Dorsal Determinants in the Xenopus Egg Are Firmly Associated with the Vegetal Cortex and Behave like Activators of the Wnt Pathway

DEVELOPMENTAL BIOLOGY 191, 69 79 (1997) ARTICLE NO. DB978710 Dorsal Determinants in the Xenopus Egg Are Firmly Associated with the Vegetal Cortex and Behave like Activators of the Wnt Pathway Yusuke Marikawa, Yu Li, and Richard P. Elinson Department of Zoology, University of Toronto, 25 Harbord Street, Toronto M5S 3G5, Canada The Xenopus egg contains maternal dorsal determinants that are specifically located at the vegetal cortex. To study physical and functional properties of the dorsal determinants, we took advantage of the animal vegetal reversed embryo. The animal vegetal reversed embryo is produced by inversion of the fertilized egg, which results in formation of ectoderm and endoderm from the unpigmented and the pigmented halves, respectively [Neff et al. (1983). Dev. Biol. 97, 103 112; Black and Gerhart (1985). Dev. Biol. 108, 310 324]. We demonstrated by cytoplasmic transplantation that the dorsal activity was specifically localized to the unpigmented cortical cytoplasm of the inverted egg, which is segregated into the future ectodermal lineage. This result suggests that the dorsal determinants are associated with the unpigmented cortex and are not dislodged by the inversion. In addition, we found that two vegetally localized transcripts, Xcat2 and Vg1 mrnas, were present in the reversed animal pole of the inverted egg, suggesting their association with the unpigmented cortex. In order to compare the dorsal determinant activity with known dorsalizing molecules, we examined the expression pattern of Xnr3 and Siamois in the reversed embryo because these two genes are activated by the Wnt-pathway activators (Xwnt- 8, b-catenin, etc.) but not by other dorsalizing molecules (noggin, BVg1, etc.). Animal cap of the reversed embryo, which received the unpigmented cortex of the egg, expressed Xnr3 and Siamois. However, Mix.1, a marker expressed in endoderm and mesoderm in the normal embryo in response to mesodermal inducers, was not detected in the animal cap of the reversed embryo. In addition, we found that b-catenin protein accumulated in nuclei of unpigmented animal pole cells of the reversed embryo. These results suggest that the maternal dorsal determinants behave more similarly to the Wntpathway activators than noggin or BVg1. 1997 Academic Press INTRODUCTION Maternal information localized in particular regions of the egg cytoplasm is responsible for future determination of developmental fates in early embryos. In the Xenopus egg, the cytoplasmic factor that is responsible for determining dorsal structures is specifically located in the vegetal cortex. Microinjection of vegetal cortical cytoplasm into ventral blastomeres of a 16-cell recipient embryo generates a complete secondary dorsal axis (Fujisue et al., 1993; Holo- wacz and Elinson, 1993). Removal of the cortical cytoplasm from the vegetal pole of the early 1-cell embryo results in a dorsal deficiency (Kikkawa et al., 1996; Sakai, 1996). The dorsal determinants in the vegetal cortex alter the response of animal cap cells to mesoderm inducers and are unlikely to be mesoderm inducers (Holowacz and Elinson, 1995), although the molecular nature of the dorsal determinants is unknown. Several molecules have been identified that, like vegetal cortical cytoplasm, are able to produce a secondary axis when expressed in early embryos. Activation of the Wnt-signaling pathway by Xwnt-8 (Sokol et al., 1991; Smith and Harland, 1991), Xwnt-8b (Cui et al., 1995), Xdsh (Sokol et al., 1995), a dominant-negative form of Xgsk3 (Dominguez et al., 1995; He et al., 1995; Pierce and Kimelman, 1995), XAPC (Vlem- inckx et al., 1997), and b-catenin (Funayama et al., 1995) causes full axis formation, although none of their mrnas or proteins has been shown to be localized at the vegetal cortex of the unfertilized egg. The mrna for another Wnt family member, Xwnt-11, is localized at the oocyte vegetal cortex, but Xwnt-11 RNA is unable to produce a full dorsal axis (Ku and Melton, 1993). A secreted molecule noggin also has the ability to induce a complete dorsal axis, but noggin RNA is not localized to the vegetal cortex (Smith and Har- land, 1992). The RNA for the TGFb family member Vg1 is localized to the oocyte vegetal cortex (Weeks and Melton, 0012-1606/97 $25.00 Copyright 1997 by Academic Press All rights of reproduction in any form reserved. 69

