Our understanding of the molecular mechanisms underlying endoderm induction in the zebrafish originates from the genetic and molecular biological studies of five mutants: one-eyed-pinhead (oep), bonnie and clyde (bon), faust (fau), casanova (cas) and spiel-ohen-grenzen (spg) (Zhang et al. 1998b; Kikuchi et al. 2000; Dickmeis et al. 2001; Kikuchi et al. 2001; Reiter et al. 2001; Sakaguchi et al. 2001; Lunde et al. 2004; Reim et al. 2004). oep encodes a member of the EGF-CFC family of proteins and its protein product acts as cofactor of Nodal (a TGF-β super family protein). The maternal-zygotic oep (MZoep) mutant, which lacks both maternal and zygotic Oep, fails to form any endoderm cells and most mesoderm cells (Zhang et al. 1998b; Gritsman et al. 1999). In the zebrafish blastula embryo, two Nodal genes, cyclops (cyc) and squint (sqt), are expressed throughout the entire marginal domain and the double mutant (cyc; sqt) of these genes is very similar to the MZoep mutant, in that it is entirely deficient of endoderm cells (Feldman et al. 1998). These results implicate Nodal as an essential signaling molecule for endoderm induction in the zebrafish embryo.
bon and fau encode a Mix-type homeodomain transcription factor and the transcription factor Gata5, respectively. bon is expressed in both the mesendoderm and mesoderm domains where the pan-mesoderm marker no tail (ntl; homologue of Xenopus brachyury) is also expressed. In contrast, fau/gata5 expression is restricted to the mesendoderm domain along the animal–vegetal axis in late blastula and early gastrula embryos (Alexander et al. 1999). Both bon and fau/gata5 expression is completely lost in MZoep and cyc; sqt double mutants, demonstrating that their expression is regulated by Nodal signaling (Alexander & Stainier 1999; Rodaway et al. 1999; Reiter et al. 2001). bon and fau/gata5 mutants contain approximately 10% and approximately 60% of the normal number of endoderm cells, respectively, as assessed by the early endoderm marker genes sox17, a high-mobility-group (HMG) transcription factor gene (Alexander & Stainier 1999) and foxA2, a winged helix/forkhead transcription factor gene (formerly known as axial (Strähle et al. 1993)) (Kikuchi et al. 2000; Reiter et al. 2001). These genetic findings indicate that both factors are crucial downstream effectors of Nodal signaling during endoderm induction in the zebrafish.
Although the endoderm phenotype in bon or fau/gata5 disrupted embryos is severe, endoderm cells still remain in bon or fau/gata5 single and double mutants (Kikuchi et al. 2000; Reiter et al. 2001). The partial endoderm induction phenotype in these mutants is likely to be a reflection of the redundant activity of other genes such as Mezzo, which is another Mix-type transcription factor whose expression domain and stage profile overlaps with Bon. In addition, both gain-of-function and loss-of-function experiments using antisense morpholino oligonucleotide (MO) further suggest that Mezzo is a redundant factor of Bon (Poulain & Lepage 2002). In fact, based on overexpression experiments and gene expression analyses using mutant embryos, Bon, Fau/Gata5 and Mezzo are in almost parallel position with each other (Reiter et al. 2001; Poulain & Lepage 2002) (Fig. 2).
Figure 2. Molecular pathway leading to endoderm in the zebrafish and Xenopus. In the zebrafish, all hierarchical cascades are based on genetic studies. In contrast, most of the regulatory pathways in Xenopus have been elucidated using animal cap assays and knockdown experiments. (1) Maternal factor (X) upstream of Nodal (Cyc, Sqt) is unknown. (2) squint is a maternal transcript. (3) Tar; Taram-a, type-I TGFβ receptor (Renucci et al. 1996) (4) Xnrs; Xnr1, Xnr2, Xnr4, Xnr5 and Xnr6 (5) Mix-type homeobox proteins; Mixer, Mix.1, Mix.2, Bix.1, Bix.2 (Milk), Bix.3 and Bix.4.
