Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8498-8502, August 1995 Developmental Biology

Role of glycogen synthase kinase 3p3 as a negative regulator of dorsoventral axis formation in Xenopus embryos (Wnt signaling/neural induction/dorsoventral polarity) ISABEL DOMINGUEZ*, KEIJI ITOH*, AND SERGEI Y. SOKOL* Department of Microbiology and Molecular Genetics, Harvard Medical School, and Molecular Medicine Unit, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215 Communicated by Tom Maniatis, Harvard University, Cambridge, MA, May 23, 1995 (received for review March 6, 1995)

ABSTRACT The dorsoventral axis is established early in kinase, has been shown to inhibit en expression (14). Shaggy Xenopus development and may involve signaling by Wnts, a has also been shown to suppress the stimulatory effect of Wg family of Wntl-protooncogene-related . The on the accumulation of Arm and to function downstream of kinase shaggy functions in the wingless/Wnt signaling path- Dsh (14). These observations suggest that shaggy is another way, which operates during Drosophila development. To assess critical regulatory component of the Wg signaling pathway. the role of a closely related kinase, glycogen synthase kinase Homologs of shaggy have been isolated from many other 3,B (GSK-313), in vertebrate embryogenesis, we cloned a cDNA organisms, including mammals, yeast, and slime mold (15). encoding a Xenopus homolog of GSK-3,8 (XGSK-313). XGSK- The Dictyostelium homolog of shaggy appears to control cell 318-specific transcripts were detected by Northern analysis in fate determination in this slime mold (16). A mammalian Xenopus eggs and early embryos. Microinjection of the mRNA homolog of shaggy, glycogen synthase kinase (GSK-3p3), was encoding a catalytically inactive form of rat GSK-313 into a originally identified as a serine/threonine kinase regulating ventrovegetal blastomere of eight-cell embryos caused ectopic glycogen metabolism (15), but its function in vertebrate em- formation of a secondary body axis containing a complete set bryogenesis has not been studied. of dorsal and anterior structures. Furthermore, in isolated To assess a possible role for GSK-3,B in dorsoventral axis ectodermal explants, the mutant GSK-3j8 mRNA activated the formation, we cloned a Xenopus homolog of GSK-3,B/shaggy expression of neural tissue markers. Wild-type XGSK-318 (XGSK-313)t and examined its expression pattern in early mRNA suppressed the dorsalizing effects of both the mutated embryos. We also constructed several mutated forms of rat GSK-3,B and Xenopus dishevelled, a proposed upstream sig- GSK-3,3 and studied effects of their overexpression inXenopus naling component of the same pathway. These results strongly embryos. Since shaggy is a presumed negative regulator of the suggest that XGSK-3.3 functions to inhibit dorsoventral axis Wg pathway in Drosophila, we speculated that a dominant formation in the embryo and provide evidence for conserva- negative mutant of GSK-3P may mimic the effect of ectopic tion of the in Drosophila and verte- Wnt expression on embryonic axis formation. Consistent with brates. these expectations, microinjection of an enzymatically inactive form of rat GSK-3f3 carrying a point mutation in the ATP- Dorsoventral patterning of embryonic mesoderm and neural binding site triggered an ectopic body axis formation in early induction are the primary events underlying vertebrate pattern embryos. This effect could be suppressed by the wild-type formation (1). In amphibian embryos, the dorsoventral axis is XGSK-3,3. Our data strongly indicate that the endogenous established soon after fertilization (2, 3). The existence of GSK-313 is a negative regulator of dorsal development in cytoplasmic substances responsible for development of dorsal Xenopus. Interestingly, the same mutant form of GSK-3,B