Publications by authors named "Joshua R Sanes"

177 Publications

Optic nerve regeneration screen identifies multiple genes restricting adult neural repair.

Cell Rep 2021 Mar;34(9):108777

Cellular Neuroscience, Neurodegeneration, Repair, Departments of Neurology and of Neuroscience, Yale University School of Medicine, New Haven, CT 06536, USA. Electronic address:

Adult mammalian central nervous system (CNS) trauma interrupts neural networks and, because axonal regeneration is minimal, neurological deficits persist. Repair via axonal growth is limited by extracellular inhibitors and cell-autonomous factors. Based on results from a screen in vitro, we evaluate nearly 400 genes through a large-scale in vivo regeneration screen. Suppression of 40 genes using viral-driven short hairpin RNAs (shRNAs) promotes retinal ganglion cell (RGC) axon regeneration after optic nerve crush (ONC), and most are validated by separate CRISPR-Cas9 editing experiments. Expression of these axon-regeneration-suppressing genes is not significantly altered by axotomy. Among regeneration-limiting genes, loss of the interleukin 22 (IL-22) cytokine allows an early, yet transient, inflammatory response in the retina after injury. Reduced IL-22 drives concurrent activation of signal transducer and activator of transcription 3 (Stat3) and dual leucine zipper kinase (DLK) pathways and upregulation of multiple neuron-intrinsic regeneration-associated genes (RAGs). Including IL-22, our screen identifies dozens of genes that limit CNS regeneration. Suppression of these genes in the context of axonal damage could support improved neural repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2021.108777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009559PMC
March 2021

A cell atlas of the chick retina based on single-cell transcriptomics.

Elife 2021 Jan 4;10. Epub 2021 Jan 4.

Center for Brain Science and Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States.

Retinal structure and function have been studied in many vertebrate orders, but molecular characterization has been largely confined to mammals. We used single-cell RNA sequencing (scRNA-seq) to generate a cell atlas of the chick retina. We identified 136 cell types plus 14 positional or developmental intermediates distributed among the six classes conserved across vertebrates - photoreceptor, horizontal, bipolar, amacrine, retinal ganglion, and glial cells. To assess morphology of molecularly defined types, we adapted a method for CRISPR-based integration of reporters into selectively expressed genes. For Müller glia, we found that transcriptionally distinct cells were regionally localized along the anterior-posterior, dorsal-ventral, and central-peripheral retinal axes. We also identified immature photoreceptor, horizontal cell, and oligodendrocyte types that persist into late embryonic stages. Finally, we analyzed relationships among chick, mouse, and primate retinal cell classes and types. Our results provide a foundation for anatomical, physiological, evolutionary, and developmental studies of the avian visual system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.63907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837701PMC
January 2021

Molecular classification of zebrafish retinal ganglion cells links genes to cell types to behavior.

Neuron 2021 02 23;109(4):645-662.e9. Epub 2020 Dec 23.

Max Planck Institute of Neurobiology, Department Genes - Circuits - Behavior, 82152 Martinsried, Germany. Electronic address:

Retinal ganglion cells (RGCs) form an array of feature detectors, which convey visual information to central brain regions. Characterizing RGC diversity is required to understand the logic of the underlying functional segregation. Using single-cell transcriptomics, we systematically classified RGCs in adult and larval zebrafish, thereby identifying marker genes for >30 mature types and several developmental intermediates. We used this dataset to engineer transgenic driver lines, enabling specific experimental access to a subset of RGC types. Expression of one or few transcription factors often predicts dendrite morphologies and axonal projections to specific tectal layers and extratectal targets. In vivo calcium imaging revealed that molecularly defined RGCs exhibit specific functional tuning. Finally, chemogenetic ablation of eomesa RGCs, which comprise melanopsin-expressing types with projections to a small subset of central targets, selectively impaired phototaxis. Together, our study establishes a framework for systematically studying the functional architecture of the visual system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2020.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897282PMC
February 2021

A community-based transcriptomics classification and nomenclature of neocortical cell types.

Nat Neurosci 2020 12;23(12):1456-1468

Vanderbilt University, Nashville, TN, USA.

