Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system.

During development, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. In Drosophila, the Slit protein regulates midline axon crossing through repulsion. To determine the role of Slit proteins in RGC axon guidance, we disrupted Slit1 and Slit2, two of three known mouse Slit genes. Mice defective in either gene alone exhibited few RGC axon guidance defects, but in double mutant mice a large additional chiasm developed anterior to the true chiasm, many retinal axons projected into the contralateral optic nerve, and some extended ectopically-dorsal and lateral to the chiasm. Our results indicate that Slit proteins repel retinal axons in vivo and cooperate to establish a corridor through which the axons are channeled, thereby helping define the site in the ventral diencephalon where the optic chiasm forms.     

GAP-43 mediates retinal axon interaction with lateral diencephalon cells during optic tract formation

GAP-43 is an abundant intracellular growth cone protein that can serve as a PKC substrate and regulate calmodulin availability. In mice with targeted disruption of the GAP-43 gene, retinal ganglion cell (RGC) axons fail to progress normally from the optic chiasm into the optic tracts. The underlying cause is unknown but, in principle, can result from either the disruption of guidance mechanisms that mediate axon exit from the midline chiasm region or defects in growth cone signaling required for entry into the lateral diencephalic wall to form the optic tracts. Results here show that, compared to wild-type RGC axons, GAP-43-deficient axons exhibit reduced growth in the presence of lateral diencephalon cell membranes. Reduced growth is not observed when GAP-43-deficient axons are cultured with optic chiasm, cortical, or dorsal midbrain cells. Lateral diencephalon cell conditioned medium inhibits growth of both wild-type and GAP-43-deficient axons to a similar extent and does not affect GAP-43-deficient axons more so. Removal or transplant replacement of the lateral diencephalon optic tract entry zone in GAP-43-deficient embryo preparations results in robust RGC axon exit from the chiasm. Together these data show that RGC axon exit from the midline region does not require GAP-43 function. Instead, GAP-43 appears to mediate RGC axon interaction with guidance cues in the lateral diencephalic wall, suggesting possible involvement of PKC and calmodulin signaling during optic tract formation.     

Matrix metalloproteinases are required for retinal ganglion cell axon guidance at select decision points

Axons receive guidance information from extrinsic cues in their environment in order to reach their targets. In the frog Xenopus laevis, retinal ganglion cell (RGC) axons make three key guidance decisions en route through the brain. First, they cross to the contralateral side of the brain at the optic chiasm. Second, they turn caudally in the mid-diencephalon. Finally, they must recognize the optic tectum as their target. The matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) families are zinc (Zn)-dependent proteolytic enzymes. The latter functions in axon guidance, but a similar role has not yet been identified for the MMP family. Our previous work implicated metalloproteinases in the guidance decisions made by Xenopus RGC axons. To test specifically the importance of MMPs, we used two different in vivo exposed brain preparations in which RGC axons were exposed to an MMP-specific pharmacological inhibitor (SB-3CT), either as they reached the optic chiasm or as they extended through the diencephalon en route to the optic tectum. Interestingly, SB-3CT affected only two of the guidance decisions, with misrouting defects at the optic chiasm and tectum. Only at higher concentrations was RGC axon extension also impaired. These data implicate MMPs in the guidance of vertebrate axons, and suggest that different metalloproteinases function to regulate axon behaviour at distinct choice points: an MMP is important in guidance at the optic chiasm and the target, while either a different MMP or an ADAM is required for axons to make the turn in the mid-diencephalon.     

Semaphorin 3d guides laterality of retinal ganglion cell projections in zebrafish

The optic chiasm is an important choice point at which retinal ganglion cell (RGC) axons either cross the midline to innervate the contralateral brain or turn back to innervate the ipsilateral brain. Guidance cues that regulate this decision, particularly those directing the midline crossing of contralateral axons, are still not well understood. Here we show that Sema3d, a secreted semaphorin expressed at the midline, guides the crossing of RGC axons in zebrafish. Both Sema3d knockdown and ubiquitous overexpression induced aberrant ipsilateral projections, suggesting that Sema3d normally guides axons into the contralateral optic tract. Live imaging in vivo showed that RGC growth cones responded to ubiquitous Sema3d overexpression by pausing for extended periods and increasing their exploratory behavior at the midline, suggesting that Sema3d overexpression causes the midline environment to become less favorable for RGC axon extension. Interestingly, Sema3d overexpression did not affect growth cone behaviors before the midline, suggesting that RGC axons normally respond to Sema3d only upon reaching the midline. After Sema3d knockdown, growth cones grew across the midline but then paused or repeatedly retracted, impairing their ability to leave the midline region. Our results indicate that a proper balance of Sema3d is needed at the midline for the progression of RGC axons from the chiasm midline into the contralateral optic tract.     

