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Naturally occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type-the astrocyte-has rarely been studied. It is presently unclear whether astrocytes are subject to significant developmental death, and if so, how it occurs. Here, we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia engulf astrocytes during the death period to promote their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally occurring cell death and demonstrate its importance for the formation and integrity of the retinal gliovascular network.

The related transmembrane proteins MEGF10 and MEGF11 are shown to have critical roles in the formation of mosaic arrangements in the retina. Building the retinal network In the retina and other nerve tissues, neural interactions are facilitated by the arrangement of the neurons in regular patterns. This paper addresses how these patterns emerge during development. Kay et al . show that in the mouse retina, where neurons of individual subtypes form arrays called mosaics, the related transmembrane proteins MEGF10 and MEGF11 are required for the formation of mosaics by starburst amacrine cells and horizontal cells. Like the related proteins CED-1 and Draper, from Caenorhabditis elegans and Drosophila respectively, MEGF10 and MEGF11 have until now been studied predominantly as receptors for cell engulfment. In many parts of the nervous system, neuronal somata display orderly spatial arrangements 1 . In the retina, neurons of numerous individual subtypes form regular arrays called mosaics: they are less likely to be near neighbours of the same subtype than would occur by chance, resulting in ‘exclusion zones’ that separate them 1 , 2 , 3 , 4 . Mosaic arrangements provide a mechanism to distribute each cell type evenly across the retina, ensuring that all parts of the visual field have access to a full set of processing elements 2 . Remarkably, mosaics are independent of each other: although a neuron of one subtype is unlikely to be adjacent to another of the same subtype, there is no restriction on its spatial relationship to neighbouring neurons of other subtypes 5 . This independence has led to the hypothesis that molecular cues expressed by specific subtypes pattern mosaics by mediating homotypic (within-subtype) short-range repulsive interactions 1 , 4 , 5 , 6 , 7 , 8 , 9 . So far, however, no molecules have been identified that show such activity, so this hypothesis remains untested. Here we demonstrate in mouse that two related transmembrane proteins, MEGF10 and MEGF11, have critical roles in the formation of mosaics by two retinal interneuron subtypes, starburst amacrine cells and horizontal cells. MEGF10 and 11 and their invertebrate relatives Caenorhabditis elegans CED-1 and Drosophila Draper have hitherto been studied primarily as receptors necessary for engulfment of debris following apoptosis or axonal injury 10 , 11 , 12 , 13 , 14 . Our results demonstrate that members of this gene family can also serve as subtype-specific ligands that pattern neuronal arrays.

Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios of full-length isoforms encoded by individual genes. Applying this approach to neural cell-surface molecules, we identify thousands of unannotated isoforms expressed in retina and brain. By mass spectrometry we confirm expression of newly-discovered proteins on the cell surface in vivo. Remarkably, we discover that the major isoform of a retinal degeneration gene, CRB1 , was previously overlooked. This CRB1 isoform is the only one expressed by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we identify a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Therefore, our isoform identification strategy enables discovery of new gene functions relevant to disease. Here the authors present an approach that can reveal the full complement of mRNA isoforms encoded by individual genes, and they identify a major isoform of the retinal degeneration gene CRB1 which functions at the cell-cell junctions of the outer limiting membrane to promote photoreceptor survival.

SignificanceThis study addresses a fundamental question in neuroscience: how do neurons functionally specify and diversify their synaptic connections? This question is particularly relevant in the visual system in which neurons handle a broad range of stimuli requiring synapses to adjust their gain accordingly. In this work, we describe how synapses of cone photoreceptors, which all vertebrate animals utilize for daylight vision, handle this task through an elegant trans-synaptic mechanism involving a splice isoform of a cell-adhesion molecule, Latrophillin 3. Cone photoreceptors mediate daylight vision in vertebrates. Changes in neurotransmitter release at cone synapses encode visual information and is subject to precise control by negative feedback from enigmatic horizontal cells. However, the mechanisms that orchestrate this modulation are poorly understood due to a virtually unknown landscape of molecular players. Here, we report a molecular player operating selectively at cone synapses that modulates effects of horizontal cells on synaptic release. Using an unbiased proteomic screen, we identified an adhesion GPCR Latrophilin3 (LPHN3) in horizontal cell dendrites that engages in transsynaptic control of cones. We detected and characterized a prominent splice isoform of LPHN3 that excludes a element with inhibitory influence on transsynaptic interactions. A gain-of-function mouse model specifically routing LPHN3 splicing to this isoform but not knockout of LPHN3 diminished CaV1.4 calcium channel activity profoundly disrupted synaptic release by cones and resulted in synaptic transmission deficits. These findings offer molecular insight into horizontal cell modulation on cone synaptic function and more broadly demonstrate the importance of alternative splicing in adhesion GPCRs for their physiological function.

