An extracellular biochemical screen reveals that FLRTs and Unc5s mediate neuronal subtype recognition in the retina

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. DOI: http://dx.doi.org/10.7554/eLife.08149.001

[1]  Ken W. Y. Cho,et al.  Unc5B Interacts with FLRT3 and Rnd1 to Modulate Cell Adhesion in Xenopus Embryos , 2009, PloS one.

[2]  C. Goodman,et al.  The Molecular Biology of Axon Guidance , 1996, Science.

[3]  T. McLaughlin,et al.  Resistance to trophic neurite outgrowth of sensory neurons exposed to insulin , 2012, Journal of neurochemistry.

[4]  V. Tarabykin,et al.  FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons , 2011, Neuroscience Research.

[5]  Tudor C. Badea,et al.  Transmembrane semaphorin signaling controls laminar stratification in the mammalian retina , 2010, Nature.

[6]  Michal Rivlin-Etzion,et al.  On and Off Retinal Circuit Assembly by Divergent Molecular Mechanisms , 2013, Science.

[7]  F. Charron,et al.  Dscam guides embryonic axons by Netrin-dependent and -independent functions , 2008, Development.

[8]  E. Delamarche,et al.  Patterned delivery of immunoglobulins to surfaces using microfluidic networks. , 1997, Science.

[9]  Jingxia Gao,et al.  Expression of unc5 family genes in zebrafish brain during embryonic development. , 2013, Gene expression patterns : GEP.

[10]  P. de Mazancourt,et al.  Netrins and Their Roles in Placental Angiogenesis , 2014, BioMed research international.

[11]  David P. Corey,et al.  Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning , 1988, Neuron.

[12]  J. Sanes,et al.  Laminar Restriction of Retinal Ganglion Cell Dendrites and Axons: Subtype-Specific Developmental Patterns Revealed with Transgenic Markers , 2010, The Journal of Neuroscience.

[13]  E. Birney,et al.  Pfam: the protein families database , 2013, Nucleic Acids Res..

[14]  F. Bonhoeffer,et al.  On the turning of Xenopus retinal axons induced by ephrin-A5 , 2003, Development.

[15]  S. Gong,et al.  Flrt2 and Flrt3 have overlapping and non-overlapping expression during craniofacial development. , 2009, Gene expression patterns : GEP.

[16]  Jia-huai Wang,et al.  Signaling mechanism of the netrin-1 receptor DCC in axon guidance. , 2015, Progress in biophysics and molecular biology.

[17]  U. Yazdani,et al.  The semaphorins , 2006, Genome Biology.

[18]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[19]  J. C. Clemens,et al.  Alternative Splicing of Drosophila Dscam Generates Axon Guidance Receptors that Exhibit Isoform-Specific Homophilic Binding , 2004, Cell.

[20]  A. Barclay,et al.  Transient intercellular adhesion: the importance of weak protein-protein interactions. , 1994, Trends in biochemical sciences.

[21]  Yutaka Yoshida,et al.  Semaphorin Signaling in Vertebrate Neural Circuit Assembly , 2012, Front. Mol. Neurosci..

[22]  M. Poo,et al.  Plexins Are a Large Family of Receptors for Transmembrane, Secreted, and GPI-Anchored Semaphorins in Vertebrates , 1999, Cell.

[23]  R. Masland.  The Neuronal Organization of the Retina , 2012, Neuron.

[24]  Masahito Yamagata,et al.  Dscam and Sidekick proteins direct lamina-specific synaptic connections in vertebrate retina , 2008, Nature.

[25]  S. Offermanns,et al.  Semaphorins and plexins as therapeutic targets , 2014, Nature Reviews Drug Discovery.

[26]  J. N. Kay,et al.  MEGF10 AND 11 MEDIATE HOMOTYPIC INTERACTIONS REQUIRED FOR MOSAIC SPACING OF RETINAL NEURONS , 2012, Nature.

[27]  Julie M. Sahalie,et al.  An experimentally derived confidence score for binary protein-protein interactions , 2008, Nature Methods.

[28]  J. N. Kay,et al.  NEUROD6 EXPRESSION DEFINES NOVEL RETINAL AMACRINE CELL SUBTYPES AND REGULATES THEIR FATE , 2011, Nature Neuroscience.

[29]  J. Sanes,et al.  Type II Cadherins Guide Assembly of a Direction-Selective Retinal Circuit , 2014, Cell.