Intravitreal Transplantation of Human Umbilical Cord Blood Stem Cells Protects Rats from Traumatic Optic Neuropathy

Objectives

To treat traumatic optic neuropathy (TON) with transplantation of human umbilical cord blood stem cells (hUCBSC) and explore how transplanted stem cells participate in the neuron repairing process.

Methods

A total of 195 Sprague-Dawley rats were randomly assigned to three groups: sham-surgery, optic nerve injury, and stem cell transplant group. Optic nerve injury was established in rats by directly clamping the optic nerve for 30 seconds. hUCBSC was microinjected into the vitreous cavity of injured rats. Optic nerve function was evaluated by flash visual evoked potentials (F-VEP). Apoptosis in retina tissues was detected by TUNEL staining. GRP78 and CHOP gene expression was measured by RT-PCR.

Results

After injury, transplantation of hUCBSC significantly blunted a reduction in optic nerve function indicated by smaller decreases in amplitude and smaller increases in peak latency of F-VEP waveform compared to the injury alone group. Also, significant more in retinal ganglion cell (RGC) count and less in RGC apoptosis were detected after transplantation compared to injured rats. The protective effect correlated with upregulated GRP78 and downregulated CHOP mRNA expression.

Conclusion

Intravitreal transplantation of hUCBSCs significantly blunted a reduction in optic nerve function through increasing RGC survival and decreasing retinal cell apoptosis. The protective role of transplantation was associated with upregulation of GRP78 expression and downregulation of CHOP expression in retinal cells.     

Suppression of Induced microRNA-15b Prevents Rapid Loss of Cardiac Function in a Dicer Depleted Model of Cardiac Dysfunction

Background

Dicer endonuclease, critical for maturation of miRNAs, is depleted in certain forms of cardiomyopathy which results in differential expression of certain microRNAs. We sought to elucidate the mechanisms underlying the rapid loss of cardiac function following cardiac-specific Dicer depletion in adult mice.

Results

Conditional Dicer deletion in the adult murine myocardium demonstrated compromised heart function, mitochondrial dysfunction and oxidant stress. Elevated miR-15b was observed as an early response to Dicer depletion and was found to silence Pim-1 kinase, a protein responsible for maintaining mitochondrial integrity and function. Anti-miRNA based suppression of induced miRNA-15b rescued the function of Dicer-depleted adult heart and attenuated hypertrophy.

Conclusions

Anti-miRNA based suppression of inducible miRNA-15b can prevent rapid loss of cardiac function in a Dicer-depleted adult heart and can be a key approach worthy of therapeutic consideration.     

Netrin signaling breaks the equivalence between two identified zebrafish motoneurons revealing a new role of intermediate targets

Background

We previously showed that equivalence between two identified zebrafish motoneurons is broken by interactions with identified muscle fibers that act as an intermediate target for the axons of these motoneurons. Here we investigate the molecular basis of the signaling interaction between the intermediate target and the motoneurons.

Principal Findings

We provide evidence that Netrin 1a is an intermediate target-derived signal that causes two equivalent motoneurons to adopt distinct fates. We show that although these two motoneurons express the same Netrin receptors, their axons respond differently to Netrin 1a encountered at the intermediate target. Furthermore, we demonstrate that when Netrin 1a is knocked down, more distal intermediate targets that express other Netrins can also function to break equivalence between these motoneurons.

Significance

Our results suggest a new role for intermediate targets in breaking neuronal equivalence. The data we present reveal that signals encountered during axon pathfinding can cause equivalent neurons to adopt distinct fates. Such signals may be key in diversifying a neuronal population and leading to correct circuit formation.     

Forced notch signaling inhibits commissural axon outgrowth in the developing chick central nerve system

Background

A collection of in vitro evidence has demonstrated that Notch signaling plays a key role in the growth of neurites in differentiated neurons. However, the effects of Notch signaling on axon outgrowth in an in vivo condition remain largely unknown.

