Rejection of inappropriate synaptic partners in mouse retina mediated by transcellular FLRT2-UNC5 signaling.

During nervous system development, neurons choose synaptic partners with remarkable specificity; however, the cell-cell recognition mechanisms governing rejection of inappropriate partners remain enigmatic. Here, we show that mouse retinal neurons avoid inappropriate partners by using the FLRT2-uncoordinated-5 (UNC5) receptor-ligand system. Within the inner plexiform layer (IPL), FLRT2 is expressed by direction-selective (DS) circuit neurons, whereas UNC5C/D are expressed by non-DS neurons projecting to adjacent IPL sublayers. In vivo gain- and loss-of-function experiments demonstrate that FLRT2-UNC5 binding eliminates growing DS dendrites that have strayed from the DS circuit IPL sublayers. Abrogation of FLRT2-UNC5 binding allows mistargeted arbors to persist, elaborate, and acquire synapses from inappropriate partners. Conversely, UNC5C misexpression within DS circuit sublayers inhibits dendrite growth and drives arbors into adjacent sublayers. Mechanistically, UNC5s promote dendrite elimination by interfering with FLRT2-mediated adhesion. Based on their broad expression, FLRT-UNC5 recognition is poised to exert widespread effects upon synaptic partner choices across the nervous system.

MicroRNA Signatures of the Developing Primate Fovea.

Rod and cone photoreceptors differ in their shape, photopigment expression, synaptic connection patterns, light sensitivity, and distribution across the retina. Although rods greatly outnumber cones, human vision is mostly dependent on cone photoreceptors since cones are essential for our sharp visual acuity and color discrimination. In humans and other primates, the fovea centralis (fovea), a specialized region of the central retina, contains the highest density of cones. Despite the vast importance of the fovea for human vision, the molecular mechanisms guiding the development of this region are largely unknown. MicroRNAs (miRNAs) are small post-transcriptional regulators known to orchestrate developmental transitions and cell fate specification in the retina. Here, we have characterized the transcriptional landscape of the developing rhesus monkey retina. Our data indicates that non-human primate fovea development is significantly accelerated compared to the equivalent retinal region at the other side of the optic nerve head, as described previously. Notably, we also identify several miRNAs differentially expressed in the presumptive fovea, including miR-15b-5p, miR-342-5p, miR-30b-5p, miR-103-3p, miR-93-5p as well as the miRNA cluster miR-183/-96/-182. Interestingly, miR-342-5p is enriched in the nasal primate retina and in the peripheral developing mouse retina, while miR-15b is enriched in the temporal primate retina and increases over time in the mouse retina in a central-to-periphery gradient. Together our data constitutes the first characterization of the developing rhesus monkey retinal miRNome and provides novel datasets to attain a more comprehensive understanding of foveal development.

CRL5-dependent regulation of the small GTPases ARL4C and ARF6 controls hippocampal morphogenesis.

The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.

Effects of Adult Müller Cells and Their Conditioned Media on the Survival of Stem Cell-Derived Retinal Ganglion Cells.

Retinal neurons, particularly retinal ganglion cells (RGCs), are susceptible to the degenerative damage caused by different inherited conditions and environmental insults, leading to irreversible vision loss and, ultimately, blindness. Numerous strategies are being tested in different models of degeneration to restore vision and, in recent years, stem cell technologies have offered novel avenues to obtain donor cells for replacement therapies. To date, stem cell-based transplantation in the retina has been attempted as treatment for photoreceptor degeneration, but the same tools could potentially be applied to other retinal cell types, including RGCs. However, RGC-like cells are not an abundant cell type in stem cell-derived cultures and, often, these cells degenerate over time in vitro. To overcome this limitation, we have taken advantage of the neuroprotective properties of Müller glia (one of the main glial cell types in the retina) and we have examined whether Müller glia and the factors they secrete could promote RGC-like cell survival in organoid cultures. Accordingly, stem cell-derived RGC-like cells were co-cultured with adult Müller cells or Müller cell-conditioned media was added to the cultures. Remarkably, RGC-like cell survival was substantially enhanced in both culture conditions, and we also observed a significant increase in their neurite length. Interestingly, Atoh7, a transcription factor required for RGC development, was up-regulated in stem cell-derived organoids exposed to conditioned media, suggesting that Müller cells may also enhance the survival of retinal progenitors and/or postmitotic precursor cells. In conclusion, Müller cells and the factors they release promote organoid-derived RGC-like cell survival, neuritogenesis, and possibly neuronal maturation.

A Novel Reporter Mouse Uncovers Endogenous Brn3b Expression.

