Cell, Vol. 57, 317-326,
April 21, 1989, Copyright
0 1969 by Cell Press
Involvement of the Xenopus Homeobox Gene Xhox3 in Pattern Formation along the Anterior-Posterior Axis A. Ruiz i Altaba and D. A. Melton Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02138
Summary The Xenopus homeobox gene Xhoxl shows a graded expression in the axial mesoderm, with the highest concentration in the posterior end of frog gastrula and neurula embryos. To investigate the function of the Xhox3 gene, synthetic Xhox3 mRNA was injected into different regions of developing embryos. In particular, Xhox3 was supplied in excess to anterior cells, which normally have the lowest levels of Xhox3 RNA. The results show that injection of Xhox3, but not control, mRNA into prospective anterior regions of developing embryos produces a series of graded axial defects. The injected embryos gastrulate normally but fail to form anterior (head) structures. Our findings suggest that Xhox3 is involved in establishing anterior-posterior cell identiUes during pattern formation of the axial mesoderm in early embryonic development. Introduction An effective way to test for the function of a cloned gene during development is to produce a transgenic animal in which the gene product is overexpressed and/or ectopitally expressed in vivo. This method has already provided insights into the functions of oncogenes and growth factors in mice (see for example Stewart et al., 1984; Adams et al., 1985; Leder et al., 1986; Quaife et al., 1987; Riither et al., 1987; Thompson et al., 1987; Heard et al., 1987; Lang et al., 1987) and is likely to provide important clues as to the function of vertebrate homeobox genes. In Xenopus, the creation of transgenic animals is difficult because of the low frequency of DNA integration and the mosaic expression of introduced genes (Etkin and Pearman, 1987; Kintner, Harvey, and Melton, unpublished results). As an alternative approach, we and others (Andrews and Brown, 1987; Harvey and Melton, 1988) have provided gene products to developing frog embryos by injecting synthetic mRNAs. In this paper we describe the use of direct mRNA injections to test the function of the Xenopus homeobox gene Xhox3. We have shown that Xhox3 is expressed in two periods during Xenopus embryonic development (Ruiz i Altaba and Melton, 1989). In the early period the gene is expressed in axial mesoderm as a gradient with the highest concentration at the posterior pole. To test whether this expression pattern is important for normal development, we have injected Xhox3 mRNA in order to provide high doses of the Xhox3 product in the anterior end (where it is normally found at the lowest levels). Xhox3 mRNA-injected embryos show a series of graded axial defects, and in the
extreme cases the head is missing. Examination of Xhox3 mRNA-injected embryos reveals that the timing and extent of migration of anterior mesoderm during gastrulation are normal. This finding is in contrast with treatments such as ultraviolet (UV) irradiation, which results in headless embryos but does so by disrupting mesodermal migration during gastrulation (Cooke and Smith, 1987). Our results are consistent with the idea that incorrect Xhox3 expression affects cell fates along the anteriorposterior (A-P) axis. Specifically, we find that Xhox3 overexpression in anterior mesodermal cells prevents anterior (head) development. We discuss these results in the context of the proposal that homeobox gene expression is involved in the establishment of mesodermal cell identities and patterning along the A-P axis (see Ruiz i Altaba and Melton, 1989). Results Synthetic Xhoxl mRNA Injection into Developing Embryos The synthetic full-length Xhox3 mRNA used for embryo injection experiments encodes a protein of about 50 kd as judged by in vitro translation (Figure 1A). Since our aim is the local overexpression of the Xhox3 protein, we first tested the stability of injected Xhox3 mRNA. As shown in Figure 16, much of the injected RNA is degraded at the time of the midblastula transition. Nevertheless, what remains at the late gastrula to early neurula stages (stage 13-14) is about a 20-fold excess compared with endogenous levels of Xhox3 mRNA. It is during this period that the level of the endogenous Xhox3 transcripts is at its maximum (Ruiz i Altaba and Melton, 1989). A similar stability profile has been observed for other mRNAs injected into developing frog embryos (Melton and Rebagliati, 1986; Andrews and Brown, 1987; Harvey and Melton, 1988; Harland and Misher, 1988). If Xhox3 mRNA is injected into fertilzed eggs before first cleavage, the mRNA is evenly distributed in the developing embryo and is therefore provided to regions (anterior) that normally have low levels of Xhox3 mRNA (Figure 1C). Fertilized eggs injected with synthetic Xhox3 mRNA were allowed to develop to the late neurula stage (stage ~20) and dissected into anterior, middle, and posterior sections. The results of RNAase protection assays that test for the presence of endogenous and injected Xhox3 mRNAs show that the injected RNA is still in lo-fold excess relative to endogenous levels at this late stage. Notably, the injected RNA is present at a uniform (and high) concentration along the A-P axis. Thus, the injection of synthetic Xhox3 mRNA obliterates the normally graded distribution of the endogenous transcripts. Because the fate map of Xenopus eggs is largely understood, it is possible to inject Xhox3 mRNA into cells that will form a particular portion of the embryo. For example, the first cleavage plane usually, but not always, marks the plane of bilateral symmetry (Klein, 1987; but see Danil-
Table 1. Summary
3 M 132034
I I I I I I
of mRNAs Injected into Developing
Xhox3 Xhox3BSA XhoxlA
B. Dose-Dependent Concentration
Effect of Xhox3 mRNA injection
50 100 200 C. Correlation of Localization mRNA Injection
0 0 5
% Defective 58 75 78 of Defects and Site of Xhox3
Equatorial region Animal pole region Vegetal pole region
70 74 47
Averages of defective embryos in anterior regions (%) were compiled from results obtained in several independent experiments. All head deficiencies as compared with control embryos were scored. More than 200 embryos were injected in each case. In (C) injections were at different sites in the early-cleavage embryo at the 2-, 4-, or &cell stage.
