Identification Of Multiple Cell Populations And The Regulatory Factors In The Oyster Shell Field

The larval shell formation of mollusk is emphasized for evolutional and developmental perspectivess.Despite that several potential shell-formation(p SF)genes are proposed to be involved in larval shell formation,the underlying molecular mechanisms remain unrevealed.Most of current studies focus on identification of particular p SF genes or the larval shell,paying little attention on the cell specifications in the shell field of molluscan embryos and the function of p SF genes in these processes.In present study,we identified four novel p SF genes from the Pacific oyster Crassostrea gigas,an emerging model bivalve molluscan species.Then we identified multiple cell populations from the shell field of the gastrula and early D-veliger.We further revealed that the GTPase CDC42 might regulate the specification of these cell populations and thus regulate shell formation.Though BMP signaling pathway has been proposed to regulate the molluscan larval shell formation,the genes responding to BMP signaling during this process have not been investigated.Among the four novel p SF genes that we identified in the present study: cgi-gata2/3,cgi-soxc,cgi-pax2/5/8 and cgi-lox4,cgi-gata2/3 and cgi-soxc are potential target genes of BMP signaling pathway.cgi-pax2/5/8 encodes a member of Pax family,whereas cgi-lox4 is a Hox gene.The association between the expression patterns of four p SF genes and shell field indicated that these genes may participate in the shell formation in embryogenesis.In particular,cgi-gata2/3 and cgi-pax2/5/8 were expressed in the edge of shell field,indicating that they may regulate the shape and extension of the larval shell.The m RNA of cgi-lox4 was detected in the inner cells of shell field and may be related with the construction of body plan or shell field invagination.cgi-soxc was expressed in the whole larval mantle of early larvae,which may be involved in periostracum biogenesis,biomineralization,and regulation of cell proliferation.In addition,cgi-gata2/3 is hypothesized to be involved in hematopoiesis and cgi-soxc may play a conserved role of in neurogenesis during embryonic development.The known p SF genes have different expression patterns,which can be generally divided into two types.In type I,a gene is expressed in a ring of cells surrounding the shell field,such as engrailed,bmp2/4 and gata2/3.Another type(type II)refers to a gene that is expressed in the whole shell field.These two types of expression patterns indicate that different cell populations are involved in the larval shell formation of mollusks.Using two-color hybridization,we investigated the inter-relationships among the expression of different p SF genes.Three cell populations were identified in the gastrula shell field.The m RNA of cgi-gata2/3,cgi-bmp2/4 and cgi-pax2/5/8 were detected in the outer cell population,while cgi-eng1 and cgi-eng2 were expressed in the middle cell population.In addition,the expression of cgi-lox4 were located in the inner cell population.In early D-veligers,two cell populations were identified from the larval mantle(tyr1+/soxc+/gata2/3+ cells and tyr1+/soxc+/gata2/3-cells).These results are important to study the differentiation and function of larval mantle as well as the larval shell formation of mollusks.In order to study the specification of different cell populations,we investigated the role of CDC42 in larval shell formation.The high expression of cgi-cdc42 in the shell field indicated that this gene may play a role in larval shell formation.We then used ZCL278,a specific inhibitor of CDC42,to treat oyster embryos.and the results revealed that the expression of some p SF genes were affected after ZCL278 treatment.In particular,the posterior expression of cgi-bmp2/4 disappeared in both in 1 ?mol/L or 5 ?mol/L treatment groups.The expression of the two engrailed genes in the middle cell population were both seriously affected(showing significant decline).On the other hand,the m RNA expression of cgi-gata2/3,cgi-pax2/5/8 and cgi-lox4 were not obviously affected by ZCL278 treatment.This is the first report that CDC42 plays a role in molluscan larval shell formation and is important to study the mechanisms of shell formation.In summary,we identified four p SF genes,which play important roles in larval shell formation.Further,multiple cell populations were identified in shell field of oyster gastrula and D-veliger.Finally,CDC42 was revealed to be involved in larval shell formation by affecting cell specifications.These results would enrich our knowledge on the molecular mechanisms of larval shell formation.