Patterning and lineage specification in the amphibian embryo

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1 Patterning and Lineage Specification in the Amphibian Embryo Agnes P. Chan and Laurence D. Etkin Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030

I. Introduction II. Xenopus as a Model System for Studying Early Embryogenesis III. Dorsal–Ventral Specification A. Cytoplasmic Determinants B. Cortical Rotation C. Nieuwkoop Center D. Vegetal Cortical Cytoplasm/-Catenin Signaling Pathway E. Molecular Nature of the Dorsal Determinant in Vegetal Cortical Cytoplasm F. Cooperation between TGF- Signaling and Wnt Signaling G. TGF- Receptors, Smads, and Target Genes IV. The Spemann Organizer A. Organizer Genes Expressed in the Dorsal Vegetal Region B. Organizer Genes Expressed in the Prechordal Mesoderm C. Organizer Genes Expressed in the Anterior Endomesoderm D. A Mammalian Structure Analogous to the Anterior Endomesoderm E. How Is the Organizer Formed after All? V. The Three Germ Layers A. Endoderm B. Mesoderm C. Ectoderm D. A Theoretical Model of Germ Layer Formation VI. Developmental Pathways and Tumorigenesis VII. Perspectives References

Xenopus has been widely used to study early embryogenesis because the embryos allow for efficient functional assays of gene products by the overexpression of RNA. The first asymmetry of the embryo is initiated during oogenesis and is manifested by the darkly pigmented animal hemisphere and lightly pigmented vegetal hemisphere. Upon fertilization a second asymmetry, the dorsal–ventral asymmetry, is established, with the sperm entry site defining the prospective ventral region. During the cleavage stage, a vegetal cortical cytoplasm Current Topics in Developmental Biology, Vol. 51 C 2001 by Academic Press. All rights of reproduction in any form reserved. Copyright  0070-2153/01 $35.00

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(VCC)/-catenin signaling pathway is differentially activated on the prospective dorsal side of the embryo. The overlapping of the VCC/-catenin and transforming growth factor beta (TGF-) pathways in the dorsal vegetal quadrant specifies dorsal–vental axis formation by regulating formation of the Spemann organizer, including the anterior endomesoderm. The organizer initiates gastrulation to form a triploblastic embryo in which the mesoderm layer is located between the ectoderm layer and the endoderm layer. The interplay between maternal and zygotic TGF-s and the T-box transcription factors in the vegetal hemisphere initiates the specification of germ-layer lineages. TGF- signaling originating from the vegetal region induces mesoderm in the equatorial region, and initiates endoderm differentiation directly in the vegetal region. The ectoderm develops from the animal region, which does not come into contact with the vegetal TGF- signals. A large number of the downstream components and transcriptional targets of early developmental pathways have been identified and characterized. This review gives an overview of recent advances in the understanding of the functional roles and interactions of the molecular players important for axis determination and germ-layer specification during early Xenopus embryogenesis.  2001 Academic Press. C

I. Introduction The transformation of a single-celled zygote into a highly organized adult organism requires precise regulation of cell growth and differentiation. The study of embryogenesis has been an intriguing subject for biologists. Experimental embryology has provided fundamental knowledge of early embryonic interactions by careful manipulation of embryos. Almost 80 years after the initial discovery of the amphibian gastrula organizer, our understanding of early development has grown from a cellular level to a molecular level. A number of developmental processes can now be explained in terms of activation or repression of gene expression. These data have provided a molecular basis for understanding the cascade of genetic regulation during early embryonic development. This chapter focuses on early embryonic development of Xenopus laevis. The setting up of the dorsal–ventral axis in embryos has a critical function in determining the future body plan. In Xenopus, dorsal–ventral polarity is established at the time of fertilization by the triggering of the translocation of vegetal cortical cytoplasm (VCC) to the prospective dorsal side of the embryo. Components of the VCC, via the -catenin-dependent pathway, activate target gene expression in the dorsal vegetal region in blastula-stage embryos. The VCC/-catenin signaling pathway functions cooperatively with the transforming growth factor beta (TGF-) pathway to induce specific gene expression in the Spemann organizer in gastrula-stage embryos. Induction of the three germ layers takes place concomitantly with the establishment of the dorsal–ventral axis. The endodermal and mesodermal layers of the embryo are induced as a result of active TGF- signaling initiated by maternally localized transcripts. Bone morphogenetic protein (BMP)

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and post-MBT Wnt signaling further pattern the dorsal–ventral polarity of the germ layers by specifying a ventral cell fate. Examples from other model systems are included throughout the chapter to try to provide additional data in specific areas of embryonic development. The use of different developmental model systems can complement the deficiencies of individual systems. Integration of information obtained from different systems can allow the elucidation of complex developmental pathways that are evolutionarily conserved. A better understanding of the mechanisms controlling growth and differentiation is a prerequisite to fulfilling the ultimate goal in unraveling the cause and control of malignancy during tumorigenesis. This review gives an overview of recent advances in the understanding of the functional roles and interactions of the molecular players important for patterning and lineage specification during early embryogenesis in Xenopus laevis.

II. Xenopus as a Model System for Studying Early Embryogenesis Xenopus laevis is one of several model systems used for studying early embryogenesis (Harland and Gerhart, 1997). Other developmental systems include Caenorhabditis elegans (Rose and Kemphues, 1998; Labouesse and Mango, 1999), Drosophila (Baek and Lee, 1999), zebrafish (Kodjabachian et al., 1999; Mullins, 1999), the chick (Bachvarova, 1999), and the mouse (Beddington and Robertson, 1999; Gardner, 2000). The advantages of using Xenopus as an experimental system for studying early embryonic development are the ease of maintenance of the animals, the availability of large quantities of embryos year-round, and the rapid development of the embryos in simple salt solution at ambient conditions. The embryos are easy to manipulate for studies involving tissue transplantation, recombination, and explant cultures. The relatively large size of the Xenopus embryo allows efficient isolation of specific regions of embryonic tissues and provides sufficient quantities of starting materials for the construction of cDNA libraries (Blumberg et al., 1991). Several genes have been isolated from the screening of a Xenopus dorsal-lip cDNA library (Cho et al., 1991; Sasai et al., 1994; Bouwmeester et al., 1996). These genes have subsequently been used to isolate homologs from other developmental systems. Furthermore, the capacity of Xenopus embryos for large injection volumes has made possible rapid functional screening using cDNA expression libraries (Smith and Harland, 1991; Smith et al., 1993; Lemaire et al., 1995). Overexpression of RNA in Xenopus embryos is an efficient way to assay for gain-of-function phenotypes. In addition to the wild-type gene products, dominantinterfering constructs of growth factors and receptors, and transcription factors fused with activation or repression domains have been overexpressed in embryos to assay for gene functions (Amaya et al., 1991; Conlon et al., 1996; Ryan et al.,

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1996; Horb and Thomsen, 1997). The use of hormone-inducible transcription factor fusion constructs has further aided in the identification of genes that are the immediate targets of transcription factors (Kolm and Sive, 1995; Tada et al., 1998; Melby et al., 1999; Saka et al., 2000). Knockout studies in Xenopus, performed with the use of antisense oligonucleotides or antisense RNA, are another method of assessing gene functions. Maternal RNA transcripts can be depleted using antisense oligonucleotides (Shuttleworth and Colman, 1988; Kloc et al., 1989; Heasman et al., 1991). Antisense RNA expression has been shown to repress zygotic gene expression and thus permit demonstration of gene functions during normal development (Steinbeisser et al., 1995). The introduction of transgenic techniques to Xenopus (Etkin and Pearman, 1987; Kroll and Gerhart, 1994; Chan and Gurdon, 1996; Kroll and Amaya, 1996; Fu et al., 1998; Amaya and Kroll, 1999; Marsh-Armstrong et al., 1999) has exploited the potential of this model system for use in studying the zygotic effects of transgene expression and in characterizing the regulatory sequences of promoters. A gene trap approach in which transgenic techniques were used to carry out mutagenesis in Xenopus has been successful (Bronchain et al., 1999). Introduction of mutations into the Xenopus genome is likely to lead to the identification of novel genes on the basis of the mutant phenotypes. Although there are several advantages to using Xenopus laevis as a model of embryogenesis, the system also suffers from pitfalls. A relatively long generation time of around 1 year is required for the animal to reach sexual maturity, making germline transmission of genetic alterations impractical. The pseudotetraploid nature of the animal does not favor genetic analysis. However, the introduction of Xenopus tropicalis, which is diploid and has a much shorter generation time of 5 months, is likely to circumvent some of the limitations of the Xenopus system (Amaya et al., 1998). On the other hand, model systems widely used for genetic studies include C. elegans, Drosophila, zebrafish, and the mouse. Both C. elegans and the mouse are reliable systems in which to study the effect of knocking out gene functions. RNA interference has been demonstrated in C. elegans (Fire et al., 1998), and gene targeting has been widely applied to manipulate genomic sequences of the mouse (Koller and Smithies, 1992).

III. Dorsal–Ventral Specification The basic body plan of an embryo is elaborated upon the dorsal–ventral axis established during early embryogenesis. In Xenopus, dorsal–ventral polarity is set up at the time of fertilization. In the fertilized egg, cytoplasmic determinants are translocated to the future dorsal side by a rotation of the egg cortex. The VCC/-catenin signaling pathway is activated on the prospective dorsal region as a consequence of the cortical rotation. This pathway functions cooperatively

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with TGF- signaling to activate expression of organizer genes at the mid-blastula transition (MBT), when zygotic gene expression first commences. In the past few years, the knowledge of vegetally localized maternal determinants and molecular components of this signaling pathways has grown dramatically.

A. Cytoplasmic Determinants In some embryos, cell fate is determined by the response of a cell to preexisting cytoplasmic factors. Embryonic cells containing such cytoplasmic factors can undergo autonomous differentiation according to their normal fate in the absence of cell–cell interactions. In Xenopus, the existence of cytoplasmic determinants with dorsalizing activity has been demonstrated in the vegetal cortex of unfertilized eggs (Fujisue et al., 1993; Holowacz and Elinson, 1993). Vegetal deposition of determinants in Xenopus probably results from an asymmetric distribution of maternal components such as RNAs and proteins along the animal–vegetal axis during oogenesis. Wild-type Xenopus embryos exhibit prominent external polarity beginning at mid oogenesis (stage III), when pigment granules become more highly concentrated in the animal cortex than in the vegetal cortex. However, animal–vegetal polarity can be traced back to the stage when the secondary oogonium undergoes mitotic divisions to give rise to nests of 16 oocytes. The secondary oogonium contains a large aggregate of mitochondria on only one side of the nucleus. It has been suggested that this aggregate is the precursor of the mitochondrial cloud, also known as the Balbiani body (Al-Mukhtar and Webb, 1971; Coggins, 1973). Differential localization of maternal RNAs in Xenopus follows one of two pathways, the message transport organizer (METRO or early) pathway and the late pathway (Forristall et al., 1995; Kloc and Etkin, 1995; Kloc et al., 2000). RNAs that follow the METRO pathway first localize to the mitochondrial cloud in stage I oocytes. Between late stage I and early stage II, the localized RNAs translocate together with the mitochondrial cloud to the vegetal region and become localized to the cortex, where they remain throughout oogenesis. RNAs that follow the late pathway are excluded from the mitochondrial cloud and are found throughout the cytoplasm in stage I oocytes. Between late stage II and early stage III, late-pathway RNAs localize to specific domains of the vegetal hemisphere including a crescentshaped region in proximity to the nucleus (Chan et al., 1999), a wedge-shaped structure in the vegetal cytoplasm (Kloc and Etkin, 1995), and at the vegetal cortical region (Melton, 1987). The RNAs eventually localize to the vegetal cortex and occupy a broader region than do METRO-pathway RNAs. A subtle difference during the process of vegetal localization has been observed between two late-pathway RNAs, Vg1 and fatvg. Whereas Vg1 mRNA shows no association with the mitochondrial cloud during oogenesis (Kloc and Etkin, 1995; Chan et al., 1999), fatvg mRNA has been found to localize to the mitochondrial

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cloud transiently in stage II oocytes (Chan et al., 1999). fatvg mRNA also associates with the germ plasm during early embryogenesis, similarly to METRO-pathway RNAs (A. P. Chan and L. D. Etkin, unpublished observation). Although the functional role of fatvg mRNA in the germ plasm is yet to be determined, the association of a late-pathway RNA to the germ plasm suggests that the association of localized RNA with the germ plasm in embryos is not limited to METRO-pathway RNAs. Both Vg1 and VegT mRNAs are localized by the late pathway and have been identified as possible cytoplasmic determinants. Xenopus Bicaudal-C (xBic-C) mRNA has also been found to localize to the vegetal cortex and shares a similar time course of localization as Vg1 and Veg T (Wesseley and De Robertis, 2000) Vg1 belongs to the TGF- superfamily. Processed Vg1 can induce dorsal mesoderm and secondary axis (Dale et al., 1993; Thomsen and Melton, 1993). It has been suggested that the processing of vegetally localized Vg1 mRNA is spatially regulated so that Vg1 is active only in the dorsal vegetal region of the embryo (Thomsen and Melton, 1993). VegT is a T-box transcription factor (Zhang and King, 1996). This factor is also known as Antipodean (Apod) (Stennard et al., 1996), Xombi (Lustig et al., 1996b), and Brat (Horb and Thomsen, 1997). Antisense oligonucleotide knockout experiments have indicated that VegT acts as a maternal determinant in the specification of both the endoderm and mesoderm lineages (Zhang and King, 1996; Kofron, et al., 1999). Several METRO-pathway RNAs have been identified as possible candidates of cytoplasmic determinants. These include the Xwnt-11, Xcat2, DEADSouth (formerly Xcat3), Xpat and Xdazl mRNAs, all of which follow the METRO pathway (Kloc and Etkin, 1995) and have been found to associate with the germ plasm in cleavage stage embryos. Xcat2 is a zinc-finger protein (Mosquera et al., 1993). High-resolution electron microscopic studies have shown that Xcat2 mRNA is associated with the germinal granules of the germ plasm (Kloc et al., 1998, 1999). The Xcat2 protein is related to the Drosophila morphogen Nanos, which is involved in germ cell development, including formation and migration (Kobayashi et al., 1996; Forbes and Lehmann, 1998). The DEADSouth protein is a DEAD-box RNA-dependent helicase (MacArthur et al., 2000) Xpat mRNA is expressed in the primordial germ cells until the cells enter the dorsal mesentery. The Xpat protein does not contain any identifiable functional domains (Hudson and Woodland, 1998) Xdazl is an RNA-binding protein required for spermatogenesis (Houston et al., 1998). Depletion of maternal Xdazl mRNA has resulted in defective germ cell migration within the endoderm during early differentiation (Houston and King, 2000). Xwnt-11 mRNA encodes a maternal Wnt molecule (Ku and Melton, 1993). Although the RNA is localized to the entire vegetal cortex, the protein differentially accumulates on the dorsal side of the embryo because of regulated translation of the localized RNA along the dorsal–ventral axis (Schroeder et al., 1999). The control of Xwnt-11 mRNA translation is mediated by differential polyadenylation. Xwnt-11 or other maternal Wnt molecules may be the cytoplasmic determinants required for the VCC/-catenin pathway that specifies the dorsal identity of the embryo, provided that the pathway is dependent on a maternal ligand.

