Hedgehog Signalling Flashcards
Describe the hh mutant in flies and the pathway
In flies with the hh mutant the larval body is squashed and disorganised. Gene also involved in adult wing patterning. Different concentrations of the hh protein yield different results showing the protein has morphogenic actions.
Engrailed intitiates hh. hh stimualtes patched whihc results in other genes being turned on - dpp and wnt. Normally hh only seen in posterior wing. If we use GAL4 UAS to express hh in anterior imaginal wing disc then a mirror image wing forms. Similar thing happens in limbs in vertebrates (chickens). Same pathway but different results.
What is hh important for in vertebrate neural tube development?
Neural development, specifically neural tube, kidneys and bones. INSECTS DO NOT HAVE NEURAL TUBE.
Notochord causes nearby neural tube cells to differentiate into ventral floor plate cells. These then signal slightly more dorsal neural tube cells to differentiate into motor neurons whihc sprout axons and then migrate to their targets.
Both times it is shh that results in the differentiation - stain for it. Moving the notochord to a more lateral position causes the differentiation to occur here instead. Can also acheive this by transplanting VFP cells.
Opposing gradient of shh and TGFbeta (released from dorsal cells) to induce/repress transcription of different genes.
What is Holoprosencephaly and what can cause it?
Developmental malformation ranging from one missing incisor to cyclopia. Cyclopic children are born alive but die young due to their hemispheres being fused. Accounts for many miscarriages but a dominant form exists that can be caused by mutations in shh.
Mice hets are normal but homoxygotes have cyclopia.
HPE also seen in US cattle and was attributed to corn lillies. A steroidal alkaloid resembling cholesterol was identified as the causative teratogen. Giving it to mice induced cyclopia. Was termed cyclopamine.
Rare human disease called Smith-lemli-opitz results from a dysfunction in endogenous cholesterol synthesis. 5% of these people have a mild type pf cyclopia.
How does cyclopamine intefere with neural tube development and shh function?
In the presence of cyclopamine there is no induction of the ventral floor plate cells by the notochord. Could cholesterol be needed for shh activity and cyclopamine block it? - No shh processing is unaffected by cyclopamine.
Could cyclopamine intefer with the reception of the shh signal? Maybe - shh normally induces isl1/2 and HNF-3 but when cyclopamine is present this doesn’t happen - seen with antibodies to the genes in explants.
What is the evidence that cholesterol precursors may be the teratogens involved in HPE?
If you block the last step in the synthesis of cholesterol in rats they get HPE
SLO disease in humans results from dysfunction of last step in cholesterol synthesis and they get mild HPE 5% pf the time.
So could high rate of HPE in abortions be because of high precursors of cholesterol and so women should eat more cholesterol during pregnancy to limit the amount of cholesterol precursors in their body?
What does the dominant pattern in humans and recessive pattern in mice show us about shh?
Must be important for multiple stages of development. Proved using antibodies in chick embryos.
What relation does alcohol have to HPE?
In foetal alcohol syndrome some babies get HPE. In mice ehtanol reduced shh in utero in prchordal mesendoderm and reduces shh in vitro.
Inhibition of shh in prechordal mesendoderm affects midline structures. This is because shh stimulates cell survival in PME as it has mitogenic activity.
Ehthanol effects shh via protein kinase A resulting in reduced midline cells and so HPE.
Where does cyclopamine act in the hh pathway?
Without shh patched does not interact with smoothened. As a result Gli Ci is cleaved and inactivates target genes.
When shh is present this binds to patched. This results in smoothened being activates and so prevents Gli Ci form being cleaves. As a result Gli Ci can activate genes.
Patched does have a sterol sensing domain but this is not where cyclopamine acts because in a patch -/- adding cyclopamine still inactivate the pathway. This can be shown with a Gli reporter construct. Cyclopamine acts downstream on smoothened for evidence read a quick review?
Is the patched sterol sensing domain important?
Yes, normally patched effluxes cholesterol. When shh binds it can no longer do this and the extra cholesterol helps move smoothened to the correct place in the cell membrane.
What is cancer and how is the hh pathway involved?
Cancer = de differentiation and proliferation. hh pathway can stimulate proliferation as was demonstrated by ethanol and the PME.
hh pathway sometimes mutated in cancer. Garlin syndrome caused by KO of patched = increased susceptibility to cancer, especially BCC.
40% of sporadic BCC and 25% of neuro ectodermal tumours have either LOF in ptc or GOF in smo.
Why would this pathway be a good target for drugs?
Most symptoms seen relate back to its function during development. So inhibiting it in an adult should have little effect. Where as traditional cancer treatment have lots of side effects.
What is the issue with using cyclopamine itself?
Cyclopamine could be useful but its not very well tolerated. Also oncogenic smo is resistant. So take cyclopamine and chemically alter. Doing this created KAAD cyclopamine - 10-20 x as potent.
In mice trials KAAD worked for both normal smo and oncogenically active smo (but not as well as still resistant). In ptc -/- = excess tissue growth - KAAD reduced cell replications.
How did they find a new chemical potent enought?
Use a light emitting assay for hh pathway to screen potential chemicals - all polycyclic aromatic compounds. Automate assay and test 1000s. Take best inhibitors and use in ex-plant model of a BCC.
Results showed regression of BCC and safe for normal cells.
What else is cyclopamine important for in cancer?
Pathway also seems to promtor metastasis. Cancer with highly active pathway were more likely to metastasise than low active. Also over expressing Glu promoted metastasis. So inhibitors even more useful.
Are there any approved drugs that inhibit the hh pathway.
Yes - Vismodegib
But two main issues toxicity and resistance.
