C. elegans L1 cell adhesion molecule functions in axon guidance
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1 C. elegans L1 cell adhesion molecule functions in axon guidance Biorad Lihsia Chen Dept. of Genetics, Cell Biology & Development Developmental Biology Center
2 C. elegans embryogenesis Goldstein lab, UNC-Chapel Hill
3 CELL ADHESION tissue boundaries epithelial adherens junctions cell migration axon guidance synapse targeting & adherens cell sorting Adapted from Molecular Biology of the Cell, Alberts et al.; Gumbiner et al., 2005, Nature review Mol Cell Bio.
4 IMPAIRED CELL ADHESION DEVELOPMENTAL DISORDERS tissue boundaries epithelial adherens junctions cell migration axon guidance synapse targeting & adherens cell sorting Adapted from Molecular Biology of the Cell, Alberts et al.; Gumbiner et al., 2005, Nature review Mol Cell Bio.
5 IMPAIRED CELL ADHESION Loss of cell adhesion Cell delamination inappropriate cell migration METASTASIS Adapted from Molecular Biology of the Cell, Alberts et al.
6 Cell Adhesion Receptors CADHERIN INTEGRIN IgCAM PLASMA MEMBRANE PLASMA MEMBRANE
7 Cell Adhesion Receptors CADHERIN INTEGRIN Barlovatz Meimon & Pouchelet, INSERM, FRANCE migrating cultured muscle cells
8 Cell Adhesion Receptors CADHERIN INTEGRIN IgCAM
9 Cultured rat hippocampal neurons IgCAM Dr. Lorene Lanier UMN Neuroscience
10 mammalian L1CAMs L1 NrCAM CHL1 neurofascin Ig cell-cell and cell-ecm adhesion FNIII binding sites to actin-linkers
11 Neuronal role for mammalian L1CAMs L1 NrCAM CHL1 neurofascin L1 syndrome Corpus callosum hypoplasia Retardation/intellectual disability Adducted thumbs Spastic paraplegia Hydrocephalus Fransen et al., Hum. Mol. Gen. Vits et al., Nat. Genetics
12 Neuronal role for mammalian L1CAMs L1 NrCAM CHL1 neurofascin autism addiction Sakurai et al., Psych. Genetics Yoo et al., Psych. Invest. Corpus callosum hypoplasia Retardation/intellectual disability Adducted thumbs Spastic paraplegia Hydrocephalus Fransen et al., Hum. Mol. Gen. Vits et al., Nat. Genetics
13 Neuronal role for mammalian L1CAMs L1 NrCAM CHL1 neurofascin schizophrenia autism addiction Chu & Liu, J. Human Genetics Sakurai et al., Psych. Genetics Yoo et al., Psych. Invest. Corpus callosum hypoplasia Retardation/intellectual disability Adducted thumbs Spastic paraplegia Hydrocephalus Fransen et al., Hum. Mol. Gen. Vits et al., Nat. Genetics
14 Neuronal role for mammalian L1CAMs L1 NrCAM CHL1 neurofascin schizophrenia autism addiction Chu & Liu, J. Human Genetics Sakurai et al., Psych. Genetics Yoo et al., Psych. Invest. Corpus callosum hypoplasia Retardation/intellectual disability Adducted thumbs Spastic paraplegia Hydrocephalus Neurodevelopment neurite outgrowth axon guidance Myelination
15 1. External embryonic development 2. Large embryos 3. Amenable to manipulation 4. Early chick embryonic structures are very similar to those of the human embryo. 1. Mammal 2. Reverse genetics 1. Genetically accessible 2. Short generation time ~ 2 weeks, 3.5 days 3. Small genomes 4. Small size 1. External embryonic development 2. Large number of transparent embryos 3. generation time ~3 months but quick embryonic development
16 Caenorhabditis elegans Nobel Prize in Physiology or Medicine 2002 for their discoveries concerning genetic regulation of organ development and programmed cell death Sydney Brenner Robert Horvitz John Sulston Invariant cell lineage: 302 neurons Transparent How does the nervous system develop? function? control behavior? Identified all cells
17 Invariant cell lineage of 959 somatic cells zygote 302 neurons
18 1994: 1 st showing of a GFP reporter in a multicellular organism 2008 recipients of the Nobel Prize in Chemistry for the discovery and development of GFP mechanosensory cells Martin Chalfie Osamu Shimomura Roger Y. Tsien
19 Importance of fluorescence proteins in C. elegans research
20
21 Advantages of C. elegans as model to study nervous system development & function Invariant cell lineage 302 neurons Transparent Neural circuitry completely mapped: serial electron micrographs Light microscopy Wiring Chemical and electrical synapses
22
23 C. elegans L1CAMs expressed ubiquitously SAX-7 SAX-7 LAD-2 expressed in 14 neurons Chen et al., J. Cell Biol. Wang et al., J. Cell Biol.
