Role of PKN1 in Retinal Cell Type Formation.

We recently identified PKN1 as a developmentally active gatekeeper of the transcription factor neuronal differentiation-2 (NeuroD2) in several brain areas. Since NeuroD2 plays an important role in amacrine cell (AC) and retinal ganglion cell (RGC) type formation, we aimed to study the expression of NeuroD2 in the postnatal retina of WT and Pkn1-/- animals, with a particular focus on these two cell types. We show that PKN1 is broadly expressed in the retina and that the gross retinal structure is not different between both genotypes. Postnatal retinal NeuroD2 levels were elevated upon Pkn1 knockout, with Pkn1-/- retinae showing more NeuroD2+ cells in the lower portion of the inner nuclear layer. Accordingly, immunohistochemical analysis revealed an increased amount of AC in postnatal and adult Pkn1-/- retinae. There were no differences in horizontal cell, bipolar cell, glial cell and RGC numbers, nor defective axon guidance to the optic chiasm or tract upon Pkn1 knockout. Interestingly, we did, however, see a specific reduction in SMI-32+ α-RGC in Pkn1-/- retinae. These results suggest that PKN1 is important for retinal cell type formation and validate PKN1 for future studies focusing on AC and α-RGC specification and development.

Visuomotor anomalies in achiasmatic mice expressing a transfer-defective Vax1 mutant.

In binocular animals that exhibit stereoscopic visual responses, the axons of retinal ganglion cells (RGCs) connect to brain areas bilaterally by forming a commissure called the optic chiasm (OC). Ventral anterior homeobox 1 (Vax1) contributes to the formation of the OC, acting endogenously in optic pathway cells and exogenously in growing RGC axons. Here, we generated Vax1AA/AA mice expressing the Vax1AA mutant, which is incapable of intercellular transfer. We found that RGC axons cannot take up Vax1AA protein from the Vax1AA/AA mouse optic stalk (OS) and grow slowly to arrive at the hypothalamus at a late stage. The RGC axons of Vax1AA/AA mice connect exclusively to ipsilateral brain areas after failing to access the midline, resulting in reduced visual acuity and abnormal oculomotor responses. Overall, our study provides physiological evidence for the necessity of intercellular transfer of Vax1 and the importance of the bilateral RGC axon projection in proper visuomotor responses.

Cis-p-tau plays crucial role in lysolecithin-induced demyelination and subsequent axonopathy in mouse optic chiasm.

Multiple sclerosis (MS) is an autoimmune demyelinating disease that leads to axon degeneration as the major cause of everlasting neurological disability. The cis-phosphorylated tau (cis-p-tau) is an isoform of tau phosphorylated on threonine 231 and causes tau fails to bind micro-tubules and promotes assembly. It gains toxic function and forms tangles in the cell which finally leads to cell death. An antibody raised against cis- p-tau (cis mAb) detects this isoform and induces its clearance. Here, we investigated the formation of cis-p-tau in a lysophosphatidylcholine (LPC)-induced prolonged demyelination model as well as the beneficial effects of its clearance using cis mAb. Cis -p-tau was increased in the lesion site, especially in axons and microglia. Behavioral and functional studies were performed using visual cliff test, visual placing test, and visual evoked potential recording. Cis-p-tau clearance resulted in decreased gliosis, protected myelin and reduced axon degeneration. Analysis of behavioral and electrophysiological data showed that clearance of cis-p-tau by cis mAb treatment improved the visual acuity along with the integrity of the optic pathway. Our results highlight the opportunity of using cis mAb as a new therapy for protecting myelin and axons in patients suffering from MS.

Multi-Elemental Analysis of Human Optic Chiasm-A New Perspective to Reveal the Pathomechanism of Nerve Fibers' Degeneration.

