Study On The Expression And Significance Of EphA/EphirnA In The Development Of Mouse Retina

Objective:Research in the expression of EphA/EphrinA signaling system in normal mouse during the development progress, to explore the significance of EphA/EphrinA in the process of visual development and the guiding role of the axons of retinal photoreceptor cells.Methods:1.There were60normal newborn C57BL/6mice have been raised until the postnatal1day、3day、5day、7day、9day and4week,then randomly chose10mice. Under the dissecting microscope,we can isolated20eye balls from the10anesthetized mice.Half of them were only remained the retinas and the other10maintained the integrity of the eye.2. The5eye balls(from step1) were made into paraffin sections.After the HE staining,we can find the development situation of mice retina in different stages under the light microscope,especially the formation process of outer plexiform layer during P5to P9.3.The10retinas(from step1) were extracted mRNA,then using RT-PCR method to detect the expression of the EphA/EphrinA signaling family from the retina,the highest expression of type is EphA4.4.The5eye balls(from step1)were made into frozen section,using the anti-EphA4and anti-Recoverin(expression in the photoreceptors and bipolar cells) antibodies to test the immunofluorescence staining and observe the growth of axons of photoreceptors and the formation of outer plexiform layer.Results:1.The morphological changes in the developing mouse retina of each periodThe retina structure is relatively simple during the P1to P3,the inner nuclear layer and outer nuclear layer has not separated yet,the out plexiform layer is not formed,the precursor cells differentiated photoreceptors that scattered in the neuroblastoma cells layer,and the axons is short and thin.The photoreceptors becomes mature and the axon structure tends to be complete, which is connect with the puerile bipolar cells,forming the outer pexiform layer from P5to P7.Each layers of the retina cells’structure tends to stability and the outer plexiform layer is clear to be seen in the P9.2.EphA/EphrinA expressed in the mouse retinaUsing the GraphPad Prism to plot the statistical chart that shows EphA4is the highest level in the retina,but EphA6and EphrinA1is the zero in the retina.Using ANOVA from the SPSS17.0software to statistic the EphA4in every time point,N=3,the Mean±SD:P1(0.48±0.33), P3(0.55±0.23), P5(0.76±0.11), P7(1.12±0.88), P9(0.74±0.08),4W (0.66±0.07), P<0.05, the difference was statistically significant.3.Recoverin and EphA4expressed in the mouse retinaIn the period of P1to P3,Recoverin were expressed in the cell membrane and cell body of photoreceptors,which scattered in the neuroblastoma cell layer;EphA4mainly expressed in the ganglion cell and inner plexiform layer.In the period of P5-P7,the axons of photoreceptor tends to mature that expressed Recoverin,the outer plexiform layer formed,individual photoreceptors expressed in the inner nulear layer;EphA4expressed in the ganglion cells as before,but the fluorescence is stronger.On the P9,the Recoverin strongly expressed at the membrane、body and axons of photoreceptors in the outer nuclear layer,the axons of the matured photoreceptors focus to the outer plexiform layer which is stability,the bipolar cells in the inner nuclear layer also expressed weaker fluorescence of Recoverin;EphA4expressed the most stronger fluorescence in the inner plexiform layer and outer plexiform layer,that forms two parallel band,but the outer nuclear layer’s fluorescence almost zero. Conclusion:1. The EphA4of EphA/EphrinA signaling system may palyed a role to the growth of normal mouse retina.2.When the Recoverin expressed on the outer layer of mouse retina,the Epha4expressed on the inner layer of retina,EphA4may probably inpede the inner strength of photoreceptors’development.