To understand the function of cortical circuits, it is necessary to catalog their cellular diversity. Past attempts to do so using anatomical, physiological or molecular features of cortical cells have not resulted in a unified taxonomy of neuronal or glial cell types, partly due to limited data. Single-cell transcriptomics is enabling, for the first time, systematic high-throughput measurements of cortical cells and generation of datasets that hold the promise of being complete, accurate and permanent. Statistical analyses of these data reveal clusters that often correspond to cell types previously defined by morphological or physiological criteria and that appear conserved across cortical areas and species. To capitalize on these new methods, we propose the adoption of a transcriptome-based taxonomy of cell types for mammalian neocortex. This classification should be hierarchical and use a standardized nomenclature. It should be based on a probabilistic definition of a cell type and incorporate data from different approaches, developmental stages and species. A community-based classification and data aggregation model, such as a knowledge graph, could provide a common foundation for the study of cortical circuits. This community-based classification, nomenclature and data aggregation could serve as an example for cell type atlases in other parts of the body.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41593-020-0685-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7683348PMC
December 2020

Cell Atlas of The Human Fovea and Peripheral Retina.

Sci Rep 2020 06 17;10(1):9802. Epub 2020 Jun 17.

Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA, 02138, USA.

Most irreversible blindness results from retinal disease. To advance our understanding of the etiology of blinding diseases, we used single-cell RNA-sequencing (scRNA-seq) to analyze the transcriptomes of ~85,000 cells from the fovea and peripheral retina of seven adult human donors. Utilizing computational methods, we identified 58 cell types within 6 classes: photoreceptor, horizontal, bipolar, amacrine, retinal ganglion and non-neuronal cells. Nearly all types are shared between the two retinal regions, but there are notable differences in gene expression and proportions between foveal and peripheral cohorts of shared types. We then used the human retinal atlas to map expression of 636 genes implicated as causes of or risk factors for blinding diseases. Many are expressed in striking cell class-, type-, or region-specific patterns. Finally, we compared gene expression signatures of cell types between human and the cynomolgus macaque monkey, Macaca fascicularis. We show that over 90% of human types correspond transcriptomically to those previously identified in macaque, and that expression of disease-related genes is largely conserved between the two species. These results validate the use of the macaque for modeling blinding disease, and provide a foundation for investigating molecular mechanisms underlying visual processing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-66092-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299956PMC
June 2020

Mouse Retinal Cell Atlas: Molecular Identification of over Sixty Amacrine Cell Types.

J Neurosci 2020 07 26;40(27):5177-5195. Epub 2020 May 26.

Center for Brain Science and Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138

Amacrine cells (ACs) are a diverse class of interneurons that modulate input from photoreceptors to retinal ganglion cells (RGCs), rendering each RGC type selectively sensitive to particular visual features, which are then relayed to the brain. While many AC types have been identified morphologically and physiologically, they have not been comprehensively classified or molecularly characterized. We used high-throughput single-cell RNA sequencing to profile >32,000 ACs from mice of both sexes and applied computational methods to identify 63 AC types. We identified molecular markers for each type and used them to characterize the morphology of multiple types. We show that they include nearly all previously known AC types as well as many that had not been described. Consistent with previous studies, most of the AC types expressed markers for the canonical inhibitory neurotransmitters GABA or glycine, but several expressed neither or both. In addition, many expressed one or more neuropeptides, and two expressed glutamatergic markers. We also explored transcriptomic relationships among AC types and identified transcription factors expressed by individual or multiple closely related types. Noteworthy among these were and , expressed by most GABAergic and most glycinergic types, respectively. Together, these results provide a foundation for developmental and functional studies of ACs, as well as means for genetically accessing them. Along with previous molecular, physiological, and morphologic analyses, they establish the existence of at least 130 neuronal types and nearly 140 cell types in the mouse retina. The mouse retina is a leading model for analyzing the development, structure, function, and pathology of neural circuits. A complete molecular atlas of retinal cell types provides an important foundation for these studies. We used high-throughput single-cell RNA sequencing to characterize the most heterogeneous class of retinal interneurons, amacrine cells, identifying 63 distinct types. The atlas includes types identified previously as well as many novel types. We provide evidence for the use of multiple neurotransmitters and neuropeptides, and identify transcription factors expressed by groups of closely related types. Combining these results with those obtained previously, we proposed that the mouse retina contains ∼130 neuronal types and is therefore comparable in complexity to other regions of the brain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.0471-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329304PMC
July 2020

Synaptic Specificity, Recognition Molecules, and Assembly of Neural Circuits.