structures is supported by the finding that microinjection of caused neuralization of ectodermal cells, suggesting that dorsal cytoplasm isolated from Xenopus eggs into ventral GSK-313 may also be involved in repression of neural tissue blastomeres causes them to adopt dorsal fates (4, 5). Several formation in the embryo. members of the Wnt family of developmental regulators (6, 7) were demonstrated to trigger dorsal development upon over- expression in ventral blastomeres, thereby mimicking the MATERIALS AND METHODS effect of dorsal cytoplasm (8, 9). These observations suggest DNA Constructs. Te isolate a Xenopus homolog of GSK- that Wnt signaling pathways may operate during dorsoventral 3,3/shaggy we designed degenerate oligonucleotide primers axis determination. Other molecules, including noggin (10), based on the similarity between the rat GSK3,B and shaggy goosecoid (11), chordin (12), and siamois (13), are also capable sequences: 5'-GCGGATTCTAYACNGAYACNAARGT-3' of triggering secondary body axis formation, but it remains to and 5'-CCGAATTCNGTRTARTTNGGRTTCAT-3'. PCR be demonstrated which of these factors are the crucial regu- conditions were as follows: denaturation at 93°C for 1 min; lators of early axial development. annealing at 50°C for the first 3 cycles and 55°C for the Little is known about the biochemical pathway by which a following 32 cycles for 2 min; extension at 72°C for 1 min. The Wnt signal is transmitted in embryonic cells. Based on the sequence of the 0.7-kb PCR product revealed substantial similarity of the mutant phenotypes, Drosophila melanogaster similarity to a portion of rat GSK-3f3. This XGSK-3,B fragment segment polarity genes wingless (wg, Wntl homolog), dishev- was used as a probe to screen a Xenopus laevis neurula cDNA elled (dsh), and armadillo (arm) appear to be involved in the library (a gift of R. Harland) by using standard techniques same signaling pathway (see ref. 14 for review). Whereas all (17). This screening resulted in the isolation of severalXenopus three gene products positively regulate transcription of the cDNA inserts which the for a target gene engrailed (en), the maternal product of shaggy [sgg; GSK-3,3 provided sequence synonym zeste-white 3 (zw3)], a serine/threonine protein Abbreviations: GSK-3/3, glycogen synthase kinase 313; XGSK-3,3, Xenopus GSK-3fB; NCAM, neural cell adhesion molecule. The publication costs of this article were defrayed in part by page charge *I.D., K.I., and S.Y.S. contributed equally to this work. payment. This article must therefore be hereby marked "advertisement" in tThe sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U31862). 8498 Downloaded by guest on September 30, 2021 Developmental Biology: Dominguez et aL Proc. Natl. Acad. Sci. USA 92 (1995) 8499 second set of PCR primers. A cDNA encoding a complete Capped synthetic mRNAs for microinjections were synthe- open reading frame of XGSK-3,3 was amplified from Xenopus sized in vitro by using T7 or SP6 RNA polymerase (18) and gastrula RNA by using Moloney murine leukemia virus reverse templates encoding the following proteins: XGSK-3,B, rat transcriptase (BRL) and Vent DNA polymerase (New En- GSK-3,3 constructs, Xdsh (24), and noggin (10). gland Biolabs) with the oligonucleotide primers 5'- ATGGTACCGGATCCATCATGTCGGGAAGGC-3' and 5 '-CCAGATCTAAGCTTGTTGGCTCAGGA-3' and in- RESULTS serted in-frame into the pXT7 vector, which encodes an Cloning and Characterization of XGSK-3f8. To address the N-terminal Myc epitope. pXT7 is a derivative of pGEM4Z role of GSK-3,3 in embryonic axis formation we first cloned a (Promega) and pSP64T (18) possessing several convenient Xenopus homolog of GSK-3j3 (see Materials and Methods). The cloning sites and allowing efficient in vitro RNA synthesis with deduced amino acid sequence of XGSK-33 is 77% identical to T7 RNA polymerase (R. Freeman and S.Y.S., unpublished shaggy and 94% identical to rat GSK-3f3 (Fig. 1), reflecting the work). in To substitute arginine for lysine-85 in rat GSK-3/3 cDNA high degree of conservation of GSK-313 evolution (15). (K85R mutant), PCR overlap extension (19) was carried out on If XGSK-3f3 is involved in early determination of the the rat GSK-3,B-XT7 plasmid (a gift of B. Neel, Harvard dorsoventral axis, it must be present maternally. Northern Medical School) with the following two sets of primers: (i) analysis revealed that eggs and early embryos contain two 5'-GTGGCCATCAGGAAAGTTCT-3' and 5'-CTTCCAGT- major XGSK-313 transcripts (approximately 2 and 5 kb) (Fig. GGTGTTAGCC-3' and (ii) 5'-AGAACTTTCCTGATGGC- 24), which are uniformly distributed in stage 10 gastrulae (Fig. CAC-3' and the T7 primer (Stratagene). The K85R PCR 2B). These observations are consistent with the possibility that product was digested with Kpn I andXho I and ligated with two XGSK-3, is involved in early embryogenesis. fragments from the parent GSK-313-XT7 plasmid digested with A Dominant Negative GSK-3,B Mutant Triggers Complete Xho I/EcoRI and with Kpn I/EcoRI. Mutagenesis was verified Secondary Axis Formation in Xenopus Embryos. To assess the by sequencing. role of GSK-3,B in early development, we made several mu- Embryo and Explant Manipulation. In vitro fertilization and tated forms of rat GSK-3f3 (prior to our cloning of XGSK-3,B) embryo culture were carried out in 0.1 x MMR as described and introduced them into early embryos, hoping to interfere (20). Staging was according to Nieuwkoop and Faber (21). For with endogenous GSK-3f3 function. The strong similarity RNA microinjections, eight-cell embryos were transferred to between rat GSK-303 and XGSK-3i3 and the ability of rat 3% Ficoll (Pharmacia) in 0.5x MMR and injected with 10 nl GSK-313 to rescue the shaggy mutant phenotype in Drosophila of a solution containing 0.1-4 ng of RNA. For animal cap embryos (32) indicate that the rat enzyme should retain its experiments, both blastomeres of the two-cell embryos were function in Xenopus embryos. A catalytically inactive mutant injected in the animal pole region and cultured until midblas- (K85R) was constructed by replacing Lys-85 of the ATP- tula stages. Animal caps were excised at stage 8-8.5 and binding site of GSK-3,B (33) with Arg. Similar mutations in cultured in 0.6x MMR until stage 11 or stage 28, when RNA was extracted for Northern analysis. Histology was carried out 1 50 as described (22). XGSK3B MSGRPRTTSF AzsCXPVQQP S. SFGSMXVS RDxDGSxVTT VVATPGQGPD GSK3IO MSGRPRTTSIr AzSCKPVQQP s .ArGSMWVS RDiRGSXVTT VVATPGQGPD Immune Complex Kinase Assays and Western Analysis. Six Shaggy MSGRPRTSSF AZGNK..QSP SLVLGGVXTC .SRDGSKITT VVATPGQGTD embryos injected with Myc-tagged constructs were lysed in 600 51 100 of lysis buffer B (1% Triton X-100/25 mM Tris HCl, pH XGSK3B RQQEVSYTDT xVIGNGSFGV VYQAXKLDTG ZLVAIKIVFQ DKRFILQ gl GSK3B RPQzVsYTDT XVIGNGSFGV VYQAKLCDSG ELVAIaKVLQ DtNrNLQ 8.0/0.1 M KCl/1 mM EDTA) at stage 9. Lysates (500 ,l) were Shaggy RVQzvsYTDT xVIGNGsrGV VFQAKLCDTG ZLVAIXKVLQ DRRFNRIZLQ cleared by centrifugation at 10,000 x g for 5 min and incubated 101 150 for 1.5 hr with 30 of 9E10 (anti-Myc) hybridoma supernatant XGSK3B DBINI VRLRYITYSS GEKKDZVYLN LVLDYVPIZTV YRVARIIYSRA gl GSK3O iDsECNI VLRYrrYSS GKDEVYLN LVLDYVPZTV YRVARBYSRA and for an additional 30 min with 20 ,ul of 50% suspension of Shaggy IKEIHCNI VKLLYrTYSS GZKRDZVFLN LVLEYIPZTV YKVARQYAKT staphylococcal protein A-agarose. The immunoprecipitates 151 200 were washed three times with buffer B and twice with 30 mM XGSK3B XQALPIIYVK LYMYQLUrRSL AYIHSFGICU RDIKPQNLLL DPZTAVLKLC GSK3B KQTLPVIYVK LYMYQLFRSL AYISrGICs fDIIPQNILL DPDTAVLKLC Tris-HCI, pH 7.5/10 mM MgCl2. Immune complex kinase Shaggy KQTIPINFIR LYNYQLFRSL AYIESLGICH RDIXKPQNLLL DPZTAVLKLC assays were carried out using ['y-32P]ATP and myelin basic 201 250 protein as substrate as described (23). Half of each reaction XGSK3a DFGsAXQLVR GzPNVsYICs RYYRAPzLIF GATDYTssID vWSAGCvLAz GSK3S DFGSAKQLVR GZPNVSYICS RYYRAPZLIr GATDYTssID MWSAGCVLAz mixture was subjected to SDS/12.5% PAGE followed by Shaggy DrGSAKQLLH GEPNVSYICS RYYRAPELIr GAINYTTKID VWSAGCILAZ autoradiography. 