Download figure to PowerPoint
The cas mutant completely lacks all endodermal cells and organs derived from gut tube. However, mesoderm induction appears to be normal in cas mutants (Alexander et al. 1999). cas encodes a Sox-type transcription factor, and its expression is restricted to endodermal progenitors in the marginal region in the late blastula embryo (Dickmeis et al. 2001; Kikuchi et al. 2001; Sakaguchi et al. 2001). The expression of cas around the marginal domain is absent in MZoep and cyc; sqt mutants and the number of cas-expressing cells is reduced in bon and fau/gata5 mutants, suggesting that cas is a downstream target of Nodal, Bon and Fau/Gata5 (Kikuchi et al. 2001). Cas can also transform a mesodermal fate to an endodermal fate when it is overexpressed, whereas the fate of ectodermal cells in the animal pole region cannot be changed to an endodermal fate by this overexpression (Kikuchi et al. 2001). These data indicate that cas is an essential gene, but not master gene, for endoderm induction and additional factors are necessary for converting ectodermal into endodermal fates.
Recently, the spg mutant, that was originally identified as a brain mutant, was also shown to have defects in endoderm induction (Lunde et al. 2004; Reim et al. 2004). spg, encoding the transcription factor Pou2/Oct4, is a maternal gene and zygotic spg is expressed ubiquitously prior to gastrulation (Belting et al. 2001; Burgess et al. 2002). To elucidate the function of Spg in endoderm induction, maternal-zygotic spg mutants (MZspg) were generated by injection of spg mRNA into homozygous eggs. In MZspg, expression of the early endoderm markers, sox17 and foxA2, is never initiated and although induction of cas mRNA is detected at the blastula stage, cas expression is not maintained during the gastrula stage (Lunde et al. 2004; Reim et al. 2004). Thus, spg is necessary for the induction of sox17 and foxA2, and for the maintenance of cas expression. Additionally, sox17 promoter–luciferase reporter assays have shown that Cas and Spg synergistically regulate sox17 expression to regulate endoderm induction (Reim et al. 2004).
In summary, Figure 2 illustrates the molecular regulatory cascade leading to endoderm induction in the zebrafish. Whereas Gata4, Gata5 and Gata6 are thought to be involved in heart and endoderm development in various model organisms (Charron & Nemer 1999; Molkentin 2000), zebrafish Gata4 and Gata6 function in endoderm induction is not well understood. The maternal factor (X in Fig. 2) upstream of Nodal has not yet been identified and although foxA1 and foxA3 are known to be expressed in endodermal cells and act downstream of Cas, the position of these factors in the regulatory cascade remains unclear. Analyses of five zebrafish mutants have revealed many aspects of the molecular cascade downstream of Nodal signaling. It will be interesting to investigate whether signaling other than Nodal regulates endoderm induction or if other signaling pathways cross-talk with Nodal signaling.
The Xenopus endoderm originates from the vegetal region, where the endodermal cells are intermingled with yolk cells, in blastula stage embryos. In the Xenopus embryo, the maternal transcript VegT, encoding a T-box transcription factor, is localized in the vegetal hemisphere of the egg and early embryo, and the zygotic VegT transcript is restricted to the equatorial region, where mesoderm cells are formed (Lustig et al. 1996; Stennard et al. 1996; Zhang & King 1996; Horb & Thomsen 1997). Endoderm is never induced, however, and mesoderm is mainly formed from the vegetal region, in VegT-depleted embryos (Zhang et al. 1998a). Moreover, VegT overexpression can ectopically induce the expression of some early endoderm maker genes, indicating that maternal VegT is necessary for endoderm formation and mesoderm patterning in the Xenopus embryo (Zhang et al. 1998a). A recent study using animal cap assays has reported that another maternal factor, Sox7, is also a crucial regulator of endoderm induction and that the ability of VegT to induce endoderm genes, except for sox17 and Xenopus Nodal-related genes (Xnrs; Xnr1, Xnr2 and Xnr4), appeared to depend upon Sox7 activity (Zhang et al. 2005).
Five Nodal-related genes (Xnr1, Xnr2, Xnr4, Xnr5 and Xnr6) are reported to be involved in mesendoderm induction in Xenopus (reviewed by Schier 2003). The Xnr5 and Xnr6 in the vegetal hemisphere are induced by VegT in a cell autonomous manner and are inducers of Xnr1, Xnr2 and Xnr4 (Takahashi et al. 2000). The cleavage mutant, Xnr2, suppresses some endoderm marker expression (Osada & Wright 1999). Additionally, the Nodal-related genes Xnr1, Xnr2 and Xnr4 and the Veg1-like TGF-β family member derrière can restore endoderm gene expression in VegT-depleted embryos (Xanthos et al. 2001). Furthermore, the ability of Sox7 to induce endodermal genes is inhibited by Cerberus, which is an antagonist of Nodal (Zhang et al. 2005). These data demonstrate that Nodal-related genes function downstream of Sox7 and VegT and that the Xnrs play important roles in endoderm induction. However, the function of individual Xnrs in endoderm induction remains to be elucidated.
