Finally, we are interested in understanding the role of the axial patterning gene, sonic hedgehog (ssh), in these mutants. Shh was recently found asymmetrically expressed in the chick and postulated to play a role in LR patterning of the heart. Whole mount in situ hybridization will be used to determine expression patterns of shh in normal and mutant zebrafish. The role of the gene will be tested in the presence of blocking antibody as well as by ectopic expression of its protein product in embryos injected with shh messenger RNA. The consequences of ectopic expression on LR organization of the heart will be examined and its influence on LR development studied.