251 300 For Western analysis cleared lysatos were electrophoresed XGSK3B LLGQPIFPG DSGVDQLVEI IXVLGTPTRE QIRZINPNYT ErKFPQIXAH GSK3B LLLGQPIFPG DSGVDQLVEI IKVLGTPTRZ QIRflNPNYT ZrKrPQIRAN on an SDS 10%/polyacrylamide gel and electroblotted onto Shaggy LLLGQPIFPG DSGVDQLVZV IKVLGTPTRE QIRflNPNYT ErKFPQIKSH polyvinylidene difluoride (PVDF) membranes (Millipore). 301 350 One-tenth embryo equivalent was loaded per lane. The mem- XGSK3B PWTKVrRART PPZAIALCSR LLEYTPTSRL TPLDACVHSr FDZLR.DPNL GSK33 PWPKV]rRPRT PPZAIALCSR LLEYTPTARL TPLEACAHSF FDzLR .DPNV brane was incubated sequentially with 5% skim milk, anti-Myc Shaggy PWQRV]rRIRT PTZAINLVSL LLZYTPSARI TPLKACAHPr FDELRMEGNH monoclonal of F. Harvard antibody (a gift McKeon, Medical 351 400 School), and horseradish peroxidase-conjugated goat anti- XGSK31A KLPNGREFPA LrNrTTQZLS SNPSLSSILI PAR. GSK31 KLPNGRDTPA LJNFTTQELS SNPPLATILI PHP. mouse IgG antibodies (Jackson ImmunoResearch). Incuba- Shaggy TLPNGRDMPP LrNFTEHELS IQPSLVPQLL PKHLQNASGP GGNRPSAGGA tions were 1 hr four brief washes with each, separated by 401 450 phosphate-buffered saline (PBS) plus 0.05% Tween 20. Per- XGSK33 ARNQAAVS ...... TTSNTTSTSN SNTGERGSTN GSK3S ARIQAAAS ...... PPANATAASD TNAGDRGQTN oxidase activity was visualized with an ECL system (Amer- Shaggy ASIAASGSAS VSSTGSGASV EGSAQPQSQG TAAAAGSGSG GATAGTGGAS sham). 451 500 RNA Preparation and Analysis. Total RNA was extracted XGSK3B AASASASNS S...... GSK3i NAASASASNS T...... and prepared for Northern analysis as described (24). RNA Shaggy AGGPGSGNNS SSGGASGAPS AVAAGGANAA VAGGAGGGGG AGAATAAATA were in vitro from probes prepared by transcription (18) 501 517 plasmids encoding XGSK-313, fibronectin (25), Xbra (26), XGSK3B GSK31 ...... A...... G ... Xwnt8 (27), goosecoid (11), XIF3 (28), Otx2 (29), neural cell Shaggy TGAIGATNAG' GANVTDS adhesion molecule (NCAM) (30), or XA1 (31). Hybridization with 32P-labeled antisense RNA or DNA (XAG1, ref. 31) FIG. 1. Deduced XGSK-3P3 amino acid sequence and its compar- probes was performed by using standard techniques (17). ison with the Drosophila shaggy and rat GSK-3f3 proteins. Downloaded by guest on September 30, 2021 8500 Developmental Biology: Dominguez et al. Proc. Natl. Acad. Sci. USA 92 (1995)

B AP DM VM VP

XGSK[ A 2 9 11 12 20

Fibronectin - XGSK[

- Brachyury C D K85R rGSK Control Xwnt8- D V D V D V 18S Goosecoid Fibronectin FIG. 2. XGSK-3f transcripts are present maternally and are uni-

formly distributed in the early embryo. (A) Northern analysis of Brachyury - XGSK-3,3 expression throughout embryogenesis. Developmental Xwnt8- Goosecoid - stages: 2, two-cell stage; 9, late blastula; 11, midgastrula; 12, late gastrula; and 20, late neurula. (B) Spatial distribution of the XGSK-3f3 transcripts in stage 10 gastrulae. Explants were isolated from the animal pole region (AP), the dorsal marginal zone (DM), the ventral FIG. 3. Effects of GSK-3,B RNAs on embryonic development. marginal zone (VM), and the vegetal pole region (VP). Two embryo Embryos were injected with 1 ng of K85R GSK-3,3 mRNA (A) or equivalents of RNA are loaded per lane for Northern analysis; 18S wild-type GSK-3p mRNA (B) into a ventrovegetal blastomere at the RNA and fibronectin bands reflect loading. Antisense probes: eight-cell stage. (X6.) (C) Representative transverse section of an brachyury, general mesodermal marker; Xwnt8, ventrolateral marker; embryo injected with K85R mRNA; b, brain; nc, notochord; s, somite. and goosecoid, dorsal