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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


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


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


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


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


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


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


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


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


Mutated gene not involved in growth control

-size of mutant clone will be same as wild type twin spot


Mutated gene required for cell to grow

-mutant clone will be smaller than wild type twin spot


Mutated gene is tumor suppressor

-mutant clone will be larger than wild type twin spot


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


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


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


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



-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.



-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


Yki removed from developing head and retina

-both tissues considerably smaller that w/t


Yki expressed at higher than normal levels in developing wing disc

-tissue maintains overall shape but is considerably larger than w/t counterpart


Indications of Yki removal/expression

-Yki promotes growth and Hippo pathway's role in development is negatively modulated by activity of Yki


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


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


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


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


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


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


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


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


Mst1 and Mst2

-homologs of Drosophila Hippo protein
-loss of either individually has no effect on organ size
-both genes must be simultaneously eliminated in order to see an effect on cell proliferation
-Mst1 and Mst2 are products of duplication event that occurred after split in insects and vertebrates.
-since individual mutations have no phenotype it is thought that the genes are redundant in function