Functions And Mechanisms Of SCF/cKit Signaling In The Early Development Of Human Embryonic Eye

Background:The eye is the most important sensory organ of the human body.Unlike other organs of the body,visual organs need special morphological structure and elaborate size control so that light can focus on the macular area of the retina to form a clear image.Optic cup（OC）is an important structure formed during the early development of embryonic eyes.Any abnormality in the formation of OC will lead to serious eye diseases,such as anophthalmia,microphthalmia and coloboma.The invagination of nerve retina（NR）is the most critical step in the formation of optic cup.It includes three continuous local processes:relaxation of nerve retina,contraction of hinge cells and expansion of nerve retinal tissue.The change of tissue biomechanics is the fundamental cause of tissue deformation.The core of tissue biomechanics is the tissue tension provided by cell proliferation and the contraction force provided by cell local contraction.The three local processes of cup formation occur at the junction of primitive retinal pigment epithelium（RPE）and primitive NR,namely ciliary marginal zone（CMZ）in the further development.Whether ciliary marginal cells are involved in the formation of optic cup and the cellular and molecular mechanisms involved in biomechanical regulation are not clear.cKit surface antigen,also known as SCFR or CD117,as a membrane receptor mediating extracellular signals,has been expressed at a certain level in the retina at the beginning of embryonic eye development.In vitro,the expression of cKit in retinal ciliary epithelium（CE）neural stem cells was up-regulated,suggesting that cKit-mediated signal transduction may be one of the mechanisms regulating the behavior of neuroretinal stem cells.However,the specific functions and regulatory mechanisms of cKit in the physiological and pathological processes of embryonic eye development are not clear.Objective:To study the expression of cKit surface antigen in early embryonic development and its relationship with ciliary marginal zone.To study the function and mechanism of SCF/cKit signaling pathway in the formation of optic cup in early embryonic eye development,and the function and mechanism of SCF/cKit signaling pathway in neurogenesis in early embryonic eye development.To study the function and mechanism of SCF/cKit signaling pathway in the pathological process of ocular dysplasia in fetal alcohol syndrome（FAS）.Hypothesis:SCF/cKit signaling pathway plays an important role in the early development of hESC-derived 3D optic cup:SCF/cKit signaling regulates the cell cycle of retinal stem/progenitor cells proliferation in CMZ of hESC-derived 3D optic cup,changes the biomechanics of ciliary rim tissue,and promotes the morphogenesis of optic cup.Interpretation:Elaborate the function and mechanism of SCF/cKit signaling pathway in the physiological and pathological process of embryonic eye development were elaborated.Improve the regulation mechanism of morphological formation and neurogenesis during early embryonic eye development,and provid theoretical basis for early diagnosis,treatment and research of ocular developmental diseases.Methods:In this study,human embryonic stem cells（hESCs）were used to induce 3D optic cups in vitro.Immunofluorescence staining and RT-PCR were used to verify its reliability of mimic the early development of human embryonic eyes.Immunofluorescence staining and two-photon microscopy in vivo calcium imaging were used to investigate the cell diversity and characteristics of ciliary marginal zone.The expression of cKit and its ligand SCF in hESCs-derived 3D optic cup was studied.The expression of cKit and its ligand in CMZ was investigated.The pharmacological modulation of SCF/cKit signaling by cKit-activated ligand SCF and specific inhibitor isck03 was used to study the function and mechanism of SCF/cKit signaling in the morphogenesis and neurogenesis of hESCs-derived 3D optic cups,studied by morphological measurement,BrdU labeling and immunofluorescence staining.Ethanol and hESCs-derived 3D optic cups were used to established the model of ocular developmental malformation of FAS.Immunofluorescence staining and two-photon microscopy in vivo calcium imaging technology were used to verify the pathological process of the pathological model of FAS.Combining with pharmacological intervention,the function and mechanism of SCF/cKit signaling pathway regulation in the pathological process of ocular developmental malformation of FAS were studied.Results:hESCs-derived 3D optic cups have typical cup-like structures of NR and RPE,and express retinal development-specific genes such as Rax,Chx10,Pax6 and MITF,well simulating the early development of human embryonic eyes.At the same time,hESCs-derived3D optic cups can develop a typical long wedge-shaped ciliary margin structure,and positive express the human ciliary margin specific molecular marker SSEA1.Retinal stem/progenitor cells from the CMZ of hESCs-derived 3D optic cups can be further divided into Chx10⁺Sox2^-cells and Chx10⁺Sox2⁺cells.In addition,retinal stem/progenitor cells in the CMZ of hESCs-derived 3D optic cups emitted mainly transient whole cell high-amplitude calcium waves and local low-amplitude high-frequency calcium waves.During the development of hESCs-derived 3D optic cup,the expression of cKit was specifically distributed in the NR of hESCs-derived 3D optic cup,enriched in the CMZ,and was confined to the CMZ at the 30th day of induction,and lost expression with further development.The expression of SCF mainly distributes in the bottom of the inner layer of NR and RPE.SCF/cKit signaling pathway can regulate the cell cycle progression of retinal stem cells/progenitor cells in CMZ of hESCs-derived 3D optic cup,increase the BrdU uptake and dilution rate of retinal stem cells/progenitor cells in CMZ,increase or decrease the cell proliferation rate,enhance the tangent tension provided by cell proliferation,and accelerate the invagination of the NR to form optic cup morphology.In addition,SCF/cKit signaling pathway can regulate the top tension of ciliary marginal cells,and coordinate the cytoskeleton assembling and dis assembling,so as to facilitate the deformation of NR and promote the formation of optic cup.At the same time,SCF/cKit signaling pathway is involved in regulating the migration and maturation of immature ganglion cells in the central NR of hESCs-derived 3D optic cup,regulating the apoptosis during development,and influencing the fate choice and differentiation of retinal terminal precursor cells.In vitro,1%ethanol concentration can effectively block the development of NR in the hESCs-derived 3D optic cup,increase cell apoptosis,change the developmental calcium spiking characteristics of retinal progenitor cells,hinder the migration and maturation of immature ganglion cells,and effectively simulate the pathological process of ocular developmental malformation of FAS.Activation of SCF/cKit signaling pathway can reduce cell apoptosis,reverse ganglion cell migration defection and maintain the normal tissue development of hESCs-derived 3D optic cup.Conclusions:cKit was characterized by high expression in the CMZ of hESCs-derived 3D optic cups.At the 30th day of induction,cKit⁺cells can be divided into two types according to the cKit expression level:cKit^+strong retinal stem/progenitor cells located in peripheral NR and cKit^+dim retinal progenitor cells located in inner layer of central NR.The ciliary marginal cells in the hESCs-derived 3D optic cups are involved in the formation of the optic cup through the regulation of SCF/cKit signaling pathway.SCF/cKit signaling pathway in the development of3D optic cup derived from hESCs can regulate the cell top tension of CMZ by regulating the cycle progression of ciliary marginal stem/progenitor cells,change the local cell system dynamics,promote the NR invagination and deformation,and accelerate the formation of optic cup.In addition,SCF/cKit signaling pathway can also regulate the migration of immature ganglion cells in the NR of hESCs-derived 3D optic cups,regulate the apoptosis of NR cells,affect the fate choince of retinal progenitor cells,and regulate retinal neurogenesis.The activation of SCF/cKit signaling pathway in the hESCs-derived 3D optic cups can reduce NR cell apoptosis,reverse the defective migration of immature ganglion cells,and maintain normal tissue growth and neurogenesis of NR,prividing resistance of the pressure of pathological factors in the pathological process of embryonic eye developmental diseases,such as eye developmental malformation of FAS.