Overexpression of a Homeodomain Protein Confers Axis-Forming Activity to Uncommitted Xenopus Embryonic Cells

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1 Cell, Vol. 65, 55-64, April 5, 1991, Copyright 1991 by Cell Press Overexpression of a Homeodomain Protein Confers Axis-Forming Activity to Uncommitted Xenopus Embryonic Cells Ken W. Y. Cho, Elaine A. Morita, Christopher V. E. Wright,* and Eddy M. De Robertis Department of Biological Chemistry University of California, School of Medicine Los Angeles, California Summary The anteroposterior character of mesoderm induced by a peptide growth factor (XTC-MIF) was tested by transplantation into host Xenopus gastrulae. Both retinoic acid and a homeodomain protein were able to override the anteriorizing effect of the growth factor. Microinjection of a posteriorly expressed homeobox mrna can respecify anteroposterior identity, transforming head mesoderm into tail-inducing mesoderm. Unexpectedly, overexpression of XIHbox 6 protein in the transplanted cells, without addition of growth factors, caused the formation of tail-like structures. The cells overexpressing XlHbox 6 were able to recruit cells from the host into the secondary axis. The results suggest that vertebrate homeodomain proteins are part of the biochemical pathway leading to the generation of the body axis. Introduction The question of how the anteroposterior (A-P) axis is generated has been investigated extensively in the amphibian embryos. In 1918 Hans Spemann established, by transplanting fragments of embryonic tissue, the time at which embryonic cells first become irreversibly determined to particular cell fates. Until early gastrulation all cells were totipotent (i.e., adopted the cell fate of the surrounding tissue), with the exception of the dorsal lip of the blastopore, which when transplanted resulted in the formation of a second A-P axis (Spemann, 1918). Using embryos of different pigmentation, Spemann and Hilde Mangold (1924) later found that the transplanted dorsal lip cells did not give rise to the secondary axis entirely by themselves, but were able to recruit neighboring embryonic cells from the host to form highly organized axial structures such as somites or the central nervous system. The discovery of this =organizer" activity played a central role in generating our current view of vertebrate development as a series of cell and tissue inductive events (reviewed by Spemann, 1938; Gurdon, 1987; Hamburger, 1988): The first induction to take place in the amphibian embryo is that of mesoderm. This induction is mediated by substances released by the vegetal yolky cells at the early blastula stage (Nieuwkoop, 1969; Jones and Woodland, 1987). An important recent advance was the discovery that * Present address: Department of Cell Biology, Vanderbilt University, Nashville, Tennessee purified peptide growth factors can induce mesoderm in blastula animal cap fragments cultured in vitro (Smith, 1987; Slack et al., 1987; Kimelman and Kirschner, 1987; Rosa et al., 1988). Xenopus tissue culture mesoderm-inducing factor (XTC-MIF) is a potent inducer of dorsal mesoderm (tissues such as notochord and muscle; Green et al., 1990) and has now been identified as activin A, a growth factor of the TGF-13 family (Smith et al., 1990). Basic fibroblast growth factor (bfgf) induces ventral mesoderm (such as blood, lateral plate mesenchyme, and some muscle; Green et al., 1990). Animal cap cells treated with XTC-MIF can act as Spemann's organizer after transplantation into the blastocoel cavity of an early gastrula, inducing the formation of a secondary axis including head structures (Cooke, 1989; Ruiz i Altaba and Melton, 1989). Thus, XTC-MIF is an anterodorsal inducer. Similar experiments with bfgf treatment result in secondary axes consisting only of tail structures (Ruiz i Altaba and Melton, 1989), and we have been able to confirm these observations (K. W. Y. C. and E. M. D. R., unpublished data). Thus, bfgf is considered to be an posteroventral inducer. Vertebrate homeobox genes provide useful A-P markers (reviewed by De Robertis et al., 1990; Kessel et al., 1990). Ruiz i Altaba and Melton (1989) showed that a posterior gene, Xhox 3 (which has a homeobox resembling that of Drosophila even-skipped), was preferentially induced by bfgf in animal caps. It was subsequently shown that an anterior trunk marker (XIHbox 1, probably the homolog of mouse Hox 3.3) was activated more by XTC-MIF than by bfgf (Cho and De Robertis, 1990). Conversely, in the same study a different posterior marker, XIHbox 6 (probably the homolog of mouse Hox 2.5 [Wright et al., 1990], which has a homeobox resembling that of Drosophila Abdominal-B), was shown to be preferentially activated by bfgf in cultured animal cap fragments. In addition to growth factors, retinoic acid (RA) has been implicated in providing positional information in vertebrate embryos. RA is present in the developing chick limb ('l'haller and Eichele, 1987) and causes duplication of posterior digits when RA-containing beads are implanted in the