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The presence of a dorsalizing activity in Xenopus embryos has been demonstrated by blastomere transplantation studies (Gimlich and Gerhart, 1984; Takasaki and Konishi, 1989; Kageura, 1990). The dorsal vegetal blastomeres of a 64-cell embryo can rescue axis formation in UV-irradiated embryos, which would otherwise develop with a ventralized phenotype lacking any axis (Gimlich and Gerhart, 1984). When the same blastomeres are transplanted to the ventral side of a recipient embryo, an ectopic secondary axis forms in addition to a normal primary axis. The transplanted dorsal vegetal cells therefore have a dorsalizing or axis-inducing activity. The spatial and temporal origins of the dorsalizing activity have been further determined by cytoplasmic-transfer and deletion experiments. Vegetal cytoplasm taken from activated eggs is capable of inducing an ectopic axis in recipient embryos (Fujisue et al., 1993; Holowacz and Elinson, 1993). Only the vegetal cortical cytoplasm—not the deep vegetal cytoplasm or the cortical cytoplasm from other regions—contributes to the dorsalizing activity. The cortical localization of a dorsalizing activity has also been demonstrated by the transplantation of cortical peels isolated from the vegetal region of activated eggs (Kageura, 1997). The segregation of dorsal determinants along the animal–vegetal axis has been investigated by removing cytoplasm from different regions by egg ligation and deletion experiments (Kikkawa et al., 1996; Sakai, 1996). When the vegetal region of an embryo is deleted just after fertilization, the embryo does not develop any dorsal identity. Vegetally deleted embryos can be rescued by injecting vegetal cytoplasm but not animal cytoplasm; again, this finding indicates the presence of a dorsalizing activity in the vegetal cytoplasm. The dorsalizing activity is present before oocyte maturation in prophase I oocytes (Elinson and Pasceri, 1989; Holowacz and Elinson, 1993, 1995). Exposure of prophase I oocytes to UV irradiation produces ventralized phenotypes even though cortical rotation has taken place. In these embryos, no dorsalizing activity can be detected after cytoplasmic transplantation (Elinson and Pasceri, 1989). This shows that dorsal determinants in the oocytes are destroyed by UV irradiation. UV irradiation of one-cell embryos also results in ventralized phenotypes, but the UV target is different from that in prophase I oocytes and is believed to be the microtubule array required for cortical rotation (Elinson and Pasceri, 1989). Studies in ascidian embryos have demonstrated the existence of prelocalized ooplasmic factors in different regions of the fertilized eggs. The animal, vegetal, and posterior regions of the embryo contain tissue-specific determinants for the development of epidermis, endoderm, and muscle, respectively (Nishida, 1997). Blastomeres isolated from the posterior region of ascidian embryos develop autonomously to form muscle (Deno et al., 1984; Nishida, 1992). A search for localized maternal RNAs in ascidian embryos led to the identification of such RNAs specifically localized to the myoplasm (Swalla and Jeffery, 1995) and ectoplasm (Swalla and Jeffery, 1996). A maternal transcript, pem-3, has also been shown to localize to the posterior-vegetal cytoplasm of the egg after fertilization (Satou, 1999). The protein product of pem-3 contains putative RNA-binding

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domains known as the KH-domain. These findings provide a molecular basis for studying cytoplasmic determinants in ascidians. The possible existence of cytoplasmic determinants in mouse embryos is still being vigorously investigated (Gardner, 1998, 1999). It has been shown that embryos subject to centrifugation or mechanical mixing of cytoplasm develop normally (Mulnard and Puissant, 1984; Evsikov et al., 1994). This finding argues against specific localization of components in the cytoplasm. Deletions of different regions of a one-cell mouse embryo have no effect on normal development (ZernickaGoetz, 1998). Again, this finding provides no evidence for there being early regional asymmetry in the mouse egg cytoplasm. In addition, extensive cell mixing that occurs in the mouse epiblast prior to gastrulation is inconsistent with an early segregation of cell lineages by the inheritance of cytoplasmic determinants (Beddington and Robertson, 1989). However, STAT3 and leptin have been shown to localize to the cortex of mouse and human oocytes, and potentially function to specify asymmetry during early cleavage (Antczak and Van Blerkom, 1997). The alignment of the animal–vegetal axis of the mouse zygote with the axis of bilateral symmetry in blastocysts and the proximal–distal axis in egg cylinders suggests that some degree of regional specification or polarity might already exist in the egg cytoplasm (Gardner, 1997; Weber et al., 1999).

B. Cortical Rotation A critical step in determining the prospective dorsal region of the embryo is triggered by cortical rotation. The mechanism and consequences of such movement has attracted considerable attention. An unfertilized Xenopus egg is radially symmetrical along the animal–vegetal axis. Upon fertilization, the dorsal–ventral axis is defined by the site of sperm entry in the animal region. The sperm entry site marks the future ventral side of the embryo and overlaps with the first cleavage plane, which divides the egg bilaterally into right and left halves. After fertilization, cortical rotation takes place one-third of the way through the first cell cycle (100 min), when the outer cortical layer of the fertilized egg rotates 30◦ with respect to a stationary core (Vincent et al., 1986; Vincent and Gerhart, 1987). However, this degree of rotation seems to be inconsistent with results from cytoplasmic transfer studies, which have demonstrated the presence of a dorsalizing activity around the equatorial region 90◦ away from the vegetal cortex (Yuge et al., 1990; Fujisue et al., 1993). In fact, axis-inducing activity has also been detected above the equatorial region in the animal dorsal sector (Gallagher et al., 1991; Hainski and Moody, 1992; Kageura, 1997). A possible explanation for the apparent discrepancy between the degree of cortical rotation and the localization of dorsal activity comes from studies of microtubule-dependent movement in the vegetal cortical region (Elinson and Rowning, 1988; Rowning et al., 1997). A set of parallel microtubules is found in a transport zone 4–8 m below the cortex associated with the inner cytoplasmic

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core (Larabell et al., 1996). These multiple layers of microtubules align with the direction of rotation (Elinson and Rowning, 1988). Since the rotation movement precedes the formation of the microtubule arrays, it has been suggested that the microtubules are not responsible for the rotation of the cortex (Larabell et al., 1996). However, small organelles can be propelled along the parallel array of microtubules that function independently of the cortex. Small organelles may therefore move along the microtubules by motor molecules toward the plus-end to the dorsal side of the embryo. Evidence suggests that endogeneous organelles can be translocated 60–90◦ away from the vegetal pole (Rowning et al., 1997). Transport by microtubules thus accounts for the apparent differences in the localization of dorsalizing activity as predicted from the degree of rotation of the egg cortex. In keeping with the microtubule transport model, a downstream component of the Wnt pathway, Dishevelled (Dsh), has been shown to associate with small vesicle-like organelles that are translocated to the prospective dorsal side by microtubules during cortical rotation (Miller et al., 1999). Microtubule transport is not only specific for dorsal–ventral specification in Xenopus embryos. A dynamic distribution of microtubules has also been observed in the yolk cells of zebrafish embryos (Jesuthasan and Stahle, 1996). In zebrafish embryos, a set of parallel microtubules at the vegetal pole region is required for setting up initial asymmetry at the one-cell stage. At the eight-cell stage, microtubule tracks originating from the dorsal equatorial blastomeres extend toward the vegetal pole. These microtubule tracks may function to mediate directional transport of organelles or determinants required for dorsal development.

C. Nieuwkoop Center After cortical rotation takes place, it is thought that a signaling center—the Nieuwkoop center—is activated in the dorsal vegetal region that subsequently induces the formation of the organizer in the overlying cells in a non-cell-autonomous manner. In a series of tissue recombination experiments, different regions of the yolky vegetal mass of Urodele embryos were tested for their inductive capacity on animal caps (Boterenbrood and Nieuwkoop, 1973). The dorsal vegetal region induced dorsal axial structures, whereas the lateral and ventral vegetal regions induced only ventral structures. This tissue recombination assay has been referred to as the Nieuwkoop recombinant assay. The dorsal vegetal region carrying a dorsal endomesoderm-inducing property is commonly referred to as the Nieuwkoop center (Gerhart et al., 1989). This region is a signaling center required for specifying the dorsal–ventral axis. The inductive effect of the dorsal vegetal cells is active between the early cleavage stage and the late blastula stage (Boterenbrood and Nieuwkoop, 1973). Cell progenies from the Nieuwkoop center do not contribute to the dorsal lip or axial structures formed during gastrulation. The progenies are located vegetal to the dorsal lip, in the endoderm, and are fated to become part of the anterior gut endoderm, as shown by lineage labeling (Bauer et al., 1994;

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Vodicka and Gerhart, 1995). The proposed function of the Nieuwkoop center is to induce the cells immediately above in the equatorial region to form the organizer. However, the requirement of the dorsal vegetal cells for dorsal development has not been directly demonstrated by studies in which all tier C and tier D dorsal blastomeres are removed. In fact, all blastomeres in tier D can be removed without affecting axis formation (Gimlich, 1986). The finding that small organelles can translocate 60–90◦ away from the vegetal pole suggests that the cytoplasmic dorsal determinant that orginates in the vegetal cortical cytoplasm may translocate all the way to the prospective organizer region in the equatorial region (Rowning et al., 1997). Transplantations of cytoplasm, cortical peels, and blastomeres have demonstrated that dorsalizing activity is distributed broadly on the dorsal sector of the embryo, with the highest activities around the vegetal and equatorial regions before and after cortical rotation (Kageura, 1990; Yuge et al., 1990; Kageura, 1997). Thus, the Nieuwkoop center may overlap physically with the region where the prospective organizer forms. Studies of axis induction by transplantation of blastomeres from 32-cell embryos showed that both tier C and tier D dorsal blastomeres are active in the induction assay (Gimlich and Gerhart, 1984; Gimlich, 1986; Kageura, 1990). The dorsalizing activity of tier C dorsal blastomeres and their participation in organizer formation during normal development also support the idea that the region that produces the early dorsal inductive signal overlaps with the organizer. In summary, the Nieuwkoop center is an important concept in defining early inductive signaling during dorsal–ventral specification. The Nieuwkoop center has been regarded as a physical entity spatially and temporally distinct from the gastrula organizer. However, it is also possible that the cytoplasmic dorsal determinants, after translocation to the dorsal side by cortical rotation and interaction with components in the dorsal region, activate the Nieuwkoop center at the dorsal equatorial region during cleavage stage (Kodjabachian and Lemaire, 1998; Moon and Kimelman, 1998). The Nieuwkoop center in turn directly induces formation of the organizer at the equatorial region during the blastula stage (Kodjabachian and Lemaire, 1998; Moon and Kimelman, 1998). In this model, the Nieuwkoop center and the organizer essentially occupy the same region in the embryo but remain temporally distinct. Molecular analysis of the Nieuwkoop center will help to clearly define its role in inducing the formation of the gastrula organizer.

D. Vegetal Cortical Cytoplasm/-Catenin Signaling Pathway Since vegetal cortical cytoplasm contains a dorsal determinant with the ability to induce a complete secondary axis, zygotic gene products that can produce a similar effect when they are expressed ectopically are likely to act along the same pathway as the dorsal determinant. Siamois (Lemaire et al., 1995), -catenin (Funayama et al., 1995; Guger and Gumbiner, 1995), Xwnt-8 (Smith and Harland, 1991), and

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some mouse Wnts (McMahon and Moon, 1989; Sokol et al., 1991) are able to generate a complete secondary axis containing most anterior structures. Siamois can be activated directly by Xwnt-8 and also by vegetal cortical cytoplasm (Carnac et al., 1996; Darras et al., 1997). Siamois and -catenin are components of the VCC/-catenin signaling pathway involved in defining the dorsal identity of an embryo before the MBT (Brannon and Kimelman, 1996; Fagotto et al., 1997). On the other hand, Xwnt-8 is not maternally expressed and cannot be the maternal dorsal determinant. Endogenous Xwnt-8 is expressed in the ventral–lateral marginal zone after the MBT and is involved in a Wnt pathway required for patterning of the mesoderm (Christian and Moon, 1993; Hoppler et al., 1996). The axis-inducing effect of overexpressed Xwnt-8 is probably due to activation of components of the VCC/-catenin signaling pathway. Since the possible involvement of a Wnt ligand to initiate VCC/-catenin signaling has yet to be determined, “VCC/-catenin” signaling will be used to describe the endogenous signaling event occurring in the dorsal region in cleavage stage Xenopus embryos in the following discussion. The basic components of the Wnt pathway are largely conserved between different developmental processes found in C. elegans, Drosophila, Xenopus (Cadigan and Nusse, 1997) and sea urchins (Wikramanayake et al., 1998; Logan et al., 1999; Huang et al., 2000). The wingless pathway involved in epidermal cell differentiation in Drosophila has been characterized (Klingensmith and Nusse, 1994; Siegfried and Perrimon, 1994) and provides a basis for study of the Wnt pathway in other systems. In the absence of Wnt signaling, -catenin is continuously phosphorylated and degraded by the glycogen synthase kinase-3 (GSK-3) complex via a ubiquitination-dependent pathway (Fig. 1). The resulting low level of -catenin cannot induce target gene expression. In contrast, when a secreted Wnt molecule is recognized by a transmembrane receptor of the frizzled family, a cytoplasmic component, Dsh, is activated. Dsh in turn suppresses the inhibitory effect of GSK3 on -catenin. This results in an accumulation of -catenin in the cytoplasm. -Catenin translocates into the nuclei and interacts with the high mobility group (HMG)-box transcription factor family Tcf/Lef1 to activate target gene expression (Cadigan and Nusse, 1997). In Xenopus, results from promoter studies have shown that direct transcriptional targets inducible by VCC/-catenin signaling include the genes encoding the paired-like homeobox transcription factors Siamois (Brannon et al., 1997; Fan et al., 1998) and Twin (Laurent et al., 1997) and the TGF- factor Xnr-3 (McKendry et al., 1997). Expression of the multiple-growth-factor antagonist Cerberus (Cer) has also been shown to be inducible by -catenin in the absence of protein synthesis (Nelson and Gumbiner, 1999). 1. -Catenin The first functional role identified for -catenin was its interaction with E-cadherin, a cell adhesion molecule required for homotypic cell interaction (Nagafuchi and Takeichi, 1989; Ozawa et al., 1989). The requirement of -catenin in embryonic

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Figure 1 Components of the Wnt signaling pathway. In the absence of Wnt signaling, -catenin is continuously phosphorylated and degraded by the GSK-3 complex through a ubiquitination pathway. APC, Axin, -TrCP, and PP2A are components of the GSK-3 complex. In the presence of a Wnt ligand, the Wnt receptor transduces the signal through Dsh and CKI, resulting in inactivation of GSK-3. GBP can also inhibit GSK-3 activity but is not a linear component of the pathway. The suppression of GSK3 activity results in the accumulation of -catenin, which activates the transcription of target genes, including siamois, twin, Xnr-3, and cerberus.

patterning has later been demonstrated both in Drosophila and Xenopus embryos. The Drosophila homolog of -catenin, armadillo, is involved in epidermal differentiation (Klingensmith and Nusse, 1994; Siegfried and Perrimon, 1994). In Xenopus embryos injected with antibodies against -catenin formed secondary axes (McCrea et al., 1993). Embryos depleted of maternal -catenin mRNA showed defects for the establishment of the dorsal–ventral axis (Heasman et al., 1994). -Catenin therefore plays roles in multiple processes, including cell adhesion and embryonic patterning. In Xenopus, -catenin mRNA and -catenin protein are present maternally (DeMarais and Moon, 1992). In cleavage-stage embryos, the -catenin protein is preferentially enriched in the cytoplasm on the prospective dorsal side (Larabell et al., 1997). Nuclear accumulation of -catenin specifically in dorsal blastomeres begins at the 16-cell stage and lasts until the mid-blastula stage. -Catenin contains a putative GSK-3-dependent phosphorylation site, armadillo repeats, and a

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transactivation domain, but no nuclear localization signal. -Catenin mutants with phosphorylation sites deleted at the N terminus have increased stability and are prevented from degradation (Yost et al., 1996). The N terminus is also required for ubiquitination-dependent degradation of -catenin in the proteasome (Aberle et al., 1997; Orford et al., 1997). Since -catenin contains no nuclear localization signal, it may enter the nucleus by association with other nuclear proteins or by an importin--dependent nuclear transport mechanism (Fagotto et al., 1998). The armadillo repeats have been suggested to interact with upstream and downstream components, such as adenomatous polyposis coli (APC) and the Tcf/Lef1 proteins (Huber et al., 1997). -Catenin interacts with the Tcf/Lef1 proteins and provides a transcription activation domain to activate expression of target genes (Molenaar et al., 1996). In addition to the Tcf/Lef1 proteins, other HMG-box transcription factors, such as Xsox17 and Xsox3, also associate with -catenin to repress transcription of certain -catenin target genes (Zorn et al., 1999a). This indicates that potential interaction of -catenin with other members of the HMG-box family may also be involved in the regulation of transcription. 2. The Tcf/Lef1 Proteins The mouse proteins Tcf1 and Lef1 are closely related lymphoid transcription factors and were the first proteins discovered in the Tcf/Lef1 family (referred to hereafter as the Tcf proteins) (Clevers and van de Wetering, 1997; Eastman and Grosschedl, 1999). Tcf-1, -3, and -4 and Lef1 have been identified in both mouse and human. hTcf-4 has been implicated in colon cancer (Korinek et al., 1997). XTcf3 is the only frog homolog identified so far (Molenaar et al., 1996). Tcf proteins contain binding domains for -catenin, CREB-binding protein (CBP), and Groucho. Tcf proteins also contain a HMG domain that recognizes a DNA consensus sequence within the regulatory sequence of target genes. Groucho and CBP are corepressors that associate with Tcf proteins to repress transcription (van de Wetering et al., 1997; Cavallo et al., 1998; Roose et al., 1998). Groucho has been suggested to function with a histone deacetylase complex to regulate histone acetylation on chromatin (Choi et al., 1999). In Drosophila, dCBP lowers the affinity of armadillo to dTcf by the acetylation of a conserved lysine residue in the armadillo-binding domain (Waltzer and Bienz, 1998). Tcf-3 and Tcf-4 contain two conserved sites at the C terminus for the binding of C-terminal binding protein, which is also involved in transcriptional repression. Studies with antimorphic protein have shown that the Xenopus homolog of C-terminal binding protein is involved in the regulation of dorsoanterior development (Brannon et al., 1999). The Tcf proteins have been proposed to be architectual factors controlling chromatin structure and transcription (van Houte et al., 1993; Love et al., 1995). The Tcf proteins have dual functions in that they normally act as a transcriptional repressor, and act as an activator in the presence of -catenin. The Tcf binding sites in the siamois promoter are required for the activation of siamois expression in the

