Reciprocal regulation of Wnt and Gpr177/mouse
Wntless is required for embryonic axis formation
Jiang Fu1, Ming Jiang1, Anthony J. Mirando1, Hsiao-Man Ivy Yu, and Wei Hsu2
Department of Biomedical Genetics, Center for Oral Biology, James P Wilmot Cancer Center, University of Rochester Medical Center, 601 Elmwood Avenue,
Box 611, Rochester, NY 14642
Edited by Kathryn V. Anderson, Sloan-Kettering Institute, New York, NY, and approved September 16, 2009 (received for review May 6, 2009)
Members of the Wnt family are secreted glycoproteins that trigger
cellular signals essential for proper development of organisms. Cel-
lular signaling induced by Wnt proteins is involved in diverse devel-
opmental processes and human diseases. Previous studies have
generated an enormous wealth of knowledge on the events in
signal-receiving cells. However, relatively little is known about the
making of Wnt in signal-producing cells. Here, we describe that
formation of embryonic axes. Embryos with deficient Gpr177 exhibit
defects in establishment of the body axis, a phenotype highly remi-
proteins are required for a wide range of developmental processes,
the Wnt3 ablation exhibits the earliest developmental abnormality.
This suggests that the Gpr177-mediated Wnt production cannot be
and LEF/TCF-dependent transcription. This activation alters the cellu-
lar distributions of Gpr177 which binds to Wnt proteins and assists
their sorting and secretion in a feedback regulatory mechanism. Our
findings demonstrate that the loss of Gpr177 affects Wnt production
in the signal-producing cells, leading to alterations of Wnt signaling
in the signal-receiving cells. A reciprocal regulation of Wnt and
Gpr177 is essential for the patterning of the anterior-posterior axis
during mammalian development.
A-P axis ? ?-catenin ? developmental deformities ? primitive streak ?
organisms (1, 2). Aberrant regulation of an evolutionary conserved
cancers and congenital diseases (3, 4). There is no question that
Wnt signaling is intimately involved in human health and disease.
Genetic analysis in mice has revealed the essential role of different
Wnt proteins in a wide range of developmental processes (http://
www.stanford.edu/?rnusse/wntwindow.html). Wnt3 deficiency ap-
pears to cause the earliest abnormality during embryogenesis,
suggesting the importance of Wnt signaling in axis determination
(5). Three body axes develop sequentially to generate embryo
is the first to form. The anterior-posterior (A-P) axis is established
Lastly, the left-right asymmetry is formed, followed by embryo
turning. Wnt signaling is critical for initiation of the embryonic axes in
early development (9–11). Disruptions of key molecules necessary for
Wnt signaling regulation lead to defects in axial patterning (12–15).
Studies in the past have generated an enormous wealth of
knowledge on the events in signal-receiving cells. Before initiating
their effects on the signal-receiving cells, Wnt proteins undergo
proper modification, sorting and secretion processes in the signal-
producing cells (16–19). Recent identification of Wntless (Wls/Evi/
Srt) (20–22), a regulator for Wnt production in Drosophila, has
directed attention to the maturation, sorting and secretion pro-
cesses in signal-producing cells. Given the extensive Wnt family in
whether Wls is essential for Wnt production. This study describes
embers of the Wnt family are secreted glycoproteins which
trigger cellular signals essential for proper development of
Gpr177, the mouse orthologue of Drosophila Wls, required for
embryogenesis. Disruption of Gpr177 disturbs axial patterning, a
phenotype resembling the loss of Wnt3. This disruption not only
affects Wnt production, but also interferes with Wnt signaling. As
a Wnt transcriptional target, Gpr177 is elevated to promote Wnt
production in a positive feedback loop. Our results indicate that a
reciprocal regulation of Wnt and Gpr177 is essential for the
establishment of the mammalian A-P axis.
