From the Laboratory for Developmental Neurobiology,
RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama
351-0198, Japan and the § Department of Basic Medical
Science, Division of Molecular Neurobiology, Institute of Medical
Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
Received for publication, October 30, 2000, and in revised form, January 11, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Zic and Gli family proteins are
transcription factors that share similar zinc finger domains. Recent
studies indicate that Zic and Gli collaborate in neural and skeletal
development. We provide evidence that the Zic and Gli proteins
physically and functionally interact through their zinc finger domains.
Moreover, Gli proteins were translocated to cell nuclei by coexpressed
Zic proteins, and both proteins regulated each other's transcriptional activity. Our result suggests that the physical interaction between Zic
and Gli is the molecular basis of their antagonistic or synergistic features in developmental contexts and that Zic proteins are potential modulators of the hedgehog-mediated signaling pathway.
Zic and Gli transcription factors share a highly conserved zinc
finger domain and have critical roles in multiple developmental processes. In human, mutations in ZIC2, ZIC3, and
GLI3 genes result in various developmental
abnormalities. ZIC2 results in malformation of the forebrain
(holoprosencephaly), ZIC3 in a disturbance of the left to
right body axis (heterotaxy), and GLI3 in complex anomalies
of the brain and digits (cephalopolysyndactyly syndrome) (1-3).
Studies in other vertebrates indicated that Zic1, Zic2, Zic3, Gli1, Gli2, and Gli3 are involved in multiple aspects of the
neural and skeletal development (4-14). Zic and Gli families are also
critical in invertebrate development as shown by the studies on
their Drosophila homologues, Odd-paired (15) and Cubitus interruptus (Ci) (16).
Although a number of studies suggest the importance of the two zinc
finger protein families, the relationship between them has not been
fully understood. However, recent studies have shown significant
Zic-Gli genetic interaction in neural and skeletal patterning.
Xenopus Zic2 and Gli2 are counter-active in the
patterning of neural tube along the dorsoventral axis (13). On the
other hand, the double mutation of Zic1 and Gli3 showed a synergistic disturbance in the segmentation of the vertebral lamina (17).
Gli proteins bind a consensus nonamer target DNA sequence (GLI-BS) (18)
to which Zic proteins can also bind (19). However, we recently found
that the DNA-binding affinity of the Zic proteins was lower than that
of Gli (20) and that Zic proteins significantly enhance gene expression
but less efficiently in the absence of GLI-BS. When Zic and Gli are
expressed together in cultured cells, they synergistically enhance, or
mutually suppress, GLI-BS-mediated transcription depending on the cell
type (20). Here we show that Zic and Gli proteins physically interact
through their zinc finger domains and regulate each other's
subcellular localization and transcriptional activity.
Plasmids--
To express Flag-tagged and hemagglutinin
(HA)1-tagged proteins, the
relevant sequence was amplified by PCR, verified by DNA sequencing, and
subcloned into pCMVtag2 (Stratagene) and pcDNA3HA (a gift from Dr.
T. Nakajima). Sequences cloned included the full-length mouse
Zic1-(1-447) and mouse Zic2 and Zic3 (19, 21). The deletion series of
Zic1 and GLI3 were also cloned. These deletions were as follows:
Zic1-(1-384), -(1-330), -(1-298), and -(1-447) with deletion
of residues 299-329, Zic1-(1-447) with deletion of 299-359, and
Zic1-(1-447) with deletion of 299-383; GLI3-(1-547), -(18-829), and
-(1-1596) with deletion of 548-624. Flag-tagged full-length human
GLI1 (22) and human GLI3 (3) constructs were as described by Dai
et al. (23). For Flag-tagged mouse Gli2, a Flag epitope tag
was introduced at the amino terminus of mouse Gli2 (24) by PCR, and
Flag-tagged Gli2 was subcloned into pCAGGS (25). An
Shh expression construct was made by inserting a
chick Shh cDNA clone containing the entire open reading
frame2 into pEF-BOS (26). The
CBP-HA expression plasmid was kindly provided by Dr. S. Ishii. To clone
the GST-GLI3 and GST-Zic1 fusion proteins, fragments were PCR-amplified
using primers that introduced BamHI and SalI
sites, the sequences verified, and the fragments cloned into the
BamHI-SalI sites of the pGEX-4T3 vector (Amersham Pharmacia Biotech). The regions cloned in this manner were
GLI3-(1-461), -(400-732), -(705-1140), -(1100-1350), -(1296-1596),
-(400-639), -(400-547), and -(547-639) as well as Zic1-(300-384).