The effect of metals on the functioning of the human eye is multifactorial and includes enzyme activity modulation, trace metal metabolic pathways changes, and cytotoxic activity. Functional dysfunctions appear mostly as a result of the accumulation of toxic xenobiotic metals or disturbances of micronutrients' homeostasis. So far, the affinity of selected metals to eye tissues, i.e., the cornea, choroid, lens, and anterior chamber fluid, has been most studied. However, it is known that many eye symptoms are related to damage to the optic nerve. In order to fill this gap, the aim of the study is to perform a multi-element analysis of tissue collected postmortem from optic chiasm and optic nerves. A total of 178 samples from 107 subjects were tested. The concentrations of 51 elements were quantified by inductively coupled plasma mass spectrometry (ICP-MS) after the wet-mineralization step. In terms of elemental composition, the optic chiasm is dominated by two trace elements, i.e., iron (Fe) and zinc (Zn), besides macro-elements Ca, K, Na, P, and Mg. The subjects formed a homogeneous cluster (over 70% subjects) with the highest accumulation of aluminum (Al). The remaining two departing clusters were characterized by an increased content of most of the elements, including toxic elements such as bismuth (Bi), uranium (U), lead (Pb), chromium (Cr), and cadmium (Cd). Changes in elemental composition with age were analyzed statistically for the selected groups, i.e., females, males, and subjects with alcohol use disorder (AUD) and without AUD. A tendency of women to lose Se, Cu, Zn, Fe with age was observed, and a disturbed Ca/Mg, Na/K ratio in subjects with AUD. Although the observed trends were not statistically significant, they shed new light on the risks and possible pathologies associated with metal neurotoxicity in the visual tract.

Slit2 is necessary for optic axon organization in the zebrafish ventral midline.

Slit-Robo signaling has been implicated in regulating several steps of retinal ganglion cell axon guidance, with a central role assigned to Slit2. We report here the phenotypical characterization of a CRISPR-Cas9-generated zebrafish null mutant for this gene, along with a detailed analysis of its expression pattern by WM-FISH. All evident defects in the optic axons in slit2-/- mutants were detected outside the retina, coincident with the major sites of expression at the ventral forebrain, around the developing optic nerve and anterior to the optic chiasm/proximal tract. Anterograde axon tracing experiments in zygotic and maternal-zygotic mutants, as well as morphants, showed the occurrence of axon sorting defects, which appeared mild at the optic nerve level, but more severe in the optic chiasm and the proximal tract. A remarkable sorting defect was the usual splitting of one of the optic nerves in two branches that surrounded the contralateral nerve at the chiasm. Although all axons eventually crossed the midline, the retinotopic order appeared lost at the proximal optic tract, to eventually correct distally. Time-lapse analysis demonstrated the sporadic occurrence of axon misrouting at the chiasm level, which could be responsible for the sorting errors. Our results support previous evidence of a channeling role for Slit molecules in retinal ganglion cell axons at the optic nerve, in addition to a function in the segregation of axons coming from each nerve and from different retinal regions at the medio-ventral area of the forebrain.

BMP Signaling Interferes with Optic Chiasm Formation and Retinal Ganglion Cell Pathfinding in Zebrafish.

Decussation of axonal tracts is an important hallmark of vertebrate neuroanatomy resulting in one brain hemisphere controlling the contralateral side of the body and also computing the sensory information originating from that respective side. Here, we show that BMP interferes with optic chiasm formation and RGC pathfinding in zebrafish. Experimental induction of BMP4 at 15 hpf results in a complete ipsilateral projection of RGC axons and failure of commissural connections of the forebrain, in part as the result of an interaction with shh signaling, transcriptional regulation of midline guidance cues and an affected optic stalk morphogenesis. Experimental induction of BMP4 at 24 hpf, resulting in only a mild repression of forebrain shh ligand expression but in a broad expression of pax2a in the diencephalon, does not per se prevent RGC axons from crossing the midline. It nevertheless shows severe pathologies of RGC projections e.g., the fasciculation of RGC axons with the ipsilateral optic tract resulting in the innervation of one tectum by two eyes or the projection of RGC axons in the direction of the contralateral eye.