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dorsal region (Brannon et al., 1997; Fan et al., 1998). They are also necessary for the repression of siamois expression on the ventral side of the embryo, since removal of Tcf binding sites from the siamois regulatory sequence results in ectopic expression in the ventral side of the embryo (Brannon et al., 1997). 3. Functional Roles of -Catenin and the Tcf Proteins -Catenin and the Tcf proteins are involved in dorsal–ventral axis specification. Overexpression of -catenin on the ventral side of an embryo can induce a complete axis (Funayama et al., 1995). Depletion of the maternal -catenin mRNA by antisense oligonucleotide knockout prevents formation of dorsal structures (Heasman et al., 1994). -catenin knockout mice display defects in mesoderm formation (Haegel et al., 1995). The nuclear function of -catenin is thought to involve the interaction of -catenin with Tcf proteins. Overexpression of a Tcf-3 mutant lacking the -catenin binding site blocks endogenous axis formation as well as the secondary axis induced by ectopic expression of -catenin (Molenaar et al., 1996). Mice deficient in both Tcf1 and Lef1 show defects in the formation of axial structures (Galceran et al., 1999). The concept that -catenin functions in the nucleus to regulate transcription has been challenged by a study using a membrane-tethered form of plakaglobin, a protein related to -catenin. Overexpression of a membrane-tethered form or a wild-type cytoplasmic form of plakaglobin can result in axis duplication (Merriam et al., 1997). On the basis of this result, it has been suggested that -catenin, like plakaglobin, functions in the cytoplasm to keep an inhibitor of dorsal gene expression out of the nucleus. However, such a mechanism is not consistent with the proposed nuclear function of -catenin as a transcriptional activator. The use of a membrane-tethered form of -catenin suggests that the membrane-tethered forms of -catenin or plakaglobin in fact interfere with the -catenin signaling pathway by binding to APC, an endogenous component required for -catenin degradation (Miller and Moon, 1997). The axis-inducing activity of the membrane-tethered forms is therefore likely to result from removal of the inhibitory effect of APC and accumulation of endogenous -catenin in the nucleus. Another result that appears inconsistent with the nuclear function of -catenin for axis duplication is that a secondary axis forms in embryos injected ventrally with antibodies directed against -catenin (McCrea et al., 1993). However, it is possible that, instead of blocking the function of -catenin, the injected anti--catenin antibodies stabilize -catenin and thus result in an increased level of -catenin in injected blastomeres. 4. Glycogen Synthase Kinase-3 The component upstream of -catenin in the Wnt signaling pathway is GSK-3, a serin/threonine kinase, which actively promotes -catenin degradation (Yost et al., 1996). Activation of the Wnt pathway suppresses the activity of Xgsk-3 and results

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in the accumulation of high levels of -catenin. This is demonstrated by the expression of a dominant-negative form of Xgsk-3 on the ventral side of Xenopus embryos, which blocks its endogenous function and subsequently stabilizes -catenin, and induces secondary axis formation (Dominguez et al., 1995; He et al., 1995; Pierce and Kimelman, 1995). Overexpression of wild-type Xgsk-3 on the dorsal side of the embryo suppresses Wnt signaling and inhibits endogenous axis formation (He et al., 1995; Pierce and Kimelman, 1995). Two distinct modes of Xgsk-3 regulation have been reported (Dominguez and Green, 2000). It is shown that an endogenous mechanism exists on the dorsal side of Xenopus embryos to deplete Xgsk-3 protein. A second mode of regulation occurs as a consequence of Wnt or Dsh expression, which causes a reduction in Xgsk-3 activity rather than depletion. 5. Adenomatous Polyposis Coli, Axin, Beta-Transducin Repeat-Containing Protein, and Protein Phosphatase 2A It has been proposed that GSK-3 functions in a large complex containing additional regulators of the Wnt signaling pathway, including APC, Axin, beta-transducin repeat-containing protein (-TrCP), and protein phosphatase 2A (PP2A), all of which promote downregulation of -catenin by GSK-3 (Wodarz and Nusse, 1998; Sokol, 1999). APC was originally identified as a tumor suppressor gene because loss of APC function was observed in colon cancer (Polakis, 1997). The APC protein contains armadillo repeats, -catenin binding sites, and protein domains that interact with Axin and PP2A (Bienz, 1999). The C terminus of the APC protein also contains a discs large (DLG)-binding domain that interacts with the PDZ protein domain found in the PSD-95, Discs large, and ZO-1 proteins (Fanning and Anderson, 1999). Both GSK-3 and APC are required to phosphorylate -catenin. GSK-3 may directly phosphorylate -catenin in the presence of APC; alternatively, GSK-3 may first phosphorylate APC, which binds to -catenin and leads to -catenin phosphorylation (Rubinfeld et al., 1996; Yost et al., 1996). However, the role of APC may seem to be more complex because overexpression of APC mutants that degrade catenin in cultured cells induce secondary axis formation when expressed in Xenopus embryos (Vleminckx et al., 1997). This result is in contrast to the expected function of APC, which is to lower the -catenin level. It has been suggested that APC and -catenin may be involved in an independent pathway to induce axis formation or that the mutant forms of APC stabilize -catenin by an unknown mechanism. A mouse mutation, fused, produces a truncated gene product of axin (Zeng et al., 1997). Mutant embryos exhibit duplicate axis formation implicating Axin as a negative regulator of axis formation. Axin contains a DIX (Dishevelled/Axin) domain at the C terminus and RGS (regulator of G protein signaling) domain at the N terminus. The DIX domain, also found in Dsh, has been proposed to allow interaction between Dsh and Axin or oligomerization of Axin (Hsu et al., 1999; Sakanaka and Williams, 1999). However, the DIX domain is not required for the

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ventralization of endogenous axis by axin. The RGS domain has been found in regulators of G protein signaling (Koelle, 1997). Removal of the RGS domain results in a dominant negative form of Axin (Hsu et al., 1999). Similar to the case with the GSK-3 dominant negative mutant, overexpression of the RGS-deleted form of Axin leads to axis duplication. The presence of an RGS domain suggests a possible involvement of G proteins in the regulation of the Wnt pathway. Axin may function in the GSK-3 complex to stabilize the interaction between GSK-3 and -catenin. -TrCP is a vertebrate homolog of Slimp, a F-box/WD40 protein that functions in the ubiquitination of -catenin (Jiang and Struhl, 1998). A mutant form of -TrCP induces ectopic axis formation (Marikawa and Elinson, 1998; Lagna and Hemmati-Brivanlou, 1999; Liu et al., 1999a). In Xenopus, three different transcripts of -TrCP have been isolated, two of which are localized to the vegetal cortex during oogenesis (Hudson et al., 1996). The regulatory and catalytic subunits of PP2A have been found to interact with APC and Axin, respectively. The B56 subunit interacts with APC, as shown in a yeast two-hybrid assay (Seeling et al., 1999). The catalytic subunit of PP2A can bind to Axin (Hsu et al., 1999). It is possible that PP2A mediates specific dephosphorylation of components of the APC complex to inhibit GSK-3-dependent phosphorylation of -catenin. 6. Glycogen Synthase Kinase-3 Binding Protein GSK-3 binding protein (GBP), an upstream regulator of GSK-3 activity, was isolated by a yeast two-hybrid assay (Yost et al., 1998). GBP is related to the product of a T-cell proto-oncogene, Frat1 (Jonkers et al., 1997). GBP inhibits the phosphorylation function of GSK-3 by direct interaction with GSK-3. Overexpression of GBP stabilizes -catenin and can induce a secondary axis (Yost et al., 1998). Antisense oligonucleotide knockout experiments have demonstrated that GBP is required for the establishment of the dorsal–ventral axis in Xenopus embryos (Yost et al., 1998). Evidence indicates that GBP can also induce depletion Xgsk-3 proteins, similar to the endogenous activity existed on the dorsal side of the embryo (Dominguez and Green, 2000). It appears that GBP is a regulator of Wnt signaling but is not a linear component of the pathway. 7. Casein Kinase I and II Overexpression of casein kinase I (CKI) induces an ectopic axis on the ventral side of embryos (Peters et al., 1999). CKI acts downstream of Dsh and upstream of GSK-3. CKI has been shown to phosphorylate Dsh. It has been suggested that such phosphorylation is not required for the functioning of CKI. CKII is also able to phosphorylate Dsh (Willert et al., 1997). Further studies on the regulation of Dsh by these two kinases will be required to determine their roles in transducing the Wnt signal.

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8. Dishevelled Dsh is upstream of GSK-3 and can inhibit the negative regulatory function of GSK3 in the presence of Wnt signaling. A double inhibition downstream of Dsh results in an increase in the -catenin level when Dsh is activated. Dsh is phosphorylated by active Wnt signaling (Yanagawa et al., 1995). The Dsh protein contains a DIX domain, a PDZ domain, and a DEP (Dishevelled, egl-10, and pleckstrin) domain. Removal of the central PDZ domain produces a dominant negative form of Dsh (Sokol, 1996), indicating that the PDZ domain is required for Dsh functioning. Dsh has been shown to translocate from the vegetal pole toward the prospective dorsal side (Miller et al., 1999). This suggests a possible endogenous role of Dsh during VCC/-catenin signaling. However, depletion of the maternal gene products will be required to provide definitive evidence for a role of Dsh in the establishment of dorsal–ventral axis in Xenopus. 9. Frizzled Frizzled proteins are receptors for Wnt ligands (Bhanot et al., 1996). Members of the Frizzled family contain a cysteine-rich domain, seven transmembrane domains, and sometimes a PDZ-binding domain. Overexpression of Xfrizzled-8 (Xfz8) alone on the ventral side of an embryo induces a secondary axis in the absence of exogenously supplied Wnt ligands (Deardorff et al., 1998). However, since the endogenous expression of Xfz8 is zygotically activated in the organizer region during gastrulation, Xfz8 is unlikely to function as the endogenous Wnt receptor for the dorsal specification pathway during the early cleavage of Xenopus embryos. A maternally encoded frizzled protein, Xfz7, has recently been isolated (Sumanas et al., 2000). Embryos depleted of Xfz7 mRNA are deficient in dorsal mesoderm formation. This study provides experimental evidence for the functioning of the Frizzled proteins upstream of other components of the Wnt pathway in dorsalventral mesoderm specification. 10. Wnt Molecules If an endogenous Wnt ligand is involved in the activation of the VCC/-catenin signaling pathway, such a Wnt molecule has not yet been identified. Several Wnt ligands have been studied, and these can be divided into two classes according to their axis-inducing ability. Mouse Wnt-1 (McMahon and Moon, 1989; Sokol et al., 1991), and several Xenopus Wnts, including Xwnt-8 (Smith and Harland, 1991), Xwnt-8b (Cui et al., 1995), Xwnt-2b (Landesman and Sokol, 1997), and Xwnt3A (Wolda et al., 1993), can induce a secondary axis when they are expressed ectopically in the ventral region. In contrast, Xwnt-4 (Du et al., 1995), Xwnt5A (Moon et al., 1993), and Xwnt-11 (Ku and Melton, 1993) have weak or no axis-inducing ability but cause morphogenetic defects when they are expressed ectopically. However, the Wnt molecules that have strong axis-inducing activity

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are not expressed at the right time and right place to be the dorsalizing signal (Moon and Kimelman, 1998). Overexpression of Xwnt-8 can induce a complete secondary axis, but endogenous expression is not detected until after the MBT in the ventral-lateral mesoderm. Although Xwnt-8b is maternally expressed and has strong-axis-inducing activity, it is localized to the animal region of cleavage-stage embryos (Cui et al., 1995). Xwnt-11 mRNA is localized to the vegetal cortex of the oocyte (Kloc and Etkin, 1995) and the protein is differentially translated along the dorsal–ventral axis (Schroeder et al., 1999); the possible involvement of Xwnt-11 in the VCC/-catenin signaling has yet to be demonstrated. It has recently been shown that Xwnt-11 does not signal through the canonical Wnt pathway involving GSK-3 and -catenin for the regulation of morphogenetic movements (Heisenberg et al., 2000; Tada and Smith, 2000). This result demonstrates the divergence of signal transduction cascades that Wnt molecules can elicit. 11. Integration of the VCC/-catenin Signaling Pathway with Other Pathways Some Wnt molecules have been shown to signal through pathways independent of the -catenin pathway. Wnt-5A signaling can increase the intracellular Ca2+ level in zebrafish embryos through a sequential action of the rat frizzled-2 receptor, G proteins, and stimulation of the phosphatidylinositol cycle (Slusarski et al., 1997). It has also been demonstrated that protein kinase C is a downstream component of the Xwnt-5A/rat frizzled-2 receptor G-protein-dependent pathway (Sheldahl et al., 1999). Treatment of embryos with lithium chloride during early cleavage stage produces a dorsalized phenotype. It was originally suggested that the dorsalizing effect of lithium is due to an activation of the phosphatidylinositol cycle (Maslanski et al., 1992). However, it was later demonstrated that lithium chloride treatment can inhibit GSK-3 activity, and the dorsalizing effect of the lithium ion is mediated by an activation of the -catenin pathway (Klein and Melton, 1996; Stambolic et al., 1996). The activity of GSK-3 is also regulated by the ribosomal S6 protein kinase p90(rsk) (Torres et al., 1999). p90(rsk) inhibits GSK-3 by phosphorylation, and results in an increase of the total amount of -catenin. Fibroblast growth factor (FGF) signaling has been shown to inhibit GSK-3 activity and can also activate p90(rsk). FGF signaling and p90(rsk) may play a role in modulating the activities of GSK-3 and -catenin during early development. E. Molecular Nature of the Dorsal Determinant in Vegetal Cortical Cytoplasm Like overexpression of -catenin and Wnts, overexpression of wild-type dsh induces ectopic axis formation. A dominant negative form of dsh, Xdd1, although