GST fusion proteins were affinity-purified by glutathione-Sepharose 4B
(Amersham Pharmacia Biotech). For bacterial expression of Flag-tagged
GLI3-(461-639), the EcoRI fragment encoding GLI3-(461-639)
was inserted into the EcoRI site of the pFLAG-ATS vector
(Sigma). Bacterially expressed Flag-tagged GLI3-(461-639) protein was
affinity-purified by anti-Flag M2 affinity gel (Sigma).
Immunoprecipitation and GST Pull-down Assays--
293T cells
were transiently cotransfected with appropriate expression constructs
using Superfect or Effectene (Qiagen). After 48 h, cells were
lysed in immunoprecipitation buffer (25 mM Hepes, pH 7.2, 0.5% Nonidet P-40, 150 mM NaCl, 50 mM NaF, 2 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin) at 4 °C. Immunoprecipitation was performed using anti-HA Y-11 polyclonal antibody (Santa Cruz Biotechnology). Bound material was detected by
immunoblotting with anti-Flag M2 monoclonal antiboby (Sigma). For GST
pull-down assays, GST fusion proteins were incubated with bacterially
expressed Flag-tagged GLI3-(461-639) or protein extracts from 293T
cells transfected with different expression constructs in the
immunoprecipitation buffer for 2 h at 4 °C. Bound proteins were
separated by SDS-polyacrylamide gel electrophoresis followed by
immunoblotting using the anti-Flag antibody.
Subcellular Localization Studies--
NIH3T3 cells and 293T
cells were transiently transfected with appropriate expression
constructs. 24 h after transfection, cells were fixed in 4%
paraformaldehyde in 0.1 M sodium phosphate buffer
for 20 min at room temperature and permeabilized with 0.3% Triton X-100 in phosphate-buffered saline for 2 min. The cells were
incubated in blocking buffer (1% bovine serum albumin and 0.1% Triton
X-100 in phosphate-buffered saline) for 1 h at room temperature
and then incubated with the anti-HA antibody and the anti-Flag
antibody. The bound antibodies were detected by Alexa 488-conjugated
anti-rabbit IgG or Alexa 568-conjugated anti-mouse IgG antibodies
(Molecular Probes, Inc.).
Luciferase Assay--
Cells were cultured in 24-well dishes.
NIH3T3 cells were transfected using Superfect with appropriate
expression constructs. At 30 h after transfection, luciferase
activities of the cells were measured as described (20). MNS70 cells
(27, 28) were transfected using FµGENE6 (Roche Molecular
Biochemicals) and assayed 48 h after transfection.
Physical Interaction between Zic and Gli Proteins--
To
investigate the physical interaction between Zic and Gli, 293T cells
were cotransfected with HA-tagged mouse Zic and Flag-tagged human or
mouse Gli, and the resultant cell lysates were immunoprecipitated with
anti-HA antibody and immunoblotted with anti-Flag antibody. Bands
corresponding to GLI1, Gli2, and GLI3 were detected (Fig. 1A).
To identify the Zic1-binding domain in GLI3, we prepared
glutathione S-transferase (GST) fusion proteins containing various parts of the GLI3 proteins (Fig. 1C). The fusion proteins
were used for GST pull-down experiments with the lysates from 293T cells transfected with Zic1, and the precipitates were immunoblotted to
detect Zic1 protein. The minimal region required for binding to Zic1 is
amino acids 547-639 of GLI3 (Fig. 1, D and E), a
region corresponding to the third through fifth zinc finger motifs
(ZF3-5). It is the most conserved region between Zic and Gli proteins
(19). Subsequently, the Gli-binding domain in Zic1 was determined by the GST pull-down assay using a series of deleted Zic1 mutants and
GST-GLI3-(547-639) (ZF3-5) (Fig. 1F). The results revealed that a region (residues 300-384) corresponding to Zic1 ZF3-5
was involved in the Zic-Gli interaction (Fig. 1G). All three
C2H2 zinc finger units in the Zic1 ZF3-5 domain were cooperatively involved in the interaction (Fig. 1G). GST-GLI3-(547-639)
(ZF3-5) bound Zic1, Zic2, and Zic3 proteins (Fig. 1E), and
GST-Zic1-(300-384) (ZF3-5) bound the GLI1, Gli2, and GLI3
proteins (Fig. 1H), suggesting that the association between
the Zic and Gli proteins is conserved in other Zic-Gli combinations.
