Cleavage of the Wnt Receptor Ryk
Regulates Neuronal Differentiation
during Cortical Neurogenesis
Jungmook Lyu,1Vicky Yamamoto,1and Wange Lu1,*
1Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Department of Biochemistry and Molecular Biology,
Keck School of Medicine, University of Southern California, Los Angeles, CA 90033
Ryk is a transmembrane receptor tyrosine kinase
(RTK). It functions as a receptor of Wnt proteins re-
quired for cell-fate determination, axon guidance,
and neurite outgrowth in different organisms; how-
ever, the molecular mechanism of Ryk signaling is
unknown. Here, we show that Ryk is cleaved, permit-
ting the intracellular C-terminal fragment of Ryk to
translocate to the nucleus in response to Wnt3 stim-
ulation. We also show that the cleaved intracellular
domain of Ryk is required for Wnt3-induced neuronal
differentiation in vitro and in vivo. These results dem-
onstrate an unexpected mechanism of signal trans-
duction for Ryk as a Wnt receptor, in which the intra-
cellular domain itself functions as the transducing
molecule to bring extracellular signals from the cell
surface into the nucleus, to regulate neural progeni-
tor cell differentiation.
The embryonic neocortex contains multipotent neural stem and
progenitor cells in the ventricular zone (VZ). During cortical de-
velopment, neural progenitor cells (NPCs) can self-renew, prolif-
erate, and differentiate into astrocytes, oligodendrocytes, and
neurons (Temple, 2001). Immature neurons generated from
NPCs undergo radial migration out of the VZ and form the corti-
cal plate (CP). The immature neurons further undergo maturation
side-out’’ manner, and eventually establish the six distinct neo-
cortical layers (Olson and Walsh, 2002; Kriegstein et al., 2006).
factors and extrinsic cell signaling. Wnt signaling is among the
cell signaling events that play an important role in regulating
the growth and differentiation of NPCs. However, the molecular
mechanisms remain poorly characterized.
Several of the 19 wnt genes identified in mammals are ex-
pressed in the developing cortex (Roelink et al., 1990; Parr et al.,
1993; Grove et al., 1998) and play critical roles in the proliferation
and neuronal differentiation of NPCs (Hirabayashi et al., 2004;
Israsena et al., 2004). wnt genes encode secreted glycoproteins
that activate intracellular signaling, including the b-catenin/TCF
pathway, the planar cell polarity pathway, and the Wnt/Ca2+
pathway. Signaling through the canonical b-catenin/TCF path-
way is mediated by the Frizzled (Fz) receptor and low-density
lipoprotein receptor-related protein(Lrp)families (He etal.,2004;
Kuhl et al., 2000).
Recently, many studies have elucidated the role(s) of Ryk as
a receptor for Wnt ligands (Yoshikawa et al., 2003). Ryk receptor
is required for the neurite outgrowth of dorsal root ganglion neu-
rons induced by Wnt3a (Lu et al., 2004), for the inhibition of ex-
tension of cortical axons by Wnt5a (Keeble et al., 2006), and
for repulsive axon guidance by Wnt3 (Schmitt et al., 2006). Ryk
is an atypical member of the receptor tyrosine kinase (RTK)
family (Halford and Stacker, 2001). The RTK family consists of
59 cell-surface receptors with similar structure and functional
characteristics: a ligand-binding extracellular domain, a trans-
tyrosine kinase activity. Their signaling, upon ligand binding, is
mediated by specific kinase-dependent cascades (Schles-
singer, 2000). The structure of Ryk, with a glycosylated extracel-
lular domain and an intracellular kinase domain, is consistent
with members of the RTK family. However, unlike other RTK
members, the intracellular kinase domain of Ryk contains highly
unusual subdomains and lacks tyrosine kinase activity (Katso
et al., 1999; Hovens et al., 1992), thus leaving unresolved the
mechanism whereby Ryk protein transduces signals from the
cell surface to the nucleus in response to Wnt stimulation.
Here, we report an unexpected molecular mechanism of Ryk-
mediated Wnt signaling involved in the neuronal differentiation
RESULTS AND DISCUSSION
Ryk Protein Is Cleaved, and Ryk ICD Translocates
into the Nucleus during Neuronal Differentiation
embryonic day 14.5 (E14.5) mouse embryos to investigate Ryk’s
function in vivo. Immunostaining of the immature neuronal
marker TUJ1 (bIII-tubulin) (Alexander et al., 1991) showed that
the number of TUJ1-positive cells in the CP, which are the newly
generated neurons derived from NPCs in the VZ (McConnell
et al., 1989; Kriegstein et al., 2006), is reduced in Ryk?/?fore-
brain compared to that of wild-type (Figure 1A; see Figure S1A
available online). Reduction in the number of neurons can be
caused by a defect in NPC proliferation (Heins et al., 2002) or
Developmental Cell 15, 773–780, November 11, 2008 ª2008 Elsevier Inc. 773
Within each nuclear area, fluorescent intensity of the secondary antibody for
anti-Ryk antibody was measured by using Axio Imager software. A minimum
of 100 cells randomly chosen from TUJ1-positive cells at the CP and Nestin-
positive cells at the VZ, or from differentiated cells (TUJ1-positive cells) cul-
tured in vitro, were examined. The values obtained from at least three samples
were averaged and presented as means ± SD.
Generation of Ryk Antibody
To detect the Ryk C-terminal fragment (CTF), peptides corresponding to res-
idues 314–562 of mouse Ryk in the form of a GST fusion were immunized in
rabbits, and the collected antiserum was affinity purified. The specificity to
the C terminus was confirmed by western blot analysis with lysates from
both wild-type and Ryk knockout mouse brain, NPCs, embryonic stem cells,
and HEK293T cells transfected with Ryk plasmids (Figure S9).
Supplemental References, and nine figures are available at http://www.
We thank Michael Stallcup and Martin Pera for critically reading this manu-
script. We are grateful to Steven Stacker for providing the Ryk knockout
mouse. This work was partially supported by pilot grant IRG-58-007048
from the American Cancer Society. J.L. was supported by an award from
the Korea Research Foundation (KRF-2006-214-C00076) and by a fellowship
from the California Institute of Regenerative Medicine (CIRM). V.Y. was
supported by a fellowship from the CIRM.
Received: May 6, 2008
Revised: September 10, 2008
Accepted: October 8, 2008
Published: November 10, 2008
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780 Developmental Cell 15, 773–780, November 11, 2008 ª2008 Elsevier Inc.