Cell 2020 04;181(3):536-556

Department of Biological Chemistry, HHMI, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

Developing neurons connect in specific and stereotyped ways to form the complex circuits that underlie brain function. By comparison to earlier steps in neural development, progress has been slow in identifying the cell surface recognition molecules that mediate these synaptic choices, but new high-throughput imaging, genetic, and molecular methods are accelerating progress. Over the past decade, numerous large and small gene families have been implicated in target recognition, including members of the immunoglobulin, cadherin, and leucine-rich repeat superfamilies. We review these advances and propose ways in which combinatorial use of multifunctional recognition molecules enables the complex neuron-neuron interactions that underlie synaptic specificity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.04.008DOI Listing
April 2020

Cell atlas of aqueous humor outflow pathways in eyes of humans and four model species provides insight into glaucoma pathogenesis.

Proc Natl Acad Sci U S A 2020 05 27;117(19):10339-10349. Epub 2020 Apr 27.

Center for Brain Science, Harvard University, Cambridge, MA 02138;

Increased intraocular pressure (IOP) represents a major risk factor for glaucoma, a prevalent eye disease characterized by death of retinal ganglion cells; lowering IOP is the only proven treatment strategy to delay disease progression. The main determinant of IOP is the equilibrium between production and drainage of aqueous humor, with compromised drainage generally viewed as the primary contributor to dangerous IOP elevations. Drainage occurs through two pathways in the anterior segment of the eye called conventional and uveoscleral. To gain insights into the cell types that comprise these pathways, we used high-throughput single-cell RNA sequencing (scRNAseq). From ∼24,000 single-cell transcriptomes, we identified 19 cell types with molecular markers for each and used histological methods to localize each type. We then performed similar analyses on four organisms used for experimental studies of IOP dynamics and glaucoma: cynomolgus macaque (), rhesus macaque (), pig (), and mouse (). Many human cell types had counterparts in these models, but differences in cell types and gene expression were evident. Finally, we identified the cell types that express genes implicated in glaucoma in all five species. Together, our results provide foundations for investigating the pathogenesis of glaucoma and for using model systems to assess mechanisms and potential interventions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2001250117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229661PMC
May 2020

Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

Neuron 2020 04 5;106(1):37-65.e5. Epub 2020 Feb 5.

Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2020.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377387PMC
April 2020

Binary Fate Choice between Closely Related Interneuronal Types Is Determined by a Fezf1-Dependent Postmitotic Transcriptional Switch.

Neuron 2020 02 4;105(3):464-474.e6. Epub 2019 Dec 4.

Center for Brain Science and Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address:

Many neuronal types occur as pairs that are similar in most respects but differ in a key feature. In some pairs of retinal neurons, called paramorphic, one member responds to increases and the other to decreases in luminance (ON and OFF responses). Here, we focused on one such pair, starburst amacrine cells (SACs), to explore how closely related neuronal types diversify. We find that ON and OFF SACs are transcriptionally distinct prior to their segregation, dendritic outgrowth, and synapse formation. The transcriptional repressor Fezf1 is selectively expressed by postmitotic ON SACs and promotes the ON fate and gene expression program while repressing the OFF fate and program. The atypical Rho GTPase Rnd3 is selectively expressed by OFF SACs and regulates their migration but is repressed by Fezf1 in ON SACs, enabling differential positioning of the two types. These results define a transcriptional program that controls diversification of a paramorphic pair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2019.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007373PMC
February 2020

Single-Cell Profiles of Retinal Ganglion Cells Differing in Resilience to Injury Reveal Neuroprotective Genes.

Neuron 2019 12 26;104(6):1039-1055.e12. Epub 2019 Nov 26.

Center for Brain Science and Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA. Electronic address:

Neuronal types in the central nervous system differ dramatically in their resilience to injury or other insults. Here we studied the selective resilience of mouse retinal ganglion cells (RGCs) following optic nerve crush (ONC), which severs their axons and leads to death of ∼80% of RGCs within 2 weeks. To identify expression programs associated with differential resilience, we first used single-cell RNA-seq (scRNA-seq) to generate a comprehensive molecular atlas of 46 RGC types in adult retina. We then tracked their survival after ONC; characterized transcriptomic, physiological, and morphological changes that preceded degeneration; and identified genes selectively expressed by each type. Finally, using loss- and gain-of-function assays in vivo, we showed that manipulating some of these genes improved neuronal survival and axon regeneration following ONC. This study provides a systematic framework for parsing type-specific responses to injury and demonstrates that differential gene expression can be used to reveal molecular targets for intervention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2019.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923571PMC
December 2019

Tell me a story.