Binding was also observed between purified GST-Zic1-(300-384) and
bacterially expressed, purified Flag-GLI3-(461-639) (Fig.
1B), suggesting a direct physical interaction between Zic
and Gli.
Translocation of Gli Proteins by Zic Proteins--
Next we
examined the subcellular localization of Zic and GLI proteins.
Transfected HA-tagged Zic1, Zic2, and Zic3 were located in cell nuclei
in all of the cell lines tested (NIH3T3, 293T, C3H10T1/2, COS7) (Fig.
2, A, B, I, and J,
data not shown), whereas the subcellular localization of Flag-tagged
GLI proteins has been found to vary in different contexts (8, 29). In
NIH3T3 and 293T cells, both GLI1 and GLI3 proteins were located
predominantly in the cytoplasm (Fig. 2, C, D, H, K, L, and
P). Coexpression of Zic1 resulted in GLI1 and GLI3 proteins
being translocated to the nucleus in varying levels (Fig. 2,
E and M). This tendency was clearest in the case
of GLI3 in NIH3T3 cells and GLI1 in 293T cells (Fig. 2, H
and P). A mutant GLI3 protein lacking residues 548-624 (GLI3 Zic and Gli Proteins Regulate Each Other's Transcriptional
Activity through the Zinc Finger Domains--
Zic proteins activate
transcription from the thymidine kinase (TK) promoter in a process that
is partially dependent on GLI-BS (20, Fig.
3). However Gli proteins specifically
require GLI-BS for transcriptional regulation and have essentially no
effect on the promoter in the absence of GLI-BS (Ref. 20; Fig.
3). To clarify the significance of the Zic-Gli association in
transcriptional regulation, we performed Zic-Gli cotransfection
experiments using TK promoter-luciferase reporter constructs with and
without GLI-BS (pGBS-TK-luc and pTK-luc, respectively) in NIH3T3 cells.
When GLI1 and Zic1 (Fig. 3A) or GLI1 and Zic2 (Fig.
3B) were cotransfected, reporter gene expression was
synergistically activated both in the presence and absence of GLI-BS.
This synergistic activation was also observed in a Shh-responsive cell
line (MNS70) (Fig. 3C). The level of synergistic increase
was not influenced by the presence of an Shh signal (Fig.
3C), suggesting that the Shh signal does not regulate the
Zic-Gli interaction. By contrast, full-length GLI3 enhanced reporter
gene expression when coexpressed with a general transcription cofactor,
CBP (Ref. 23; Fig. 3D). When Zic1 was coexpressed
with GLI3 and CBP, a marked increase was observed in comparison with
GLI3 and CBP coexpression (Fig. 3D). Conversely, when Zic1
was coexpressed with a carboxyl-terminally truncated GLI3 protein
(Ref. 30; GLI3 18-829 in Fig. 3D), the Zic1-mediated
reporter gene activation was suppressed both in the pGBS-TK-luc and
pTK-luc reporters (Fig. 3D). GLI3
The Zic-Gli associations may be involved in transcriptional regulation
as well as in synergistic or antagonistic effects in different
developmental contexts (13, 17). Synergistic activation can be
explained in part by enhancement of the nuclear localization of Gli
proteins by Zic proteins. In addition, the physical interaction may
contribute to the recruitment of both proteins onto the GLI-BS or core
promoter, resulting in elevation of the local concentrations of both
proteins. On the other hand, if we assume that the protein-to-protein association interferes with the ability of Gli or Zic to regulate transcription, then the presence of Zic would reduce the effect of Gli
and vice versa. This could be the molecular basis of the Zic-Gli
counter-activity. The presumptive molecular machinery of synergistic
activation or inactivation by the Zic-Gli interaction may vary
according to cell type and the developmental context.
Gli/Ci proteins function downstream of the hedgehog (hh)
signaling pathway as both transcriptional activators and repressors (30-33). On the other hand, the signal transduction pathway mediated by Zic proteins has not been well understood. Our results suggest that
Zic proteins can interact with every Gli protein including the
repressive form. We therefore speculate that Zic proteins are a
potential modulator of the hh signaling pathway in various situations
in animal development. Interestingly, expression of the Zic family
grossly overlaps that of GLI3 in neural tube, somites, and limb buds
(17, 34, 35). Further clarification of the role of Zic proteins in the
hh signaling pathway should help clarify the molecular mechanisms of
body pattern control.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (52K):
[in a new window]
Fig. 1.
