We investigate mutations in trbeta2, a splice variant of thrb, identifying changes in function, structure, and behavior in larval and adult zebrafish retinas. Two N-terminus CRISPR mutants were ...identified. The first is a 6BP+1 insertion deletion frameshift resulting in a truncated protein. The second is a 3BP in frame deletion with intact binding domains. ERG recordings of isolated cone signals showed that the 6BP+1 mutants did not respond to red wavelengths of light while the 3BP mutants did respond. 6BP+1 mutants lacked optomotor and optokinetic responses to red/black and green/black contrasts. Both larval and adult 6BP+1 mutants exhibit a loss of red-cone contribution to the ERG and an increase in UV-cone contribution. Transgenic reporters show loss of cone trbeta2 activation in the 6BP+1 mutant but increase in the density of cones with active blue, green, and UV opsin genes. Antibody reactivity for red-cone LWS1 and LWS2 opsin was absent in the 6BP+1 mutant, as was reactivity for arrestin3a. Our results confirm a critical role for trbeta2 in long-wavelength cone development.
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Excitatory and inhibitory neurons in the CNS are distinguished by several features, including morphology, transmitter content, and synapse architecture 1. Such distinctions are exemplified in the ...vertebrate retina. Retinal bipolar cells are polarized glutamatergic neurons receiving direct photoreceptor input, whereas amacrine cells are usually monopolar inhibitory interneurons with synapses almost exclusively in the inner retina 2. Bipolar but not amacrine cell synapses have presynaptic ribbon-like structures at their transmitter release sites. We identified a monopolar interneuron in the mouse retina that resembles amacrine cells morphologically but is glutamatergic and, unexpectedly, makes ribbon synapses. These glutamatergic monopolar interneurons (GluMIs) do not receive direct photoreceptor input, and their light responses are strongly shaped by both ON and OFF pathway-derived inhibitory input. GluMIs contact and make almost as many synapses as type 2 OFF bipolar cells onto OFF-sustained A-type (AOFF-S) retinal ganglion cells (RGCs). However, GluMIs and type 2 OFF bipolar cells possess functionally distinct light-driven responses and may therefore mediate separate components of the excitatory synaptic input to AOFF-S RGCs. The identification of GluMIs thus unveils a novel cellular component of excitatory circuits in the vertebrate retina, underscoring the complexity in defining cell types even in this well-characterized region of the CNS.
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•GluMIs provide a novel route of light-driven excitation to retinal ganglion cells•GluMIs are monopolar cells but express synaptic features of bipolar cells•GluMIs have light-response properties that differ from type 2 bipolar cells•GluMIs and bipolar cells may provide functionally distinct input to ganglion cells
Della Santina et al. identify a new neuron type in the mouse retina. These cells (GluMIs) resemble inhibitory interneurons morphologically but make glutamatergic ribbon synapses in the inner retina. GluMIs have light-response features that are distinct from known excitatory interneurons and thus provide a novel pathway for excitation in the retina.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Proper functioning of sensory systems requires the generation of appropriate numbers and proportions of neuronal subtypes that encode distinct information. Perception of color relies on signals from ...multiple cone photoreceptor types. In cone-dominated retinas, each cone expresses a single opsin type with peak sensitivity to UV, long (L) (red), medium (M) (green), or short (S) (blue) wavelengths. The modes of cell division generating distinct cone types are unknown. We report here a mechanism whereby zebrafish cone photoreceptors of the same type are produced by symmetric division of dedicated precursors. Transgenic fish in which the thyroid hormone receptor β2 (trβ2) promoter drives fluorescent protein expression before L-cone precursors themselves are produced permitted tracking of their division in vivo. Every L cone in a local region resulted from the terminal division of an L-cone precursor, suggesting that such divisions contribute significantly to L-cone production. Analysis of the fate of isolated pairs of cones and time-lapse observations suggest that other cone types can also arise by symmetric terminal divisions. Such divisions of dedicated precursors may help to rapidly attain the final numbers and proportions of cone types (L > M, UV > S) in zebrafish larvae. Loss-and gain-of-function experiments show that L-opsin expression requires trftë activity before cone differentiation. Ectopic expression of trβ2 after cone differentiation produces cones with mixed opsins. Temporal differences in the onset of trβ2 expression could explain why some species have mixed, and others have pure, cone types.
