| Background: Hematopoietic stem cells(HSCs)refer to a rare amount of cell population with the ability to replenish themselves through self-renewal,to differentiate into a complete series of blood cells and immune cells and to maintain hematopoiesis during the lifetime of an organism.The formation and development of HSCs is regulated by sorts of endogenous signaling pathways and various physical and chemical factors in their microenvironment.Any imbalance or abnormality occurred in the regulatory signaling network may lead to developmental disorders or functional defects in HSCs which may further cause pathological hematopoiesis,or even hematological malignancies.Although HSCs have been discovered and widely used for transplant treatment of a variety of hematological diseases including leukemia for more than 50 years,it remains to be an unfinished task to produce enough HSCs with long-term reconstruction ability in vitro,which limits their effective clinical application.The fundamental reason is that our understanding of the developmental process and the underlying regulatory network and molecular mechanism of embryonic HSCs are still incomplete.Therefore,more systematic and complete investigation into the dynamic regulatory mechanisms in the production and development of HSCs will contribute to our understanding and treatment of hematological diseases as well as the early realization of induced differentiation and acquisition of HSCs in vitro.For a long time,most studies on the development and function of HSCs only focused on the role of biochemical signals.Until recent years,multiple independent research teams have confirmed that biomechanical signaling are also essential for the production and development of embryonic HSCs.The development of embryonic HSCs not only needs the stimulation of external biomechanical factors such as blood flow-derived mechanical forces but also involves the role of internal forces,both of which are closely related to the cytoskeleton.Therefore,we speculate that cytoskeleton and cytoskeletonrelated regulatory factors may be involved in the regulation of embryonic HSCs development.Glia maturation factor(gmf)has been established as an important regulator of cytoskeleton remodeling.Vertebrates express two distinct gmf subtypes,namely glia maturation factor g(gmfg)and glia maturation factor b(gmfb).gmfb is mainly expressed in the nervous system,while gmfg is highly expressed in the hematopoietic system,including adult HSCs.gmfg expression was also detected in the mouse dorsal aorta(DA)where embryonic HSCs emerge.In addition,gmfg regulates the cellular activities of diversified mature blood cells such as the migration and adhesion of adult T lymphocytes,monocytes and neutrophils,suggestive of an essential role of gmfg in adult hematopoietic system.However,the function and potential regulatory mechanism of gmfg in the formation,maintenance and differentiation of embryonic HSCs remains to be discovered.In recent years,zebrafish has been widely used in the study of hematopoiesis due to its unique characteristics as follows: a great power in reproduction,ectogenesis,transparent embryos,easy to be genetically modified and high conservation with mammals in hematopoietic development and key signaling pathways.Therefore,zebrafish was used as the primary experimental model in this study to systematically explore and clarify the role and mechanism of gmfg in the production and development of embryonic HSCs,providing a new perspective for enriching the regulatory network of vertebrate HSCs.Chapter 1: gmfg regulates the production and development of embryonic HSCsObjective: To explore the dynamic expression of gmfg and gmfb during hematopoietic differentiation in vitro and embryonic hematopoietic development,and to investigate the role of gmfg and gmfb in the regulation of HSCs production and development.Methods: The dynamic expression of gmfg and gmfb was detected during the hematopoietic differentiation of human embryonic stem cells(h ESCs);Whole-Mount In Situ Hybridization(WISH)was used to investigate the spatio-temporal expression pattern of gmfg;vascular endothelium and hemogenic endothelium(HE)of zebrafish embryos were sorted to detect the expression of gmfg and gmfb;two distinct types of targeted morpholinos(MOs)including translation blocking MO(m RNA target)and splicing modifying MO(pre-m RNA target)were used to knockdown gmfg;translation-blocking MO were used to knockdown gmfb;by using WISH,fluorescence microscope and laser scanning confocal microscope,the expression of runx1 and cmyb,the number of kdrl+ cmyb+ double positive cells and cd41 positive cells within the ventral wall of the dorsal aorta(DA)or in the caudal hemopoietic tissue(CHT),the rag1 expression in the thymus and the number of lcr positive cells and lyz positive cells in the whole embryo,were detected in the experimental groups and control group at different time points during embryonic development.Moreover,lcr: e GFP transgenic line,l-plastin and mpx probes were used to detect lcr