Evolution of Intron/Exon Organization in β-Catenin
The lack of any shared intron positions between Drosophila and human β-catenins led to the suggestion that either the gene in the common ancestor completely lacked introns or that there was a combination of intron insertion and loss during evolution that erased the original intron/exon organization (Nollet et al., 1996). Our analysis suggests that neither scenario is accurate. The high degree of conservation of intron positions between β-catenins of the cnidarian Nematostella and the deuterostomes argues for an ancestral bilaterian β-catenin gene with at least eight introns. In the deuterostome lineage, only a few introns have been gained. One, that shared among β-catenins of Drosophila and the two invertebrate deuterostomes, may have evolved de novo in the ancestral bilaterian, although a loss in Nematostella cannot be ruled out. Similarly, four introns shared between the invertebrate deuterostomes that are not present in Nematostella or Drosophila may have been gained after the cnidarian/deuterostome split, while the two intron positions common only to amphioxus and humans may have arisen de novo in their common ancestor. Loss appears to be equally uncommon in deuterostomes. Only one intron has evidently been lost from sea urchin and amphioxus β-catenin and only three in the human. In contrast, in the Drosophila lineage there has been a large loss of introns. None of the eight introns shared between the cnidarian and the deuterostome β-catenins is present in Drosophila. The only conserved intron position in Drosophila β-catenin is one that is shared with sea urchin and amphioxus. This massive loss of introns has been observed in a number of other genes not only in Drosophila but in other ecdysozoans as well (Roy and Gilbert, 2005). Our results suggest that this loss occurred after the divergence of protostomes from the cnidarian and deuterostome lineages. However, where in the protostome lineage this loss of introns occurred is not certain, since genome sequences of basal ecdysozoans such as onychophorans and of lophotrochozoans such as annelids and molluscs are not yet available.
The Distribution of β-Catenin During Early Development Is Not Conserved Among Deuterostomes
The distribution of β-catenin after fertilization and during cleavage stages differs considerably amongst the deuterostomes, suggesting that the roles of this gene in early development are not evolutionarily conserved. In amphioxus, cytoplasmic β-catenin becomes concentrated at the animal pole at the first cleavage, suggesting a possible role in maintaining animal/vegetal polarity during early cleavage, but by the 8-cell stage, distribution is apparently uniform. The pattern of cytoplasmic β-catenin in a second species of amphioxus, Branchiostoma belcheri, is the same as in B. floridae through the two-cell stage (Yasui et al., 2002). However, these authors described a persistent asymmetry of cytoplasmic β-catenin until the mid-gastrula, the latest stage examined. Although this could reflect a species difference, it may be due to differences in affinity of the antibodies used in the two studies for the phosphorylated versus the non-phosphorylated forms of β-catenin or to the lack of affinity purification of the antiserum used for B. belcheri, which on Western blots appears to recognize some additional proteins (Yasui et al., 2002; Oda et al., 2003). Indeed, in the B. belcheri study compared to ours, there was a much higher level of cytoplasmic label, which tended to mask the nuclear label (Yasui et al., 2002). In sea urchins, the distribution of cytoplasmic β-catenin is also uniform at the 8-cell stage, although a few embryos appear to have higher levels of cytoplasmic β-catenin in vegetal blastomeres at the 16-cell stage (Logan et al., 1999). In contrast to both sea urchins and amphioxus, in Xenopus, cytoplasmic β-catenin, together with other components of the canonical Wnt-signaling pathway and microtubules, is localized to the future dorsal side of the embryo by the cortical rotation.