Gpr177 Is Essential for Mouse Embryogenesis. We investigated how
many Wls exist, and whether Wls regulates the Wnt pathway
essential for mammalian development. By protein sequence
analysis (NCBI HomoloGene) we found that Gpr177, which
shows high percentages of identity with Wls of human (96.1%),
Drosophila (44.9%), and C. elegans (40.9%), is likely the mouse
orthologue. No additional gene product shared significant ho-
mology with fly and human counterparts. We then performed
whole mount RNA in situ hybridization to examine Gpr177
expression in early embryogenesis. Gpr177 was expressed in the
proximal epiblast at the junction between the embryonic and
extraembryonic tissue, and later was more restricted to the
primitive streak and mesoderm extending to the distal tip of the
embryo (Fig. 1 A–D). Its strong presence was found in both
posterior visceral endoderm and epiblast at the prestreak, but
switched to the mesoderm at late-streak (Fig. 1 E–H). The
expression pattern of Gpr177 is reminiscent of Wnt3, which is
required for axial patterning (5, 23).
To determine whether Gpr177 regulates Wnt production and
this regulation is essential for mouse development, we created a
mutant strain Gpr177lacZ, carrying an insertion of ?-geo into the
disrupted the seven transmembrane domain which results in
generation of a fusion transcript (Fig. S1C). PCR analyses
further confirmed that the Gpr177 locus was altered by trans-
gene-mediated mutagenesis (Fig. S1D). The mutant lacking the
carboxyl-terminal region of Gpr177 disabled its function as the
truncation interrupts its subcellular distribution and protein
interaction (see below). Mice heterozygous for Gpr177lacZap-
peared normal and were fertile. We were not able to recover
Gpr177 homozygous (Gpr177-/-) newborns or embryos after
E10.5, suggesting that they died during early embryogenesis.
From E6.5 to E8.5, Gpr177?/? and ?/- embryos consisted of
three germ layers and underwent gastrulation to form organized
structures, including extraembryonic ectoderm, chorion, allan-
tois, head folds, and primitive streak (Fig. 1 I, K, M, N, and T).
Author contributions: J.F., M.J., A.J.M., H.-M.I.Y., and W.H. designed research; J.F., M.J.,
A.J.M., H.-M.I.Y., and W.H. performed research; J.F., M.J., A.J.M., H.-M.I.Y., and W.H.
analyzed data; and A.J.M. and W.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1J.F., M.J., and A.J.M. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
November 3, 2009 ?
vol. 106 ?
However, Gpr177-/- embryos did not exhibit distinct structures
but remained to grow as egg cylinders (Fig. 1 J and O). The
mutants consisted of two layers of tissue, ectoderm and visceral
endoderm. In addition, the mesoderm and primitive streak were
missing and ectoderm was composed of a thick layer of cells.
Later, the ectoderm and visceral endoderm continue to grow
and the excess ectoderm becomes irregular and folded (Fig. 1 L
recognizing its carboxyl terminus, which was deleted in the
mutants. Immunostaining detected a strong presence of Gpr177
in the control mesoderm at E7.5 (Fig. 1 P–R). However, the
Gpr177-positive mesoderm was absent in the mutants (Fig. 1S).
Disruption of Gpr177 Impairs Development of the Body Axis. We next
analyzed Gpr177 mutants for the expression of specific markers
during gastrulation (Fig. 2). BMP4 is expressed in the extraem-
bryonic ectoderm where its signals induce the proximal epiblast
to acquire posterior cell fates and restrict the formation of distal
visceral endoderm (DVE), which are precursors of anterior
visceral endoderm (AVE) to the distal end (7, 24). The expres-
sion of BMP4 apparently is not affected by the mutation,
implying proper positioning of extraembryonic ectoderm during
the proximal-distal (P-D) axis formation (Fig. 2 A and B; n ? 5).
To examine the formation of AVE, we examined the expression
of Cer1 (25), an AVE marker in early to mid-streak stage
embryos and later in the definitive endoderm emanating from
the node (Fig. 2C). In Gpr177-/- embryos, we detected Cer1 in
of Gpr177 in E6.25 (A, E, and F) and E7 (B), and E7.25 (C, D, G, and H) embryos.
