Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center

Cell. Vol. 67, 753-765,

November

15, 1991, Copyright

0 1991 by Cell Press

Injected Xwnt-8 RNA Acts Early in Xenopus Embryos to Promote Formation of a Vegetal Dorsalizing Center William C. Smith and Richard M. Harland Department of Molecular and Cell Biology Division of Biochemistry and Molecular Biology University of California Berkeley, California 94720

Summary Expression cloning from a pool of gastrula cDNAs identified the Wnt family member Xwnt-8 as having dorsal axis-inducing activity in Xenopus embryos. Microinjected Xwnt-8 mRNA was able to rescue the development of a dorsally complete anterior-posterior axis in embryos ventralized by exposure to UV light. Axis induction was observed in embryos injected in either marginal or vegetal blastomeres at the 32tell stage. Vegetal blastomeres receiving Xwnt-8 mRNA contributed progeny not to the induced dorsal axis, but to the endoderm, a result consistent with Xwnt-8 causing cells to act as a Nieuwkoop center (the vegetal-inducing component of normal dorsal axis formation), rather than as a Spemann organizer (the induced dorsal marginal zone component that directly forms the dorsal mesoderm). Xwnt-8, which is normally expressed ventrally in mldgastrula and neurula embryos, appears to mimic, when injected, maternally encoded dorsal mesoderm-inducing factors that act early in development. Introduction The unfertilized amphibian egg is radially symmetrical around an animauvegetal axis that is established during oogenesis. In Xenopus, the axis is characterized by animal and vegetal hemispheres differing in the materials they contain (Gerhart, 1980). The key early event in the establishment of dorsal/ventral polarity occurs during the first cell cycle, and involves a rotation of the cortical layers of the cytoplasm relative to the deeper cytoplasm, such that the vegetal cortex moves away from the point of sperm entry and toward the future dorsal midline (Vincent et al., 1986; reviewed in Gerhart et al., 1989). By an unknown mechanism, this early cortical rotation leads to differences in the dorsal and ventral cells, resulting later in their different abilities to induce a dorsal axis (Gimlich and Gerhart, 1964; Gimlich, 1986; Kageura, 1990). Mesoderm is induced in the marginal zone of the blastula stage embryo at the border of the animal hemisphere (which is fated to make ectoderm) and the vegetal hemisphere (which is fated to make endoderm). Factors responsible for mesoderm induction originate from the vegetal cells (Nieuwkoop, 1973; Boterenbrood and Nieuwkoop, 1973; Gimlich and Gerhart, 1984; Dale et al., 1985) and act on the marginal zone. Induction by the vegetal dorsalizing region (also known as the Nieuwkoop center; Gerhart et al., 1989) causes the overlying marginal zone cells to de-

velop as dorsal mesoderm, and this dorsal mesoderm in turn has the organizer properties discovered by Spemann and Mangold (reviewed in Spemann, 1938). The Spemann organizer forms the dorsal lip of the blastopore. Gastrulation movements are not only seen earlier at the dorsal lip, but are more extensive than on the ventral side (Gerhart and Keller, 1986). The cells that move into the embryo on the dorsal side differentiate into notochord and somites and are responsible for the induction of neural tissue. Cells invaginating on the ventral side form ventral mesoderm (e.g., blood islands). These early inductive events can be manipulated by very simple treatments that result in exaggerated ventral or dorso-anterior development. These treatments have proved to be very useful in the study of mesoderm induction. Cortical rotation can be blocked by a number of agents (UV irradiation of the vegetal hemisphere, cold, pressure), all of which act by disturbing microtubule function (Scharf and Gerhart, 1983; Elinson and Rowning, 1988). Embryos that have not undergone cortical rotation develop normal amounts of mesoderm (Cooke and Smith, 1967) but all of the mesoderm is of ventral character. It thus appears that cortical rotation promotes the formation of the Nieuwkoop center in the vegetal hemisphere and that in the absence of any cytoplasmic rearrangements, induction of ventral mesoderm still takes place throughout the marginal zone. Treatments that do not completely block microtubule function, such as less than maximal doses of UV light, result in embryos with anterior dorsal defects. The extent of cortical rotation is directly related to the amount of organizer formed, which is in turn related to the anterior extent of dorsal development (Stewart and Gerhart, 1990). Although ventralized embryos have lost the ability to form dorsal mesoderm on their own, transplantation of vegetal or marginal zone cells from the dorsal quadrant of blastula stage embryos into ventralized recipients can restore dorsal axis formation (Gimlich and Gerhart, 1964; Gimlich, 1986) showing that the ventralized embryos merely lack proper inductive signals. An important distinction between transplanted Nieuwkoop center and Spemann organizer is that cells derived from the Nieuwkoop center do not populate the rescued axis, but only provide the necessary inducing factor(s); in contrast, cells derived from the Spemann organizer directly populate the most dorsal mesoderm of the body axis (Gimlich and Cooke, 1983). Treatment of blastula stages with lithium ion can promote the formation of dorsal mesoderm, resulting in hyperdorsalized, excessively anteriorized embryos (Kao et al., 1986; Kao and Elinson, 1988). Localized injection of lithium ion can thus restore dorsal axis formation to UV-ventralized embryos (Kao and Elinson, 1989). Lithium ion is thought to act to increase the responsiveness of cells to vegetal inductive signals, so that both ventral and dorsal types of inductions result in the formation of dorsal marginal zone, i.e., an expanded organizer (Kao and Elinson, 1986, 1989; Slack et al., 1988). At the molecular level, it is thought that mesoderm induc-

Cd 754

Table 1. Dorsal Size-Fractionated

Axis-Rescuing LiCI-Treated

Activity of Microinjected Stage 11 Poly(A)+ RNAs Stage 11 Embryos in UV-Ventralized Embryos

A.

Injection

DAI

n

Percent

1.

None (n = 66)

o-1 2-3 4-5

65 3 0

9.5 5 0

2.

HP0 (n = 56)

o-1 2-3 4-5

46 6 2

67 11 2

3.

Stage

11 poly(A)+

o-l 2-3 4-5

45 6 0

66 12 0

4.

Stage

11 LiCl poly(A)’

O-l 2-3 4-5

26 22 0

54 46 0

5.

