The Roles Of BMP-Chordin In Molluscan Dorsal-ventral Patterning

The setting up of body axes is one of most important steps of body patterning in bilaterian animals.Generally,body axes include anterior-posterior(AP)axis,dorsal-ventral(DV)axis and left-right(LR)axis.It is widely accepted that the cooperation of two conserved molecules,a BMP ligand(BMP2/4)and its antagonist(Chordin),establishes a gradient of BMP signaling along the DV axis and induces cells at different positions to differentiate in the correct manner.Such a DV patterning mechanism has been revealed in a wide range of bilaterian animals,including both deuterostomes and ecdysozoans.However,a study revealing BMP5-8 and Gremlin patterning DV axis of the leech Helobdella robusta belonging to annelida of lophotrochozoa calls into question the conservation of a common DV patterning mechanism among bilaterian animals.Therefore,studies on the DV patterning in other lophotrochozoan species are required.In the present study,we screened bmp and antagonist genes that may be involved in DV patterning and investigated their expression pattern in the bivalve mollusk Crassostrea gigas and the gastropod mollusk Lottia goshimai.Then we employed a specific BMP inhibitor(dorsomorphin)to explore the functions of BMP signaling in early development of C.gigas.Finally,we inhibited the expression of chordin by injecting antisense morpholino in L.goshimai,and confirmed Chordin played a key role in the DV pattering of Lottia goshimai.We firstly screened related genes,including bmp2/4,chordin,bmp5-8,and gremlin,in the early embryos of C.gigas.Searching of the genome and phylogenetic analysis revealed that C.gigas possesses single orthologs of bmp2/4,chordin,and bmp5-8 and no gremlin homolog was found.Whole mount in situ hybridization revealed mRNA localization of bmp2/4 and chordin on the opposite sides of both C.gigas and L.goshimai embryos,suggesting the potential involvement of a BMP2/4-Chordin antagonism in DV patterning in those two species.Furthermore,universal bmp5-8 expression and the absence of a gremlin homolog in the C.gigas genome suggest that the DV patterning mechanism in H.robusta may be a derived trait.BMP signaling is initiated when a BMP ligand binds to the cell-surface TGF-βreceptor heterotetramer complex,and complex interactions between BMP ligand and receptors make TGF-β receptors an important node in regulating BMP signaling.To provide support for investigating the role of BMP signaling in early molluscan development,we performed a genome-based analysis of TGF-β receptors and revealed that C.gigas possessed all five canonical members of TGF-β receptors.Whole mount in situ hybridization revealed that four receptor genes exhibited universal expression at the gastrula stage but cgi-bmprII mRNA was only expressed at the anterior lip of blastopore.Though the roles of BMP signaling in development is studied extensively in insects and vertebrates,our knowledge of BMP signaling in molluscan development is limited,which BMP signaling may be involved in DV pattering and larval shell formation.To investigate the role of BMP signaling in the early molluscan development,we firstly screened several potential developmental regulator genes that were expressed asymmetrically along the DV axis,including three dorsal expressed genes(engrailedl,engrailed2,gata2/3 and goosecoid)and four ventral expressed genes(forkhead,brachyury,and vasa).Then we treated oyster embryos using a BMP signaling inhibitor(dorsomorphin)and the results revealed obvious expression changes of developmental regulatory genes.Three dorsally expressed genes were inhibited and two genes expressed at the opposite side showed increased expression.These results indicated that BMP signaling may function in dorsal-ventral(DV)patterning in oyster embryo.Chordin is determinant in establishing a gradient of BMP signaling along the DV axis,therefore to clarfy the function of Chordin is an essential step to elucidate molluscan DV patterning.Due to technical restriction,oyster is inappropriate material to achieve this purpose.To obtain more direct evidences regarding to the functions of BMP and Chordin in molluscan DV pattering,we inhibited the expression of chordin in L.mai embryos by injecting morpholino.A "twin" embryo with duplicated bodies and connected abdomen was obtained after MO injection,as indicated by scanning electronic observation and the expression changes of several marker genes.In the MO-injected embryo,which exhibited apparently duplicated body,the genes expressed dorsally(gata2/3 and engrailedl)exhibited two symmetric expression regions assembling a mirror image,indicating two dorsal sides were developed in the"twin" embryo.On contrast,the mRNA of two ventrally expressed genes was only detected in the cells located at the middle line of the embryo,indicating the existence of the ventral side in the middle line.Muscle systems staining further supported the development of "twin" larvae with duplicated bodies,which emerged at later developmental stages.In the MO-injected embryo,distributions of pSMAD1/5/8 was detected on two opposite sides of the embryo,while there were only signals on one side of the normal embryo.Altogether,our results confirm that chordin is a master gene modulate DV pattering of mollusk.This study would contribute to revealing the detailed formation mechanism of molluscan DV axis.In conclusion,we identified the orthologs of bmp and chordin in C.gigas and L.goshimai and proposed a conserved BMP2/4-Chordin DV patterning mechanism in mollusk based on gene expression patterns.Further,experiments using a BMP inhibitor indicated roles of BMP signaling in DV patterning and cell specification in the shell field.Finally,the Chordin-knockdown experiment confirmed the crucial role of Chordin in molluscan DV patterning for the first time.These results would enrich our knowledge on the molecular mechanisms of molluscan DV patterning,and also support a conserved DV patterning mechanism among bilaterian animals.