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A major challenge in regenerative medicine is replacing cells lost through injury or disease. While significant progress has been made, much remains unknown about the accuracy of native regenerative ...programs in cell replacement. Here, we capitalized on the regenerative capacity and stereotypic retinal organization of zebrafish to determine the specificity with which retinal Müller glial cells replace lost neuronal cell types. By utilizing a targeted genetic ablation technique, we restricted death to all or to distinct cone photoreceptor types (red, blue, or UV‐sensitive cones), enabling us to compare the composition of cones that are regenerated. We found that Müller glia produce cones of all types upon nondiscriminate ablation of these photoreceptors, or upon selective ablation of red or UV cones. Pan‐ablation of cones led to regeneration of the various cone types in relative abundances that resembled those of nonablated controls, that is, red > green > UV ~ blue cones. Moreover, selective loss of red or UV cones biased production toward the cone type that was ablated. In contrast, ablation of blue cones alone largely failed to induce cone production at all, although it did induce cell division in Müller glia. The failure to produce cones upon selective elimination of blue cones may be due to their low abundance compared to other cone types. Alternatively, it may be that blue cone death alone does not trigger a change in progenitor competency to support cone genesis. Our findings add to the growing notion that cell replacement during regeneration does not perfectly mimic programs of cell generation during development.
The regenerative capacity of the zebrafish, and the stereotypic organization of its retinal cell populations, enable examination of the specificity by which retinal Müller glial cells replace neurons that are lost. Zebrafish have four cone types, with peak sensitivities to red, green, blue, and ultraviolet wavelengths. Using a targeted cell ablation approach, we found that Müller glia produced cones of all types when all cones, red cones and UV cones were ablated. In contrast, blue cone ablation largely failed to generate cones. The relative proportions of regenerated cells of each type depended on the cone types that were ablated, and was biased toward the major cone type that was lost.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Whether neurons can restore their original connectivity patterns during circuit repair is unclear. Taking advantage of the regenerative capacity of zebrafish retina, we show here the remarkable ...specificity by which surviving neurons reassemble their connectivity upon regeneration of their major input. H3 horizontal cells (HCs) normally avoid red and green cones, and prefer ultraviolet over blue cones. Upon ablation of the major (ultraviolet) input, H3 HCs do not immediately increase connectivity with other cone types. Instead, H3 dendrites retract and re-extend to contact new ultraviolet cones. But, if regeneration is delayed or absent, blue-cone synaptogenesis increases and ectopic synapses are made with red and green cones. Thus, cues directing synapse specificity can be maintained following input loss, but only within a limited time period. Further, we postulate that signals from the major input that shape the H3 HC's wiring pattern during development persist to restrict miswiring after damage.
Neuronal identity has long been thought of as immutable, so that once a cell acquires a specific fate, it is maintained for life.1 Studies using the overexpression of potent transcription factors to ...experimentally reprogram neuronal fate in the mouse neocortex2,3 and retina4,5 have challenged this notion by revealing that post-mitotic neurons can switch their identity. Whether fate reprogramming is part of normal development in the central nervous system (CNS) is unclear. While there are some reports of physiological cell fate reprogramming in invertebrates,6,7 and in the vertebrate peripheral nervous system,8 endogenous fate reprogramming in the vertebrate CNS has not been documented. Here, we demonstrate spontaneous fate re-specification in an interneuron lineage in the zebrafish retina. We show that the visual system homeobox 1 (vsx1)-expressing lineage, which has been associated exclusively with excitatory bipolar cell (BC) interneurons,9–12 also generates inhibitory amacrine cells (ACs). We identify a role for Notch signaling in conferring plasticity to nascent vsx1 BCs, allowing suitable transcription factor programs to re-specify them to an AC fate. Overstimulating Notch signaling enhances this physiological phenotype so that both daughters of a vsx1 progenitor differentiate into ACs and partially differentiated vsx1 BCs can be converted into ACs. Furthermore, this physiological re-specification can be mimicked to allow experimental induction of an entirely distinct fate, that of retinal projection neurons, from the vsx1 lineage. Our observations reveal unanticipated plasticity of cell fate during retinal development.
