A Study Of The Expression Profile Of ADAMTS18 In Early Embryonic Development Of Mice And Its Role In Angiogenesis

ADAMTSs(A disintegrin-like and metalloproteinase with thrombospondin motifs)are a group of 19 secreted metalloproteinases with major roles in assembly and degradation of the extracellular matrix(ECM).They play crucial roles in development,tissue morphogenesis,vascular biology and tumor occurrence.ADAMTS18 is an "orphan" metalloproteinase whose functions and substrates remain unclear.Our group begins to study ADAMTS18 functions since 2010,and develops Adamts18 knockout(KO)mouse model in 2017.Our previous studies have shown that ADAMTS18 deficiency is associated with visceral fat development,carotid artery development,and dendrite morphogenesis.These results suggest that ADAMTS18 is crucial for early embryonic development.However,expression profile of ADAMTS18 in early embryonic development and its role in angiogenesis remains to be characterized.In this study,we determined the expression profiles of Adamts18 in mouse embryo tissues at early embryonic development stages by using in situ hybridization(ISH)methods.We also investigated the association between ADAMTS18 and angiogenesis in mice by retinal angiogenesis experiments and Matrigel plug angiogenesis study.Finally,we created Adamts18 deficient zebrafish using morpholino antisense oligonucleotides(MO)to generate exon 3 skipped adamts18 mRNA transcripts.The effects of Adamts18 on organogenesis and angiogenesis in the early stage of zebrafish embryo development and its molecular mechanism were studied by experimental methods such as microscopic observation,RT-PCR and qRT-PCR.The results showed that in the head Adamts18 mRNA was mainly expressed in pseudostratified epithelium of optic vesicle and olfactory placode,pterygoid epithelium of midbrain at E10.5,and the subcommissural organ(SCO)at E11.5.In the neck,Adamts18 mRNA was mainly expressed in pharyngeal epithelium of the first branchial arch at E9.5-E11.5,specialized mesenchymal cells of the second branchial arch at E10.5-E11.5,and vascular epithelium of the third branchial arch at E11.5.In the trunk,Adamts18 mRNA was mainly expressed in epithelium of duodenum and dorsal aortic at E10.5,and pulmonary bud epithelium and mesenchymal cells,hepatic primordial epithelium,and mesonephric vesicle specialized mesenchymal cells and dorsal aortic epithelium at E11.5.Adamts18 mRNA was also highly expressed in dental epithelium and outer enamel epithelium at E14.5-E16.5,and tongue foliate papilla at E16.5.In addition,Adamts18 mRNA was transiently expressed in E13.5 interlobular vein,E14.5 vertebral-basilar artery(V-BA),and postnatal(P1)common carotid vein.ADAMTS18 deficiency leads to defective formation of retinal capillary network in mice.Mouse subcutaneous Matrigel plug study shows that ADAMTS18 deficiency leads to reduced angiogenesis in mice.In addition,Adamts18 deficiency in zebrafish embryos caused developmental defects,including expanded brain ventricle and hindbrain edema,eye defects,and accumulation of blood in the caudal vein.Adamts18 deficiency also led to impaired trunk angiogenesis and formation of the caudal vein plexus(CVP).Consequently,Adamts18 deficient zebrafish embryos exhibited incomplete formation of intersegment vessels(ISVs)and dorsal longitudinal anastomotic vessel(DLAV),disruption of the honeycomb structure of CVP,and reduced CVP area and loop number.Furthermore,Adamts18 deficiency resulted in impaired blood circulation in major trunk,caudal vein(CV),and common cardinal vein(CCV).These aberrant vascular phenotypes in mutant zebrafish embryos were shown to be associated with a decreased expression of multiple angiogenesis-related signaling genes,including TGF-β,Slit/Robo,Dll4/Notch,Cox2,and Fgfr.These findings indicate the critical role of ADAMTS18 in the early embryonic development and angiogenesis.In summary,we demonstrated for the first time that Adamts18 is widely expressed during mouse organogenesis,and indicate the critical role of ADAMTS18 in the early stages of vascular network development.This study provides a new experimental basis for further study on the biological function of ADAMTS18 and a new idea for the treatment of related diseases.