Figure 1. Translation of Xhox3 mRNA In Vitro and Stability and Distribution of Synthetic Xhox3 mRNAs in Embryos (A) 35S in vitro translation products of Xhoxd mRNA (lane X) and Bromo mosaic virus RNA used as positive control (lane P). A no-RNA negative control is also shown (lane N). The sizes of the protein markers (lane M) are given in kd. The line at right depicts a ~50 kd Xhox3 translation product. (B) Northern blot analysis of total RNA samples from embryos injected with Xhox3 mRNA. Lane 0, sample taken right after injection; lane 3, 3 hr after injection; lane M, midblastula transition (stage 819); other numbers refer to stages according to Nieuwkoop and Faber (1987). The line at right indicates the position of the intact injected Xhox3 mRNA. Five embryo equivalents of total RNA were used per lane. The hybridization probe was the whole Xhox3 cDNA insert of pcXhox3 (Ruiz i Altaba and Melton, 1989). (C) Distribution of injected Xhox3 RNAs along the A-P axis of the embryo. Control and injected embryos at the neurula stage (-stage 20) were fixed and cut into thirds as shown in the diagram. Total RNA was extracted and assayed for the presence of injected Xhox3 RNA and endogenous Xhox3 mRNA by RNAase protection. The protected fragments are shown after denaturing gel electrophoresis. A, anterior; M, middle; P, posterior. All samples were also assayed for the presence of EF-la RNAs (Krieg and Melton, 1989) as a control for RNA recovery. A 24 hr exposure of the endogenous assay is shown and compared with a 4 hr exposure for the injected RNA. Overall, there is about 10 times more injected than endogenous Xhoxd RNA at this stage (-stage 20). Note particularly that injection of Xhox3 RNA obliterates the endogenous gradient, and the prospective anterior region has a high level of Xhox3 RNA. (D) Spatial distribution of injected material as visualized by fluorescence of FLDx (Gimlich and Cooke, 1983). In this case, FLDx was injected only into one blastomere at the 2-cell stage. An anterior view of the resulting neurula is shown.
chick and Black, 1988). Thus, by injecting the mRNA after the first cleavage furrow has formed, the Xhox3 mRNA is provided to either the right or left half of the embryo. This can be demonstrated by injecting a fluorescent lineage tracer as shown in Figure 1D. If mRNA is injected at a later stage, e.g., the 4-, 8-, or 18-cell stage, the RNA is deposited
into more restricted regions in the embryo. In all, the results on the stability and distribution of injected mRNAs suggest that it is possible to provide an excess of Xhox3 gene product to some or all regions of gastrula and early neurula stage embryos depending on when and where the mRNA is injected. Phenotype of Embryos Injected with Xhox3 mRNA Xhox3 and control mRNAs were injected into developing embryos at different locations and stages in order to test the possible function of Xhox3 during embryonic development. The results of these injection experiments are summarized in Table 1. The main conclusion is that injection of Xhox3, but not control, mRNAs into the whole embryo causes defects in the development of anterior structures. The controls include injection of equivalent amounts of synthetic P-globin mRNA (Xpm) and an engineered mutant Xhox3 mRNA (Xhox3BSA) that renders the homeobox and sequences downstream of it out of frame. These controls show that the defects obtained by injecting Xhox3 mRNA into developing embryos are not the result of an increased amount of generic mRNA. Another Xenopus homeobox gene, XhoxlA, was used as a second type of control. Injection of XhoxlA mRNA (Harvey et al., 1988) did not cause the specific anterior defects that are observed following Xhox3 mRNA injection. This rules out the possibility that injection of mRNA coding for a DNA binding (homeobox) protein per se results in anterior defects. The percentage of embryos showing anterior axial deficiencies varies from one experiment to the next. Moreover, within the same batch of embryos, different concentrations of Xhox3 mRNA cause varying degrees of axial
Gene in Pattern Formation
tures, these would seem to be secondary to the earliest observed defect: the disruption of the normal patterning of the axial mesoderm (see below). Because injected embryos show defects principally in the anterior region and because this part of the embryo has the lowest levels of endogenous Xhox3 mRNA, a series of injections were performed to deposit the mRNA preferentially in the anterior region. This was accomplished by injecting Xhox3 mRNA at the animal end of the developing egg after the first, second, or third cleavage divisions. Since RNA does not cross cell boundries, these injections localize the injected RNA to prospective anterior cells. Conversely, injections at the vegetal end localize the mRNA to more posterior structures. It must be noted, however, that the fate map of the egg shows that these distinctions are not complete; animal pole material can give rise to some posterior structures and vegetal pole material can contribute to some anterior structures (Moody, 1987; Dale and Slack, 1987). The results show that injecting Xhox3 mRNA into the animal end of developing embryos produces a higher proportion of anterior defects than observed following vegetal injections (see Table 1).
