Identification Of Hematopoietic Sites And Study On The Hematopoietic Regulatory Mechanism In The Pacific Oyster Crassostrea Gigas

Haematopoiesis,one of the most important activities for maintaining life,is an intricate complex process through which the production of distinct blood cell types in hematopoietic tissues and terminal differentiation into circulating blood cells.The hematopoietic tissue,the site for producing and releasing blood cells,is not only the tissue supporting the growth,dif-ferentiation,and survival of blood cells but is also a key immune organ.With bioinformatics,molecular biology,cell biology,and immunology methods,several hematopoiesis-related molecules and the hematopoiesis-related sites were identified,and the regulation of cytokines in hematopoiesis for Crassostrea gigas was explored in the present study.1.the molecular features of hematopoiesis-related molecules and their functions in hematopoiesisThe hematopoiesis-related molecules were identified and characterized from C.gigas,including proliferating cell nuclear antigen（PCNA）,ATP-binding cassette transporter G2（ABCG2）,vascular endothelial growth factor（VEGF）and vascular endothelial growth factor receptor（VEGFR）.Compared with the homologous molecules,their corresponding domains were highly conserved.All of the above molecules were constitutively expressed in the man-tle,gonad,adductor muscle,gills,digestive gland,haemocytes,and labial palp.Cg PCNA and Cg ABCG2 were highly expressed in the gills and gonad,they were distributed mainly in the nucleus and on the surface of membranes for haemocytes.Cg VEGF and Cg VEGFR were highly expressed in the gills and haemocytes,and distributed mainly in the cytoplasm and on the surface of membranes for haemocytes.In the oysters treated with the recombinant protein Cg Astakine（r Cg Astakine）,the protein abundance of Cg PCNA was enhanced in agranulo-cytes and gills,and especially colocalized with the Ed U signals in gills vessels.After Cg ABCG2 expression was interfered with by specific si-RNA injection,the number of cells with Ed U-positive signals was reduced and the cell cycle of haemocytes was arrested at G0/G1 phase.After the injection of recombinant Cg VEGF protein（r Cg VEGF）,the propor-tions of Ed U-positive cells in haemocytes and gills were significantly increased.After being stimulated by V.splendidus,the expression of the above molecule in haemocytes was in-creased.These results suggest that the above molecules may be involved in proliferative pro-cesses.2.the characterization of gills’structural features and the identification of hematopoie-sis-related sitesThe m RNA expression levels of hematopoiesis-related transcriptional regulators,specif-ic marker molecules,and immune-related molecules in different sectors of gills were investi-gated by q RT-PCR analysis.The gills of oysters were divided into eight sectors.The m RNA transcripts of hematopoietic transcription factors（Cg SCL and Cg Runx）,hematopoiesis-related cytokines（Cg Astakine,Cg IL17-1）,cell cycle markers（Cg PCNA,Cg CDK2）and the marker-molecule of hematopoietic stem and progenitor cells（Cg SOX₂,Cg ABCG2）were relatively highly expressed at the proximal region in gills.And immune effector molecules such as defensin（Cgdefensin-1,Cgdefensin-2,Cg Bigdefensin）and inflammatory factors（Cg IL17-3,Cg IL17-6）were the relatively low expression there.At the proximal region of the hinge in gills,a large number of spherical cells with a big nucleus and DNA replication were detected in some cells by the Ed U labeling.Ed U-positive cells are mainly distributed at the apical of common filaments in internal gills.And the number of Ed U-positive cells increased following Vibrio splendidus stimulation.A few long-term labels retaining cells（LRCs）were positive for stem cell marker SOX₂ quiescently located at the proximal region in gills.These results suggest that the gills nearby the hinge may be the potential hematopoiesis sites for oys-ters.3.the regulatory role of the VEGF-VEGFR-MAPKs pathway in cell proliferation and migration at hematopoiesis-related sitesThe m RNA expression level of Cg VEGF and Cg VEGFR in the hematopoietic sites in-creased significantly after the stimulation of V.splendidus.The Ed U-positive cells among the haemocytes and the hematopoietic sites increased significantly after the stimulation with r Cg VEGF in vivo.And the number of migrated hematopoietic sites tissue cells increased sig-nificantly after the stimulation with r Cg VEGF in vitro.When the Cg VEGFR expression was interfered by specific si-RNA injection,the percentage of Ed U-positive cells among the hae-mocytes,the m RNA expressions of cell proliferation-related genes（Cg GATA and Cg Runx）and cell migration-related genes（Cg MMP）,the phosphorylated Cg Erk and Cg JNK protein in the hematopoietic sites all declined dramatically post r Cg VEGF and taurodeoxycholate sodi-um salt（STC,VEGFR activator）stimulation.And the m RNA expressions of cell migration-related genes（Cg TIMP）increased significantly,Ed U-positive cells migrated and aggregated close to the hematopoietic sites.After treatment with the VEGFR inhibitors（Brivanib and Semaxanib）,the percentage of Ed U-positive cells among the haemocytes,the m RNA expres-sions of cell proliferation-related genes（Cg GATA and Cg Runx）and cell migration-related genes（Cg MMP）,the phosphorylated Cg VEGFR,Cg Erk and Cg JNK protein in the hemato-poietic sites all declined dramatically post r Cg VEGF stimulation.And the m RNA expressions of cell migration-related genes（Cg TIMP）increased significantly,Ed U-positive cells migrated and aggregated close to the hematopoietic sites.The results indicated that the hematopoietic sites in gills was the hematopoiesis sites in adult oysters,and Cg VEGF signaling induced the migration of newborn hemocytes by activating the MAPK pathway at the potential hemato-poietic sites of oyster.In conclusion,the hematopoiesis-related molecules Cg PCNA,Cg ABCG2,Cg VEGF,and Cg VEGFR are highly conserved which regulate the proliferation of haemocytes in C.gigas.The proximal region of the hinge in gills is more likely to be the hematopoiesis sites in adult oysters.Cg VEGFR mediated the Cg VEGF-induced newborn hemocyte migration by activat-ing MAPK pathway at the potential hematopoietic sites in oysters.These results provide theo-retical insights for future studies on the regulatory mechanisms during hematopoiesis in inver-tebrates.