Stage

11 UV poly(A)t

O-1 2-3 4-5

23 1 1

92 4 4

0.

Injection

DAI

n

Percent

1.

None

O-l 2-3 4-5

63 16 0

64 16 0

2.

Fraction

6 library

O-l 2-3 4-5

26 16 1

56 40 2

3.

Fraction

IO library

o-1 2-3 4-5

36 9 0

61 19 0

RNA (n = 51)

RNA (n = 46)

RNA (n = 25)

(n = 99)

RNA (n = 45)

RNA (n = 47)

and In Vitro Transcripts

from Two Libraries

of

(A) Dorsal axis-rescuing activity of microinjected stage 11 poly(A)+ RNAs in UV-ventralized embryos. (6) Dorsal axis-rescuing activity of microinjected in vitro transcripts from two libraries of size-fractionated LiCI-treated stage 11 embryos in UV-ventralized embryos. Fraction 6 library was made from RNAs that were approximately 1.3 to 1.9 kb and fraction 10 library was made from RNAs that were approximately 0.7 to 0.95 kb. As controls, UV-ventralized embryos were either not injected (%one”) or were microinjected with water only (“H&“). The degree of dorsal axis rescue is graded according to the dorso-anterior index (DAI) described previously (Kao and Elinson, 1966).

tion is mediated by polypeptide growth factors, including members of the transforming growth factor p (TGFj3) and fibroblast growth factor (FGF) families (reviewed in Smith, 1989; Dawid et al., 1990). It was originally observed that treatment of explanted blastula stage animal caps with media conditioned by the Xenopus cell line XTC could induce dorsal mesoderm (Smith, 1987). The factor in this conditioned medium was later identified as activin A (Smith et al., 1990). Animal explants (animal caps) treated with activins can differentiate into miniature embryos with well-defined axes (Thomsen et al., 1990; Sokol and Melton, 1991). Several TGFj3 family members have been identified in Xenopus embryos (Weeks and Melton, 1987; Thomsen et al., 1990). FGFs in these assays appear to induce ventral and lateral mesoderm, while activins induce more dorsal mesoderm (Green et al., 1990). As a strategy for identifying endogenous RNAs involved in dorsal patterning (dorsalizing RNAs), we have used RNA injections to rescue dorsal development in embryos that were ventralized by UV irradiation. Although our initial motivation was to identify neural-inducing factors, the assay would also identify molecules that act earlier to induce dorsal mesoderm. We have constructed a plasmid cDNA library from a size-selected fraction of RNA from dorsalized embryos (UC1 treated). The library was con-

strutted to allow the in vitro synthesisof synthetic messenger RNA using SP6 RNA polymerase. Transcripts made from pools of library plasmid were microinjected into UVventralized embryos. By a process of sib selection, single clones with dorsalizing activity were isolated. Here we report on the identification and characterization of one such clone, which is identical to the previously isolated Xwnt-8 (Christian et al., 1991 a, 1991 b). Xwnt-8 RNA has also been found to possess axis-rescuing activity by Sokol et al. (1991). Two classes of dorsalizing RNAs can be imagined, those that mediate the formation of a “Nieuwkoop center” and those that mediate the formation of the Spemann organizer. As described earlier, the Nieuwkoop center and Spemann organizer populate different areas of the mature embryo; thus, the category of axis-rescuing activity can be determined by tracing the fate of injected blastomeres. The data presented here suggest that Xwnt-8 can mimic the effects of maternal factors that act early in the embryo as part of the Nieuwkoop organizer. Results Identification and Cloning of Dorsalizing mRNAs In initial experiments we assayed poly(A)+ RNAs from a

;;;t-8

A

RNA Promotes

Vegetal

Dorsalizing

Center

Formation

1 2 3 4 5 6 7 8 9 1011 12131415t6 ,21,200 ) 5.000 ~~~ - 4.270 - 3,500

Figure

1. Size-Fractionated

Poly(A)’

RNA from LiCl Embryos

and N-CAM

-

1,950 1,580 1,330

-

980 830

-

125

Induction

(A) Size-fractionated poly(A)t RNA from LiCl embryos. Sixty micrograms of poly(A)t RNA from LiCl embryos was size fractionated on a lO%-30% sucrose gradient in the presence of methylmercuric hydroxide. One-twentieth of each of the 16 fractions collected was electrophoresed on a formaldehyde-containing agarose gel. Nylon filter blots were made from the gel and probed with a “P end-labeled deoxythymidine 17-mer. An autoradiograph of the resulting blot is shown. (6) N-CAM induction by size-fractionated LiCl embryo poly(A)t RNA. Four-cell stage UV-ventralized embryos were microinjected with approximately 10 ng of size-fractionated poly(A)+ RNA. Total RNA was prepared from pooled embryos (32-62 per pool) at the tailbud stage. The RNA equivalent of 2.5 embryos was electrophoresed on agarose/formaldehyde gels. Nylon filter blots were probed both for N-CAM and for EFl-a mRNAs. RNA prepared from UV-ventralized embryos microinjected with RNA from fractions 7-9 is shown. Also shown is RNA from noninjected UV-ventralized and normal embryos.

number of sources for their dorsalizing effects after injection into UV-ventralized embryos. Embryos were injected in one cell at the 4-cell stage with approximately 10 ng of RNA and allowed to develop to the tailbud stage (stage 28-32; Nieuwkoop and Faber, 1987). The embryos were then examined for the presence of dorsal/anterior structures, and the results are summarized in Table 1A. The dorsal axial defects in our injected embryos could be described on a scale of progressive anterior truncations, as has been reported previously in a number of studies. Embryos were graded for degree of axis rescue using the dorso-anterior index (DAI) of Kao and Elinson (1988). According to this scale, the most severely affected embryos (those having no dorsal structures) are given a DAI score of 0, while normal embryos are given a DAI score of 5. No dorsal axis-rescuing activity was observed with injected poly(A)’ RNA from oocytes or eggs, nor from control or UV-treated gastrulae. However, poly(A)‘RNA from gastrulae that had earlier been exposed to LiCl (LiCI gastrula RNA) was able to rescue part of the dorsal axis in a number of embryos (Table 1A). This rescue was consistent in several experiments and was apparent as an increase in the percentage of embryos showing an intermediate degree of dorso-anterior development (DAI 2 and 3), accompanied by a decrease in the percentage of the most severely ventralized embryos (DA1 0 and 1). The DAI 2-3 embryos had extensive development of dorsal trunk and hindbrain