anterior wing bud (Tickle et al., 1982). In Xenopus, treatment of embryos with RA at about the time of gastrulation causes truncation of anterior head structures. Furthermore, it was shown that RA-treated neuroectoderm becomes posteriorized, but the question of whether RA had any effect on the mesoderm was not addressed in these studies (Durston et al., 1989; Sive et al., 1990). In isolated Xenopus animal cap cells RA does not induce mesoderm or activate homeobox genes when added alone. However, RA can potentiate considerably induction of the posterior homeobox gene XIHbox 6 when added together with growth factors (Cho and De Robertis, 1990). Thus RA and growth factors seem to cooperate with each other in the induction of mesoderm. The initial aim of this study was to test whether RA could override the anteriorizing effect of XTC-MIF on animal caps using the transplantation method (also known as Ein-

2 Cell 56 O / ~ I nsteck RA Q Blastula [donorl XTC-MIF "~ Early Gastrula Ihost] Figure 1. RA Acts on Mesoderm Induced by XTC-MIF Animal cap tissue isolated from stage 8 blastula embryos was treated with XTC-MIF growth factor and/or RA and transplanted into the ventral side of the blastocoel of early gastrula embryos (stage 10.5). (A) Untreated animal cap transplant; (B) RA-treated animal cap transplant: no structures are formed; (C) XTC-MIF-treated animal cap transplant: note the formation of head structure with eyes and cement gland; (D) animal cap transplant treated with both XTC-MIF and RA: a tail structure is formed. The locations of the transplanted tissue are indicated by arrows. steckung, meaning sticking-in in German) of Otto Mangold (1933). Having found this hypothesis to be true, we next tested whether microinjection of XlHbox 6 mrna (a gene whose transcripts are increased in animal caps by RA treatment) could substitute for RA in this assay. XIHbox 6 was indeed able to posteriorize XTC-MIF-treated animal caps, leading to the formation of tails instead of heads. The most interesting result, however, came from the controis for this experiment. Overexpression of the XIHbox 6 protein in animal caps was able to supplant the action of growth factors and RA in conferring axis-forming activity after transplantation, leading to the recruitment of host cells into secondary tails. The results suggest that homeobox genes not only provide A-P markers, but are important components of the biochemical pathway generating the vertebrate A-P axis. Results RA Overrides the Anteriorizing Effec~t of XTC-MIF Previous work has shown that treatment with RA affects predominantly the head region of the Xenopus embryo (Durston et al., 1989; Sive et al., 1990), and we could fully confirm these findings in our laboratory (data not shown). Although the truncations of head structures caused by RA are very striking, similar phenotypes can be obtained in Xenopus by treatments that interfere with gastrulation movements. For example, microinjection of trypan blue into the blastocoel of an early gastrula or interference with the egg cortical rotation that takes place after fertilization (by agents as diverse as UV irradiation, nocodazole, or cold treatment) can result in truncation of anterior structures (reviewed by Gerhart et al., 1989). To eliminate this

3 Homanprotein Confers Axis-Forming Activity 57 Table 1. Structures Induced by Transplantation of Growth Factor- and RA-Treated Animal Caps Treatment of Induced Secondary Structures Animal Caps Head Trunk/Tail Vesicle Normal XTC-MIF 17 (27%) 38 (61%) 3 (5%) 4 (7%) 62 XTC-MIF + RA" 0 (0%) 23 (64%) 3 (8%) 10 (28%) 36 RA 0 (0%) 0 (0%) 3 (18%) 14 (82%) 17 Untreated 0 (0%) 0 (0%) 1 (5%) 20 (95%) 21 Animal caps from stage 8 blastulae were treated with XTC-MIF and/or RA and transplanted into the blastocoel of early gastrula embryos (stage 10.5). After 2 days, secondary axes were scored. Head structures can be morphologically identified by the presence of eyes, auditory vesicles, and cement gland. Secondary tail structures were identified by the presence of spinal cord, notochord, and dorsal or ventral fin. "The experimental data were compiled from three independent experiments. Total possibility, we utilized the method developed by Ruiz i Altaba and Melton (1989), who showed that transplanted XTC-MIF-treated animal cap cells will induce secondary axes containing head structures. We used this system to directly test for the effect of RA on the A-P polarity of mesoderm. Figure 1 summarizes the result from this experiment. Animal caps are isolated at midblastula, cultured for 3 hr in vitro with or without XTC-MIF and RA, washed, and then implanted into the ventral side of the blastocoel of a host sibling embryo at the early gastrula stage. When untreated animal caps are implanted, the cells appear later as a dark patch, which is located usually in the ventral part of the embryo (Figure 1A). The untreated transplanted cells become integrated into the host embryo; lineage-tracing experiments showed that the uncommitted animal cap cells can become part of the ectoderm, mesoderm, and