The approximate positions of E–H are shown by the dotted line in A and C.
Gross morphological analysis of Gpr177?/? (I and K) and Gpr177-/- (J and L)
embryos at E7.5 (I and J) and E8.5 (K and L). Sections of the Gpr177?/? (M, N,
were analyzed by histology (M–O, T, and U) and immunostaining of Gpr177
(P–S). Arrowheads and arrows indicate the anterior and posterior mesoderm,
ectoplacental cone; NE, neural ectoderm; PS, primitive streak; VE, visceral
100 ?m (E–H and P–S); 500 ?m (K and L); and 200 ?m (M–O, T, and U).]
Disruption of Gpr177 impairs embryogenesis in mice. (A–H) In situ
axis. (A–N and P–T) Molecular marker analysis of control (?/? and ?/-) and
Gpr177 mutant (-/-) littermates at E6.5 (A and B) and E7.0–7.5 (C–N and P–T)
of BMP4 (A and B), Cer1 (C and D), Otx2 (E and F), Hesx1 (G and H), Gsc (I and J),
Axin2 (R–T). The control embryos are shown with the anterior facing to the left.
E6.5 and E7.5 embryos shows the level of Gpr177, Wnt3/3a, and ?-catenin pro-
number represents the relative protein level of Wnt3/3a and ?-catenin between
Gpr177?/?, Gpr177?/-, and Gpr177-/-. (R–T) Axin2GFPmouse strain, expressing
GFP in the Axin2-expressing cells, was used to examine the activation of Wnt/?-
catenin signaling in the Gpr177?/? (R and S) and Gpr177-/- (T) embryos during
gastrulation. AVE, anterior visceral endoderm; DE, definitive endoderm; PS,
primitive streak. [Scale bars, 200 ?m (A and B) and 300 ?m (C–N and P–T).]
Fu et al.PNAS ?
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the visceral endoderm although its expansion into the definitive
endoderm was absent, suggesting that AVE was established
during initial regional patterning (Fig. 2D; n ? 3). However, the
subsequent development of the anterior ectoderm did not occur.
Otx2 is expressed ubiquitously in all germ layers at the early
streak-stage, but maintained only in the anterior region at the
mid-streak stage (Fig. 2E). We detected Otx2 throughout the
entire ectoderm of Gpr177 mutants (Fig. 2F; n ? 4). In addition,
the expression of Hesx1 was not affected in the AVE (Fig. 2 G
and H; n ? 3), but was altered in the forebrain of Gpr177-/-
embryos. The uniform presence of Otx2 reflected an undiffer-
entiated ectoderm rather than expansion of anterior neural cell
fates. These data indicate that the posterior development of the
embryo might also be defective. Gsc was expressed in the node
adjacent to the anterior region of primitive streak and the newly
formed axial mesoderm (Fig. 2I). At the late-streak stage, the
located at the anterior end of the primitive streak, possess the
28). However, Gsc expression was abolished in the mutant, sug-
gesting the lack of gastrulation organizer activity in the Gpr177
mutant (Fig. 2J; n ? 3). Furthermore, mesoderm specification
requires T (Brachyury), which is expressed in the primitive streak
and axial mesoderm (Fig. 2K). Its expression in the most anterior
streak region is dependent on Wnt signaling (29). At E7.5, we did
not detect its presence in the mutant, confirming the lack of
primitive streak and mesoderm formation (Fig. 2L; n ? 3).
Gpr177 Deficiency Alters Wnt Production and Signaling. The above
phenotypes are highly reminiscent of the ablation of Wnt3, which
is required for establishment of the primitive streak (5). We
therefore examined the expression of Wnt3 whose transcripts were
found in the primitive streak, proximal epiblast, and visceral
endoderm, at the junction between the embryonic and extraem-
bryonic ectoderm with higher levels localized to the posterior
5). Although the expression pattern of Wnt3 transcript does not
be affected by Gpr177 deficiency. We therefore determined
whether Gpr177 is essential for Wnt3 production and signaling in
early embryogenesis. Previous reports indicated that ?-catenin
levels of ?-catenin were drastically reduced by the mutation (Fig.