Methodology/Principal Findings

In this study, the neural tubes of HH10-11 chick embryos were in ovo electroporated with various Notch transgenes of activating or inhibiting Notch signaling, and then their effects on commissural axon outgrowth across the floor plate midline in the chick developing central nerve system were investigated. Our results showed that forced expression of Notch intracellular domain, constitutively active form of RBPJ, or full-length Hes1 in the rostral hindbrain, diencephalon and spinal cord at stage HH10-11 significantly inhibited commissural axon outgrowth. On the other hand, inhibition of Notch signaling by ectopically expressing a dominant-negative form of RBPJ promoted commissural axonal growth along the circumferential axis. Further results revealed that these Notch signaling-mediated axon outgrowth defects may be not due to the alteration of axon guidance since commissural axon marker TAG1 was present in the axons in floor plate midline, and also not result from the changes in cell fate determination of commissural neurons since the expression of postmitotic neuron marker Tuj1 and specific commissural markers TAG1 and Pax7 was unchanged.

Conclusions/Significance

We first used an in vivo system to provide evidence that forced Notch signaling negatively regulates commissural axon outgrowth.     

VEGF signaling through neuropilin 1 guides commissural axon crossing at the optic chiasm

During development, the axons of retinal ganglion cell (RGC) neurons must decide whether to cross or avoid the midline at the optic chiasm to project to targets on both sides of the brain. By combining genetic analyses with in vitro assays, we show that neuropilin 1 (NRP1) promotes contralateral RGC projection in mammals. Unexpectedly, the NRP1 ligand involved is not an axon guidance cue of the class 3 semaphorin family, but VEGF164, the neuropilin-binding isoform of the classical vascular growth factor VEGF-A. VEGF164 is expressed at the chiasm midline and is required for normal contralateral growth in vivo. In outgrowth and growth cone turning assays, VEGF164 acts directly on NRP1-expressing contralateral RGCs to provide growth-promoting and chemoattractive signals. These findings have identified a permissive midline signal for axons at the chiasm midline and provide in vivo evidence that VEGF-A is an essential axon guidance cue.     

Estimated Rates of Retinal Ganglion Cell Loss in Glaucomatous Eyes with and without Optic Disc Hemorrhages

Purpose

To evaluate whether optic disc hemorrhages are associated with faster rates of estimated retinal ganglion cell (RGC) loss in glaucoma.

Methods

A longitudinal observational cohort study of 222 eyes of 122 patients with glaucoma recruited from the Diagnostic Innovations Glaucoma Study (DIGS) followed for an average of 3.74±0.85 years. All subjects had optical coherence tomography and standard automated perimetry during follow up. Optic disc hemorrhages were detected by masked evaluation of stereophotographs. Rates of change in estimated numbers of RGCs were determined using a previously described method. A random coefficients model was used to investigate the relationship between disc hemorrhages and rates of change in estimated RGC counts over time.

Results

19 eyes of 18 subjects had at least one disc hemorrhage during follow up. At baseline, average estimated RGC counts in eyes with and without disc hemorrhages were 677,994 cells and 682,021 cells, respectively (P = 0.929). Eyes with optic disc hemorrhages during follow-up had significantly faster rates of estimated RGC loss than eyes without disc hemorrhages (22,233 cells/year versus 10,704 cells/year, P = 0.020). The effect of disc hemorrhages on the rates of estimated RGC loss remained significant after adjusting for confounding variables.

Conclusion

Eyes with disc hemorrhages showed faster rates of RGC loss compared to eyes without disc hemorrhages. These results provide further evidence that disc hemorrhages should be considered as an indicator of increased risk for faster neural loss in glaucoma.     

Sonic hedgehog is indirectly required for intraretinal axon pathfinding by regulating chemokine expression in the optic stalk

Successful axon pathfinding requires both correct patterning of tissues, which will later harbor axonal tracts, and precise localization of axon guidance cues along these tracts at the time of axon outgrowth. Retinal ganglion cell (RGC) axons grow towards the optic disc in the central retina, where they turn to exit the eye through the optic nerve. Normal patterning of the optic disc and stalk and the expression of guidance cues at this choice point are necessary for the exit of RGC axons out of the eye. Sonic hedgehog (Shh) has been implicated in both patterning of ocular tissue and direct guidance of RGC axons. Here, we examine the precise spatial and temporal requirement for Hedgehog (Hh) signaling for intraretinal axon pathfinding and show that Shh acts to pattern the optic stalk in zebrafish but does not guide RGC axons inside the eye directly. We further reveal an interaction between the Hh and chemokine pathways for axon guidance and show that cxcl12a functions downstream of Shh and depends on Shh for its expression at the optic disc. Together, our results support a model in which Shh acts in RGC axon pathfinding indirectly by regulating axon guidance cues at the optic disc through patterning of the optic stalk.     

