Pilot Study Of Mmp21 In Left-right Asymmetric Development Of Zebrafish Embryos

Objective: To investigate the effect of matrix metallopeptidase 21(mmp21)on left-right asymmetric development of zebrafish embryos.Methods: The expression of mmp21 was down-regulated by injecting morpholino antisense oligonucleotides(MO)in zebrafish embryos at 1 ~ 2 cell stage.(1)The effectiveness of mmp21-MO was detected by reverse transcription PCR(RT-PCR)at 10 somite stage.(2)In order to evaluate the left-right asymmetric development of zebrafish embryos,the markers of cardiac and extracardiac organs(intestinal,brain)were analyzed by whole embryo in situ hybridization(WISH).(3)Cardiac morphology,heart rate and ventricular shortening fraction(VSF)of living embryos were observed under stereoscopic microscope.(4)Kupffer's vesicle(KV)was observed under stereoscopic microscope and the size of KV was measured by ImageJ software.(5)The length and number of cilia were quantified by whole embryo in situ immunofluorescence with acetylated-tubulin antibody.(6)The markers of midline barrier were analyzed by WISH.Results: mmp21 MO microinjection effectively knock-down the expression level of mmp21.The expressions of cardiac and extracardiac organs markers detected by WISH became left-right randomized in a dose-dependent manner.The position of atria and ventricles showed left-right randomized.Heart rate and ventricular shortening fraction were significantly decreased(P <0.05).The size of KV as well as cilia number and length were reduced(P <0.05).The expression of midline molecular barrier lefty1 was partially or totally missing.Conclusions: Knockdown of mmp21 results in abnormal left-right asymmetric development of cardiac and other cardiac organs(gut,brain)and caused cardiac dysfunction.The changes of left-right asymmetric development may result from the left-right key structure KV and cilia defects,as well as the missing of midline molecular barrier.In this study,the effect of mmp21 on the asymmetric development of embryos was studied in zebrafish embryos,which is the basis for further research of mmp21 and also is an important enlightenment of the role of mmp21 in human embryonic development.