Authors:
Joshua R Sanes

Elife 2019 08 6;8. Epub 2019 Aug 6.

Center for Brain Science, Harvard University, Cambridge, United States.

Many authors start with the figures when writing a scientific paper, but it is easier to tell a good story if you start with the Introduction and the Results, and leave the figures to later.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.50527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684263PMC
August 2019

Molecular Classification and Comparative Taxonomics of Foveal and Peripheral Cells in Primate Retina.

Cell 2019 02 31;176(5):1222-1237.e22. Epub 2019 Jan 31.

Center for Brain Science and Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address:

High-acuity vision in primates, including humans, is mediated by a small central retinal region called the fovea. As more accessible organisms lack a fovea, its specialized function and its dysfunction in ocular diseases remain poorly understood. We used 165,000 single-cell RNA-seq profiles to generate comprehensive cellular taxonomies of macaque fovea and peripheral retina. More than 80% of >60 cell types match between the two regions but exhibit substantial differences in proportions and gene expression, some of which we relate to functional differences. Comparison of macaque retinal types with those of mice reveals that interneuron types are tightly conserved. In contrast, projection neuron types and programs diverge, despite exhibiting conserved transcription factor codes. Key macaque types are conserved in humans, allowing mapping of cell-type and region-specific expression of >190 genes associated with 7 human retinal diseases. Our work provides a framework for comparative single-cell analysis across tissue regions and species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424338PMC
February 2019

Expression and Roles of the Immunoglobulin Superfamily Recognition Molecule Sidekick1 in Mouse Retina.

Front Mol Neurosci 2018 9;11:485. Epub 2019 Jan 9.

Department of Molecular and Cellular Biology, Center for Brain Science, Harvard University, Cambridge, MA, United States.

Processes of >100 types of interneurons (bipolar and amacrine cells) and projection neurons (retinal ganglion cells, RGCs) form specific and stereotyped patterns of connections in the inner plexiform layer (IPL) of the mouse retina. Four closely related homophilic immunoglobulin superfamily recognition molecules (Sidekick [Sdk] 1, Sdk 2, Dscam, and DscamL1) have been shown to play roles in patterning neuronal arbors and connections in chick retina, and all but Sdk1 have been shown to play related roles in mice. Here, we compare patterns of Sdk1 and Sdk2 expression in mouse retina and use genetic methods to assess roles of Sdk1. In adult retina, 3 neuronal types express but not at detectable levels, 5 express but not and 3 express both. Patterns of gene expression and protein localization at or near synapses are established during the first postnatal week. Dendrites of amacrine cells and RGCs that express but not arborize in the same narrow stratum in the center of the IPL. In the absence of Sdk1, this laminar restriction is degraded. Overexpression of in developing cells that normally express reorients their dendrites to resemble those of endogenously Sdk1-positive cells, indicating that Sdk1 plays an instructive role in patterning the IPL. Sdk1 fails to affect arbors when introduced after they are mature, suggesting that it is required to form but not maintain laminar restrictions. The effect of ectopically expressed sdk1 requires the presence of endogenous Sdk1, suggesting that the effect requires homophilic interactions among Sdk1-positive neurites. Together with previous results on Sdk2, Dscam, DscamL1, as well as the related Contactins, our results support the idea that an elaborate immunoglobulin superfamily code plays a prominent role in establishing neural circuits in the retina by means of tightly regulated cell type-specific expression and homophilically restricted intercellular interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnmol.2018.00485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333872PMC
January 2019

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes.

Front Mol Neurosci 2018 23;11:385. Epub 2018 Oct 23.

Center for Brain Science and Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, United States.