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able to block ectopic axis formation by the activation of Wnt signaling, has no effect on endogenous axis formation (Sokol, 1996). Similar results have been obtained for Wnt molecules in dominant negative forms (Hoppler et al., 1996), frizzled proteins (Xu et al., 1998), and Wnt antagonists (Glinka et al., 1998). One interpretation of these results is that the VCC/-catenin pathway is not triggered by the presence of a Wnt ligand, but that a component of the Wnt pathway initiates the signaling cascade. Evidence supporting this model came from the study in which Dsh is shown to translocate from the vegetal pole toward the prospective dorsal side (Miller et al., 1999). This finding provides a link between the dorsal activity localized to the vegetal cortical cytoplasm of the egg and the activation of the VCC/-catenin signaling pathway on the dorsal sector of the embryo. However, the possibility that translocation of dsh toward the future dorsal side is preceded by the signaling of a Wnt ligand at the vegetal cortex of the egg should not be ruled out. The effect of Xdd1 overexpression has also been examined in prospective ectoderm transplanted with VCC (Marikawa and Elinson, 1999). In keeping with the findings in embryos, overexpression of Xdd1 in prospective ectoderm has no effect on the activity of VCC, as demonstrated by the expression of target genes siamois and Xnr3. Overexpression of different components of the Wnt pathway has shown that the activity of VCC is not inhibited by Xdd1 or Xgsk-3 but is inhibited by Axin. On the basis of these findings, it has been proposed that VCC may in fact act on Axin instead of Xgsk-3 to mediate its dorsalizing activity. This model is distinct from the general concept that an inhibition of Xgsk-3 activity by the endogenous dorsalizing signal is a prerequisite for -catenin-mediated gene expression in the dorsal region. F. Cooperation between TGF- Signaling and Wnt Signaling Activation of the VCC/-catenin pathway alone is not sufficient for specification of the dorsal–ventral axis. In UV-irradiated embryos, which lack any dorsal structures, the VCC/-catenin pathway is still activated in the vegetal pole and siamois, a transcriptional target of the Wnt pathway, is expressed in the vegetal pole region (Darras et al., 1997). Furthermore, transplantation of vegetal cortical cytoplasm, or overexpression of siamois, only induces ectopic axis formation when the recipient site is the equatorial region but not when the recipient site is the animal region (Kageura, 1997). Thus, the VCC/-catenin pathway has to synergize with other components in the equatorial region to specify axis formation. One distinct possibility is that there is a cooperation between the VCC/-catenin and TGF- signaling pathways. Evidence for this includes the induction of notochord in prospective ectoderm by Xwnt-8 and the TGF- signaling molecule, Vg1 (Cui et al., 1996). Overexpression of Vg1 alone induces dorsal mesoderm but no notochord, and Xwnt-8b alone does not induce mesoderm formation. Coexpressing components of the Wnt and TGF- pathways enhances expression of both the Wnt-responsive gene siamois and the activin/TGF--responsive genes goosecoid (gsc) and chordin (Crease et al., 1998). Both TGF- response elements and Wnt

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response elements are present in the promoter of the organizer gene gsc. In addition, it is also shown that both Wnt and TGF- signaling are required for specifying formation of anterior endoderm. All these findings further strengthen the idea of cooperativity between the Wnt and the TGF- pathways (Watabe et al., 1995). In overexpression experiments, several TGF-s have been shown to induce dorsal development. For dorsal development to occur, the endogenous ligand has to be active in the embryo at the right time and place. Spatial restriction of Vg1 mRNA to the vegetal cortex is suggested to provide a localized source of maternal protein to act as a dorsal inducer (Thomsen and Melton, 1993). Overexpression of a mutant form of Vg1 cannot perturb axis formation, although dorsal mesoderm and endoderm formation are affected (Joseph and Melton, 1998). This finding indicates that Vg1 is required for dorsal development, but expression of Vg1 alone is not sufficient in specifying formation of the dorsal–ventral axis. Activin protein is present maternally and may be a candidate for the endogenous TGF- signal involved in dorsal–ventral axis formation (Fukui et al., 1994). Results from the overexpression of a dominant negative activin type II receptor in Xenopus embryos, although they have demonstrated the requirement of TGF- signaling, have not been conclusive because of the inhibition of additional TGF- members (Hemmati-Brivanlou and Melton, 1992). The use of a specifically designed dominant negative activin type II receptor, containing only the extracellular domain and lacking the transmembrane domain and intracellular domain, has circumvented the problem of nonspecific interference with other receptors at the cell surface (Dyson and Gurdon, 1997). This dominant negative receptor selectively blocks the function of activin but not that of Vg1 and nodal-related factors Xnr1 and Xnr2, although BMP signaling is slightly inhibited. Overexpression of this dominant negative activin type II receptor in Xenopus embryos has demonstrated the requirement of activin for the development of dorsal structures and the initiation of mesoderm induction. Furthermore, although knockout mice deficient in different activin subunits also show no defects in early development (Matzuk et al., 1995a), a type I activin receptor knockout does produce a defect in gastrulation (Gu et al., 1998). Therefore, it is likely that an activin-like TGF- is required for early development. The zygotically activated Nodal-related TGF-s are the best candidates for the endogenous TGF- signals involved in mesoderm and endoderm formation.

G. TGF- Receptors, Smads, and Target Genes Members of the TGF- superfamily are involved in a wide variety of cellular processes, including cell growth and differentiation (Massague, 1998). Two major types of serine/threonine kinase receptors, types I and II, are required for TGF- signaling. Upon ligand binding of TGF- or activin, type II receptors recruit and phosphorylate type I receptors (Fig. 2). The signal is transduced from the activated type I receptors by Smad proteins, which shuttle between the cytoplasm and the

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Figure 2 Components of the TGF- signaling pathway. TGF- ligands are first recognized by the type II receptor, which recruits and phosphorylates the type I receptor upon ligand binding. The signal is transduced by the R-Smad, which is activated by the type I receptor. The R-Smad associates with a co-Smad and translocates into the nucleus. The R-Smad and co-Smad complex with additional transcriptional factors to activate expression of target genes such as mix2 and gsc.

nucleus to control target gene expression (Massague, 1998). Specific Smad proteins transduce signals from different subgroups of TGF- molecules. Several Smads function as transcription factors. Smads proteins specific for TGF- signaling can bind to activin-response elements in the promoter of activin responsive genes. Three classes of Smad proteins have been identified (Christian and Nakayama, 1999). Receptor Smads (R-Smads) are phosphorylated by the intracellular domain of type I receptor. The activated R-Smads escort a second class of Smads, the coactivator Smads (co-Smads), into the nucleus to control target gene expression. The third class of Smads, the inhibitory Smads, prevent the R-Smads from binding to the type I receptor or the co-Smads. The Smad proteins contain three domains: a MH1 domain, a linker region, and a MH2 domain. The activity of Smad proteins is under constitutive inhibition by interaction between the MH1 and MH2 domains (Hata et al., 1997). Such autoinhibition is released by the phosphorylation of the MH2 domain at a C-terminal motif of R-Smads. The MH1 domain is required for DNA binding, whereas the MH2 domain plays a role in transcriptional activation and interaction with type I receptors, binding with co-Smads and other DNA-binding

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proteins (Baker and Harland, 1996; Liu et al., 1996, 1997; Meersseman et al., 1997). Smad2 and Smad3 are the R-Smads downstream of TGF-1 and activin signaling (Baker and Harland, 1996; Chen et al., 1996b; Graff et al., 1996; Lagna et al., 1996; Nakao et al., 1997b). Smad1, Smad5, and Smad8 are downstream of BMP activation (Graff et al., 1996; Hoodless et al., 1996; Liu et al., 1996; Thomsen, 1996; Chen et al., 1997; Suzuki et al., 1997a). Smad4 is a co-Smad (Lagna et al., 1996). Smad6 is an inhibitory SMAD for BMP signaling (Imamura et al., 1997; Hata et al., 1998). Smad 7 is an inhibitory Smad for BMP and TGF- signaling (Hayashi et al., 1997; Nakao et al., 1997a). In addition to the inhibitory Smads, other mechanisms regulating TGF- signaling have been reported in Xenopus embryos. BAMBI, a transmembrane protein related to TGF- type I receptors but lacking an intracellular kinase domain, has been identified as a pseudoreceptor (Onichtchouk et al., 1999). Expression of BAMBI is induced by BMP signaling. BAMBI has been shown to associate with TGF- family receptors to inhibit BMP, activin, and TGF- signaling. Smurf1, a ubiquitin ligase, can interact with and trigger ubiquitination and consequently the inactivation of Smad proteins specific for the BMP pathway (Zhu et al., 1999a). Overexpression of Smurf1 can enhance the cellular responses to Smad2, which mediates the activin/TGF- pathway. Smurf1 may conrol the competence of cells to respond to different TGF- signals by regulating the levels of Smad proteins specific for BMP signaling (Zhu et al., 1999a). A maternal forkhead domain DNA-binding protein, FAST-1, was first identified as a component of a transcriptional complex containing Smad2 and Smad4 involved in the activation of Mix.2, an immediate response gene of activin signaling (Chen et al., 1996a). The transcriptional complex that binds to the gsc promoter contains FAST-2 (Labbe et al., 1998). Overexpression of FAST-1 fusion constructs containing either a transcriptional activation domain or a repressor domain has shown that FAST-1 is in fact involved in the induction of a set of activin/Vg1 responsive mesodermal and endodermal genes. This finding suggests that FAST-1 is a key maternal regulator of transcriptional responses to mesoderm inducers (Watanabe and Whitman, 1999).

IV. The Spemann Organizer In amphibians, the organizer forms at the dorsal lip of the blastopore of the gastrula embryo. The organizer is known as the Spemann organizer because the axisinducing activity of this region was demonstrated for the first time in 1921 using urodelean amphibians by Hilda Mangold, a student of Hans Spemann (Hamburger, 1988). Mangold transplanted dorsal blastoporal lips of advanced gastrulae of unpigmented newts to the flanks of pigmented host newts at the same stage of development (Spemann and Mangold, 1924). In the most successful case, the

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resulting embryo had a secondary body axis containing anterior structures that included hindbrain and otic vesicles. In the secondary axis, the neural tube was composed entirely of cells that originated from the pigmented host embryo; the notochord contained cells that originated from the unpigmented transplanted tissue; and the somites were derived from a mixture of cells from both origins. The ability of the transplanted dorsal blastoporal lip to recruit cells from the host embryo to participate in the formation of the secondary axis demonstrated that the transplaned tissue had an organizing ability. The inability of the dorsal lip of advanced gastrula to generate a complete secondary axis including the head prompted Spemann to test the hypothesis that the head and trunk were induced by different regions of the mesodermal tissue of the organizer. Since the organizer is a highly dynamic structure and cells at the blastoporal lip continously undergo involution, the cell population at the blastoporal lip is distinct during different stages of gastrulation. These cells assume a progressively more posterior identity as gastrulation proceeds. By transplanting dorsal lip tissues to the ventral side of the blastocoel, Spemann demonstrated that blastoporal lip from the early gastrula induced head and brain structures while blastoporal lip from the late gastrula induced spinal cord and tail structures. Spemann introduced the terms “head organizer” and “trunk organizer” to specify the regional and temporal differences of the organizer (Spemann, 1927). Regional specification of organizer-derived tissue was further demonstrated by Otto Mangold (Mangold, 1933). O. Mangold showed that, at the neurula stage, the organizer-derived mesodermal tissue layer had assumed an anteroposterior identity and could be divided into regions including the anterior endomesoderm, the prechordal mesoderm, and the anterior and posterior chordamesoderm. Of these, only the prechordal mesoderm, fated to form the head mesenchyme, demonstrated head-inducing ability. The anterior endomesoderm, the most anterior region containing cells of endodermal and mesodermal origins and fated to give rise to the liver, showed little or no head-inducing activity. The anterior region of the chordamesoderm gave rise to the hindbrain and spinal cord, whereas the posterior region of the chordamesoderm formed only the spinal cord. In Xenopus, the organizer is formed in the dorsal vegetal region of the embryo as a consequence of signaling by TGF- and VCC/-catenin (Harland and Gerhart, 1997; Heasman, 1997; Niehrs, 1999, 2000). Molecular characterization of the organizer has led to the identification of genes that are specifically expressed in the organizer and can induce secondary axes by acetopic expression. These organizer genes can be classified into three groups on the basis of the type of secondary axis generated. However, it should be noted that the organizer tissue is a dynamic structure. The organizer is not a constant population of cells, and considerable cell movement and rearrangement take place during gastrulation. The first group of organizer genes are transcriptional targets of the VCC/-catenin pathway, and some are expressed in the dorsal vegetal region well before the appearance of the dorsal lip during the blastula stage. The products of some of these genes can induce

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Figure 3 Expression domains of organizer genes in the head and trunk organizers. The organizer consists of four expression domains: anterior endomesoderm, prechordal mesoderm, superficial layer, and chordamesoderm. The head organizer is made up of the prechordal mesoderm and anterior endomesoderm. The chordamesoderm possesses trunk organizer activity. (modified from Harland and Gerhart, 1997; with permission, from the Annual Review of Cell and Developmental Biology, Volume c 13, 1997, by Annual Reviews www.AnnualReviews.org)

complete secondary axes containing head and trunk when the genes are overexpressed on the ventral side of the embryo. The second group of organizer genes are expressed in the prechordal mesoderm region of the head organizer at the gastrula stage and can generate ectopically incomplete secondary axes lacking anterior structures. The last group of organizer genes are expressed in the anterior endomesoderm of the head organizer at the gastrula stage. The product of some of these genes can produce an ectopic head without a trunk by overexpression. The expression domains of the organizer genes at the gastrula stage are summarized in Fig. 3.

A. Organizer Genes Expressed in the Dorsal Vegetal Region siamois and twin are paired-like homeobox genes that are transcriptional targets of VCC/-catenin signaling (Brannon et al., 1997; Laurent et al., 1997; Fan et al., 1998). Overexpression of either siamois or twin induces ectopic secondary axis with a complete head (Lemaire et al., 1995; Laurent et al., 1997). siamois is expressed in the dorsal vegetal region soon after the MBT. Vegetal cortical cytoplasm can induce siamois expression ectopically, indicating that siamois is a downstream target of the dorsal determinant in the vegetal cortical cytoplasm (Darras et al., 1997). Transplantation of vegetal cortical cytoplasm to the animal hemisphere activates the expression of chordin, whereas transplantation of vegetal cortical

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cytoplasm to the vegetal hemisphere activates the expression of cer. This shows that different regions along the animal–vegetal axis have diferent abilities to respond to the cytoplasmic dorsal determinant. Activation of cer expression in the vegetal hemisphere by siamois induces the formation of anterior endomesoderm, whereas the activation of chordin in the animal hemisphere induces the formation of the head organizer (Darras et al., 1997). siamois is therefore able to induce the formation of the anterior endomesoderm and the head and trunk organizers. Since overexpression of siamois mRNA in either the ventral vegetal region or the equatorial region results in ectopic axis formation (Laurent et al., 1997), the action of siamois may be mediated by a diffusible growth factor that instructs cells in the equatorial region to participate in axis formation. twin has axis-inducing properties similar to those of siamois. Like Siamois, Twin has been shown to bind to and activate the Wnt-responsive regulatory elements of the gsc promoter (Laurent et al., 1997). In zebrafish, a paired-like homeobox gene, nieuwkoid/dharma encoded by the bozozok locus, expressed in dorsal blastoderm and dorsal yolk syncytial layer has demonstrated organizer gene activity (Koos and Ho, 1998; Yamanaka et al., 1998; Fekany et al., 1999). The dorsal yolk syncytial layer forms at the blastula stage when cells in the deep marginal blastoderm release their nuclei into the yolk cells. bozozok mutants are deficient in activity and exhibit a loss of shield derivatives, equivalent of the Xenopus organizer, and anterior neural structures. Although sequence comparison does not suggest the existence of an ortholog for nieuwkoid/dharma, the involvement of a paired-like homeobox gene in organizer formation both in frogs and in teleofish indicates a potential conserved role of the paired-like homeobox transcription factor in other developmental systems.

B. Organizer Genes Expressed in the Prechordal Mesoderm Organizer genes expressed in the prechordal mesoderm include genes that code for transcription factors (Gsc, Xlim-1, Xanf-1, Xotx2), growth factor antagonists (noggin, chordin, follistatin, frzb, dickkopf-1 [dkk-1]), and growth factors (Xnr14 and anti-dorsalizing morphogenetic protein [ADMP]). Most of these genes are expressed above the dorsal lip in the prechordal mesoderm, which contributes to the head mesenchyme. dkk-1, Xnr-1, and Xnr-2 are also expressed in the anterior endomesoderm. chordin is expressed in the prechordal mesoderm, including the superficial layer. Xnr-3 is most strongly expressed in the superficial layer of the dorsal lip, which gives rise to the pharyngeal endoderm. ADMP is related to BMP3. Overexpression of ADMP inhibits dorsal mesodermal markers, including organizer genes, and induces ventral markers (Moos et al., 1995). It has also been shown that ADMP is expressed in the trunk organizer (Dosch and Niehrs, 2000). Since the function of ADMP cannot be blocked by a dominant-negative BMP receptor or other BMP antagonists except follistatin, it may function in the trunk organizer to antagonize head formation by a distinct pathway (Dosch and Niehrs, 2000).

