How cells are arranged in three dimensions to produce functional organs such as the heart is a fundamental question in biology that has not been fully addressed. Once cells are instructed to become heart tissue, they must coordinate their movements to organize into a multi-layered functional structure. One critical aspect of this process is how cells arrange differently on the left versus the right side of the heart, commonly referred to as left-right patterning. Previous work has determined that the FoxH1 transcription factor plays an important role in integrating multiple TGFbeta signals in heart cells so that they respond appropriately to left-right patterning information. Utilizing the powerful genetics available in the zebrafish system, the roles of FoxH1 in coordinating TGFbeta signaling and directing left-right cell migrations in the developing heart will be explored. The outcome of this work will be a better understanding of how the heart forms, especially with regards to left-right patterning. This information will have significant impact on stem cell studies aimed at producing functional organs for transplant. This project is well suited to be conducted by graduate students and undergraduates and will be the framework for recruiting women and underrepresented minorities into research through thesis work at Princeton and through the Summer Research Experience program conducted by the Department of Molecular Biology.
The goal of our research is to understand how the heart forms during embryonic development. We use zebrafish embryos as our model system because the heart has many similarities in structure and physiology to the human heart. One critical aspect of heart formation involves asymmetry both in the placement of the heart to the left and in the formation of left and right chambers within the heart. A failure to correctly establish asymmetry in the heart can lead to congenital heart defects. We are studying how asymmetries are generated during the formation of the zebrafish heart to increase our understanding of heart development in vertebrates as a whole. During our preliminary studies for this project, we demonstrated that loss of the gene foxH1 in the heart resulted in defects in asymmetries during heart development. Instead of the heart being positioned to the left, it remained symmetrically placed in the middle of the embryo. We also found that both the myocardial layer (muscle) of the heart and the endocardium (internal lining) were affected by loss of FoxH1. We are conducting experiments to see if FoxH1 needs to be present in both tissues, or if presence in one tissue can influence the other to generate asymmetries. FoxH1 is a transcription factor, which means that it functions to control the expression of other genes. To determine which genes FoxH1 controls, we have undertaken genomic analysis, identifying all genes that are expressed in the embryo, to see which genes are no longer expressed when FoxH1 is absent. We will accomplish this by comparing the genes expressed in hearts with FoxH1 to hearts that do not have FoxH1. We are also exploring the roles of two proteins that signals to cells to change their behavior. One of these proteins is called Southpaw and is critical for generating asymmetry in the heart. The other is called TGFbeta3 and our preliminary work suggests this protein cooperates with Southpaw in generating asymmetries. Over the course of the grant period, we have made significant progress in our genomic analysis to find genes that are controlled by FoxH1. We have perfected our technique of isolating heart cells from embryos and identifying all the genes that are expressed when FoxH1 is active and hyperactive. We are still working on identifying genes expressed when FoxH1 is absent. New technological advances in CRISPR/Cas9 genome editing have given us a better approach to this experiment, and we are generating the necessary tools to complete the proposed work. We were also fortunate that mutants in the genes encoding southpaw and tgfbeta3 became available in the last year, allowing us to design and execute our experiments in a more rigorous fashion. While we have worked to develop better tools for our proposed experiments, we have continued to explore how Southpaw controls asymmetric heart development. We made an exciting discovery that Southpaw appears to drive the formation of a small structure in the cell called a podosome. These structures are thought to act in cell movement, but to our knowledge there are no good systems to study their formation and function in the living vertebrate embryo. Thus we have the first potential system to further our understanding of these structures. Podosomes, also called invadosomes, were first discovered as being formed in high number in cells isolated from metastatic cancers. Thus our work will provide a system for studying how these form, and thus, how to prevent them to possibly reduce metastasis in cancer. I would like to point out, that the results we obtained that appear to be the most exciting and have direct relevance for human health, we obtained while we were struggling to accomplish what we had originally proposed. This is one of the amazing moments that often happens in basic research; you start out with the question you want to answer, and yet find answers to questions you didn’t realize you were asking. In many cases, these fortuitous discoveries are the ones that drive translational research but are not possible without funding from agencies like the NSF which focus on outstanding basic science. The work we have accomplished over the three year funding period could not have happened without the efforts of multiple graduate students and undergraduates. Through this grant and this project, I have mentored three graduate students, and four undergraduates. Of the seven, six are women, four are African-American, and one is Latino, increasing the diversity in our research science community. We have also disseminated our work through attendance at meeting from the regional to international level, through invited seminars at Universities, and to school aged children through outreach activities such as the Princeton Science EXPO.