marginal zone marker. The bar on the right of (Scale bar = 150 ,um.) (D) Effect of K85R RNA on early mesodermal A indicates position of 28S rRNA. markers in the marginal zone. Dorsal (D) and ventral (V) halves were dissected from normal embryos or from embryos injected with K85R protein kinase CC and in Raf kinase were reported to behave or rat GSK-3,B RNAs and subjected to Northern analysis (RNA from in a dominant negative manner (34, 35). four explants is in each lane). Microinjection of K85R mRNA into a ventrovegetal blas- tomere of eight-cell embryos resulted in the formation of a stage. Analysis of embryonic lysates revealed significant kinase secondary body axis (Fig. 3A; Table 1), similar to the effects activity in immunoprecipitates from late blastulae injected of Wntl and Xwnt8 mRNAs (8). In contrast, injection of a with Myc-tagged wild-type XGSK-3,B or rat GSK-313 RNAs, wild-type GSK-3j3 mRNA into a ventrovegetal blastomere, or but not with K85R mRNA (Fig. 4,4). Lack of kinase activity in injection of K85R mRNA into a dorsovegetal blastomere, did immunoprecipitates from Xenopus embryos injected with not affect axial structures (Fig. 3B). Several other mutants of K85R RNA did not result from poor expression of the mutant GSK-3,3, including two C-terminal deletions and a point protein, as evident from Western analysis (Fig. 4B, lane 3). mutation of conserved Tyr-216, failed to interfere with em- Comparison of protein levels with their relative enzymatic bryonic axis formation (data not shown). These observations activities (Fig. 4) indicates that XGSK-3,B has a lower specific indicate that the effect of the K85R mutant is reproducible and activity than rat GSK-313, and, thus, could be down-regulated very specific. The induced axes frequently (42%) contained the in the embryo. These observations suggest that the K85R most anterior structures, including eyes and cement glands mutant is catalytically inactive and may inhibit the endogenous (Fig. 3A; Table 1). Histological sections revealed two properly XGSK-3,3 function in a dominant negative manner by non- organized axes, each including notochord, neural tube, and productively sequestering its in vivo substrates or regulators. somites (Fig. 3C). In addition, Northern analysis showed Wild-Type GSK-3p Counteracts the Effects of the K85R induction of goosecoid, a Spemann organizer marker, in Mutant and Inhibits the Dorsalizing Effect of Xenopus Di- ventral marginal zone cells (Fig. 3D). Together, these obser- shevelled. If K85R protein competitively interferes with en- vations suggest that the mutated form of GSK-3j3 is biologi- dogenous GSK-313 function, then the induction of a secondary cally active and induces an ectopic Spemann organizer. axis by K85R should be suppressed by wild-type GSK-3/3. K85R GSK-3p Is Enzymatically Inactive. To verify that the Whereas overexpression of K85R mRNA in a ventrovegetal K85R form of GSK-3f3 is catalytically inert, we used immune blastomere induced complete secondary axes in 42% of in- complex kinase assays using Myc-tagged variants of GSK-3f3. jected embryos, coinjection of the same dose of K85R mRNA To evaluate expression levels and enzymatic activities of (0.5 ng) with the wild-type XGSK-3f3 mRNA led to a sub- exogenous GSK-3f3, in vitro synthesized mRNAs were injected stantial reduction in the number of embryos with complete into the animal pole region of both blastomeres at the two-cell secondary axes (7%; Table 1). Many of the injected embryos Table 1. Effects of GSK3J3 RNAs on embryonic axis No. (%) with phenotype No. ofAxsdpiaon injected Axis duplications Single RNA injected embryos Complete Partial axis Other K85R 183 77 (42) 44 (24) 24 (13) 38 (21) K85R + GSK3,B 148 10 (7) 46 (31) 60 (40) 32 (22) K85R + XGSK3, 66 5 (7) 27 (41) 21 (32) 13 (20) GSK3,B 93 0 0 73 (78) 20 (22) XGSK3f3 42 0 0 40 (95) 