Multiple Wnt Signaling Pathways Converge to Orient

the Mitotic Spindle in Early C. elegans Embryos

Walston T. et al.Developmental Cell, Vol. 7, 831–841, December, 2004

ABpl

The fate of the EMS daughters is controlledby a Wnt/b-cat pathway.

The orientation of the EMS division is controlled by a different Wnt pathway involving Wnt(MOM-2), Porcupine(Porc;MOM-1), Fz(MOM-5), GSK-3(GSK-3b) and CK1(KIN-19).

A pathway involving MES-1, a receptor tyrosine kinase, and SRC-1, a Src family tyrosine kinase, acts redundantly with Wnt signaling with respect to the fate of EMS daughters and the orientation of the EMS spindle.

mom-1 (Porc), mom-2 (Wnt), mom-5 (Fz), and mom-3 (uncloned), cause spindle alignment defects in the ABar blastomere of the 8-cell embryo.

Background

Background

Although many Wnt signaling components have been identified that participate in spin

dle orientation, the role of the Dsh family has not been clearly characterized.

(dsh-1, dsh-2, mig-5)

Loss of function of the CKI homolog, kin-19, causes defects in the fate of EMS daughter

cells. Although the role of CKI in spindle alignment has not been examined, CKI localizes t

o centrosomes and mitotic spindles in vertebrate systems.

The nontranscriptional Wnt spindle alignment pathway requires contact from the C blas

tomere to align the spindle of ABar.

Wnt/b-catenin pathway regulates the timing of spindle rotation in ABar, presumably by

specifying the fate of neighboring blastomeres.

We demonstarate...

dsh-2(or302)

dsh-1(RNAi); dsh-2(or302); mig-5(RNAi)

Defects in Alignment of the EMS and ABar Spindles.

Defects in EMS

KIN-19/CKⅠ localizes to centrosomes and DSH-2 accumulates between P2 and EMS.

microtubule condensed chromosome

~4cell stage 4~6cell stage 6~32cell stage

cytoplasm boundary between P2 and EMS

cortex of all cells

Consistent with P2 signaling to EMS to specify endoderm fate and EMS spindle orientation.

KIN-19 RNAi → does not affect Dsh-2 localization.

Fz

APCAxin

b-catNEMO

TCFRNA pol.

Positive : Dsh, CK , GSK-3, Src (Dsh background)ⅠNegative : JNK, APC, Axin, b-cat, NEMO, TCF, RNA pol.

Spindle defects in ABar.

Contact with the C blastomere aligns the spindle in ABar.

Caudalhomolog

laserkilled

Mom-2(Wnt)

Mom-5(Fz) Mom-5(Fz)

canonical non-canonical

C

EMS ABar

ABar spindle defects visualized by -tubulin::GFP

dsh-2(RNAi); mig-5(RNAi) wrm-1(RNAi)

TBB-2/ -tubulin::GFP

Three Wnt signaling pathways operate in the Early C. elegans embryos.

G signaling in spindle orientation ??

GSK-3 in spindle orientation ??

Discussion

Heterotrimeric G-protein in spindle orientation

G : GPB-1, GPB-2G : GPC-1, GPC-2

Gotta M et al, Nat Cell Biol, 2001.

A P

D

V

ABaABp

P2

EMS

Heterotrimeric G-protein in spindle orientation

C. elegans has 20G genes. → GOA-1, GPA-16

Conclusion : G signaling, not G, participates spindle orientation in C. elegans.

Gotta M et al, Nat Cell Biol, 2001.

Cdc42 regulates GSK-3 and APC to control cell polarity.

There is a larger complex containing GSK-3, Par6, PKC.

Scratch-induced cell migration assay : Cdc42, p-GSK-3-cat and APC localize at the leading edge of migrating cell.

Etienne-Manneville S et al. Nature, 2003.

Astrocyte

MTOC : microtubule organizing centre

Activation of G signaling downstream of Wnt-11/Xfz7 regulates Cdc42 activity.

Penzo-Mendez A et al. Dev Biol, 2003.

During xenopus gastrulation

Cdc42, p-GSK-3-cat, APC

Leading edge

Dvl-1 ?Dvl-2 ?Dvl-3 ?

Src

Canonical Wnt

Fz

Non-canonical Wnt

FzG

Dvl

GSK-3

Axin

APC

-cat

-cat-cat

CKⅠ

-cat

-catTcf/Lef

Dvl

RhoA

MEKK, SEK

JNK

PKC

Cdc42

Par-6PKC

Polarity

Cell fate,spindle rotation

Mes-1