Retinal Development In Stmn4 Deficient In Zebrafish Embryos

The optic nerve（mainly the retina nerve）is a special nerve of body in the central nervous system（CNS）,which can conduct and process visual impulses.Since the main functional cells in the retina are neuronal cells,when the development of the optic nerve is damaged,it not only causes visual damage,but also associates with epilepsy,dementia and other brain diseases.Microtuble（MT）is a major component of the neuronal cytoskeleton.It not only supports cell morphology and transports neurotransmitters,but also plays a crucial role in maintaining neuronal protrusion extension and synaptic development.Stathmin family proteins can mediate microtubule depolymerization by participating in the regulation of microtubule dynamic balance,and have a strong correlation with tubulin.Meanwhile,all of genes encoding Stathmins are highly expressed in brain and neurons.Our previous study found that copper overload of zebrafish embryos and juveniles showed retinal cell development defects in the nervous system.At the same time,the expression of stmn4 gene in copper overload of zebrafish embryonic cells was significantly reduced.Studies have shown that the absence of stmn4Stathmin 4/RB3)specifically affects the development of retinal ganglion cells（RGCs）,but the detailed mechanism is still unclear.These results indicated that copper overload may influence the development of fish retinal cells by affecting the expression of stmn4gene.Therefore,this study constructed a stmn4 deletion mutant in zebrafish(stmn4^-/-),identified the function of stmn4 in regulating RGCs,and further revealed its molecular regulating mechanism affecting the development of retinal nerve cells,thus improving the theoretical basis of copper overload affecting the development of fish retinal nerve cells.In the study,we first detected the phenotype of the stmn4^-/-mutant zebrafish and observed that the morphological development of the eye was changed,and the outer nuclear layer（ONL）cells of the cone cells and rod cells were significantly sparse.Subsequently,the movement behavior of the stmn4^-/-zebrafish was detected.It was observed that the sensitivity to light did not change as we thought,but its response ability decreased in stmn4^-/-zebrafish.The expression of optic nerve cell marker genes was significantly down-regulated in the mutant,but the expression of retinal progenitor cells（RPCs）was up-regulated.The expression of microtubule and cell cycle related genes was down-regulated and the expression of apoptosis related genes was up-regulated in the mutant.Mechanistically,we found that the retinal cells of optic nerve progenitor cells in stmn4^-/-mutants could not exit the mitosis and complete the whole cell cycle process during the differentiation of retinal cells,and then apoptosis occurred and the number of differentiated and mature retinal cells decreased.Our experimental methods mainly include hematoxylin-eosin staining（H&E）,immunofluorescence（IF）,whole-mount in situ hybridization（WISH）,qRT-PCR and so on,comprehensively elaborated our research results,and also combined with transcriptome sequencing data to further confirm the above findings and conclusions.This paper reveals the mechanism of stmn4 in affecting the development of zebrafish optic nerve cells,identify the cell differentiation stages and processes that mainly participates in and affects.At the same time,the mechanism of its influence is verified by transcriptome data,which provides a new way of thinking for modulating the development process of optic nerve cells.