2O). Using an antibody recognizing Wnt3/3a, the protein level was
Wnt3a). Note that the mutants were fairly normal at the prestreak
where Wnt3a is not present. Because of technical limitation, we
developing embryo. Nonetheless, the results suggest that Wnt
expression was not affected in the embryos. However, ?-catenin
was not activated likely due to a secretion defect. Furthermore,
tion, and has been widely used as a downstream target of Wnt (32,
33). Its expression in the posterior region of the E7.5 embryo (Fig.
2P) appeared to be missing in the mutant (Fig. 2Q; n ? 2). Using
the Axin2GFPmouse strain to label the Axin2-expressing cells, we
detected GFP signals in the Gpr177?/? but not Gpr177-/- embryos
during gastrulation, indicating that the canonical Wnt pathway is
Gpr177 acts downstream of Wnt3 and regulates its signaling in early
patterning of the A-P axis. In addition to Wnt production, Gpr177
is required for Wnt signaling during embryonic axis formation.
Subcellular Distribution of Gpr177. To determine the role of Gpr177
in production of Wnt, we examined its localization within the cell.
First, immunostaining showed that endogenous Gpr177 is mainly
localized to cellular vesicles in the majority of cell types (n ? 10)
Cut view (E–I) and 3-D imaging (J–O) analyses show that Gpr177 co-localizes
coloring of the co-localization signal in white (I and M–O). NEP cells were
immunostained with Gpr177 (E and J) and GM130 (F and K), and counter-
stained with DAPI (G–O). Three serial sections along the front-view (Co Level
1, 2, and 3) reveal the co-localization signal on the surface of Golgi (M–O).
(P–T) High levels of Gpr177 lead to its accumulations in Golgi. Three-
dimensional imaging of the endogenous Gpr177 in the vesicles of C3H10T1/2
cells (P). Expression of a myc-tag full length Gpr177 (MT-Gpr177) shows
alteration in subcellular distribution of Gpr177 (Q), co-localizing with GM130
(R–T). Co-immunostaining of a MT-Gpr177 mutant lacking the carboxyl ter-
minal region (U) and Calnexin (V) reveals their co-localization (W).
Wnt regulates cellular distribution of Gpr177. (A–D) Three-
www.pnas.org?cgi?doi?10.1073?pnas.0904894106Fu et al.
examined with an exception of neural stem cells (Fig. 3 A–D). In
from E12.5 neural tube and forebrain, respectively, Gpr177 was
highly concentrated in the perinuclear area resembling Golgi in
addition to the vesicles (Fig. 3 A and B). Consistent with a previous
study of Wls in Drosophila (34), the endogenous Gpr177 was
co-localized with a Golgi marker GM130 (Fig. 3 E–I) in cut view
analysis. Using a combined 3-D imaging and co-localization anal-
ysis, Gpr177 was also co-localized with GM130 on Golgi (Fig. 3
J–L). In serial sectioned views of the 3-D images (Fig. 3 M–O), the
co-localization signal (white color) appeared to be on the Golgi
apparatus between the Gpr177-positive vesicles (green color) and
Golgi (red color). The budding of the Gpr177 containing vesicles
seems to occur on the surface of Golgi and continue their intra-
cellular trafficking routes.