In spite of differences in cytoplasmic distribution of maternal β-catenin, nuclear localization of β-catenin occurs in all three of the above deuterostomes after the 4th cleavage (16-cell stage), although in tunicates, it occurs one cleavage later (Imai et al., 2000). Not surprisingly, nuclear β-catenin is most prominent in the cells with the highest concentration of cytoplasmic β-catenin at the 8-cell stage. Thus, in sea urchins, β-catenin becomes preferentially localized to vegetal nuclei (micromeres and macromeres), where it functions in specification of the vegetal plate and formation of mesendoderm (Wikramanayake et al., 1998; Logan et al., 1999; reviewed in Brandhorst and Klein, 2002). Nuclear β-catenin induces secondary mesenchyme by signaling through Notch, which in turn becomes concentrated at the boundary between secondary mesenchyme and presumptive endoderm (Sherwood and McClay, 1997; Sweet et al., 1999; McClay et al., 2000). In contrast, in amphioxus, there is uniform nuclear localization of β-catenin in all cells from the 16-cell stage through the late blastula/early gastrula, which correlates with the evident absence of either a dorsalizing or vegetalizing effect of Li+ when applied before the mid-blastula stage. While a negative result may be due to incorrect timing or concentration of Li+, this result together with the lack of preferential localization of β-catenin to dorsal or vegetal nuclei suggests that in amphioxus, neither dorso/ventral polarity nor mesendodermal identity is established by signaling via β-catenin. In fact, the sharp reduction of both cytoplasmic and nuclear β-catenin in presumptive mesendoderm suggests that, in contrast with sea urchins, absence of β-catenin signaling may be necessary for mesendoderm specification in amphioxus. The down-regulation of β-catenin in presumptive mesendoderm at the late blastula stage correlates with up-regulation of the Wnt/β-catenin inhibitor Dickkopf1/2/4 in the same cells (our unpublished data), suggesting that, as in vertebrates (González-Sancho et al., 2005), induction of Dickkopf down-regulates the Wnt/β-catenin pathway. In ascidian tunicates, as in sea urchins, β-catenin accumulates in nuclei of vegetal blastomeres, specifying them as endoderm (Imai et al., 2000; Satou et al., 2001), although it does not seem to function in patterning either the dorso/ventral or anterior/posterior axis. Instead, endodermal cells with nuclear β-catenin induce the notochord, which can be respecified as endoderm by application of Li+ (Yoshida et al., 1998; Imai et al., 2001). In Xenopus, unlike other deuterostomes, β-catenin becomes preferentially incorporated at the 16-cell stage into dorsal nuclei where it functions in specification of dorsal identity and activation of organizer genes (Schneider et al., 1996; Larabell et al., 1997; Miller et al., 1999; Kelly et al., 2000; Schohl and Fagotto, 2002; Xanthos et al., 2002). Blockage of signaling via β-catenin in early embryos prevents formation of dorsal/anterior structures (Heasman et al., 2000), while upregulation of β-catenin signaling with Li+ has the opposite effect. This role of nuclear β-catenin in dorsal/anterior specification in Xenopus does not appear to be comparable to the role of β-catenin signaling in oral/aboral axis patterning in sea urchins, which is evidently mediated by localized β-catenin in the vegetal plate (Wikramanayake and Klein, 1997; Brandhorst and Klein, 2002).
