Retinoic Acid Lack The Mechanism Of Development Of The Zebrafish Heart

PartⅠ:The establishment of retinoid acid deficiency zebrafish embryo modelTo set up a zebrafish model with retinoid acid(RA) deficiency for further investigation.Retinal dehydrogenase type 2(raldh2) is the key synthetase for RA, both chemogenetic approach and gene knock-down method were used to inhibit raldh2,and block the synthesis of RA,and then make zebrafish embryos with RA deficiency.First,we treated zebrafish embryos with exogenous 4-diethylaminobenzaldehyde (DEAB),an inhibitor of raldh2,at various concentrations from different developmental stage.The effects of DEAB on the embryonic development were dose-dependent and time-dependent.The embryos treated with 5×10^-6mol/L DEAB from gastrula stage exhibit swollen pericardial cavity,abnormal cardiac development, decreased heart rate,curved tail,small head and pectoral fin defect.These abnormal phenotype could be rescued by exogenous retinoid acid treatment,which demonstrated DEAB treatment could effectively block RA synthesis.Next,we designed morpholino antisense oligonucleotides(MO) targeting zebrafish raldh2 gene to knock-down its expression.The mortality and abnormal embryo rate rose with the increase of raldh2-MO dosage.The embryos with raldh2-MO exhibit similar phenotype to those with DEAB treatment,while the con-MO group had no obvious developmental defects.The construction of raldh2-EGFP plasmid verified the effectiveness and specificity of raldh2-MO.The RA deficiency zebrafish embryo model could be effectively established by both exogenous DEAB treatment and raldh2-MO microinjection. PartⅡ:The effect of retinoic acid deficiency on the embryonic cardiac development of zebrafishAfter the establishment of RA deficiency zebrafish model,we observed the whole cardiac developmental process and analyzed the effect of insufficient RA on the cardiac development.First,we analyzed and compared the cardiac phenotype among the wild type embryos,DEAB treated group and raldh2-MO group.Both DEAB treated embryos and raldh2-MO embryos exhibit similar abnormal cardiac phenotype,including swollen pericardial cavity,tubular heart,incomplete D-loop,abnormal atria and ventricle development,blood regurgitation,slow blood flow and weak heart beat, which implied RA signaling played a critical role in several key stages of cardiac development.Second,we observed the influence of RA deficient to embryonic cardiac function by analysis of heart rate and ventricular systolic fraction among various groups.In wild type embryos,the heart beat powerfully with regular rhythm,and the heart rates increased gradually with cardiac development.While in DEAB treated group and raldh2-MO group,the rhythm was regular,but heart beat became weaker and slower. The difference of heart rate and ventricular systolic fraction between wild type and RA deficiency embryos was of statistical significance(P＜0.05),which revealed cardiac dysfunction in embryos lack of RA signaling.Then,we analyzed the regulation of specific cardiac gene expression by whole-mount in situ hybridization.The results showed the vmhc(ventricular myosin heavy chain) expression cell domain was expanded while amhc(atrial myosin heavy chain) expression cell domain was reduced in RA deficient embryos.The nppa (natriuretic peptide precursor A) expression remained in the ventricle,but reduced obviously in the atria.The alteration of vmhc,amhc and nppa expression demonstrated that the occurrence of RA deficiency in the early stage of cardiac development may act as a contributor to ventricle development,and an inhibitor to atria development.RA played an important role in the cardiac diversification along the anterior-posterior axis.Other characters of RA deficiency embryos were analyzed by phenotype,Alcian Blue staining and whole-mount in situ hybridization of HuC probe.In RA deficiency embryos,their pectoral fins were either seriously reduced in size or completely absent, and the cartilages in pharyngeal arches 3-7 were obviously reduced both in size and in number.HuC expression was down-regulated in the brain,especially in the hindbrain when RA signaling was insufficient.Conclusively,RA deficiency embryos were characterized by defects in the pectoral fins,pharyngeal arches and hindbrain.PartⅢ: Cardiac related tbxl,tbx5 and tbx20 gene expression are down-regulated in raldh2 knock-down embryosRA signaling may interact with cardiac related transcriptional factors to regulate cardiogenesis and development.Tbx1,tbx5 and tbx20 are members of T-box transcriptional factors,which play important roles during embryogenesis in the vertebrate.We described the expression pattern of tbx1,tbx5 and tbx20,and the regulation of their expression when raldh2 gene was knocked down to elucidate the underlying mechanism of RA signaling in the cardiac development.The results of whole-mount in situ hybridization of tbx1,tbx5 and tbx20 probes revealed that these genes had distinct expression patterns,but all of them expressed in the embryonic heart.In wild type embryos,tbx5 expressed in the atrium,the posterior part of heart,and pectoral fin buds,tbx1 took part in the development of cardiac outflow tract,pharyngeal arches and otic vesicle,while tbx20 expression was found in the heart,including the atrium,ventricle and outflow tract,branchio motor neuron, tegmentum and retina.Tbx5,tbx1 and tbx20 may contribute to different roles in the cardiac development according to their various expression patterns.To some extent, the expression of tbx5,tbx1 and tbx20 was reduced in raldh2 knock-down embryos.The expression of tbx5,tbx1,tbx20 can be regulated by RA signaling during cardiac development,and the underlying mechanism still needs further investigation.