Members of the Cadm/SynCAM/Necl/IGSF/TSLC family of cell adhesion molecules are known to have diverse functions during development of the nervous system, but information regarding their role during central nervous system (CNS) development in vivo is scarce. The rapid development of a relatively simple nervous system in larval zebrafish makes them a highly tractable model organism for studying gene function during nervous system development. An essential prerequisite for functional studies is a description of protein localization. To address this we have generated subtype-specific antibodies to two members of the zebrafish cell adhesion molecule family: cadm2a and cadm3. Using these novel antibodies we show that cadm3 and cadm2a are expressed throughout the nervous system of larval stage zebrafish. Particularly striking, and largely nonoverlapping expression of cadm2a and cadm3 is observed in the developing retina and spinal cord. Using in vitro binding assays we show that cadm2a and cadm3 bind heterophilically and preferentially to cadm1 and cadm4, respectively. These binding preferences are very similar to those seen for tetrapod Cadms but our study of protein localization suggests novel and diverse functions of cadms during nervous system development.

Neurons in layer VI of visual cortex represent one of the largest sources of nonretinal input to the dorsal lateral geniculate nucleus (dLGN) and play a major role in modulating the gain of thalamic signal transmission. However, little is known about how and when these descending projections arrive and make functional connections with dLGN cells. Here we used a transgenic mouse to visualize corticogeniculate projections to examine the timing of cortical innervation in dLGN. Corticogeniculate innervation occurred at postnatal ages and was delayed compared with the arrival of retinal afferents. Cortical fibers began to enter dLGN at postnatal day 3 (P3) to P4, a time when retinogeniculate innervation is complete. However, cortical projections did not fully innervate dLGN until eye opening (P12), well after the time when retinal inputs from the two eyes segregate to form nonoverlapping eye-specific domains. In vitro thalamic slice recordings revealed that newly arriving cortical axons form functional connections with dLGN cells. However, adult-like responses that exhibited paired pulse facilitation did not fully emerge until 2 weeks of age. Finally, surgical or genetic elimination of retinal input greatly accelerated the rate of corticogeniculate innervation, with axons invading between P2 and P3 and fully innervating dLGN by P8 to P10. However, recordings in genetically deafferented mice showed that corticogeniculate synapses continued to mature at the same rate as controls. These studies suggest that retinal and cortical innervation of dLGN is highly coordinated and that input from retina plays an important role in regulating the rate of corticogeniculate innervation.

In many species, neurons are unevenly distributed across the retina, leading to nonuniform analysis of specific visual features at certain locations in visual space. In recent years, the mouse has emerged as a premiere model for probing visual system function, development, and disease. Thus, achieving a detailed understanding of mouse visual circuit architecture is of paramount importance. The general belief is that mice possess a relatively even topographic distribution of retinal ganglion cells (RGCs)-the output neurons of the eye. However, mouse RGCs include ∼30 subtypes; each responds best to a specific feature in the visual scene and conveys that information to central targets. Given the crucial role of RGCs and the prominence of the mouse as a model, we asked how different RGC subtypes are distributed across the retina. We targeted and filled individual fluorescently tagged RGC subtypes from across the retinal surface and evaluated the dendritic arbor extent and soma size of each cell according to its specific retinotopic position. Three prominent RGC subtypes: On-Off direction selective RGCs, object-motion-sensitive RGCs, and a specialized subclass of nonimage-forming RGCs each had marked topographic variations in their dendritic arbor sizes. Moreover, the pattern of variation was distinct for each RGC subtype. Thus, there is increasing evidence that the mouse retina encodes visual space in a region-specific manner. As a consequence, some visual features are sampled far more densely at certain retinal locations than others. These findings have implications for central visual processing, perception, and behavior in this prominent model species.