70 Marikawa et al. 1987). Vg1 RNA has no dorsalizing activity, but BVg1, a modified version of Vg1 designed to promote efficient secretion of mature Vg1, has the ability to induce a full dorsal axis (Dale et al., 1993; Thomsen and Melton, 1993). Expression of a transcription factor Siamois is induced in animal caps by Xwnt-8, a dominant-negative form of Xgsk3, XAPC, and b-catenin, but not by BVg1 or noggin (Brannon and Kimelman, 1996; Carnac et al., 1996; Vleminckx et al., 1997). Expression of the TGFb family member Xnr3 is also induced in animal caps by Xwnt-8 but not by noggin or a dorsal mesoderm inducer, activin (Smith et al., 1995). Thus, it is likely that expression of Siamois and Xnr3 is a specific marker for activation of the Wnt pathway. In addition, b- catenin, acting as a downstream element of the Wnt pathway, translocates into the nucleus when activated (Funayama et al., 1994; Schneider et al., 1996; Yost et al., 1996; Larabell et al., 1997). Therefore, it is of particular interest to examine whether the dorsal determinants in the vegetal cortical cytoplasm are able to induce transcriptional activation of Xnr3 and Siamois and a nuclear accumulation of b- catenin protein. For this purpose, we exploited the reversed embryo, which is derived from fertilized eggs that are inverted to orient the unpigmented vegetal hemisphere upward (Neff et al., 1983). In the inverted egg, heavy yolky cytoplasm, which is originally located in the unpigmented half, is displaced into the opposite side by the gravity, while the surface pigmentation pattern is unchanged (Neff et al., 1984). The inverted egg is able to develop into an apparently normal embryo except for the pigmentation pattern. The unpigmented side gives rise to ectodermal tissues and the pigmented side gives rise to endodermal tissues (Neff et al., 1983). Thus, the embryo derived from the inverted egg displays a reversal of the animal vegetal axis with regard to the cortical pigmentation. In this study, we have determined the location of the dorsal determinants in the in- verted egg. Then, we have examined the correlation be- tween the dorsal determinants and expression pattern of Xnr3 and Siamois and the distribution of b-catenin protein in the reversed embryo. MATERIALS AND METHODS Eggs and Embryos Adult Xenopus laevis females were injected with 650 IU of human chorionic gonadotropin (Sigma, St. Louis, MO) to induce ovulation and kept at 15 18 C overnight. Eggs were squeezed into sperm suspension, which was prepared by homogenizing testes in 100% Steinberg s saline. Fertilized eggs were dejellied in 2.6% L- cysteine solution (ph 8.0) and washed extensively in 20% Steinberg s saline. Inversion and Centrifugation of Eggs Neff et al. (1983, 1984) prepared inverted eggs by placing fertilized eggs upside down under natural gravity, and Black and Gerhart (1985) added a centrifugation process to promote efficient displace- FIG. 1. A schematic diagram of the procedure of inversion and centrifugation of eggs (see Materials and Methods). SS, Steinberg s saline. ment of inner cytoplasm. We have obtained inverted eggs according to the centrifugation procedure of Dr. Atsunori Shinagawa, Yamagata University, Japan (reported at the Annual Meeting of the Japanese Society of Developmental Biologists, 1995). The procedure is schematically represented in Fig. 1. Dejellied eggs were placed in 5% Ficoll dissolved in 100% Steinberg s saline to remove water in the perivitelline space. Each egg was placed in a hole made in Paraplast coating the bottom of a dish that was filled with 5% Ficoll in 100% Steinberg s saline. Eggs were inverted with the aid of a hair loop to orient the unpigmented vegetal hemisphere up- ward. At 28 32 min after insemination (approximately 35% of the first cell cycle: 0.35NT), the dish was placed on the top of a centri- fuge bucket and centrifuged at 10 30g for 5 10 min. After centrifugation, eggs were transferred to 20% Steinberg s saline and cultured at 15 C. Normal looking blastulae were developed from 40 90% of inverted eggs. Cytoplasmic Transplantation Transplantation of cortical cytoplasm was carried out according to Holowacz and Elinson (1993). The tip of a glass microneedle was