To determine whether the differential localization of Gpr177 is
Gpr177 (MT-Gpr177) protein in a variety of cell types (n ? 6). In
293T, mouse embryonic fibroblasts, and mouse mesenchymal stem
cells, MT-Gpr177 displayed localization to Golgi in addition to
cellular vesicles (Fig. 3 P–T). Furthermore, a MT-Gpr177 mutant,
lacking the carboxyl-terminal region similar to the Gpr177-lacZ
fusion, exhibited a dislocated distribution pattern. Co-localization
of an ER marker Calnexin with this mutant suggests that it fails to
enter the secretory pathway (Fig. 3 U–W). The results suggest that
The expression level of Gpr177 might dictate its cellular distribu-
tion. It is conceivable that neural stem cells, expressing high levels
of Gpr177, are the only Wnt-secreting cell type we have examined.
because of its role in assisting the production of Wnt proteins.
Gpr177 Is a Transcriptional Target of Wnt/?-catenin Signaling.To test
whether expression of Wnt might alter the cellular level and
distribution of Gpr177, we studied the transcriptional regulation
of Gpr177. We isolated and characterized the Gpr177 regulatory
region which contains 7 potential LEF/TCF binding sites to
determine whether the Gpr177 transcription is regulated by Wnt
(Fig. 4A). The results indicated that Gpr177 transcription is
stimulated by the dominant-activated mutant, ?N?-cat or cat-
CLEF1 (Fig. 4B). Deletion analysis of the Gpr177 regulatory
region further showed that four potential LEF/TCF-binding sites
(nos. 4–7) might be responsible for the transcriptional stimula-
tion (Fig. 4C). Chromatin immunoprecipitation (ChIP) analysis
demonstrated that the expressed catCLEF1 bound to these four
LEF/TCF consensus sites in cells (Fig. 4 D and E).
Next, we determined which of these four sites is most critical
for the activation of Gpr177 by the ?-catenin and LEF/TCF-
dependent transcription. Because the sites 4 and 5, as well as 6
and 7, are closely linked to each other, a point mutation strategy
(Fig. 4A) was used to disrupt each of these four sites (M4, M5,
M6, and M7). Disruption of number 4 or 7 site, but not 5 or 6,
significantly diminished the transcriptional stimulation (Fig. 4F).
mutation (cross) of LEF/TCF binding consensus sequences. (B) Expression of dominant activated catCLEF1 or ??-cat protein stimulates the transcription activity
of a 3-kb Gpr177 promoter (P1) in 293T cells. Relative luciferase activity (RLA) determined the transcriptional activation of the Gpr177 promoter-luciferase
construct. The analysis of pGL3, a parent vector, shows background activity. (C) Fold of induction shows the effect of catCLEF1 on transcriptional activation of
the deletion mutants. (D) 293T cells were transfected by increasing amounts of DNA plasmid (1, 3, and 9 ?g) to express catCLEF1, analyzed by immunoblot (IB).
(E) ChIP analysis reveals high affinity LEF/TCF binding sites (nos. 4–5 and 6–7) in the Gpr177 promoter. The order number of these sites is color coded with those
shown in A. NC is a negative control, which analyzes the regulatory region of Gpr177 without LEF/TCF binding sequence. The controls are direct PCR analyses
and LEF/TCF-dependent transcription. (G) IB analysis indicates the Gpr177 level elevated in the primary MEC cells by the MMTV-Wnt1 transgene. The expression
level of Actin was analyzed as a loading control. (H–K) ?-gal staining of the virgin 2-month mammary glands in whole mounts (H and I) and sections (J and K)
reveals the Wnt-dependent activation of Gpr177 in the mammary glands. The reporter expression from the Gpr177-lacZ knock-in allele was detected in the
MMTV-Wnt1 transgenics (I and K) but not the controls (H and J). Three-dimensional imaging of the immunostained control (L) and MMTV-Wnt1 transgenic (M)
MEC reveals distinct localization patterns of endogenous Gpr177. The endogenous Gpr177 distribution in C3H10T1/2 cells (N) is also altered by high levels of
HA-Wnt3A (O). The insets show co-immunostaining of Gpr177 with Golgi markers, GM130 (M) and GS28 (O). Immunostained cells were counterstained by DAPI
(blue). G, Golgi; V, vesicle. [Scale bars, 500 ?m (H and I) and 50 ?m (J and K).]