Conventionally, neuronal development is regarded to follow a stereotypic sequence of neurogenesis, migration, and differentiation. We demonstrate that this notion is not a general principle of neuronal development by documenting the timing of mitosis in relation to multiple differentiation events for bipolar cells (BCs) in the zebrafish retina using in vivo imaging. We found that BC progenitors undergo terminal neurogenic divisions while in markedly disparate stages of neuronal differentiation. Remarkably, the differentiation state of individual BC progenitors at mitosis is not arbitrary but matches the differentiation state of post-mitotic BCs in their surround. By experimentally shifting the relative timing of progenitor division and differentiation, we provide evidence that neurogenesis and differentiation can occur independently of each other. We propose that the uncoupling of neurogenesis and differentiation could provide neurogenic programs with flexibility, while allowing for synchronous neuronal development within a continuously expanding cell pool.

Reactive astrocytes have been implicated in the pathogenesis of neurodegenerative diseases, including a non-cell autonomous effect on motor neuron survival in ALS. We previously defined a mechanism by which microglia release three factors, IL-1α, TNFα, and C1q, to induce neurotoxic astrocytes. Here we report that knocking out these three factors markedly extends survival in the SOD1G93A ALS mouse model, providing evidence for gliosis as a potential ALS therapeutic target.

Glaucoma is a neurodegenerative disease that features the death of retinal ganglion cells (RGCs) in the retina, often as a result of prolonged increases in intraocular pressure. We show that preventing the formation of neuroinflammatory reactive astrocytes prevents the death of RGCs normally seen in a mouse model of glaucoma. Furthermore, we show that these spared RGCs are electrophysiologically functional and thus still have potential value for the function and regeneration of the retina. Finally, we demonstrate that the death of RGCs depends on a combination of both an injury to the neurons and the presence of reactive astrocytes, suggesting a model that may explain why reactive astrocytes are toxic only in some circumstances. Altogether, these findings highlight reactive astrocytes as drivers of RGC death in a chronic neurodegenerative disease of the eye.

Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853∗]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage.

Ribbon synapses transmit information in sensory systems, but their development is not well understood. To test the hypothesis that ribbon assembly stabilizes nascent synapses, we performed simultaneous time-lapse imaging of fluorescently-tagged ribbons in retinal cone bipolar cells (BCs) and postsynaptic densities (PSD95-FP) of retinal ganglion cells (RGCs). Ribbons and PSD95-FP clusters were more stable when these components colocalized at synapses. However, synapse density on ON-alpha RGCs was unchanged in mice lacking ribbons (ribeye knockout). Wildtype BCs make both ribbon-containing and ribbon-free synapses with these GCs even at maturity. Ribbon assembly and cone BC-RGC synapse maintenance are thus regulated independently. Despite the absence of synaptic ribbons, RGCs continued to respond robustly to light stimuli, although quantitative examination of the responses revealed reduced frequency and contrast sensitivity.

The potential gap junction forming mouse connexin29 (Cx29) protein is concomitantly expressed with connexin32 (Cx32) in peripheral myelin forming Schwann cells and together with both Cx32 and connexin47 (Cx47) in oligodendrocytes of the CNS. To study the genomic structure and functional expression of Cx29, either primary cells or cell culture systems might be selected, from which the latter are easier to cultivate. Both structure and expression of Cx29 is still not fully understood. In the mouse sciatic nerve, brain and the oligodendroglial precursor cell line Oli-neu the Cx29 gene is processed in two transcript isoforms both harboring a unique reading frame. In contrast to Cx32 and Cx47, only Cx29 protein is abundantly expressed in undifferentiated as well as differentiated Oli-neu cells but the absence of Etbr dye transfer after microinjection concealed the function of Cx29-mediated gap junction communication between those cells. Although HeLa cells stably transfected with Cx29 or Cx29-eGFP neither demonstrated any permeability for Lucifer yellow nor for neurobiotin, blocking of Etbr uptake from the media by gap junction blockers does suppose a role of Cx29 in hemi-channel function. Thus, we conclude that, due to its high abundance of Cx29 expression and its reproducible culture conditions, the oligodendroglial precursor cell line Oli-neu might constitute an appropriate cell culture system to study molecular mechanisms or putative extracellular stimuli to functionally open Cx29 channels or hemi-channels.

