Anastasia Nabatkhorian 3 6 2015 Developmental Neurobiology Exam 2 Review Retroviral labeling Retrovirus o But these don t replicate o o Inject a virus that selectively infects cells in S phase actively dividing Not active virus so it doesn t replicate BUT it gets integrated in genome o Can label the virus ex green fluorescence and then track them so you see every cell that comes from the ones you injected o If only transfecting a few cells clonal analysis One stem cell is tagged and then every cell that is labeled thereon is a clone of that cell o Chick example inject virus in spinal cord and see what gets labeled Motor neurons form in ventral part If you only infect one cell but you end up with all typed of labeled cells you know it was a stem cell or a multipotent progenitor Interkinetic nuclear migration moving up and down its processes Ventricular surface Neural tube cells have a bipolar shape Interkinetic nuclear migration o inside lining the ventricle where arrow is pointing ventricular surface Dark pink neural crest cells Light pink neural tube o Neuroepithelial cells have two processes inside and outside of spinal cord with nucleus somewhere in middle They are not static and move around depending on cell cycle moving up and down these processes is called interkinetic nuclear migration Multipotent progenitor o Can give rise to different cell types but is more specified than a stem cell and only has set amount of divisions before differentiating unlike stem cells which can continuously self renew BrdU labeling birthdating analysis o Pulse chase experiment expose cells with label for certain amount of time pulse then remove label for extended amount of time chase o BrdU thymidine analog or radioactive thymidine H3 thymidine can be integrated into DNA by replaceing T pulse Then use a stain specific to BrdU and see all derivatives of originally tagged cells lineage labeling o See all dividing cells cells dividing really quickly will dilute it out quickly but stem cells which divide slowly will retain the BrdU longer determine how fast cells are dividing and what they re turning into o Inject pregnant female with BrdU and collect embryo at particular stages to see what cells turn into and hwere they reside o E11 stem cells around ventricular surface o E13 process of migrating out o E15 migrated out and differentiated Inside out growth o o Development goes from inside to outside as development goes on o Each layer is born at a different time of development o Radial glial cells span from ventricular to pial surface and provide scaffolding for neurons to migrate outwards More newly born neurons migrate out past the old ones o First born neurons actually create layer VI because the newer born neurons migrate past the deep layers and make the upper layers Q fraction Quit Fraction o o In the expansion phase a lot of symmetric cell division takes place to make progenitors high P fraction o In the neurogenic phase many cells are exiting the cycle by asymmetric cell division meaning there is a high Q quit fraction P fraction Proliferative Factor o Higher amount of cells continue to proliferate by symmetric division and make progenitors o P Q 1 Ventricular zone o Inside surface lining the ventricle o Neuroepithelial cells progenitor cells neurons and glial cells glial cells are radially oriented and span from ventricular zone to outer pial surface and provide scaffolding for migration of neurons outwards o Cells along base of ventricular surface usually have more potential Clonal analysis o Very few cells are retrovirally transfected labeled studied so all future labeled cells are clones of that first one Pro astrocytic factors EGF CNTF and BMPs o These factors all try to turn cell into astrocyte inhibit pro neural o EGF epidermal growth factor just the name works on other cell types too targets S phase cyclins to go into cell cycle bHLH factors what do they do o o HLH transcription factors drive neural progenitors to become differentiated neurons o Helix loop helix transcription factor pro neural o When turned on the feedback loop turns cells next to it into NON neuronal types so they aren t all the same in the population In neighboring cell HLH expresses delta activates notch cascade turns on hes1 transcription factor maintains progenitors Hes5 pro astrocytic Similar to p27kip where different levels can affect cortex size increasing HLH signaling it will increase cortex size by maintaining progenitor pool Generation of Purkinje cells versus granular cells o o o Purkinje primary cerebellum neurons generated at ventricular surface just like cerebral cortex but then migrate into Purkinje cell layer o Granule cells are generated at external granule cell layer then migrate to granule cell layer along Bergmann glia passing across the Purkinje layer and going down send out axons horizontally in classic T shape o Shh acts as mitogen for generation of granule cells o Used by granule cells to migrate across from EGC to granule cell layer Bergman glia Reeler mouse o o Reeler mouse mutation mouse that waves back and forth First phenotype was observed then reelin gene was identified o This mutation caused outside in pattern where newer neurons don t go back the older ones so newer neurons are closer to ventricular layer and older ones are at pial surface cortex is flipped therefore reelin must have to do with migration of neurons generation of the inside out pattern P27kip Cyclin dependent kinase inhibitor o o Stops or slows down cell cycle o Mitogens FGF EGF Shh Cyc D inhibits p27kip o Expression of this gene determines when cell will exit cell cycle o Extracellular ligands signal transduction pathway turn on transcription factors upregulate cyclin D Rb retinoblastoma protein transcription factor turns on genes that tell cell to re enter cell cycle o If you get rid of p27kip there are major effects on amounts of neurons generated o It codes for Cdk protein which binds to and prevents the activation of cyclin D E complexes which then slows down or stops cell cycle o Increase CdkI 27 more p27 inhibit cell cycle smaller cortex Higher Q fraction o Decrease CdkI 27 less p27kip larger cortex Lower Q higher P fractions Extracellular granular cell o Where granule cells are made migrate from here across Bergmann cells and past Purkinje cells to the granule cell layer Directed differentiation o o directing stem cell differentiation into a cell of interest by recapitulating development o o Turn pluripotent