Chiasmal herniation following treatment of pituitary macroadenoma.

PURPOSE: To evaluate whether the occurrence of chiasmal herniation coincides with visual field (VF) deterioration and to compare the course of VF defects in patients with and without radiological chiasmal herniation following treatment of pituitary adenoma. METHODS: This retrospective cohort study included 48 pituitary macroadenoma patients with chiasm compression, divided into three groups: Group 1 (N = 12), downward displaced optic chiasm and deteriorated VFs; Group 2 (N = 16), downward displaced optic chiasm; Group 3 (N = 20), control-group matched for tumour size and follow-up VFs, in mean deviation (dB). VFs were compared over time and a severity index, Chiasm Herniation Scale (CHS), for herniation based on radiological parameters was designed. RESULTS: After treatment, all groups showed improvement of VFs (Gr1: 2.97 dB p = 0.097, Gr2: 4.52 dB p = 0.001 and Gr3: 5.16 dB p = 0.000), followed by long-term gradual deterioration. The course of VFs between patients with and without herniation was not significantly different (p = 0.143), neither was there a difference in the course before and after herniation (p = 0.297). The median time till onset of herniation was 40 months (IQR 6 month-10 years) and did not significantly differ (p = 0.172) between the groups. There was no relation between VFs and the degree of herniation (p = 0.729). CONCLUSION: Herniation does not appear to have clinical relevance with respect to VF outcome. The newly designed CHS is the first scoring system to quantify the severity of herniation and, in the absence of alternatives, may be useful to describe MRI findings to serve future added value in larger sized outcome studies.

Arbutin Improves Functional Recovery and Attenuates Glial Activation in Lysolecethin-Induced Demyelination Model in Rat Optic Chiasm.

Neuroinflammation, glial activation, and oxidative injury are the main pathological mechanisms of demyelination in multiple sclerosis (MS). Arbutin, a natural polyphenol compound, possesses antioxidant, anti-inflammatory, and neuroprotective properties whose therapeutic potential has not been studied in the experimental animal models of MS. In the present study, the efficiency of arbutin on lysolecthin (LPC)-induced local demyelination model was investigated. Demyelination was induced by micro-injection of 2 µl LPC (1%) into the rat optic chiasm and the treated group received daily injection of arbutin (50 mg/kg, i.p) during 2 weeks. Visual-evoked potential (VEP) recordings were used to functionally assess the visual pathway. Gene expression analysis was done to evaluate the arbutin effect on the inflammatory, stress oxidative-related mediators, and myelin markers. The myelin-specific staining was performed to assess demyelination and GFAP staining as an astrocyte marker. We found that arbutin significantly reduced P1-latency of VEPs waves and demyelination at 7 and 14 days post-demyelination. Arbutin decreased inflammatory cytokines (IL-1B, IL-17, TNF-α) and iNOS mRNA expression level. In addition, the expression level of anti-inflammatory cytokine (IL-10) and antioxidant mediators (Nrf-2 and HO-1) was enhanced by arbutin treatment. Arbutin increased MBP and Olig2 expression levels in demyelination context. Finally, arbutin attenuated GFAP as an astrocyte marker. Finally, this study demonstrates that arbutin improves functional recovery and myelin repair in the demyelinated optic chiasm through attenuation of inflammation, astrocyte activation, and oxidative stress. These findings might open new promising avenues for treating demyelinating disorders such as multiple sclerosis. Graphical abstract.

Analysis of axon divergence at the optic chiasm in nogo-a knockout mice.