Gogo Receptor Contributes to Retinotopic Map Formation and Prevents R1-6 Photoreceptor Axon Bundling

Background

Topographic maps form the basis of neural processing in sensory systems of both vertebrate and invertebrate species. In the Drosophila visual system, neighboring R1–R6 photoreceptor axons innervate adjacent positions in the first optic ganglion, the lamina, and thereby represent visual space as a continuous map in the brain. The mechanisms responsible for the establishment of retinotopic maps remain incompletely understood.

Results

Here, we show that the receptor Golden goal (Gogo) is required for R axon lamina targeting and cartridge elongation in a partially redundant fashion with local guidance cues provided by neighboring axons. Loss of function of Gogo in large clones of R axons results in aberrant R1–R6 fascicle spacing. Gogo affects target cartridge selection only indirectly as a consequence of the disordered lamina map. Interestingly, small clones of gogo deficient R axons perfectly integrate into a proper retinotopic map suggesting that surrounding R axons of the same or neighboring fascicles provide complementary spatial guidance. Using single photoreceptor type rescue, we show that Gogo expression exclusively in R8 cells is sufficient to mediate targeting of all photoreceptor types in the lamina. Upon lamina targeting and cartridge selection, R axons elongate within their individual cartridges. Interestingly, here Gogo prevents bundling of extending R1-6 axons.

Conclusion

Taken together, we propose that Gogo contributes to retinotopic map formation in the Drosophila lamina by controlling the distribution of R1–R6 axon fascicles. In a later developmental step, the regular position of R1–R6 axons along the lamina plexus is crucial for target cartridge selection. During cartridge elongation, Gogo allows R1–R6 axons to extend centrally in the lamina cartridge.     

Human induced pluripotent stem cells differentiation into oligodendrocyte progenitors and transplantation in a rat model of optic chiasm demyelination

Background

This study aims to differentiate human induced pluripotent stem cells (hiPSCs) into oligodendrocyte precursors and assess their recovery potential in a demyelinated optic chiasm model in rats.

Methodology/Principal Findings

We generated a cell population of oligodendrocyte progenitors from hiPSCs by using embryoid body formation in a defined medium supplemented with a combination of factors, positive selection and mechanical enrichment. Real-time polymerase chain reaction and immunofluorescence analyses showed that stage-specific markers, Olig2, Sox10, NG2, PDGFRα, O4, A2B5, GalC, and MBP were expressed following the differentiation procedure, and enrichment of the oligodendrocyte lineage. These results are comparable with the expression of stage-specific markers in human embryonic stem cell-derived oligodendrocyte lineage cells. Transplantation of hiPSC-derived oligodendrocyte progenitors into the lysolecithin-induced demyelinated optic chiasm of the rat model resulted in recovery from symptoms, and integration and differentiation into oligodendrocytes were detected by immunohistofluorescence staining against PLP and MBP, and measurements of the visual evoked potentials.

Conclusions/Significance

These results showed that oligodendrocyte progenitors generated efficiently from hiPSCs can be used in future biomedical studies once safety issues have been overcome.     

Matrix Metalloproteinase 14 in the Zebrafish: An Eye on Retinal and Retinotectal Development

Background

Matrix metalloproteinases (MMPs) are members of the metzincin superfamily of proteinases that cleave structural elements of the extracellular matrix and many molecules involved in signal transduction. Although there is evidence that MMPs promote the proper development of retinotectal projections, the nature and working mechanisms of specific MMPs in retinal development remain to be elucidated. Here, we report a role for zebrafish Mmp14a, one of the two zebrafish paralogs of human MMP14, in retinal neurogenesis and retinotectal development.