Figure 2. Phenotype, Specificity, Defects in Xhoxd mRNA-injected
and Dose Embryos
(A) and (8) show a series of embryos (stages 33-36) with a range of axial defects. The embryo on the top in both (A) and (B) was injected with I)-globin mRNA; the rest were injected with Xhoxd mRNA. Embryos in (A) and (B) were injected with RNAs at concentrations of 100 and 200 nglml, respectively. Some embryos show microcephaly and anophthalmia; others are more severly affected, exhibiting acephaly and severe trunk reductions associated with reduced neural develop ment. (C) shows representative examples of tadpole stage embryos that were injected with either Xhoxd mRNA (bottom) or mutant Xhoxd mRNA (top) at 200 nglml.
defects (Table lb and Figure 2). In slightly defective embryos only the more anterior structures of the head are missing, while in more severely affected embryos there are no anterior structures and the embryo is reduced in size. Thus, there is variation not only in the percentage of embryos with defects but also in the severity of the defects (see Table 1 and Figure 2). It seems likely that the number of embryos with anterior defects and the severity of these are functions of both the dose of injected RNA and distribution of the RNA to different cell lineages. The most obvious and consistent defect observed in Xhox3 mRNA-injected embryos is the lack of anterior structures. However, as shown in Figure 2, tadpole stage injected embryos are somewhat reduced in overall length and some are kinked along the A-P axis. There is also a noticeable lack of melanophores that is more pronounced in severely affected embryos. While it is possible that Xhox3 has a direct effect on posterior and neural struc-
Xhox3 mRNA-Injected Embryos Gestrulate Normally Headless embryos that resemble the Xhox3 overexpression phenotype can be obtained by other treatments. In particular, injection of trypan blue into the blastocoel of early gastrula stage embryos (Gerhart et al., 1984) or UV irradiation of the vegetal pole of fertilized eggs (Chung and Malacinski, 1980; Scharf and Gerhart, 1983) can both cause anterior defects. In both of these latter cases, the headless phenotype is the result of a disruption of the normal migration of mesodermal cells to the anterior end during gastrulation. In short, anterior development is defective because there are no mesodermal cells in the anterior region of the embryo (Gerhart et al., 1984; Cooke and Smith, 1987). We have therefore examined the timing and extent of mesoderm migration during gastrulation in Xhox3 mRNA-injected embryos. Comparison of the timing of mesoderm invagination in uninjected embryos and in embryos injected with Xhox3 or XhoxlA mRNA (Harvey et al., 1986) shows that there is no significant difference in the time at which gastrulation starts (the formation of the dorsal lip of the blastopore) or in the time at which gastrulation gastrulation is completed (blastopore closure) (not shown). Injected embryos were also sectioned to compare the extent of mesoderm migration from posterior to more anterior positions during gastrulation. These results (Figure 3) failed to show any significant difference between Xhox3 mRNA-injected and control embryos. Injection of Xhox3 or XhoxlA synthetic mRNA before first cleavage does not prevent mesodermal cells from reaching the prospective anterior (head) region. This is in clear contrast with UV-treated or trypan blueinjected embryos (Figures 30 and 3E), in which mesoderm migration is retarded or disrupted. This analysis shows that the anterior defects observed following Xhox3 mRNA injection are not caused by a disruption in the morphogenetic movements of presumptive anterior meso-
derm. Excess Xhox3 mRNA does not affect mesodermal invagination and/or migration. Headless embryos can be obtained not only by disruption of mesoderm migration during gastrulation but also by removal of prospective anterior mesodermal cells (Cooke, 1985; Takasaki, 1987). Therefore, the presence and localization of the descendent of Xhox3 mRNA injected cells were monitored in tadpole stage embryos. To this end, the fluorescent lineage tracer fluorescein-lysine-dextran (FLDx; Gimlich and Cooke, 1983) was coinjetted with Xhox3 and control mRNAs to follow the fate of the injected ceils. Sufficient amounts of FLDx were coinjetted so that living labeled cells could be visualized by direct UV illumination with a dissecting microscope. Injection of FLDx alone or with control RNAs has no deleterious effects on the recipient blastomeres and allows normal development to proceed (see Figure 1C). As shown in Figure 4, the axial defects and the presence of FLDx are colocalized in affected tadpoles coinjected with Xhox3 mRNA and FLDx. In tadpoles derived from embryos injected with Xhox3 mRNA and FLDx into animal pole blastomeres, the FLDx is found in the anterior defective regions (Figure 4A). In no case were defective tadpoles found to contain FLDx only in posterior structures. In tadpoles derived from embryos injected into one blastomere at the 2-cell stage, the lateral defects coincide with the localization of the fluorescent marker (Figure 46). These results show that the defects are produced in regions containing the descendents of cells that had large amounts of Xhox3 mRNA and that there is not a general necrosis of prospective anterior mesodermal cells in Xhox3 mRNA-injected embryos.
