Our lab’s work has revealed molecular programs of transcription and axon guidance factors that distinguish the ipsilateral and contralateral retinal ganglion cell (RGC) pathways through the optic chiasm and to thalamic targets in the mouse brain. Currently, we aim to uncover additional factors that determine and specify the ipsi- and contralateral RGC populations through studying spatiotemporal aspects of their generation, namely regulation of RGC neurogenesis by the cell cycle regulator Cyclin D2, and probing the ciliary margin zone (CMZ) as a source of RGC subpopulations. In parallel, we are investigating pre-target axon organization and mechanisms of axon fasciculation in the developing pathway from eye to thalamus, revealing additional ipsi-contralateral RGC differences. We are also interrogating how axon targeting and cell specification go awry in the albino visual system, in which the lack of melanin leads to a reduction of the ipsilateral projection through altered timing of neurogenesis. Current projects are detailed below.
Retinal Ganglion Cell neurogenesis and specification
With the characterization of axon guidance factors in the last two decades, the field has aimed to characterize transcriptional programs that regulate the expression of guidance factors and their receptors on growing axons and in the terrain through which they grow. We identified Zic2 as a master gene for the ipsilateral RGC program of differentiation. Through promoter analysis of NrCAM and Plexin-A1, key to contralateral axon guidance, we identified the SoxCs (Sox4, 11, 12) as transcriptional regulators of contralateral but not ipsilateral RGC differentiation (Kuwajima et al., Neuron, 2017).
In addition, we analyzed genes expressed in the two subpopulations, identified in a novel unbiased screen (Wang et al., eNeuro, 2016) that revealed that the cell cycle regulator Cyclin D2 is enriched in ventral retina, and that Cyclin D2 is critical for output of RGCs from this zone (Marcucci et al., Cell Reports, 2016). This work has rekindled the controversy that the CMZ may a source of progenitors of retinal cells in higher vertebrates as in lower vertebrates. Live imaging indicated that cells translocate laterally from the CMZ to populate the neural retina (Marcucci et al., 2016). Next steps are to fate-map the ipsi- can contralateral cells from the CMZ, dissect the biology of Cyclin D2, and study how the CMZ is patterned.
Axon guidance in the optic tract
Organization of RGC axons post-chiasm before targets: Using dye-labeling and a genetic model in which ipsilateral axons are labeled (Sert, the serotonin transporter), we observed that RGC axons are organized by eye-specificity and topography in the optic nerve and tract. Ipsilateral RGC axons are offset laterally in the tract relative to contralateral axon topographic position, suggesting molecular differences between the two cohorts. In an in vitro assay, we demonstrated that ipsilateral RGC neurites have a greater preference for self-fasciculation, or bundling, compared to contralateral neurites. In addition, we examined RGC axon organization and fasciculation in the EphB1 KO mutant in which a subset of ipsilateral RGC axons aberrantly crosses the midline, but still targets the ipsilateral zone in the dorsal lateral geniculate nucleus. The aberrantly crossing axons retain their association with the remaining ipsilateral axons in the contralateral tract, further supporting the in vitro and wild type in vivo pattern of cohort-specific axon organization throughout the pathway. These data pave the way for tests of molecular candidates specific to ipsi- or contralateral RGCs, as indicated in our gene profiling of these cells, that underlie the segregation of the axons and their ultimate targeting - an unanswered question in the field.
The albino visual system
In albinos across mammalian species, disruption of pigmentation in the RPE results in a reduced ipsilateral RGC projection. Our previous work demonstrated that neurogenesis is perturbed in the albino embryonic retina - ipsilateral RGC genesis is delayed, production of Zic2-positive RGCs is reduced, and production/specification of contralateral RGCs is increased, with concomitant alterations in retinogeniculate connectivity. In Marcucci et al., 2016, we observed that Cyclin D2 expression is also reduced, placing the focus on this factor and the CMZ as an earlier developmental phase during which neurogenetic perturbations in the albino can be studied.
We also chronicled the pathology of the embryonic albino RPE: cell shape, melanosome disposition, and gap junction protein (Cx43) expression, localization and phosphorylation state are aberrant (Iwai-Takekoshi et al., J Comp Neurol, 2016). We hypothesize that perturbation of RPE integrity could alter transmission of factors from RPE to neural retina that in turn regulate RGC neurogenesis and specification.
Under review is a study on gene profiling of albino and pigmented RPE that indicates that cytoskeleton, junctional and patterning genes (Wnts) are dysregulated in the albino RPE. In experiments activating the Wnt pathway (lithium chloride injections into pigmented mice), ipsilateral (Zic2+) cells are reduced, mimicking the albino phenotype. We hypothesize that perturbation of RPE integrity could alter transmission of factors from RPE to neural retina that in turn regulate RGC neurogenesis and specification. New studies will focus on how the CMZ and early phases of eye and RGC development proceed in the absence of melanin.
Glial phagocytosis in the dlgn
In collaboration with Dr. Mimi Shirasu-Hiza, we utilize the visual system of the Fragile X Syndrome model mouse (Fmr1 KO) to study glial-mediated synaptic refinement. In a Drosophila model of Fragile X Syndrome, Dr. Shirasu-Hiza has shown that glia engulfment of neuronal inputs is defective in elimination of supernumerary synapses during development and after injury. In mice, work from other labs implicates glia in the developmental refinement of ipsilateral and contralateral RGC axon arbors and their synaptic connections. We are extending Dr. Shirasu-Hiza’s work into the mouse retinogeniculate system by examining the developmental refinement of ipsilateral and contralateral RGCs in the Fmr1 KO mouse compared with WT mice, and assaying glial number, morphology, and cellular behavior during visual system development.