Interaction of Zic proteins with Gli
proteins. A, Flag-tagged GLI1, Gli2, and GLI3 were
cotransfected with an empty HA-tagged expression vector, HA-Zic1
and HA-Zic2, in 293T cells (top). Cell lysates were
immunoprecipitated and immunoblotted using the antibodies indicated.
Middle and bottom panels, expression
analysis of the Flag- and HA-tagged proteins, respectively, by
immunoblotting of the cell extracts. B, direct binding
assay. Bacterially expressed Flag-tagged GLI3-(461-639) was incubated
with GST-Zic1-(300-384), and a GST pull-down assay was performed.
Bound material was detected by immunoblotting. C-H, mapping
of the binding domains. C, mapping of the Zic-binding region
in GLI3 protein. The various regions of the GLI3 protein indicated were
prepared as GST fusion proteins. The numbers refer to amino
acids. D and E, GST pull-down assays. Extracts
from 293T cells expressing Flag-Zic1 (D) or Flag-Zic
proteins (E) were incubated with the recombinant GST fusion
proteins indicated. The elutions from glutathione-Sepharose beads were
analyzed by immunoblotting. F, mapping of the GLI-binding
region in Zic1 proteins. Expression vectors for Zic1 deletion proteins
tagged with Flag epitope were constructed. The numbers refer
to amino acids. G and H, GST pull-down assays.
Cell lysates containing the indicated Flag-Zic1 deletion proteins
(G) or Flag-Gli proteins (H) were subjected to
GST pull-down assay using the GST fusion proteins indicated.
ZF3-5) was not translocated by the coexpressed Zic1
(Fig. 2H). Enhancement of the nuclear translocation of GLI proteins was also observed with any combinations of Zic-/Gli proteins (data not shown) and in other cell lines (C3H10T1/2, HeLa, and COS7,
data not shown).
View larger version (28K):
[in a new window]
Fig. 2.
Zic proteins promote nuclear
translocation of Gli proteins. A-G and
I-O, subcellular localization of Zic and GLI in NIH3T3
(A-G) and 293T cells (I-O). The cells were
transfected with HA-Zic1 alone (A, B, I, and J),
Flag-GLI3 alone (C and D), Flag-GLI1 alone
(K and L), HA-Zic1/Flag-GLI3 (E-G),
and HA-Zic1/Flag-GLI1 (M-O). Nuclei were counter-stained
with DAPI (4',6-diamidino-2-phenylindole) (blue)
(B, D, G, J, L, and O). Note that GLI3 and GLI1
translocated to the nucleus when coexpressed with Zic1 (E
and M). H and P, summaries of the
subcellular localization of GLI1 and GLI3 proteins in NIH3T3
(H) and 293T cells (P) in the presence and
absence of coexpressed Zic1. Cells were classified depending on the
expression of the GLI protein in the nucleus (N), cytoplasm
(C), or both the nucleus and cytoplasm (N+C). The
numbers of cells counted are indicated by n. The percentages
are shown.
ZF3-5 and a
carboxyl-terminally truncated construct lacking ZF3-5 (GLI3-(1-547)) had no effect on reporter gene expression (Fig. 3D),
suggesting an essential role for the ZF3-5 domain in the
transcriptional interaction between Zic1 and GLI3. These findings
therefore indicate that the Zic proteins enable the GLI proteins to
participate in transcriptional regulation through a
protein-to-protein association.
View larger version (37K):
[in a new window]
Fig. 3.
