Going Over Homework 1 or 2 questions on the test about this 03 04 2015 Example mouse line 1 Driver expresses Cre Aldh1l1 molecule expressed in astrocytes Looking at what is expressed in ifferentc ell types and saw that Aldh1l1 is expressed only in astrocytes Put Cre recombinase after it expresees Cre every tme it expresses your molecule of interest Aldh1l1 Reporter mouse secondary sema3a is normal with no defects When you mate the two all astrocytes express Cre recombinase and it finds those two pieces of DNA floxp and cuts out gene Made knockout mice only in astrocytes only astrocytes don t have the gene but all other cells have it so they still function fine EXPERT GROUP 2 GFAP marker for stem cell when expressed Cre is expressed and disrupts Sema3a Want to knockout Sema3a so they put loxp site on either side come cut me with Cre Can t knockout in embryo because they need it so one mouse has flox and one has Cre so they both can live then they mate so one has flox sites to knowout the site you want and one with Cre to knowkcout Sema3a in ones with GFAP targets Time based Instead of knocking out sema when gfap was on it was with aldh Alpha MN drives flexion major one to flex muscles Gamma MN keeps it so they can respond again keeps it tight keeps it constricted so you can maintain it and respond again Staining purposes differentiate between them Protoplasmic AS in grey matter Fibrous in white matter MN in grey matter sema probably coming from protoplasmic AS why they knockout there GFAP not expressed in neurons good so you don t knock it out there they can survive Results Alpha MN were disoriented without sema but not gamma ones They need it to live so eventually they died that s why P30 didn t have differences anymore Sema sirectly repels MN so that s how it works They are fine early in development but then they can t maintain orientation at early postnatal stages AS derived sema3a is regulating where axons orient on just alpha MN particular neuronal subtypes respond to inhibitors and others don t o Took section and drew line from body to ventral root where axons exit SP see orientation of where it goes out to where it needs to go without Sema3a not oriented correctly Only ones that ended up in right orientation survived AS that should be secreting sema na dcoculturing with MN axons went other way repels it o Infect with Cre cuts out sema no ligand o Do the same with receptor loses ability no repel o Shows that it s really what they knocked out Neuropilin receptor is on alpha not gamma result Or already known Going over paper in Groups Section 1 Split SC intro dorsal and ventral halves they purified the astrocytes They used microarray to show where genes where if in specific astrocutes or not also if in specific region sorsal or ventral 38 genes specific to dorsal or ventral Semaphorin 3a was most ventrally specific one They showed that the sema3a plays role in positioning ventral molecules Showed expression gradient that went from dorsal to ventral area and they oriented towards MN cell bodies To compare the non AS and the AS they did quantitative PCR of complementary DNA Basically showed many genes are regionally specific but sema was most prominent Section 2 First used sema knockout to show that alpha MN get disoriented but not gamma Used Cre recombinase to cut out gene later on rather then creating a mutant Next showed that early in development they were fine but then they fail to maintain the orientation in early postnatal stages Finally they showed that sema directly repels MN axons Cocultured in dish Section 3 Studied how alpha and gamma MN survived depending on amount of sema created knockout where AS didn t have sema At P7 there were normal amounts of alpha but by P30 alpha decreased by half and the interneurons and gamma stayed the same In order to test if alpha could be increased by sema through Nrp pathway receptors sema works thorough they cultures alpha on plate and found that the alpha MN were dose dependent on sema So more sema more survival Section 4 Functioning of different vesicles carrying glutamate and GABA to MN First looked at V Glut 1 one of the vesicles that carried glutamate In the mutants sema knockout huge decrease in molecules carrying glutamate specifically VGlut1 In Vglut2 no change They discovered that vglut1 is dependent on sensory input from DRG but v2 is not as dependent V2 is actually upstream from the sensory inputs from DRG but V1 is directly dependent on DRG Electrophysiology looked at EPSPs and IPSPs increase in IPSPs and decrease in EPSPs MN able to make up for shift decreasing the threshold so MN could get to where they used to be regardless of lack of sema Section 5 First in vivo they created sema3a mutant and with that they showed that the knockouts had abnormal ventrally positioned axons compared to control Sema is essential t opositioning in ventral par of SC Next in vitro PV marker for particular sensory neurons think of it as TrkA op of spinal cord and PV go to bottom come from same source but only go to base or top of SC sema3a tells them to go to either spot Same source from same cells but affects different cell types differently to ly down the pattern Co cultured dorsal and ventral AS with dissociated DRG and deleted sema3a by adding cre recombinase AS cultures retained distinct regional expression Testing for differences between regional SC AS so they found that AS has more intrinsic signaling Bring it all together AS encode subtype specific sensory axon guidance signals 03 04 2015 03 04 2015