Transgenic mouse lines are routinely employed to label and manipulate distinct cell types. The transgene generally comprises cell-type specific regulatory elements linked to a cDNA encoding a reporter or other protein. However, off-target expression seemingly unrelated to the regulatory elements in the transgene is often observed, it is sometimes suspected to reflect influences related to the site of transgene integration in the genome. To test this hypothesis, we used a proximity ligation-based method, Targeted Locus Amplification (TLA), to map the insertion sites of three well-characterized transgenes that appeared to exhibit insertion site-dependent expression in retina. The nearest endogenous genes to transgenes HB9-GFP, Mito-P, and TYW3 are , and , respectively. For two lines, we demonstrate that expression reflects that of the closest endogenous gene ( and ), even though the distance between transgene and endogenous gene is 550 and 680 kb, respectively. In all three lines, the transgenes decrease expression of the neighboring endogenous genes. In each case, the affected endogenous gene was expressed in at least some of the cell types that the transgenic line has been used to mark and study. These results provide insights into the effects of transgenes and endogenous genes on each other's expression, demonstrate that mapping insertion site is valuable for interpreting results obtained with transgenic lines, and indicate that TLA is a reliable method for integration site discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnmol.2018.00385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6206269PMC
October 2018

Isozyme-Specific Role of SAD-A in Neuronal Migration During Development of Cerebral Cortex.

Cereb Cortex 2019 08;29(9):3738-3751

Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan.

SAD kinases regulate presynaptic vesicle clustering and neuronal polarization. A previous report demonstrated that Sada-/- and Sadb-/- double-mutant mice showed perinatal lethality with a severe defect in axon/dendrite differentiation, but their single mutants did not. These results indicated that they were functionally redundant. Surprisingly, we show that on a C57BL/6N background, SAD-A is essential for cortical development whereas SAD-B is dispensable. Sada-/- mice died within a few days after birth. Their cortical lamination pattern was disorganized and radial migration of cortical neurons was perturbed. Birth date analyses with BrdU and in utero electroporation using pCAG-EGFP vector showed a delayed migration of cortical neurons to the pial surface in Sada-/- mice. Time-lapse imaging of these mice confirmed slow migration velocity in the cortical plate. While the neurites of hippocampal neurons in Sada-/- mice could ultimately differentiate in culture to form axons and dendrites, the average length of their axons was shorter than that of the wild type. Thus, analysis on a different genetic background than that used initially revealed a nonredundant role for SAD-A in neuronal migration and differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cercor/bhy253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335017PMC
August 2019

Cadherin Combinations Recruit Dendrites of Distinct Retinal Neurons to a Shared Interneuronal Scaffold.

Neuron 2018 09 6;99(6):1145-1154.e6. Epub 2018 Sep 6.

Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA 02138, USA. Electronic address:

Distinct neuronal types connect in complex ways to generate functional neural circuits. The molecular diversity required to specify this connectivity could be supplied by multigene families of synaptic recognition molecules, but most studies to date have assessed just one or a few members at a time. Here, we analyze roles of cadherins (Cdhs) in formation of retinal circuits comprising eight neuronal types that inform the brain about motion in four directions. We show that at least 15 classical Cdhs are expressed by neurons in these circuits and at least 6 (Cdh6-10 and 18) act individually or in combinations to promote specific connectivity among the cells. They act in part by directing the processes of output neurons and excitatory interneurons to a cellular scaffold formed by inhibitory interneurons. Because Cdhs are expressed combinatorially by many central neurons, similar interactions could be involved in patterning circuits throughout the brain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2018.08.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284407PMC
September 2018

A method for single-neuron chronic recording from the retina in awake mice.

Science 2018 06;360(6396):1447-1451

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.

The retina, which processes visual information and sends it to the brain, is an excellent model for studying neural circuitry. It has been probed extensively ex vivo but has been refractory to chronic in vivo electrophysiology. We report a nonsurgical method to achieve chronically stable in vivo recordings from single retinal ganglion cells (RGCs) in awake mice. We developed a noncoaxial intravitreal injection scheme in which injected mesh electronics unrolls inside the eye and conformally coats the highly curved retina without compromising normal eye functions. The method allows 16-channel recordings from multiple types of RGCs with stable responses to visual stimuli for at least 2 weeks, and reveals circadian rhythms in RGC responses over multiple day/night cycles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aas9160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047945PMC
June 2018

Heterogeneity of retinogeniculate axon arbors.

Eur J Neurosci 2019 04 7;49(7):948-956. Epub 2018 Aug 7.

F.M. Kirby Neurobiology Center, Department of Neurology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts.