[30]  Cheng Li,et al.  Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application , 2001, Genome Biology.

[31]  N. Lewin-Koh,et al.  A secreted protein microarray platform for extracellular protein interaction discovery. , 2012, Analytical biochemistry.

[32]  Y. Rao,et al.  DSCAM functions as a netrin receptor in commissural axon pathfinding , 2009, Proceedings of the National Academy of Sciences.

[33]  Masahito Yamagata,et al.  Retinal Ganglion Cells with Distinct Directional Preferences Differ in Molecular Identity, Structure, and Central Projections , 2011, The Journal of Neuroscience.

[34]  Joel S. Bader,et al.  Precision and recall estimates for two-hybrid screens , 2008, Bioinform..

[35]  J. Sanes,et al.  Design Principles of Insect and Vertebrate Visual Systems , 2010, Neuron.

[36]  C. Niehrs,et al.  The transmembrane protein XFLRT3 forms a complex with FGF receptors and promotes FGF signalling , 2004, Nature Cell Biology.

[37]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[38]  Z. Pan,et al.  Characterization of multiple bistratified retinal ganglion cells in a purkinje cell protein 2‐Cre transgenic mouse line , 2013, The Journal of comparative neurology.

[39]  T. Blundell,et al.  Flexibility and small pockets at protein e protein interfaces: New insights into druggability Progress in Biophysics and Molecular Biology , 2022 .

[40]  Inji Park,et al.  EphB/ephrinB Signaling in Cell Adhesion and Migration , 2014, Molecules and cells.

[41]  Qifa Zhang,et al.  Genetic basis of sRNA quantitative variation analyzed using an experimental population derived from an elite rice hybrid , 2015, eLife.

[42]  Peer Bork,et al.  SMART 7: recent updates to the protein domain annotation resource , 2011, Nucleic Acids Res..

[43]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[44]  R. Stacy,et al.  Developmental relationship between cholinergic amacrine cell processes and ganglion cell dendrites of the mouse retina , 2003, The Journal of comparative neurology.

[45]  J. Ting,et al.  Immune plexins and semaphorins: old proteins, new immune functions , 2013, Protein & Cell.

[46]  A. Kolodkin,et al.  Mechanisms and molecules of neuronal wiring: a primer. , 2011, Cold Spring Harbor perspectives in biology.

[47]  J. Sanes,et al.  Expanding the Ig Superfamily Code for Laminar Specificity in Retina: Expression and Role of Contactins , 2012, The Journal of Neuroscience.

[48]  M. Taniguchi,et al.  Congenital diseases and semaphorin signaling: Overview to date of the evidence linking them , 2015, Congenital anomalies.

[49]  S. Brunak,et al.  SignalP 4.0: discriminating signal peptides from transmembrane regions , 2011, Nature Methods.

[50]  Plexin structures are coming: opportunities for multilevel investigations of semaphorin guidance receptors, their cell signaling mechanisms, and functions , 2012, Cellular and Molecular Life Sciences.

[51]  Thomas M. Jessell,et al.  Semaphorin 3E and Plexin-D1 Control Vascular Pattern Independently of Neuropilins , 2005, Science.

[52]  Robert D. Finn,et al.  InterPro in 2011: new developments in the family and domain prediction database , 2011, Nucleic acids research.

[53]  Gavin J. Wright,et al.  A cell surface interaction network of neural leucine-rich repeat receptors , 2009, Genome Biology.

[54]  Benjamin Sivyer,et al.  Direction selectivity in the retina: symmetry and asymmetry in structure and function , 2012, Nature Reviews Neuroscience.

[55]  P. Rigby,et al.  Regulated expression of FLRT genes implies a functional role in the regulation of FGF signalling during mouse development. , 2006, Developmental Biology.

[56]  E. Smythe,et al.  TAG1 Regulates the Endocytic Trafficking and Signaling of the Semaphorin3A Receptor Complex , 2012, The Journal of Neuroscience.

[57]  Raquel Manzano,et al.  Amyotrophic Lateral Sclerosis: A Focus on Disease Progression , 2014, BioMed research international.

[58]  K. Garcia,et al.  An Extracellular Interactome of Immunoglobulin and LRR Proteins Reveals Receptor-Ligand Networks , 2013, Cell.

[59]  G. Neufeld,et al.  The semaphorins: versatile regulators of tumour progression and tumour angiogenesis , 2008, Nature Reviews Cancer.