Xenopus Dorsal Determinants Activate Wnt Pathway 71 beveled with an inclination of 30 by a microneedle grinder (Model overnight at 020 C in 80% methanol, 20% DMSO. After several PB-7; Narishige, Tokyo). Cortical cytoplasms were withdrawn from washes in TBST (140 mm NaCl, 10 mm Tris, ph 7.4, 0.1% Triton), a normal egg and an inverted egg and injected into two ventral the embryos were blocked in 1% BSA in TBST at room temperature vegetal blastomeres of a 16-cell recipient embryo. Dorsoanterior for half an hour, followed by incubation at 4 C overnight in anti- index (DAI) of the secondary axis was scored according to Kao and b-catenin antibodies (1:100 dilution; a generous gift from Dr. Peter Elinson (1988). Hausen, Tübingen, Germany) in 1% BSA in TBST. After washing in 0.2% BSA in TBST for three times, 2 hr per wash, the embryos were incubated at 4 C overnight in AP-conjugated goat anti-rabbit Northern Analysis IgG (1:700 dilution; BIO/CAN Sci. Inc., Mississauga, Ontario) in 1% BSA in TBST. After extensive wash in 0.2% BSA in TBST, RNA was extracted from whole embryos and dissected tissue the embryos were visualized by the NBT/BCIP method (Boerhinger pieces by the Proteinase K/LiCl method according to Sambrook et Mannheim) and cleared in benzyl benzoate/benzyl alcohol. al. (1989), with a slight modification. Specimens were digested in 1 mg/ml Proteinase K, 20 mm Tris (ph 7.6), 100 mm NaCl, 30 mm EDTA, and 1% SDS at 37 C for 1 hr to overnight. After three extractions with phenol/chloroform, RNA was precipitated in 4 M RESULTS LiCl and washed in 70% ethanol. The RNA was electrophoretically separated on a 1% agarose gel containing formaldehyde and Mops buffer, blotted onto a nylon Development of the Reversed Embryo membrane (Boehringer Mannheim) by capillary transfer, and UV The centrifugally inverted eggs developed into embryos crosslinked. The membrane was prehybridized in hybridization that looked similar to those described in Neff et al. (1983). buffer (HB: 50% formamide, 51 SSC, 101 Denhardt s solution, 1 They displayed a reversal of the small/large blastomere patmg/ml torula RNA, 1% SDS, 10 mm sodium phosphate, ph 6.8) tern: smaller blastomeres developed in the unpigmented at 65 C for 1 hr and hybridized in HB containing 100 500 ng/ml hemisphere and larger blastomeres developed in the pigof digoxygenin (DIG)-labeled RNA probes at 65 C overnight. DIGmented hemisphere. At the blastula stage, a blastocoel was labeled RNA probes for chordin (Sasai et al., 1994), Xwnt-8 (Chrisformed near the unpigmented hemisphere of the reversed tian et al., 1991), Vg1 (Weeks and Melton, 1987), Xcat2 (Mosquera et al., 1993), Xnr3 (Smith et al., 1995), Mix.1 (Rosa, 1989), and the embryo. At the early gastrula stage, the first involution was fibroblast growth factor receptor (FGF-R; Musci et al., 1990) were observed subequatorially as an accumulated pigmentation. made by in vitro transcription using DIG-UTP according to the The blastopore lip of the reversed embryo expanded later- manufacturer s instructions (Boehringer Mannheim). After hybridization, ally in the pigmented hemisphere (Fig. 2A). Some of the the membrane was washed at 68 C twice in 21 SSC, 0.1% reversed embryos (20 90% of normal looking blastulae deally SDS and twice in 0.11 SSC, 0.1% SDS, 15 min per wash. The pending on the batches of the eggs used) developed into membrane was then rinsed in maleic acid buffer (MAB: 0.1 M ma- apparently normal tadpoles except for the pigmentation leic acid, 0.15 M NaCl, ph 7.5), blocked in MAB containing 1% pattern: the anterior portion of the reversed embryo was blocking reagent (Boehringer Mannheim), and incubated in MAB less pigmented compare to that of the normal tadpole (Fig. containing 1% blocking reagent plus alkaline phosphatase (AP)- 2C). In most cases, the cement gland of