A Posterior Wnt/β-Catenin Signaling Center Around the Blastopore Is Conserved Among Deuterostomes and Acts in Patterning the Anterior/Posterior Axis
Although early roles of nuclear β-catenin in deuterostomes appear to be quite divergent, later roles in patterning along the anterior/posterior (animal/vegetal) axis are evolutionarily conserved except in the tunicates, in which development is considerably modified in association with determinate cleavage. In all the other deuterostomes studied including amphioxus, a Wnt/β-catenin signaling center develops around the blastopore. It is characterized by elevated levels of nuclear β-catenin as well as Notch and brachyury and functions in specification of posterior identity. At the onset of gastrulation in amphioxus, cells at the mesendoderm/ectoderm boundary have high levels of nuclear β-catenin and express Brachyury and Wnt8 (Zhang et al., 1997; Holland et al., 2000a; Schubert et al., 2001; Yasui et al., 2001). By the mid-gastrula, Wnt1 and then Notch, Wnt3 and Wnt5 also turn on around the blastopore (P.W.H. Holland et al., 1995; L.Z. Holland et al., 2001; Schubert et al., 2001). Similarly, in the late sea urchin blastula, primary mesenchyme and veg2 cells lose nuclear β-catenin leaving a high concentration in a ring of endoderm cells at the endoderm/ectoderm boundary (Logan et al., 1999). Wnt-8 and Wnt-1 are both expressed in vegetal cells: Wnt-8 in the vegetal plate and Wnt-1 in cells adjacent the archenteron (Wikramanayake et al., 1998; Ferkowicz and Raff, 2001). Notch, which controls the localization of nuclear β-catenin in this region, and brachyury are also concentrated in cells at the endoderm/ectoderm boundary (Sherwood and McClay, 1997, 1999, 2001; Gross and McClay, 2001; reviewed in Brandhorst and Klein 2002).
Posterior Wnt/β-catenin signaling is also present throughout the vertebrates. Wnt/β-catenin signaling is concentrated at the posterior end of the zebrafish gastrula, and in the mouse and chick, in the primitive streak and node (Dorsky et al., 2002; Mohamed et al., 2004; Schmidt et al., 2004). In the late Xenopus blastula, there is a ring of high levels of nuclear β-catenin around the marginal zone (the future mesoderm) that later localizes to the dorsal and ventral lips of the blastopore (Schohl and Fagotto, 2002). The patterns of XWnt-8, brachyury (Xbra) and XWnt-11 are similar to those of nuclear β-catenin (Smith and Harland, 1991; Christian and Moon, 1993; Lemaire and Gurdon, 1994). During the neurula stage, expression of XWnt-8 ceases, while XWnt-3a and caudal also turn on around the blastopore (Epstein et al., 1997; McGrew et al., 1997; Beck and Slack, 1998, 1999; Ikeya and Takada, 2001; Lickert and Kemler, 2002). Brachyury, directly regulated by Wnt/β-catenin, stays turned on posteriorly during the neurula and tailbud stages (Gont et al., 1993; Yamaguchi et al., 1999; Arnold et al., 2000; Vonica and Gumbiner, 2002). Components of Notch-signaling (XNotch and X-Delta-1) are also expressed in posterior tissues from gastrulation onwards (Beck and Slack, 1998).
Manipulation of Wnt/β-catenin signaling shows that in amphioxus, sea urchins, and vertebrates, Wnt/β-catenin signaling functions in conferring posterior identity to tissues around the blastopore and in patterning the anterior/posterior axis (reviewed in Huelsken and Birchmeier, 2001; Kiecker and Niehrs, 2001; Brandhorst and Klein, 2002). For amphioxus, we have shown that a pulse of Li+ at the late blastula stage slows the loss of nuclear β-catenin from the mesendoderm and increases its concentration in posterior tissues, which elongate greatly and express the posterior markers Wnt1, Wnt5, Wnt11, brachyury. The failure of β-catenin to remain expressed in the mesendoderm after Li+ treatment is probably due to the concomitant upregulation of Dickkopf 1/2/4 in these cells (our unpublished data), which, after removal of the Li+, down-regulates β-catenin signaling. Enhanced degradation of β-catenin by Dickkopf-1 on withdrawal of Li+ has similarly been observed in human mesenchymal stem cells in culture (Gregory et al., 2005). Li+-treated amphioxus embryos also lose anterior identity as shown by the lack of expression of FoxQ, an exclusive marker of the anteriormost ectoderm in normal embryos (Yu et al., 2003). In addition, these embryos lose neural plate identity as shown by lack of expression of the neural plate marker Sox1/2/3 and continued expression of β-catenin in the dorsal ectoderm. The extreme elongation of amphioxus embryos treated with Li+ at the late blastula is reminiscent of Xenopus animal caps treated with Li+ or overexpressing XWnt8, β-catenin, or XWnt-11, which preferentially signals through the Wnt/JNK pathway (Tada and Smith, 2000; Kühl et al., 2001; Tada et al., 2002), and could be due to the induction of convergent extension movements. Conversely, Wnts preferentially signaling through the Wnt/Ca++ pathway inhibit convergent extension (Kühl et al., 2001; Choi and Han, 2002). In amphioxus, Wnt(s) signaling though all three pathways—the Wnt/β-catenin (Wnt-1), Wnt/Ca++ (Wnt-5), and Wnt/JNK (Wnt-11)—are expressed around the blastopore of normal embryos and at the elongating tip of the Li+-treated ones. Thus, it is likely that convergent-extension is involved in elongation of both Li+-treated and normal embryos.