Our earlier studies have shown that the axon growth inhibitory molecule Nogo affects axon routing at the optic chiasm likely through a differential regulation of Nogo receptor on the optic axons. Using isoform specific antibodies, we further showed that Nogo-A was predominantly expressed by retinal ganglion cells and their axons, while Nogo-B was highly localized on the radial glia at the midline of the chiasm, suggesting a role of Nogo-B in regulating turning of uncrossed axons. To further investigate the roles of Nogo-A in axon divergence, we analyzed the routing of axons in the chiasm of Nogo-A knockout mice during the growth of axons across the midline. At E13 to E16, there was no significant difference in the contralateral projection (P = 0.6943 for E13; P = 0.9867 for E14; P = 0.4121 for E15 and P = 0.3402 for E16). The results also showed the absence of Nogo-A did not cause any obvious change to the ipsilateral projection at the optic chiasm, both for the early generated uncrossed axons at E13 and E14 and the late cohorts at E15-E16, when compared with the wild-type mice (P = 0.4788 for E13; P = 0.188 for E14; P = 0.3152 for E15 and P = 0.432 for E16). These findings support that Nogo-A is not the major isoform to guide the axon divergence in the mouse optic chiasm.

Expression of Developmentally Important Axon Guidance Cues in the Adult Optic Chiasm.

Purpose: Regeneration of optic nerve axons after injury can be facilitated by several approaches, but misguidance at the optic chiasm is often observed. We characterized guidance cues in the embryonic visual system and adult optic chiasm before and after optic nerve crush (ONC) injury to better understand barriers to optic nerve regeneration in adults. Methods: Radial glial (RC2/BLBP/Slit1), developmental (Pax2) and extracellular markers (CSPG: H2B/CS-56) were assessed in C57BL/6J mice by immunohistochemistry. RC2, BLBP, Slit1, and CSPG are known inhibitory guidance cues while Pax2 is a permissive guidance cue. Results: At embryonic day 15.5 (E.15.5), RC2 and BLBP were identified superior to, and extending through, the optic chiasm. The optic chiasm was BLBP-ve in adult uninjured mice but BLBP+ve in adult mice 10 days after ONC injury. The reverse was true for RC2. Both BLBP and RC2 were absent in adult mice 6 weeks post-ONC. Slit1 was present in the optic chiasm midline and optic tracts in embryonic samples but was absent in uninjured adult tissue. Slit1 was observed superior to and at the midline of the optic chiasm 10 days post-ONC but absent 6 weeks after injury. Pax2 was expressed at the junction between the optic nerve and optic chiasm in embryonic brain tissue. In embryonic sections, CS-56 was observed at the junction between the optic chiasm and optic tract, and immediately superior to the optic chiasm. Both 2H6 and CS-56 staining was absent in uninjured and ONC-injured adult brains. Conclusion: Differences in guidance cue expression during development, in adulthood and after injury may contribute to misguidance of regenerating RGC axons in the adult optic chiasm.

Fingolimod (FTY720) improves the functional recovery and myelin preservation of the optic pathway in focal demyelination model of rat optic chiasm.

It has been shown that fingolimod (FTY720) possesses beneficial effects on remyelination in the central nervous system (CNS). In this study, the effects of FTY720 and sodium valproate (VPA) as histone deacetylase inhibitor (HDAC) on the conductivity of visual signals, extent of demyelination area, glial activation, and expression levels of HDAC1and S1PR1 have been evaluated in the optic chiasm of lysolecithin (LPC)-induced demyelination model. In order to produce this demyelination model, LPC (1%, 2 µL) was injected into the rat optic chiasm. Latency of visual waves was measured by visual evoked potential (VEP) recording. The extent of demyelination area and level of glial activation were assessed using immunostaining. Gene expression analysis was performed to evaluate the expression levels of HDAC1, S1PR1, Olig2, and MBP in the optic chiasm. Analysis of electrophysiological data showed that LPC administration increased the latency of visual signals. FTY720 improved the functional recovery of the visual pathway and reduced the level of glial activation in the optic chiasm. FTY720 enhanced myelin repair and up-regulated the expression levels of Olig2 and MBP. Additionally, the expression levels of HDAC1 and S1PR1 were significantly reduced in animals treated with FTY720. In contrast to FTY720 treated animals, administration of VPA could not significantly improve the functional recovery of optic pathway following LPC injection. Cumulatively, the results of the present study demonstrate that FTY720 application improves the functional recovery of the optic pathway by reducing demyelination levels, amelioration of glial activation, and down-regulating of S1PR1 and HDAC1.