structures, but were lacking anterior head structures such as eyes and cement gland. The embryos injected with LiCl gastrula RNA were compared with the other groups of injected embryos by Northern blot analysis and showed an increase in neural cell adhesion molecule (N-CAM) mRNA (not shown), a marker of dorso-anterior development (Kintner and Melton, 1987). LiCl RNA-injected embryos also showed occasional expression of En-2 (engrailed) protein (data not shown; En-2 is a marker of the midbrain/ hindbrain boundary; Hemmati-Brivanlou and Harland, 1989; Hemmati-Brivanlou et al., 1991). The rescuing activity could be a single species of mRNA or a complex mixture, so to test whether the active mRNA fell into a single size class, 60 ug of LiCl gastrula RNA was size fractionated on asucrose gradient (Figure 1A). The 16 fractions from the gradient were concentrated and injected into UV-ventralized embryos. A single peak of rescuing activity was found that contained RNA of sizes approximately 1.3-l .9 kb (fraction 8). By Northern blot assay, the peak of activity was sharp, with only fraction 8 able to rescue significant amounts of N-CAM expression (Figure IB). TWO plasmid cDNA libraries were made from the sizefractionated RNA, one from fraction 8, which contained the axis-rescuing activity, and the other from fraction 10, which contained RNAs of -0.7 to 0.95 kb, to be used as a negative control. The cDNA was synthesized to allow

for directional cloning into the vector pGEMdZf(-), which placed the 5’end of the cDNAs next to the SP6 RNA polymerase promoter (see Experimental Procedures). After amplification, DNA was extracted from a portion of the library, with the rest being saved for replating. Capped RNA transcripts were synthesized from starting pools of 100,000 independent clones from the two libraries. The axis-rescuing activity of the two pools of RNA transcripts was assessed also as described above, and the results are summarized in Table 1 B. The RNA transcripts made from the fraction 8 cDNA library were able to rescue dorsal axis formation in a portion of the ventralized embryos, as was seen by an increase in the number of embryos having DAI scores of 2-3. No difference was seen between those embryos injected with RNA transcripts made from the fraction 10 library and those receiving no injection. Single clones with dorsalizing activity were isolated by a process of sib selection. Both the degree of axis rescue and the percentage of embryos rescued increased as progressively smaller pools of RNA transcripts were injected (see Experimental Procedures). Sequence analysis of the isolated active clone showed it to be identical to the previously identified Wnt family member Xwnt-8 (Christian et al., 1991a), starting at nucleotide 15 of the Xwnt-8 sequence (J. L. Christian et al., personal communication; submitted to GenBank). Is Xwnt-8 the Only Axis-Rescuing Activity in LiCl Gastrula RNA? The fact that we could only detect axis-rescuing activity with total poly(A)+ RNA from LiCI-treated embryos was surprising since LiCl treatment actually reduces the abundance of Xwnt-8 transcripts relative to control and UV gastrulae (Christian et al., 1991a, 1991 b and below). To test whetherxwnt-8 issolelyresponsiblefor rescue, wespecifitally degraded Xwnt-8 RNA using short anti-sense Xwnt-8 oligonucleotides to direct in vitro RNAase H cleavage of the coding region. Oligonucleotide-mediated destruction was verified by Northern blot analysis. Removal of Xwnt-8 mRNA from either the starting pool of fraction 6 library transcripts, or poly(A)+ RNA from stage 11 LiCI-treated embryos, did not significantly reduce their axis-rescuing activity in injection experiments (not shown). In control experiments, ovary poly(A)+ RNA (which alone has no axis-rescuing activity) was supplemented with synthetic Xwnt-8 mRNA; oligonucleotide/RNAase H cleavage destroyed axis-rescuing activity in this sample (data not shown). These experiments indicated that Xwnt-8 was not the sole component of the dorsal axis-rescuing activity in poiy(A)+ RNA from stage 11 embryos. The reason why Xwnt-8 was isolated in this screen of the library rather than another (apparently more potent or abundant) dorsalizing activity (activities) is not clear. However, since Xwnt-8 apparently was not the sole dorsalizing activity in our library we should be able to isolate dorsalizing activities up-regulated in LiCI-treated embryos. Quantitative Effects of Xwnt-8 mRNA Injection Purified Xwnt8 mRNA in amounts from 1 to 1000 pg was injected into UV-ventralized embryos at the 4-cell stage,

Figure

2. Dorsal

Axis

Rescue

by Xwnt-8

UV-ventralized embryos were microinjected with increasing amounts of Xwnt-8 encoding mRNA at the Qcell stage. Representative tailbud stage embryos are shown. (A) Normal embryo (no UV treatment or injection). (B) Uninjected UV-ventraked embryo. (C) UV-ventralized embryo injected with water alone. (D-G) UV-ventralized embryos microinjected with 1, 10, 100, and loo0 pg of Xwnt-g-encoding mRNA, respectively. Open arrow in (D) indicates posterior dorsal fin. Closed arrows in (F) and (G) indicate enlarged cement glands.

and representative tailbud stage embryos for each dose are shown in Figure 2. A control embryo of the same stage is shown in Figure 2A, and an uninjected UV-irradiated embryo is shown in Figure 26. Injection of water alone had no effect on the axis (Figure 2C). At 1 pg per embryo, Xwnt-8 RNA gave a range of effects from either no apparent dorsal axis rescue (DAI 0), to induction of posterior dorsal axial structures (DAI l-2) (Figure 2D). In no cases were anterior head structures apparent. Xwnt-8 RNA microinjected at 10 pg resulted in embryos either nearly normal in appearance or with slightly reduced heads (DAI 4-5) (Figure‘2E). Embryos receiving 100 pg of Xwnt-8 mRNA ranged from nearly normal in appearance to slightly hyper-dorsoanteriorized (DAI 5-6). The embryo pictured in Figure 2F, having received 100 pg of mRNA, showed the bent axis characteristic of mildly affected LiCI-treated embryos (Kao and Elinson, 1988). Injection of the highest dose of RNA (1000 pg) resulted in dorsoanteriorized em-