Gpr177 is regulated by the canonical Wnt pathway. (A) Graphs illustrate the luciferase reporter constructs for Gpr177 promoter with the wild-type or
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A combinatorial mutation of number 4 and 5 (M45) or number
6 and 7 (M67) caused a reduction similar to the effect of M4 or
M7 (Fig. 4F). However, the loss of both number 4 and 7 sites
(M47), but not number 5 and 6 sites (M56) drastically abolished
the ?-catenin and LEF/TCF-dependent transcription (Fig. 4F).
The transcriptional activity of M47 is about the same as M4567
where all four sites are disrupted (Fig. 4F). The results strongly
suggest that Gpr177 is a direct Wnt downstream target whose
expression is regulated by the ?-catenin and LEF/TCF-
Wnt Expression Alters the Cellular Level and Distribution of Gpr177.
We examined whether the Gpr177 expression is stimulated by
Wnt that might provide a feedback mechanism to regulate its
production and signaling. Indeed, immunoblot analysis showed
that the steady state level of the endogenous Gpr177 increased
compared to the controls (Fig. 4G). To test the stimulation of
Gpr177 by Wnt in animals, we crossed the MMTV-Wnt1 trans-
gene into mice carrying the Gpr177lacZallele. This allele, which
contains the ?-geo reporter controlled by the Gpr177 locus,
permits an examination of its endogenous expression activity. In
virgin mammary glands heterozygous for the Gpr177lacZallele,
no ?-gal staining could be detected (Fig. 4 H and J). In contrast,
the MMTV-Wnt1 transgenic littermates showed strong ?-gal
stained signals, suggesting that the Gpr177 expression is stimu-
lated (Fig. 4 I and K). Next, the cellular localization of Gpr177
affected by Wnt was analyzed to further our investigation on
their interactions. We examined primary MEC cells isolated
from MMTV-Wnt1 transgenic females and their control litter-
mates. Compared to the controls, Gpr177 was located to Golgi
in addition to cellular vesicles in the MMTV-Wnt1 cells (Fig. 4
L and M). Furthermore, transient expression of HA-Wnt3A in
C3H10T1/2 cells also led to an accumulation of endogenous
Gpr177 in the Golgi (Fig. 4 N and O). These data support the
hypothesis that Wnt proteins modulate the cellular level and the
expression of Gpr177. As a direct target of Wnt, Gpr177 might
facilitate their maturation, sorting, and secretion processes in a
feedback regulatory loop.
Interactions of Gpr177 and Wnt Proteins.To investigate whether the
Wnt-dependent elevation of Gpr177 assists the Wnt production
through a feedback regulatory mechanism, we examined their
subcellular localizations. Immunostaining showed that the ele-
vated Gpr177, caused by expression of Wnt1 (Fig. 5 A–D),
became co-localized together in the Golgi and vesicles. Co-
immunoprecipitation further identified complexes containing
assay indicated that Gpr177 associates with Wnt1, Wnt3, and
Wnt5a proteins, which are highly expressed in neural stem cells
(Fig. 5H). This association was not detectable in other cell types
without high levels of Wnt expression. Analysis of the GST-
Gpr177 deletion mutants (Fig. 5G) further revealed that a short
N-terminal domain (amino acids 101–232) is required for Wnt
proteins to associate with Gpr177 (Fig. 5H). Thus, the Wnt-
mediated elevation of Gpr177 interacts with Wnt proteins in a
feedback loop. Although it remains possible that Gpr177 does
not bind directly to Wnt, they appear to regulate each other in
a reciprocal manner.
This study demonstrated an essential role of Gpr177, the mouse
orthologue of Drosophila Wls, in establishment of the A-P axis.