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1. Genes Encoding Transcription Factors The transcription factor Gsc contains a paired class homeodomain and is able to generate an incomplete secondary embryonic axis when overexpressed (Cho et al., 1991; Steinbeisser et al., 1995). Dissociated Xenopus embryos express the same level of gsc as undissociated embryos, indicating that the expression of gsc is cell autonomous—that is, independent of cell–cell interaction (Lemaire and Gurdon, 1994). Analysis of isolated organizer explants has demonstrated that by the early gastrula stage, gsc expression is already limited to a discrete region of the organizer (Zoltewicz and Gerhart, 1997). gsc is expressed in the anterior half of isolated organizer explants. The gsc expression domain is spatially distinct from the Xnot expression domain in the posterior half, which normally gives rise to the chordamesoderm. In the mouse, gsc is expressed in the anterior primitive streak and the anterior mesoderm, which gives rise to the head process (Blum et al., 1992). gsc knockout mice have craniofacial and rib-cage defects, but their early development is largely unaffected (Rivera-Perez et al., 1995; Yamada et al., 1995). However, the neural-inducing strength of the mouse node from gsc knockout mice is impaired, as demonstrated by studies in which gsc-deficient mouse node was transplanted to chick embryos (Zhu et al., 1999b). The presence of gsc-related genes in different systems, including Drosophila (Goriely et al., 1996; Hahn and Jackle, 1996), chicken (Lemaire et al., 1997) and human (Gottlieb et al., 1997) suggests that a related family member may compensate for gsc function in gsc knockout mice. A hydra homolog of gsc, Cngsc, has also been identified (Broun et al., 1999). Cngsc is expressed in tissues with organizer activity and is able to induce a secondary axis when expressed in Xenopus embryos. This suggests that the function of the Gsc protein has been conserved during evolution. In Xenopus, the requirement of gsc function has been demonstrated by studies involving overexpression of antimorphic forms of gsc, containing transcription activation domain or multiple copies of the myc epitope at the N terminus (Ferreiro et al., 1998). The transcriptional activation effect of the myc epitope is not expected, and caution should be taken when the myc epitope is used in the context of a putative transcription factor (Ferreiro et al., 1998). Antimorphic gsc is expected to inhibit the function of endogenous Gsc as well as other closely related family members, if they do exist in Xenopus. When antimorphic gsc is expressed on the dorsal side of the embryo, ventral genes are activated ectopically, and embryos exhibit dorsoanterior defects. This finding shows that Gsc normally acts as a transcriptional repressor inhibiting ventral gene expression in the organizer region, and is consistent with the suggestion that Gsc functions as a transcriptional repressor to directly suppress the transcription of Xbra (Artinger et al., 1997; Latinkic et al., 1997). Overexpression of antimorphic Gsc also activates expression of endogenous gsc, indicating a possible self-repression of gsc in embryos. Like gsc, the mutant form of the LIM-domain-containing homeobox gene Xlim-1 (Taira et al., 1994) and the homeobox genes Xanf-1 (Zaraisky et al., 1995) and Xotx2 (Pannese et al., 1995) also produce an incomplete secondary embryonic

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axis lacking anterior structures when these genes are overexpressed in embryos. In addition, Xotx2 induces ectopic cement gland formation in embryos (Blitz and Cho, 1995; Pannese et al., 1995). Mice deficient in lim1 have anterior truncations, including a missing forebrain and midbrain (Shawlot and Behringer, 1995). A study of otx2 knockout mice demonstrated a loss of anterior neural structures in these animals (Ang et al., 1996). Xanf-1 is related to mouse Hesx-1/Rpx, which may be involved in anterior development (Hermesz et al., 1996; Thomas and Beddington, 1996). Gain-of-function studies in frogs and loss-of-function studies in mice together suggest an important role of Xlim-1, Xanf-1, and Xotx2 in development of the most anterior part of the head.

2. Genes Encoding Growth Factor Antagonists The organizer genes noggin (Smith et al., 1993), chordin (Sasai et al., 1994), and follistatin (Hemmati-Brivanlou et al., 1994) encode for protein factors originating from the organizer. When these genes are ectopically expressed on the ventral side of frog embryos, a secondary axis with an incomplete anterior structure is generated. The protein products of these genes are structurally distinct and have been shown to function through their ability to antagonize, and thus inhibit, BMP signaling by direct binding to BMP molecules. Noggin is a glycoprotein secreted as a homodimer. Mouse noggin (McMahon et al., 1998) is expressed in the node, which gives rise to prechordal mesoderm and participates in head formation. noggin knockout mice do not show the early developmental defects that would be expected when an organizer gene is disabled (Brunet et al., 1998; McMahon et al., 1998). noggin-deficient mice have defects in somites and neural tube formation. This suggests that although noggin is not required for neural induction, it is important for later events, including patterning of somites and neural tube. Three noggin genes have been isolated from zebrafish (Furthauer et al., 1999). noggin 1 and noggin 2 are expressed in the organizer and the notochord, respectively, whereas noggin 3 is involved in a later stage of development for chondrogenesis. Although only a single noggin gene has been reported so far in other species, the finding of multiple noggin genes in zebrafish suggests that the functional redundancy of related genes should be taken into consideration. Chordin contains cysteine-rich domains (CRs) and is also secreted. The CRs have been shown to be novel protein modules for BMP binding and therefore confer the biological activity of chordin (Larrain et al., 2000). The zebrafish chordino mutant, originally known as dino, displays a partially ventralized phenotype with reduced head formation and, often, lacks a notochord, indicating an effect of chordin in organizer function (Hammerschmidt et al., 1996). It is therefore informative to study mice deficient in either chordin or noggin or both of the genes to determine their requirement in specifying anterior development and a possible functional redundancy of these genes during early development. Double-homozygous mutant mice for chordin and noggin have been generated (Bachiller et al., 2000). These

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embryos showed severe defects in the development of the forebrain. Chordin and Noggin are indeed required for anterior development in the mouse. BMP signaling has been shown to play a role in embryonic patterning (Dale and Jones, 1999). Inhibition of BMP signaling by a cleavage mutant generates secondary axes (Hawley et al., 1995), whereas activation of the BMP pathway by overexpression of BMPs or constitutively active BMP receptors results in a ventralized phenotype (Jones et al., 1996b). bmp-2, -4, and -7 mRNAs are maternally expressed and are detected in the entire animal hemisphere. Only bmp-4 mRNA, but not bmp-4 or bmp-7 mRNA, is downregulated at the organizer region when the organizer forms. Chordin and Noggin have been shown to mediate a dorsalizing or axis-inducing effect by binding to BMPs and inhibiting BMP signaling in the organizer (Holley et al., 1996; Piccolo et al., 1996; Zimmerman et al., 1996). Chordin can bind BMP-2, BMP-4, and BMP-4/7 heterodimer but not TGF-. The antagonistic effect between Chordin and BMP is evolutionarily conserved, as demonstrated by the Drosophila homologs Sog and Dpp and the functional substitution of Chordin and BMP by Sog and Dpp in frog embryos (Sasai et al., 1995). Noggin can bind BMP-2 and BMP-4, and can bind BMP-7 less tightly, but is not able to bind TGF-. Noggin has a higher affinity to BMPs (K D = 20 pM) than does Chordin (K D = 300 pM, 1 nM for inducing neural response and dorsalization of mesoderm). The binding affinities between BMPs and their receptors are in the same range as the binding affinities between BMPs and Noggin or Chordin. An additional level of regulation of BMP signaling is carried out by a metalloprotease of the astacin family (Dumermuth et al., 1991), Tolloid, which can inhibit the antagonistic function of Chordin on BMP signaling. Genetic studies in Drosophila have shown that tolloid can potentiate the effects of Dpp (Shimell et al., 1991; Ferguson and Anderson, 1992). Xolloid and BMP-1, related proteins of the tolloid family, contain a metalloprotease domain that is followed by CUB (Cls and Clr/Uegf/BMP1) domains and EGF-like domains (Piccolo et al., 1997; Goodman et al., 1998; Wardle et al., 1999b). The CUB domains may be required for protein–protein interaction—for example, to interact with the substrate. Xolloid has been demonstrated to cleave Chordin within specific sites. This suggests that Xolloid can negate the inhibitory effects of chordin on BMP signaling. In Xenopus embryos, overexpression of Xolloid and XBMP-1 results in a ventralized phenotype as expected from the removal of Chordin from the embryo. Dominant negative forms of Xolloid and XBMP-1 produce embryos with a dorsalized phenoptype with enlarged heads and anterior structures, although the deletion mutant inhibits the activities of both Xolloid and XBMP-1. In zebrafish, Xolloid is encoded by the mini fin (mfn) gene (Connors et al., 1999). mfn mutants exhibit a loss of ventroposterior tissues, including the ventral fin, that is due to a dorsalization resulting from diffusion of Chordin to the most ventral marginal regions at the end of gastrulation. In wild-type embryos, the presence of Mfn may negatively regulate Chordin activity and promote BMP signaling in the ventral marginal cells. A related sea-urchin metalloprotease, SpAN, when expressed in Xenopus embryos, can block the dorsalizing activity of both noggin and chordin

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(Wardle et al., 1999a). Since SpAN does not cleave noggin or chordin directly, binding of Noggin or Chordin to BMPs may be a prerequisite for SpAN to function (Wardle et al., unpublished observations, 1999a). It has also been suggested that SpAN may trigger the release of BMPs from other BMP-binding proteins or the extracellular matrix. The mechanism by which SpAN inactivates the dorsalizing function of noggin and chordin has yet to be determined. A recently identified member of the BMP-4 synexpression group, Xenopus Twisted gastrulation (xTsg), encodes a secreted BMP-binding protein. xTsg functions as antagonist to provide a permissive signal for BMP signalling (Oelgeschlager et al., 2000). Follistatin was originally suggested to bind Activin directly and thereby control the amount of free Activin (Tashiro et al., 1991). However, in vitro data have demonstrated an affinity of Follistatin for BMP-4 (Fainsod et al., 1997; Iemura et al., 1998). The Follistatin/BMP-4 complex can bind to BMP receptors, suggesting that Follistatin regulates BMP signaling through a mechanism different from that by which Noggin and Chordin regulate BMP signaling (Iemura et al., 1998). The organizer function of Follistatin is further complicated by the fact that follistatin is not expressed in the equivalent of the organizer in zebrafish (Bauer et al., 1998) or mouse (Albano et al., 1994) and is only weakly expressed in chicken (Levin, 1998). In addition, no axial defects are detected in follistatin knockout mice (Matzuk et al., 1995b). This result argues against a requirement for follistatin during early development. The role of follistatin in the inhibition of BMP signaling and organizer activity requires further investigation. frzb, also known as frzb1, is expressed in the organizer region (Leyns et al., 1997; Wang et al., 1997). The expression pattern of frzb1 is complementary to endogenous Xwnt-8 expression in the ventral lateral mesoderm. Frzb functions as a growth factor antagonist by direct binding to Wnts and thus inhibition of Wnt signaling. Frzb belongs to a clas of proteins that are known as frizzled-related proteins (FRPs) because their structure is similar to that of the membrane-bound Wnt receptor of the frizzled family, except that FRPs lack the transmembrane domain. Overexpression of frzb generates a partial secondary axis at a low frequency, and overexpression of frzb in whole embryos causes dorsalization, with embryos showing enlarged heads and shortened body axes. Frzb also inhibits the effect of ectopic Xwnt-8 expression in a non-cell-autonomous manner, suggesting that Frzb functions extracellularly to suppress Wnt function. A related protein, FrzA, when overexpressed in embryos, shows a phenotype similar to that produced by overexpression of frzb in embryos (Xu et al., 1998). However, frzA is not involved in organizer function because expression commences at the neurula stage in the somitic mesoderm.

C. Organizer Genes Expressed in the Anterior Endomesoderm The identification of genes that are specifically expressed during gastrulation in the anterior endomesoderm has suggested a crucial role of this region as part of the head organizer for the generation of all the anterior structures of an axis.

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The anterior endomesoderm includes the deep dorsoanterior endodermal cells that do not undergo cell involution. The prechordal mesoderm, on the contrary, does involute when the dorsal lip forms. Several genes have been identified in the anterior endomesoderm of the Xenopus head organizer. Xnr-1, -2, and -4, Xblimp, and XHex can regulate specific gene expression in the anterior endomesoderm. The cer and dkk-1 genes encode growth factor antagonist expressed in the anterior endomesoderm and have been suggested to be involved in anterior development. 1. Genes That Regulate Anterior Endomesoderm Formation Xnr-1 and Xnr-2 are expressed in the anterior endomesoderm as well as the prechordal mesoderm (Jones et al., 1995). The involvement of the Xnrs in axis induction has been suggested by the observation that a complete secondary axis forms when Xnr-1 is coexpressed with noggin (Lustig et al., 1996a). noggin alone can only generate an incomplete secondary axis. Xnr-1 and Xnr-2 can induce the expression of anterior endomesoderm markers, including XHex, cer, frzb1, and Xsox17, in prospective ectodermal explants (Zorn et al., 1999b). The use of a cleavage mutant form of Xnr-2, cmXnr2, permits loss-of-function analysis of Xnr-2. Overexpression of cmXnr2 results in anterior truncation and delayed or suppressed expression of dorsoanterior endodermal genes (Osada and Wright, 1999). A similar phenotype has also been observed with the overexpression of a mutant Activin type II receptor containing the extracellular domain (Dyson and Gurdon, 1997) or a Smad2 dominant negative construct (Hoodless et al., 1999). In zebrafish, a similar phenotype is observed in the cyclops/squints and MZoep mutants (Feldman et al., 1998; Gritsman et al., 1999). cyclops and squint are nodal-related genes. MZoep is a mutant lacking both maternal and zygotic activities of oep, which is a member of the EGF-CFC (epidermal growth factor-Cripto/Frl-1/Cryptic) family. Oep is membrane-bound and is produced extracellularly in cells responsive to Nodal. Oep functions as an essential cofactor to facilitate Nodal signaling. The mutant phenotype of MZoep can be rescued by overexpressing activin, activated activin receptor, and Smad2. This suggests that Nodal signaling activates an activin-like pathway during early embryonic patterning. In the frog, a temporal and spatial regulation of Nodal signaling is required for the development of the most anterior structures. Maternal factors (Vg1, VegT, or both) may activate the expression of Xnrs, which induce the formation of the anterior endomesoderm through an activin-like pathway. It is thought that one of the functions of the anterior endomesoderm is then to create a Nodal-free zone, by the expression of growth factor antagonists, in the anterior endomesoderm within the head organizer for anterior development (Piccolo et al., 1999). The VCC/-catenin pathway is also involved in inducing expression of genes that regulate anterior endomesoderm formation. When a N-Tcf3 mutant is overexpressed in embryos, expression of XHex and cer is inhibited (Zorn et al., 1999b). Overexpression of a dominant negative Siamois mutant also represses cer

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expression in embryos (Darras et al., 1997). This indicates that a functional Wnt pathway is required for anterior endomesoderm formation. It is also shown that overexpression of bmp inhibits XHex and cer expression in embryos (Zorn et al., 1999b). Although overexpression of chordin or noggin cannot induce XHex or cer expression, they are required to suppress BMP signaling to maintain XHex and cer expression in the anterior endomesoderm. Xblimp1 encodes a zinc-finger transcription repressor and is similar to the mammalian PRDI-BF1/Blimp-1 gene (de Souza et al., 1999). Expression of Xblimp1 is detected in the anterior endomesoderm and prechordal mesoderm. Overexpression of Xblimp1 mRNA on the ventral side of the embryo induces dorsoanterior marker genes, including cer, gsc, and Xotx2, but not frzb or dkk-1. This shows that Xblimp is able to regulate expression of specific genes in the anterior endomesoderm. A complete secondary axis can be generated by the overexpression of Xblimp1 and the BMP-antagonist chordin in embryos. XHex is a transcription factor expressed in the anterior endomesoderm (Newman et al., 1997; Jones et al., 1999). The expression domain of XHex largely overlaps with that of cer in the deep dorsal marginal cells in blastula- and gastrula-stage embryos (Zorn et al., 1999b). Overexpression of XHex induces cer expression in explants derived from ventral endoderm. The mouse homolog Hex is expressed in the primitive endoderm of mouse blastocysts and later in the visceral endoderm at the distal tip of the egg cylinder (Thomas et al., 1998). Hex is one of the earliest markers of the anterior visceral endoderm (AVE) in mouse embryos and is initially detected in the primitive endoderm of blastocysts. The AVE is involved in setting up an early asymmetry of the mouse embryo before the node is formed and has been suggested to be analogous to the anterior endomesoderm in Xenopus (Bouwmeester and Leyns, 1997; Beddington and Robertson, 1998). In both mouse and frog embryos, expression of Hex is also detected in the angioblasts, which are precursors for the blood cells and endothelium during vasculogenesis (Newman et al., 1997; Thomas et al., 1998). It has been suggested that Hex could be a marker gene for stem cell populations of endodermal origin. 2. Growth Factor Antagonists Expressed in the Anterior Endomesoderm Cer is a growth factor antagonist expressed in the anterior endomesoderm of Xenopus embryos (Bouwmeester et al., 1996). Cer contains a single cysteine-rich domain containing conserved cysteine residues and is secreted. In Xenopus, although the anterior endomesoderm does not show any head-inducing property, overexpression of cer can generate ectopic head structures in the absence of a trunk. Such overexpression of cer may activate additional organizer genes that function in conjunction with cer to induce an ectopic head. Cer also demonstrates a neuralizing activity as shown by the induction of the forebrain marker Xotx2 and neural marker NCAM in prospective ectoderm (Bouwmeester et al., 1996). Several mammalian

