Exam 5: Lecture 7 Flashcards
1
Q
Hippo Pathway-General
A
- 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
2
Q
Hippo Pathway-First Identified and Associations
A
- via efforts to understand how a tissue or organ knows when to stop growing
- since then closely associated with tumor formation and cancer
3
Q
Mutations in Hippo Pathway
A
- 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
4
Q
Founding Member of Hippo Tumor Suppressor Pathway (Shar-pei)
A
- 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
5
Q
Removal of Shar-pei
A
- 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
6
Q
Why tissues appear larger: 1
A
-number of cells in wild type and mutant tissue can be the same, but the cells can be bigger in the mutant
7
Q
Why tissues appear larger: 2
A
-size of wild type and mutant cells can be the same but the number of these cells can be significantly higher in mutant tissue
8
Q
Fly Ommatidium
A
- 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
9
Q
Mutant Clones
A
- 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
10
Q
Mutated gene not involved in growth control
A
-size of mutant clone will be same as wild type twin spot
11
Q
Mutated gene required for cell to grow
A
-mutant clone will be smaller than wild type twin spot
12
Q
Mutated gene is tumor suppressor
A
-mutant clone will be larger than wild type twin spot
13
Q
Drosophila Eye and Tumor Suppressors
A
- 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
14
Q
Why create mutant clones instead of looking at entire animals?
A
- 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
15
Q
Hippo Pathway Componets
A
- 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
16
Q
Removal of hippo gene
A
- 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
17
Q
Yorkie-inhibition
A
- 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.
18
Q
Yorkie
A
- 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
19
Q
Yki removed from developing head and retina
A
-both tissues considerably smaller that w/t
20
Q
Yki expressed at higher than normal levels in developing wing disc
A
-tissue maintains overall shape but is considerably larger than w/t counterpart
21
Q
Indications of Yki removal/expression
A
-Yki promotes growth and Hippo pathway’s role in development is negatively modulated by activity of Yki
22
Q
Yki and Cell Proliferation
A
- 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
23
Q
Bantam miRNA
A
- 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
24
Q
Results of bantam/Yki experiments
A
- 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
25
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
26
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
27
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
28
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
29
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
30
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
