Lecture 9 Differentiation 02 18 2015 Don t memorize all the genes from the chapter go though slides Intrinsic factors the instructions for differentiation of a cell is determined from within Extrinsic factors the instructions for differentiation of a cell is determined from the cell s environment Usually a balance of both Transcription factor network turned on intrinsic Ligands extracellular signals extrinsic Get notes for this is most often controlled by extracellular signals ligands see ppt for rest of sentence from slide Cut out things by dominant negatives etc How do you make knockouts Will learn this Forward phenotype genotype screening whole ton of animals to ask for particular phenotype natural mutagenesis is inefficient so irradiate animals or add mutagenic drugs into housing medium or inject with mutagenizing agents to mutanize and get phenotype then look for genotype Flies often used quick lifespan short generation time Reverse genotype phenotype suspect particular transcription factor involved in neurogenesis from some background knowledge mutate that candidate to see if it s playing the role you think it is OR screen tons of genes t get a smaller amount of candidates then mutate the genes and see what the phenotypes are longer generation time mouse axolotl etc lineage map of c elegans know how many cells found in differentiated adult AND the lineage of those cells Invariant process get rid of one stem cell and that whole lineage line from that stem cell is eliminated Random mutagenesis and then screening for particular phenotypes see what kind of genes regulate that phenotype forward Invariant check Mechanosensation phenotype see book in c elegans used simple screening approach by touching front and moves back or touching back and moves forward see if it moves when you touch it if it doesn t move look at it and determine which genes regulate differentiation of mechanosensors Mutagen Screen for phenotype of interest See slide for more images Upsteam neural progentitor touch cell Identified unc 86 gene that led to loss of particular step and then instead of touch cell it made a sensory neuron Lost ability to make next decision step to make a touch cell Broke down entie network to see all the steps that are taken This works really well in c elegans where there is a lot of conservation in cell differentiation process and where there is NO PLASTICITY another gene doesn t have redundant function another cell doesn t come take over that was all forward genetics This guy will likely win nobel prize first person to show CRISPR work in an animal Cut and paste DNA where and when we want it https www youtube com watch feature player detailpage v 2pp17E 4E O8 Inject viral DNA into bacteria makes short copy and incorporates into its own genome in particular location incorporates into CRISPR array kind of like its own immune system made into guide RNAs that float into cell and screen for possible infection has RNA ready to bind to any invading virus cas9 complex will cut up the DNA Watch video again in bacteria Powerful because all you need is short guide RNA to target any gene of interest Easy to make this in lab today Can target many genes at once Method of reverse genetics It s actually just cutting the DNA but THEN it tries to repair itself but is very error prone so you can make mutations or you can incorporate what you want 2 out of 3 times you make a knockout when cutting DNA because it is so error prone when trying to repair itself Naturally occurring CRISPR Cas Merge crRNA and tracrRNA for genome editing Efficient genome editing in embryos have used to make many transgenic animals most recently monkeys so cheap to make these pieces of RNA that you can actually use to do forward genetics too Haven t done it animals YET but going to 64 000 different guide RNAs to target 18 000 genes Many labs adopting this now modeling complicated disease Go over those two approaches and make sure you understand Gap genes pair rule genes etc turno on particular hox genes which tell what to become head and tail Gastrulation neuroblast comes from ventral surface of drosophila also there is a dorsal ventral code of transcription factors Code of anterior posterior AND dorsal ventral transcription factors Lay down a kind of zip code on neural stem cell population make grid to tell what to turn into what Whole section in book about this but this is most important Symmetric vs symmetric cell division is also important in mammalian neurobiology not just drosophila Basal next couple of slides get confusing Apical lining ventricle Basal lining outside of any epithelial cell Stem cells on apical surface different between symmetric and asymmetric determine what cell will become gastrulation neuroblast dilaimnates comes down neural stem cell neural progenitor cell called GMC ganglion mother cell New neuroblast vs neural progenitor cell stem cell vs more differentiated intracellular components that tells whether or not they will differnetiatie ino progenitor cell Par complex conserved in mammalian cells stays on apical surface and where these genes are located determines which part of mother cell stays stem cell ad what will differentiate into GMC and progenitor cell Mitotic spindle apical basal cell division Par complex determines the axis of cell division Green is apical surface Yellow orange is basal surface green genetically labeled numb protein basal red progeny chromosomes goes with GMC from that point on Actual proteins within cytoplasm that determine what will stay stem cell and what will turn into differentiated progeny https www youtube com watch feature player detailpage v py kqa8BsjZI neural crest cells more plastic in some areas of body intrinsic in some areas extrinsic see book for neural crest intro ch3 and ch4 Truly multipotent brain Neural crest cells totally unique in ability to give rise to bone AND Controlled by both extrinsic and intrinsic factors depending on where you arise from head or spinal cord Neuro crest cells give rise to Sensory neurons dorsal root ganglia Cells in the gut Cornea Aorta of the heart Portions of bone marrow multipotent stem cells found there Portions of skin what gives us pigment melanocytes osteoblasts and osteoclasts bone In his lab they usually use white not dark green axolotls that have mutation that naturally arose in Mexico City lake 1864 that white animal was brought back to Paris museum of natural history that one single animal mutation gave rise to