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proteins have been identified that are related to Cer and the product of the tumor suppressor gene DAN (Ozaki and Sakiyama, 1993). These proteins form the Cerberus/DAN family, which is also known as the Can family (Pearce et al., 1999). A C. elegans homolog of the Can family, CeCan1, has also been identified, suggesting that the protein is highly conserved throughout evolution (Pearce et al., 1999). Overexpression of cer inhibits the functions of Xnr-1, BMP, and Xwnt-8 through direct binding of different regions of the Cer protein to these growth factors (Piccolo et al., 1999). A truncated form of the Cer protein, Cer-S, retains only the site for Nodal binding and can specifically inhibit Nodal signaling. The ventral marginal zone can be induced to show headlike properties by inhibiting BMP and Nodal signaling with dominant negative BMP receptors and Cer-S. It has been suggested that expression of cer in the anterior endomesoderm results in a zone free of Nodal, BMP, and Wnt molecules and is required for head development. However, ablation of cer-expressing tissue in the endoderm affects formation of the heart, but has no effect on head development (Schneider and Mercola, 1999). Furthermore, mouse deletion mutants with the cer1 locus deleted have been shown to develop normally without any defects in anterior patterning (Simpson et al., 1999). Mouse homozygous embryos deficient in a cerberus-like gene also showed the same result. One interpretation of these results is that functional redundant protein products of the same family may compensate for the requirement of Cer during early development (Belo et al., 2000). Dkk-1 is a Wnt antagonist expressed in the anterior endomesoderm, prechordal mesoderm, and anterior chordamesoderm (Glinka et al., 1998). Members of the Dkk protein family are secreted proteins containing two cysteine-rich regions with conserved cysteine residues in each region. Several related proteins have been identified in chicken and mouse (Monaghan et al., 1999). A family of Dkk-related proteins have been identified in human, including hDkk-1, -2, -3, and -4 and Soggy (Krupnik et al., 1999). Soggy is related to Dkk-3 but lacks the cysteine domains. hDkk-2 and hDkk-4 undergo processing resulting in removal of the second cysteine-rich region. Both hDkk-1 and hDkk-4 can suppress the secondary axis generated by ectopic expression of Wnt but not that generated by downstream components such as dsh and frizzled-8. This suggests that Dkk is likely to function upstream of the Wnt receptor to antagonize Wnt signaling. The frog homolog dkk-1 has been shown to antagonize the early axis-inducing ability and the late ventralizing effect of Xwnt8 (Glinka et al., 1998). dkk-1 cannot induce a secondary axis when it is expressed alone. It can do so only when BMP signaling is inhibited by a dominant-negative BMP receptor or BMP antagonists. This demonstrates that inhibition of Wnt and BMP activity alone is sufficient for head induction. However, this apparently conflicts with the requirement of the antagonistic function of Cer as well in inhibiting Nodal signaling during head formation. In fact, cer expression is induced by an inhibition of both BMP and Wnt signaling, thus indicating that Cer, and therefore a repression of Nodal activity in addition to that of BMP and Wnt, is involved in the development of anterior structures (Piccolo et al., 1999).

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Xfz8, a membrane-bound Wnt receptor, is also expressed in the anterior endomesoderm and dorsal lip region (Deardorff et al., 1998; Itoh et al., 1998). In late gastrula and neurula-stage embryos, Xfz8 is expressed in the most anterior ectoderm. Both wild-type Xfz8 and a dominant negative construct, ECD8, containing only the extracellular region, induce secondary axes when expressed ventrally in embryos (Itoh and Sokol, 1999). The in vivo function of Xfz8 has yet to be elucidated. Lineage labeling shows that cells expressing ECD8 mostly contribute to the head ectoderm, which induces the secondary notochord in a noncell-autonomous manner. ECD8 suppresses the activity of several Wnts, including ones that are not inhibited by other Wnt antagonists, such as Frzb. It has been suggested that a Wnt ligand required to suppress a dorsal cell fate in the ventral region is inhibited by ECD8. The mode of action of Xfz8 in the anterior endomesoderm requires further studies.

D. A Mammalian Structure Analogous to the Anterior Endomesoderm In frogs, the anterior endomesoderm is required for the formation of a complete axis including the most anterior structure, and anterior endomesoderm formation is regulated by TGF-, Wnt, and BMP signaling. Studies of the mouse AVE, which may be functionally analogous to the anterior endomesoderm in frog, have demonstrated the role of TGF- signaling in specifying AVE formation during early embryonic development. In the mouse, the AVE is derived from the extraembryonic lineage and does not contribute to the formation of the embryo proper. During gastrulation, cells in the AVE are progressively displaced by the definitive endoderm that originates from the node (Lawson et al., 1991). The definitive endoderm in the AVE then differentiates into liver and gut endoderm. The generation of chimeric mouse embryos consisting of wild-type and mutant cells in specific cell lineages—such as the embryonic epiblast and the extraembryonic visceral endoderm—has provided a useful tool for determining the role of AVE during early patterning (Beddington and Robertson, 1999). Mice deficient in Nodal cannot gastrulate and show no anteroposterior specification (Conlon et al., 1991; Varlet et al., 1997). Introduction of wild-type cells into nodal-deficient blastocysts gives rise to chimeric embryos in which wild-type cells are included in the epiblast. In these embryos, the gastrulation defect but not the anterior defect is rescued, indicating that Nodal signaling is required in the epiblast for primitive streak formation and therefore gastrulation. Introduction of nodal-deficient cells into wild-type blastocysts gives rise to chimeric embryos in which the visceral endoderm is composed entirely of wild-type cells and the epiblast is composed of nodal-deficient cells and wild-type cells. Both the gastrulation defect and anterior–posterior patterning are rescued in these embryos. This finding suggests that Nodal signaling is required in the visceral endoderm during gastrulation for the specification of the anteroposterior axis of early embryos (Varlet et al., 1997).

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Studies in mice deficient in Smad2 have also demonstrated a role for Smad2 in early anteroposterior patterning and mesoderm formation (Nomura and Li, 1998; Waldrip et al., 1998; Weinstein et al., 1998). The extreme alleles show a complete lack of mesoderm formation. One of the studies demonstrated the formation of extraembryonic mesoderm, but more important, the study also showed the absence of anterior visceral marker expression, indicating a possible involvement of Smad signaling in anterior specification (Waldrip et al., 1998). The phenotype of Smad4 knockout mice is similar to the extreme phenotypes of Smad2 knockouts in terms of defective gastrulation and mesoderm formation (Sirard et al., 1998). Gastrulation is rescued in chimeric embryos containing wild-type extraembryonic tissue and defective Smad4 embryonic tissue. This indicates that Smad4 is required in the visceral endoderm to mediate gastrulation in the epiblast in a non-cell-autonomous manner. These findings substantiate the role of Nodal and Smad signaling in specifying the AVE during early axis formation. In Xenopus, some components of the Wnt pathway are required to specify the dorsal–ventral axis, but there is no evidence for the involvement of a maternal Wnt molecule. In contrast, studies in mouse has revealed early Wnt signaling during axis formation by the Wnt3 knockout study (Liu et al., 1999b). Mice deficient in Wnt3 do not gastrulate or form mesoderm. However, expression of the AVE marker genes tested is not affected. The involvement of a Wnt pathway in mesoderm formation in mouse embryos has also been demonstrated by -catenin knockout mutants and Tcf1/Lef1 double mutants, which are defective in mesoderm formation (Haegel et al., 1995; Galceran et al., 1999). This indicates that Wnt signaling is required for mesoderm formation and gastrulation in mouse, but Wnt3 expression is not required for AVE formation. In the mouse embryo, it appears that mesoderm and primitive streak formation are regulated by both TGF- and Wnt signaling and that anteroposterior specification of the visceral endoderm is mainly dependent on TGF- signaling.

E. How Is the Organizer Formed after All? One possible model of organizer formation is the following: The vegetal hemisphere of the egg contains localized maternal components that are able to activate TGF-/Nodal signaling (Fig. 4). Fertilization, followed by cortical rotation, displaces a cytoplasmic dorsal determinant, an activator or a component of the Wnt pathway, to the dorsal vegetal region, where Wnt signaling interacts with the TGF signaling pathway to induce the formation of the anterior endomesoderm and the organizer. TGF-/Nodal signaling alone is not sufficient for axis induction but requires interaction with the Wnt pathway to initiate early patterning. Thus, TGF and Wnt signaling may be activated in parallel before the MBT. After the MBT, zygotic transcription provides an integration point when a combined effect from both pathways regulates expression of organizer genes and induces axis formation.

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Figure 4 VCC/-catenin signaling sets up dorsal–ventral differences. The sperm entry point (SEP) marks the future ventral side of the embryo. Upon fertilization, the egg cortex rotates toward the future dorsal side. The movement displaces cytoplasmic dorsal determinants, originally localized to the vegetal cortex, to the dorsal side of the embryo. The dorsal determinants activate the Wnt signaling pathway, resulting in the nuclear localization of -catenin in dorsal cells. Wnt signaling and TGF- signaling superimpose in the dorsal vegetal quadrant to initiate expression of organizer genes, such as gsc, during the MBT. Both Gsc and Vox are transcriptional repressors. gsc activates chordin expression indirectly through a double inhibition. The organizer expresses a number of growth factor antagonists to keep the region free of BMPs, Wnts, and Nodal signaling. These antagonists are also required to pattern the mesoderm and ectoderm, but it is not known if they are also required for endodermal patterning. BMP and Wnt signaling are required outside the organizer region for a ventral cell fate. Maternal TGF-s or VegT may be involved in the early phase of mesoderm induction. TGF-s are also zygotically activated in the vegetal region for the specification of mesoderm and endoderm.

V. The Three Germ Layers In almost all metazoans, the basic body plan is derived from three germ layers: the endoderm, mesoderm, and ectoderm. In Xenopus embryos, it has been suggested that the vegetal endoderm secretes a source of mesoderm-inducing signal that can direct neighboring ectodermal cells toward the mesodermal lineage. The mesoderm forms in the equatorial region in the presence of TGF- signaling. The endoderm forms in the vegetal region, and formation of the endoderm requires a high level of TGF- signaling. The ectoderm forms in the animal region in the absence of TGF- stimulation from the vegetal region. A. Endoderm The endoderm arises from the yolky cells of tier D in the vegetal region of a 32-cell embryo. These vegetally derived blastomeres are fated to form the lining of the gastrointestinal and respiratory tracts and liver and pancreas (Chalmers

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and Slack, 1998; Wells and Melton, 1999). In tissue recombination experiments, Nieuwkoop demonstrated the induction of endodermal cell lineages in animal cap cells that had been in contact with vegetal regions (Nieuwkoop, 1997). Pharyngeal and dorsal endoderm were induced in the animal caps. It was suggested that the animal cap tissue contained two major layers, including an inner sensorial layer and an outer epithelial layer. The outer layer can be induced by the vegetal cells to form endoderm. Treatment of prospective ectodermal tissues with Activin or processed Vg1 induces expression of the XlHbox8 and IFABP marker genes, which are specifically expressed in the endodermal lineage (Wright et al., 1989; Gamer and Wright, 1995; Henry et al., 1996). Studies with a cleavage mutant of Vg1 have demonstrated that Vg1 is required for development of endoderm and dorsal mesoderm (Joseph and Melton, 1998). Treatment of prospective ectodermal tissues with bFGF does not induce XlHbox8 expression, although a functional FGF signaling pathway is required for endodermal differentiation. During the tailbud stage, XlHbox8, a homeodomain transcription factor, is expressed in cells that give rise to the pancreas. XlHbox8 is expressed in dorsal vegetal explants isolated from blastula embryos and is a marker of anterior endoderm. The response of XlHbox8 expression to lithium chloride treatment and UV irradiation is similar to what has been seen with organizer genes: Lithium chloride treatment increases XlHbox8 expression and UV irradiation abolishes XlHbox8 expression in whole embryos. IFABP, a cysteine-rich protein, is expressed in the epithelium of the small intestine. IFABP is a marker of general mesoderm since it is expressed in both dorsal and ventral vegetal explants. Endodermin (edd) has also been used as an endoderm differentiation marker (Sasai et al., 1996). In gastrula-stage embryos, edd expression is expressed in the endodermal mass and is also activated in precursor cells giving rise to the prechordal mesoderm and notochord and the superficial layer of the dorsal blastopore lip. By the tailbud stage, edd expression is restricted to the endoderm. edd encodes a novel member of the 2-macroglobulin protein family, a proposed function of which is to inactivate proteases. Both Mixer (Henry and Melton, 1998) and Xsox17 and  (Hudson et al., 1997) have been shown to initiate endodermal differentiation in prospective ectoderm. Processed Vg1 induces expression of Mixer, which can activate the endodermal markers XlHbox8, IFABP, LFABP, and edd in prospective ectodermal tissues in the absence of mesoderm. The induction of endodermal marker genes by Vg1 is blocked by a Mixer-engrailed repressor fusion construct, Mixer-ENR, indicating that endoderm differentiation induced by the overexpression of Vg1 is mediated by Mixer. Whereas the mesodermal marker Xbra is induced in cells adjacent to Vg1-expressing cells, Mixer expression is activated in the same cells in which Vg1 is overexpressed. This is consistent with the idea that a high concentration of TGF is required for endodermal development and a lower concentration of TGF- is required for mesoderm induction. The HMG-box transcription factors Xsox17 and  are specifically expressed in the endodermal mass at the early gastrula stage.

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Figure 5 The maternal transcription factor VegT is required for both endoderm and mesoderm formation. Maternal determinants, and to some extent VegT, can activate cell-autonomous expression of endoderm-specific genes and TGF--related growth factors. The TGF-s activate further expression of endoderm-specific genes and reinforce the expression of TGF-s during endoderm formation in the vegetal region. TGF-s activated by maternal VegT are also required for mesoderm formation. VegT may activate its zygotic isoform to maintain TGF- expression during mesoderm formation.

