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HEART PROGENITOR CELL MOVEMENT IN DEVELOPING FROG EMBRYOS Timothy Simon1, Tim Jackson1, Lance Davidson1,2 Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15260 2 Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA 15260 1 INTRODUCTION Congenital heart defects (CHD) include some of the most prevalent birth defects in the US, affecting nearly 40,000 infants each year [1]. CHD is the incomplete or abnormal formation of the heart or intrathoracic vessels leading to a significant effect on the subject’s functionality [2]. While the causes of CHD are poorly understood in humans, much effort has been placed in understanding the molecular causes of CHD in model organisms. For instance, missense mutations in phosphatase proteins, specifically SHP-2, can cause this structural irregularity during the formation of the heart in the developing frog embryo [3]. In this study, the specific mutation SHP-2N308D will be explored extensively Figure 1: Schematic of normal SHP-2 protein and mutation constructs, including SHP-2N308D [3] Y27632 on a frog embryo through the analysis of time lapse movies. We intend to calculate the area of the heart region, comparing across several varying concentrations of Y27632. In order to understand the mechanics of heart progenitor cell migration, we will track the heart progenitor cells and determine the strains within the tissues they travel through. We must also calculate principal strain on the epithelial layer determining whether it contracts or expands during the period of progenitor cell migration. MATERIALS AND METHODS To visualize cell movements within the embryo we must stabilize the tissues through which heart progenitors migrate. We mount the embryos rigidly on their dorsal surface so that we can follow cell movements on the embryo's ventral surface where heart progenitors migrate and the two clusters of cells join to form the heart. First, we remove the vitelline membrane of post-gastrulation and pre-neurulation Xenopus laevis embryos to allow for surgical manipulation. Microsurgery is then performed on the post-gastrula embryos (stage 12.5). Small incisions are made along the dorsal side of the embryo. These incisions will expose the deep cells Behind the incomplete migration of the heart progenitor cells is the mutated SHP-2N308D pathway. The mutant phosphatase protein SHP-2N308D induces hyper-activation of the Rho-associated coiled protein kinase (ROCK). Changes in ROCK activity will alter the stiffness of the tissue through the reorganization of the Xenopus Laevis cytoskeletal tissue. As the cytoskeletal structure is altered and the tissue stiffness increases, the heart progenitor cells are unable to migrate from the lateral plate mesoderm to the ventral midline and fuse to form a complete, fully functional heart [3]. The pharmacological compound, Y27632, inhibits ROCK activity. The complete effect that this compound has on the development on the SHP-2 mutated embryo and the normal, non-mutated embryo has not been fully characterized [3]. However, no connection has been made between the inhibition of ROCK and the rescue of the mutated SHP2N308D pathway in the Xenopus Laevis embryo. It was hypothesized that the introduction of Y27632 to a normally developing embryo will cause abnormal formation of the heart thus proving that compound Y27632 is capable of inhibiting ROCK hyperactivity and rescuing SHP-2 mutated embryos. To substantiate this hypothesis, we must characterize the effect of ROCK-inhibiting compound beneath the epithelial layer. Figure 2: Diagram of the procedure described above. The red X indicates the position of the small incisions made after de-vitellinizing the embryo. [4] Embryos are plated, incisions down, onto a fibronectincoated dish and treated with varying concentrations of the compound Y27632. 24 hour time-lapse movies centered on the heart-forming region on the ventral side of the embryo were collected. These images were processed and analyzed in ImageJ. The area of the heart-beating region was normalized then calculated. A principal strain map of the epithelial layer, detailing the specific areas of contraction and expansion, was also created using ImageJ software. The embryo was then fixed and stained and imaged using a laser scanning confocal microscope (Leica SP5). 