the reversed embryo conjugated anti-dig antibody (1:5000 dilution; Boehringer Mannwas totally unpigmented. Therefore, as reported by Neff et heim). The membrane was then washed twice in MAB containing 0.15% Tween 20, 20 min per wash, and rinsed in alkaline phosphaanimal vegetal axis with regard to the cortical pigmenta- al. (1983), the inversion of eggs resulted in the reversal of tase buffer (APB: 0.1 M Tris HCl, 0.1 M NaCl, ph 9.5). After incution bation in APB containing chemiluminescent substrate CSPD (1:100 pattern. dilution; Boehringer Mannheim), the membrane was exposed to an To see whether the site of the first involution of the X-ray film. reversed embryo corresponds to a dorsal lip, the expression pattern of chordin, whose transcript is found exclusively RT PCR Analysis on the dorsal lip in the normal embryo (Sasai et al., 1994), was examined. Chordin transcript was present at the site RNA was isolated from whole embryos or animal caps using of first involution but not on the side opposite to the first Trizol reagent (Gibco BRL, Gaithersburg, MD) following the manu- involution (Fig. 2C). In agreement, when the expression of facturer s instructions. DNA was removed by digestion with RQ1 the ventral lateral mesoderm marker Xwnt-8 (Christian et DNase (Promega, Madison, WI) at 37 C for half an hour. Reverse al., 1991) was examined, it was found more strongly on the transcription and PCR conditions were according to Hemmati-Bri- side opposite to the first involution (Fig. 2C). Thus, the vanlou and Melton (1994). EF-1a primers were as described by Hemfirst involution of the reversed embryo corresponds to a mati-brivanlou and Melton (1994). Siamois primers were as dedorsal lip. scribed by Brannon and Kimelman (1996). PCR products were analyzed on a 5% acrylamide gel. Association of Dorsal Determinants with the Immunocytochemistry Cortical Cytoplasm of the Unpigmented Hemisphere Whole mount immunostaining for b-catenin was carried out according to Schneider et al. (1996) with the following modification. Dorsal determinants are present in the vegetal cortical Embryos were fixed for 2 3 hr with 3.7% formaldehyde in 0.1 cytoplasm of the normal egg (Fujisue et al., 1993; Holowacz M Mops (ph 7.4), 2 mm EGTA, 1 mm MgSO 4 and permeabilized and Elinson, 1993; Kikkawa et al., 1996; Sakai, 1996). To

72 Marikawa et al.

Xenopus Dorsal Determinants Activate Wnt Pathway 73 nal mrnas, Vg1 and Xcat2. Vg1 RNA is localized at the vegetal cortex of oocytes. The RNA is released from its tight localization after maturation but remains in the vegetal hemisphere (Melton, 1987; Weeks and Melton, 1987). Xcat2 RNA is associated with the vegetal cortex and remains associated with the cortex after maturation (Mosquera et al., 1993; Forristall et al., 1995). To examine the distribution of Vg1 and Xcat2 RNAs in the inverted egg, cytoplasmic fragments (each approximately one-fifth of total egg volume) were cut from the reversed animal and the reversed vegetal poles and analyzed by Northern blotting (Fig. 4A). A significant amount of Vg1 RNA was found in the reversed animal fifth as well as in the reversed vegetal fifth (Fig. 4B), suggesting that a certain amount of Vg1 RNA may be still associated with the cortical cytoplasm even after the release from its tight localization during oocyte maturation. Xcat2 RNA was also found in the reversed animal fifth. The amount of Xcat2 RNA in the reversed animal fifth was almost as much as in the normal vegetal fifth (Figs. 4C and 4D). However, the presence or absence of Xcat2 RNA in the reversed vegetal fifth was variable. In one case, less Xcat2 RNA was found in the reversed vegetal fifth (Fig. 4D). But in another case, Xcat2 RNA was not detected in the reversed vegetal fifth (Fig. 4C). A similar difference in Xcat2 RNA distribution was observed when the reversed animal and the reversed vegetal halves were examined: the RNA was exclusively found in the reversed animal half in one case, but the RNA was also found in the reversed vegetal half in the other case (data not shown). The reason for this difference is not clear, although variation in cytoplasmic fluidity among batches of eggs may affect