Zic and Gli regulate each other's
transcriptional activity. A and B,
luciferase activity in NIH3T3 cells transfected with expression vectors
for Flag-GLI1 (100, 150, 200, 250 ng) alone or for Flag-GLI1 (100, 150, 200, 250 ng) and Flag-Zic1 (100 ng) together (A) and for
Flag-GLI1 (150 ng) alone or for Flag-GLI1 (150 ng) and Flag-Zic2 (100 ng) together (B). C, luciferase activity in MNS70
cells transfected with expression vectors for Shh (100 ng), Flag-GLI1
(50 ng), Flag-Zic1 (50 ng), and Flag-Zic2 (50 ng) in the indicated
combinations. A total of 200 ng of reporter plasmid (pGBS-TK-luc,
black; pTK-luc, white) was included in each
transfection experiment. The total amount of DNA was adjusted to 400 ng
with control vector pCMVtag2. D, NIH3T3 cells were
transfected with expression vectors for Flag-Zic1 (50 ng), Flag-GLI3
(350 ng), Flag-GLI3 lacking residues 548-624 (GLI3
ZF3-5, 350 ng),
Flag-carboxyl-terminally truncated GLI3 (GLI3 18-829, 100 ng), Flag-carboxyl-terminally truncated GLI3 lacking ZF3-5 (GLI3
1-547, 350 ng), and CBP-HA (100 ng) in the indicated
combinations. The numbers refer to amino acids. The total
amount of DNA was adjusted to 500 ng with control vector pCMVtag2. The
reporter plasmid with (pGBS-TK-luc, black) and without
(pTK-luc, white) GLI-BS was included in each transfection
experiment. The luciferase gene without the TK promoter was not
activated by either Gli or Zic. The error bars represent
standard deviations. Luciferase activities are indicated as values
relative to those of cells transfected with reporter plasmids and empty
expression vectors.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. B. Vogelstein, H. Sasaki, S. Ishii, P. Dai, H. Akimaru, J. Miyazaki, and T. Nakajima for the plasmids, Dr. M. Nakafuku for MNS70 cells and for helpful advice, and Dr. T. Tamura for helpful advice.
![]() |
FOOTNOTES |
---|
* This work was supported by Special Coordination Funds for Promoting Science and Technology, grants from the Japanese Ministry of Education, Science and Culture, Takeda Science Foundation, the Naito Foundation, Senri Life Science Foundation, and the Japan Society for Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 81-48-467-9745; Fax: 81-48-467-9744; E-mail; jaruga@brain.riken.go.jp.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.C000773200
2 J. Aruga, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HA, hemagglutinin; PCR, polymerase chain reaction; Shh, Sonic hedgehog; GST, glutathione S-transferase; TK, thymidine kinase; luc, luciferase; CBP, CREB (cAMP-response element-binding protein)-binding protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Brown, S. A., Warburton, D., Brown, L. Y., Yu, C. Y., Roeder, E. R., Stengel, R. S., Hennekam, R. C., and Muenke, M. (1998) Nat. Genet. 20, 180-183[CrossRef][Medline] [Order article via Infotrieve] |
2. | Gebbia, M., Ferrero, G. B., Pilia, G., Bassi, M. T., Aylsworth, A. S., Penman-Splitt, M., Bird, L. M., Bamforth, J. S., Burn, J., Schlessinger, D., Nelson, D. L., and Cassey, B. (1997) Nat. Genet. 17, 305-308[Medline] [Order article via Infotrieve] |
3. | Vortkamp, A., Gessler, M., and Grzeschik, K. H. (1991) Nature 352, 539-540[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Aruga, J.,
Minowa, O.,
Yaginuma, H.,
Kuno, J.,
Nagai, T.,
Noda, T.,
and Mikoshiba, K.,.
(1998)
J. Neurosci.
18,
284-293 |
5. |
Nagai, T.,
Aruga, J.,
Minowa, O.,
Sugimoto, T.,
Ohno, Y.,
Noda, T.,
and Mikoshiba, K.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1618-1623 |
6. |
Mo, R.,
Freer, A. M.,
Zinyk, D. L.,
Crackower, M. A.,
Michaud, J.,
Heng, H. H.,
Chik, K. W.,
Shi, X. M.,
Tsui, L. C.,
Cheng, S. H.,
Joyner, A. L.,
and Hui, C. C.
(1997)
Development
124,
113-123 |
7. |
Park, H. L.,
Bai, C.,
Platt, K. A.,
Matise, M. P.,
Beeghly, A.,
Hui, C. C.,
Nakashima, M.,
and Joyner, A. L.
(2000)
Development
127,
1593-1605 |
8. | Ding, Q., Fukami, S-i., Meng, X., Nishizaki, Y., Zhang, X., Sasaki, H., Dlugosz, A., Nakafuku, M., and Hui, C. C. (1999) Curr. Biol. 9, 1119-1122[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Matise, M. P.,
Epstein, D. J.,
Park, H. L.,
Platt, K. A.,
and Joyner, A. L.