Modulating proteoglycan receptor PTPσ using intracellular sigma peptide improves remyelination and functional recovery in mice with demyelinated optic chiasm.

Multiple sclerosis (MS) is an autoimmune disease characterized by myelin and axonal damage in the central nervous system (CNS). Glial scar which is a hallmark of MS contains repair inhibitory molecules including chondroitin sulfate proteoglycans (CSPGs). CSPGs inhibit repair of damaged area through various receptors including protein tyrosine phosphatase sigma (PTPσ). In the current study we use intracellular sigma peptide (ISP), an inhibitor of PTPσ signaling, in LPC-induced focal demyelination of mouse optic chiasm. ISP treatment resulted in decreased demyelination, reduced astrogliosis, and increased newly generated oligodendrocytes which subsequently led to enhanced remyelination. Analyzing of electrophysiological (as performed by visual evoked potential recording) and behavioral (performed by visual cliff test) outcomes showed that ISP-treatment improved the integrity of optic pathway as well as the visual acuity. When ISP was administrated only during the repair phase, histological, electrophysiological and behavioral studies showed its regenerative effect. Our results demonstrated the possibility of using ISP as a new strategy to inhibit PTPσ for myelin protection, myelin repair in demyelinated axons, and functional neural pathway conductivity restoration in patients suffering from MS.

Length of myelin internodes of individual oligodendrocytes is controlled by microenvironment influenced by normal and input-deprived axonal activities in sensory deprived mouse models.

Oligodendrocytes myelinate neuronal axons to increase conduction velocity in the vertebrate central nervous system (CNS). Recent studies revealed that myelin formed on highly active axons is more stable compared to activity-silenced axons, and length of the myelin sheath is longer in active axons as well in the zebrafish larva. However, it is unclear whether oligodendrocytes preferentially myelinate active axons compared to sensory input-deprived axons in the adult mammalian CNS. It is also unknown if a single oligodendrocyte forms both longer myelin sheaths on active axons and shorter sheaths on input-deprived axons after long-term sensory deprivation. To address these questions, we applied simultaneous labeling of both neuronal axons and oligodendrocytes to mouse models of long-term monocular eyelid suturing and unilateral whisker removal. We found that individual oligodendrocytes evenly myelinated normal and input-deprived axons in the adult mouse CNS, and myelin sheath length on normal axons and input-deprived axons formed by a single oligodendrocyte were comparable. Importantly, the average length of the myelin sheath formed by individual oligodendrocytes did change depending on relative abundance of normal against sensory-input deprived axons, indicating an abundance of deprived axons near an oligodendrocyte impacts on myelination program by a single oligodendrocyte.

Retinal ganglion cell axon sorting at the optic chiasm requires dystroglycan.

In the developing visual system, retinal ganglion cell (RGC) axons project from the retina to several distal retinorecipient regions in the brain. Several molecules have been implicated in guiding RGC axons in vivo, but the role of extracellular matrix molecules in this process remains poorly understood. Dystroglycan is a laminin-binding transmembrane protein important for formation and maintenance of the extracellular matrix and basement membranes and has previously been implicated in axon guidance in the developing spinal cord. Using two genetic models of functional dystroglycan loss, we show that dystroglycan is necessary for correct sorting of contralateral and ipsilateral RGC axons at the optic chiasm. Mis-sorted axons still target retinorecipient brain regions and persist in adult mice, even after axon pruning is complete. Our results highlight the importance of the extracellular matrix for axon sorting at an intermediate choice point in the developing visual circuit.