endoderm (data not shown). When the animal cap cells are treated with RA alone, no secondary axial structures are induced, although sometimes small protrusions or vesicles were observed (Figure 1B). Histological analysis of these vesicles revealed only ectodermal cells, confirming that RA on its own is unable to induce mesoderm (Cho and De Robertis, 1990). When XTC-MIF-treated animal cap cells were transplanted, head structures were formed (Figure 1C) in 27 /0 of the embryos (Table 1). However, when animal caps were incubated with XTC-MIF and RA before transplantation, the formation of head structures was entirely abolished, so that only tail structures were induced (Figure 1 D; Table 1). The RA concentration used for the experiments shown in Table 1 was 10 -s M, but similar results were obtained at 10-6 M. RA at 10-7 M did not prevent the formation of XTC-MIF-induced heads (data not shown), nor did it cause any teratogenic effects in intact embryos when administered as a 30 rain pulse at the beginning of gastrulation (data not shown). It should be noted that these RA concentrations are applied externally to animal caps and that the effective concentrations within cells are unknown. However, the endogenous concentration of RA in early Xenopus embryos is known and has been determined to be 1.5 x 10-7 M (Durston et al., 1989). To verify that RA at those concentrations does not cause cell death, animal caps were cultured for 24 hr and examined histologically for signs of nuclear picnosis. No signs of cell death were observed in RA-treated animal caps, even at 10 -s M, either in the presence or absence of XTC-MIF. The only difference found was that RA significantly inhibits the elongation movements induced by XTC-MIF in animal caps (data not shown). From the experiments described in this section we conclude that RA can override the head-forming (anteriorizing) activity of XTC-MIF-induced mesoderm. Overexpreseion of a Homeodomain Protein Can Change the A-P Fate of Embryonic Mesoderm In a previous study we showed that the level of XIHbox 6 mrna is increased by addition of RA to animal caps treated with growth factors (Cho and De Robertis, 1990). Because XIHbox 6 is a posteriorly expressed gene, it was conceivable that the effect of RA on the A-P character of mesoderm was mediated by the activation of homeobox genes. We directly tested this hypothesis by overexpressing XIHbox 6 protein in Xenopus embryonic cells, which were then treated with XTC-MIF. Because a full-length cdna clone was not available, an expression vector was constructed by joining cdna and genomic fragments of XIHbox 6 to Xenopus 13-globin 5' leader sequences, as described in Experimental Procedures. When capped synthetic mrna (Krieg and Melton, 1984) is transcribed from this construct and microinjected into both blastomeres of two-cell stage embryos, large amounts of XIHbox 6 protein are synthesized and migrate into the nucleus, as tested by immunostaining with specific antibodies. When these embryos are permitted to develop up to the tailbud stage, a high proportion display developmental defects, in particular heads (and eyes) of smaller size (880/0) and split (spina bifida) tails (420/0) (data not shown). The split tail phenotype is almost always accompanied by incomplete closure of the blastopore. Because small heads and split tails can also result from nonspecific interference with gastrulation movements (Gerhart et al., 1989; Holtfreter and Hamburger, 1955), simple overexpression of XIHbox 6 protein did not permit us to draw any conclusions regarding whether this protein is able to impart more posterior values to embryonic cells. To test this, we investi-

4 Cell 58 Figure 2. Expression of a Posterior Homeodomain Protein in Animal Caps Transforms Head-Inducing Transplants into Tail-Inducing Ones Animal caps incubated in XTC-MIF growth factor induce anterior (head) structures when implanted into the blastocoel of early gastrulae (A) (three embryos shown). When the animal caps are derived from embryos microinjected bilaterally with XIHbox 6 mrna at the two-cell stage and treated in the same way, tails are produced instead of anterior structures (B) (three embryos shown). Secondary axes are indicated by arrows. Quantitation of this A-P transformation experiment is shown in Table 2. gated whether X I H b o x 6 protein was able to c h a n g e t h e A - P values of XTC-MIF-induced mesoderm. Figure 2 and Table 2 show the result of this experiment. Transplanted animal cap cells that were treated with XTCMIF and that w e r e from e m b r y o s that had been microinjected at the two-cell stage with XIHbox 6 gave a high incidence of tail structures when c o m p a r e d with similarly treated animal caps from uninjected embryos. When exa m i n e d histologically, these tails displayed axial structures including neural tube with a central canal, notochord, and m y o t o m e s throughout the length of the tail (data not shown). We conclude that overexpression of a posterior h o m e o b o x g e n e can respecify A - P identity, in