Figure 3. Gastrulation bryos
Embryos were injected with Xhox3 or XhoxlA mRNA or with trypan blue, or treated with UV light, and allowed to develop to the end of gastrulation (stage 13) as marked by uninjected control sibling embryos. At this point some embryos were fixed and sectioned, while the rest were allowed to develop to the tadpole stage for assessment of phenotype. Panels at right show representative examples of tadpole stage embryos displaying the appropiate phenotypes. Panels at left show representative examples of sagittal sections of late gastrula stage embryos treated in different ways. (A) Uninjected control embryos. (B) Xhox3 mRNA-injected embryos. (C) XhoxlA mRNA-injected embryos. (D) UV-treated embryos. (E) Trypan blue-injected embryos. In (D) and (E), extreme cases are shown in the left panels. In these two cases the severity of the phenotype depends on the extent of mesoderm migration. In all cases anterior is to the right. Arrows in the left panels demarcate the anteriormost boundary of mesoderm migration. a, archenteron; b, blastocoel; d, dorsal blastopore lip; ec, ectoderm; en, enoderm; m. mesoderm; v, ventral blastopore lip.
Xhox3 Overexpression Affects Anterior Cells Lithium treatment of early blastulae produces a patterning defect that is superficially opposite to that produced by Xhox3 mRNA injection. Lithium treatment results in anteriorized embryos with greatly reduced posterior structures, whereas Xhox3 injection produces anterior deficiencies. Moreover, we have shown that treatment with lithium reduces the overall level of Xhox3 expression (Ruiz i Altaba and Melton, 1989). Unlike Xhox3 mRNA injection, lithium adversely affects pattern by interfering with the normal migration of mesodermal cells during gastrulation (Kao and Elinson, 1989). While both exposure to lithium and UV irradiation interfere with the gastrular movements of mesodermal cells, they do so in opposite ways with opposing effects: the former produces posterior defects whereas the latter produces anterior defects. Given that lithium treatment reduces the level of Xhox3 expression and that it produces posterior defects, one might naively assume that Xhox3 injection could reverse the effects of lithium treatment. Embryos injected with Xhox3 mRNA before first cleavage and treated with lithium chloride at the blastula stage were allowed to develop to the tadpole stage. The results (Figure 5) show that these injected embryos are not rescued from the effects of lithium and in fact lack both posterior and anterior structures. We interpret this phenotype to be due to the effects of lithium (lackof posterior structures) and Xhox3 injection (failure to develop normal anterior structures). The results
Gene in Pattern Formation
Figure 4. Localization of the Descendents Cells Injected with Xhox3 and FLDx The injected ized in living with UV light. same embryo liaht and the
FLDx lineage tracer was visualembryos by direct illumination In each case, two pictures of the are shown, one under visible other under UV illumination. In
stage, and the FLDx is present in the left anterior side, while that in (A) was injected before first cleavage and has the injected material in the whole anterior area. In both cases injection was into the animal hemisphere. In these embryos the FLDx is found in mesodermal and neural structures.