(1998)
Development
125,
2759-2770 |
10. | Hui, C. C., and Joyner, A. L. (1993) Nat. Genet. 3, 241-246[Medline] [Order article via Infotrieve] |
11. |
Nakata, K.,
Nagai, T.,
Aruga, J.,
and Mikoshiba, K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11980-11985 |
12. | Nakata, K., Nagai, T., Aruga, J., and Mikoshiba, K. (1998) Mech. Dev. 75, 43-51[CrossRef][Medline] [Order article via Infotrieve] |
13. | Brewster, R., Lee, J., and Ruiz i Altaba, A. (1998) Nature 393, 579-583[CrossRef][Medline] [Order article via Infotrieve] |
14. | Ruiz i Altaba, A. (1999) Trends Genet. 15, 418-425[CrossRef][Medline] [Order article via Infotrieve] |
15. | Benedyk, M. J., Mullen, J. R., and DiNardo, S. (1994) Genes Dev. 8, 105-117[Abstract] |
16. | Orenic, T. V., Slusarski, D. C., Kroll, K. L., and Holmgren, R. A. (1990) Genes Dev. 4, 1053-1067[Abstract] |
17. | Aruga, J., Mizugishi, K., Koseki, H., Imai, K., Balling, R., Noda, T., and Mikoshiba, K. (1999) Mech. Dev. 89, 141-150[CrossRef][Medline] [Order article via Infotrieve] |
18. | Kinzler, K. W., and Vogelstein, B. (1990) Mol. Cell. Biol. 10, 634-642[Medline] [Order article via Infotrieve] |
19. | Aruga, J., Yokota, N., Hashimoto, M., Furuichi, T., Fukuda, M., and Mikoshiba, K.,. (1994) J. Neurochem. 63, 1880-1890[Medline] [Order article via Infotrieve] |
20. |
Mizugishi, K.,
Aruga, J.,
Nakata, K.,
and Mikoshiba, K.
(2001)
J. Biol. Chem.
276,
2180-2188 |
21. |
Aruga, J.,
Nagai, T.,
Tokuyama, T.,
Hayashizaki, Y.,
Okazaki, Y.,
Chapman, V. M.,
and Mikoshiba, K.
(1996)
J. Biol. Chem.
271,
1043-1047 |
22. | Kinzler, K. W., Bigner, S. H., Bigner, D. D., Trent, J. M., Law, M. L., O'Brien, S. J., Wong, A. J., and Vogelstein, B. (1987) Science 236, 70-73[Medline] [Order article via Infotrieve] |
23. |
Dai, P.,
Akimaru, H.,
Tanaka, Y.,
Maekawa, T.,
Nakafuku, M.,
and Ishii, S.
(1999)
J. Biol. Chem.
274,
8143-8152 |
24. |
Sasaki, H.,
Hui, C. C.,
Nakafuku, M.,
and Kondoh, H.
(1997)
Development
124,
1313-1322 |
25. | Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene 108, 193-199[CrossRef][Medline] [Order article via Infotrieve] |
26. | Mizushima, S., and Nagata, S. (1990) Nucleic Acids Res. 18, 5322[Medline] [Order article via Infotrieve] |
27. | Nakafuku, M., and Nakamura, S. (1995) J. Neurosci. Res. 41, 153-168[Medline] [Order article via Infotrieve] |
28. |
Nakagawa, Y.,
Kaneko, T.,
Ogura, T.,
Suzuki, T.,
Torii, M.,
Kaibuchi, K.,
Arai, K.,
Nakamura, S.,
and Nakafuku, M.
(1996)
Development
122,
2449-2464 |
29. | Kogerman, P., Grimm, T., Kogerman, L., Krause, D., Unden, A. B., Sandstedt, B., Toftgard, R., and Zaphiropoulos, P. G. (1999) Nat. Cell Biol. 1, 312-319[CrossRef][Medline] [Order article via Infotrieve] |
30. | Wang, B., Fallon, J. F., and Beachy, P. A. (2000) Cell 100, 423-434[Medline] [Order article via Infotrieve] |
31. | Alexandre, C., Jacinto, A., and Ingham, P. W. (1996) Genes Dev. 10, 2003-2013[Abstract] |
32. | Marigo, V., Johnson, R. L., Vortkamp, A., and Tabin, C. J. (1996) Dev. Biol. 180, 273-283[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Lee, J.,
Platt, K. A.,
Censullo, P.,
and Ruiz i Altaba, A.
(1997)
Development
124,
2537-2552 |
34. | Hui, C. C., Slusarski, D., Platt, K. A., Holmgren, R., and Joyner, A. L. (1994) Dev. Biol. 162, 402-413[CrossRef][Medline] [Order article via Infotrieve] |
35. | Nagai, T., Aruga, J., Takada, S., Gunther, T., Sporle, R., Schughart, K., and Mikoshiba, K. (1997) Dev. Biol. 182, 299-313[CrossRef][Medline] [Order article via Infotrieve] |