positive cells in the intermediate cell mass(ICM)region at 24 hours post fertilization(24hpf)and the expression of l-plastin and mpx in the trunk and posterior blood island(PBI)region at 28 hpf,respectively.Results: The expression of gmfg increased gradually with the hematopoietic differentiation of h ESCs;compared to undifferentiated h ESCs,the expression of gmfg was sharply increased in HE(CD31+CD34+)on day 6 while no significant changes in the expression of gmfb was observed during the hematopoietic differentiation process;WISH results showed that the spatio-temporal expression patterns of gmfg in zebrafish embryo was very similar to the trajectory of HSC emergence,maintenance and migration;both these two distinct types of gmfg MOs can effectively reduce the protein level of endogenous gmfg;the expression of runx1 and cmyb in the DA region at 36 hpf was significantly reduced after gmfg knockdown;the runx1 expression in the DA region at 24 and 28 hpf was also significantly decreased;the cmyb expression in the DA region at 48 hpf and in the CHT region at 52 hpf and the cmyb positive cells in the CHT region at 75-77 hpf were also significantly decreased;compared with control group,no significant changes in the expression of 24 hpf runx1 and 30 hpf cmyb in the DA region were observed in the gmfb knockdown embryos;the number of kdrl+cmyb+ HE cells in the ventral wall of DA at 48 hpf was significantly reduced in the gmfg-deficient embryos;at 3 days post fertilization(3dpf),the number of cd41 positive cells in the CHT region was significantly reduced and the ratio of cd41 positive cells decreased to about half of the control group;rag1 expression in the thymus at 4dpf was significantly decreased or even absent,and the number of lcr positive cells and lyz positive cells in the whole embryo at 3dpf were significantly reduced;compared with the non-injected p53 mutants,the expression of 26 hpf runx1 and 36 hpf cmyb in the DA region remained decreased in the gmfg-atg MO and gmfg-sp MO-injected p53 mutants;the number of lcr positive cells in the ICM region at 24 hpf,the expression of l-plastin and mpx in the trunk and PBI region at 28 hpf were significantly decreased in the gmfg-deficient embryos.Conclusion: EHT was blocked,the production of HSCs is defective,the maintenance and differentiation of HSCs also are impaired after gmfg knockdown,which suggest that gmfg signaling is required for HSC production,maintenance and differentiation.gmfb knockdown does not affect the production and development of HSCs.The HSCs defects caused by the knockdown of gmfg by MOs are independent of non-specific activation of p53 pathway.gmfg also is involved in the regulation of primitive hematopoiesis.Chapter 2: The effects of gmfg on HSC apoptosis and proliferation,PLM formation and arterial developmentObjective: To explore whether gmfg indirectly regulates HSC production and development through its effects on HSC apoptosis and proliferation,posterior lateral mesoderm(PLM)formation and arterial development.Methods: Apoptosis in DA region was detected by TUNEL immunofluorescence at 26 and 32hpf;proliferation in the DA region was detected by p H3 immunofluorescence at 26 and 36hpf;fli1a probe was used to detect PLM formation at 12 hpf by WISH;ephrin B2 a and dlc probes were used to investigate the arterial development by WISH.Results: The number of fli1a+TUNEL+double positive apoptotic cells at 26 and 32 hpf and fli1a+p H3+double positive proliferative cells at 26 and 36 hpf in the DA region showed no big difference compared with the control group;the 12 hpf fli1a expression in the gmfg-atg MO and gmfg-sp MO groups was also comparable to that of control group;however,the expression of ephrin B2 a and dlc in the trunk region at 28 hpf was significantly reduced in the gmfg-atg MO group.Conclusions: HSC apoptosis and proliferation as well as PLM formation at the early stage of the embryo remained unaffected after gmfg knockdown,indicating that the effects of gmfg on HSC production and development are independent of these processes.However,arterial defects were observed,therefore,it cannot be excluded that the effects of gmfg on HSC is a result of its effect on arterial development.Chapter 3: gmfg mediates the effect of blood flow on HSC maintenanceObjective: To explore the functional relationship between gmfg and blood flow and the underlying molecular mechanisms in the regulation of HSC maintenance.Methods: The coding sequence(CDS)of zebrafish gmfg was constructed to pcs2+ plasmid via homologous recombination;the plasmid was linearized by the Not I restriction endonuclease;the linearized plasmid was used as the template for transcription of gmfg-m RNA in vitro;the synthesized gmfg-m RNA was coinjected with gmfg-atg MO according to a concentration gradient of 50ng/μl,100ng/μl,150ng/μl and 200ng/μl into the single cell of zebrafish embryos,the injection volume was 1nl,then the embryos were collected at 28 hpf for subsequent WISH analysis of runx1/cmyb to explore the optimal concentration for gmfg overexpression;tnnt2a-MO