Overexpression of Xsox17 or  in prospective ectoderm initiates endodermal differentiation by inducing the expression of XlHbox8 and IFABP. Expression of Mixer-ENR in vegetal explants inhibits the expression of Xsox17 and , XlHbox8, and IFABP, but not edd. It appears that the induction of XlHbox8 and IFABP by Mixer is mediated through the action of Xsox17 and that Mixer regulates edd expression independently of Xsox17 by a different pathway. The vegetally localized maternal mRNA, xBic-C, has also been shown to lead to endoderm formation when over expressed in ectodermal explant (Wessely and De Robertis, 2000). This result demonstrate that endoderm formation in Xenopus embryos is also governed by maternal determinant. It has been suggested that endoderm formation takes place in two distinct steps involving maternal determinants and zygotically activated TGF-s (Yasuo and Lemaire, 1999). This is consistent with the demonstration that specification of the endodermal lineage occurs during the mid-blastula stage (Heasman et al., 1984). Some early genes that are expressed in the endoderm, such as the genes encoding the transcription factors Xsox17 and Mix.1 and the genes encoding the TGF-s Xnr-1, -2, and -4, Activin B, and Derriere, can be activated cell-autonomously by maternal determinants in dissociated embryonic cells (Clements et al., 1999; Yasuo and Lemaire, 1999). TGF- signaling by zygotically expressed Xnr-1, Xnr-2, Derriere, and possibly other TGF-s subsequently activates the zygotic expression of Mixer and GATA-4 together with an upregulation of Xsox17, Mix.1, Xnr-1, and Xnr-2 expression (Fig. 5). VegT is a maternal determinant, one of its function is involved in endoderm differentiation (Lustig et al., 1996b; Stennard et al., 1996; Zhang and King, 1996; Horb and Thomsen, 1997). Maternal VegT mRNA is localized to the oocyte vegetal cortex, which is fated to give rise to the endodermal lineage. Endoderm differentiation is inhibited in embryos in which maternal VegT mRNA has been depleted

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by antisense oligonucleotides (Zhang et al., 1998a). These embryos express mesodermal and neural markers instead of endodermal markers in the vegetal region. Overexpression of VegT in prospective ectoderm activates early endodermal genes, including Xnr-2, Xsox17, and Mix.1, similar to the endogenous maternal determinants discussed previously (Yasuo and Lemaire, 1999). Activation of Xnr-1 expression may require other factors in addition to VegT (Yasuo and Lemaire, 1999). In zebrafish embryos, the molecular components of the TGF- signaling pathway leading to endoderm formation is similar to that observed in Xenopus. During zebrafish endoderm development, the nodal-related growth factors Cyclops and Squint activate a type I TGF- receptor TARAM-A in the presence of Oep activity (Renucci et al., 1996; Schier et al., 1997; Peyrieras et al., 1998; Zhang et al., 1998b). Activation of TARAM-A signaling induces Mixer expression, which acts through the casanova gene locus for the expression of sox17 (Alexander et al., 1999; Alexander and Stainier, 1999). Some of the molecular components involved in the formation of endoderm are therefore conserved between frogs and teleofish. This is a good example to demonstrate the advantages of studying different model systems for the elucidation of a signaling pathway. Ectopic expression of the organizer genes chordin and noggin also induce endoderm formation in prospective ectoderm (Sasai et al., 1996). Inhibition of BMP signaling by Chordin, Noggin, or a dominant negative BMP receptor can induce neural as well as endodermal gene expression. In prospective ectodermal explants, FGF treatment in addition to chordin or noggin overexpression induces neural markers with a posterior character. However, inhibition of FGF signaling in prospective ectodermal tissues overexpressing chordin or noggin shifts the induction from a neural toward an endodermal fate. In embryos, FGF signaling has been demonstrated in all three germ layers (LaBonne and Whitman, 1997). The endogenous requirement for FGF signaling in the induction of endoderm by chordin and noggin requires further analysis. It is conceivable that the organizer emits anti-BMP signals such as Chordin and Noggin to pattern the endodermal layer, similar to the processes of neural induction in the ectoderm and dorsalization in the mesoderm. Transcription factors such as Mix.1, milk, and Bix1-4 are involved in endoderm formation. Mix.1 (Rosa, 1989; Lemaire et al., 1998) and milk (Ecochard et al., 1998) are immediate-early genes expressed in response to activin treatment. The expression pattern and the function of milk are similar to those of Mix.1. Mix.1 is initially expressed in the entire vegetal hemisphere. The expression domains of Mix.1 and Xbra become mutually exclusive during gastrulation. Ectopic expression of Mix.1 in the marginal zone downregulates Xbra expression in the mesoderm. Mix.1 induces edd expression in prospective ectodermal tissues, but only activates the endodermal markers XlHbox8 and IFABP when Mix.1 is coexpressed with siamois. edd is also activated in milk- or Mix.1-expressing cells, indicating a cellautonomous activation of edd expression by these transcription factors. With the use of a mutant form of Mix.1, namely m11, it has been found that Mix.1 mediates the ventralization effect of bmp-4 during mesoderm formation

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(Mead et al., 1996). However, a different study suggested that bmp-4 and Mix.1 are expressed in different domains and that wild-type Mix. 1 represses both dorsal and ventral mesoderm formation (Lemaire et al., 1998). These suggestions argue against the function of Mix .1 as a ventralizing agent. One suggested explanation is that the mutation introduced into m11 may have altered its specificity to affect functions of genes other than Mix.1. The Bix genes (Bix1-4) were identified by searching for Xbra target genes using a hormone-inducible glucocorticoid receptor fusion construct, Xbra-GR (Tada et al., 1998; Casey et al., 1999). The Bix proteins are similar to the Mix proteins, including Mix.1, Mix2, Mixer, and Milk. The Bix2 protein is identical to milk. The Bix genes are expressed at the onset of gastrulation in the mesoderm and endoderm, and their expression precedes that of Xbra. Bix1 is also an immediateearly gene in response to Activin, Xbra, and VegT. A low level of Bix1 expression in the prospective ectoderm induces ventral mesoderm formation, and a high level of Bix1 expression induces endoderm. The T-box transcription factors Xbra and VegT can activate the promoter sequence of the Bix1 gene. The promoter of the Bix4 gene also contains three T-box binding sites. Through the use of transgenic embryos, it has been demonstrated that two T-box binding sites of the Bix4 promoter are sufficient for mesodermal and endodermal expression. Expression of Bix4 requires maternal VegT. In VegT-depleted embryos, expression of Bix4 rescues endodermal markers but not mesodermal markers or mesoderm-inducing activity, suggesting a role of Bix4 in endoderm formation.

B. Mesoderm The blastomeres from tier B and tier C of a 32-cell embryo contribute to the prospective mesoderm lineage, which is further patterned into dorsal, intermediate, and ventral mesodermal derivatives (Dale and Slack, 1987a). Fate mapping studies show that cells from the dorsal marginal zone contribute to dorsal mesoderm, including head mesoderm, notochord, and somites, whereas cells from the ventral marginal zone form somites and blood cells. Specification studies show that the lateral marginal zone gives rise to intermediate mesoderm, such as somites and pronephros, only as a result of interaction between the dorsal and ventral mesoderm, a process known as dorsalization (Dale and Slack, 1987b). By combining vegetal and animal regions from embryos at different developmental stages, it has been determined that the vegetal region emits mesoderminducing signals between stage 6 and stage 10.5 (Jones and Woodland, 1987). The responsiveness of the animal cap region to the mesoderm-inducing signal has a similar timing. This indicates that mesoderm induction can be initiated as early as the early cleavage stage before the MBT. By combining vegetal masses from early and late blastula embryos with prospective ectodermal tissues, it has been shown that post-MBT vegetal masses have a much stronger induction potential for both

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the mesoderm-inducing and endoderm-inducing activities than those derived from pre-MBT embryos (Wylie et al., 1996; Yasuo and Lemaire, 1999). The increase in inducing in activity of the vegetal mass is probably due to the zygotic activation of TGF- molecules. These molecules are likely to produce amplified inducing signals after the MBT to reinforce mesoderm formation that may have begun before the MBT. Activins can induce the formation of different mesodermal derivatives in prospective ectodermal tissues, including both dorsal and ventral mesoderm, in a dose-dependent fashion (Smith, 1995). It has been suggested that a morphogen gradient is involved in the specification of mesodermal fates in developing embryos (Neumann and Cohen, 1997; Gurdon et al., 1998). Two different mechanisms have been suggested for the setting up of a TGF- morphogen gradient in Xenopus blastula-stage embryos: sequential short-range signaling by a relay mechanism and long-range signaling by passive diffusion. In the relay model, secondary inducing signals are involved in the propagation of the TGF-1 signal across cell boundaries (Reilly and Melton, 1996). In the diffusion model, the Activin protein diffuses among cells and directly activates gene expression in a concentrationdependent manner (Gurdon et al., 1994, 1996). This long-range diffusion is a unique property of Activin and is not observed with other TGF-s such as Xnr-2 and BMP4, which evoke mesoderm gene expression in a cell-autonomous manner. Protein secretion and processing and the extracellular matrix are constraints affecting the diffusion of different TGF- molecules (Jones et al., 1996a). This finding indicates that different mechanisms are involved in controlling the distribution of TGF- molecules within embryonic tissues. Overexpression of constitutively active activin type I receptors activates cell-autonomous gene expression, again arguing against the relay mechanism and supporting a direct action of Activin in the responding cells (Jones et al., 1996a). It has also been shown that responding cells activate expression of different mesoderm genes, according to their position in the morphogen gradient, by sensing the absolute number of occupied receptors, but not the relative number of occupied versus unoccupied receptors (Dyson and Gurdon, 1998). Furthermore, cell– cell interaction is not necessary for cultured blastula cells to respond to the activin morphogen gradient in a concentrationdependent way (Gurdon et al., 1999). This finding suggests that individual blastula cells can respond to Activin, arguing against the requirement for interaction between neighboring cells to refine the response to a morphogen gradient. Whereas Activin is required for the activation of mesodermal gene expression, FGF is required for maintaining the expression of these genes (LaBonne and Whitman, 1994). FGFs induce the formation of mesodermal derivatives of ventral characters in prospective ectodermal tissues (Smith, 1995). FGF induces Xbra expression through the activation of a MAP kinase pathway (LaBonne et al., 1995; Umbhauer et al., 1995). A substantial increase in MAP kinase activity has been demonstrated in dissected embryos (LaBonne and Whitman, 1997). This suggests that wounding introduced by dissection may cause the release of growth factors and a subsequent activation of MAP kinase signaling. It should be noted that such

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artefactual activation of MAP kinase can be circumvented by performing dissection at a low temperature (LaBonne and Whitman, 1997). Overexpression of dominant negative receptor constructs for activin and FGF results in embryos that have no mesoderm and embryos that have mesodermal defects, respectively (Amaya et al., 1991, 1993; Hemmati-Brivanlou and Melton, 1992). These phenotypes suggest a role of TGF- and FGF in mesoderm formation. Studies with Vg1 mutant have demonstrated the role of Vg1 in the formation of dorsal mesoderm (Joseph and Melton, 1998). The maternal store of Vg1 mRNA and Vg1 protein, the potent mesoderm-inducing activity of processed Vg1, and the requirement of Vg1 for dorsal mesoderm formation are all consistent with a role for Vg1 as a maternal TGF- required for mesoderm induction. Members of the Nodal-related family, a class of proteins related to TGF-, are zygotically expressed in the marginal zone. Xnr-1, Xnr-2, and Xnr-4 are expressed in the marginal zone, with higher concentration in the dorsal mesoderm (Jones et al., 1995; Joseph and Melton, 1997). Overexpression of Xnr-1 and Xnr-2 induces both dorsal and ventral mesoderm in prospective ectodermal tissues and, like that of organizer genes such as noggin, chordin, and siamois, can dorsalize tissue explants of ventral marginal zone. A number of T-box transcription factors are involved in mesoderm formation (Stennard et al., 1997). Xbra is the prototype of the Xenopus T-box family, which also includes VegT and Eomesodermin (Eomes) (Smith et al., 1991; Ryan et al., 1996). Overexpression of Xbra or different FGFs induce ventral mesodermal cell types in prospective ectoderm. Although basic FGF (bFGF) has been used as an inducing source for animal cap assays, the endogenous source of FGF is likely to be eFGF (Isaacs et al., 1994). Embryonic FGF (eFGF), but not bFGF, is secreted and is expressed in the equatorial region of gastrula-stage embryos. eFGF and Xbra can activate the expression of each other in an autoregulatory loop. Xbra expression is suppressed in dissociated embryos, indicating that Xbra expression is non-cell-autonomous (Lemaire and Gurdon, 1994). The promoter of Xbra2, a pseudoallele of Xbra, is regulated by FGF and Activin (Latinkic et al., 1997). The Xbra promoter is also subject to regulation by gsc in response to different levels of Activin. A high level of Activin activates gsc expression, which in turn suppresses Xbra expression. A low level of activin activates Xbra but not gsc expression. The demarcation between the endoderm and mesoderm regions may be regulated in a similar manner, such that a high level of endogenous TGF- induces Mix.1 expression, which represses Xbra expression in the endoderm. Studies involving the depletion of maternal VegT transcripts has demonstrated a role of VegT for the establishment of the germ layers in Xenopus embryos. An antisense oligonucleotide knockout study showed that maternal VegT mRNA is required for the formation of endoderm (Zhang et al., 1998a). With an increased efficiency of depletion by using an increased dose of antisense oligonucleotides and HPLC-purification of the oligonucleotides, maternal VegT has been demonstrated to be required for formation of 90% of the mesodermal tissues (Kofron et al., 1999). In VegT-depleted embryos, the expression of TGF- factors, including Xnr-1,-2,

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and -4 and Derriere, is not detected. Overexpression of the same TGF-s in VegTdepleted embryos rescues mesoderm and body axis formation with the use of Cer-S, which can antagonize Xnr factors, the existence of an endogenous gradient of Xnrs in the endoderm has been demonstrated (Agius et al., 2000). Both studies demonstrated that TGF-s such as Xnr-1, -2, and -4 and derriere, which are activated by the maternal transcription factor VegT, are involved in mesoderm development. An isoform of maternal VegT is zygotically expressed not in the endoderm, but in the equatorial region. This isoform contains a different N-terminal region as a result of alternative splicing (Stennard et al., 1999). Unlike the maternal VegT isoform, expression of the zygotic VegT isoform can be activated by Activin and is non-cell-autonomous in dissociated embryos. In contrast, expression of the maternal VegT isoform is not responsive to Activin treatment. Overexpression of zygotic VegT induces both dorsal and ventral mesoderm in prospective ectodermal tissues in a dose-dependent manner. In whole embryos, overexpression of zygotic VegT induces ectopic dorsal lip formation and morphogenetic cell movement (Lustig et al., 1996b). Zygotic VegT is first expressed above the dorsal lip region; the expression domain subsequently extends laterally and ventrally in the marginal zone and marks the lateral and ventral mesoderm similar to the expression pattern of Xbra. Overexpression of zygotic VegT also upregulates Xbra expression in prospective ectodermal tissues. The expression of Xbra and zygotic VegT overlaps in the marginal zone, but not in the notochord, in which only Xbra is expressed. Another T-box gene, eomes, has an expression pattern similar to that of zygotic VegT in the early gastrula (Ryan et al., 1996). eomes is an early T-box gene expressed at the MBT. Expression of eomes in prospective ectodermal tissues can be induced by growth factors, including Activin and BMP-4, but not eFGF or Xwnt-8. Like overexpression of zygotic VegT, eomes overexpression can induce expression of dorsal and ventral mesodermal marker genes. A dominant-interfering construct containing eomes and the engrailed repressor domain produces a gastrulation defect that can be rescued by wild-type eomes. Zygotically expressed VegT, eFGF, and derriere, a novel member of the TGF-like family, regulate the expression of each other (Sun et al., 1999). derriere is expressed in the prospective endoderm and mesoderm during the blastula stage and is later restricted to the posterior mesoderm. The expression pattern of derriere is similar to that of zygotic VegT. derriere activates mesodermal markers of both dorsal and ventral character, as well as some ectodermal, neural, and endodermal marker genes. derriere can also activate zygotic VegT and eFGF expression. The expression of derriere can be induced by growth factors, including Activin, BVg1, bFGF, Derriere itself, and the transcription factors encoded by T-box genes such as Xbra and zygotic VegT. Ectopic expression of wild-type derriere in embryos induces a partial axis on the ventral side or microcephaly on the dorsal side, similar to the phenotypes produced by overexpression of the zygotic VegT isoform. Expression of a cleavage mutant of Derriere results in embryos with posterior