•Spontaneous fate reprogramming in the vsx1 lineage of the zebrafish retina•Nascent vsx1 bipolar cells are re-specified to an amacrine cell fate•Notch signaling plays a major role in vsx1 bipolar cell re-specification•Over-stimulating Notch signaling enhances reprogramming in the vsx1 lineage
Engerer, Petridou et al. find evidence for spontaneous cell fate reprogramming during retinal development. Newly post-mitotic vsx1 bipolar cells switch to an amacrine cell fate. Notch signaling plays a major role, conferring plasticity to nascent bipolar cells, allowing suitable transcription factors to instruct the amacrine cell fate.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The outer epithelial layer of zebrafish retinae contains a crystalline array of cone photoreceptors, called the cone mosaic. As this mosaic grows by mitotic addition of new photoreceptors at the rim ...of the hemispheric retina, topological defects, called "Y-Junctions", form to maintain approximately constant cell spacing. The generation of topological defects due to growth on a curved surface is a distinct feature of the cone mosaic not seen in other well-studied biological patterns like the R8 photoreceptor array in the Drosophila compound eye. Since defects can provide insight into cell-cell interactions responsible for pattern formation, here we characterize the arrangement of cones in individual Y-Junction cores as well as the spatial distribution of Y-junctions across entire retinae. We find that for individual Y-junctions, the distribution of cones near the core corresponds closely to structures observed in physical crystals. In addition, Y-Junctions are organized into lines, called grain boundaries, from the retinal center to the periphery. In physical crystals, regardless of the initial distribution of defects, defects can coalesce into grain boundaries via the mobility of individual particles. By imaging in live fish, we demonstrate that grain boundaries in the cone mosaic instead appear during initial mosaic formation, without requiring defect motion. Motivated by this observation, we show that a computational model of repulsive cell-cell interactions generates a mosaic with grain boundaries. In contrast to paradigmatic models of fate specification in mostly motionless cell packings, this finding emphasizes the role of cell motion, guided by cell-cell interactions during differentiation, in forming biological crystals. Such a route to the formation of regular patterns may be especially valuable in situations, like growth on a curved surface, where the resulting long-ranged, elastic, effective interactions between defects can help to group them into grain boundaries.
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Highly polarized cells such as photoreceptors require precise and efficient strategies for establishing and maintaining the proper subcellular distribution of proteins. The signals and molecular ...machinery that regulate trafficking and sorting of synaptic proteins within cone inner segments is mostly unknown. In this study, we show that the polyphosphoinositide phosphatase Synaptojanin 1 (SynJ1) is critical for this process. We used transgenic markers for trafficking pathways, electron microscopy, and immunocytochemistry to characterize trafficking defects in cones of the zebrafish mutant, nrc(a14) , which is deficient in phosphoinositide phosphatase, SynJ1. The outer segments and connecting cilia of nrc(a14) cone photoreceptors are normal, but RibeyeB and VAMP2/synaptobrevin, which normally localize to the synapse, accumulate in the nrc(a14) inner segment. The structure of the Endoplasmic Reticulum in nrc(a14) mutant cones is normal. Golgi develop normally, but later become disordered. Large vesicular structures accumulate within nrc(a14) cone photoreceptor inner segments, particularly after prolonged incubation in darkness. Cone inner segments of nrc (a14) mutants also have enlarged acidic vesicles, abnormal late endosomes, and a disruption in autophagy. This last pathway also appears exacerbated by darkness. Taken altogether, these findings show that SynJ1 is required in cones for normal endolysosomal trafficking of synaptic proteins.
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Neuronal output is modulated by inhibition onto both dendrites and axons. It is unknown whether inhibitory synapses at these two cellular compartments of an individual neuron are regulated ...coordinately or separately during in vivo development. Because neurotransmission influences synapse maturation and circuit development, we determined how loss of inhibition affects the expression of diverse types of inhibitory receptors on the axon and dendrites of mouse retinal bipolar cells. We found that axonal GABA but not glycine receptor expression depends on neurotransmission. Importantly, axonal and dendritic GABAAreceptors comprise distinct subunit compositions that are regulated differentially by GABA release: Axonal GABAAreceptors are down-regulated but dendritic receptors are up-regulated in the absence of inhibition. The homeostatic increase in GABAAreceptors on bipolar cell dendrites is pathway-specific: Cone but not rod bipolar cell dendrites maintain an up-regulation of receptors in the transmission deficient mutants. Furthermore, the bipolar cell GABAAreceptor alterations are a consequence of impaired vesicular GABA release from amacrine but not horizontal interneurons. Thus, inhibitory neurotransmission regulates in vivo postsynaptic maturation of inhibitory synapses with contrasting modes of action specific to synapse type and location.
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Classic cadherins represent a family of calcium-dependent homophilic cell-cell adhesion molecules. They confer strong adhesiveness to animal cells when they are anchored to the actin cytoskeleton via ...their cytoplasmic binding partners, catenins. The cadherin/catenin adhesion system plays key roles in the morphogenesis and function of the vertebrate and invertebrate nervous systems. In early vertebrate development, cadherins are involved in multiple events of brain morphogenesis including the formation and maintenance of the neuroepithelium, neurite extension and migration of neuronal cells. In the invertebrate nervous system, classic cadherin-mediated cell-cell interaction plays important roles in wiring among neurons. For synaptogenesis, the cadherin/catenin system not only stabilizes cell-cell contacts at excitatory synapses but also assembles synaptic molecules at synaptic sites. Furthermore, this system is involved in synaptic plasticity. Recent studies on the role of individual cadherin subtypes at synapses indicate that individual cadherin subtypes play their own unique role to regulate synaptic activities.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
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