Hesperetin reduces myelin damage and ameliorates glial activation in lysolecithin-induced focal demyelination model of rat optic chiasm.

Visual impairment is considered as the most common initial manifestation of multiple sclerosis (MS) patients. It has been shown that hesperetin (Hst), a flavonoid of citrus fruit, possesses anti-inflammatory and antioxidant effects both in vitro and in vivo. The present study was designed to evaluate the pharmacological/medicinal effects of Hst treatment on myelin repair and glial activation in lysolecithin (LPC)-induced focal demyelination model. In order to induce local demyelination model, LPC 1% (2 µL) was injected into the optic chiasm of rats. Animals received oral administration of Hst at dose of 20 mg/kg for 14 or 21 days post lesion induction. Visual evoked potential (VEP) recordings were conducted before and also on days 7, 14 and 21 post LPC injection. Glial activation and myelination of optic chiasm were evaluated by immunostaining on brain sections. Analysis of VEPs data revealed that oral administration of Hst effectively reduced the latency of N1 waves. Immunostaining results showed the reduced number of astrocytes and microglia in animal which were treated with Hst. Furthermore, the extent of demyelination area was decreased in animals treated by Hst. Taken together; our results suggest that Hst treatment significantly protects and repairs myelin sheath, therefore it might be regarded as effective supplementary agent in demyelinating disorders, particularly MS.

Sonic Hedgehog Is a Remotely Produced Cue that Controls Axon Guidance Trans-axonally at a Midline Choice Point.

At the optic chiasm choice point, ipsilateral retinal ganglion cells (RGCs) are repelled away from the midline by guidance cues, including Ephrin-B2 and Sonic Hedgehog (Shh). Although guidance cues are normally produced by cells residing at the choice point, the mRNA for Shh is not found at the optic chiasm. Here we show that Shh protein is instead produced by contralateral RGCs at the retina, transported anterogradely along the axon, and accumulates at the optic chiasm to repel ipsilateral RGCs. In vitro, contralateral RGC axons, which secrete Shh, repel ipsilateral RGCs in a Boc- and Smo-dependent manner. Finally, knockdown of Shh in the contralateral retina causes a decrease in the proportion of ipsilateral RGCs in a non-cell-autonomous manner. These findings reveal a role for axon-axon interactions in ipsilateral RGC guidance, and they establish that remotely produced cues can act at axon guidance midline choice points.

VEGF-A and neuropilin 1 (NRP1) shape axon projections in the developing CNS via dual roles in neurons and blood vessels.

Visual information is relayed from the eye to the brain via retinal ganglion cell (RGC) axons. Mice lacking NRP1 or NRP1-binding VEGF-A isoforms have defective RGC axon organisation alongside brain vascular defects. It is not known whether axonal defects are caused exclusively by defective VEGF-A signalling in RGCs or are exacerbated by abnormal vascular morphology. Targeted NRP1 ablation in RGCs with a Brn3bCre knock-in allele reduced axonal midline crossing at the optic chiasm and optic tract fasciculation. In contrast, Tie2-Cre-mediated endothelial NRP1 ablation induced axon exclusion zones in the optic tracts without impairing axon crossing. Similar defects were observed in Vegfa120/120 and Vegfa188/188 mice, which have vascular defects as a result of their expression of single VEGF-A isoforms. Ectopic midline vascularisation in endothelial Nrp1 and Vegfa188/188 mutants caused additional axonal exclusion zones within the chiasm. As in vitro and in vivo assays demonstrated that vessels do not repel axons, abnormally large or ectopically positioned vessels are likely to present physical obstacles to axon growth. We conclude that proper axonal wiring during brain development depends on the precise molecular control of neurovascular co-patterning.

SoxC Transcription Factors Promote Contralateral Retinal Ganglion Cell Differentiation and Axon Guidance in the Mouse Visual System.

