HH ligand binds —> HH signal active
HH acts as inhibitor of rezeptor Patched
Patched when active (without HH) inhibits Smoothened and targets it for degradation
Vertebrates
Ci = Gli
Pimary Cilia are essential
1) How would you visualize, in which cells of an embryo or tissue the hedgehog pathway is activated?
By using Reporter Gene assay for example the LacZ/Galactosidase assay -> a target gene of active HH signaling is Gli1 the galactosidase expression could be engineered under control of that promotor and now whenever HH is active ß-galactosidase is expressed and can be visualized by X-gal staining
GFP or luciferase reporters could be used as reporters in the same manner
Immunochemistry -> antibodys specific to HH pathway components e.g. smoothened would be a good component because it is degraded when HH is not active and only upon HH binding it is activated
Fluorescent In Situ Hybridization (FISH): mRNA Detection with FISH can be used to detect the spatial distribution of Hh target gene mRNAs within cells and tissues using fluorescently labeled probe
Transgenic Models with Hh Pathway Reporters: Creating transgenic animals that carry a reporter gene under the control of Hh-responsive elements can provide a whole-organism view of Hh pathway activation. For example, mice with a Gli1-GFP reporter
2) How can you determine the fate of cells expressing Shh at a particular stage of mouse development?
genetic labelling: Cre under control of shh promoter, reporter e.g. lacZ or GFP in non-essential locus. floxed stop cassette in front of reporter, gets excised upon activation of cre. Usage of mutant loxP sites (e.g. RE, LE loxP sites) allows irreversible reporter activation.
barcoding also via genetic labelling. non-endogenous sequence instead of reporter cassette
3) Patched is the Hedgehog receptor. What effect on Hedgehog signaling do you expect if you overexpress Patched in a tissue?
Patched receptor is inhibiting and degrading Smoothend and by that inhibiting the hedgehog pathway -> an overexpression would result in inhibition of the HH signal and because there are so many receptors HH ligand wouldnt be able to diffuse further so the effect on surrounding tissue is inhibited too (potentially)
4) What effects of Patched loss of function mutations do you expect on the expression of HH responsive genes?
This would result in permanent activation of HH responsive genes because Smoothened is no longer inhibited or degraded -> permanent activation could result in tumor formation
-> Gorlin Syndrome: heterozygous mutation in Ptch1 -> carzinomas and hh overactivation phenotypes
5) Explain the function of cubitus interuptus and its three vertebrate homologues in Hedgehog signaling.
Ci Protein (or the Gli proteins in vertebrates) is the nuclear effector that is translocated into the nucleus in its activator or repressor from without HH signal Ci is phosphorylated and cleaved to become a transcriptional repressor in the nucleus
But when HH signal is active the Phosphorylation will not happen and Ci acts as a activator in the nucleus!
The 3 different Gli proteins are a little special
Gli 1 can only act as activator!
Gli2/3 act just like Ci as repressor or activator
6) How can HH signaling control gene expression in a concentration dependent manner?
HH ligand acting as a morphogen -> different diffusion rates also controled by Patched and Smoothened availability
Concentration-dependent Activation: Different concentrations of Hh activate Smo to varying extents, leading to a graded response.
Gli Transcription Factors: The activation of Smo regulates the Gli family of transcription factors (Gli1, Gli2, Gli3 in vertebrates). The balance between full-length activator forms and truncated repressor forms of Gli proteins determines the transcriptional output.
-> Gli binding site affinity, how many binding sites repression and interaction with other TFs
Low Hh Concentration: Low levels of Hh lead to partial activation of Smo, resulting in a mix of Gli activators and repressors. This can result in the expression of some target genes while repressing others.
High Hh Concentration: High levels of Hh lead to full activation of Smo, resulting in predominantly Gli activator forms and robust activation of Hh target genes
7) Which cells carry primary cilia? What is the structure of cilia?
-> most eukaryotic cells e.g. epithelial cells in kidney, liver, pankreas, olfactory neurons …
Cilia consist of 9 microtubuli, on them Motorproteins can transport cargo
8) What functions of cilia do you know?
motile
cilia in respiratory epithilia to get rid of mucus
liquid transport in kidney
left right axis formation
Non motile
primary cilia -> sensory organelles
9) What phenotypes do you expect in patients with mutations in ciliary genes?
-> dysfunctional HH pathway = inactive
-> defect midline separation
-> defect mucus transport
-> left right axis disruption
-> no sperm movement
10) Smoothened belongs to the G protein–coupled receptor super-family. How does the signaling of G-proteins coupled receptors work?
GPCR
7 transmembrane domain
upon ligand binding (extracellular) conformational change intracellular and interaction with the G-Protein exchanging GDP for GTP releasing the activated Galpha subunit that can now affect other proteins e.g. adenyl cyclase or phospholipase C
when the Galpha subunit hydrolyzes the GTP to GDP it is recycled and returnes to the inactive state reassociating with the Gßy dimer
11) What approaches can you think of to inhibit hedgehog signaling?
Vismodegib and Sonidegib: Smoothened inhibitors used to treat advanced basal cell carcinoma -> inhibit smoothened
Trap HH ligands
Stableize Patched rezeptor so it can inhibit smoothened
Get the Ci-Protein into the repressor form and keep it like this -> Phosphorylation of Ci (normally done bei PKA and Slimb -> stabelize them)
12) What is holoprosencaphaly?
Defect in midline formation due to blocked HH signaling
Inhibition of HH signaling by jervine or cyclopamine, inhibitors of Smo, or by mutating Shh results in holoprosencephaly and cyclopia. Cyclopamine and jervine are steroidal alkaloid found in the corn lily (Veratrum californicum).
SHH signaling -> splitting of the single eye field, formation of retinal primordia
SUMMARY POINTS
1. The Hedgehog ligand is modified with both palmitoyl and cholesteryl moieties, causing it to attach strongly to membranes.
2. The Hedgehog ligand requires the membrane protein Dispatched and extracellular chaperones for release from producing cells and for long-range signaling.
3. The Hedgehog ligand binds heparan sulfate proteoglycans, which are required for its extracellular movement.
4. Extracellular distribution of the Hedgehog ligand is negatively regulated by its binding to its membrane receptor, Patched, and to the secreted antagonist Hhip.
5. In the absence of ligand, Patched inhibits Hedgehog signaling by repressing the downstream seven-transmembrane protein Smoothened.
6. The Hedgehog ligand activates signaling by binding and inhibiting Patched, thus derepressing Smoothened.
7. The coreceptors Cdon/Ihog, Boc/Boi, and Gas1 cooperate with Patched for ligand reception and are collectively necessary for Hedgehog pathway activation.
8. Smoothened is activated by an endogenous ligand via its extracellular domain; in vertebrates, the Smoothened ligand is cholesterol. S
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