this case transforming head m e s o d e r m into tail mesoderm, as demonstrated by the results shown in Table 2. Overexpression of XlHbox 6 Protein Is Sufficient to Induce Tail-like Structures The most surprising result from these studies c a m e when Table 2. Overexpression of XIHbox 6 Homeodomain Protein Posteriorizes the Embryonic Axis Treatment of Animal Caps Exp. 1 XTC-MIF XIHbox 6 + XTC-MIF Exp. 2 XTC-MIF XIHbox 6 + XTC-MIF None Induced Secondary. Structures Head Trunk/Tail Normal 6 (46%) 5 (39%) 1 (7%) 11 (73%) 2 (15%) 3 (20%) Total (65%) 9 (31o/0) 1 (4%) 29 1 (4%) 25 (92 /0) 1 (4%) 27 0 (0%) 0 (0%) 9 (100o/0) 9 Two-cell stage embryos were microinjected with XIHbox 6 mrna, and animal caps were isolated from stage 8 blastula embryos. Isolated animal caps were treated with XTC-MIF for 3 hr and transplanted into host gastrulae. The higher percentage of head structure formation in these two experiments is probably due to the use of early host embryos at stages in which the dorsal lip is first visible. Note that implants that would have normally formed heads produce tails as a consequence of overexpression of a posterior homeodomain protein.

5 Homeoprotein Confers Axis-Forming Activity 59 microinjection, Einsteck 2 cell stage Blastula [donor] Early aastrula [host} Figure 3. Transplantation of Animal Caps Overexpressing XIHbox 6 Homeodomain Protein Induces Tail-like Structures A schematic diagram describing the experimental manipulation is shown on the left. Fertilized two-cell stage embryos were microinjected with sense XIHbox 6 RNA or mutant XIHbox 6 RNA (devoid of most of the homeobox sequence). Microinjected embryos were allowed to develop to stage 8. Animal cap tissues were isolated, incubated until sibling embryos reached the gastrula stage, and transplanted into the ventral side of the blastocoel. (A) Transplantation of animal caps ovaraxpressing XIHbox 6 protein; (B) transplantation of animal caps microinjected with mutant XIHbox 6 protein. Arrows indicate the transplanted donor tissues. control embryos in which XTC-MIF treatment had been omitted were examined. It was found that microinjection of XIHbox 6 mrna is sufficient to induce tail-like structures. Figure 3 depicts the experimental design. XIHbox 6 mrna is microinjected into both blastomeres of a two-cell embryo. The animal cap is dissected at midblastula, cultured in saline solution (1 x MMR; see Experimental Procedures) for 3 hr until the sibling embryos start gastrulation, and then transplanted into the blastocoel cavity of host embryos. An example of the resulting tail-like structures is shown in Figure 3A. Table 3 shows the results of three independent experiments in which the effect of XIHbox 6 mrna was tested. The frequency of tail formation varies somewhat from experiment to experiment. It is possible that the precise stage at which the transplantation is performed, unequal distribution of the microinjected mrna, or the batch of embryos plays a role in this variability. Table 3 shows that the formation of tail-like structures is dependent on functional XIHbox 6 message. When untreated animal caps are implanted, secondary axial structures are only rarely induced. Similarly, microinjection of a "mutant" sense XIHbox 6 mrna in which the homeobox is deleted does not induce axial structures (Figure 3B). Antibody staining showed that the protein lacking the homeodomain is stable in embryos but fails to enter nuclei. The latter result is in agreement with a previous study indicating that the homeodomain is required for nuclear migration of another homeobox gene product (Harvey et al., 1986). Microinjection of the antisense XIHbox 6 transcript or of an unrelated mrna encoding Drosophila type IV collagen (Blumberg et al., 1988) also failed to produce secondary structures (Table 3). XIHbox 6 overexpression per se does not cause mesoderm induction in animal caps. After 24 hr animal caps remain rounded and do not show histological signs of me- Table 3. Transplantation of Animal Cap Cells Overexpressing XIHbox 6 Homeodomain Protein Induced Secondary Structure Type of RNA Vesicle/ No Injected Tail Other Structure Total XIHbox 6 (sense) 20 (80%) 3 (12%) 2 (8%) 25 exp 1 XIHbox 6 (sense) 11 (44%) 4 (18%) 10 (40%) 25 axp 2 XIHbox 8 (sense) 15 (75%) 1 (5%) 4 (20%) 20 axp 3 XIHbox 6 (antisense) 0 (0%) 1 (9%) 10 (91%) 11 Mutant XIHbox 6 0 (0%) 5 (11%) 39 (89%) 44 Drosophila collagen 0 (0%) 0 (0%) 8 (100%) 8 Uninjected 2 (4%) 4 (9%) 40 (87%) 46 Two-cell stage embryos ware injected with RNAs and allowed to develop to stage 8 blastula. The concentration of RNA was 100 p,g/ml, and 10 nl was injected into each blastomere. Animal caps were isolated and transplanted into the blastocoel of early gastrulae, After 2 days, formation of the secondary structure was scored. Only structures projecting from the site of the transplanted animal caps in the ventral region are scored as tails (see Experimental Procedures). Three experiments are shown here. In two other experiments the frequency of tail structures by XIHbox 6 mrna was lower (15% and 23%).

