Study On The Mechanism Of Tbx5a Interaction With Kctd10 And Nkx2.5 During Cardiac Development In Zebrafish

Congenital heart disease（CHD）is one of the most common birth defects.In 2015,it was found that the incidence rate in Europe and United States reached 2‰,and about 150,000 children with congenital heart disease were born each year in China.Cardiac is one of the earliest organs that begin to function during vertebrate development.25%to 30%of congenital heart disease appears a heart valve abnormality,among which Holt-Oram syndrome（heart-hand syndrome,HOS）is a valve defect congenital heart disease.One of the main causes is the absence of Tbx5（T-box 5）,leading to severe heart valve dysplasia,resulting in cardiac dysfunction.The Tbx5 gene belongs to the T-box family,whose members all contain a unique DNA-binding domain.Transcription factors encoded by individual members of T-box family play an important role in embryonic development.The T-box gene family plays an important role in vertebrate heart development during early embryonic development.The T-box gene family primarily acts on early cardiac lineage determination s in vertebrate,such as ventricular specialization,atrioventricular division,and embryonic conduction system differentiation.In recent years,Tbx5is a gene that has been further studied in the field of cardiac development.Deficiency of Tbx5 is one of the most important causes of HOS congenital heart disease,but its pathogenesis,as well as studies on the binding factors of Tbx5 during cardiac development,are still not well understood.In this thesis,the study of the interaction between Tbx5 with Kctd10 and Nkx2.5 in cardiac development is of great significance for demonstrating the intervention of heart-hand syndrome and understanding the pathogenesis of congenital heart disease.The research on tbx5 in this thesis is mainly divided into two aspects.On the one hand,in 2014,Peking University found that the over activation of Tbx5a protein in Kctd10 mutants affected the expression of has2（hyaluronan synthase 2）in the glial heart pectin layer,which led to cardiac malformation.This article has done further research on this basis.Firstly,the tbx5a gene was successfully knocked out in zebrafish using CRISPR/Cas9 gene-editing system,and the F₁mutant was screened.Double mutants were obtained by hybridizing both F₁generation of tbx5a mutants and kctd10 mutants.Through phenotypic analysis,it was found that the double mutant relieved not only the contraction abnormality of the kctd10 mutant heart but also the elongation of the tbx5a mutant heart.The kctd10 and tbx5a genes were segmentally cloned according to different functional domains,and the binding positions of Kctd10 and Tbx5a were verified by Co-IP assay to be△BTB domain and TAD2 domain,respectively.On the other hand,both Tbx5 and Nkx2.5 are highly conserved transcription factors that play a crucial role in early embryonic development.Both genes have highly homologous structures and functions in humans and mice.The expression of the Nkx2.5 gene is also one of the earliest signs of early embryonic development.Gene nppa is a downstream cardiac target gene associated with cardiac development.We designed and synthesized the nppa promoter and inserted it into the p GL3 vector to construct the nppa-luc plasmid.Meanwhile,we also found that Nkx2.5 can successfully combine with the two structural domains of Tbx5a to form a polymer that activates nppa.In summary,on the one hand,the Tbx5a gene was successfully knocked out with CRISPR/Cas9 system,and the double mutants of kctd10 and tbx5a were obtained through hybridization.By observing the phenotype,it was found that the phenotype of two single mutants,tbx5a and kctd10,were alleviated in the double mutant.Besides,two functional domains with different genes were cloned in sections,and the binding positions of the two genes were found and verified.This study preliminarily revealed the interaction mechanism and binding position of Kctd10 and Tbx5a,which provided a theoretical basis for those who continued to study the early heart development signal transduction network.On the other hand,the promoter of nppa was designed and synthesized,and it was concluded that Tbx5a can effectively activate the downstream target gene nppa promoter with Nkx2.5 in the presence of both domains.This study provides a preliminary analysis of the interaction mechanism between Tbx5a and Nkx2.5,which sets a theoretical basis for the follow-up study of Tbx5a and Nkx2.5.Moreover,the construction of nppa-luc expression vector also provides a certain experimental material basis for the subsequent research of cardiac signal transduction network.