Lecture 12 Axon Guidance 02 27 2015 Assignment Groups of 5 each person becomes an expert on one part On Wednesday all the experts meet then you go back to your group and discuss Shown for first time that astrocytes in certain developing regions of spinal cord secrete attractive repulsive molecules that tell neurons where to go Read the whole paper and then be an expert on one of the 5 results sections 1 Microarray 2 Knockout reporter animals 3 Cell culture 4 Electrophysiology 5 brings it all together HW 3 sentences 1 paragraph summary of your section before class on Wednesday on TurnItIn Group quiz at the end Nature article that just came out Astrocyte encoded positional cues maintain sensorimotor circuit integrity http www nature com nature journal v509 n7499 full nature13161 html Notes Axons use multiple guides to find their way Through the retina to the tectum Remove just one cue cut out swuare of neuroepithelim where they know the axons grow over and they rotate it 90 degrees so it will be getting wrong cues So they start going worng way then switch Even within this small area axons are going along a predetermined path In that particular square if you rotate it 90 degress axon will also rotate 90 degress shows that neuroepithelium contains LOCAL cues not just long distance because even that small square has an effect Chick spinal cord 3 days and 4 days Sensory neurons green represent developing dorsal root ganglia will make their way to peripheral tissues Commissural neurons send axons out ventrally and on day 4 cross over midline and innervate tissue on other side That might actually have been motor neurons Check Netrin acts same way as pipette tip we talked about last class where it came towards the substance Go towards floor plate so take molecules from there and put elsewhere and see if they work Netrin happens to work put in other areas and the dorsal commissural interneurons will go towards netrin BMP in dorsal region of spinal cord repels these axons also helping them be driven towards ventral floorplate Shh is also attractive we know this is expressed in ventral floorplate Once they get to floorplate they cross over and take sharp turn towards the head Open book assay cut open the neural tube and lay it open like a book so you can watch the axons grow There must be some switch to make the axons turn there is an AP gradient of Wnt goes up a Wnt gradient Wnt is a Mitogen morphogen AND a chemoattractant Higher towards head axons like Wnt go towards higher gradient in head RA highest in posterior and lower in anterior bt in spinal cord development higher in anterior and lower in posterior opposite Earlier in development laying down pattern of spinal cord highest concentration of Wnt FGF and RA in head to lay down AP axis Then later it switches Once you ve determine what is head and what is posterior gradients change Salamander that he published on Green RA expression Hindbrain anterior spinal cord boundary the arrow gradient going down posteriorly Hindbrain is laid down by expression of these molecules Cyp enzymes breaking it down generating the gradient Same thing takes place in the Wnt pathway Early in develop higher in posterior and lower in anterior Later switches Wnt gradient is laid down by inhibition from other molecules Higher in anterior and lower in posterior is laid down in this manner opposite from early in development Dorsal comm interneur attracted to Shh on bottom then once they get there and cross over they start being repelled by Shh and grow towards Wnt towards head Anterior Wnt highest in anterior region is attracting commissural neurons they go towards head up Wnt gradient The ones coming down the spinal cord are REPULSED by high Wnt signals Corticospinal axons are going down spinal cord towards posterior and are repelled by Wnt Gastrulation of early patterning first stong signals then switches After neural tube closure after axons have extended started to move out an dthe rhomobomeres axis has been laid down it switches Wnt is the ligand binding to the fz3 receptor Fz3 frazzled make knockout of that receptor and they cross over but don t make the turn Floorplate expresses both chemoattractants AND chemorepulsants Comparative biology learn a lot Invertebrates don t have floorplate but they have ventral midline expressing the same things Invertebrates are conserved at this level Comm mutant grows up gradient but doesn t cross over Robo mutant crosses over but doesn t shut off its ability to listen to netrin signal so it keeps recrossing midline because its attracted to netrin even though it knows where to go Midline aka floorplate expreses Slit Crosses floor plate and turns doesn t go back in Precrossing at growth cone slit is repulaive and netrin is attractive High levels of Robo3A which turns off the inhibitory abilities of slit on that axon It makes it so that by stopping the repulsion you re allowing the attraction to work High expression of Robo3A turns off Robo1 which turns off the repulsion of Slit Postcrossing Turn off Robo3A and nstead more Robo3B is expressed which allows Robo1 to be expressed and allows slit to repulse it which also inhibits DCC meaning netrin won t attract it anymore Isoforms of the same gene same genes but expressed differently High levels of Shh allow the switch to happen inactivates the attraction Very clea experimentally Take eFP and graft onto open book assay of spinal cord Black on right is cell bodies Go right into floor plate replace by adding netrin and they go towards netrin Wait until after they have crossed over floorplate and they come across eFP they aren t attracted at all Go from being attracted to netrin to NOT being attracted to netrin AND being repulsed by slit Comm neurons up on dorsal crossing floorplate are attracted to netrin but at the same time netrin is repulsive to other axons in same area Ventral neurons sending their axons dorsally are REPELLED by netrin1 opposite of the comm neurons Eyes to brain second axon growth path Learned a lot from this pathway Optic tectum target in the brain Optic cup forms which has progenitor cells which turn into retinal ganglion cells Cross over on ventral surface of brain Left eye right and left side of brain How do axons from one eye go to opposite or same side of brain ipsilateral same and contralateral opposite Leave optic cup enter retinal nerves and then decide which side to go Step 1 once you start growing our of cell body once you get near region of optic