Xwnt-8 757

RNA Promotes

stage

Normal

r

8

Vegetal

Dorsalizing

Center

Formation

9 10 11 11.5 12 13 14 15 20 25 Xwnt-8

lstage

8

9 10 1111.5 12 13 14 15 20 25

r :

/‘

Xwnt-8

LiCl embryos EFl-a lstage

8

9 10 11 11.5 12 13 14 15 20 25 Xwnt-8

EFl-u

Figure 3. Northern Slot Analysis LiCI-. and W-Treated Embryos

of Developmentally

Staged

Normal,

RNA was prepared from ten embryos from the indicated stages and treatments. The RNA from 2.5 embryos was loaded on each lane. An autoradiograph of a filter hybridized with random-primed Xwnt-8 and EFl-a probes is shown.

bryos (DA1 7-8) (Figure 2G). These embryos had greatly reduced trunk structures and many had small rudimentary tails. In addition, anterior head structures (e.g., cement gland) were enlarged. Expression of Xwnt-8 mRNA in Normal, LiCI-Treated, and UV-Treated Embryos We were surprised to have isolated Xwnt-8 in our screen for dorsalizing factors since a previous report had shown a complete absence of Xwnt-8 in LiCI-treated Xenopus embryos at stage 11 (Christian et al., 1991 b). The results of Northern blot analyses for Xwnt-8 mRNA of developmentally staged normal, UV-, and LiCI-treated embryos are shown in Figure 3. Each RNA sample represented a pool of ten embryos. The average DAIS for the UV- and LiCI-treated embryos were 1.08 and 9.13, respectively. Filters were hybridized with both Xwnt8 and EFl-a probes. The EFl-a probe allowed for comparison of RNA loading between samples from the different treatments (i.e., normal, UV treated, and LiCl treated) at the same developmental stages (Krieg et al., 1989). The autoradiograph shown in Figure 3 was intentionally overexposed to show the EFl-a signal at stages 8 and 9. Lighter exposure of the blot showed that later stages are equally loaded. The Xwnt8 probe identified an RNA of approximately 1.5 kb. Detectable levels of Xwnt-8 mRNA were first seen for all types of embryos (i.e., normal, UV treated, and LiCl treated) beginning at approximately stage 9. The amount of Xwnt8 mRNA in UV-treated embryos was higher than

in normal or LiCI-treated embryos, at all stages. Xwnt-8 mRNA was detectable in LiCI-treated embryos, although at lower levels at all stages analyzed. Hybridization of an Xwnt-8 probe to other filters loaded with LiCI, UV, and control RNAs confirmed these results. The peak of expression of Xwnt-8 mRNA in normal embryos was seen at the gastrula and early neurula stages, and was substantially reduced in older embryos. The apparent increase in expression in normal embryos at stage 20 was not reproducible and was not reflected by in situ analysis. The effects of LiCl and UV on Xwnt-8 expression are consistent with the observation that Xwnt-8 is normally expressed in the ventral mesoderm of gastrulae (Christian et al., 1991 b). We have confirmed and extended these observations using hybridization of digoxigenin-labeled RNA probes to control and perturbed embryos. In particular, we have focused on the extent of Xwnt-8 expression around the circumference of the marginal zone. A series of whole-mount in situ hybridizations is shown in Figure 4. For a detailed description of gastrulation movements see Keller (1975), Gerhart and Keller (1986), and Hausen and Riebesell(l991). Figures 4A, 48, and 4C show views from the vegetal pole. Xwnt-6 is not detected in stage 8 blastulae (Figure 4A; these also serve as a negative control for hybridization background). In the late blastula (stage 9-l 0; Figure 48) Xwnt-8 expression is detected as a band of cells in the marginal zone. Specific staining was apparent as discrete soots distinct from the patchy background staining seen in the blastocoel. Higher magnifications showed that specific staining was localized to nuclei. At this stage staining extends around most of the marginal zone, but is excluded from a narrow zone whose size is consistent with the measured size of the organizer (Stewart and Gerhart, 1990). Shortly afterward, expressing cells become restricted to the ventral portion of the marginal zone (Figure 4C) and subsequently to posterior ventral tissue (Figures 4D and 4E, viewed from the left side; Figure 4F, viewed from the dorsal side). In the midneurula, expression falls but persists in the posterior ventral region and two small areas lateral to the anterior neural plate (square in Figure 4F). Expression is detected at a low level until stage 26, where it forms a band of cells that extend around the belly, anterior to the blastopore. A second phase of expression then starts in the brain, a few scattered cells are detected in the brain at stage 28, and by stage 36 a small number of cells are detected in the forebrain (Figure 46) and the posterior tip of the spinal cord (not shown). These cells are variable in number and position, but were detected in all animals examined. In situ hybridization to LiCI- and UV-treated embryos confirms the findings of the Northern blot experiments. Figure 4H shows a vegetal view of a stage 11 LiCl embryo with no detectable expression. Figure 41 shows a UVtreated embryo at stage 11. As would be predicted from the ventralizing consequences of UV irradiation, Xwnt-8 staining of the mesoderm extends around the entire marginal zone of the animal. Not all embryos show such extreme effects, presumably reflecting the range of phenotypes that result from dorsalizing and ventralizing treatments.