6 Cell 60 NT B 0 Figure 4. Lineage-TracingAnalysisof SecondaryTail StructuresResultingfromTransplantationof AnimalCap Cells ExpressingXIHbox6 Protein (A) Low powermagnificationof a transversesectionof a secondarytail structure, (B) Schematicdiagramof sectionshownin (A). Abbreviations:NC, notochord;i SM, somiteof primaryaxis NT neuraltube EN endodermal~,olk; 2 sin, somiteof secondaryaxis. (C) High powermagnificationof secondarystructure after Hoechst33258 nuclearstaining. (D) TRITC labelingof the same secondarystructure shown in (C). Notethat the somiteof the secondarystructure is heavilystainedwith TRITC, but that most of the tail-like projectionis derivedfrom unlabeledhost cells. sodermal induction. We also measured cardiac actin and N-CAM mrna levels and found them to be absent from XlHbox 6-injected animal caps (data not shown). Therefore, the results suggest that the environment of the host embryo is providing additional factors to the transplanted cells, such as growth factors and RA, in order to induce axial structures. Overexpression of the homeodomain protein presumably changes the way in which the transplanted tissue responds to signals from the host. Overexpression of Homeodomain Protein Can Recruit Cells from the Host Lineage-tracing experiments were carried out to determine whether the implanted cells form the tail structures entirely by themselves or whether they recruit neighboring host cells to participate in axis formation. The animal caps were labeled by immersion in TRITC (trimethyl rhodamine isothiocyanate-r; Heasman et al., 1984) or by coinjecting with a rhodamine-dextran conjugate (Gimlich and Braun, 1985). Both procedures give fairly uniform staining, but TRITC gives a stronger signal. Eleven embryos ~'l'able 3, experiment 2) that produced secondary tails after overexpression of XIHbox 6 in lineage-traced animal caps were embedded in paraffin, serially sectioned, and stained with Hoechst A typical transverse section is shown in Figure 4. Comparison of Figures 4C (nuclear DNA staining) and 4D (rhodamine fluorescence that marks cells derived from the transplanted cells) clearly shows that most of the cells in the secondary tail are not labeled with rhodamine and have therefore been recruited from the host embryo. The implanted animal caps contribute mostly to somitic mesoderm at the base of the secondary tail. From this we conclude that cells that are overexpressing XIHbox 6 acquire the ability to induce and organize cells from the host embryo to participate in the formation of a secondary axis. The secondary tails induced by overexpression of XIHbox 6 appear to be quite normal by external examination. As shown in Figure 3A, they have a well-formed anterior edge with nervous tissue (determined by N-CAM immunostain-

7 Homeoprotein Confers Axis-Forming Activity 61 ing of sectioned embryos), including rows of melanocytes, and well-developed fin structures. However, detailed histological examination of these secondary axes shows that the neural tissue does not form a tubular structure with a central ependymal canal and that a well-defined notochord is lacking. The somites, which are well segmented, are usually restricted to the proximal part of the secondary axis and do not extend throughout the tail-like structure. When secondary axes induced by XTC-MIF plus XIHbox 6 protein were similarly examined, more complete structures were formed, with neural tube, somites, and notochord extending throughout the length of the tail, as described above. Thus while XIHbox 6 overexpression can confer axis-organizing activity, the secondary axes formed are not entirely normal and less complete than those induced in the presence of XTC-MIF. Discussion In this study we used the Mangold transplantation method to study the influence of RA and of the homeodomain protein XIHbox 6 on mesodermal cell fate. Making use of the observation of Ruiz i Altaba and Melton (1989) that animal caps treated with XTC-MIF (activin) transplanted into host gastrulae induce secondary head structures at a significant frequency, we were able to show that both RA and XIHbox 6 protein (which is posteriorly expressed) are able to override the anteriorizing (head-forming) effect of XTC- MIF. As a control to these experiments we transplanted animal cap cells microinjected with XIHbox 6 mrna without XTC-MIF treatment. It was unexpectedly found that overexpression of a homeodomain protein in the donor tissue can induce tail-like structures in the host embryo. RA and XlHbox 6 Can Influence the A-P Fate of Mesoderm RA treatment of Xenopus embryos results in anterior truncations, and previous studies had shown that RA can posteriorize the neuroectoderm (Durston et al., 1989; Sive et al., 1990). To investigate a possible effect of RA on the mesodermal component, we