Phylogenetic Analysis Of Wnt Genes And The Expression Patterns In Pacific Oyster Crassostrea Gigas

Wnt genes encode a kind of highly conserved secretory glycoprotein ligands,which usually have length with 350-400 amino acids together with one or more N terminal glycosylation sites.They play important roles in biological processes of embryonic and adult cells in multicellular animals,including the embryonic body axis formation,organogenesis,cell fate decisions,proliferation,migration and regeneration,maintaining of cell homeostasis etc.Study on the distribution of Wnt gene subfamilies and their functional evolution provides insight into the origin of life and the gene evolution trajectory.As widely interested signaling molecules,they have been studied in various species.The gene sequences in most subfamilies are conserved among different species,and the gene functions have also been shown to be similar.However,studies on molluscan Wnt genes are limited.Studying on molluscan Wnt genes will help to better understand their functional evolution in invertebrates,especially the roles involved in the process of embryonic development and adult tissue.In this thesis,we systematically studied Wnt genes from metazoan genomes,with emphasis on the poorly studied superphylum Lophotrochozoa(four annelids,fourteen mollusks,eight platyhelminths,one bdelloid rotifer,and one brachiopod species)and with the other seven model animal species(sponges and cnidarians,ecdysozoans and deuterostomes).The Wnt gene members were studied and classified based on the phylogenetic analysis of Wnt conservative domain.The clustering of some Wnt genes was also studied.We further collected the publicly available RNA-seq or microarray data on different developmental stages from six species and analyzed the correlation of Wnt genes expression pattern.Expression pattern of oyster Wnt genes were then studied on different tissues,while Cgi Wnt6 was verified by immunofluorescence and in situ hybridization methods.?.Wnt gene distribution,and the structure characteristics in typical metazoansA total of 478 Wnt sequences from 35 species were analyzed in this study.Comparing with former reports,we identified four new in Nematostella vectensis,one loss in Danio rerio and Strongylocentrotus purpuratus,no difference in the Drosophila melanogaster,Caenorhabditis elegans and Daphnia pulex,one new in Lottia gigantea and Schistosoma mansoni,four new in Helobdella robusta;the Wnt genes in the Lingula anatina,Aplysia californica,Biomphalaria glabrata,Lymnaea stagnalis(great pond snail),Radix auricularia,Modiolus philippinarum,Bathymodiolus platifrons,Mytilus galloprovincialis(Mediterranean mussel),Patinopecten yessoensis,Crassostrea virginica,Crassostrea gigas,Octopus bimaculoides,Pinctada fucata,Adineta vaga,Spirobranchus(Pomatoceros)lamarcki,Schistosoma japonicum,and Taenia solium were first systematically reported.Between 3 and 39 Wnt loci were identified in each genome,and the protostome-specific loss of Wnt3 subfamily was confirmed.Wnt9 only existed in the bilateral animal suggesting the similar lost during evolution.Three sponges Wnt genes that used to be unclassified were classified.We identified gastropod-specific loss of Wnt8 subfamily,rather than the previously proposed mollusk-specific loss.Further,there have more sequence polymorphisms in the Platyhelminthes species,i.e.a unclassified subfamily were designed beside the normal Wnt1,Wnt2,Wnt5,and Wnt11 subfamilies.A molecular phylogenetic tree were constructed containing 311 Wnts of 13 subfamilies except the unclassified members.Three highly closed relationships within subfamilies: Wnt1-Wnt6(BS=76%,BI=100%),Wnt4-Wnt11(BS=51%,BI=96%)and Wnt9-Wnt10(BS=46%,BI=71%)exhibited extensive tandem duplication,indicating that this phenomenon is common to several different Wnt subfamilies.Some duplicated Wnt genes belonging to a same subfamily or closely related subfamilies showed a tandem distribution in the lophotrochozoan genomes,indicating tandem duplication events during Wnt family evolution.Gene clustering analysis showed that,besides the well-studied Wnt10-Wnt6-Wnt1-Wnt9 and Wnt7-Wnt5 clusters,there are also other ones like the Nve Wnt8aa-Nve Wnt8 ab,Sha Wnt2b-Sha Wnt2 a,suggesting that these subfamilies were separated by tandem duplication early in the animal evolution.These linkages were retained in their descendants with possible function similarity and complementarity.?.Wnt gene spatio-temporal expression analysis in different speciesBased on six different developmental RNA-seq and microarray data analysis in oysters,fruit flies,nematodes,zebrafish,lingula and sea anemones,we found that the Wnt subfamily were obviously up-regulated expression at different stages,such as maternal expression of Wnt5 and Wnt9;expression in embryogenesis of Wnt1,Wnt2,Wnt3,Wnt4 and Wnt8;the early,middle and lately up-regulated expression of Wnt6,Wnt11 and Wnt A.These diverse expression patterns suggest the possible characteristics of diversification function of the Wnt members.Further,members of the conserved Wnt10-Wnt6-Wnt1-Wnt9 cluster showed highly correlated time-course expression patterns in two assayed lophotrochozoans,the oyster C.gigas and the brachiopod L.anatina,reflecting the possible similar function of the clustered Wnt genes,such as Lan Wnt10 aa,Lan Wnt10 ab and Lan Wnt6,as well as the Cgi Wnt10,Cgi Wnt6,Cgi Wnt1 and Cgi Wnt9 a.Expression diversities in different oyster tissues suggested possible differentiation function of each Wnt genes.Finally,based on expression in the spatio-temporal expression diversity,we can speculate that parts of the Wnt10-Wnt6-Wnt1-Wnt9 cluster are likely to be involved in the intestinal system formation in the process of embryonic development.?.In situ hybridization and immunofluorescence analysis in oyster larvaeThe in situ hybridization experiments and immunofluorescence experiments of different developmental oysters embryo and larvae showed that: Cgi Wnt6 expressed high expression level in the developmental process of stomach and intestine in oyster,suggesting it may participate in the intestinal formation.Comparing the exons and introns of the Wnt6 family in different species,we found the structure of highly conservation 4 exons structure in bilaterian.At the same time,the RNA expression level correlation results suggest that the oyster clustered genes Cgi Wnt1,Cgi Wnt9 a and Cgi Wnt10 in the genome highly expressed in the intestinal system and may also be involved in the process.In all,combining the understanding of the Wnt genes in different animals,especially of the model animals,we suspect that they may participate in the oyster intestinal system developmental process.