Cdl 750

Figure

4. In Situ Hybridization

to Normal,

LiCI-, and UV-Treated

Embryos

Plain arrows indicate the edge of the blastocoel; arrowheads indicate the dorsal lip of the blastopore (or the lip of the blastopore for LiCl and UV embryos); open arrows show the anterior limit of the archenteron. (A) Stage 8 blastula; no staining. (6) Stage 9-10 blastula. Dorsal is at the top. Nuclei around the marginal zone are stained. The dorsal quadrant lacks detectable staining (limits of staining region shown with bars). Some background staining is evident in the blastocoel of (B) and (D) (see Harland, 1991). (C) Stage 11 gastrula. Dorsal is at the top. Staining is limited to the ventral side in the marginal zone. Lateral views of such embryos are consistent with the sectioned embryo presented in Christian et al. (1991 b). (D) Stage 12 late gastrula, lateral view. The anterior limit of the archenteron is indicated. Staining is absent from the yolk plug and endoderm, but otherwise extends in two “wings” around the closing blastopore. (E) Stage 14 early neurula, lateral view. The blastocoel collapses during neurulation. Staining is limited to the posterior ventral tissue. (F) Stage 16 neurula, dorsal view. Head is to the left. Arrowhead points to the remnant of the blastopore. Photograph is in the plane of the notochord, which extends between the arrowhead and the open arrow. Staining is excluded from the mesoderm below the neural plate, but residual staining is widespread in the posterior ventral mesoderm. Two patches of staining (one of which is marked with a square), are evident lateral to the anterior neural plate. (G) Head of stage 36 tadpole. Staining cells are visible next to the x. (H) Stage 11 gastrula from LiCI-treated batch of embryos. The blastopore (arrowhead) closes vigorously in such embryos, leading to a puckered appearance. No staining is evident (compare with [Cl). (I) Vegetal view of a stage 11 UV-treated gastrula. The blastopore appears late in UV embryos. Staining surrounds the marginal zone of this embryo (compare with [Cl).

Xwnt-8 759

RNA Promotes

Vegetal

Dorsalizing

Center

Formation

The apparent nuclear localization of Xwnt-8 transcripts at early stages has been noted before (A. McMahon, personal communication), but is particularly clear using the whole-mount stain. This raises the question whether nuclear localization may have some regulatory role. Other transcripts appear to have nuclear restriction in the marginal zone at early stages (XMyoD, Frank and Harland, 1991; Mix-l, Ft. M. H., unpublished data; Rosa, 1989) but others do not show nuclear restriction (e.g., DG42, R. M. H., unpublished data; Rosa et al., 1988). We suppose that the rate of processing of the primary transcript will determine the cellular location of transcripts at this stage. Xwnt-8 Injection Acts Early to Rescue the Axis, and Injected Cells Behave as a Nleuwkwp Center The expression patterns observed for Xwnt-8 mRNA appeared to be very much at odds with the ability of microinjetted exogenous Xwnt-8 mRNA to induce dorsal axis formation. Specifically, the elevated levels of Xwnt8 mRNA in UV-treated embryos and the ventral location of the transcript are not expected of a dorsalizing RNA. Because detectable levels of Xwnt-8 mRNA are not seen until approximately stage 9, while the injection of Xwnt-8 was at the 4cell stage, it was conceivable that the time of action of Xwnt-8 is important in dorsal axis rescue. As speculated previously, it is possible that the normal role of Xwnt-8 is not in axis formation, but that injection of Xwnt-8 mimics the effects of a maternal Wnt-related molecule (Christian et al., 1991 a, 1991 b). A series of experiments was undertaken to examine the effects of varying the time and location of injection of Xwnt-8 mRNA. UV-treated embryos were microinjected with Xwnt-8 mRNA at stage 8 (the 32cell stage) and stage 8. At stage 8 the blastomeres are arranged in four tiers of 8 cells each. To follow the fates of cells injected with Xwnt-8 mRNA, mRNA encoding bacterial 8galactosidase was injected along with the Xwnt-8 mRNA. Injected mRNA should act as a more faithful lineage tracer for the Xwnt8 than would fluoresceinated dextran, since it is known that dextrans diffuse quickly to fill the injected cell, whereas RNA diffuses relatively slowly (Vize et al., 1991). To facilitate the identification of P-galactosidase activity encoded by the injected mRNA, the protein carried a nuclear localization signal (Picard and Yamamoto, 1987). We illustrate the results of control injections of 8-galactosidase mRNA into single blastomeres of normal stage 8 embryos in Figures 5A-5C. Significantly higher mortality was seen in embryos injected in vegetal blastomeres, owing to the leakage of cytoplasm from the site of injection. The injected embryos were allowed to develop to the tailbud stage, and the presence of 8-galactosidase protein was visualized by staining whole-mounted embryos with X-gal. A light blue background in the endoderm was seen in all embryos as a result of X-gal staining. However, because the bacterial P-galactosidase protein was localized to the nucleus, the specific staining was easy to distinguish. The staining patterns are entirely consistent with the fate maps of the 32-Cell stage embryo (Dale and Slack, 1987a; Moody,

1987). Injection of 8-galactosidase mRNA into the animal tier (tier 1) of cells resulted in embryos with X-gal staining cells in the anterior ectoderm, although occasionally some staining cells were seen in the mesoderm (Figure 5A). X-gal staining was observed in endodermal cells from embryos injected in vegetal blastomeres (tier 4) while mesodermal cell staining was predominant in embryos injected in either of the two marginal tiers of cells (tiers 2 and 3) (Figures 58 and 5C). Because no distinction was made between dorsal and ventral blastomeres in these injections, the location of staining cells within the tissue layers among embryos was somewhat variable. The results from coinjection of 10 pg of Xwnt-8 and 0.5 ng of f3-galactosidase mRNAs into these three locations in stage 8 UV-ventralized embryos are summarized in Table 2, and representative embryos are shown in Figure 5. Only those embryos with X-gal staining cells are included in the numbers presented in Table 2 (with the exception of the uninjected controls). The results from two separate experiments are presented in Table 2. Control UV embryos were injected in either of the two middle tier blastomeres with 8-galactosidase mRNA alone (Figure 5D). The average DAI of these embryos was no different from uninjected embryos. Very little dorsal axis rescue was seen in embryos injected with Xwnt-8 mRNA in single animal pole (tier 1) blastomeres. X-gal staining confirmed that these embryos had been injected with the mRNAs at levels comparable with the other groups of embryos (Figure 5E). Injection of Xwnt-8 mRNA into either single marginal or vegetal pole blastomeres was very effective in rescuing dorsal axis formation (Table 2). Many embryos with normal, or nearly normal, dorsal axis formation were observed. X-gal staining cells were observed in the rescued dorsal mesoderm for embryos receiving marginal blastomere injections (Figure 5F). However, X-gal staining was not restricted to dorsal mesoderm, and in many cases staining extended into the ventral side of the animal. This type of result was much more striking for embryos that were injected in thevegetal cells. In embryos rescued byvegetal blastomere injections, X-gal staining was almost exclusively in the endoderm, and no staining was found in the dorsal mesoderm (Figure 5G). This pattern of staining is what would be expected for formation of a Nieuwkoop center, orvegetal dorsalizing region, from the injected cell. The effectiveness of rescue after injection into later stage embryos was also tested. Stage 8 embryos were coinjected with Xwnt-8 and 8-galactosidase mRNA in vegetal and marginal blastomeres. The blastomeres at stage 8 are significantly smaller than at stage 8. For these injections a group of 4-5 blastomeres in a small area was injected with approximately0.25 nl, rather than a single blastomere with approximately 1 nl as was done at stage 6. Because of the particularly small size of the blastomeres in the marginal zone, marginal blastomeres were generally injected at positions further below the equator than they were at stage 6. Again, X-gal staining of these embryos showed that the amount of the injected mRNAs was comparable with embryos injected at stage 6 (Figures 5H and 51). Injections of Xwnt-8 at stage 8 were much less effective