used transplantation of XTC- MIF-treated animal caps, which can induce head structures (Figure 1). Because RA was known to increase the steady-state levels of XIHbox 6 mrna in XTC-MIF-treated animal caps (Cho and De Robertis, 1990), we also tested the effect of XIHbox 6 overexpression in this system. Both RA and XIHbox 6 protein were able to override anterior inductions by XTC-MIF-induced mesoderm. The low percentage of head inductions found in Table 1 (27%) does not permit us to conclude that RA transforms heads into tails. However, the results shown in Table 2 concerning the posteriorizing effect of XIHbox 6 protein on XTC-MIFinduced mesoderm clearly show that in this case head structures are indeed transformed into tail structures. This type of change from heads to tails can be considered a homeotic transformation, although the term posteriorization describes the action of XIHbox 6 protein equally well. The effect of RA on embryonic mesoderm, which results in abolishment of head inductions, is in keeping with its mode of action in chicken wing bud, where it has been shown to act on the mesodermal component (Tickle et al., 1989) and with the proposal that RA and growth factors cooperate with each other to set the level of expression of homeobox genes in Xenopus mesoderm (Cho and De Robertis, 1990). The recent report that genes of the Hox 2 complex are sequentially activated by RA in teratocarcinoma cells (Simeone et al., 1990) strengthens the view that RA could provide positional information via its effects on homeobox genes. Homeodomain Proteins and the Organization of the Body Axis During the course of this study we found that animal caps overexpressing XIHbox 6 protein induced tail-like structures after transplantation into host embryos (Figure 3). This finding was unexpected because overexpression of XIHbox 6 per se does not induce mesoderm in cultured animal caps. Presumably, the host embryo provides additional signals (such as growth factors and RA) that act upon the transplanted cells. Lineage-tracing studies showed that the transplanted animal cap cells, which themselves contribute mainly to somitic mesoderm, are able to induce neighboring host cells to form tail-like structures. This ability to recruit uncommitted cells to form part of a secondary axis is what defines organizer activity (Spemann, 1938). Spemann distinguished between head and tail organizers, which were dorsal blastopore lips isolated from early or late gastrulae, respectively (Spemann, 1931); the XIHbox 6 inductions could constitute a type of tail organizer. The original embryo (called Um 132) in which the organizer phenomenon was discovered had a tail organizer (Spemann and Mangold, 1924). One would predict that other, more anteriorly expressed, homeobox genes should lead to the induction of head organizer activity. Because XIHbox 6 does not induce head structures, it is not possible to say whether its overexpression transforms animal cap cells into Spemann's dorsal lip organizer. Similar tail structures could be obtained if animal cap cells became lateral or ventral blastopore lip. Indeed, preliminary experiments suggest that the entire marginal zone (i.e., the prospective mesoderm at the blastula stage) can induce axial structures. Lateral and ventral marginal zone fragments were dissected from midblastula embryos, cultured for 3 hr, and then transplanted into host gastrulae. We found that ventral and lateral marginal zone could induce tail-like structures and that dorsal marginal zone induced more anterior structures similar to those induced by XTC-MIF (K. W. Y. C. and E. M. D. R., unpublished data). This result is in agreement with the finding that bfgf, a ventral mesoderm inducer, is able to induce formation of tail structures in this sensitive einsteckung assay (Ruiz i Altaba and Melton, 1989; K. W. Y. C. and E. M. D. R., unpublished data). It should also be kept in mind that in normal development the formation of lateral marginal zone requires a signal emanating from the dorsal organizer (Dale and Slack, 1987; Gerhart et al., 1989), which could be provided by the host embryo in these experiments. Thus our results do not distinguish whether animal cap cells overexpressing XIHbox 6 are transformed into Spemann's tail organizer or into lateral or ventral marginal

