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Joe Kim

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MIDTERM 2 LIFESCI 2A03 Quicknotes November 26, 2013 Stem Cell Niche  Adult Stem Cells – cells that can divide to create two cells (another stem cell or differentiated cell)  Symmetric Cell Division – two identical daughter cells are created (both stem cells)  Asymmetric Cell Division – two daughter cells are different from one another (one stem cell and a progenitor cell with limited self-renewal potential) o Maintains homeostasis; balance between stem cells and undifferentiated cells  Cancer tissue homeostasis tissue aging/degeneration o Differences between cells after asymmetric division 1. Differential segregation of mRNA, cystolic proteins, cell membrane proteins 2. Differences in surrounding environments o Two mechanisms 1. Extrinsic fate determinants – one cell stays in cell niche (maintains contacts), other leaves 2. Intrinsic fate determinants – asymmetric localization of components within the dividing cell that results in their differential segregation (one cell has determinants, other doesnot)  Stem Cell Niche – microenvironment that maintains stem cell identity  Adult stem cell categories – i) somatic stem cells (go on to create or replenish body cells) ii) ASC (create germline cells; sperm and egg)  Drosophila Ovaries (model stem cell niche system) – consist of ovarioles made up of string of follicles (developing eggs); contains germline cells that go on to form egg and somatic cells that support egg development o Germ line stem cell niche at anterior end of ovariole o Stem Cell Niche – terminal filament, cap, inner sheath cells; express molecules for maintenance and self renewal of female GSCs; formed by somatic cells o Germ Line Stem Cells (GSC) – undergo asymmetric division within niche; results in GSC and cystoblast (CB)  GSC remains in niche; retains stem cell identity  CB leaves niche and forms cyst (group of germline due to mitotic division; one cell in cyst will become egg, other cells become supporting tissue o Cellular marker used to identify GSC – use antibody to proteins within fusome/spectrosome (only found in germ line cells)  Spectrosome while in niche (in GSC); fusome when leaves niche, grows (CB; cyst) o GSC and CB differ in intrinsic and extrinsic factors that determine identity  Intrinsic – expression of bam, differentiation-promoting gene  CB require expression of two genes (bam (bag of marbland bgcn (benign gonial cell ne)  needs to differentiate  GSC does not transcribe bam; Bmp signals from niche repress transcription of bam; Dpp signaling silences bam transcription to establish asymmetric divisions of GSC  GSC remains undifferentiated to undergo asymmetric cell division  Extrinsic – GSC in niche, CB out of niche  Can visualize bam gene by fusing bam promoter with GFP coding sequence (bamP-GFP) o Promoter – specific DNA sequences required for binding TFs (that recruit RNA polymerase) and RNA polymerase o P[bamP-GFP] – P element (mobile DNA used to introduce exogenous sequences into fly genome); Promoter sequence of bam; GFP downstream of promoter  Experiments 1. Remove bam gene function  germ cells do not develop; all CBs, no cysts or GSCs  Info – bam only present in CB; outside of niche  Hypothesis – the bam gene is required for germline cell development  Null Hypothesis – there is no significant difference in the development of germline cells in the presence or absence of bam gene expression  Control – wt bam; CB form, differentiated and developing cysts (Figure B)  Experiment – bam[86] = deletion, non-functional gene; P[bamP-GFP] activated for visualization  Result – Symmetric division (GSC not developing), many CB; CB do not form cysts; germ cells do not differentiate (Figure A)  Conclusion – Bam protein required for germ cell development 2. Overexpression of dpp  no CBs, all GSCs  Info – dpp from niche; receptor on stem cells  Hypothesis – if Dpp is required to maintain stem cells, then too much Dpp should increase number of stem cells  Null Hypothesis – there is no significant difference in the number of stem cells in the presence of normal and the presence of elevated Dpp  Experiment – induce overexpression of dpp; 6 days elevated temperature; {hs-Gal4} and {UAS-dpp}  Heat shock promoter for gal4 expression  Gal4 protein binds upstream activating sequence (UAS)  UAS for dpp gene activation  Result – cell shape looks the same as lack of bam, but P[bamP-GFP} not expressed; all GSC, no CB  Conclusion – dpp can maintain stem cell identity 3. Dpp over expression + removal of SE on bam  over proliferation of GSC, but CB also produced  Hypothesis – regulation of ban expression by Dpp occurs through the silencer element with the bam promoter  Null Hypothesis – there is no significant difference between bam expression in the presence of absence of the bam SE 1 MIDTERM 2 LIFESCI 2A03  Experiment – P[bamPSE-GFP]= deletion of SE on bam promoter; 6 days elevated T and overexpression of Dpp  Result – over proliferation of GSC but bam promoter is active (CB present; can see GFP)  Conclusion – Dpp controls bam expression through SE on bam promoter 4. Dpp may signal to Mad to bind to SE on bam promoter to regulate bam expression (inhibit)  Info – Mad (transcriptional regulator protein) binds to silencer element (SE) on bam promoter to inhibit expression  Hypothesis – SE of bam promoter contains DNA sequences recognized by Mad  Experiment – sequence analysis  Result – Site A bound by Med; Site B allows Mad to bind promoter sequence  Conclusion – bam regulatory sequences may bind to Mad 5. Overexpression of dpp + overexpression of bam  CB can form and differentiate into cysts  Hypothesis – the defect that occurs when Dpp is over expressed (inhibited bam, too many GSC, no CB)can be reversed by increasing bam expression  Experiment – overexpress bam while overexpressing dpp (using heat shock promoters)  Result – cysts form  Conclusion – bam expression causes cells to act like CB (even in the presence of Dpp; Dpp represses bam in the niche, when cells leave niche, can express bam and become CBs) 6. Dpp signaling  no bam; transition phase expresses both  Info – Dad = dpp target; indicates Dpp signal is active (marker)  Detect Dad using Dad-lacZ (cleaves -galatosidase)  Hypothesis – dpp signaling activity is restricted to GSCs and some CBs where bam transcription is actively repressed  Experiment – visualize location of dpp expression by dad-lacZ expression; visualize CB by bamP-GFP  Result – GSC in niche expressing dad, not bam (B); CB and cyst expressing bam, not dad (C), transition from GSC to CB express both  Conclusion – dpp signaling, then no bam; as dpp signaling reduces, bam expression increases 7. Dpp signaling  results in pMad  no bam  Hypothesis – if an end result of Dpp signaling pathway is phosphorylation of Mad, then pMad should be seen in cells that are not expressing bam and no pMad in cells expressing bam  Result – high pMad (pink), no bam expression; low pMad, increased bam expression  Conclusion – further support that dpp signaling pathway activated in GSCs at high levels, bam transcription activity repressed 8. Remove dpp function  bam transcription occurs in GSC  GSCs not maintained hr56  Info – dpp mutation prevents dpp signaling at high T (29˚C)  Hypothesis – dpp is essential for repressing bam transcription in GSC  Null Hypothesis – there is no significant difference in bam transcription in the presence or absence of dpp  Control – normal dpp at 29˚C  no effect (no bam in GSCs)  Experiment – identify germ cells by labeling fusome with fluorescent Hts (protein in fusome) antibody; identify all cells by labeling DNA with DAPI; visualize bam-GFP  Result – bam-GFP expression increases gradually in GSCs; number of GSC reduce; eventual GSC elimination (not renewing)  Conclusion – dpp signal required to repress bam transcription in GSCs and maintain GSCs 9. Absence of pMad  expression of bam  Hypothesis – if pMad repressesbam in dpp pathway in GSCs then in the absence of pMad, bam will be expressed  Control – wt dpp; pMad present in GSC, no bam-GFP expression (Figure E) hr56  Experiment - dpp at 29˚C˚, 4 days  Result – pMad low in GSC, bam-GFP expression (Figure G)  Conclusion – pMad represses bam expression in GSCs (?) 10. Overexpression of dpp  no bam expression in CB (make the niche larger)  Control – no heat; GSC with round fusome at tip, no bam-GFP; CB and cyst cells express bam-GFP  Experiment – heat shock-Gal4:UAS-dpp system; heat  Result – more single germ cells with round fusome; do not express bam-GFB  Conclusion – dpp overexpression is sufficient for repressing bam transcription in CB 11. Empty niche (?)  Hypothesis – if parts are interdependent; if component removed, whole niche collapses 2 MIDTERM 2 LIFESCI 2A03  Experiment – turn off niche (extrinsic signal dpp); turn off GSC response (internal signal); briefly express bam protein using hs-Bam to force resident GSC to differentiate and exit niche; determine stability of niche  Result – GSC disappearing, cap cells disappear, niche degenerates  Conclusion –  Summary o In Niche – Dpp signaling leads to production of pMad TF in SCpMAd represses bam transcription by physically binding to SE with transcription regulatory sequence of bam gene o Outside Niche - No dpp signaling  no pMad  no repression of bam transcription o Model – one GSC in niche asymmetrically divides; pushes one daughter cell out of ichdifferentiation requires bam transcription Molecular Basis of Regeneration – Planaria as a Model System  Distinguishing stem cells – i) Morphology i) Cell-surface or intracellular protein markers iii) Cell proliferation  Techniques 1. Labeling actively dividing cells  determine which cells are stem cells/neoblasts a. BrdU (bromodeoxyuridine) = thymine analog – can be detected using an antibody when incorporated into DNA  Grow planaria in medium containing BrdU; cells entering S-Phase will use BrdU instead of thymine in DNA  Neoblast cells (actively dividing cells needed for regeneration; stem cells) in S-phase will be visualized; differentiated cells will not contain BrdU b. H3P = phosphorylated form of histone H3 – can be detected using an antibody when bound to DNA  Histone H3 = chromosomal protein bound to DNA to help in folding; only seen in mitosis on condensed chromosomes 2. Labeling mRNA using fluorescent in situ hybridization  determine which genes are expressed in stem cells/neoblasts o Identify cell expressing particular gene using a probe – assay directly for presence of