1 Area of Heartbeat (mm2) RESULTS After treating the embryos with a solution of 50 μM Y27632 and collecting a time lapse video of their development they were fixed and stained for fibrillin. Confocal stacks were maximally projected into a single image. A noticeable difference was evident upon comparing the Y27632 treated embryo and the embryo left in control media. The Y27632-treated embryo’s heart formed a bifid heart or incompletely fused heart. The control embryo’s heart progenitor cells successfully merged. Because the Y27632 inhibits ROCK, the heart progenitor cells were incapable of migrating all the way to the ventral midline and fusing together fully. We retrospectively identified the regions of the embryo contributing to the heart in long term time-lapse sequences that culminate in the beating heart. Time-lapse images were analyzed using ImageJ software to calculate the area of the heart-beating region in the embryos. Once the general area is determined through a t-projection image, the precise area is thresholded to accurately calculate the pixel area where the heart cells contract. The pixel area can they be converted to square mm. This procedure was done for embryos treated with 50 μM Y27632 and the control with 0 μM Y27632. emerges of enabling successful heart progenitor cell migration in a previously mutated Xenopus Laevis frog embryo. In the future, the connection between congenital heart disease and the hyper-activity of ROCK must be solidified. In order to conclusively determine that lessening ROCK hyper-activity with Y27632 will rescue the mutated SHP-2 pathway and allow for complete heart formation, the molecular and cellular mechanics behind heart migration must be explored further. It must also be effectively confirmed that the hyper-activity of ROCK is in fact downstream of the SHP-2N308D mutant and will allow for the normal formation of the heart. Preliminary data suggests that Y27632-induced ROCK inhibition prevents correct fusion of the bilateral heart progenitor cell populations. However, in order to make a statistically significant conclusion regarding the hypothesized outcome, more experiments and data is needed. The creation of a cardiogenic fate map is another tool that can be utilized to understand better the mechanics of heart progenitor cell movement. Using DiI, a fluorescent dye that localizes in the cell membrane, heart-forming cells can be tracked from the early stages of the Xenopus Laevis embryo up until the beginning of heart contraction. The far-reaching goal of this project is to relate the development of the Xenopus Laevis heart to the human heart and thus be able to better understand the causes of Congenital Heart Disease. This long-term goal is far off in the future, however, understanding of the mechanics of heart progenitor cell migration is needed to reach it. 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 50 μM 50 μM 0 μM ACKNOWLEDGMENTS I would like to acknowledge Tim Jackson and Dr. Lance Davidson for their help throughout. I would also like to thank Jesse Lowe and Dr. Borovetz for helping me through the entire process this semester. 0 μM Concentration of Y27632 Figure 2: Graph concentrations of heartbeat areas and solution REFERENCES 1. Hoffman, J. I. E., Kaplan, S. The Incidence of Congenital Heart Disease. JACC. 2002;39(12):1890-1900. 2. Mitchel, S. C. et al. Congenital Heart Disease in Births. 1971;43(3):323-32. 3. Langdon, Y. et al. SHP-2 acts via ROCK to regulate the cardiac actin cytoskeleton. 2012; 139(5):94857. 4. Drawings after Nieuwkoop and Faber.(1967) Normal Tables of Xenopus laevis (Daudin). Amsterdam: Elsevier North-Holland Biomedical. A previously made algorithm that utilized Insight Toolkit (ITK) was used to calculate the principal strain on the epithelial layer and create a tracking grid on the epithelial cells as the heart progenitor cells migrated. The algorithm displays contraction and expansion of the epithelial layer of the developing embryo. DISCUSSION Confocal imagery of the Y27632-treated embryo shows the incomplete fusion of the heart progenitor cells from the opposite lateral plate mesoderm. The treated embryo forms a bifid heart . The contractile area of the embryo treated with the compound is larger than that of the embryo treated with the control carrier, DMSO. The larger area in the Y27632 treated cells attests to the partial migration and merging of the heart progenitor cells as the Xenopus Laevis embryo develops. The abnormal heart formation in the treated embryo indicates the inhibition of ROCK. If ROCK hyper-activity could be inhibited or diminished in a SHP-2 mutant frog embryo, the possibility 2