Flashcards in Exam 5: Lecture 7 Deck (30)
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Hippo Pathway-General
-Tumor suppressor pathway used to control growth of tissues and organs
-Entire pathway has been elucidated in both flies and mammals
-Nearly all components are conserved across 500 million years of evolution
-tasked with repressing activity of Yki homolog (YAP)
-represents universal mechanism for controlling growth
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Hippo Pathway-First Identified and Associations
-via efforts to understand how a tissue or organ knows when to stop growing
-since then closely associated with tumor formation and cancer
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Mutations in Hippo Pathway
-Fat4 and NF2 are underlying causes of breast cancer and schwannomas
-main target of this pathway is Yki TF and is expressed at higher than normal levels in several breast, colorectal, and liver cancers
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Founding Member of Hippo Tumor Suppressor Pathway (Shar-pei)
-documented effects of removal of this gene from entire head and retina
-wild type control animals have flat head cuticle surface
-mutant tissue over proliferates making undulating folds of head capsule tissue
-resembles shar-pei dog so that's where it gets it's name
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Removal of Shar-pei
-ability to suppress cell proliferation is not limited to head and retina
-remove shar-pei within clone of cells within torax leads to tumor formation
-loss of shar-pei throughout haltere leads to significant increase in size
-in every tissue examined, shar-pei controls organ size throughout all developing Drosophila tissues
-same shown for mammalian Hippo pathway
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Why tissues appear larger: 1
-number of cells in wild type and mutant tissue can be the same, but the cells can be bigger in the mutant
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Why tissues appear larger: 2
-size of wild type and mutant cells can be the same but the number of these cells can be significantly higher in mutant tissue
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Fly Ommatidium
-each ommatidium separated from neighbors by single cell
-in shar-pei mutant there are more cells between ommatidia
-results indicate Hippo pathway is a true tumor suppressor pathway and that its role in development is to suppress cellular proliferation
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Mutant Clones
-there are tricks one can employ to generate patches of cells that will be mutant for both copies of a single gene (-/-)
-when mutant clones are generated, twin clone that is wild type for both copies of gene of interest also generated
-growth features of two clones can be analyzed against each other
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Mutated gene not involved in growth control
-size of mutant clone will be same as wild type twin spot
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Mutated gene required for cell to grow
-mutant clone will be smaller than wild type twin spot
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Mutated gene is tumor suppressor
-mutant clone will be larger than wild type twin spot
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Drosophila Eye and Tumor Suppressors
-w/t tissue marked with red pigment and mutant tissue lacks pigment
-control: approximately equal amounts of red and white tissue
-experimental: mutant tissue completely taken over entire retina
-the gene that is mutant in this example is member of Hippo suppressor pathway
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Why create mutant clones instead of looking at entire animals?
-tumor suppressor genes usually expressed in all cells of developing organism
-mutation that removes tumor suppressor gene from entire animal will die early in development due to tumor formation
-generation of mutant clones in eye allow for rest of animal to develop normally
-since eye is dispensable organ, fly containing retinal clones that are mutant for a tumor suppressor will survive to be analyzed
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Hippo Pathway Componets
-isolation of Hippo pathway components have been achieved due to variety of genetic and biochemical screens
-with one exception, loss of any member of Hippo pathway leads to phenotypes seen in shar-pei mutants
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Removal of hippo gene
-leads to over-proliferation, tissue undulations, and increase in overall organ size
-additional defects in planar cell polarity, cell junction integrity, and cell migration seen in some pathway members associated with the membrane
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Yorkie-inhibition
-entire cytoplasmic hippo pathway focused on inhibiting activity of Yki TF
-it encodes transcriptional co-activator that cannot bind to DNA on its own but interacts with DNA BP scalloped
-Sd-Yki composite TF bind to enhancers of genes involved in promoting cell proliferation and tissue growth
-Hippo pathway regulates Yki so tissues and organs stop growing when appropriate size has been achieved.
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Yorkie
-member of Hippo pathway isolated in screens looking for growth regulators
-however, mutant clones grew poorly when compared with w/t tissue
-gene called Yorkie
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Yki removed from developing head and retina
-both tissues considerably smaller that w/t
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Yki expressed at higher than normal levels in developing wing disc
-tissue maintains overall shape but is considerably larger than w/t counterpart
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Indications of Yki removal/expression
-Yki promotes growth and Hippo pathway's role in development is negatively modulated by activity of Yki
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Yki and Cell Proliferation
-generate clones of w/t tissue these cells grow happily in wing disc
-overexpress Yki clones are bigger
-remove Yki then clones hardly grow at all
-support idea that Yki promotes cell proliferation
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Bantam miRNA
-one of the targets of Yki-Sd complex
-remove bantam miRNA cells grow poorly
-now remove bantam from clone that is overexpressing Yki, clones grow more poorly than if bantam is present
-overexpress bantam miRNA in cells lacking yki, clones grow much better than clones that just lack yki
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Results of bantam/Yki experiments
-suggest bantam is genetic target of Yki-Sd complex
-idea that in cells that are growing Yki-Sd activates expression of bantam which in turn will bind to 3' UTR of a set of target mRNAs
-resulting degradation of target mRNA or the blockage of translation of that mRNA results in enhanced growth
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hid and 3' UTR
-miRNAs bind to 3' UTR of mRNAs and stimulate degradation or block translation
-search for putative targets identified head involution defective (hid) mRNA is putative target
-within 3' UTR of hid are five potential binding sites for bantam miRNA
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miRNA sensor assay
-determine if particular 3' UTR is true target of any given miRNA
-fused 3' UTR of hid to coding sequences of GFP
-w/t wing disc mRNA translated normally and disc glows
-Using UAS-GAL4 system forcibly expressed bantam miRNA along A/P axis
-level of GFP expression dropped along A/P axis
-indicates bantam miRNA was able to bind to 3' UTR and block production of GFp
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Hid and Cell Death
-member of programmed cell death pathway
-in response to apoptotic stimuli hid and other proteins initiate cascade of events leading to death of cell
-different versions of pathway
-in fly embryo significant amount of programmed cell death
-caused by overproduction of cells during early stages of development
-excess cells must be pruned away prior to hatching of embryo into larva
-in mutants that lack hid gene all cell death is blocked
-overexpress hid within developing eye, high levels of cell death induced and only tiny eye is produced
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Cells that are growing
-Yki-Sd complex activates expression of bantam miRNA which in turn binds to hid mRNA and blocks production of Hid protein
-without this key cell death inducer apoptosis is blocked and cells can proliferate
-when it's time for cells to stop growing, Hippo pathway blocks activity of Yki-Sd
-results in loss of bantam expression
-without bantam miRNA hid mRNA can be translated
-Hid protein can then induce cell death and prevent tissue or organ from growing out of control
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Hippo Pathway and Cancer
-several tumors and cancers in humans are associated with loss of several different Hippo pathway elements
-oss of Hippo signaling in mouse liver leads to dramatic increase in size of organ
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