Brn3b (Pou4f2) is a class-4 POU domain transcription factor known to play central roles in the development of different neuronal populations of the Central Nervous System, including retinal ganglion cells (RGCs), the neurons that connect the retina with the visual centers of the brain. Here, we have used CRISPR-based genetic engineering to generate a Brn3b-mCherry reporter mouse without altering the endogenous expression of Brn3b. In our mouse line, mCherry faithfully recapitulates normal Brn3b expression in the retina, the optic tracts, the midbrain tectum, and the trigeminal ganglia. The high sensitivity of mCherry also revealed novel expression of Brn3b in the neuroectodermal cells of the optic stalk during early stages of eye development. Importantly, the fluorescent intensity of Brn3b-mCherry in our reporter mice allows for noninvasive live imaging of RGCs using Scanning Laser Ophthalmoscopy (SLO), providing a novel tool for longitudinal monitoring of RGCs.

Retinal Ganglion Cell Replacement: Current Status and Challenges Ahead.

The neurons of the retina can be affected by a wide variety of inherited or environmental degenerations that can lead to vision loss and even blindness. Retinal ganglion cell (RGC) degeneration is the hallmark of glaucoma and other optic neuropathies that affect millions of people worldwide. Numerous strategies are being trialed to replace lost neurons in different degeneration models, and in recent years, stem cell technologies have opened promising avenues to obtain donor cells for retinal repair. Stem cell-based transplantation has been most frequently used for the replacement of rod photoreceptors, but the same tools could potentially be used for other retinal cell types, including RGCs. However, RGCs are not abundant in stem cell-derived cultures, and in contrast to the short-distance wiring of photoreceptors, RGC axons take a long and intricate journey to connect with numerous brain nuclei. Hence, a number of challenges still remain, such as the ability to scale up the production of RGCs and a reliable and functional integration into the adult diseased retina upon transplantation. In this review, we discuss the recent advancements in the development of replacement therapies for RGC degenerations and the challenges that we need to overcome before these technologies can be applied to the clinic. Developmental Dynamics 248:118-128, 2019. © 2018 Wiley Periodicals, Inc.

RBX2 maintains final retinal cell position in a DAB1-dependent and -independent fashion.

The laminated structure of the retina is fundamental for the organization of the synaptic circuitry that translates light input into patterns of action potentials. However, the molecular mechanisms underlying cell migration and layering of the retina are poorly understood. Here, we show that RBX2, a core component of the E3 ubiquitin ligase CRL5, is essential for retinal layering and function. RBX2 regulates the final cell position of rod bipolar cells, cone photoreceptors and Muller glia. Our data indicate that sustained RELN/DAB1 signaling, triggered by depletion of RBX2 or SOCS7 - a CRL5 substrate adaptor known to recruit DAB1 - causes rod bipolar cell misposition. Moreover, whereas SOCS7 also controls Muller glia cell lamination, it is not responsible for cone photoreceptor positioning, suggesting that RBX2, most likely through CRL5 activity, controls other signaling pathways required for proper cone localization. Furthermore, RBX2 depletion reduces the number of ribbon synapses and disrupts cone photoreceptor function. Together, these results uncover RBX2 as a crucial molecular regulator of retina morphogenesis and cone photoreceptor function.

Nonautonomous Roles of MAB-5/Hox and the Secreted Basement Membrane Molecule SPON-1/F-Spondin in Caenorhabditis elegans Neuronal Migration.

Nervous system development and circuit formation requires neurons to migrate from their birthplaces to specific destinations.Migrating neurons detect extracellular cues that provide guidance information. In Caenorhabditis elegans, the Q right (QR) and Q left (QL) neuroblast descendants migrate long distances in opposite directions. The Hox gene lin-39 cell autonomously promotes anterior QR descendant migration, and mab-5/Hox cell autonomously promotes posterior QL descendant migration. Here we describe a nonautonomous role of mab-5 in regulating both QR and QL descendant migrations, a role masked by redundancy with lin-39 A third Hox gene, egl-5/Abdominal-B, also likely nonautonomously regulates Q descendant migrations. In the lin-39 mab-5 egl-5 triple mutant, little if any QR and QL descendant migration occurs. In addition to well-described roles of lin-39 and mab-5 in the Q descendants, our results suggest that lin-39, mab-5, and egl-5 might also pattern the posterior region of the animal for Q descendant migration. Previous studies showed that the spon-1 gene might be a target of MAB-5 in Q descendant migration. spon-1 encodes a secreted basement membrane molecule similar to vertebrate F-spondin. Here we show that spon-1 acts nonautonomously to control Q descendant migration, and might function as a permissive rather than instructive signal for cell migration. We find that increased levels of MAB-5 in body wall muscle (BWM) can drive the spon-1 promoter adjacent to the Q cells, and loss of spon-1 suppresses mab-5 gain of function. Thus, MAB-5 might nonautonomously control Q descendant migrations by patterning the posterior region of the animal to which Q cells respond. spon-1 expression from BWMs might be part of the posterior patterning necessary for directed Q descendant migration.