Genetic analysis in mice has revealed the requirement of many
different Wnt proteins in a wide range of developmental processes.
abnormality. The Gpr177 mutation causes defects in the primitive
(5). This suggests an indispensible role of Gpr177 in Wnt sorting
and secretion. The Gpr177-mediated Wnt production is essential
for mammalian development.
Golgi accumulations of endogenous Gpr177 occur in neural
stem cells. This is in agreement with previous analyses of
exogenous Wls, over-expressed in transfected cells (20, 35–37).
rather the expression level within the cells. Indeed, high levels of
Wnt proteins are found in the neural stem cells where their
association with Gpr177 can be detected. The results imply that
Wnt-producing cells. Indeed, cells expressing Wnt exhibit ele-
vated levels of Gpr177, leading to Golgi accumulation. Wnt
expression might modulate the distribution of Gpr177 that
dictates the trafficking routes in signal-producing cells. This
to be activated for the secretion of Wnt proteins (34–38). In
contrast, the main role of Gpr177 expressed at low levels in non
Wnt-producing cells could help in generating a morphogen
gradient for long range effects through endocytosis and exocy-
tosis. Further analysis on the trafficking routes of Gpr177-
containing vesicles will elucidate the mechanism underlying the
process of Wnt maturation, sorting and secretion.
We hypothesize that a reciprocal interaction between Wnt and
Gpr177 plays a key role in the regulation of their expression,
subcellular distribution, binding, and organelle-specific association.
The mechanisms underlying the reciprocal regulation in the Wnt-
requirement of Wnt signaling in development of various organs
suggests that Gpr177 might be required for these developmental
processes (1, 2). Indeed, we have found that Gpr177 is expressed in
dimensional imaging analysis of immunostained C57MG cells expressing MT-
Wnt1 reveals co-localization (C) of endogenous Gpr177 (A) with MT-Wnt1
the sectioned level is shown by purple rectangle. (E and F) IP-IB analysis
identifies protein complexes containing Gpr177 and Wnt in cells transfected
by HA-Wnt1 (E) or MT-Gpr177 (F). (G) A scheme of GST-Gpr177 fragments.
Numbers indicate positions of amino acids. Endoplasmic reticulum signal
sequence and transmembrane domains are highlighted in green and blue,
respectively. (H) GST pull-down assay analyzes the association of Gpr177 with
the Wnt1, Wnt3, or Wnt5a protein complex.
Wnt proteins bind to and co-localized with Gpr177. Three-
www.pnas.org?cgi?doi?10.1073?pnas.0904894106 Fu et al.
As Wnt signaling is intimately involved in a variety of cancers and Download full-text
for these processes. Creation of mouse strains permitting genetic
inactivation of Gpr177 in a spatiotemporal-specific fashion prom-
ises important insights into the reciprocal regulation of Wnt and
Gpr177 in development and disease. As there are 19 different
members of Wnt in mammals, it is not known whether Gpr177 is
in cells expressing a specific Wnt is likely to gain knowledge on the
generality of its function.
Details for experimental materials and analyses are described in the SI Meth-
ods. In brief, the Gpr177 mutant strain was generated using an ES clone (Bay
Genomics). For embryo genotyping, yolk sacs or embryonic materials recov-
ered from paraffin sections were used in PCR analysis. Axin2GFPstrain permits
of experimental animals described in this work comply with guidelines and
policies of the University Committee on Animal Resources at the University of
Rochester. Isolation and culture of primary neurospheres, NEP, MEC, calvarial
MSC, and other cell lines are described in the SI Methods (41–44). RNA probes
(5, 25) were generated to analyze the gene expression pattern by in situ
hybridization (45). Histology, ?-gal staining, immunostaining, immunoblot,
protein precipitation, chromatin immunoprecipitation, and various DNA vec-
tors used are described in the SI Methods (39, 40, 42, 45–47).
Brigid Hogan for reagents, C.-S. Victor Lin and Chris Proschel for technical
assistance, and the reviewers for comments and suggestions. This work was
supported by National Institutes of Health Grants DE15654 and CA106308
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no. 44 ?