2 (5) Embryos were injected at the four- to eight-cell stage into a single ventrovegetal blastomere with 0.5-1 ng of mRNA(s) and allowed to develop until control sibling embryos reached stage 36. Axis duplications were considered complete when both cement gland and eyes were duplicated, and partial, when secondary axes lacked head structures. Other phenotypic abnormalities include incomplete blastopore closure and defects in the primary axis. Downloaded by guest on September 30, 2021 Developmental Biology: Dominguez et aL Proc. Natl. Acad. Sci. USA 92 (1995) 8501 B B Mr 1 2 3 4 5 xlo-3 1 2 3 4 ...CA A 68- NCAM 1 2 3 4 g _ 43- *. :i MBP

FIG. 4. Enzymatic activities of different forms of GSK-3f3. Lysates Otx2- were prepared at late blastula stages from embryos injected with Myc-tagged RNA constructs: lanes 1, Xenopus GSK-3,B; lanes 2, rat GSK-3p3; lanes 3, K85R GSK-3p3; and lanes 4, no injection. (A) Immune XA1- complex kinase assays with anti-Myc antibodies and with myelin basic A protein (MBP) as substrate. (B) Western analysis of embryonic lysates 12 3 4 5 with anti-Myc antibodies. XIF3- Fibronectin - had a single body axis (Fig. SB; Table 1). Both XGSK-3f3 and XAG1- rat GSK-313 mRNAs inhibited the effect of the K85R mRNA. Brachyury - The ability of the wild-type GSK-3f to rescue the K85R Xwnt8- mutant argues against nonspecific or toxic effect of K85R on Goosecoid - 28S- embryogenesis. Our findings suggest that endogenous GSK-3f3 functions as FIG. 6. Neuralization of ectodermal explants by K85R GSK-303. an inhibitor of dorsoventral axis formation in the embryo. Both blastomeres of the two-cell embryos were injected with different that the Wnt/Wg pathway is mRNAs as indicated. Animal cap explants were isolated at stage 8 and Assuming cultured until stage 11 (A) for early marker analysis or until stage 28 conserved in evolution, one would expect that GSK-313 acts (B) for late marker analysis. Early mesodermal markers are the same downstream of Dsh, another component of the Drosophila Wg as in Fig. 2. Otx2 and XIF3 are anterior neural markers, XAG1 is a signaling pathway. We have recently cloned a Xenopus ho- cement gland marker, XA1 is an anterior ectoderm marker, and molog of Dsh (Xdsh) and demonstrated that its overexpression NCAM is a panneural marker. Fibronectin mRNA and 28S rRNA induces a secondary body axis in Xenopus embryos (24). To bands reflect loading. Animal cap RNA was derived from uninjected assess whether wild-type GSK-3,B could act as a direct antag- embryos (lane 4) or from embryos injected with 1 ng of K85R mRNA onist of dorsoventral axis formation and to attempt to order it (lane 1), with 1 ng of Xdsh mRNA (lane 2), with 100 pg of noggin in the vertebrate Wnt signaling pathway, we coinjected 1 ng mRNA (lane 3). Sibling embryo RNA is in lane 5. Total RNA from 10 animal caps or from 2 embryos is loaded per lane. The same blot each of XGSK-313 and Xdsh mRNAs. Whereas injection of with different probes. Xdsh mRNA alone produced axis duplications in 59% of the was stripped and reprobed injected embryos (n = 51), only 5% ofembryos coinjected with cultured until midgastrula or tailbud stages. Northern analysis Xdsh and XGSK-313 mRNAs had partial secondary axes (n = of total RNA isolated from injected caps showed that early 38). These observations are consistent with the idea that the mesodermal markers Xbra, Xwnt8, and goosecoid (36) were main elements of the Wnt/Wg signal transduction pathway are not activated in injected animal caps, suggesting that K85R functionally conserved from Drosophila to vertebrates. does not induce mesoderm (Fig. 6A). In some experiments we Neuralization of Ectodermal Explants Overexpressing observed that larger doses of K85R RNA are capable of K85R GSK-3j3. Formation and patterning of embryonic me- inducing muscle actin RNA in animal caps (not