Publicação
Fate of the earliest retinal ganglion cells during development of the visual system
| Resumo: | During development of the mammalian eye, the first retinal ganglion cells (RGCs) that extend to the brain are located in the dorsocentral retina, in the mouse, during mid-embryogenesis. These RGCs extend to either ipsilateral or contralateral targets, but the ipsilateral projections from the central retina do not survive into postnatal periods. The function and means of disappearance of the transient ipsilateral projection are not known. We have followed the course of this transient early ipsilateral RGC cohort, paying attention to how far they extend, whether they enter target regions in the brain and if so, which ones, and the time course of their disappearance. Several different techniques were tested for labeling the axonal projection from the central retina. While the use of a viral vector and a conditional Brn3b knock-out mouse presented difficulties in specifc labeling of RGCs and of ipsi- or contralateral projections, respectively, both the application of the lipophilic tracer DiI and in utero electroporation of GFP into central retina allowed the analysis of the growth and position of the ipsilateral and contralateral retinal projections. The proportion of ipsi- versus contralateral projections was calculated at E13.5 and 15.5 using DiI. In utero electroporation of E12.5 retina with GFP constructs was used to label axons prospectively into postnatal ages. Our results show that the earliest ipsilateral axons are segregated from the laterally-positioned contralateral axons in the optic tract. In agreement with previous reports, we found that the number of central retina ipsilateral RGCs declines after E16.5. Nonetheless, some ipsilateral axons from the central retina enter the superior colliculus (SC) and arborize minimally, but very few enter the dorsal lateral geniculate nucleus (dLGN). To understand whether caspases are involved in the disappearance of the ipsilateral projection from the central retina, immunohistochemistry experiments were performed but without conclusive results. To identify candidate genes expressed in ipsilateral vs contralateral RGC axons in the central retina at E13.5, a technique that combines retrograde labeling with DiI, applied retrogradely, with immunohistochemistry was developed. To date, no molecular marker was found that selectively labeled the transient ipsilaterally-projecting RGC axons from central retina. The results of this work and the methods developed will be useful to better understand the elimination of transient axonal projections, and their role in establishing neuronal circuits. |
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| Autores principais: | Soares, Célia Márcia Azevedo |
| Assunto: | Ciências Médicas::Medicina Básica |
| Ano: | 2016 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | During development of the mammalian eye, the first retinal ganglion cells (RGCs) that extend to the brain are located in the dorsocentral retina, in the mouse, during mid-embryogenesis. These RGCs extend to either ipsilateral or contralateral targets, but the ipsilateral projections from the central retina do not survive into postnatal periods. The function and means of disappearance of the transient ipsilateral projection are not known. We have followed the course of this transient early ipsilateral RGC cohort, paying attention to how far they extend, whether they enter target regions in the brain and if so, which ones, and the time course of their disappearance. Several different techniques were tested for labeling the axonal projection from the central retina. While the use of a viral vector and a conditional Brn3b knock-out mouse presented difficulties in specifc labeling of RGCs and of ipsi- or contralateral projections, respectively, both the application of the lipophilic tracer DiI and in utero electroporation of GFP into central retina allowed the analysis of the growth and position of the ipsilateral and contralateral retinal projections. The proportion of ipsi- versus contralateral projections was calculated at E13.5 and 15.5 using DiI. In utero electroporation of E12.5 retina with GFP constructs was used to label axons prospectively into postnatal ages. Our results show that the earliest ipsilateral axons are segregated from the laterally-positioned contralateral axons in the optic tract. In agreement with previous reports, we found that the number of central retina ipsilateral RGCs declines after E16.5. Nonetheless, some ipsilateral axons from the central retina enter the superior colliculus (SC) and arborize minimally, but very few enter the dorsal lateral geniculate nucleus (dLGN). To understand whether caspases are involved in the disappearance of the ipsilateral projection from the central retina, immunohistochemistry experiments were performed but without conclusive results. To identify candidate genes expressed in ipsilateral vs contralateral RGC axons in the central retina at E13.5, a technique that combines retrograde labeling with DiI, applied retrogradely, with immunohistochemistry was developed. To date, no molecular marker was found that selectively labeled the transient ipsilaterally-projecting RGC axons from central retina. The results of this work and the methods developed will be useful to better understand the elimination of transient axonal projections, and their role in establishing neuronal circuits. |
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