The retinogeniculate synapse transmits information from retinal ganglion cells (RGC) in the eye to thalamocortical relay neurons in the visual thalamus, the dorsal lateral geniculate nucleus (dLGN). Studies in mice have identified genetic markers for distinct classes of RGCs encoding different features of the visual space, facilitating the dissection of RGC subtype-specific physiology and anatomy. In this study, we examine the morphological properties of axon arbors of the BD-RGC class of ON-OFF direction selective cells that, by definition, exhibit a stereotypic dendritic arbor and termination pattern in the retina. We find that axon arbors from the same class of RGCs exhibit variations in their structure based on their target region of the dLGN. Our findings suggest that target regions may influence the morphologic and synaptic properties of their afferent inputs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/ejn.13986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286704PMC
April 2019

Cadherins Interact With Synaptic Organizers to Promote Synaptic Differentiation.

Front Mol Neurosci 2018 30;11:142. Epub 2018 Apr 30.

Center for Brain Science and Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, United States.

Classical cadherins, a set of ~20 related recognition and signaling molecules, have been implicated in many aspects of neural development, including the formation and remodeling of synapses. Mechanisms underlying some of these steps have been studied by expressing N-cadherin (), a Type 1 cadherin, in heterologous cells, but analysis is complicated because widely used lines express endogenously. We used CRISPR-mediated gene editing to generate a Human embryonic kidney (HEK)293 variant lacking Cdh2, then compared the behavior of rodent cortical and hippocampal neurons co-cultured with parental, mutant and -rescued 293 lines. The comparison demonstrated that Cdh2 promotes neurite branching and that it is required for three synaptic organizers, neurologin1 (NLGL1), leucine-rich repeat transmembrane protein 2 (LRRtm2), and Cell Adhesion Molecule 1 (Cadm1/SynCAM) to stimulate presynaptic differentiation, assayed by clustering of synaptic vesicles at sites of neurite-293 cell contact. Similarly, Cdh2 is required for a presynaptic organizing molecule, Neurexin1β, to promote postsynaptic differentiation in dendrites. We also show that another Type I cadherin, Cdh4, and a Type II cadherin, Cdh6, can substitute for Cdh2 in these assays. Finally, we provide evidence that the effects of cadherins require homophilic interactions between neurites and the heterologous cells. Together, these results indicate that classical cadherins act together with synaptic organizers to promote synaptic differentiation, perhaps in part by strengthening the intracellular adhesion required for the organizers to act efficiently. We propose that cadherins promote high affinity contacts between appropriate partners, which then enable synaptic differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnmol.2018.00142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936767PMC
April 2018

Tbr1 instructs laminar patterning of retinal ganglion cell dendrites.

Nat Neurosci 2018 05 9;21(5):659-670. Epub 2018 Apr 9.

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA.

Visual information is delivered to the brain by >40 types of retinal ganglion cells (RGCs). Diversity in this representation arises within the inner plexiform layer (IPL), where dendrites of each RGC type are restricted to specific sublaminae, limiting the interneuronal types that can innervate them. How such dendritic restriction arises is unclear. We show that the transcription factor Tbr1 is expressed by four mouse RGC types with dendrites in the outer IPL and is required for their laminar specification. Loss of Tbr1 results in elaboration of dendrites within the inner IPL, while misexpression in other cells retargets their neurites to the outer IPL. Two transmembrane molecules, Sorcs3 and Cdh8, act as effectors of the Tbr1-controlled lamination program. However, they are expressed in just one Tbr1 RGC type, supporting a model in which a single transcription factor implements similar laminar choices in distinct cell types by recruiting partially non-overlapping effectors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41593-018-0127-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920715PMC
May 2018

Role for Wnt Signaling in Retinal Neuropil Development: Analysis via RNA-Seq and In Vivo Somatic CRISPR Mutagenesis.

Neuron 2018 04 22;98(1):109-126.e8. Epub 2018 Mar 22.

Department of Biological Chemistry, HHMI, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

Screens for genes that orchestrate neural circuit formation in mammals have been hindered by practical constraints of germline mutagenesis. To overcome these limitations, we combined RNA-seq with somatic CRISPR mutagenesis to study synapse development in the mouse retina. Here synapses occur between cellular layers, forming two multilayered neuropils. The outer neuropil, the outer plexiform layer (OPL), contains synapses made by rod and cone photoreceptor axons on rod and cone bipolar dendrites, respectively. We used RNA-seq to identify selectively expressed genes encoding cell surface and secreted proteins and CRISPR-Cas9 electroporation with cell-specific promoters to assess their roles in OPL development. Among the genes identified in this way are Wnt5a and Wnt5b. They are produced by rod bipolars and activate a non-canonical signaling pathway in rods to regulate early OPL patterning. The approach we use here can be applied to other parts of the brain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2018.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930001PMC
April 2018

Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents.

