1. Requirement of the nkx2.5 Gene In Heart Development Martin Liberman
2. Introduction Background Genetic disorders are caused by abnormalities in an organisms genome. Over 20% of infant mortality is caused by genetic and birth defects (Hoffman et al.). Affecting over 40,000 newborns per year in the United States, congenital heart defects are the most common genetic defects (CHD; 2014). Congenital heart defects can present in many forms including, atrial and ventricular septal defect (ASD and VSD), aortic and pulmonary valve stenosis (AVS and PVS), single ventricle defects, and many others.
3. Introduction (cont.) Literature Review When the formation of the heart in a developing fetus is disrupted it results in congenital heart disease. Mutations in NKX2.5 result in CHD in humans. (Benson et al., 1999). In order to fully understand how mutations in nkx2.5 lead to cardiac malformations, it was essential to discover the roles this transcription factor plays during cardiogenesis (Targoff et al., 2008). nkx2.5 is essential in limiting atrial cell number, promoting ventricular cell number, and preserving chamber-specific identity in zebrafish (Targoff et al., 2013). Loss of nkx2.5 leads to substantial impairment in proliferation of cardiomyocyte precursors in zebrafish (Prall et al., 2007). To analyze the early and late functions of nkx2.5, I evaluated the influence of timing of its expression on cardiac chamber formation, through the use of an nkx2.5 transgene.
4. Methodology Transgene, Tg(hsp70l:nkx2.5-EGFP) expressed nkx2.5 tagged to Green Fluorescent Protein (GFP) All embryos were taken out of their protective shell before each experiment. The in situ hybridization method allowed for the visualization of transcribed genes through the use of probes specific to their particular mRNA molecules The immunofluorescent staining method enabled the study of translated genes by using antibodies for specific protein molecules After the experimentation was complete, the fish samples were imaged Genotyping was performed immediately after each method and embryos were classified based on their phenotype and genotype.
5. In situ and Immunofluorescent images of transgenic and non-transgenic zebrafish embryos. Results
6. Results (cont.) Non-transgenic phenotypes post in situ and immunofluorescence. The images above illustrate the imaged embryos after in situ and immunofluorescence procedures. The nkx2.5 deficient embryos (D-F) show a bulbous atrium and a shrunken ventricle.
7. Results (cont.) Transgenic phenotypes post in situ and immunofluorescence. The phenotypes of nkx2.5 deficient embryos with activated transgene (M-R) show the embryos rescued by the transgene. These embryos hearts are similar to that of wild type embryos (G-L).
8. Results (cont.) Transgenic and non-transgenic phenotypes. This image shows the comparison of the phenotypes of transgenic (B, D, G, H), non-transgenic (A, C, E, F), wild type (A, B, E, G) and nkx2.5 deficient (C, D, F, H) embryos. The graphs show the percent of embryos with wild type and nkx2.5 phenotypes per heat shock group. During 7 somites (11hpf) to 26hpf, the percent of visible mutant phenotype was diminished.
9. Discussion The discoveries demonstrate that nkx2.5 expression is only essential for about 15 hours during embryogenesis for normal heart development. The transgene is only effective for a period of eight hours and would have to be activated at a specific moment in development for the heart to express nkx2.5, and be able to rescue an nkx2.5 deficient embryo. The results show that the time period in which the transgene is most effective for rescuing nkx2.5 deficient embryos is within 7 somites (11 hpf) and 26hpf. The mutant fish did not express the gene during that time were not able to survive to adulthood.
10. Discussion (cont.) Limitations: The results demonstrated that transgenic nkx2.5 mutant embryos were able to survive to adulthood, however, the morphology of the rescued nkx2.5 mutants has yet to be fully analyzed. It is unknown if the transgenic mutant hearts are impaired during the adult stages of development. The research was limited to analysis of nkx2.5 gene function only during embryonic heart development.
11. Conclusion The results illustrate the specific time interval during which nkx2.5 is essential and most effective. These findings can help to further advancements in treating congenital heart defects brought about through mutations in NKX2.5 for humans. The discovery of the effectiveness of the transgene can contribute to future research and provide a more precise way of analyzing the effects of nkx2.5 at more specific time periods in development.
12. Acknowledgements I would like to thank my research advisor and mentor Dr. Vladimir Shapovalov.
13. References 1) Benson, D., et al., 1999. Mutations In The Cardiac Transcription Factor NKX2.5 Affect Diverse Cardiac Developmental Pathways. Journal of Clinical Investigation. 1567-573. 2) Boston Childrens Hospital. 2014. Congenital Heart Defects in Children. http://www.childrenshospital.org/health- topics/conditions/congenital-heart-defects 3) Buckingham, M., et al., 2005. Building the mammalian heart from two sources of myocardial cells. Nat. Rev. Genet. 6, 826835. 4) Centers for Disease Control and Prevention. 2014. Congenital Heart Defects. http://www.cdc.gov/ncbddd/heartdefects/index.html 5) Hoffman JL, Kaplan S. The incidence of congenital heart disease. J Am Coll Cardiol. 2002;39(12):1890-1900. 6) Inoue, D., & Wittbrodt, J. 2011, May 13. One for All-A Highly Efficient and Versatile Method for Fluorescent Immunostaining in Fish Embryos. PLoS ONE, 6(5), 1-7. doi:10.1371/journal.pone.0019713 7) Prall, O.W., et al., 2007. An Nkx2-5/Bmp2/Smad1 negative feedback loop controls heart progenitor specification and proliferation. Cell 128, 947959. 8) Targoff, K. L., Colombo, S., George, V., Schell, T., Kim, S. H., Solnica-Krezel, L. and Yelon, D. (2013). Nkx genes are essential for maintenance of ventricular identity. Development 140, 42034213. 9) Targoff, K.L., et al. (2008). Nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number, Dev. Biol. doi:10.1016/j.ydbio.2008.07.037 10) Thisse, C., & Thisse, B. (2007, December 20). High-resolution in situ hybridization to whole-mount zebrafish embryos. Nature Protocols, 3(1), 59-69. doi:10.1038/nprot.2007.514 11) Tu, Shu, and Neil C. Chi. (2012) "Zebrafish Models in Cardiac Development and Congenital Heart Birth Defects." Differentiation: 4-16.