Cdl 760

Xwnt-8 761

RNA Promotes

Table 2. Results

from

Vegetal

Coinjection

Dorsalizing

Center

of Xwnt-8

Formation

and f3-Galactosidase

mRNAs

in Stage

6 UV-Ventralized

Embryos

DAI n

Stage 6

0

1

2

3

4

5

Average

41 43

31 36

7 2

2 2

1 0

0 1

0 0

0.34 0.23

28 40

24 32

1 3

1 3

2 2

0 0

0 0

0.32 0.37

11 21

9 11

1 3

0 3

0 0

0

3

0.27 1.09

9 42

0 0

0 3

1 0

7 18

0 8

5

3.00 3.09

4 36

0 0

0 2

0 4

3 9

1 9

0 12

3.25 3.69

8 43

6 29

0 6

2 5

0 3

0 0

0 0

0.50 0.58

12 23

5 10

0 4

2 7

2 2

2 0

1 0

1.92 1.04

1. Uninjected (1)

(2)

2. 6-Galactosidase

only/marginal

(1)

(2) 3. Xwnt-8

+ t%galactosidase/animal

(1)

(2) 4. Xwnt-8

+ f3-galactosidaselmarginal

(1)

(2) 5. Xwnt-9

+ 6-galactosidaselvegetal

(1)

(2) Stage

0

6. Xwnt-6 (1)

+ j3-galactosidaselvegetal

(2) 7. Xwnt-8 (1)

+ j%galactosidase/marginal

(2)

UV-ventralized embryos were injected at stages 6 and 8 with IO pg of Xwnt-8 and 0.5 ng of 5-galactosidase mRNAs either in animal (tier l), vegetal (tier 4) or marginal (tier 2 or 3) blastomeres. Stage 6 embryos were injected with mRNAs in a volume of approximately 1 nl in a single blastomere. A group of 4-5 blastomeres in small area were injected with approximately 0.25 nl each in stage 6 embryos. Control stage 6 embryos were injected with j3-galactosidase mRNA alone into marginal blastomeres. The degree of dorsal axis rescue is graded according to the dorso-anterior index (DAI) described previously (Kao and Elinson, 1988). A few mildly hyperdorsalized embryos (DAI 6) were observed and were included in the DAI 5 column. The results of two separate experiments are shown.

in rescuing dorsal axis formation than were injections at stage 6 (Table 2). Marginal zone injections appeared to be somewhat more effective then vegetal injections. Discussion Xwnt-8 Injection Rescues Anterior Dorsal Structures in UV-Ventralired Embryos Starting with a messenger RNA expression library, we have isolated an activity that induces formation of dorsoanterior structures in ventralized embryos. Similar expression cloning strategies have been used to clone a number of cDNAs by injection of mRNAs from libraries into Xenopus oocytes (Noma et al., 1986). This activity is Xwnt-6, which was previously isolated in a screen for new Writ genes (Christian et al., 1991a, 1991b). Here we have shown that Xwnt-6 injection does not simply act on an existing dorsal organizer to yield split axes (McMahon and Moon, 1989) or to increase the amount of dorsal tissue

Figure

5. Xwnt-8/B-Galactosidase

mRNA

formed (Christian et al., 1991 b). Instead, Xwnt-8 injection can rescue axis formation in ventralized embryos, and therefore must cause formation of a new axis. The completeness of the anterior-posterior series of tissues formed depends on the amount of organizer made in the embryo (Stewart and Gerhart, 1990; Gerhart et al., 1989). Our results suggest that Xwnt8 causes formation of organizer in a dose-dependent fashion. The degree of rescue depends on the dose of Xwnt-6 RNA injected; not only can a complete axis be rescued, but injection can result in embryos with excessive dorso-anterior structures. In contrast to Xwnt-8, activin alone cannot rescue a full complement of dorso-anterior structures; treatment of ventral animal caps with soluble activin does not induce eyes (Sokol and Melton, 1991) though it is sufficient to induce neural structures up to the hindbrain level (M. E. Bolce, A. Hemmati-Brivanlou, and R. M. H., unpublished data). Injection of activin RNA can give partial axis duplication (Thomsen et al., 1990) or partial rescue of ventralized

Coinjection

(A-C) Animal (tier 1) (A), marginal (tier 2 or 3) (S), and of f%galactosidase mRNA. (D-G) Injections of mRNAs into UV-ventralized stage blastomeres with 0.5 ng of P-galactosidase plus 10 pg Xwnt-8 mRNAs. (G) Vegetal blastomeres with 0.5 ng of with bent axis. (H and I) Injection of mRNAs into UV-ventralized stage or vegetal (I) blastomeres.

vegetal

(tier 4) (C) blastomeres

of normal

stage

6 embryos

were microinjected

with 0.5 ng

6 embryos. (D) Marginal blastomeres with 0.5 ng of 6-galactosidase mRNA. (E) Animal of Xwnt-6 mRNAs. (F) Marginal blastomeres with 0.5 ng of 6-galactosidase plus 10 pg of 6-galactosidase plus 10 pg Xwnt-8 mRNAs. Figure shows slightly hyperdorsalized embryo 8 embryos

with 0.5 ng of 5-galactosidase

plus 10 pg of Xwnt8

mANAs

into marginal

(H)