how molecules are redistributed by centrifugation (Neff et al., 1990). Alter- natively, there may be a certain variation in the RNA binding capability of the cortex depending on the batches of eggs. In any case, the reversed animal cortex of the inverted egg contained a significant amount of Vg1 and Xcat2 RNAs. examine the location of the dorsal determinants in the inverted egg, cytoplasmic transplantation was performed. To avoid confusion, parts of the reversed egg are named as shown in Fig. 1. The unpigmented hemisphere of the reversed egg is called the reversed animal hemisphere because it is fated to become ectoderm, and the pigmented hemisphere of the reversed egg is called the reversed vegetal hemisphere because it is fated to become endoderm. The animal and vegetal hemispheres of the normal egg are called the normal animal and normal vegetal hemispheres, respectively. In both the normal and the reversed cases, the vegetal hemisphere contains the mass of large yolk platelets (Fig. 1). Cortical cytoplasms were injected into two ventral vegetal blastomeres of a normal 16-cell recipient embryo and assayed for the ability to induce a secondary axis (Fig. 3). The cortical cytoplasm of the normal vegetal hemisphere induced a secondary axis (6/13). In contrast, the cortical cytoplasm of the reversed vegetal hemisphere did not induce a secondary axis (0/14), while the reversed animal cortical cytoplasm induced an axis (12/16). The axis forming activity of the reversed animal cortical cytoplasm (average DAI Å 2.1) was as strong as the normal vegetal cortical cytoplasm (average DAI Å 1.4). The presence of dorsal determinants in the animal cortical cytoplasm of the reversed egg may indicate that the dorsal determinants are associated with the unpigmented cortex firmly enough to resist the centrifugal force. Alternatively, the dorsal determinants may be associated with cytoplasmic components that had low specific gravity and end up in the centripetal (reversed animal) side of centrifuged eggs. In order to discriminate these possibilities, eggs were centrifuged in the uninverted orientation (Fig. 3C, bottom row). The cortical cytoplasm of the pigmented hemisphere, located at the centripetal side of these centrifuged eggs, did not induce a secondary axis (0/9 cases). However, the cortical cytoplasm of the unpigmented hemisphere of these centrifuged eggs induced a secondary axis (8/12; average DAI Å 1.1). These results indicate that the dorsal determinants are firmly associated with the unpigmented cortex. Distribution of Localized Maternal Messenger RNAs in the Inverted Egg Because the dorsal determinant activity was found exclusively in the reversed animal cortex, it is of particular interest to see what kind of molecules are associated with the reversed animal cortex. We focused on two localized mater- Expression of Xnr3 and Siamois in Ectodermal Cells of the Reversed Embryo The TGFb family member Xnr3 is expressed in the superficial layer of Spemann organizer (Smith et al., 1995), and the homeobox-containing transcription factor Siamois is expressed within the Nieuwkoop Center (Lemaire et al., 1995). Xnr3 and Siamois are expressed in animal cap cells in response to several dorsalizing genes which are components and activators of the Wnt pathway, but they are not induced by noggin or dorsal mesoderm inducers including FIG. 2. Development of the reversed embryos. (A) A vegetal view of a normal (N) and a reversed (R) embryo at gastrula stage. An expanding blastopore lip is seen in the pigmented area of the reversed embryo. (B) A normal (N) and a reversed (R) embryo at late tailbud stage. Note that the anterior portion, especially the cement gland, of the reversed embryo is much less pigmented than that of the normal embryo. (C) Expression pattern of Chordin and Xwnt-8 in the reversed embryo. Marginal tissues were taken from early gastrulae as shown in the diagram and analyzed by Northern blot. Chordin RNA is expressed exclusively at the site of the first involution (N-DM and R- DM), while Xwnt-8 RNA is expressed more on the side opposite to the first involution (N-VM and R-VM). FGF-R is used as a loading control.

