Abstract
Purpose:
Retinal axon divergence at the optic chiasm is key to establishing the binocular visual pathway. Specific axon guidance receptors and their ligands, expressed in retinal ganglion cells (RGCs) and at the chiasm, tightly regulate 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. To identify additional genes important for ipsi vs. contra RGC identity, we developed a method of purifying these RGC subtypes based on their anatomical projection and investigated the function of candidate genes following gene profiling.
Methods:
Ipsi and contra RGCs were isolated from embryonic day (E)16 mouse embryos via retrograde labeling from the optic tract followed by fluorescence-activated cell sorting. Microarray data was analyzed using GeneSpring GX11. Expression of select candidates was validated by qPCR, in situ hybridization, and immunohistochemistry in E13-16 wild-type embryos. Ccnd2-/- embryos were analyzed for spatiotemporal expression changes of cell cycle and ipsi, contra, or pan-RGC markers. Retinal axon projections were labeled anterogradely with DiI or CTB and retrogradely with rhodamine-dextran.
Results:
We identified and validated a number of membrane proteins, transcription factors, signaling molecules, and cell cycle genes that may regulate axon guidance, cell differentiation, and fate specification of ipsi vs. contra RGCs. Interestingly, ipsi but not contra RGCs retain some expression of RGC precursor markers. We also identified a novel role for cyclin D2 in the production of ipsi RGCs. Cyclin D2 is highly expressed in ventral peripheral retina preceding and coincident with ipsi RGC genesis unlike cyclin D1, which shows consistent expression levels in progenitors throughout the retina. Cyclin D2-/- embryos show a decrease in ipsi RGCs and their corresponding retinal projections.
Conclusions:
Ipsi and contra RGCs are defined by distinct molecular signatures during development, and mechanisms directing cell differentiation may influence RGC fate decisions. One such factor, cyclin D2, is important for the production of ipsi RGCs. These developmental pathways can be harnessed in cell replacement therapy of RGC subtypes and regeneration of their axons.
Keywords: 698 retinal development •
531 ganglion cells •
500 differentiation