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truncation missing somites and tail. This mutant phenotype is distinct from the dorsoanterior defects produced by the Vg1 mutant and the activin cleavage mutant, thus demonstrating the requirement of different TGF-s in both dorsal and ventral mesoderm development. The similarity of the Derriere cleavage mutant phenotype to overexpression of dominant negative forms of FGFR, Xbra, and VegT further confirms the regulation between Derriere, FGF, and VegT. When does mesoderm induction occur? Tissue recombination assays have demonstrated an early mesoderm-inducing signal during cleavage stage. However, it has also been shown that a mesoderm-inducing signal is zygotically activated. Can these data be reconciled with each other? On the basis of the available information, it is possible that mesoderm induction occurs in two phases, one before and one after the MBT. During the early cleavage stage, TGF- signaling is initiated, albeit at a low level. At the MBT, maternal VegT activates expression of transcriptional targets, such as Xnr-1, Xnr-2, and derriere, to amplify and reinforce TGF- signaling in the prospective endoderm to initiate endodermal differentiation. In addition, these factors signal to cells in the marginal zone to induce the expression of the zygotic isoform of VegT, and other T-box transcription factors, such as Xbra and Eomes. Zygotic VegT specifies mesoderm formation by further activating the transcription of TGF-s and other T-box members in the marginal zone. For example, zygotic VegT can activate derriere, which is able to induce FGF expression. Zygotic VegT is also able to induce the expression of eomes and Xbra. In this model, maternal determinants and zygotic TGF-s are both required for mesoderm induction. An intricate regulation cascade between TGF- and the T-box transcription factors therefore functions in the vegetal and marginal zones to bring about mesoderm formation. In addition to TGF-s and FGF, bmp overexpression induces ventral mesoderm formation in prospective ectodermal tissues, including blood and mesenchyme (Dale and Jones, 1999). It has been suggested that only heterodimers between BMP-2 or BMP -4 and BMP-7, but not homodimers, are potent inducers of ventral mesoderm (Nishimatsu and Thomsen, 1998). Homodimers are only able to ventralize mesoderm that has already been induced by activin. Inhibition of homoand heterodimers of BMPs by the BMP antagonists noggin and gremlin in the ventral marginal region shows an activation of organizer genes, but the expression of panmesodermal markers is unaffected (Eimon and Harland, 1999). This result suggests that BMP signaling is required for the patterning of mesoderm rather than primary mesoderm induction. A high level of BMP in the ventral marginal zone induces blood, the most ventral mesoderm, and the expression of both Xvent-1 (Gawantka et al., 1995) and Vox (also known as Xvent-2, Xom, and PV.1) (Ault et al., 1996; Ladher et al., 1996; Onichtchouk et al., 1996; Schmidt et al., 1996). A lower level of BMP in the lateral marginal zone leads to muscle formation and only induces the expression of Vox (Onichtchouk et al., 1998). Vox is expressed in the marginal zone and animal cap region excluded from the organizer and is later excluded from the

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notochord and neural plate. The expression pattern of Vox is complementary to that of chordin and Xnot (Schmidt et al., 1996). Studies using a dominant-interfering construct Vox have shown that Vox is a transcriptional repressor for a subset of organizer genes, including chordin, gsc, Xotx2, XFD’-1, and Xnr-1, but not noggin (Melby et al., 1999). Since gsc overexpression represses Vox (Ferreiro et al., 1998), the finding that Vox represses chordin explains the observation that gsc activates chordin expression. The activation of chordin by gsc is therefore indirect and is mediated by a suppression of the inhibitory effect of Vox. This demonstrates a repression of chordin by bmp-4 at the transcriptional level. The regulation between chordin and bmp-4 is not limited to protein–protein interaction. Xnf7 is a maternally expressed transcription factor that shows a differential cytoplasmic localization before and after the MBT (El-Hodiri et al., 1997). Overexpression of an engrailed-xnf7 fusion construct has been shown to induce expression of the BMP-4 antagonist chordin and the formation of incomplete secondary axes. This indicates the possible involvement of a maternal transcription factor for the regulation of BMP4 signaling in axial patterning (H. El-Hodiri and L. D. Etkin, unpublished observations). Members of the EGF-CFC family may be required for Nodal signaling. One of the family members, FRL-1, induces mesoderm and neural differentiation in prospective ectoderm (Kinoshita et al., 1995). Another family member, Cryptic, is expressed in mesoderm and midline neuroectoderm and is thought to be required for mesoderm and neural patterning (Shen et al., 1997). Similar to the case with the BMPs, Wnts are also involved in mesoderm development. However, Wnts are not mesoderm inducers, because they do not induce mesoderm formation in prospective ectoderm. Xwnt-8 is expressed in the ventral lateral marginal zone in gastrula-stage embryos. Wnt antagonists such as cer, dkk-1, and frzb are expressed in the dorsal mesoderm, whereas sizzled is expressed in the ventral mesoderm. frzb and sizzled contain the extracellular region of the Wnt receptor frizzled family. Wnt signaling is involved in patterning the mesodermal layer. Ectopic expression of Xwnt-8 after the MBT in the dorsal marginal zone exerts a ventralizing effect and increases the amount of somitic tissues formed at the expense of the notochord (Christian and Moon, 1993; Hoppler et al., 1996). In contrast, suppression of Wnt-signaling with a dominant negative Xwnt-8 construct or the Wnt antagonist frzb inhibits ventral gene expression (Hoppler et al., 1996; Leyns et al., 1997; Wang et al., 1997). It has also been suggested that Xwnt-8 is required to pattern and sharpen the boundary between notochord and somites in the dorsal and dorsolateral marginal zone (Hoppler and Moon, 1998). In the ventral marginal zone, the Wnt antagonist sizzled, which is activated by BMP-4, may restrict Wnt signaling to the lateral region (Salic et al., 1997; Marom et al., 1999). In the marginal zone, Wnt signaling is therefore eliminated in the dorsal and ventral mesoderm by different Wnt antagonists. As a consequence, Wnt signaling is active only in the lateral marginal region to specify formation of lateral mesodermal derivatives.

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C. Ectoderm Blastomeres from tier A of a 32-cell embryo give rise to the prospective ectoderm, which differentiates into neural and nonneural ectodermal lineages (Chang and Hemmati-Brivanlou, 1998). At the gastrula stage, the dorsal ectoderm in proximity to the organizer develops into the neural plate, and the ventral ectoderm gives rise to the epidermis. The cement gland, various placodes, and neural crest are formed at the boundary between the neural plate and the epidermal region. By embryonic cell dissociation experiments, it has been shown that BMP signaling mediates the decision between neural and epidermal cell fates (Wilson and Hemmati-Brivanlou, 1995). Whereas intact ectodermal explants develop into atypical epidermis and express epidermal keratin, dissociated ectodermal cells follow a neural fate and express neural markers. Epidermal cell fate can be rescued in dissociated animal cap cells by treatment with BMP-4 but not activin. BMP signaling is therefore required to repress neural development in the ectoderm. A number of organizer genes, including chordin, noggin, follistatin, Xnr-3, cer, and dkk-1, can induce prospective ectoderm to adopt a neural character by exerting a dorsalizing activity. Chordin, Noggin, Follistatin, Xnr-3, and Cer possess anti-BMP activities, whereas Cer and Dkk-1 have anti-Wnt activities. Therefore, a suppression of the BMP or Wnt pathway results in induction of a neural fate in the ectoderm. FGF signaling has been suggested to be involved in neural induction and anterior neural patterning. Studies using a dominant negative FGF receptor provide evidence that FGF signaling is not required for the early event of neural induction but is involved in the posterior development of the mesoderm and neuroectoderm layers (Kroll and Amaya, 1996; Holowacz and Sokol, 1999). However, in a different study, it has also been demonstrated that FGF signaling is involved in anterior neural induction (Hongo et al., 1999). Chordin and Noggin can both interact with BMPs physically and inhibit binding of BMPs to the receptor, albeit with different affinities (Piccolo et al., 1996; Zimmerman et al., 1996). Follistatin inhibits BMP function through a mechanism different from that of Noggin and Chordin because the Follistatin/BMP-4 complex is able to bind to BMP receptors (lemura et al., 1998). It has been suggested that follistatin may direct specific degradation of BMPs by mediating binding to receptors. Xnr-3 induces neural marker expression in prospective ectoderm (Hansen et al., 1997). Unlike Xnr-1, -2, and -4, Xnr-3 has no mesoderm-inducing activity, but can dorsalize ventral mesoderm. The activation of neural genes by Xnr-3 can be inhibited by overexpression of BMP-4 or an activated form of the BMP receptor. This suggests that Xnr-3 also inhibits BMP activity during neural induction. The mechanism by which Xnr-3 inhibits BMP functions is unclear. It has been proposed that Xnr-3 may dimerize with BMP-4 to produce a nonfunctional complex or act as a BMP receptor antagonist that competes with BMP for binding to the receptor. Cer also has neural-inducing activity in prospective ectoderm (Bouwmeester et al., 1996). Since the neuralizing effect of Cer can be blocked by overexpression of

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BMP-4, Cer also mediates its neurogenic effect by inhibiting BMP signaling. The demonstration of a BMP-4-binding site in the N terminus of the Cer protein further substantiates a physical interaction between Cer and BMPs. Xenopus brain factor 2 (XBF-2) has been shown to be a common downstream target of noggin and cer (Mariani and Harland, 1998). Expression of noggin or cer in prospective ectoderm induces XBF-2 expression, which acts as a transcriptional repressor that inhibits BMP-4 transcription. Thus, noggin and cer play a role in neural induction by antagonizing BMP signaling at both the protein level and the transcriptional level. The control of neural and nonneural cell fate provides the basis for neural and ectodermal differentiation. However, there is not yet any evidence for the diffusion of any of the secreted growth factors expressed in the organizer. It appears that additional positional information is required to further pattern the ectoderm. It has been proposed that different levels of BMP generate a morphogen gradient across the dorsal–ventral axis to specify different cell fates in the ectoderm (Dale and Wardle, 1999). A dose-dependent effect of BMP-4 overexpression in inducing neural and epidermal gene expression in dissociated animal cap cells has been observed (Wilson et al., 1997). A low level of BMP-4 induces neural marker expression, and intermediate and high doses activate cement gland and epidermal keratin expression, respectively. A similar dose-dependent effect has been observed with a dominant-negative BMP receptor, tBR, and the signal transducer of BMP signaling Smad1. It is not clear how such a gradient is established in embryos. The msx1 gene is a target of BMP-4 that can mediate epidermal induction (Suzuki et al., 1997b). Msx1 is a homeobox transcription factor and is an immediate early gene expressed in response to BMP-4 induction. Expression of msx1 is detected in ventral ectoderm and mesoderm. In whole embryos, overexpression of msx1 causes ventralization. Overexpression of msx1 rescues epidermal differentiation in dissociated ectoderm, which would otherwise follow a neural fate. It has also been shown that the Xmsx2 gene is involved in the anterior–posterior patterning of dorsal mesoderm in Xenopus embryos (Gong and Kiba, 1999). The induction of neural marker expression by the Wnt antagonist, dkk-1, suggested a possible involvement of Wnt signaling in the regulation of ectoderm differentiation (Glinka et al., 1998). It has been suggested that a Wnt signal may be required during the cleavage stage to suppress BMP signaling in the dorsal region of the embryo during neural induction. The dorsal ectoderm is consequently sensitized to respond to the neuralizing signals emanating from the organizer (Baker et al., 1999). The neural-inducing organizer genes display different expression domains during gastrulation. At mid-gastrula stage, cer is expressed in a broad area of the anterior endomesoderm, including the leading edge mesoderm. A gap of cer expression is observed along the prechordal plate region, where chordin and noggin are expressed (Bouwmeester et al., 1996). These results show that different neural inducers occupy distinct positions within the mesodermal layer. Therefore, a combination of differentially expressed neural inducers and neural-inducing activity in the mesodermal layer, and a spatial restriction of epidermal inducers in

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the ectoderm, may play a role in the specification of neural and nonneural regions within the ectoderm.

D. A Theoretical Model of Germ Layer Formation On the basis of what is known about the different signaling pathways active during germ layer formation, a picture of this process is emerging. In this model, the specification of the three germ layers is governed by the TGF-/Nodal signaling pathway, whereas the BMP and Wnt pathways pattern the germ layers along the dorsal–ventral axis. Vegetal cells are under the influence of a high level of TGF-/Nodal signaling and differentiate into prospective endoderm. Cells in the marginal zone are subject to a moderate level of TGF-/Nodal signaling and are induced to form the prospective mesoderm. The animal region develops into the prospective ectoderm, since it receives little or no TGF-/Nodal signal from the vegetal cells. Although a VCC/-catenin pathway is required before the MBT in the dorsal region to set up the dorsal–ventral axis, Wnt signaling is inhibited in the dorsal marginal zone after the MBT by Wnt antagonists. In addition, Nodal and BMP signaling are inhibited in the dorsal marginal zone by growth factor antagonists. The dorsal marginal zone, free of Wnt, Nodal, and BMP signaling, gives rise to the head organizer. Outside the organizer region, Wnt and BMP signaling are actively required to pattern the marginal zone into lateral and ventral mesoderm. In the ectoderm, Wnt and BMP signaling are required for specification of neural versus epidermal differentiation. The dorsal ectoderm follows a neural cell fate in the absence of Wnt and BMP signaling, and the ventral ectoderm exhibits active Wnt and BMP signaling and undergoes epidermal differentiation.

VI. Developmental Pathways and Tumorigenesis The study of embryogenesis in different developmental systems has provided a basic knowledge of the signaling pathways controlling normal growth and differentiation. A deregulation, or improper activation, of components such as ligands, receptors, intracellular components, and target genes sometimes results in the development of cancer. Mutations in APC and -catenin have been reported in colon tumorigenesis (Kinzler and Vogelstein, 1996). APC is a tumor suppressor gene mutated in familial colon cancer and most cases of sporadic colon cancers. The mutation in APC results in a truncated form of the protein that lacks the ability to maintain a low -catenin level by degradation. The mutated forms of -catenin escape regulation by other components, such as GSK-3 phosphorylation, leading to increased stability. An increased level of -catenin triggers cellular transformation in colon cells. Frat1 is a homolog of GBP that can suppress the inhibitory effect of the GSK-3 complex and

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results in the accumulation of -catenin (Yost et al., 1998). Activation of the Frat1 gene has been reported to contribute to T-cell lymphomas (Jonkers et al., 1997). Smad4 functions together with receptor Smads to transduce downstream signals originating from members of the TGF- superfamily. The DPC4 gene is the human homolog of Smad4. DPC4 is deleted in the majority of pancreatic carcinomas (Hahn et al., 1996). Loss of function of DPC4 has been reported in both pancreatic and colorectal carcinomas. Members of the EGF-CFC protein family, required for Nodal signaling, have been shown to have transformation potential. Overexpression of cripto can transform mammalian cell lines and stimulate proliferation of breast cancer cell lines (Ciccodicola et al., 1989; Normanno et al., 1994). Finally, DAN, a gene that encodes a growth factor antagonist belonging to the Cerberus/DAN family, has been identified as a potential tumor suppressor gene (Ozaki and Sakiyama, 1993). DAN suppresses cell growth in nontransformed cells, and expression of DAN is downregulated in transformed fibroblasts. Deregulation of the Wnt or TGF- pathways has also been reported in other carcinomas that have not been discussed earlier. The elucidation of the basic components of signaling pathways, mechanisms of regulation, and target gene activation during early development will provide a key to understanding the molecular basis of human cancer. The more we understand the regulatory mechanisms of cell growth and differentiation, the more likely it is that we will be able to unravel and control the causes of malignancy.

VII. Perspectives Xenopus embryos offer an efficient system in which to unravel gene function by the overexpression of RNA. Both gain-of-function and dominant-interference assays have provided valuable information regarding the functional roles and requirement of genes for embryonic axis determination and pattern formation. However, more emphasis on protein distribution is needed to further substantiate the endogenous role of gene products during early development. The depletion of maternal transcripts by antisense oligonucleotides has demonstrated the requirement of maternal gene products for early specification of cell lineages. Additional roles of maternal genes in controlling cell fate determination and early development are likely to be elucidated through the use of antisense oligonucleotides knockout experiments. The cloning and characterization of the promoter sequence of genes activated during early development will shed light on the genetic pathways controlling embryogenesis. In addition, these characterized promoter sequences will increase the selection of regulatory sequences for use in controlling the spatial and temporal expression of transgenes in transgenic Xenopus embryos. Although Xenopus laevis does not favor genetic manipulation, such techniques are under rapid development with the use of Xenopus tropicalis. This will certainly provide an additional advantage for the use of Xenopus as a developmental

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system. In addition, genetics analyses have also been well established in other developmental models which provide additional experimental systems. For example, the mouse has been used for knocking out gene functions by gene targeting to determine the requirements for gene products during development. In studying early embryogenesis, the use of a combination of different developmental models is likely to complement the deficiency of individual systems. The integration of unlimited information from different developmental systems will definitely provide perspectives in the elucidation of developmental gene functions and aid in the understanding of early embryonic development.

Acknowledgments APC and LDE thank Drs. Patrick Lemaire, Fiona Stennard, Aaron Zorn, Malgozata Kloc, and Maki Wakamiya for helpful discussions, suggestions, and comments on the manuscript. Work from the author laboratory has been supported by grants from NSF, NIH and March of Dimes.

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