9/29/2011 Retinal Ganglion Cells with Distinct The Journal of Neuroscience Directional Preferences Differ in Molecular Identity, Structure, and Central • Published by the Society for Neuroscience Projections • First issue: 1981 Jeremy N. Kay, Irina De la Huerta, In-Jung Kim, Yifeng Zhang, • Impact Factor: 10.21 Masahito Yamagata, Monica W. Chu, Markus Meister, and Joshua R. Sanes The Journal of Neuroscience The Journal of Neuroscience 31 (21), 7753 – 7762 Volume 31, Issue 21 Date published: May 25, 2011 Presented by: Michelle Lee Primary Authors Authors • Jeremy N. Kay • Harvard U., Department of MCB • Research Associate in Sanes ’ Lab • Masahito Yamagata • University of San Francisco • Harvard U., Dept. of MCB • In-Jung Kim • Postdoctoral Fellow in Sanes ’ lab • Harvard U., Dept. of MCB • Irina De la Huerta • Postdoctoral Fellow in Sanes ’ lab • Discovered OFF marker for RGC • …………. • Monica W. Chu • Harvard U., Dept. of MCB • Yifeng Zhang • Ph.D. Student at UCSD • Harvard U., Dept. of MCB • Technician in Sanes ’ lab • Postdoctoral Fellow in Meister lab Primary Authors What’s the research is about? • Markus Meister • This is not a theory-/ experiment-based • Harvard U., Dept. of MCB research • Principle Investigator • This is a research of exploration and • Retina and the Olfactory system observation • Joshua R. Sanes • Harvard U., Dept. of MCB • Principle Investigator • Molecules and structures that regulate synapse formation 1
9/29/2011 Background information What’s the research is about? Retina • Exploring Retinal Ganglion Cells with • A light-sensitive tissue lining the inner surface of the Directional Preferences in: eye – Molecular Identity (genes! surface markers!) – Structure (dendritic asymmetry!) – Central Projections (where do axons project to?) • The 4 directions: – Dorsal – Ventral – Nasal – Temporal Background information Background information Retina Retina • 3 layers of neurons • 2 layers of synapses Light! Background information Retinal Ganglion Cells So… What have been done so far? • Signaling to brain- large diameter axons • Since Cajal … • On/ Off center cells – Dendritic morphology • VERY diverse structures and sizes – Cell body and dendritic tree size – Arborized sub- layer in … • 5 main types; 60-100 subtypes • Directional Preferences (1964!) 2
9/29/2011 ooDSGC What we don’t know yet… • On-Off direction-selective retinal ganglion cells • Detailed differences between the 4 subtypes! • Bistratified: bipolar & amacrine cell input • DIRECTIONAL SELECTIVITY: 4 directions • It’s ASSUMED that there are 4 subtypes of ooDSGCs • They “know” they are different D: Cdh6 Transgenic technology N: DRD4/ T: ? Methods W9 V: BD/Cdh6 • Purpose: to mark different lines of ooDSGC • Transgenic technology that might have different directional • Electrophysiology preferences • Histology • Use markers that only express in certain lines of RGCs – BD-RGCs: Fstl4 : (ventral) – W9-RGCs: Thy : (nasal) – Cdh6-expressing RGCs: Cdh6 : ( dorsal & ventral) – DRD4-RGCs: DRD4 : (nasal) Transgenic technology Transgenic technology Courtesy of Dr. Hopkins X 3
9/29/2011 Transgenic technology Transgenic technology No fluorescence because of the STOP sequence CreER + the specific marker integrated into cells that ALSO have the marker sequence Specific marker Gene Fstl4/ Thy/ cdh6/ DRD4… Tamoxifen turns CreER on: Red Fluorescent label: BD CreER cut off STOP: Green fluorescent label: DRD4 YFP expresses! Electrophysiology So again… What is this CRAZY thing for? • Measure directional preference • Isolate retinas out • Cells recorded with cell-attached patch microelectrodes • Light stimuli delivered in different directions • Spikes recorded with light moving through different directions Red Fluorescent label: BD Green fluorescent label: DRD4 D: Cdh6 N: DRD4/ T: ? Electrophysiology Histology- In situ Hybridization W9 V: BD/Cdh6 • Purpose: Mark out WHERE and WHICH gene is expressing in a whole tissue Because we need to know which cell to look at! BD-RGC BD-RGC W9-RGC Cdh6 4
9/29/2011 Histology- In situ Hybridization How were the genes selected? • Use RNA/ DNA probes • Microarray shows lots of genes to choose • Probes Hybridize with mRNA (precursor of from • Products are cell surface or secreted proteins : Protein) might be involved in development or function • Antibodies that carries fluorescent dye reveal of ooDSGCs hybridized molecules – Cdh6, Col25c1, Mmp17, CART D: Cdh6 N: DRD4/ T: ? Histology- Lucifer Yellow Staining Results W9 V: BD/Cdh6 • http://webvision.med.utah.edu/movies/electr • Molecular identity ode.mov • Dendritic/ cell body structures • Axonal projections D: Cdh6 D: Cdh6 N: DRD4/ N: DRD4/ T: ? Molecular identity T: ? Molecular identity W9 W9 V: BD/Cdh6 V: BD/Cdh6 In situ marks out the In situ marks out the target gene expression: target gene expression: Cdh6 Cdh6 GFP marks out target GFP marks out target RGC line: BD RGC line: DRD4 2 markers did not 2 markers merged! merge no expression Confirm of gene on DRD4 expression on this cell line 5
9/29/2011 D: Cdh6 N: DRD4/ T: ? Molecular identity Molecular identity W9 V: BD/Cdh6 14 days after birth 7 days after birth Key point here: the development is + + + + + experience-independent! Molecular identity- CART Molecular identity- CART • CART: specific marker for ooDSGC! • Morphologically matched! – Bistratified, ON/OFF, Merged markers! Dendritic/ cell body structures • W9/DRD4 cells: Morphology does not correlate with their preferred direction • Dendritic/ cell body structures D: Cdh6 N: DRD4/ T: ? W9 V: BD/Cdh6 6
9/29/2011 Dendritic/ cell body structures Dendritic/ cell body structures • BD cells: Morphology DOES correlate with • Perhaps dendrite directions play a role in their preferred direction directional selectivity? • • Maybe… BUT… D: Cdh6 N: DRD4/ T: ? W9 V: BD/Cdh6 Axonal projections • ooDSGC projections V.S. RGC – Absent from many retinal targets Axonal projections – Arbors are confined to the upper portion in SC – Nearly NO ipsilateral porjections D: Cdh6 D: Cdh6 Axonal projections N: DRD4/ T: ? Last slide, I promise! N: DRD4/ T: ? W9 W9 V: BD/Cdh6 V: BD/Cdh6 • Differences in targets among ooDSGC subsets – Only vertical GC (BD and Cdh6) project to the MTN and NOT – DRD4-RGCs do not project to MTN and NOT 7
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