The Roles Of BMP/Smad Pathway And Tyrosinase In The Early Development Of The Pacific Oyster Crassostrea Gigas

The larval shell formation of mollusk is emphasized for evolutional and practicalconcerns. However, the underlying molecular mechanisms remain unrevealed.Although several preliminary researches indicated that Dpp (a BMP2/4-homolog)regulated this process; the details of the signal pathway are not clear. Beside theregulatory genes such as Dpp, the effect genes of larval shell formation are alsounknown. In this study, we cloned and analyzed three regulatory genes, which weremembers of the BMP/Smad pathway, and one effect gene, a tyrosinase gene, from thePacific oyster Crassostrea gigas, which is a world-wide economic species and apotential model organism of bivalves. The results confirmed that they play importantroles in larval shell formation.Firstly we cloned the Dpp gene, which acted as the ligand in BMP/Smadpathway. As an important member of the transforming growth factor (TGF) β family,Dpp acted as a morphogen in many animals. The dpp-homolog from C. gigas (cgdpp)exhibited typical characteristics of TGF-β members. The expression pattern of cgdppindicated cgdpp might participate in the cell motility and differentiation duringdevelopment of the shell field in embryogenesis. In trochophore, the cgdpp mightregulate the shape and extension of the shell. Only trace mount of cgdpp was detectedin the early D-veliger, indicating the cgdpp was not involved in the later developmentof the shells since D-veliger. Smad family plays a crucial role in TGF-β signaling pathway. To further explorethe roles of TGF-β signal pathways in development of bivalve mollusks, we clonedand characterized two smad genes from C. gigas. Sequence analyses of the two genes,designated cgsmad1/5and cgsmad4, revealed conserved functional characteristics,including two MH domains and other functional motifs. Their expression patternsduring early development revealed that the two genes were both maternal effect genesand they might contribute to body patterning, gastrulation and shell formation duringdevelopment of C. gigas. The different expression patterns of the two genes in theshell field indicated different TGF-β pathways might dominate in different phases ofshell formation.Beside the regulatory genes as mentioned above, we also cloned an effect gene (atyrosinase gene, cgtyr) involved in larval shell formation. Sequence analysis of cgtyrrevealed it had typical copper-binding domains and a signal peptide. Through wholemount in situ hybridization and a scanning microscopic observation, the expression ofcgtyr was firstly revealed in the saddle-shaped shell field in trochophore, whichconfirmed cgtyr participated in the shell formation. In the following development toearly D-veliger, cells in the central region of shell field shut off cgtyr expression; andcgtyr expression was sustained only in the edge of the shell field and the hinge region,which might role the shell growth from trochophore to early D-veliger. Moreover, itwas unexpected to observe cgtyr expression was shut off after D-veliger was formed.This indicated that other molecules functioned in the following shell developmentsince then.In summary, we analyzed not only the ligand gene (dpp) but also thesignal-transducing genes (smads) from the BMP/Smad pathway, which playedimportant roles in larval shell formation. Further, to our best knowledge, thetyrosinase gene reported here is the first known effect gene in larval shelldevelopment. These results would enrich our knowledge on the molecularmechanisms of larval shell formation, and might also be helpful for researches on body planning and gastrulation of mollusks.