Driever WNT-Signaling

• Canonical WNT pathway (ß-Catenin-dependent):

  WNT Ligand -> Frizzled Receptor + LRP5/6 (lipoprotein receptor-related protein) -> Dishevelled Activation -> Inhibits ß-Catenin Destruction Complex (Axin, APC, GSK-3, CK1) -> unphosphorylated ß-Catenin translocates to nucleus -> binds TCF/LEF-1 6 displaces Corepressor Groucho -> Target gene expression

  No WNT: ß-Catenin Destruction complex phosphorylates ß-Catenin-> polyubiquitinated -> Proteasomal degradation -> Co-repressor Groucho inhibit TCF/LEF-1 TFs -> Inhibition of target gene expression

• non canonical pathways:

  • Ca2+ dependent:

    Non Canonical WNTs (e.g. Wnt5a) bind Fz Receptors & Coreceptors (Ror 1/2) -> Activation of Phophoslipase C (PLC) -> converts PIP2 to IP3 & DAG -> Calcium release activates Ca2+ dependent signaling pathways (e.g PKC, CamKII - calmodulin-dependent protein kinase II)

  • Planar cell polarity (PCP): Non-canonical Wnt ligands bind Fz & Coreceptors (Vangl, Celsr) -> activates Dishevelled -> activates Rho/Rac GTPases -> Activation Rho kinase (ROCK) & JNK kinase -> Cytoskeletal Reorganization & Planar cell Polarity establishment

    Prickle negative regulator of Dsh

No Wnt: Co-repressor Groucho bound to TCF TF - Histone deacetylases are recruited -> Removal of Acetyl groups -> Chromatin Closed configuration -> Transcription inhibition

Wnt: ß-catenin accumulates and translocates into the nucleus

-> here it binds to TCF releasing it from Corepressor groucho and the coupled HDAC -> instead histone acetylation occurs by a CBP acyltransferase and when open chromatin structure RNA polymerase can bind and start transcription assisted by a complex with ß-catenin as mediator

Fertilization: Sperm entry at animal pole triggers cytoplasm reorganization = Cortical rotation crucial for dorsal-ventral axis establishment

Dorsalizing factors: at vegetal side of egg upon sterm entry -> upon cortical rotation transported with direction of rotation to the future dorsal pole.

Fast transport on microtubules: GSK-3 Binding protein (GBP) & Dishevelled

Slow transport with cortical rotation of Wnt11

Stabilization ß-Catenin Signaling:

Inhibition ß-Catenin Destruction complex

Wnt signals activate pathway

accumulation and nuclear translocation of ß-catenin

on dorsal side

-> formation of Spemann-MANGOLD Organizer

Positive:

WNT ligands bind Frizzled receptor -> Initial pathway activation step, activation of Dishevelled inhibits ß-Catenin destruction complex leading to ß-catenin accumulation & Wnt target gene activation

Inhibitors of destruction complex components are also positive regulators for WNT signaling

Negative:

WNT ligand inhibitors like FRZB / Dally / Kny

anti WNTs that stabilize ß-catenin destruction complex (AXIN) or GSK-3ß which is the kinase phosphorylating ß-catenin for ubiquitination / degradation

Dickkopf protein that internalize the WNT receptors LRP

E.g Intestinal Crypt

WNTs are produced by niche cells and act as niche factor -> maintains stem cell status & lead to formation of progenitor cells

Wnt signal induce asymmetric cell division-> lineage decisions ->

Cells exposed to Wnt distribute signaling components to Wnt exposed side -> Orients mitotic spindle & centrosomes during division - daughter cell close to Wnt source maintains nuclear ß-Catenin & stem cell gene expression (niche cell) , distal cell loses expression (progenitor cell)

Diffusion regulated easily & specifically

-> Clear Lineage Decision:

β-catenin is present in only one daughter cell, it provides a decisive signal that can determine the fate of that cell, ensuring that only one of the daughter cells continues as a stem cell while the other differentiates

-> local acting signal:

produced by cells in close proximity to the stem cells, allowing precise spatial control of signaling within the niche

-> slow distribution

-> ß-catenin staining with a antibody (immunochemistry)

-> transcriptional reporter (GFP on target genes) for example GFP tagging Axin (big central mediator in the ß-catenin destruction complpex)

-> dishevelled kinase activity reporter (phospho antibody)

-> mRNA analysis

-> proximity ligation assay: visualize protein-protein interactions for example ß-catenin binding to the destruction complex (Fluorescence Resonance Energy Transfer (FRET))

-> cell that can distinguish between two sides

-> spatial asymmetry of cellular components

-> in the case of WNT signaling ß-catenin is transported along the mitotic spindle to only one side of the dividing cell —> polarization / cell fate

PCP = Planar Cell polarity

-> Wnt signaling induces Polarized distribution of protein complexes

WNT receptor cluster at different poles of the cell:

• distal = Frizzled - Dishevelled

• proximal = Strabismus/Vangl - Prickle (Pk)

Inhibition of each other on their side -> polarize the cell by interacting with the cytoskeleton (which is polarized by having a growth direction / physical law) -> directed transport of Frizzled to one side and Strbismus to the other -> activated downstream targets activate Rho/Rac GTPases -> Activation Rho kinase (ROCK) & JNK kinase -> Cytoskeletal Reorganization & Planar cell Polarity establishment

Bsp:

• Cilia Orientaton

• Hair Cells Inner Ear

3 different pathways in literature with distinct outcomes

most accurate to view Wnt signaling as a highly interconnected network rather than strictly separate pathways. Separation for Better understanding

• Shared components (e.g Fz, Dsh) + crosstalk

• same activators just context dependent (Cell type, developmental stage…)

-> PCP important for the upfolding of the neural folds to the neural tube & closure of the neural tube control actomyosin contraction -> mediolateral contraction

-> alteration in cytoskeletal components -> contraction of actomyosin to form a closed tube (Fz -> Dsh -> contraction signal to actomyosin)

-> if not proper signaling function severe developemental defects with flat head, open spine, douple brain …

-> APC important part of ß-Catenin destruction complex backbone

-> Impaired APC -> No ß-catenin & Axin binding

-> colorectal cancer: ß-Catenin binding region & degradation is deleted

Result: Loss of ß-Catenin degradation ability, reduced ß-Catenin binding, Increased Wnt sensitivity

-> permanent proliferation signal out of the stem cell niche -> cancer

Increased Wnt antagonists like DKK involved in can promote neurodegenerative diseases

Disregulation of components of the ß-Catenin destruction complex can lead to various degenerative disorders (E.G GSK3)

Wnts are key synaptic organizers impaired Wnt signaling affects synaptic development

Wnt signaling regulates synaptogenesis through both canonical and non-canonical pathways. Canonical Wnt/β-catenin signaling influences gene expression and synaptic assembly, while non-canonical pathways like PCP and Wnt/Ca²⁺ pathways modulate dendritic development, synaptic strength, and plasticity

• Wnt signals guide incoming axons & induce axon growth by modulating the cytoskeleton

• recruitment of presynaptic proteins -> synaptic bouton formation

• Wnt signals directly to the postsynaptic dendrite stimulate spine morphogenesis, postsynaptic protein recruitment and synaptic strength

• At mature synapses, Wnt signaling regulates synaptic function and maintenance.

-> high regulatory presicion to keep WNT signaling in homeostatic range

• negative feedback

• complex regulation network -> positive and negative regulators (+ WNT ligand, receptor, Dsh / - Dally, FRZB, GSK-3ß, Axin, receptor inhibitors …)