of this experiment are consistent with our interpretation of the assays on gastrulation shown in Figure 3. Because Xhox3 injection does not have an effect on the migration of mesodermal cells whereas treatment with lithium does, Xhox3 cannot reverse the effects of lithium. All these results support the idea that excess Xhox3 expression does not allow normal anterior development. Defective Neural-Plate Formation in Xhox3 mRNA-Injected Embryos The earliest defects detected in Xhox3 mRNA-injected embryos are found at the neural plate stage when neural folds fail to form properly. This problem is accentuated as development proceeds and results in an abnormal cement gland and anterior neural folds (Figure 6). In weakly affected cases, only slight defects in the position of these
Figure 5. Effects of Xhox3 Overexpression
in Anterior Cells
Control uninjected (A) or Xhox3 mRNA-injected (B) embryos were treated with lithium chloride and allowed to develop to the early tadpole stage. Representative examples of severely anteriorized uninjected (A) or Xhox3 mRNA-injected (B) embryos obtained after lithium chloride treatment are shown. Note that in the latter case the embryo lacks posterior structures (tails and trunks) because of the restrictive effect of lithium treatment but also lacks normal anterior structures because of the effects of Xhox3 overexpression (anterior neural folds and cement gland). anf, anterior neural folds; cg, cement gland; tr, trunk rudiment.
markers are seen, while in the extreme cases anterior dorsal differentiation is absent. The specificity of these defects is demonstrated by injecting Xhox3 mRNA into one blastomere at the 2-cell stage, in which case one-half of the neurula is derived from the injected cell. Figure 6B shows that the injected half of the embryo fails to form normal neural folds and a cement gland whereas the contralateral control is normal. Injection of f3-globin mRNA has no effect (Figure 6A). Supplying excess Xhox3 mRNA to the whole embryo (by injection before first cleavage) leads to an overall reduction of neural plate size and the absence of anterior neural folds and cement gland, both of which are dependent on the dose of injected Xhox3 mRNA (Figure 6C). Not surprisingly, defective neural plate formation leads to deficiencies in subsequent development. Anterior central nervous system (CNS) structures (brain and anterior spinal cord) are abnormal as is the development of melanophores (neural crest cell derivatives) in Xhox3 mRNA-injected embryos (Figures 6D and 6E). Internal Characteristics of Xhox3 mRNA-Injected Embryos Histological analyses of defective regions in Xhox3 mRNAinjected embryos show that all tissue types including notochord, muscle, and nerve seem to acquire their normal differentiated state, albeit in an abnormal pattern. Injection of Xhox3 mRNA has no obvious effects in the development of nonneural ectoderm or endoderm. Clear defects are observed, in varying degrees, in the mesoderm and neural ectoderm of Xhox3 mRNA-injected embryos. Figures 7A and 78 show the axial regions of cross sections from embryos injected with either P-globin of Xhox3 mRNAs before the first cleavage, so that the RNA is uniformily distributed in the developing embryo. In the latter case (Figure 78) the mesoderm (somites and notochord) shows a pattern defect; the notochord is severely reduced in size and the somites are fused along the midline. In severely affected cases the notochord is missing
Figure 6. The Early Phenotype of Xhoxd Overexpression Is a Defective Neural Plate (A-C) Anterior-dorsal views of late neurulaearly tailbud stage embryos injected with Xhox3 mRNA. (A) Injection with 6-globin mRNA. (6) Injection of Xhox3 RNA into the blastomere that formed the right side of the embryo. (C) lnjection of Xhox3 mRNA into the whole embryo. In Xhox3 mRNA-injected cases neural differentiation is impaired to varying degrees. The size of the cement gland (darkly pigmented anterior area with crescent shape) is greatly reduced in Xhox3 mRNA-injected (B, C) embryos. (D) and (E) show higher magnifications of the head region of stage 45 swimming tadpoles that were injected with 6-globin (D) or Xhox3 mRNA (E). Note the lack of anterior structures that are derived from secondary inductions such as the eyes and the abnormal appearance of melanophores. (E) shows an acephalic embryo where normal heart, blood, internal gills, and gut can be seen along with a defective CNS, unusually large (fused) somites, and small, dotlike melanophores.
