EGF-CFC proteins are essential coreceptors for the TGF-β signals Vg1 and GDF1

EGF-CFC proteins are membrane-bound extracellular factors with essential roles during vertebrate development. Members of the EGF-CFC family consist of One-eyed pinhead (Oep) in zebrafish, FRL1 in Xenopus, Cryptic in chick, and Cripto and Cryptic in mouse and human (for review, see Shen and Schier 2000). Genetic studies in zebrafish and mouse have shown that EGF-CFC proteins are required for mesoderm and endoderm induction and left-right axis formation. Zebrafish embryos lacking both the maternal and zygotic contribution of Oep (MZoep) are defective in mesendoderm induction (Gritsman et al. 1999). Similarly, mouse cripto mutants fail to form a primitive streak and lack embryonic mesoderm (Ding et al. 1998). During later stages of development, EGF-CFC genes are required for proper left-right axis formation. Loss of late Oep activity results in embryos that lack expression of left-side-specific genes and display randomization of left-right laterality (Yan et al. 1999). Similarly, mouse cryptic mutants do not express left-side-specific genes and have left-right defects such as heterotaxia and right isomerism (Gaio et al. 1999; Yan et al. 1999). In addition, mutations in cryptic/CFC1 are associated with laterality defects in humans (Bamford et al. 2000). Genetic studies have shown that EGF-CFC proteins are essential for signaling by TGF-β signals of the Nodal family (Gritsman et al. 1999). Double mutants for the zebrafish nodal-related genes cyclops and squint are phenotypically identical to MZoep mutants (Feldman et al. 1998; Gritsman et al. 1999). Moreover, Nodal signals are inactive in MZoep mutants (Gritsman et al. 1999). Biochemically, the EGF-CFC protein Cripto can act as a coreceptor for Nodal signaling (Reissmann et al. 2001; Yeo and Whitman 2001; Bianco et al. 2002; Sakuma et al. 2002; Yan et al. 2002). Current evidence suggests that Cripto binds to the Activin type I receptor Alk4 and forms a complex with Nodal and the type II Activin receptor ActRIIB (for review, see Whitman 2001). Upon receptor activation, the intracellular kinase domain of the type I receptor phosphorylates the signal transducers Smad2 and/or Smad3 (for review, see Massague and Chen 2000). During mesoderm induction, this leads to the expression of downstream genes such as goosecoid and brachyury/T/no tail (for review, see Schier and Shen 2000; Whitman 2001). Further support for an essential role of EGF-CFC proteins in Nodal signaling is provided by the observations that some hypomorphic or conditional mouse nodal mutants display left-right defects resembling cryptic mutants (Lowe et al. 2001; Brennan et al. 2002; Norris et al. 2002), and strong nodal mutants share aspects of the cripto phenotype (Conlon et al. 1994; Lowe et al. 2001; Norris et al. 2002). In light of analyses of other TGF-β signals, the requirement for EGF-CFC proteins as Nodal coreceptors has appeared unusual (Massague and Chen 2000). With the exception of the TGF-β type III receptor in TGF-β2 signaling, and of endoglin in Alk1-mediated signaling, no coreceptors have been implicated in TGF-β signaling (Massague and Chen 2000). The use of EGF-CFC coreceptors also seems uncommon because genetic and biochemical studies have shown that Activin utilizes the same receptors, Alk4 and ActRIIB, as Nodal but does not require EGF-CFC coreceptors (Massague and Chen 2000; Schier and Shen 2000; Whitman 2001). In contrast to Nodal, Activin can activate downstream signaling and induce mesoderm formation in both wild-type and MZoep mutant embryos (Gritsman et al. 1999). Moreover, Activin can bind to Alk4 and ActRIIB in the absence of EGF-CFC proteins (Massague and Chen 2000). It has thus been unclear whether the Nodal/EGF-CFC interaction is unusual or whether other TGF-β signals rely on coreceptors such as EGF-CFC. A third class of TGF-β ligands exhibits similar biological activities as members of the Nodal and Activin families. Signals belonging to the Vg1/GDF1 family (Vg1 in Xenopus, zebrafish and chick; GDF1 in mouse) share only 35%–55% identity with Nodal or Activin in the mature domain, but can also act as mesoderm inducers (Schier and Shen 2000; Whitman 2001). Processed bVg1 and bGDF1 (chimeras between the N-terminal prodomain of BMP and the C-terminal mature domains of Vg1 and GDF1) induce mesodermal markers in Xenopus (Thomsen and Melton 1993; Kessler and Melton 1995; Wall et al. 2000). Similarly, grafts of cells expressing native cVg1 or chimeric BMP-cVg1 initiate formation of ectopic primitive streaks in chick (Seleiro et al. 1996; Skromne and Stern 2002). It has been proposed that Vg1 acts upstream of Nodal signals in this process (Wall et al. 2000; Skromne and Stern 2002). Xenopus Vg1 is expressed maternally before the transcription of nodal genes (Weeks and Melton 1987) and misexpression of bVg1 induces ectopic Xnr1 (Xenopus nodal related 1) expression (Hyatt et al. 1996; Hyatt and Yost 1998; Wall et al. 2000). Similarly, cVg1 is expressed before nodal during chick embryogenesis and misexpression of cVg1 in the anterior marginal zone during gastrulation induces ectopic nodal expression (Skromne and Stern 2002). GDF1 and Vg1 have also been implicated upstream of Nodal signals during left-right axis formation. GDF1 mouse mutants have left-right laterality defects and lack nodal expression in the left lateral plate (Rankin et al. 2000). Moreover, bVg1 can induce leftness in Xenopus upstream of Nodal signals (Hyatt et al. 1996; Hyatt and Yost 1998; Ramsdell and Yost 1999). Despite the pivotal roles of Vg1/GDF1 during embryogenesis, the signaling pathway activated by these TGF-β signals has not been defined molecularly. Several lines of evidence suggest that Vg1 and GDF1 may activate the same or related pathways as Activin and Nodal. First, Vg1 signaling results in the phosphorylation of Smad2 (Lee et al. 2001). Second, mesoderm induction by bGDF1 and bVg1 can be inhibited by the Smad2-interaction domain (SID) of FAST1, which blocks formation of Smad2–Smad4 complexes (Wall et al. 2000). Third, a truncated form of ActRIIB (ΔXAR1; Hemmati-Brivanlou and Melton 1992) missing the cytoplasmic kinase domain, can block Vg1 and GDF1 signaling (Kessler and Melton 1995; Wall et al. 2000). However, direct binding of Vg1 to ActRIIB has not been detected (Kessler and Melton 1995). Here, we present genetic and biochemical studies in zebrafish and Xenopus that indicate that Vg1 and GDF1 act similarly to Nodal and depend on EGF-CFC coreceptors for the interaction with and activation of Activin receptors.