Cell 762

embryos(Sokol et al., 1991). Thus, only Wnt products have been shown to rescue a complete axis (Sokol et al., 1991; this work). Xwnt-8 Acts Early to Form a Vegetai Dorsaiizing Center (Nieuwkoop Center) Xwnt-8 could mimic an activity that is normally fnade by Spemann organizer cells or could act early to promote formation of the organizer. The results of our lineage tracing experiments with coinjected Xwntd and P-gaiactosidase mRNAs clearly show that Xwnt-8 is effective in dorsal axis induction when present in vegetal cells. These injected vegetal cells fate map to the endoderm and not to the rescued dorsal axis itself. This is the normal fate of dorsal vegetal blastomeres in embryos (Gimlich, 1986; Dale and Slack, 1987a; Moody, 1987). These results suggest that Xwnt8 injection can directly cause cells to become a vegetal dorsalizing center (Nieuwkoop center, Gerhart et al., 1969). Formation of the Nieuwkoop center results in the induction of the overlying marginal blastomeres to form a Spemann organizer. Xwnt-8 mRNA was also effective in rescuing dorsal axis formation when injected into marginal zone blastomeres. These injected cells did fate map to the rescued axial mesoderm. The most likely explanation is that these marginal zone cells were able to stimulate themselves to form a Spemann organizer, and that the continued expression of Xwnt-8 in the marginal zone was not necessary for the further development and elaboration of the dorsal mesoderm. However, it is possible that Xwnt8 could also be mimicking factors present in the Spemann organizer, and thus mediate the action of aspemann organizer. The latter possibility is made unlikely by our finding that Xwntd is much less effective at rescuing dorsal structures when injected into the midblastula rather than the 32-cell stage. A normal organizer can induce a considerable amount of dorsal tissue when transplanted at this stage (Stewart and Gerhart, 1990), and Xwnt-6 cannot mimic this process. This conclusion is also supported by reported differences in the effects of early biastula stage injection of Xwnt-1 mRNA versus a Xwnt-1 expression plasmid. While the injection of Xwnt-1 mRNA results in axial duplications, the microinjected Xwnt-1 expression plasmid resulted only in anterior neural defects (see McMahon, 1991). The differing effects may result from the delay in onset of transcription from the expression plasmid until the late blastula stage. In experiments combining explants of animal and vegetal pole cells, the loss of competence of the animal pole ceils to form mesoderm was found to occur at approximately stage 10% (Jones and Woodland, 1987), although the ability to form dorsal mesoderm may be lost even earlier (Green et al., 1990). A loss of competence may account for the decreased responsiveness to microinjected Xwnt-8 mRNA. We are currently unable to account for the inability of microinjected animal pole blastomeres to rescue dorsal axis formation. If secreted Xwnt-8 protein from injected vegetal blastomeres directly induces dorsal mesoderm formation in the overlying marginal zone cells, it is not clear why a similar signal could not travel from the animal

pole to the marginal zone. It is possible that Xwnt8 does not act directly to cause formation of the Spemann organizer, but some intermediate step is required. Only vegetal and marginal zone cells might be competent to complete the next step in the pathway to organizer formation. is Xwnt-8 the Endogenous Agent That Induces the Spemann Organizer? As pointed out previously (Christian et al., 1991 b), the normal time and place of expression of Xwnt8, in ventral mesoderm of the gastrula and neurula, effectively rules out its participation in early dorsal mesoderm induction. It is most likely that Xwnt8 can mimic the effect of a maternally encoded dorsal mesoderm-inducing factor. This factor may be a member of the Wnt family. However, it is also conceivable that Xwnt-8 can activate overlapping signaling pathways used by “true” dorsal mesoderm inducers. The normal role of Xwnt-8 is still not understood. Clearly, cells lose their competence to respond to Xwnt8 and make dorsal structures. Perhaps a new period of competence to respond to Xwnt-8 arises, and results in formation of ventral structures (Christian et al., 1991 b). In this connection it is interesting that Xwnt8 is initially expressed in a large fraction of the marginal zone, subsequently narrowing its expression to posterior ventral tissue. The early phase is similar to the expression of XMyoD in the marginal zone (Frank and Harland, 1991), and supports the idea that there are only two cell states in the marginal zone, organizer and ventral mesoderm (Dale and Slack, 1987b). As gastrulation proceeds, the lateral marginal zone is influenced by organizer (dorsalized) to make mesodermal cell types between notochord and blood islands (Dale and Slack, 1987b; Frank and Harland, 1991). The process of dorsalization restricts Xwnt-8 expression progressively to more ventral and posterior cells. Expression Cloning of Other Dorsaiizing Activities We were surprised to have cloned Xwnt8 from a LiCl gastrula RNA expression library. Both in situ hybridization and Northern blot analysis show that Xwnt-8 RNA is least abundant in LiCl (dorsalized) gastrula RNA and most abundant in UV (ventralized) gastrula RNA. Yet we were unable to identify dorsalizing activity in UV or control gastrula RNA. Two possibilities are evident. First, Xwnt-6 might not be the critical dorsalizing RNA in the LiCl embryos and was isolated fortuitously, and, second, there may be suppressors of dorsalizing activity in the normal and ventralized populations. We have preliminary evidence that there is at least one other dorsalizing activity in LiCl RNA, and this is currently being purified. Given appropriate assays the expression cloning approach could be applied to other questions in vertebrate development. Experimental Procedures Embryos Embryos were obtained as described (Condie and Harland, 1967). Ventralized embryos were produced by irradiating vegetal hemispheres with UV light 20-30 min following fertilization. The embryos were supported on a quartz slide supported directly above the bulbs of an inverted Stratalinker (Stratagene). The meter was set to deliver a dose of IO5 pJ/cm2 ultraviolet light (h max 254 nm).