A common strategy by which developing neurons locate their synaptic partners is through projections to circuit-specific neuropil sublayers. Once established, sublayers serve as a substrate for selective synapse formation, but how sublayers arise during neurodevelopment remains unknown. Here, we identify the earliest events that initiate formation of the direction-selective circuit in the inner plexiform layer of mouse retina. We demonstrate that radially migrating newborn starburst amacrine cells establish homotypic contacts on arrival at the inner retina. These contacts, mediated by the cell-surface protein MEGF10, trigger neuropil innervation resulting in generation of two sublayers comprising starburst-cell dendrites. This dendritic scaffold then recruits projections from circuit partners. Abolishing MEGF10-mediated contacts profoundly delays and ultimately disrupts sublayer formation, leading to broader direction tuning and weaker direction-selectivity in retinal ganglion cells. Our findings reveal a mechanism by which differentiating neurons transition from migratory to mature morphology, and highlight this mechanism’s importance in forming circuit-specific sublayers. Our experience of the world relies on circuits spanning the sense organs and the brain that process information received through our senses. These circuits are made up of many different types of nerve cells that form connections with each other while the brain is developing. For these circuits to be set up properly, nerve cells have to be selective about how they connect with each other. However, researchers know little about how exactly nerve cells form the right connections, or about which genes and proteins are involved. One of the better understood circuits in the body is known as the ‘direction-selective circuit’. Found in the retina at the back of the eye of all backboned animals, this circuit’s task is to detect the direction that objects are moving. In the case of mice, scientists have identified all of the cells that make up the circuit, and know how they are all supposed to be connected together. This is a useful starting point for researchers to look in more detail at how nerve cells make the right connections during development to set up a working circuit. Ray et al. looked at how the direction-selective circuit forms in the retinas of young mice by genetically engineering cells to carry fluorescent proteins, or staining them with chemicals. This allowed the cells to be examined under a microscope at different points in their development. It turns out that one type of cell, known as the ‘starburst amacrine cell’ because of its firework-like shape, coordinates the formation of the whole direction-selective circuit. First, starburst cells branch out and touch each other. Next, they build a scaffold for the circuit with their branch-like extensions. Finally, other cell types follow this scaffold to form connections and complete the circuit. Ray et al. identified a protein called MEGF10 on the surface of starburst cells that tells the cells when they have made contact with each other. When starburst cells had MEGF10 taken away, or were prevented from contacting each other, they did not build a scaffold properly, and the circuit was less effective at detecting movement. It is possible that cells in other brain circuits use a similar method to form connections. Understanding more about how nerve cells form circuits will help researchers to work out what goes wrong in developmental disorders that affect vision, memory and learning. This knowledge would be helpful for designing new treatments for these conditions.

This study demonstrates the existence of a novel retinal narrow-field amacrine cell subtype that is neither GABAergic nor glycinergic. These cells arise from a late-born glycinergic population and are specified by expression of the transcription factor Neurod6. Most regions of the CNS contain many subtypes of inhibitory interneurons with specialized roles in circuit function. In the mammalian retina, the ∼30 subtypes of inhibitory interneurons called amacrine cells (ACs) are generally divided into two groups: wide/medium-field GABAergic ACs and narrow-field glycinergic ACs, which mediate lateral and vertical interactions, respectively, within the inner plexiform layer. We used expression profiling and mouse transgenic lines to identify and characterize two closely related narrow-field AC subtypes. Both arise postnatally and one is neither glycinergic nor GABAergic (nGnG). Two transcription factors selectively expressed by these subtypes, Neurod6 and special AT-rich-sequence-binding protein 2 (Satb2), regulate a postmitotic cell fate choice between these subtypes. Satb2 induces Neurod6, which persists in nGnG ACs and promotes their fate but is downregulated in the related glycinergic AC subtype. Our results support the view that cell fate decisions made in progenitors and their progeny act together to diversify ACs.

