Neuronal Diversification Within the Retina: Generation of Crossed and Uncrossed Retinal Ganglion Cells

摘要

Recent advances in the field of axon guidance have revealed complex transcription factor codes that regulate neuronal subtype identity and their corresponding axon projections. Retinal axon divergence at the optic chiasm midline is key to the establishment of binocular vision in higher vertebrates. In the visual system of binocular animals, the ipsilaterally and contralaterally projecting retinal ganglion cells are distinguished by the laterality of their axonal projections. Specific axon guidance receptors and their ligands are expressed in retinal ganglion cells (RGCs) and at the chiasm, tightly regulating the development of the ipsilateral (uncrossed) and contralateral (crossed) retinal projections. Though many factors are known, their dysfunction leads to only partial misrouting of RGC axons. Moreover, the complex transcription factor codes that regulate RGC subtype identity are only beginning to be uncovered. Numerous gaps remain in our understanding of how these guidance molecules are transcriptionally regulated and how they are induced by the patterning genes that set up the different domains in which these RGC subtypes reside. An even more elusive question within the field is how the ipsilateral and contralateral RGC subpopulations acquire their different cell fates. In this thesis, I present my work on dissecting out the molecular signatures of the ipsilateral and contralateral RGC populations during embryonic development through gene profiling followed by the functional characterization of one candidate from this screen. In Chapter 2, I developed a cell purification method based on retrograde labeling of these two cell populations from their divergent axonal projections followed by cell sorting. This method can be used in studies requiring purified populations of embryonic RGCs. In Chapter 3, I conducted a microarray screen of purified ipsilateral and contralateral RGCs using the above method. Through subsequent validation of the in vivo expression patterns of select candidates, I identified a number of genes that are differentially expressed in ipsilateral and contralateral RGCs. Subsequent functional characterization of these genes has the potential to uncover novel mechanisms for regulating axon guidance, cell differentiation, fate specification, and other regulatory pathways in ipsilateral and contralateral RGC development and function. The results of this screen also revealed that ipsilateral and contralateral RGC may have distinct developmental origins and utilize different strategies for differentiation. In Chapter 4, I demonstrate a novel role for cyclin D2, one of the above candidates, in the production of ipsilateral RGCs. The G1-active cyclin D2 is highly expressed in the ventral peripheral retina preceding and coincident with the developmental window of ipsilateral RGC genesis. I further found that ipsilateral RGC production is disrupted in the cyclin D2 null mouse. The expression of cyclin D2 in a distinct proliferative zone that has evolutionary significance in ipsilateral RGC production and its subtype-specific requirement during retinal development suggest that cyclin D2 may mark a distinct progenitor pool for ipsilateral RGCs. Thus, these studies offer an important advance in our understanding of neuronal subtype diversification within the retina.