SPNS2 Regulates Retinal Morphogenesis By Activating S1P Receptor 3 Signaling

Background:Spinster homolog 2?SPNS2?is recently discovered as a membrane transporter mediating sphingosine-1-phosphate?S1P?secretion.S1P,synthesized intracellularly and transported out of cells by SPNS2,binds five S1P receptors?S1P receptors1-5,S1PR1-5?to exert various kinds of biological activities.SPNS2 is the only transporter identified so far to mediate S1P secretion in vivo,which helps clarify the underlying mechanisms that how physiological S1P levels are established and maintained.Previous studies showed that ATP-binding cassette?ABC?transporters may be able to mediate S1P secretion in vitro.Given no overt changes of S1P levels are detected in abc mutant animals,there is no direct evidence supporting that ABC transporters participate in the process of S1P secretion in vivo.In contrast,several studies demonstrated SPNS2affects various pathophysiological processes by regulating in vivo S1P levels in different conditions such as the migration of myocardial precursors,the maintenance of the circulating levels of S1P,angiogenesis,the migration of lymphocytes,the maintenance of auditory function,the pathophysiological regulation of alveolar epithelial cells,and the process of liver fibrosis.However,no evidence shows that SPNS functions in the central nervous system.Objectives:Our current studies use a rat strain that shows a global spns2 deficiency caused by the insertion of the egfp sequence in its 1^st intron.The spns2-deficient rats display serious periocular phenotypes,such as eyelid-open at birth?EOB?,corneal opacity,and symblepharon.In addition,we discovered intraocular phenotypes including retinal laminar structure disorder and angiogenesis defect,which indicate SPNS2 likely participates in the retinal morphogenesis.Therefore,the ocular phenotypes are analyzed in this study to clarify the functions and underlying mechanisms of SPNS2 in the process of retinal development,which provides a theoretical basis for exploring the potential value in the treatment of associated diseases.Results:These defects are first visible at birth in spns2-deficient retinas as tiny wrinkles,which rapidly develop into rosette formations during the later stages of development.We demonstrated that EOB phenotype does not account for early retinal laminar disorganization by using two different approaches:either establishing an artificial EOB model using spns2^+/+rats or performing a rescue experiment for the EOB phenotype in spns2^-/-animals.The abnormal morphogenesis is the result of excessive proliferation of retinal progenitor cells?RPCs?,which is caused by both higher frequency of symmetric division and inefficient cell-cycle exit.This phenomenon is associated with the aberrant expression and distribution of apical junction?AJ?complex.Firstly,the disrupted distribution of polarity proteins in mutant retinas occurs before the morphology of the retina is affected,indicating that maintaining the polarity of RPCs is one of the fundamental mechanisms for SPNS2 to regulate retinal development.Loss of cell polarity in spns2-deficient retinas,which is considered as the primary cause of laminar disorganization,leads to the emergence of abundant ectopic dividing cells.In most ectopic dividing cells,the adhesion proteins distribute around the nuclei and are equally allocated into both daughter cells.It has been established for dividing RPCs that the inheritance of adhesion molecules after mitosis is related to the cell fate.Thus,the equal distribution of adhesion proteins leads to the production of more daughter cells that remain the progenitor cell fate,rather than entering the neuronal fate after division,which accounts for the over-proliferation found in spns2-deficient retinas.Secondly,in physiological conditions,the levels of adhesion molecules in RPCs drop dramatically during late retinal development,with the distribution changing from previous cell-body-surrounding pattern to a limited linear distribution along the apical membrane to prompt the cell-cycle exit.The SPNS2/S1PRs signaling pathway plays an important role in the processes of down-regulating the expression levels of adhesion proteins and inducing cell-cycle exit.The absence of SPNS2 causes high expression levels of adhesion molecules at the late stage of retinal development,which interrupts the cell-cycle exit of RPCs and their differentiation into mature neurons.This provides another potential mechanism for the over-proliferation of RPCs occurs in spns2-deficient retinas.Thirdly,SPNS2 transports endogenous S1P out of cells to activate Rac1 signaling pathway,which induces post-mitotic RPCs migrating to different locations and differentiating into appropriate cell types.The migration of post-mitotic RPCs is compromised in spns2-deficient retinas,which causes the improper stay of these cells in the outer nuclear layer?ONL?.The redundant cells further differentiate into rod photoreceptors,which eventually leads to the significantly increased cell number in the ONL and the disorder of retinal structure.S1PR3 is probably one key functioning receptor accounting for the postnatal stage of retinal development,since down-regulating of s1pr3 at neonatal stage in spns2^+/+retinas can mimic the phenotypes of retinas lack of functional SPNS2.Conclusions:In this study,we firstly establish that SPNS2/S1PR3 signaling participates in the regulation of postnatal stage of retinal development,which plays important roles in maintaining the cell polarity of RPCs,promoting cell-cycle exit,and inducing the migration of post-mitotic newborn neurons.Our findings shed light on the development of a novel therapeutic target for the treatment of various kinds of ophthalmic diseases such as retinal tumors,ocular trauma and blood vessel associated oculopathy.Meanwhile,these data provide a theoretical basis for exploring the functions of SPNS2 in the central nervous system.