8 Cell 62 zone. This does not alter the fact, however, that they acquire the ability to recruit host cells to form part of the induced secondary axis. Since the induction of axial structures by XIHbox 6 resembles somewhat the formation of a second blastopore, one should then ask whether XIHbox 6 is involved in the normal process of gastrulation. The answer to this is clearly negative, because XIHbox 6 is first transcribed at the early neurula stage (Sharpe et al., 1987). It is quite possible, however, that XIHbox 6 protein might act upon the target genes of other, earlier-acting homeobox genes normally expressed in the dorsal blastopore lip. It should be kept in mind that XIHbox 6 has a homeobox of the Antennapedia type and that all genes of this family bind to similar DNA target sequences, frequently containing a CAATTAAA sequence (Desplan et al., 1988; Hoey and Levine, 1988; Cho et al., 1988). One might expect that overexpression of a number of Xenopus homeodomain proteins of this type could induce secondary axes. Some of the target genes activated by XIHbox 6 might include, for example, blastopore lip-specific growth factors involved in the normal gastrulation process. This would be analogous to what happens in Drosophila, in which the Ultrabithorax homeodomain protein is required for expression of the growth factor decapentaplegic in visceral mesoderm (ImmergEick et al., 1990; Reuter et al., 1990). We have recently prepared a Xenopus dorsal lip cdna library and screened it for homeoboxes (B. Blumberg et al., unpublished data). One of the genes isolated from the dorsal lip was a labial homolog. When this gene was overexpressed in animal cap cells and tested by transplantation, extensive secondary inductions were observed, consisting of tail-like structures and somite and neural inductions located anteriorly and proximally to the secondary axes (unpublished data). Another homeobox gene expressed in the dorsal lip has an in vitro DNA-binding specificity similar to that of the Drosophila head determinant bicoid (K. W. Y. C. et al., unpublished data) and will be interesting to test in this assay in the future. The experimental results are consistent with a scenario in which during the course of early development positional information signals, such as growth factors and RA, lead to expression of certain homeobox genes in the region of the future dorsal lip. These homeodomain proteins would in turn activate target genes involved in gastrulation and formation of the body axis. There is experimental evidence suggesting that vertebrate homeobox genes, like their fruit fly counterparts, act as true homeotic genes specifying the identity of body regions (Wright et al., 1989; Kessel et al., 1990; McGinnis et al., 1990). The results reported here suggest that, in addition, vertebrate homeobox genes are active participants in the biochemical pathway leading to the generation of the A-P axis. In this view, the expression of homeobox genes would be a cause, rather than a mere consequence, of axis formation. The experimental approach used here provides a direct assay for the function of homeobox genes in vertebrate development. SP6 r-~ N P S E BSE - ~1 ' I ~,? "' - I I I I genomlc c DNA genomic I ' t O.lkb psp64 vector Figure 5. The XIHbox 6 Plasmid Used to Make Synthetic mrna Details of its construction are described in Experimental Procedures. The XIHbox 6 coding region is indicated by an open box; the homeobox is in black. The stippled box indicates Xenopus 13-globin leader sequences. The direction of transcription from the SP6 promoter is marked by an arrow. The genomic and cdna coding regions are indicated. Various restriction enzyme sites are indicated: E, EcoRI; S, Smal; P, Pstl; B, BamHI; N, Ncol. Experimental Procedures Fertilization and Microln ecflon of Embryos Xenopus laevis eggs laid in high salt modified Barth solution (MBS; Laskey et al., 1977) were fertilized in vitro. They were dejellied using 2o/o cysteine (ph 7.8), washed, and transferred to 0.1 x MBS (Gurdon, 1976) in a petri dish with a t h agarose bottom. Embryos were staged according to Nieuwkoop and Faber (1967). Two-cell stage embryos were microinjected with in vitro synthesized mrna as described (Wright et al., 1989). The concentration of RNA was 100 p.g/ml, and 10 nl was injected into each blastomere. RA Treatment All trans-ra (Sigma) was dissolved in dimethyl sulfoxide at 2 x 10-2 M and stored at - 20 C. RA was stored in the dark and diluted, immediately prior to use, with vigorous stirring. Growth Factor XTC-MIF conditioned medium was heated at 95 C for 5 min prior to use in order to activate the growth factor (Smith, 1987) and then diluted 1:3 in MMR medium (100 mm NaCI, 2 mm KCI, 1 mm MgSO,, 2 mm CaCI=, 5 mm HEPES [ph 7.6], 0.1 mm EDTA; Kimelman and Kirschner, 1987). Construction of psp64-xihbox 6 Expreuion Vector Figure 5 depicts the construction of the XIHbox 6 expression vector. The protein coding region of the XIHbox 6 gene was cloned into the psp64-x~m vector (Krieg and Melton, 1984), such that the Xenopus 13-globin 5' leader sequence lies immediately upstream of the initiator methionine. In brief, the protein coding region was initially cloned into Bluescript KS* (Stratagene) in three parts: the N-terminal 49 amino acids from a genomic clone, the