was used to intercept blood flow and construct a silent heart(sih)model,then kdrl+cmyb+HE was sorted by flow cytometry for subsequent q PCR analysis of the m RNA level of gmfg within HE;in addition,the trunk and tail tissue were dissected to detect the protein level of gmfg after blood flow interception;100ng/μl gmfg-m RNA was coinjected with tnnt2a-MO into zebrafish embryos,then 36 hpf runx1/cmyb and 48 hpf cmyb were detected by WISH and cd41 positive cells were analyzed by using cd41:e GFP transgenic line to verify whether gmfg overexpression could rescue the decrease of runx1 and cmyb expression in the DA region and cd41 positive cells in the CHT region caused by loss of blood flow.The expression of klf2a、yap and yap target gene was detected by q PCR,western blot and WISH after gmfg knockdown;gmfg sh RNA was used to knock down gmfg in HUVEC,then the protein level of YAP in the nucleus and cytoplasm and the protein level of intracellular CTGF were detected by western blot.Results: gmfg-m RNA has been transcribed in vitro and the optimal injection concentration of gmfg-m RNA for gmfg overexpression was 100ng/μl confirmed by WISH,because this concentration was enough to rescue the decrease of runxl/cmyb expression in the DA region caused by gmfg knockdown without causing obvious embryo deformity;loss of blood flow resulted in significant decreased m RNA level of gmfg within HE and protein level in the trunk and tail tissue,respectively;overexpression of gmfg can partially rescue the defects in the expression of 36 hpf runx1/cmyb and 48 hpf cmyb in the DA and the decrease of cd41 positive cells in the CHT caused by loss of blood flow;klf2a expression remained unaffected in gmfg-deficient embryos;knockdown of gmfg did not cause changes in the transcription and protein levels of yap,but the m RNA level of ctgfa and cyr61 within HE and the protein level of CTGF in the trunk and tail tissue significantly decreased;knockdown of gmfg in HUVEC led to the decreased protein level of nuclear YAP but increased protein level of cytoplasmic YAP as well as the decreased protein level of intracellular CTGF.Conclusion: Blood flow acts as upstream of gmfg in the regulation of HSC maintenance.gmfg mediates the effect of blood flow on HSC maintenance through impacting the nuclear entry of YAP.Chapter 4: gmfg regulates HSC production through Notch signalingObjective: To explore the functional relationship between gmfg and the classical hematopoietic regulatory pathways of Notch,Hedgehog,BMP and c AMP in the regulation of HSC production.Methods: Targeted inhibitors or agonists of Notch,Hedgehog,BMP and c AMP signaling were customized according to published literature and were added into holt buffer at 12-14 hpf,combined with or without gmfg overexpression or gmfg knockdown,the control group was treated with 0.1% DMSO,embryos were collected and fixed at 28-30 hpf and then subjected to WISH analysis of runx1/cmyb expression;by using Notch signal reporter line tp1:GFP,the changes of tp1 signal in the DA region were observed by laser scanning confocal microscope,and the percentage of tp1 positive cells were analyzed by flow cytometry in control and gmfg knockdown groups;Further,by using tp1:m Cherry and fli1a:e GFP double transgenic line,the number of fli1a+tp1+ double positive cells in the DA region at 28 hpf was counted in control and gmfg knockdown groups;kdrl+cmyb+HE cells from control and gmfg knockdown group at 48 hpf were sorted by flow cytometry and then were subjected to q PCR analysis for the expression of Notch target genes hey1 and hey2;Control-MO and gmfg-atg MO was injected into hsp70l:Gal4;UAS:NICD embryos,then the embryos were put into a 38℃ incubator for 1h for heat treatment at 12-14 hpf,then these embryos were collected and fixed at 28 hpf and subjected to WISH analysis of runx1 expression.Results: Overexpression of gmfg was insufficient to rescue defects in the expression of runx1/cmyb in the DA region caused by inhibition of Notch,Hedgehog and BMP pathway;activation of c AMP pathway was also insufficient to rescue defects in runx1/cmyb expression caused by gmfg deficiency;compared with control group,gmfg-deficient embryos exhibited weak and discontinuous tp1 signal in the DA region and significantly decreased percentage of tp1+ cells in the whole embryo;further,the number of fli1a+tp1+double positive cells in the DA region at 28 hpf was significantly reduced,the expression of hey1 and hey2 within 48 hpf HE were also significantly reduced;moreover,activation of Notch signaling after heat shock treatment could partially rescue the phenotype of decreased runx1 positive cells caused by gmfg deficiency.Conclusion: Notch signaling acts downstream of gmfg in the regulation of HSC production.The relationship between Hedgehog or BMP or c AMP signaling and gmfg may be mutually independent in the regulation of HSCs generation,or,Hedgehog and BMP signaling may act downstream of gmfg and c AMP signaling act upstream of gmfg. |
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