shown). These soderm is thought to be an essential part of dorsoventral axis results may reflect a synergy between K85R and low doses of determination (1, 36). Several growth factors from the trans- mesoderm-inducing signals in the animal caps similar to what forming growth factor 3 and fibroblast growth factor families was described for Xwnt8 and for noggin (37, 38). can induce mesoderm de novo when added to ectodermal In several experiments animal caps injected with K85R explants (36). In contrast, competence modifiers (37, 38), such mRNA developed cement glands (data not shown). Since as Wnts and noggin, induce dorsal mesoderm only in cooper- cement glands are often associated with neural tissue, we set ation with mesoderm-inducing factors. To determine whether out to evaluate neural development in the explants. Neural K85R can induce or modify mesoderm formation, we studied tissue markers, including NCAM (30), XIF3 (28), and Otx2 the differentiation of animal pole explants injected with K85R and mRNA. (29) [also called OtxA (38)], as well as anterior ectodermal K85R mRNA (1 ng) was microinjected into the animal pole cement gland markers XA1 and XAG1 (31), were all activated of both blastomeres of two-cell Xenopus embryos. Animal cap in animal caps overexpressing K85R mRNA compared with explants were isolated at the midblastula stage (stage 8) and uninjected controls (Fig. 6B). Neuralizing properties of K85R were quite similar to those of Xdsh (24) and noggin (Fig. 6). A These findings indicate that K85R mRNA can neuralize animal cap explants in the absence of early mesodermal markers. DISCUSSION We have cloned a Xenopus homolog of shaggy/GSK-3,B and found that it is expressed in eggs and early embryos, suggesting that, like its Drosophila counterpart, XGSK-3P3 plays an im- portant role in embryogenesis. Overexpression of a presumed FIG. 5. Wild-type XGSK-3f overcomes the effect of the K85R dominant negative form of GSK-3,B (K85R) leads to induction mutant on dorsoventral axis formation. Embryos were injected with in the ventral 0.5 ng of K85R RNA in the ventrovegetal blastomere at the eight-cell of an ectopic set of dorsal structures marginal stage (A) or with a mixture of 0.5 ng of K85R RNA and 0.25 ng of the zone and activates neural markers in ectodermal explants, wild-type XGSK-3,B RNA (B) and allowed to develop until stage 39. consistent with the GSK-3f3 role as a negative regulator of (x5.) dorsal mesoderm and neural tissue development. Downloaded by guest on September 30, 2021 _~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... ,...,_ t l 8502 Developmental Biology: Dominguez et al. Proc. Natl. Acad. Sci. USA 92 (1995) The effects of the K85R mutant are similar to those of 6. Nusse, R. & Varmus, H. E. (1992) Cell 69, 1073-1087. noggin and of Xenopus dishevelled (Fig. 6), which can both 7. Dickinson, M. E. & McMahon, A. P. (1992) Curr. Opin. Genet. dorsalize ventral mesoderm and neuralize ectodermal cells Dev. 2, 562-566. (24, 38). In contrast, mesoderm-inducing factors induce neural 8. Sokol, S., Christian, J. L., Moon, R. T. & Melton, D. A. (1991) Cell 67, 741-752. tissue via an obligatory formation of the mesodermal inter- 9. Smith, W. C. & Harland, R. M. (1991) Cell 67, 753-765. mediate (36). Interestingly, whereas low doses of K85R RNA 10. Smith, W. C. & Harland, R. M. (1992) Cell 70, 829-840. activated neural markers in animal caps in the absence of early 11. Cho, K. W. Y., Blumberg, B., Steinbeisser, H. & De Robertis, mesodermal markers (Fig. 6), higher doses of K85R were able E. M. (1991) Cell 67, 1111-1120. to trigger synthesis of cardiac actin transcripts in animal caps 12. Sasai, Y., Lu, B., Steinbeisser, H., Geissert, D., Gont, L. K. & De (not shown). Such properties of K85R suggest that presump- Robertis, E. M. 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