and the somitic mesoderm and neural ectoderm are extremely aberrant in their pattern (Figure 7C). As with external defects, the earliest defects that can be detected by histological analysis are in the abnormal morphology of the neural folds in Xhox3 mRNA-injected embryos (not shown). Defects in neural structures are obvious in late-stage embryos with a severe Xhox3 phenotype (Figure 7C). Neural defects are only found in embryos with an abnormal axial mesoderm. These findings raise the possibility that Xhox3 overexpression primarily interferes with the normal development of the axial mesoderm and that abnormal neural development is a secondary effect. Histological analysis of older tadpole stage embryos (stages 33-36) injected with Xhox3 .mRNA suggests that the developmental defects are best ,described as graded axial defects. While control embryos injected with 6-globin (Xpm) mRNA show normal A-P pattern and bilateral symmetry (Figures 7D and 7E), anterior (head) development is severely repressed in Xhox3 mRNA-injected embryos. The CNS is deformed, the notochord is missing, the somites fuse along the midline of the embryo, and the overall axial length is significantly reduced. Melanophores appear as small black dots and lack their normal star-like appearance. The extent of the notochord varies depending on the severity of the phenotype. In some cases only parts of the notochord are seen. In the most extreme cases the embryos have an overall rounded shape with only some small axial structures. Discussion During normal development, Xhox3 mRNA first appears at the midblastula transition and its levels increase during gastrulation. As development proceeds, the highest levels
of Xhox3 mRNA are found in the posterior pole, near the blastopore. By the late gastrula stage Xhox3 transcripts form a gradient along the A-P axis. Thus, at this early stage the anterior regions of the embryo have relatively less Xhox3 mRNA (and presumably less Xhox3 protein) than their posterior counterparts. During this early period of expression, Xhox3 is expressed in the axial mesoderm (Ruiz i Altaba and Melton, 1969). In the present experiments we have supplied embryos with excess Xhox3 mRNA, particularly in the prospective anterior tissues, by direct injection of synthetic mRNA. The main conclusion from these experiments is that Xhox3 mRNA-injected embryos develop with obvious and drastic axial deficiencies. The head and other anterior structures are severely deformed and are often missing in embryos injected with Xhox3 mRNA. Specificity of the Defects Observed following Xhox3 mRNA Injection There are a variety of ways, some trivial, to produce headless Xenopus tadpoles. In addition to simple injection damage, UV irradiation and other treatments that disrupt gastrulation (see below) can give rise to anterior defects. Since our Xhox3 injection experiments also produce anterior defects, it is important to demonstrate the specificity of this response. We have done so in two ways. First, we have injected three control mRNAs, none of which leads to the same type or extent of anterior deficiency observed with Xhox3 mRNA. The injection of mRNAs for f&globin, XhoxlA, and Xhox3BSA (a mutated Xhox3 gene that does not code for the homeodomain) rules out any injection artifacts. Moreover, the XhoxlA control shows that injection of a DNA binding protein per se does not produce headless embryos. Secondly, we note that anterior defects similar to those
Figure 7. Histological
Gene in Pattern Formation
Analysis of Neurula and Tail bud Stage Embryos
Injected with Xhox3 mRNA
Cross sections of the axial region of neurula stage embryos (closed neural tube stage) injected with 6-globin (A) or Xhox3 mRNA (B, C). In (B) the neural tube is disorganized and the notochord is severely reduced in size, allowing the somites to fuse across the midline. (C) shows a cross section of an early tail bud stage embryo severely affected by Xhoxd mRNA injection. Note that in Xhox3 mRNA-injected embryos, the notochord is severely reduced in size, or absent. The somites are disorganized and eventually fuse across the midline, and the CNS is clearly deformed. In all cases only axial deficiencies were found. ec, ectoderm; en, endoderm; n, notochord; nf, neural fold; nt, neural tube; s, somite. (D) through (G) show horizontal sections of stage 33-36 tadpole embryos. In (D) and (E) two sections at different levels of a 6-globin-injected embryo are shown as a control. (F) and (G) show two sections at different levels of a severely affected embryo injected with Xhox3 mRNA. The A-P axis is reduced overall. The most severe defects are in the anterior dorsal regions. The brain is reduced and the spinal cord is deformed. There is abnormal melanophore development, the notochord is absent, and fused somites are obvious(G). b, brain; e, eye; en, endoderm; ev. ear vesicle; n, notochord; s, somite; SC, spinal cord.