Transcription factors control cell identity by regulating diverse developmental steps such as differentiation and axon guidance. The mammalian binocular visual circuit is comprised of projections of retinal ganglion cells (RGCs) to ipsilateral and contralateral targets in the brain. A transcriptional code for ipsilateral RGC identity has been identified, but less is known about the transcriptional regulation of contralateral RGC development. Here we demonstrate that SoxC genes (Sox4, 11, and 12) act on the progenitor-to-postmitotic transition to implement contralateral, but not ipsilateral, RGC differentiation, by binding to Hes5 and thus repressing Notch signaling. When SoxC genes are deleted in postmitotic RGCs, contralateral RGC axons grow poorly on chiasm cells in vitro and project ipsilaterally at the chiasm midline in vivo, and Plexin-A1 and Nr-CAM expression in RGCs is downregulated. These data implicate SoxC transcription factors in the regulation of contralateral RGC differentiation and axon guidance.

DSCAM promotes axon fasciculation and growth in the developing optic pathway.

Although many aspects of optic pathway development are beginning to be understood, the mechanisms promoting the growth of retinal ganglion cell (RGC) axons toward visual targets remain largely unknown. Down syndrome cell adhesion molecule (Dscam) is expressed by mouse RGCs shortly after they differentiate at embryonic day 12 and is essential for multiple aspects of postnatal visual system development. Here we show that Dscam is also required during embryonic development for the fasciculation and growth of RGC axons. Dscam is expressed along the developing optic pathway in a pattern consistent with a role in regulating RGC axon outgrowth. In mice carrying spontaneous mutations in Dscam (Dscamdel17 ; Dscam2J), RGC axons pathfind normally, but growth from the chiasm toward their targets is impaired, resulting in a delay in RGC axons reaching the dorsal thalamus compared with that seen in wild-type littermates. Conversely, Dscam gain of function results in exuberant growth into the dorsal thalamus. The growth of ipsilaterally projecting axons is particularly affected. Axon organization in the optic chiasm and tract and RGC growth cone morphologies are also altered in Dscam mutants. In vitro DSCAM promotes RGC axon growth and fasciculation, and can act independently of cell contact. In vitro and in situ DSCAM is required both in the RGC axons and in their environment for the promotion of axon outgrowth, consistent with a homotypic mode of action. These findings identify DSCAM as a permissive signal that promotes the growth and fasciculation of RGC axons, controlling the timing of when RGC axons reach their targets.

Ipsilateral and Contralateral Retinal Ganglion Cells Express Distinct Genes during Decussation at the Optic Chiasm.

The increasing availability of transcriptomic technologies within the last decade has facilitated high-throughput identification of gene expression differences that define distinct cell types as well as the molecular pathways that drive their specification. The retinal projection neurons, retinal ganglion cells (RGCs), can be categorized into distinct morphological and functional subtypes and by the laterality of their projections. Here, we present a method for purifying the sparse population of ipsilaterally projecting RGCs in mouse retina from their contralaterally projecting counterparts during embryonic development through rapid retrograde labeling followed by fluorescence-activated cell sorting. Through microarray analysis, we uncovered the distinct molecular signatures that define and distinguish ipsilateral and contralateral RGCs during the critical period of axonal outgrowth and decussation, with more than 300 genes differentially expressed within these two cell populations. Among the differentially expressed genes confirmed through in vivo expression validation, several genes that mark "immaturity" are expressed within postmitotic ipsilateral RGCs. Moreover, at least one complementary pair, Igf1 and Igfbp5, is upregulated in contralateral or ipsilateral RGCs, respectively, and may represent signaling pathways that determine ipsilateral versus contralateral RGC identity. Importantly, the cell cycle regulator cyclin D2 is highly expressed in peripheral ventral retina with a dynamic expression pattern that peaks during the period of ipsilateral RGC production. Thus, the molecular signatures of ipsilateral and contralateral RGCs and the mechanisms that regulate their differentiation are more diverse than previously expected.