central 133 amino acids from a cdna clone (pg ls; Sharpe et al, 1987), and the C-terminal 49 amino acids plus a 1.35 kb 3' trailer from a second genomi(~clone (Wright et al., 1990), resulting in the construct called px4. Ultimately, this was released from the Bluescript KS* and inserted into the psp64-x~m vector. The detailed construction of Bluescript KS* px4 was as follows. A 398 nucleotide PstI-EcoRI fragment from cdna pg t s was cloned into Bluescript KS + digested with Pstl and EcoRI. Next, a 1.5 kb piece of DNA from a genomic clone with EcoRI and blunted Pstl ends was inserted into the Bluescript containing cdna, which had been digested with EcoRI and EcoRV. The resulting construct contained the C-terminal 182 amino acids plus over 1 kb of 3' trailer sequence. Finally, the N-terminal amino acids plus 5' leader sequence were added onto the Bluescript construct. A 300 bp Pstl fragment of DNA was removed from a plasmid that contained the 5' end of the coding sequence. This was then inserted in the correct orientation into the Blueecript construct that had been digested with Pstl. This completed the Bluescript construct, px4. To clone the protein coding sequence into the psp64-xi~m

9 Homeoprotein Confers Axis-Forming Activity 63 vector immediately adjacent to the I~-globin 5' leader sequence, px4 was digested with Ball at the ATG sequence of the initiator methionine. A Ncol linker was added to the Ball-digested end. The XIHbox 6 sequences were excised from the px4 construct with Ncol and BamHI and cloned into psp64-xi3m digested with Ncol and BamHI (Figure 5). Preparation of RNA for Mlcrolnjectlon Capped RNAs were synthesized as described (Krieg and Melton, 1984; Wright et el., 1989). XIHbox 6 sense RNA was synthesized by linearizing the psp64-xihbox 6 DNA with BamHI and transcribing with SP6 RNA polymerase. Mutant XIHbox 6 RNA was made by digesting the psp64-xihbox 6 DNA with EcoRI (thus deleting the majority of the homeodomain coding region; see Figure 5) and transcribing with SP6 RNA polymerase. Antisense XIHbox 6 RNA was synthesized by digesting the px4 DNA with Hind lll and transcribing with T3 RNA polymerase. Drosophila type IV collagen was prepared by digesting the pbb127 DNA with Hindlll and transcribing with T3 RNA polymerase (Blumberg et al., 1988). Synthesized RNAs were precipitated twice with ethanol, washed in 70% ethanol, and resuspended in a buffer containing 88 mm NaCI, 1 mm KCI, and 15 mm Tris-HCI (ph 7.5) (Gurdon, 1976). Elnsteckung Stage 8 blastula embryos were dechorionated manually, and animal cap fragments were dissected out in an agar dish containing 1 x MBS as described previously (Cho and De Robertis, 1990). The dissected animal cap fragments were allowed to stand for 15 min to remove loosely attached cells, transferred into medium containing XTC-MIF growth factor and/or RA, and incubated for 3 hr until the sibling embryos reached the early gastrula stage. Animal caps were then washed in 1 x MBS for 10 min and used for transplantation. Stage 10.5 early gastrula embryos were manually dechorionated in 1 x MBS, and a small slit was cut in the animal pole using a tungsten needle (California Fine Wire Company). Half an animal cap was transplanted into the blastocoel of each early gastrula embryo, and the transplanted tissue was pressed against the interior of the ventral side of.the host embryo. The transplanted embryos were left to heal for 15 to 30 min in 1 x MBS and then transferred into 0.1 x MBS. They were allowed to develop to tailbud or swimming tadpole stage (2 to 3 days), photographed, and sectioned. Care should be paid to heal the transplanted embryos properly. If the slit at the animal pole region did not heal properly, the transplanted embryos were discarded. This is because filiform ectodermal projections can occasionally arise from these embryos. The projections can be easily identified since they are very thin and appear at random locations in the embryos, even in mock-operated gastrulae that have not received transplanted cells. Histologically, these protrusions, which are formed only in some batches of embryos, consist of a cylindrical projection of ectoderm and mesenchyme lacking axial structures such as somites or neural tissue. On the other hand, true secondary axes are structures found in the ventral region of embryos in close proximity to the final location of transplanted animal cap, which can be recognized by its darker pigmentation; only these were scored as tails. Lineage Tracing Animal cap explants (from stage 8 blastulae) were incubated for 10 min in 1 x MMR containing 200 p.g/ml TRITC isomer R (Sigma) as described previously (Heasman et al., 1984). The explants were rinsed three times in 1 x MMR and used for transplantation. 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