Proc Natl Acad Sci U S A 2018 02 13;115(9):2126-2131. Epub 2018 Feb 13.

Center for Brain Science, Harvard University, Cambridge MA, 02138;

Sensitive and specific antibodies are essential for detecting molecules in cells and tissues. However, currently used polyclonal and monoclonal antibodies are often less specific than desired, difficult to produce, and available in limited quantities. A promising recent approach to circumvent these limitations is to employ chemically defined antigen-combining domains called "nanobodies," derived from single-chain camelid antibodies. Here, we used nanobodies to prepare sensitive unimolecular detection reagents by genetically fusing cDNAs encoding nanobodies to enzymatic or antigenic reporters. We call these fusions between a reporter and a nanobody "RANbodies." They can be used to localize epitopes and to amplify signals from fluorescent proteins. They can be generated and purified simply and in unlimited amounts and can be preserved safely and inexpensively in the form of DNA or digital sequence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1722491115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834735PMC
February 2018

Combinatorial Effects of Alpha- and Gamma-Protocadherins on Neuronal Survival and Dendritic Self-Avoidance.

J Neurosci 2018 03 8;38(11):2713-2729. Epub 2018 Feb 8.

Program for Neurosciences and Mental Health, Hospital for Sick Children, Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 0A4, Canada and

The clustered protocadherins (Pcdhs) comprise 58 cadherin-related proteins encoded by three tandemly arrayed gene clusters, -α, -β, and -γ (, , and , respectively). Pcdh isoforms from different clusters are combinatorially expressed in neurons. They form multimers that interact homophilically and mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, axonal tiling, and dendritic self-avoidance. Most studies have analyzed clusters individually. Here, we assessed functional interactions between and clusters. To circumvent neonatal lethality associated with deletion of , we used Crispr-Cas9 genome editing in mice to combine a constitutive mutant allele with a conditional allele. We analyzed roles of Pcdhas and Pcdhgs in the retina and cerebellum from mice (both sexes) lacking one or both clusters. In retina, Pcdhgs are essential for survival of inner retinal neurons and dendritic self-avoidance of starburst amacrine cells, whereas Pcdhas are dispensable for both processes. Deletion of both and clusters led to far more dramatic defects in survival and self-avoidance than deletion alone. Comparisons of an allelic series of mutants support the conclusion that Pcdhas and Pcdhgs function together in a dose-dependent and cell-type-specific manner to provide a critical threshold of Pcdh activity. In the cerebellum, Pcdhas and Pcdhgs also cooperate to mediate self-avoidance of Purkinje cell dendrites, with modest but significant defects in either single mutant and dramatic defects in the double mutant. Together, our results demonstrate complex patterns of redundancy between Pcdh clusters and the importance of Pcdh cluster diversity in postnatal CNS development. The formation of neural circuits requires diversification and combinatorial actions of cell surface proteins. Prominent among them are the clustered protocadherins (Pcdhs), a family of ∼60 neuronal recognition molecules. Pcdhs are encoded by three closely linked gene clusters called -α, -β, and -γ. The Pcdhs mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, and spatial patterning of axons and dendrites. Most studies to date have been limited to single clusters. Here, we used genome editing to assess interactions between -α and -γ gene clusters. We examined two regions of the CNS, the retina and cerebellum and show that the 14 α-Pcdhs and 22 γ-Pcdhs act synergistically to mediate neuronal survival and dendrite patterning.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.3035-17.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852656PMC
March 2018

Building Bridges through Science.

Neuron 2017 Nov;96(4):730-735

The Scripps Research Institute, La Jolla, CA 92037, USA.

Science is ideally suited to connect people from different cultures and thereby foster mutual understanding. To promote international life science collaboration, we have launched "The Science Bridge" initiative. Our current project focuses on partnership between Western and Middle Eastern neuroscience communities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2017.09.028DOI Listing
November 2017