[60]  R. Wong,et al.  Functional architecture of the retina: Development and disease , 2014, Progress in Retinal and Eye Research.

[61]  Jonathan B Demb,et al.  Cellular Mechanisms for Direction Selectivity in the Retina , 2007, Neuron.

[62]  L. Brass,et al.  The role of semaphorins and their receptors in platelets: Lessons learned from neuronal and immune synapses , 2011, Platelets.

[63]  T Pawson,et al.  Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity. , 1994, Science.

[64]  C. Niehrs,et al.  A role for fibronectin‐leucine‐rich transmembrane cell‐surface proteins in homotypic cell adhesion , 2006, EMBO reports.

[65]  J Schultz,et al.  SMART, a simple modular architecture research tool: identification of signaling domains. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[66]  T. Kumada,et al.  Ion channels, guidance molecules, intracellular signaling and transcription factors regulating nervous and vascular system development , 2015, The Journal of Physiological Sciences.

[67]  E. Giraudo,et al.  The role of semaphorins and their receptors in vascular development and cancer. , 2013, Experimental cell research.

[68]  J. N. Kay,et al.  Development of dendritic form and function. , 2015, Annual review of cell and developmental biology.

[69]  D. Snow,et al.  Comparison of sensory neuron growth cone and filopodial responses to structurally diverse aggrecan variants, in vitro , 2013, Experimental Neurology.

[70]  T. Südhof,et al.  Structural Basis of Latrophilin-FLRT-UNC5 Interaction in Cell Adhesion. , 2015, Structure.

[71]  D. Feldheim,et al.  Tbr2 Is Required to Generate a Neural Circuit Mediating the Pupillary Light Reflex , 2014, The Journal of Neuroscience.

[72]  R. Klein,et al.  FLRT Structure: Balancing Repulsion and Cell Adhesion in Cortical and Vascular Development , 2014, Neuron.

[73]  Marla B. Feller,et al.  Organization and development of direction-selective circuits in the retina , 2011, Trends in Neurosciences.

[74]  Alex Bateman,et al.  Large-scale screening for novel low-affinity extracellular protein interactions. , 2008, Genome research.

[75]  L Shapiro,et al.  Linking molecular affinity and cellular specificity in cadherin-mediated adhesion , 2009, Proceedings of the National Academy of Sciences.

[76]  C. Freitag,et al.  A common variant of the latrophilin 3 gene, LPHN3, confers susceptibility to ADHD and predicts effectiveness of stimulant medication. , 2010, Molecular psychiatry.

[77]  Z. Pan,et al.  Characterization of transgenic mouse lines expressing Cre recombinase in the retina , 2010, Neuroscience.

[78]  D. Baker,et al.  A Vast Repertoire of Dscam Binding Specificities Arises from Modular Interactions of Variable Ig Domains , 2007, Cell.

[79]  Masahito Yamagata,et al.  Sidekicks Synaptic Adhesion Molecules that Promote Lamina-Specific Connectivity in the Retina , 2002, Cell.

[80]  M. Tessier-Lavigne,et al.  DSCAM Is a Netrin Receptor that Collaborates with DCC in Mediating Turning Responses to Netrin-1 , 2008, Cell.

[81]  M. Feller,et al.  Genetic Identification of an On-Off Direction- Selective Retinal Ganglion Cell Subtype Reveals a Layer-Specific Subcortical Map of Posterior Motion , 2009, Neuron.

[82]  Shiaoching Gong,et al.  A gene expression atlas of the central nervous system based on bacterial artificial chromosomes , 2003, Nature.

[83]  J. Vielmetter,et al.  In vitro assay to test differential substrate affinities of growing axons and migratory cells , 2004, Experimental Brain Research.

[84]  H. Wässle,et al.  Immunocytochemical analysis of the mouse retina , 2000, The Journal of comparative neurology.

[85]  G. Neufeld,et al.  Semaphorins in angiogenesis and tumor progression. , 2012, Cold Spring Harbor perspectives in medicine.

[86]  H. Baier,et al.  In Vivo Imaging Reveals Dendritic Targeting of Laminated Afferents by Zebrafish Retinal Ganglion Cells , 2006, Neuron.

[87]  M. Tessier-Lavigne,et al.  Netrins and their receptors. , 2007, Advances in experimental medicine and biology.