observed as a result of Xhox3 overexpression are also obtained by treatments that prevent prospective anterior mesodermal cells from migrating during gastrulation to their normal position (see Figure 3; Gerhart and Keller, 1986). These treatments include gastrulation arrest by trypan blue (Gerhart et al., 1984) extirpation of prospective anterior endomesodermal cells (Cooke, 1985; Takasaki, 1987) and UV irradiation (Chung and Malacinski, 1980; Scharf and Gerhart, 1983). UV irradiation of the vegetal pole of fertilized eggs (Grant and Wacaster, 1972; Malacinski et al., 1977; Youn and Malacinski, 1981; Malacinski and Youn, 1981; Scharf and Gerhart, 1980, 1983; Cooke 1985; reviewed by Gimlich, 1985) or oocytes (Holwill et al., 1987) results in embryos that develop axial deficiencies that resemble those caused by Xhox3 overexpression except that in the latter case no extreme grade 5 (ventralized) embryos (Malacinski et al., 1977; Scharf and Gerhart, 1983) have been observed. It is therefore significant that Xhox3 mRNA-injected embryos gastrulate normally. Both the timing and extent of mesodermal invagination are indistinguishable from those in control embryos. It is important to emphasize that the Xhox3 mRNA-
injected embryos gastrulate normally and that the descendants of injected cells are not necrotic (Figures 3 and 4). The injected embryos have mesodermal cells that are correctly positioned at the anterior end of the embryo (Figure 3), and yet head structures are not formed. From this we conclude that a high level of Xhox3 expression in the anterior region is incompatible with normal anterior fates. Correlation of High Levels of Xhox3, Posterior Fates, and Anterior Defects Interestingly, endogenous Xhox3 expression in UVtreated embryos dramatically increases during the gastrula and neurula stages to a maximum of about 5-fold above the normal level (Ruiz i Altaba and Melton, 1989). Thus, in both UV-treated and Xhox3 mRNA-injected embryos the levels of Xhox3 mRNA are above normal. In both cases the embryos develop a series of axial deficiencies that can be correlated with the UV dose or the amount of Xhox3 mRNA injected. Even though UV treatment and Xhox3 mRNA injection produce similar phenotypes, the mechanisms by which these are produced are different, as noted above. Whereas, UV treatment affects the migra-
tion of prospective anterior mesodermal cells during gastrulation, Xhox3 overexpression affects the normal development of anterior mesodermal cells that do reach their normal anterior positions. These differences lead us to conclude that the cells in an anterior position of a Xhox3 mRNA-injected embryo are unable to form normal anterior mesoderm and cannot induce anterior epidermall neural structures. Thus, while cells occupy an anterior position they are no longer capable of normal anterior differentiation. In contrast, UV-irradiated embryos lack mesodermal cells in anterior positions. The fact that Xhox3 expression is enhanced in UV-treated embryos may reflect the overabundance of “posterior” cells in these embryos (Cooke and Smith, 1987), since Xhox3 is expressed at the highest levels in posterior cells (Ruiz i Altaba and Melton, 1989). Late Xhox3 Expression Tadpole stage embryos show a second period of Xhox3 expression in the CNS (mainly in the brain) and in the posterior tail bud (Ruiz i Altaba and Melton, 1989). Since the tail bud region is responsible for the growth and morphogenesis of the posterior axial structures of tadpole stage embryos (Spofford, 1948; Bijtel, 1958; Elsdale and Davidson, 1983; Woodland and Jones, 1988), it is possible that the mild posterior defects that we observe in injection experiments with high doses of Xhox3 mRNA (Figure 2) result from overexpression of Xhox3 in the tail bud. High doses of Xhox3 mRNA are probably required to produce these posterior defects, since only after injection of large amounts of Xhox3 mRNA is excess RNA likely to be present in the tail bud region. Defects in the CNS and melanophores in Xhox3 mRNA-injected embryos are likely to be secondary to defects in the axial mesoderm and related to abnormal neural induction. Nevertheless, these could also be related to ectopic expression of Xhox3 and/or deregulation of a possible late function of Xhox3 in the CNS (Ruiz i Altaba and Melton, 1989). We have not tried to provide an exhaustive description of the defects following Xhox3 mRNA injection for two reasons. First, as mentioned above, the extent of the defects depends on the time, place, and concentration of mRNA injected. Second, the analysis of the defects observed in late-stage embryos is complicated by the fact that most of these are secondary and likely to be a direct consequence of early problems in the patterning of the axial mesoderm. Conclusion In the simplest view, our results suggest that high levels of Xhox3 expression are incompatible with specification and further development of mesodermal cells as ‘anterior:’ High levels of Xhox3, above a threshold, may be necessary for the specification of cells as posterior. Since we do not observe an overall transformation of anterior to posterior structures, the Xhox3 overexpression phenotype cannot be considered a homeotic transformation. Furthermore, this suggests that low and high levels of Xhox3 expression may be necessary but not sufficient for anterior and posterior specification and development, respectively. Consistent with this idea is the fact that in anteriorized em-