Results

Whole mount in situ hybridization and immunohistochemical stainings for Mmp14a in developing zebrafish embryos reveal expression in the optic tectum, in the optic nerve and in defined retinal cell populations, including retinal ganglion cells (RGCs). Furthermore, Mmp14a loss-of-function results in perturbed retinoblast cell cycle kinetics and consequently, in a delayed retinal neurogenesis, differentiation and lamination. These Mmp14a-dependent retinal defects lead to microphthalmia and a significantly reduced innervation of the optic tectum (OT) by RGC axons. Mmp14b, on the contrary, does not appear to alter retinal neurogenesis or OT innervation. As mammalian MMP14 is known to act as an efficient MMP2-activator, we also explored and found a functional link and a possible co-involvement of Mmp2 and Mmp14a in zebrafish retinotectal development.

Conclusion

Both the Mmp14a expression in the developing visual system and the Mmp14a loss-of-function phenotype illustrate a critical role for Mmp14a activity in retinal and retinotectal development.     

Nogo Receptor Inhibition Enhances Functional Recovery following Lysolecithin-Induced Demyelination in Mouse Optic Chiasm

Background

Inhibitory factors have been implicated in the failure of remyelination in demyelinating diseases. Myelin associated inhibitors act through a common receptor called Nogo receptor (NgR) that plays critical inhibitory roles in CNS plasticity. Here we investigated the effects of abrogating NgR inhibition in a non-immune model of focal demyelination in adult mouse optic chiasm.

Methodology/Principal Findings

A focal area of demyelination was induced in adult mouse optic chiasm by microinjection of lysolecithin. To knock down NgR levels, siRNAs against NgR were intracerebroventricularly administered via a permanent cannula over 14 days, Functional changes were monitored by electrophysiological recording of latency of visual evoked potentials (VEPs). Histological analysis was carried out 3, 7 and 14 days post demyelination lesion. To assess the effect of NgR inhibition on precursor cell repopulation, BrdU was administered to the animals prior to the demyelination induction. Inhibition of NgR significantly restored VEPs responses following optic chiasm demyelination. These findings were confirmed histologically by myelin specific staining. siNgR application resulted in a smaller lesion size compared to control. NgR inhibition significantly increased the numbers of BrdU+/Olig2+ progenitor cells in the lesioned area and in the neurogenic zone of the third ventricle. These progenitor cells (Olig2+ or GFAP+) migrated away from this area as a function of time.

Conclusions/Significance

Our results show that inhibition of NgR facilitate myelin repair in the demyelinated chiasm, with enhanced recruitment of proliferating cells to the lesion site. Thus, antagonizing NgR function could have therapeutic potential for demyelinating disorders such as Multiple Sclerosis.     

Up-regulated Dicer expression in patients with cutaneous melanoma

Background

MicroRNAs (miRNAs) are small non-coding RNAs (18–24 nucleotides) that have recently been shown to regulate gene expression during cancer progression. Dicer, a central enzyme in the multi-component miRNA biogenesis pathway, is involved in cutting precursor miRNAs to functionally mature forms. Emerging evidence shows that Dicer expression is deregulated in some human malignancies and it correlates with tumor progression, yet this role has not yet been investigated in skin cancers.

Methods and Findings

Using an anti-human monoclonal antibody against Dicer and immunohistochemistry, we compared the expression of Dicer protein among 404 clinically annotated controls and skin tumors consisting of melanocytic nevi (n = 71), a variety of melanomas (n = 223), carcinomas (n = 73) and sarcomas (n = 12). Results showed a cell-specific up-regulated Dicer in 81% of cutaneous, 80% of acrolentiginous and 96% of metastatic melanoma specimens compared to carcinoma or sarcoma specimens (P<0.0001). The expression of Dicer was significantly higher in melanomas compared to benign melanocytic nevi (P<0.0001). In patients with cutaneous melanomas, Dicer up-regulation was found to be significantly associated with an increased tumor mitotic index (P = 0.04), Breslow''s depth of invasion (P = 0.03), nodal metastasis (P = 0.04) and a higher American Joint Committee on Caner (AJCC) clinical stage (P = 0.009). Using western blot analysis, we confirmed the cell-specific up-regulation of Dicer protein in vitro. A pooled-analysis on mRNA profiling in cutaneous tumors showed up-regulation of Dicer at the RNA level in cutaneous melanoma, also showing deregulation of other enzymes that participate in the biogenesis and maturation of canonical miRNAs.

Conclusions

Increased Dicer expression may be a clinically useful biomarker for patients with cutaneous melanoma. Understanding deregulation of Dicer and its influence on miRNA maturation is needed to predict the susceptibility of melanoma patients to miRNA-based therapy in the future.