| The asymmetrical deformity of the heart affects the quality of life,even lead to death.At present around the heart asymmetric molecular mechanism involved in the process of development is still not clear,therefore,research about the molecular mechanism of candidate genes witch regulate cardiac function and asymmetric development has important theoretical and clinical significance.In this paper,use zebrafish as a model to study the role of Had31 gene in the heart left-right asymmetric development.First we get the spatiotemporal expression profiles of Had31 in zebrafish embryos.Results show that when the embryos developed to the 14 segments,The expression of Had31 on the left side of the heart primordial was significantly stronger than that on the right side of the heart.When the embryos developed to the 16 segments Had31 was significantly stronger on the right side of the heart primordial than the left.When embryonic development to 17 segments Had31 in expression quantity on either side of the heart primordial,suggest Had31 related to heart left-right asymmetric development.In the early stage of our laboratory,Had31 knockout strains were established.The Had31 homozygous embryo from heterozygous parents show enlarged pericardial cavity,heart can’t normal looping,homozygous can lay eggs,but the eggs can’t develope.using myocardial marker Myl7,asymmetric development marker(Spaw,Lefty2,Foxa3,Trypsin)show that the heart looping to be abnormal.Since Had31 Homozygous sterility,we convert use morpholino oligonucleotides,(MOs)to knockdown the expression of Had31.At first we use Had31-gfp transgenic strains to analysis the efficiency of Had31 MO to knockdown the expression of Had31 protein-green fluorescent.The results show that the Had31 MOs is effective.We then compared the phenotype of Had31 knockdown embryo to the knockout homozygous embryo.The experimental results show that they have similar phenotype.It is reported that the abnormality of Heart cyclization may be related to the migration of precursor cells in the early stage of the heart.We use Myl7 as the probe to detect the effect of knockdown Had31 expression on the early cell migration of myocardial cells.The results show that Had31 leads to the heart of the cyclization abnormalities may be associated with early cardiac progenitor cell migration.Had31 is expressed on both sides of the heart,suggesting that Had31 may regulate the asymmetrical development of the heart on both sides of the midline.The Nodal-Pitx2 signal on the left side of the midline is known to regulate the left-right asymmetrical development of heart.The transmembrane protein Nomo is the antagonist of Nodal.We verified the inhibitory effect of Nomo on Nodal/Spaw expression through the experiment of in-situ hybridization.Our lab early results showed that Had31 located in the nucleus and cytoplasm,Had31 and Nomo,were interacting with each other.Based on the above results,we assume that Had31 is located downstream of Nomo in the Nodal/Spaw signal in the left signal of the middle line.To test this hypothesis,we knockdown or over express Nodal/Spaw or Nomo to analysis Had31,experimental results show that Nodal/Spaw activate the expresstion of Had31.Since Nomo is the antagonist of Nodal/Spaw,Knockdown Nomo should induced the expression of Had31 in both sides of cardiac primordia,and over express of Nomo should suppressed the expression of Had31 in both sides of cardiac primordial.Our in situ hybridization results consistent with the expected results.Namely Nomo inhibition Had31 expression on both sides of the midline.These experimental results show that Had31 is located downstream of the Nodal signaling pathway on the left of the zebrafish embryo.The downstream member of Nodal signal Pitx2 is a homeobox transcription factor.So,what is the upstream and downstream relationship between Had31 and Pitx2?Our experiment results show that Had31 activate the expression of Pitx2,suggest Pitx2 may located in the downstream of Had31.If this inference is correct,over expresse Pitx2 should be able to save the mutant phenotype of Had31.We over expressed Pitx2 proved that it can indeed rescue the cardiac cyclomatic abnormality caused by knockdown Had31,indicating that Had31 is located upstream of Pitx2.Through the above experiment known Had31 activate Pitx2,Nodal/Spaw activate Had31,so knockdown Nodal/Spaw should suppress the expression of Pitx2,and over express of Nodal/Spaw should induced the expression of Pitx2.Our embryo in situ hybridization results consistent with the expected results.Since Had31 activates the expression of Pitx2,Nomo inhibits the expression of Had31,so it is expected that knockdown of Nomo should increase the expression of Pitx2,and over express of Nomo should suppress the expression of Pitx2.In situ hybridization of our embryos was consistent with the expected results.To sum up,our results Preliminary showed that Had31 is a new member of the left signal of the midline,which forms Nodal-Had31-pitx2 signal to regulate the left-right asymmetry development of heart.Recent studies have shown that the BMP4-prrx1a signal regulates asymmetrical development of the heart on the right side of the dorsal midline.Therefore,we assume that Had31 is located downstream of BMP4 signal on the right signal of the middle line.To test this hypothesis,we knockdown or over express BMP4 to analysis Had31,in situ hybridization experiment results show that BMP4 suppress the expresstion of Had31.Prrx1a is a member of the homeobox transcription factor and is the downstream gene of the BMP4-Prrx1a signaling axis,and BMP4 activates the expression of Prrx1a.So,what is the upstream and downstream relationship between Had31 and Prrx1a?Our results show that Had31 suppress the expression of Prrx1a,suggest Prrx1a may located in the downstream of Had31.We knockdown Prrx1a proved that it can indeed rescue the cardiac cyclomatic abnormality caused by knockdown Had31,indicating that Had31 is located upstream of Prrx1a.The above research results show that Nomo inhibiting Had31 expression,and Had31 supress Prrx1a expression,so it is expected that knockdown of Nomo should supress the expression of Prrx1a,and over express of Nomo should induce the expression of Prrx1a.Our in situ hybridization results was consistent with the expected results.In summary,Had31 may be a new member of the BMP4-Prrx1a signal,and may regulate the asymmetric development of the heart by the BMP4-Had31-Prrx1a signaling axis on the right side of the dorsal midline.The heart valve is an important component of the heart organ.A lot of congenital heart disease is known to be associated with valve defects.Currently the molecular mechanism of heart valve development is limited.The effects of Had31 and fhl1A gene on the development of zebrafish valve were also studied in this paper.First,we studied the effect of Had31 gene on the development of zebrafish valve.In situ hybridization results show that:when the Had31 knockout homozygous embryo to 52 hours post-fertiliaztion(52hpf)the expresstion of Versican and Has2 are iup-regulated,the expresstion of Notch1b,BMP4 and Tbx2b are remains unchanged.It is suggested that Had31 can regulate the development of zebrafish heart valve.The other gene of this paper studied was fhl1A.The early results of our research group showed that fhl1A was expressed in the heart chamber,Atrioventricular junction and skeletal muscle.In this paper,we use the fhl1A homozygous knockout strain witch established by our lab to study the effect of fhl1A on the development of heart valve.The results show that the express of the valve marker genes Versican and BMP4 are down-regulated,the expresstion of Has2 is up-regulated.It is suggested that fhl1A can regulate the development of zebrafish heart valve. |
PDF Full Text Request