mRNA  Probe = small sequence of DNA that is complementary to a target sequence; will hybridize in base pair specific manner and fluoresce  visualize only labeled cells o Whole organism in situ hybridization – visualize which genes are expressed in specific cells of the organism  Forward Genetic Screen – create random mutations in genome; observe to identify effect of mutation in certain gene o Mutation  observe effect  discover gene  Reverse Genetic Screen – mutate identified gene that is suspected to have an effect of desired function (regeneration) o Known gene  make mutation  observe whether there is an effect 3. RNA Interference (RNAi) to eliminate gene function  determine the role of a gene o Process  dsRNA introduced into cell  Dicer cuts ds RNA into siRNA (still double stranded)  Proteins assemble into RISC complex  RISC picks up siRNA breaks strands apart, keeping single strand complementary to target  Single stranded siRNA binds mRNA target  RISC cuts mRNA where hybridization has occurred  Ribosome cannot translate mRNA into functional protein  gene expression is silenced o Net Result – genes are silenced post-transcriptionally; mRNA destroyed  no gene expression  Same result as deleting gene from genome (used to use premature stop codon) o Goal in Lab – create dsRNA complementary to target gene o Incorporate dsRNA into bacteria – i) can feed to many animals ii) generate permanent reagent(can freeze bacteria) 4. RNAi Screen to identify subset of genes in the genome involved in a specific process (regeneration) o Screen = large-scale experiment in which many genes in the genome are disrupted and the effects (phenotypes) are observed Sanchez Lab  Identify Neoblast cells – i) morphology of cells ii) BrdU labeling iii) antibody to H3P  Eliminate neoblast cells by irradiation, see if markers are still present to demonstrate that these markers are neoblast specific o After irradiation – planaria incapable of regenerating, no cells labeled by H3 antibody  neoblast functioning correlates with regeneration  Neoblasts are required for regeneration – irradiation prevents normal blastema formation after amputation o Experiment – irradiate, amputate; 3 days later, un-irradiated fragment has blastema formation; regional and whole irradiation has no normal blastema formation  Determine genes expressed in neoblasts by i) tracking gene expression (in situ hybridization) ii) manipulating gene function (RNAi)  RNAi Screen identified putative candidates in regeneration pathway  found smedwi genes; similar to piwi gene o Piwi – encode regulatory proteins responsible for maintaining incomplete differentiation in stem cells o BrdU identified mitotically active cells (neoblasts)  in situ hybridization to smedwi-1 and smedwi-2 (same location as neoblasts) o RNAi by irradiation eliminated neoblasts and smedwi-2 expressing cells  no longer see smedwi genes  Smedwi-1 RNAi did not produce robust effects (regeneration occurred); smwedwi-2 RNAi produced similar effects o irradiation (no regeneration)  Effects of RNAi – placed into distinct categories identifying genes involved in each step in the regenerative process 1. Wound Healing and Initiation of Regeneration (Blastema Initiation) 2. Neoblast Maintenance (Homeostasis) 3. Proliferation (Growing the Blastema) 4. Differentiation and Patterning 3 MIDTERM 2 LIFESCI 2A03 5. Morpholaxis – integrating new tissues with old 6. Non-regenerative homeostasis  Experiments – to determine which step in regeneration smedwi-2 is involved in 1. Wound Healing and Initiation of Regeneration (Blastema Initiation)  Null Hypothesis – the elimination of smedwi-2 expression hasno effect on the ability to form a blastema  Negative Control – blastema formation; RNAi against unc-22  blastema forms, regeneration is normal  Negative Control – no phenomenon is expected; ensure that there is no effect where there should be no effect  Unc-22 – expressed in differentiated muscle cells; no role in regeneration  Positive Control – eliminate blastema formation; irradiate to eliminate neoblasts  no blastema, no regeneration  Positive Control – phenomenon is expected; ensure that there is an effect when there should be an effect  Experiment – smedwi-2 RNAi  Result – no regeneration, no blastema formation  Reject null hypothesis – smedwi-2 seems to be involved in blastema formation 6. Non-regenerative homeostasis  Null Hypothesis – the elimination of smedwi-2 expression hasno effect on the ability to maintain homeostasis i.e. maintain net cell number  Homeostasis – the ability of an animal to maintain its cells; differentiated cells are lost (due to aging, cell death etc)  Negative Control – homeostasis function; RNAi against unc-22  normal homeostasis  Positive Control – eliminate homeostasis; irradiate to eliminate neoblasts  curling indicative of cell loss  Irradiation disrupts homeostasis; cell loss overtime in absence of functional neoblasts  Irradiation + amputation – no regeneration at 6 days; whole morphology changing (curling) at 15 days  Irradiation + intact organism – indents at 4 days, decreasing size at 8 days, regression and cell loss across anterior surface at 11 days, similar to amputated animal at 14 days  Experiment – smedwi-2 RNAi
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