Sensory processing can be tuned by a neuron's integration area, the types of inputs, and the proportion and number of connections with those inputs. Integration areas often vary topographically to sample space differentially across regions. Here, we highlight two visual circuits in which topographic changes in the postsynaptic retinal ganglion cell (RGC) dendritic territories and their presynaptic bipolar cell (BC) axonal territories are either matched or unmatched. Despite this difference, in both circuits, the proportion of inputs from each BC type, i.e., synaptic convergence between specific BCs and RGCs, remained constant across varying dendritic territory sizes. Furthermore, synapse density between BCs and RGCs was invariant across topography. Our results demonstrate a wiring design, likely engaging homotypic axonal tiling of BCs, that ensures consistency in synaptic convergence between specific BC types onto their target RGCs while enabling independent regulation of pre- and postsynaptic territory sizes and synapse number between cell pairs.

Retrieval of synaptic vesicles can occur 1-10 s after fusion, but the role of clathrin during this process has been unclear because the classical mode of clathrin-mediated endocytosis (CME) is an order of magnitude slower, as during retrieval of surface receptors. Classical CME is thought to be rate-limited by the recruitment of clathrin, which raises the question: how is clathrin recruited during synaptic vesicle recycling? To investigate this question we applied total internal reflection fluorescence microscopy (TIRFM) to the synaptic terminal of retinal bipolar cells expressing fluorescent constructs of clathrin light-chain A. Upon calcium influx we observed a fast accumulation of clathrin within 100 ms at the periphery of the active zone. The subsequent loss of clathrin from these regions reflected endocytosis because the application of a potent clathrin inhibitor Pitstop2 dramatically slowed down this phase by ~3 fold. These results indicate that clathrin-dependent retrieval of synaptic vesicles is unusually fast, most probably because of a "priming" step involving a state of association of clathrin with the docked vesicle and with the endosomes and cisternae surrounding the ribbons. Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) showed that the majority of clathrin is moving with the same kinetics as synaptic vesicle proteins. Together, these results indicate that the fast endocytic mechanism operating to retrieve synaptic vesicles differs substantially from the classical mode of CME operating via formation of a coated pit.

The dorsal lateral geniculate nucleus (dLGN) of the mouse has been an important experimental model for understanding thalamic circuit development. The developmental remodeling of retinal projections has been the primary focus, however much less is known about the maturation of their synaptic targets, the relay cells of the dLGN. Here we examined the growth and maturation of relay cells during the first few weeks of life and addressed whether early retinal innervation affects their development. To accomplish this we utilized the math5 null (math5 (-/-) ) mouse, a mutant lacking retinal ganglion cells and central projections.

The maintenance of synaptic transmission requires that vesicles be recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, including a fast "kiss-and-run" mechanism that releases neurotransmitter through a fusion pore. Using an improved fluorescent reporter comprising pHluorin fused to synaptophysin, we find that only a slow mode of endocytosis (tau = 15 s) operates at hippocampal synapses when vesicle fusion is triggered by a single nerve impulse or short burst. This retrieval mechanism is blocked by overexpression of the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi, and it is associated with the movement of clathrin and vesicle proteins out of the synapse. These results indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of vesicle retrieval after physiological stimuli.