74 Marikawa et al. FIG. 3. Dorsalizing activity of cortical cytoplasm. (A) A schematic diagram showing the experimental procedure used to assay for the ability of cortical cytoplasm to induce a secondary axis. Cortical cytoplasms from the animal pole or the vegetal pole were withdrawn from donor eggs and microinjected into two ventral vegetal blastomeres of a 16-cell recipient embryo. (B) Examples of double axis embryos that were produced by injection of the cortical cytoplasm from the reversed animal hemisphere of the inverted egg. (C) A table summarizing the result of the cytoplasmic transfer experiment. Top row: Cortical cytoplasm taken from the vegetal hemisphere of the normal egg had dorsal axis forming activity. Middle row: Cortical cytoplasms of the inverted egg were taken from the unpigmented (reversed animal) and the pigmented (reversed vegetal) hemispheres. Dorsal axis forming activity was found mostly in the reversed animal cortical cytoplasm. Bottom row: Cortical cytoplasms were taken from the pigmented and the unpigmented hemispheres of the egg that is centrifuged without inversion. Dorsal axis forming activity was found exclusively in the unpigmented cortical cytoplasm. Dorsoanterior index (DAI) of the secondary axis was scored according to Kao and Elinson (1988). that the agent functions similarly to the activators of the Wnt pathway. In the inverted eggs, the dorsal determinants were located in the reversed animal hemisphere, which was fated to be- BVg1 and activin (Smith et al., 1995; Carnac et al., 1996; Brannon and Kimelman, 1996; Fagotto et al., 1997). Therefore, if an unidentified dorsalizing agent induces the expression of Xnr3 and Siamois in animal cap cells, it is likely

Xenopus Dorsal Determinants Activate Wnt Pathway 75 FIG. 4. Distribution of the Xcat2 and Vg1 RNAs in the reversed egg. (A) Cytoplasmic fragments (each approximately one-fifth of total egg volume) were dissected from eggs that were fixed in 98% ethanol and 2% acetic acid and digested in the Proteinase K solution for RNA extraction. (B) A Northern blot of Vg1. The RNA is present in the reversed animal fifth as well as the reversed vegetal fifth. (C) A Northern blot of Xcat2. The RNA is seen exclusively in the reversed animal fifth. (D) Another Northern blot of Xcat2. The RNA is present more in the reversed animal fifth and less in the reversed vegetal fifth. Each lane contains total RNA extracted from five cytoplasmic fragments. FGF-R was used as a loading control. come ectoderm. This situation offers an opportunity to examine whether the dorsal determinants in ectodermal cells induce Xnr3 and Siamois. Animal caps were taken from reversed embryos at the early gastrula stage and assayed for the expression of these genes (Fig. 5A). Both Xnr3 and Siamois were expressed in the reversed animal cap, but neither of them was expressed in the normal animal cap (Figs. 5B and 5C), suggesting that the dorsal determinants behave like activators of the Wnt pathway. Mix.1 is expressed in endodermal and mesodermal cells in normal development and is induced in animal caps by dorsal mesoderm inducers including BVg1 and activin (Rosa, 1989; Cornell et al., 1995). Mix.1 was strongly expressed in the reversed vegetal cap but not expressed in the reversed animal caps (Fig. 5D). Therefore, the dorsal determinants are unlikely to be dorsal mesoderm inducers like BVg1 and activin, consistent with the previous study (Holowacz and Elinson, 1995). Furthermore, the Mix.1 expression pattern confirmed a reversal of the animal vegetal axis in the reversed embryo with respect to the endoderm. Accumulation of b-catenin Protein in Nuclei of Animal Pole Cells of the Reversed Embryo Schneider et al. (1996) and Larabell et al. (1997) have shown that b-catenin protein, acting as a component of the Wnt-signaling pathway, accumulates in nuclei on the dorsal side of the blastula. Several treatments which result in Wnt- pathway activation, such as mrna injections of Xwnt-8, a dominant-negative mutant of Xgsk3, and b-catenin, induce a nuclear accumulation of b-catenin protein (Funayama et al., 1995; Schneider et al., 1996; Yost et al., 1996; Larabell et al., 1997). Since the dorsal determinants behave like the Wnt-pathway activators with regard to the transcriptional activation of Xnr3 and Siamois in the animal cap, it is of interest to examine whether b-catenin protein accumulates in nuclei of cells in the reversed animal hemisphere. At stage 8 9, whole reversed and normal embryos were immunocytochemically stained with anti-b-catenin polyclonal antibody. b-catenin was found along the plasma membrane of all cells of both the reversed and the normal embryos, probably due to the cadherin/catenin interaction. In 