bryos, obtained by lithium treatment (Figure 5A) (Masui, 1961; Kao et al, 1986; Breckenridge et al., 1987; Cooke and Smith, 1988), Xhox3 expression during the gastrula and neurula stages is severely reduced (Ruiz i Altaba and Melton, 1989). Our results support the idea that the level of Xhox3 expression in the axial mesoderm is an important determinant of positional value along the A-P axis. The refinement of positional values may involve the expression of other region-specific homeobox genes such as Xhox36 and XlHbox 1 (Condie and Harland, 1987; Carrasco and Malacinski, 1987; Oliver et al., 1988). Further investigation of when and where the Xhox3 protein is normally expressed and of the effects of interference with the normal pattern of Xhox3 expression should help clarify these issues. It has been proposed that determination of mesodermal cell identities along the A-P axis could be the result, in part, of the organizer region, or posterior pole (Spemann, 1938), acting as a source of a diffusible signal (Pasteels, 1951). We have extended this idea to suggest that a response to this diffusible signal may be the regulation of homeobox gene expression in the axial mesoderm, and this, in turn, could have a direct role in the establishment of A-P cell fates in the mesoderm (see Ruiz i Altaba and Melton, 1989). Here we have shown that incorrect expression of the homeobox gene Xhox3 results in abnormal patterning of the axial mesoderm along the A-P axis. In this context it is interesting to note that the molecular basis of the signal of the organizer may be related or responsive to growth factors. Indeed, Cooke et al., (1988) have shown that injection of a Xenopus XTC mesoderm-inducing factor (Smith, 1987) into the blastocoele of frog embryos results in tadpoles with axial deficiencies that are similar to those observed following Xhox3 mRNA injection. Thus, we are led to the possibility that a diffusible signal with a graded distribution (see Pasteels, 1951; Toivonen and Sax&n, 1968; Wolpert, 1969), perhaps a growth factor-like molecule(s), establishes A-P cell identities and therefore polarity in the frog embryo by regulating the expression of homeobox genes. Experimental
Materials Female Xenopus laevis were induced to lay eggs, using human chorionic gonadotropin. Embryos were obtained by fertilization of eggs with testis homogenates. Tadpoles were staged according to Nieuwkoop and Faber (1967). UV-treated embryos were obtained by UV illumination of the vegetal pole of fertilized eggs as described in Scharf and Gerhart (1983). Lithium-treated embryos were obtained by incubating blastula stage embryos in 0.25 M LiCl in water for S-10 min. Restriction enzymes, T7 RNA polymerase, RNasin, RQl RNAase-free DNAase, and E. coli DNA polymerase I were from Promega Biotech. RNAase A was from Sigma. 32P-radionucleotides were purchased from Amersham. Hybridization Experiments RNA extraction from embryos and Northern blotting experiments were as described in Ruiz i Altaba et al. (1987). RNAase protection assays were as described in Melton et al. (1984). The Xhox3 probe was synthetized by transcribing Alul-cut pcXhox3BA (Ruiz i Altaba and Melton, 1989) with T7 RNA polymerase. In Vitro Synthesis and Translation of Synthetic RNAs Sense RNAs were synthetized in vitro as described in Melton et al.
Gene in Pattern Formation
(1984) and Krieg and Melton (1964). Xhoxd mRNA was synthetized by transcription of Hindlll-cut pcXhox3 (Rub i Altaba and Melton. 1989) with T7 RNA polymerase. The mutant Xhoxd mRNA was synthetized by transcription of Hindlll-cut pcXhox3BSA with T7 RNA polymerase. pcXhox3BSA is a BamHI-Smal deletion subclone of pcXhox3. 8-Globin mRNA was synthetized by transcription of EcoRl cut pSP6C8-globin with SP6 polymerase (Melton, 1985). XhoxlA mRNA was synthetized by transcription of BamHI-cut p64T-CIA with SP6 polymerase (Harvey et al., 1986). In vitro translation of synthetic Xhoxl mRNA in the rabbit reticulocyte system was done following the procedures described by the manufacturer (Promega Biotec). RNasin was added to the translation reaction to inhibit RNA breakdown. About 1 ug of RNA was used per reaction. Samples were run in 10% SDS-polyacrylamide gels, and the translation products were visualized by autoradiography. Embryo Injections and Histology Embryos were injected with a volume of about 30 nl. RNAs were dissolved in 10 m M Tris (pH 7.5). RNAs were mixed with the fluorescent tracer FLDx just before injection. The FLDx-injected embryos were kept in the dark during their development. FLDx fluorescence was visualized through UV filters by direct UV illumination of living injected embryos. Trypan blue-injected embryos were obtained by injecting a 1% solution of trypan blue in IO m M Tris (pH 7.5) into the blastocoel of developing gastrula embryos. This treatment usually causes immediate gastrulation arrest. Injected and control embryos were fixed in Bouin’s fixative (75% aqueous picric acid, 25% formaldehyde [370/o], 5% glacial acetic acid) overnight, rinsed extensively with 70% ethanol, and transferred to Paraplast through an ethanol and xylene series. Sections were cut with a microtome, applied to gelatin-treated slides, and stained with eosin and hematoxylin. Photographs of living embryos and sections were taken with TriX or T-Max film (Kodak).
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Acknowledgments We are grateful to C. Kintner, R. Harvey, J. C. Smith, D. Tannahill, and all the members of this laboratory for discussion and/or comments on the manuscript. This work was supported by 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 ‘adverfisemenf” in accordance with 16 USC. Section 1734 solely to indicate this fact. Recerved July 25, 1988; revised February
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