Previous studies in developing Xenopus and zebrafish reported that the phosphate transporter slc20a1a is expressed in pronephric kidneys. The recent identification of SLC20A1 as a monoallelic candidate gene for cloacal exstrophy further suggests its involvement in the urinary tract and urorectal development. However, little is known of the functional role of SLC20A1 in urinary tract development. Here, we investigated this using morpholino oligonucleotide knockdown of the zebrafish ortholog slc20a1a. This caused kidney cysts and malformations of the cloaca. Moreover, in morphants we demonstrated dysfunctional voiding and hindgut opening defects mimicking imperforate anus in human cloacal exstrophy. Furthermore, we performed immunohistochemistry of an unaffected 6-week-old human embryo and detected SLC20A1 in the urinary tract and the abdominal midline, structures implicated in the pathogenesis of cloacal exstrophy. Additionally, we resequenced SLC20A1 in 690 individuals with bladder exstrophy-epispadias complex (BEEC) including 84 individuals with cloacal exstrophy. We identified two additional monoallelic de novo variants. One was identified in a case-parent trio with classic bladder exstrophy, and one additional novel de novo variant was detected in an affected mother who transmitted this variant to her affected son. To study the potential cellular impact of SLC20A1 variants, we expressed them in HEK293 cells. Here, phosphate transport was not compromised, suggesting that it is not a disease mechanism. However, there was a tendency for lower levels of cleaved caspase-3, perhaps implicating apoptosis pathways in the disease. Our results suggest SLC20A1 is involved in urinary tract and urorectal development and implicate SLC20A1 as a disease-gene for BEEC.

Classic bladder exstrophy represents the most severe end of all human congenital anomalies of the kidney and urinary tract and is associated with bladder cancer susceptibility. Previous genetic studies identified one locus to be involved in classic bladder exstrophy, but were limited to a restrict number of cohort. Here we show the largest classic bladder exstrophy genome-wide association analysis to date where we identify eight genome-wide significant loci, seven of which are novel. In these regions reside ten coding and four non-coding genes. Among the coding genes is EFNA1, strongly expressed in mouse embryonic genital tubercle, urethra, and primitive bladder. Re-sequence of EFNA1 in the investigated classic bladder exstrophy cohort of our study displays an enrichment of rare protein altering variants. We show that all coding genes are expressed and/or significantly regulated in both mouse and human embryonic developmental bladder stages. Furthermore, nine of the coding genes residing in the regions of genome-wide significance are differentially expressed in bladder cancers. Our data suggest genetic drivers for classic bladder exstrophy, as well as a possible role for these drivers to relevant bladder cancer susceptibility.

Myelination is crucial for the development and maintenance of axonal integrity, especially fast axonal action potential conduction. There is increasing evidence that glutamate signaling and release through neuronal activity modulates the myelination process. In this study, we examine the effect of manipulating glutamate signaling on myelination of oligodendrocyte (OL) lineage cells and their development in zebrafish (zf). We use the "intensity-based glutamate-sensing fluorescent reporter" (iGluSnFR) in the zf model (both sexes) to address the hypothesis that glutamate is implicated in regulation of myelinating OLs. Our results show that glial iGluSnFR expression significantly reduces OL lineage cell number and the expression of myelin markers in larvae (zfl) and adult brains. The specific glutamate receptor agonist, L-AP4, rescues this iGluSnFR effect by significantly increasing the expression of the myelin-related genes, plp1b and mbpa, and enhances myelination in L-AP4-injected zfl compared to controls. Furthermore, we demonstrate that degrading glutamate using Glutamat-Pyruvate Transaminase (GPT) or the blockade of glutamate reuptake by L-trans-pyrrolidine-2,4-dicarboxylate (PDC) significantly decreases myelin-related genes and drastically declines myelination in brain ventricle-injected zfl. Moreover, we found that myelin-specific ClaudinK (CldnK) and 36K protein expression is significantly decreased in iGluSnFR-expressing zfl and adult brains compared to controls. Taken together, this study confirms that glutamate signaling is directly required for the preservation of myelinating OLs and for the myelination process itself. These findings further suggest that glutamate signaling may provide novel targets to therapeutically boost remyelination in several demyelinating diseases of the CNS.