In the inner plexiform layer (IPL) of the mouse retina, ~70 neuronal subtypes organize their neurites into an intricate laminar structure that underlies visual processing. To find recognition proteins involved in lamination, we utilized microarray data from 13 subtypes to identify differentially-expressed extracellular proteins and performed a high-throughput biochemical screen. We identified ~50 previously-unknown receptor-ligand pairs, including new interactions among members of the FLRT and Unc5 families. These proteins show laminar-restricted IPL localization and induce attraction and/or repulsion of retinal neurites in culture, placing them in an ideal position to mediate laminar targeting. Consistent with a repulsive role in arbor lamination, we observed complementary expression patterns for one interaction pair, FLRT2-Unc5C, in vivo. Starburst amacrine cells and their synaptic partners, ON-OFF direction-selective ganglion cells, express FLRT2 and are repelled by Unc5C. These data suggest a single molecular mechanism may have been co-opted by synaptic partners to ensure joint laminar restriction. A nervous system comprises complex circuits of neurons connected by junctions called synapses. These connections need to develop in a highly specific manner, which means neurons need to be able to recognize one another and ‘figure out’ with which neighboring neuron or neurons they should form connections. Neurons do this by physically interacting with one another via proteins on their cell surfaces; these proteins essentially provide instructions to each of the neurons. However, for most neurons, details remain unclear about how they recognize and ‘talk’ to one another to form the connections needed to develop into working neural circuits. To form precise connections, neurons must navigate their way to the appropriate location that places them close to the other neurons with which they need to connect (also known as their “synaptic partners”). In many regions of the nervous system, neurons become organized in parallel layers during development such that synaptic partners reside within the same layer. This process is called lamination and it occurs in the retina in the back of the mammalian eye. Now Visser et al. have searched for the cell surface proteins that are involved in lamination in the mouse retina. This search involved a number of different gene expression, biochemistry and cell biology-based techniques. Visser et al. identified two families of proteins that might control the lamination of many different subtypes of neurons. The findings reveal some of the molecular mechanisms that underlie the formation of neural circuits in the developing retina and suggest that a pair of synaptic partners may use the same recognition proteins to ensure that they target to the same layer. The next step will be to confirm whether the proteins identified are indeed responsible for organizing neurons into distinct layers during the development of the mouse retina.

Loss-of-function mutations in MEGF10 lead to a rare and understudied neuromuscular disorder known as MEGF10 -related myopathy. There are no treatments for the progressive respiratory distress, motor impairment, and structural abnormalities in muscles caused by the loss of MEGF10 function. In this study, we deployed cellular and molecular assays to obtain additional insights about MEGF10 -related myopathy in juvenile, young adult, and middle-aged Megf10 knockout (KO) mice. We found fewer muscle fibers in juvenile and adult Megf10 KO mice, supporting published studies that MEGF10 regulates myogenesis by affecting satellite cell differentiation. Interestingly, muscle fibers do not exhibit morphological hallmarks of atrophy in either young adult or middle-aged Megf10 KO mice. We next examined the neuromuscular junction (NMJ), in which MEGF10 has been shown to concentrate postnatally, using light and electron microscopy. We found early and progressive degenerative features at the NMJs of Megf10 KO mice that include increased postsynaptic fragmentation and presynaptic regions not apposed by postsynaptic nicotinic acetylcholine receptors. We also found perisynaptic Schwann cells intruding into the NMJ synaptic cleft. These findings strongly suggest that the NMJ is a site of postnatal pathology in MEGF10 -related myopathy. In support of these cellular observations, RNA-seq analysis revealed genes and pathways associated with myogenesis, skeletal muscle health, and NMJ stability dysregulated in Megf10 KO mice compared to wild-type mice. Altogether, these data provide new and valuable cellular and molecular insights into MEGF10 -related myopathy.

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders.

In response to injury, progenitor cells in the adult brain can proliferate and generate new neurons and/or glia, which may then participate in injury-induced compensatory processes. In this study, we explore the ability of young adult mice to generate new cells in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesions, which selectively kill nigrostriatal dopaminergic neurons. Using the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU), we labeled dividing cells 3, 10, and 15 days after MPTP lesion. A robust proliferative response was seen specifically in the substantia nigra (SN) and the dorsal striatum 3 days postlesion; the response persisted 10–14 days. To explore the fate of proliferative cells, we administered BrdU 3 days postlesion and examined the phenotype of BrdU + cells at various times thereafter, using double immunolabeling. In the striatum, nearly all newly-generated cells rapidly differentiated into GFAP + astrocytes that participated in the injury-induced glial reaction. In the SN, however, reactive astroglia were not BrdU + . Some midbrain cells co-immunostained for BrdU and Mac-1, a microglial marker. However, most BrdU + cells in the SN failed to express markers for microglia, astroglia, oligodendroglia, or neurons, suggesting that they may remain as uncommitted progenitors. Thus, progenitors in the vicinity of the degenerating dopaminergic cell bodies respond differently to lesion than progenitors in the vicinity of the degenerating axon terminal. Although the putative midbrain progenitors appear uncommitted 22 days after their birth, it is possible that they may adopt neural or glial fates if allowed to survive longer, or if exposed to exogenous factors.