The seven Mix-type homeodomain factors (Mixer, Mix.1, Mix.2, Bix1, Bix2/Milk, Bix3 and Bix4) and three Gata factors (Gata4, Gata5 and Gata6) function downstream of VegT and Nodal-related factors (Xanthos et al. 2001) and are thought to be involved in endoderm induction (Weber et al. 2000; Afouda et al. 2005). The Mix-type genes are expressed in the vegetal region and some are also expressed in the marginal domain which gives rise to mesoderm cells. When overexpressed in animal caps, these Mix-type genes exhibit different abilities to activate endoderm gene expression: Mix.1 can activate endoderm gene expression only when co-expressed with the homeobox gene siamois (Lemaire et al. 1998); Bix1 overexpression at low and high levels induces mesoderm and endoderm gene expression, respectively (Tada et al. 1998); Bix2/Milk appears to promote endoderm gene expression at the expense of mesoderm gene expression (Ecochard et al. 1998) and Bix4 can rescue endoderm, but not mesoderm, gene expression in VegT-depleted embryos (Casey et al. 1999). In addition, Mixer is a strong endoderm inducer when overexpressed (Henry & Melton 1998) and MO-mediated knockdown data shows that Mixer plays an essential role in controlling the amount of mesoderm induction by the vegetal cells (Kofron et al. 2004).
The three zygotic Gata genes (gata4, gata5 and gata6) initiate their expression in vegetal cells fated to form endoderm at the onset of gastrulation. The functional analyses of three these genes demonstrate that Gata5 and Gata6 function as activators of endoderm genes such as sox17 and HNF1β, whereas the function of Gata4 to induce endoderm genes is dependent upon gata6 induction (Afouda et al. 2005). Additionally, Gata5 enhances the ability of Mixer to induce sox17α expression and acts upstream of Xhex and gata4 in cooperation with Mixer (Xanthos et al. 2001).
In summary, Figure 2 illustrates the molecular regulatory cascade leading to endoderm formation in Xenopus embryos. The hierarchical relationship, however, between individual factors in this cascade such as the Mix-type homeobox proteins, the Xnrs and the Gata proteins is still not yet fully clear due to their functional redundancies.
Our understanding of endoderm formation in vertebrates originates mainly from studies of the zebrafish and Xenopus. Although there are several differences between the molecular regulatory cascades in these species, as shown in Figure 2, it seems that the factors involved in endoderm induction and its associated regulatory pathways are conserved between these two vertebrates (Fig. 2). In addition to these species, recent studies have shown that analyses of knockout mice have also contributed to our understanding of endoderm development. In mouse, the hypomorphic allele of Nodal is associated with a complete lack of a definitive endoderm, indicating that Nodal is also an essential factor in the murine embryo, as has been observed in zebrafish and Xenopus (Lowe et al. 2001). Additionally, the Mix-type homeodomain protein, Mixl1, and the Sox-type HMG domain protein, Sox17, are also necessary for definitive endoderm formation in the mouse embryo, as is the case for the zebrafish and Xenopus (Hart et al. 2002; Kanai-Azuma et al. 2002).
In contrast to these factors, it is not yet clear whether the Gata family of transcription factors (Gata4, Gata5 and Gata6) function in endoderm induction in the mouse (Molkentin 2000). The knockout mice for each individual Gata factor do not show any defects in endoderm induction, presumably because of the functional redundancy among family members. However, these Gata genes are expressed in extraembryonic tissues, heart and definitive endoderm and transfection of Gata genes into non-endodermal cells can induce specific endoderm gene expression such as IFABP, gastric H+/K+-ATPase and HNF4 (Maeda et al. 1996; Gao et al. 1998; Morrisey et al. 1998). These data suggest that Gata transcription factors are involved in endoderm differentiation in mouse. In addition, although the molecular regulatory pathways leading to endoderm induction in the mouse have not yet been analyzed, it is interesting to note that Nodal, Mix-type, Sox and Gata factors regulate endoderm development in three vertebrates: the zebrafish, Xenopus and mouse.