23 of 58 reversed embryos from four experiments, a patch of cells near the animal pole showed strong nuclear staining, while surrounding animal cells did not (Fig. 6A). Cells with nuclear staining often also had some cytoplasmic staining. An additional 14 reversed embryos had nuclear staining in animal pole cells, and the nuclear staining extended down into the marginal zone. In contrast, animal cells of normal embryos did not show strong nuclear staining, although weak dumbbell-shaped staining was observed in most cells (Fig. 6B). The latter staining was probably perinuclear, as noted by Schneider et al. (1996). Nuclei were stained on one side of the marginal zone of the normal embryo (25 out of 47), confirming that our immunostaining procedure was appropriate (Fig. 6C). The observation of nuclear staining

76 Marikawa et al. FIG. 5. Expression pattern of Xnr3, Siamois, and Mix.1 in the reversed embryos. (A) Animal caps and vegetal caps were taken from the normal embryo and the reversed embryo at early gastrulae stage (stage 10/). (B) A Northern blot showing the expression pattern of Xnr3, in a stage 10/ normal whole embryo, five normal animal caps, and five reversed animal caps. Xnr3 is strongly expressed in the reversed animal cap but not expressed in the normal animal cap. FGF-R was used as a loading control. (C) An RT PCR showing the expression pattern of Siamois. Siamois is expressed in the reversed animal cap but not expressed in the normal animal cap. EF-1a was used as a control. (D) A Northern blot showing the expression pattern of Mix.1. Each lane contains total RNA corresponding to a single whole embryo or five caps. Mix.1 is expressed in both the normal vegetal cap and the reversed vegetal cap, but in neither the normal animal cap nor the reversed animal cap. FGF-R was used as a loading control. in about half of both reversed and normal embryos is in part due to the lumping of data from stage 8 through stage 9 embryos. The older embryos with smaller cells tended not to have nuclear staining. DISCUSSION The egg vegetal cortex contains the dorsal determinants that are responsible for establishing dorsoventral patterning FIG. 6. Nuclear accumulation of b-catenin protein in the animal hemisphere of the reversed embryo. (A) A reversed embryo (stage 8.5) had a patch of cells near the animal pole in which nuclei are stained strongly compare to the surrounding cells. The dark area at the bottom is displaced animal pigment, much of which is visible because the embryo is cleared. (B) A normal albino embryo (stage 8.5) which was injected with the mrna for a dominant negative mutant of XGSK3 into one animal blastomere at the 8-cell stage as a positive control (Dominguez et al., 1995; Yost et al., 1996). A patch of cells in the animal hemisphere had strong nuclear staining (arrow), while the surrounding animal region did not. (C) A normal albino embryo (stage 8.5) had strong nuclear staining on one side of the marginal zone.

Xenopus Dorsal Determinants Activate Wnt Pathway 77 direct synthesis of a processed mature form of Vg1 and induce axial structures in injected embryos (Dale et al., 1993; Thomsen and Melton, 1993). It may be that endogenous Vg1 precursor protein is processed specifically on the dorsal side of normal embryos, thus allowing Vg1 to act in setting up the dorsal axis. However, in the reversed egg, a signifi- cant amount of Vg1 mrna is present at the animal pole, which contains the dorsal axis forming activity. Nonethe- less, Mix.1, which is induced in normal animal caps by BVg1 (Cornell et al., 1995), is expressed at the vegetal pole but not at the animal pole of the reversed embryo. This implies that Vg1 is not processed in the animal region of the reversed embryo, even though the dorsal determinants are present in the same place. The dorsal determinants are located in the future ectodermal region in the inverted egg, raising the question how the reversed embryo gives rise to a normal tadpole. In normal development, the dorsal determinants are thought to estab- lish the Nieuwkoop Center, which is located at the dorsal vegetal region and has endodermal fate. The Nieuwkoop Center induces the Spemann organizer in the overlying marginal region in turn. We do not know whether the ectoder- mal tissue, which has received the dorsal determinants in the reversed embryo, behaves like the Nieuwkoop Center to induce Spemann organizer in the underlying marginal region. Wylie et al. (1996) has shown that a dorsal axis in the b-catenin-depleted embryo is recovered by injection of b-catenin mrna into the most animally located blasto- mere, even though the injected blastomere gives rise to the anterior epidermis. In the present study, we showed