The Functional Study Of Embryonic Cardiac Development Genes In Zebrafish And The Transcriptomic Analysis Of Its Developmental Key Points

Heart is one of the first formed functional organs in vertebrates.During its development,the heart tube undergoes looping to form atrioventricular canal(AVC)and finally develops into multi-chambered functional organ.It is regulated by multiple signaling pathways.Cardiac valves are highly organized with delicate structures that ensure unidirectional blood flow through the cardiac chambers and large vessels.Tbx20,Tbx2 and Tbx5 are the key genes of Atrial Septal formation in TBX family.From previous reported,we know Tbx2 inhibited the differentiation of the chambers.It codes transcription inhibitor to repress the differentiation of chambers and atrioventricular canal specific gene expression.Deficiency Tbx2 could activate the chamber myocardial gene programmer in AVC,fail to formal chambers.Tbx5 is associated with Holt-Oram syndrome(HOS).Mutation with Tbx20 in mice has no impact on early heart tube development,but fail to looping.The heart looping can form the functional heart chambers.Different transcription factors through series of signal pathway to regulate the division,migration,differentiation,apoptosis in cells,then to finish the construction of cardiac morphology.AVC is the earliest and most important process in looping.Parts of the Congenital heart disease(CHD)are associated with abnormal heart looping.It is one of the most common birth defects which threats the human's healthy.Zebrafish is the ideal animal model to study the early heart development and CHD pathogenesis.The transparent embryos is easy to observation.This study used CRISPR/Cas9 system,by microinjection successfully knock out tbx2b?tbx5a?tbx20 genes to provide material for the follow-up studies.The efficiency of T7E1 in F0 mutation is about 57.5 %?38%?42.1%.Sequencing analysis show that there are two types in tbx2 b F1 mutant carriers including-11 bp and-20 bp and in tbx5 a F1 mutant carriers including-10 bp and +10bp.In tbx20 F1 mutant carriers including-8 bp.In tbx20 F2 mutants,some cardiac phenotypes were observed: embryo development to 48 hpf,The mutant heart turn to looping abnormal and the structure of the heart is stretched and deformed with the enlargement of the pericardial cavity.Embryo development to 3 dpf,all phenotypes are obvious and the heart stretched into thin lines.Realtime Fluores-cence Quantitative PCR results showed that the expression of lef1,nppa and has2 in the tbx20 mutantion are down regulation.We use the zebrafish as animal model to separate heart gene myl7 marker with GFP+/+ in different development zebrafish heart with 24 hpf?30 hpf?36 hpf?48 hpf.Using transcriptomics we find there are 488 different transcription factors genes expression including T-box gene family ?GATA gene family ?Nkx gene family and several related genes in bHLH gene family.The KEGG shows that differential gene mainly enriched on signal pathways such as Notch?Wnt?TGF-beta and so on.This study successfully produced tbx2b?tbx5a ?tbx20 mutant fish lines in zebrafish and find that tbx20 mutant can affect the development of AVC in heart which can provide deep research in early heart development about the function of the tbx2b?tbx5a?tbx20.This paper using systematic analysis in 4 key cardiac developmental phases with differentially expressed genes which provide the foundation for the study of the regulation in early cardiac development.