Functional Studies Of ADAM23 In Zebrafish Embryogenesis

The A Disintegrin And Metalloprotease(ADAM)family is a type of membraneanchored proteins consisting of a signal peptide,a precursor protein,a metalloproteinase domain,a disintegrin domain,a cysteine-rich domain,the epidermal growth factor domain,the transmembrane domain,and the cytoplasmic tail.The family is composed of thirty-four members.In humans and mice,ADAMs are widely expressed in many tissues.Studies have shown that members of this protein family are involved in cell-cell or cell-matrix interactions,including physiological processes such as fertilization,neural cell development,and tumor cell migration.ADAM23 is a member of the ADAM family and has a completely similar structure of the ADAM family.The protein's size is about 75 kDa and is widely expressed in the central nervous system.In 1998,a homologous gene of ADAM23 was cloned from mouse cells and then cloned from the human genome database.The protein was highly conserved between humans and mice.Analysis of sequences of the ADAM23 protein showed that it lacks metalloproteinase activity and its dis-integrin domain is highly conserved,suggesting that its function may be combined with an integrin as a cell surface receptor.Studies have shown that the ADAM23 gene has an important role in human brain development,the migration and elongation of neurons,and the function of exercise and perception systems.ADAM23 knockout mice showed a phenotype of epilepsy.At the same time,the silencing of ADAM23 gene is involved in the occurrence of various cancers.The ADAM23-integrin complex can change the cell adhesion,and the loss of this function affects the migration and differentiation of cancer cells.However,most of these studies were carried out at the cellular level,and much more studies are needed to define the in vivo physiological roles of ADAM23.In particular,the effect of the ADAM23 gene in the development of zebrafish is unknown.Zebrafish is a type of vertebrates,and has organs very similar to humans in structure and physiology and at the molecular level.We can easily observe the development of internal organs and systems due to the transparence of zebrafish embryos.It is suitable for studying developmental processes as one of the best models.Using BLAST analysis with human amino acid sequences,we found that theADAM23 gene has only one homologous gene,adam23 a,in zebrafish.The adam23 a sequences are highly conserved by about 75%,and expressed in 17 tissues or organs in zebrafish embryos.To date,there are few studies on the ADAM23 gene in zebrafish.Functionally,the ADAM23 protein was reported as a receptor binding to LGI1.The interaction between the two proteins regulates the intensity of synaptic transmission,suggesting that ADAM23 is involved in brain development.However,it is still unknown whether the ADAM23 gene has other significant effects on the development of zebrafish embryos.In this thesis project,we aimed to identify the physiological role of ADAM23 in development using zebrafish as an experimental animal model.First,we cloned the full-length wild type human ADAM23 into the pCS2+ vector(pCS2+-ADAM23).pCS2+-ADAM23 and negative control plasmid mCherry-pSP64 were linearized and used for preparation of ADAM23-WT mRNA and mCherry mRNA using an in vitro transcription kit.Second,capped ADAM23-WT mRNA or mCherry mRNA was injected into 1-2 cell embryos by microinjection,and allowed them to develop to different stages.Third,the embryos at different developmental stages were collected and subjected to whole-mount in situ hybridization.The probes used for whole-mount in situ hybridization included mlc2 f,smyhc1,myf5,mef2 ca and myogenin formuscle differentiation and development,sox9 a,osterix,dlx2 a and col1a1 a for bone differentiation and development,eve1,elavl3,shha,krox20 and neurog1 nervous system differentiation and development,and etsrp,fli 1,cmyb and runx1 for cardiovascular system differentiation and development of zebrafish embryos.In each tissue,multiple developmental marker genes were selected to detect their developmental changes.The data of whole-mount in situ hybridization showed that compared with mCherry control,overexpression of ADAM23 significantly increased the expression levels of muscle marker gene myogenin,skeletal marker genes dlx2 a and col1a1 a,and the nervous system marker gene neurog1.These results suggest that ADAM23 plays an important role in the differentiation and development of muscles,bones and the nervous system.Our study is the first to show that ADAM23 regulates the development of zebrafish embryos,which provides important insights into the zebrafish embryogenesis and the physiological role of ADAM23.