Xwnt-8 763

RNA Promotes

Vegetal

Dorsalizing

Center

Formation

Hyperdorsalized embryos were produced by a 1 hr treatment with 0.1 M LiCl in 1/3x MR beginning at the 32-cell stage. The extent of ventralization or dorsalization was scored according to the scale given by Kao and Elinson (1988). RNA Isolation/Synthesis and Injection Total cellular RNA was isolated from Xenopus embryos using a hot phenol extraction/LiCI precipitation protocol. Five to ten milliliters of packed embryos was homogenized in 10 vol of 1% SDS, 50 mM TrisHCI (pH 8.0), 10 mM EDTA, and 0.2 M LiCI. An equal volume of aqueous phenol (pH 6) was then added. The extract was heated to 65’C for 20 min and then centrifuged at 5000 rpm for 15 min in the Sorvall GSA rotor. After a second phenol extraction, the aqueous phase was precipitated with an equal volume of isopropanol. The resulting pellet was redissolved in 8 M urea (l/10 the volume of the initial extraction buffer). RNA was then precipitated by the addition of l/3 vol of 10 M LiCI. The precipitate was collected by a 10 min centrifugation at 10,000 rpm (Sorvall SA600 rotor). The resulting pellet was washed in 70% ethanol and then resuspended in distilled water. Poly(A)’ RNA was isolated from the total RNA by oligo(dT)-cellulose chromatography (Aviv and Leder, 1972). For injection, poly(A)’ RNAs were dissolved in distilled water at 1 mglml. Capped RNAs were synthesized in vitro using SP6 RNA polymerase (Harland and Weintraub, 1985; Krieg and Melton, 1984). Pools of plasmids containing mixtures of cDNAs generated by sib selection (see below) were linearized with Notl and transcribed with SP6 RNA polymerase. Following two ethanol precipitations, the resulting pools of RNAs were resuspended in distilled water at concentrations of 5 to 0.05 mg/ml (depending on the number of clones in the particular pool). Messenger RNA encoding nuclear 8-galactosidase was synthesized with SP6 RNA polymerase from the plasmid pSPGnucj3Gal linearized withXhol(Vizeetal., 1991; R. M. H. and A. Hemmati-Brivanlou, unpublished data). This plasmid was constructed by ligating into the Hindlll site of pGEM Blue (Promega), a6 kb Hindlll fragment from the plasmid 497-524-z. The Hindlll fragment contains the 8-galactosidase gene fused in frame with the glucocorticoid nuclear localization signal and a translation initiation signal from the HSV TK gene (Picard and Yamamoto, 1987). RNA encoding 8-galactosidase was dissolved in water at a concentration of 0.5 mglml for injection. Embryos were injected with RNAs in volumes of about 10 nl (2- to 4-cell stage), 1 nl (stage 6) or 0.25 nl (stage 8). Embryos were kept in 5% ficoll before injection to encourage regular cleavage patterns (Kao and Elinson, 1989). 6Galactosidase protein was visualized in embryos using X-gal (Sanes et al., 1966). Following staining the embryos were refixed, bleached for 2 hr in 70% methanol, 10% HzOz under fluorescent light, and cleared (Hemmati-Brivanlou and Harland, 1969). Construction of cDNA Libraries and Sib Selection Sixty micrograms of poly(A)’ RNA from stage 11 LiCI-treated embryos was size fractionated on a 10% to 30% sucrose gradient in the presence of methylmercuric hydroxide (Sambrook et al., 1989). First strand cDNA was synthesized from 2 pg of the size-fractionated poly(A) RNAs primed with oligo(dT) oligonucleotide containing the recognition site for Not1 (5’~CAGACACGTAGCGGCCGCITJ,,-3’). After synthesis of the second strand (Gubler and Hoffman, 1983) cDNAs were treated with EcoRl methylase, ligated with EcoRl linkers, digested with EcoRl and Notl, and finally ligated to 125 ng of modified pGEM-5Zf(-) (Promega). The pGEM5Zf(-) used here was modified by the addition of the oligonucleotide B’CGAATTCGTGCA-3 into the Nsil site to create an EcoRl site. The vector was not treated with alkaline phosphatase, but the excised polylinker sequence was removed on a Sepharose 4BCL column (Condie and Harland, 1967). The ligated products were used to transform XL-I Blue cells (Stratagene), and plated to give 100,000 colonies per 15 cm plate. A total of 106 independent colonies was obtained. Of six randomly picked plasmids, all had inserts, ranging from 1000 to 2500 nucleotides. Plasmid DNAs were isolated from plate cultures by the alkaline-lysislpolyethylene glycol precipitation protocol (Sambrook et al., 1969). Dorsalizing activity in the library was assayed by injecting RNA transcripts made from pooled plasmid DNA (see above). Single clones were isolated by a process of sib selection. In this procedure the plasmid library was replated on 12 plates with IO-fold fewer colonies per plate. RNA was synthesized from pooled plasmid DNAs isolated from

each plate and tested for dorsalizing activity by injection into UV-ventralized embryos. Those pools with dorsalizing activity were replated and screened as described above. This process was repeated until single clones were isolated. In the second round of sib selection, 2 of 10 pools of 104 clones were positive; in the third, 2 of 12 pools of 103 clones were positive; in the fourth, 8 of 12 pools of 100 clones were positive. The fifth yielded four positive pools of ten clones, and finally one clone of these ten was positive. Northern Blot Analysis RNA was extracted from pools of ten developmentally staged embryos (Condie and Harland, 1987); the RNA from two and one-half embryos per sample was electrophoresed on formaldehyde-containing agarose gels (Sambrook et al., 1989). Nylon filter blots were hybridized also as described previously (Condie and Harland, 1967). Probes were prepared from N-CAM (Kintner and Melton, 1987) and EFl-a (Krieg et al., 1969) plasmids. For an Xwnt-8 probe, an EcoRl fragment was excised from the plasmid isolated from the LiCl RNA library. Whole-Mount In Situ Hybridlzatlon Hybridization using a digoxigenin-labeled RNA probe was done as described (Harland, 1991). A plasmid containing Xwnt-8 (Christian et al., 1991b) was linearized with BamHl and transcribed with T7 RNA polymerase.

We thank Jan Christian for aXwnt-8 plasmid and the Xwnt-6 sequence. R. M. H. thanks Bob Gimlich for helping to attempt an earlier version of the rescue experiment in 1964. We thank D. A. Melton for communicating results priorto publication, and J. C. Gerhartfor critically reading the manuscript. This work was supported by an American Cancer Society fellowship to W. C. S. and a grant from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

September

3, 1991; revised

September

23, 1991

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Xwnt-6 765

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Formation

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