Describe the possible sites of regulation between the expression of a cell signal and the responses of a target cell

including Hedgehog and/or canonical Wnt signalling as examples.

What is a signalling pathway?

This is the mechanism by which an external ligand or signalling molecule can induce a change in the gene expression profile of a cell.

Wnt and Shh signalling pathways

The Wnt pathway mechanism

• Only looking at the canonical Wnt pathway, involved in beta-catenin stabilisation.

• What does the pathway look like when it is on? When it is off?

• Place arrows pointing to where regulation can occur, and which points we are considering in this presentation.

The components of the Wnt pathway

• Only the canonical pathway is considered here, although there are other pathways involving Wnt.

• Wnt binds to Frizzled.

• Activation requires Frizzled and LRP5/6 binding

• Dickkopf (Dkk) proteins = Wnt Antagonist• Secreted - Indirect Inhibition - Bind LRP5/6

Mao, Wu et al., 2001

Antagonist - Dkk

Mao, Wu et al., 2001

Antagonist - Dkk

• High LRP6 + Dkk1 = Little Inhibition• Kremens – single-pass membrane protein• Kremens Binds with LRP– form ternary complex

• Induces Endocytosis– Prevents further signalling– No longer binds Wnt

• Increases Inhibition

Wang, Zang et al., 2008

Antagonist - Dkk

Without HH signal

-patched keeps Smoothened inactive in intracellular vesicles

-Ci is ubiquitinylated and proteolytically processed in proteasomes by multiprotein complex

-Processed Ci acts as transcriptional repressor of the HH responsive gene

With HH signal

-HH binds to Patched and iHog, endocytosis of Patched and degradtion in lysosomes

-activation of Smoothened by phosphorylation of PKA and CK1

-Translocation of Smoothened to cell surface, and recruit fused, Ci and Costal 2

-Ci not phosphorylated and enter nucleus as transcriptional activator

What happens in vertebrates- Gli proteins (Gli 1,2 and 3) are Ci-like gene regulatory proteins- Downstream of Smoothened- Gli 3 acts as a transcriptional repressor or activator- Gli 1 and Gli 2 are not processed, act as activators exclusively

Regulation of HH pathway by Gli code

What affects the gli code?

Different concentration of GliA/GliR can regulate the cell fate

-Wnt signaling can increase Gli activity

-alteration in activities of PKA, GSK3, CK1 and ligase by other pathways (affect Gli activities)

-SuFu inhibition of Gli (prevent Gli from entering nucleus)

Auto-regulation by the negative feedback loop

• Cells exposed to a high concentration of Shh sustain high levels of Gli activity for a prolonged period of time

• Exposure to a lower concentration of Shh is initially able to induce high levels of Gli activity, these levels are not maintained and the cells express Olig2

• The decrease in Gli activity indicates that cells adapt to the concentration of Shh over time

• Up-regulation of Ptc1 contributes to the gradual adaptation• The transcriptional up-regulation of Ptc1 by Shh signalling results in the

gradual accumulation of Ptc1 protein in responding cells• If the cells are exposed to a high concentration of Shh, sufficient ligand

will be available to bind the induced Ptc1 allowing signal transduction to be maintained

• In cells exposed to low concentration of Shh, unliganded Ptc1 will begin to build up; this will inhibit signal transduction

Primary cilia as hedgehog signalling centre

• Mutations in different proteins required for cilia formation have distinct effects on Hh signalling: some block responses to Hh ligands, others cause ligand-independent pathway activation and yet others change the spatial organization of pathway activation.

• Initially it was proposed that cilium acts as an antenna however it is now shown that transport machinery within the cilium provides an apparatus that is essential for mammalian Hh signal transduction

• Mutations in IFT proteins required for ciliary assembly results in dysfunctional Hh signalling and severe developmental disorders in mammals

• Forward genetic screens where neural tube patterns were analysed leading to the conclusion that cilia was essential to the hedgehog signalling pathway

Localization of core components of the Hh signalling pathway to cilia

• Ptch1, the mouse Hh receptor, is enriched within the primary cilium in the absence of ligand and moves out of the axonemal shaft to surround the base of the cilium after exposure to ligand

• Smo protein becomes enriched in primary cilia in response to ligand• Gli transcription factors are found at the tip of the cilium both in the presence and absence of ligand.

This localization to the ciliary tip is thought to be required for the activation and/or processing of the Gli transcription factors

• Sufu, a negative regulator of the pathway, also localizes to the tip of the cilium in both the presence and absence of ligand. Sufu is able to act as a repressor of Hh pathway even in the absence of cilia

IFT proteins regulate the Hh pathway

• Mouse IFT proteins are required for transmission of information from Shh, Ptch and Smo to the Gli transcription factors that mediate activity of the Hh pathway

• For example, double mutant embryos that lack both an IFT-B protein (IFT172 or IFT88) and an upstream signalling component (Shh, Ptch or Smo) have phenotypes very similar to those seen in the IFT mutants

• IFT172, IFT88 and Kif3a are required for processing of Gli3 repressor• IFT172 has been shown to be required for Gli activator function• Intra-flagellar transport is required for transduction of Hh signals

from the membrane proteins Ptch and Smo to the downstream Gli transcription factors