that the dorsal determinants in animal cap cells induce expression of two Wnt-target genes, Xnr3 and Siamois, and nuclear accumulation of b-catenin. Therefore, dorsal determinants located at the animal pole in the reversed embryo may direct formation of the Spemann organizer in the underlying marginal region by a mechanism similar to the rescue of the embryonic axis with animal b-catenin injections. Alternatively, although we could only detect dorsal determinants in the reversed animal hemisphere by cytoplasmic transplantation, the dorsal determinants may spread into the marginal zone, as suggested by our observation that some reversed embryos had b-catenin nuclear staining at the mar- ginal zone as well as at the animal pole. Further experiments, such as deletions of regions of the inverted egg, are required to determine where the active dorsal center is in the reversed embryo. of the Xenopus embryo. To understand the molecular nature of the dorsal determinants, we exploited the animal vegetal reversed embryo that is derived from the inverted egg. The dorsal determinants in the inverted egg were located at the unpigmented cortex, which was segregated into ectodermal tissues. Animal cap cells of the reversed embryos revealed the expression of Xnr3 and Siamois, as well as the nuclear accumulation of b-catenin, while they did not express Mix.1. Our results on the endogenous dorsal determinant are concordant with a variety of results in which members of the Wnt/b-catenin pathway are overexpressed or underexpressed (Heasman et al., 1994; Wylie et al., 1996; Fagotto et al., 1996, 1997). Therefore, we conclude that the dorsal determinants of the egg behave more like the Wnt-pathway activators rather than noggin or BVg1. The reversed embryo provides a direct correlation between the dorsal determinants of the vegetal cortical cytoplasm, the transcriptional activation of Xnr3 and Siamois, and the nuclear accumulation of b-catenin protein. A similar correlation between the dorsal determinants and activation of the Wnt pathway is seen in embryos that are derived from UV-treated eggs. The UV-treated egg retains the dorsal axis forming activity at the vegetal pole, as demonstrated by cytoplasmic transfer (Fujisue et al., 1993; Holowacz and Elinson, 1993). Vegetal pole cells of the UV-treated embryo also express Siamois (Brannon and Kimelman, 1996; Cui et al., 1996) and have b-catenin protein translocated into nuclei (Schneider et al., 1996). Therefore, the dorsal determinants may activate the Wnt-signaling cascade regardless of their location in the embryo. Our cytoplasmic transfer results indicate that the cortical dorsal activity remained with the unpigmented cortex in the reversed embryos. This result suggests that the dorsal determinants are firmly associated with the vegetal cortex and allows us to estimate their distance from the surface. Work by others has shown that after inversion of the egg, the germ plasm remained localized at the unpigmented surface and is partitioned into reversed animal cap cells. The reversed embryos contained a reduced number of the primordial germ cells (PGCs), probably as a result of the displacement of the germ plasm away from its original vegetal pole location (Neff et al., 1983; Wakahara et al., 1984; Cleine and Dixon, 1985; Cleine, 1986; Jurand and Dixon, 1986). Xcat2 mrna appears to be associated with the germ plasm (Forristall et al., 1995; Kloc and Etkin, 1995). In our inverted eggs, Xcat2 mrna stayed mostly at the reversed animal pole but some did not, indicating that the germ plasm tended to remain with the unpigmented surface. Since the germ plasm is distributed within 25 30 mm from the egg surface (Ressom and Dixon, 1988), the bulk of the cortical dorsal determinants must be distributed within this distance from the egg surface. Kageura s (1997) recent dem- onstration of dorsal determinants in isolated cortex also indicates that the determinants are localized very close to the surface. Injection of Vg1 mrna directs synthesis of Vg1 precursor protein but not a mature product, resulting in no effect on normal development. In contrast, the BVg1 mrna can ACKNOWLEDGMENTS We thank A. Shinagawa, who presented experiments on the centrifugally reversed embryo at the Annual Meeting of the Japanese Society of Developmental Biologists (1995), for inspiring Y.M. We are grateful for the invaluable gift of the anti-b-catenin antibody from P. Hausen. We also thank M. Brannon and D. Kimelman for their technical advice on the RT PCR conditions, E. Amaya for FGF-R plasmid, E. M. DeRobertis for chordin plasmid, D. Kimelman for Mix.1 plasmid, M. L. King for Xcat2 plasmid, D. A. Melton

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