The visual system transmits information about fast and slow changes in light intensity through separate neural pathways. We used in vivo imaging to investigate how bipolar cells transmit these signals to the inner retina. We found that the volume of the synaptic terminal is an intrinsic property that contributes to different temporal filters. Individual cells transmit through multiple terminals varying in size, but smaller terminals generate faster and larger calcium transients to trigger vesicle release with higher initial gain, followed by more profound adaptation. Smaller terminals transmitted higher stimulus frequencies more effectively. Modeling global calcium dynamics triggering vesicle release indicated that variations in the volume of presynaptic compartments contribute directly to all these differences in response dynamics. These results indicate how one neuron can transmit different temporal components in the visual signal through synaptic terminals of varying geometries with different adaptational properties.

Retinal ganglion cell (RGC) loss is a hallmark of glaucoma and the second leading cause of blindness worldwide. The type and timing of cellular changes leading to RGC loss in glaucoma remain incompletely understood, including whether specific RGC subtypes are preferentially impacted at early stages of this disease. Here we applied the microbead occlusion model of glaucoma to different transgenic mouse lines, each expressing green fluorescent protein in 1-2 specific RGC subtypes. Targeted filling, reconstruction, and subsequent comparison of the genetically identified RGCs in control and bead-injected eyes revealed that some subtypes undergo significant dendritic rearrangements as early as 7 d following induction of elevated intraocular pressure (IOP). By comparing specific On-type, On-Off-type and Off-type RGCs, we found that RGCs that target the majority of their dendritic arbors to the scleral half or "Off" sublamina of the inner plexiform layer (IPL) undergo the greatest changes, whereas RGCs with the majority of their dendrites in the On sublamina did not alter their structure at this time point. Moreover, M1 intrinsically photosensitive RGCs, which functionally are On RGCs but structurally stratify their dendrites in the Off sublamina of the IPL, also underwent significant changes in dendritic structure 1 week after elevated IOP. Thus, our findings reveal that certain RGC subtypes manifest significant changes in dendritic structure after very brief exposure to elevated IOP. The observation that RGCs stratifying most of their dendrites in the Off sublamina are first to alter their structure may inform the development of new strategies to detect, monitor, and treat glaucoma in humans.

Understanding how neural circuits transmit information is technically challenging because the neural code is contained in the activity of large numbers of neurons and synapses. Here, we use genetically encoded reporters to image synaptic transmission across a population of sensory neurons-bipolar cells in the retina of live zebrafish. We demonstrate that the luminance sensitivities of these synapses varies over 10(4) with a log-normal distribution. About half the synapses made by ON and OFF cells alter their polarity of transmission as a function of luminance to generate a triphasic tuning curve with distinct maxima and minima. These nonlinear synapses signal temporal contrast with greater sensitivity than linear ones. Triphasic tuning curves increase the dynamic range over which bipolar cells signal light and improve the efficiency with which luminance information is transmitted. The most efficient synapses signaled luminance using just 1 synaptic vesicle per second per distinguishable gray level.

Redox regulation of specific cysteines via oxidoreductases of the thioredoxin family is increasingly being recognized as an important signaling pathway. Here, we demonstrate that the cytosolic isoform of the vertebrate-specific oxidoreductase Glutaredoxin 2 (Grx2c) regulates the redox state of the transcription factor SP-1 and thereby its binding affinity to both the promoter and an enhancer region of the CSPG4 gene encoding chondroitin sulfate proteoglycan nerve/glial antigen 2 (NG2). This leads to an increased number of NG2 glia during in vitro oligodendroglial differentiation and promotes migration of these wound healing cells. On the other hand, we found that the same mechanism also leads to increased invasion of glioma tumor cells. Using in vitro (human cell lines), ex vivo (mouse primary cells), and in vivo models (zebrafish), as well as glioblastoma patient tissue samples we provide experimental data highlighting the Yin and Yang of redox signaling in the central nervous system and the enzymatic Taoism of Grx2c.