The Functions Of Lrp5 And Lrp6 In Self-renewal And Differentiation Of Hematopoietic Stem Cells

Hematopoietic stem cells(HSCs)are the adult stem cells that give rise to mature blood cells through the process of hematopoiesis.HSCs have the capacity of self-renewal to maintain the HSCs pool and the ability for multi-lineage differentiation,which are responsible for sustained production of multiple blood lineages throughout lifetime.HSCs develop to multipotent progenitors(MPPs),and then differentiate to common myeloid progenitor cells(CMPs)and common lymphoid progenitor cells(CLPs),subsequently,replenish myeloid(granulocyte,macrophage),erythroid,platelet/megakaryocyte and lymphoid(B,T and NK cell)lineages.In short,HSCs are responsible for sustaining the individual's hematopoiesis and the balance of immune system homeostasis.The regulation of HSCs development is tightly controlled by complex signal networks and hematopoietic micro-environment,termed the HSCs niche.A variety of signaling pathways have been implicated in HSCs self-renewal and maintenance,such as Wnt,Notch,BMP,and mTOR signaling pathways.Two characterized Wnt signaling pathways are canonical(or Wnt/?-catenin pathway)and non-canonical pathways,which have been identified for essential roles in the embryonic development,tissue regeneration,cell proliferation and migration,and various diseases.The canonical Wnt signaling pathway also has been demonstrated as a critical role in the self-renewal,proliferation and differentiation of HSCs.The canonical Wnt signaling pathway is activated by binding a Wnt protein ligand to a Frizzled(Fz)family receptors,and Low-density Lipoprotein Receptor-related Proteins 5 and 6(Lrp5/6)act as a co-receptors.The previous studies are controversial on the regulation of HSCs upon canonical Wnt signaling pathway,due to the different experimental systems using overexpression and knockout mice as gain-or loss-functions models.In this study,the mice models were established via conditional ablation of Lrp5 and Lrp6 in the HSCs and all blood cells by Vav-Cre Loxp systerm,in order to analyze the differentiation and development of HSCs after blocking the canonical Wnt signaling pathway.We aimed to demonstrate the functions and molecular mechanisms of canonical Wnt signaling pathway in HSCs using the new strategies.In the present study,Vav-Cre mice were crossed with Lrp5fl/fl and Lrp6fl/fl mice to obtain Vav-Cre Lrp5fl/fl Lrp6+/+(Lrp5KO),Vav-Cre Lrp5+/+Lrp6fl/fl(Lrp6KO)and Vav-Cre Lrp5fl/fl Lrp6fl/fl(dKO)mice.The results showed that the development of mature lymphoid and myeloid cells were not affected after deletion of Lrp5 and Lrp6 genes in hematopoietic system.However,the proportion of HSCs in the bone marrow(BM)of dKO mice was significantly diminished compared with littermate wild-type(WT)controls.Subsequently,the reconstitution capacity of HSCs from dKO mice were detected by competitive repopulation assay,and the frequecy of peripheral blood cell reconstituted by dKO HSCs in recipients were significantly reduced at both short-and long-term(8 weeks and 16 weeks after BM transplantation,respectively).Meanwhile,the in vivo splenic colony-forming unit assay showed that colonies derived from dKO HSCs in the recipient spleens were significantly diminished.We further measured HSCs self-renewal by performing serial transplantations.In the 4th recipients,the contribution from dKO HSCs was markedly decreased.Taken together,these results revealed a critical requirement for Lrp5 and Lrp6 in HSCs self-renewal and blood lineage reconstitution capacity.We next investigated the proliferation,apoptosis and cell cycle of HSCs from different mice.The results of Annexin V staining showed no difference in HSCs from all genotype mice.Neverthless,BrdU+cells were markedly decreased in dKO HSCs,which implied that cell proliferation of dKO HSCs were slower than WT HSCs.Meanwhile,cell cycle analysis with Hoechst 33342 and Ki-67 showed that the number of dKO HSCs was increased in G0 phase,but decreased in G1 phase,indicating the reduced cycling of HSCs after abolition of both Lrp5 and Lrp6.These data suggested that Lrp5 and Lrp6 affect the differentiation and self-renewal of HSCs through inhibiting the proliferation and decelerating the cell cycle of HSCs.To explore the molecular mechanism,RNA-seq and qPCR analysis were performed using sorted HSCs.The different genes between dKO and WT HSCs can be enriched in DNA replication and cell cycle signaling pathways through GO,KEGG and GSEA analysis.We next verified the different genes by qPCR detection,a series of genes can be confirmed with high-throughput results.The data showed that many different genes involved in the regulation of HSCs,such as Egr1,Cdkn1a,Nr4a1,Gata2,Junb,and Btg2 were upregulated(dKO V.S.WT HSCs),whereas Ccna2,Ranbp1,and Ran were downregulated after the deletion of Lrp5 and Lrp6.According to the evidences from litterature,these upregulated and downregulated genes lead to the same phenotypes of HSCs in cell cycle and proliferation.In the following study,we will focuse on these genes,and perform the rescue experiments using shRNA and retroviral overexpression vectors,in order to reveal major downstream genes and understand the refined mechanisms of canonical Wnt signaling pathway in regulating HSCs self-renewal and differentiation.In summary,we constucted the mice models by conditionally deleting Lrp5 and Lrp6 to block the canonical Wnt signaling pathway.We found the deficency of self-renewal and differentiation in dKO HSCs,because of the impaired proliferation and the decelarated cell cycle.Combined RNA-seq and qPCR results,we found a group of genes upregulated or downreglatued in dKO HSCs,which resulted in defects in HSCs homing,proliferation and cell cycle.In the following study,it will merit further investigation to dig out the major regulator at the downstream of canonical Wnt signaling pathway in controlling HSCs fate.Together,this study provides not only new experimental evidence on the regulatory network of HSCs,but also a key rationale for exploring the downstream genes administered by canonical Wnt signaling pathway as therapeutic targets to clinical blood diseases.