In sea urchins, nuclear Wnt/β-catenin signaling from vegetal cells acts together with Notch and brachyury to pattern the animal/vegetal axis and control the position of the ectoderm/endoderm boundary (Emily-Fenouil, et al., 1998; Wikramanayake et al., 1998; Huang et al., 2000; Vonica et al., 2000; Howard et al., 2001). Up-regulating Wnt/β-catenin signaling with Li+ or injection of dominant/negative forms of GSK3-β vegetalizes sea urchin embryos, reducing the number of animal cells, increasing the proportion of endoderm, and shifting expression patterns of several genes including Brachyury toward the animal pole (Ghiglione et al., 1993; Cameron and Davidson, 1997; Emily-Fenouil et al., 1998; Gross and McClay, 2001). Blocking Wnt/β-catenin signaling has the opposite effect, inhibiting formation of endoderm (Logan et al., 1999; Huang et al., 2000) and blocking brachyury expression (Gross and McClay, 2001).
In vertebrates, Wnt/β-catening signaling is also essential for proper anterior/posterior patterning. Mouse embryos deficient in β-catenin express neither the anterior markers Hex and Otx nor the posterior marker brachyury (Huelsken et al., 2000). In Xenopus, the effects of Li+-application at the late blastula (Fredieu et al., 1997) are remarkably like those of comparable experiments in amphioxus. Forebrain and midbrain markers are not expressed, the archenteron does not form, and blastopore closure is delayed or incomplete (Fredieu et al., 1997). Experimental evidence shows that the Wnt/β-catenin and Notch pathways together with brachyury and caudal constitute a posterior signaling center that specifies and maintains posterior identity (Beck and Slack, 1998, 1999; reviewed in Gamse and Sive, 2000; Kiecker and Niehrs, 2001). After the mid-blastula transition, up-regulating β-catenin signaling (i.e., injection of XWnt-8, Li+, β-catenin overexpression) inhibits dorsal-anterior development, resulting in embryos with a shortened dorsal axis, no heads or notochords, and enlarged somites (Yamaguchi and Shinagawa, 1989; Cui et al., 1995; Kinoshita and Asashima, 1995; Fredieu et al., 1997; Kao and Elinson, 1998; Domingos et al., 2001; Hamilton et al., 2001). Gene markers of ventral and lateral mesoderm are concomitantly up-regulated (Hamilton et al., 2001). In addition, over-expression of XWnt-3a together with active Notch induces ectopic tails in animal cap explants grafted onto the neural plate (Beck and Slack, 1998, 1999), while over-expression of Disheveled, a component of both the Notch and Wnt-signaling pathways, posteriorizes neural tissue and activates posterior markers such as brachyury (Itoh and Sokol, 1997). Conversely, blocking Wnt/β-catenin or Notch signaling (e.g., injection of a dominant-negative XWnt-8, antisense Wnt-8 morpholino oligonucleotide) expands anterior neural fates and/or causes posterior defects (Takada et al., 1994; McGrew et al., 1997; Beck and Slack, 2002; Erter et al., 2001).