24 lad-2 animals show axon guidance defects A/P SDQL D/V Wang et al., J. Cell Biol.
25 Which axon guidance pathway does LAD-2 mediate? Netrins Slits Semaphorins Ephrins
26 Semaphorin family and Receptor MAB-20 PLX-2 PLEXIN RECEPTOR Adapted from Pasterkapm and Kolodkin, Curr. Opin. Neurobiol.
27 mab-20/sema2 & plx-2 have axon guidance roles male tail development MAB-20 PLX-2 MAB-20 X X PLX-2 guidance axon guidance defects SDQL SDQL Wang et al., J. Cell Biol.
28 mab-20/sema2 & plx-2 have axon guidance roles male tail development MAB-20 PLX-2 MAB-20 X X PLX-2 no enhanced defects SDQL SDQL X MAB-20 ligand Y receptor X X PLX-2 guidance axon guidance defects Wang et al., J. Cell Biol.
29 mab-20/sema2 & plx-2 have axon guidance roles male tail development MAB-20 PLX-2 MAB-20 X X PLX-2 no enhanced defects SDQL SDQL MAB-20 X ligand Y receptor X X PLX-2 enhanced defects Wang et al., J. Cell Biol.
30 mab-20/sema2 & plx-2 have axon guidance roles SDQL MAB-20 X MAB-20 X protein Y X PLX-2 protein X X PLX-2 no enhanced defects enhanced defects Does LAD-2 mediate axon guidance via MAB-20/Sema2? Wang et al., J. Cell Biol.
31 Does LAD-2 mediate axon guidance via MAB-20/Sema2? MAB-20 X X PLX-2 X LAD-2 no enhanced defects MAB-20 X Ligand Y X X PLX-2 LAD-2 enhanced defects
32 LAD-2 functions in MAB-20/Sema2-mediated axon guidance Wang et al., J. Cell Biol.
33 Does MAB-20/Sema2 act as a repellent to SDQL? mab-20
34 Does MAB-20/Sema2 act as a repellent to SDQL? mab-20
35 Does MAB-20/Sema2 act as a repellent to SDQL? mab-20 Ectopic ventral MAB-20 expression mab-20 mab-20
36 MAB-20/Sema2 acts as a repellent mab-20 Ex[mab-20] P < 0.01 mab-20; Ex[mab-20] P < 0.01 P < 0.05 Wang et al., J. Cell Biol.
37 Can dorsal MAB-20/Sema2 induce ventral migration of SDQL axon? WT WT Ex[mab-20] P < 0.01 P < 0.01 P < 0.01 lad-2; Ex[mab-20] Wang et al., J. Cell Biol.
38 MAB-20/Sema2 acts as a repellent WT WT Ex[mab-20] P < 0.01 P < 0.01 P < 0.01 lad-2; Ex[mab-20] Wang et al., J. Cell Biol.
39 Is LAD-2 required to mediate MAB-20/Sema2 signal? lad-2 WT Ex[mab-20] P < 0.01 P < 0.01 P < 0.01 lad-2; Ex[mab-20] Wang et al., J. Cell Biol.
40 LAD-2 is required to mediate MAB-20/Sema2 signal! lad-2 WT Ex[mab-20] P < 0.01 P < 0.01 P < 0.01 lad-2; Ex[mab-20] Wang et al., J. Cell Biol.
41 Can MAB-20/Sema2 interact with PLX-2?
42 MAB-20/Sema2 interacts with PLX-2 Myc::PLX-2 FLAG::MAB-20 Myc::PLX-2 FLAG::MAB LYSATE IP Wang et al., J. Cell Biol.
43 Can LAD-2 interact with MAB-20 & PLX-2?
44 LAD-2 interacts with MAB-20/Sema2 & PLX-2 LAD-2 Myc::PLX-2 FLAG::MAB-20 LAD-2 _ LAD-2 SAX-3 Myc::PLX-2 FLAG::MAB LYSATE IP Wang et al., J. Cell Biol.
45 LAD-2 secures the MAB-20/Sema2 - PLX-2 interaction SAX-3 LAD-2 _ Myc::PLX-2 FLAG::MAB-20 SAX-3 LAD-2 Myc::PLX-2 FLAG::MAB LYSATE IP Wang et al., J. Cell Biol.
46 Model for how LAD-2 mediates MAB-20/Sema2 function nematostella Vectensis Neuropilin present (Putnam et al., 2007) No L1CAMs (C. Magie, Pers. comm) LAD-2 L1 Wang et al., J. Cell Biol.
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