Lecture 20

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Human Biology
Maria Papaconstantinou

30 MAR 2011 HMB321 L20: Zebrafish & Cardiovascular Disease (CVD) Why Study Heart Development? -We are born with only a set number of heart muscles, which, over time, will senesce and we eventually encounter issues (e.g.CVD) Need a greater understanding of heart development -We need to understand how heart cells are in the embryo => can we learn something like this to add something endogenous into the heart to aid these issues -Adult heart muscle cells have different components than embryonic -Understanding gene programming can tell us how to turn off certain functions that are detrimental,though occur in adult heart cells Heart development: Complex Morphological Process -Once cells are specified,must organize into a tube -While the embryo is growing and pumping blood through, looping will occur (hence the fact that our blood pumps through left side first) Ventricular Septal Defect -In some cases,incomplete septation occurs leaving an obvious hole in the septum -Unlike in wt heart,deoxygenated and oxygenated blood can be mixed -Can decrease survival due to reduction of oxygenated blood perfusing through body Model Organisms to Study Heart Development -Fruit flies -Have a single pulsating tube that moves lymphatic fluids throughout the body (analogous to the heart and the blood that flows through) -Mammalian heart (mice) -Analogous to our heart,i.e.Atria,ventricles,etc What can the fruit fly “heart” (simple tube) teach us? -Tinman gene (unlike wt heart => heartless flies;no D/V structures) -Tried to find vertebrate homologs ofTinman using RNA in situ hybridization -Nkx2.5 is the vertebrate homolog => used for migration of specified cells to the midline to form a heart tube,etc -In mouse,fly,vertebrates Nkx2-5 KO mice -Tried KO mice of the Tinman homolog => found that there was severe defects in mice cardiovascular development (prenatal death) --/- form no left/right side ventricle -Tinman homologs is also essential in mouse development and is important for higher organisms,e.g.Mice Elaboration of “beating tube” -Gene that was first discovered int eh fly => important in vertebrates -Mutation of this gene in children => congenital heart defects -Important thing is that all these genetic pathways from Tinman => development of heart, is conserved across different taxa and species Mutations in many genes lead to Congenital Heart Defects (CHDs) -How do these genes work?What can we do with this? Need model systems Usefulness of Zebrafish to study CV development -Embryos are transparent,allowing visualization of GFP for heart development -Unlike mouse embryo,can visualize development from embryonic stage -Can visualize the early stages of division (e.g.Blastula => morula => etc) -At the 14h time point => all these things have already formed (2 chambers vs. Human 4,but nonetheless similar) Forward Genetic Screens in the Zebrafish -Can raise large numbers of embryos in petri dishes (using eggs) -Can undergo large sampling of the genome to find genes related to heart development pathways -Forward genetic screening => randomly mutagenize the genome via ENU (random point mutations,including in the germline of the fish) -Give them enough to be sick => breed them with female fish (wt) -Create 2 generations to create 2 families of fish -Interbreed siblings of F2 => get F3 to get recessive mutation (often lethal) -Allows for unbiased gene discovery of genes; can find effects in organ systems that are related to diseases that are significant to us -However,difficult to identify the mutated gene Valve formation mutants can be distinguished by blood toggling -Blood toggling => valve defects => blood pushed back into the atrium;improper blood flow, as opposed to pushing blood out of the atrium Zebrafish models of heartstrings/tbx mutant (human CVD gene) -tbx5 is humanTF => mutations cause Holt Oram Syndrome (heart,limb abnormalities) -In the fish, they similarly have heart defects, i.e. Large adema (heart is also not formed; no blood circulation;no looping;stringy looking heart tube) -Fish’s pectoral fins (analogous to our arms) are not elaborated/stump-like formations, akin to what is found in HOS patients Can the 2-chambered fish heart be used to study the CHDs and heart failure? -Same genes deleted in both humans and fish => give you same phenotypes -Heartstrings => mutation in tbx5 (HOS;limb and heart defects) -Pickwik => titin (sudden heart failure;associated with improper contractile apparatus in heart) -Silent heart => troponin t (heart defects) Micr
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