Subtractive hybridization identifies chick-cripto, a novel EGF-CFC ortholog expressed during gastrulation, neurulation and early cardiogenesis

EGF-CFC genes encode a novel class of extracellular, membrane-associated proteins that notably play an important role during vertebrate gastrulation. Whereas the two cysteine-rich domains that characterize these proteins, namely the extracellular EGF-like and the CFC domain, are known to be encoded by two evolutionarily conserved exons, it is generally assumed, based on weak primary sequence identity, that the remaining parts of the protein differ among vertebrates, suggesting that known members of the EGF-CFC family do not represent true orthologs. Here, by characterizing the full cDNA and genomic sequences of a new EGF-CFC gene in chick, and by comparing them with their counterparts in human (CRIPTO), mouse (cripto and cryptic), Xenopus (FRL-1) and zebrafish (one-eyed pinhead), we show that all EGF-CFC genes share an identical genomic organization over the entire coding region. Not only are the central two exons (coding for the EGF-like and CFC motifs) conserved, but also conserved are the total number of exons, their size, their intron phase and their correlation with discrete protein modules, in particular those modules that allow the EGF-CFC motif to become membrane-associated. Therefore, despite apparent divergence between their 5' and 3'-terminal exons, all known CRIPTO-related genes are structurally orthologous. We named this novel ortholog in bird, chick-cripto. We report the mRNA distribution of chick-cripto, which begins in the epiblast of the gastrula, with a pattern similar to EGF-CFC genes of other vertebrates.     

Chick CFC controls Lefty1 expression in the embryonic midline and nodal expression in the lateral plate

Members of the EGF-CFC family of proteins have recently been implicated as essential cofactors for Nodal signaling. Here we report the isolation of chick CFC and describe its expression pattern, which appears to be similar to Cfc1 in mouse. During early gastrulation, chick CFC was asymmetrically expressed on the left side of Hensen's node as well as in the emerging notochord, prechordal plate, and lateral plate mesoderm. Subsequently, its expression became confined to the heart fields, notochord, and posterior mesoderm. Implantation experiments suggest that chick CFC expression in the lateral plate mesoderm is dependent on BMP signaling, while in the midline its expression depends on an Activin-like signal. The asymmetric expression domain within Hensen's node was not affected by application of FGF8, Noggin, or Shh antibody. Implantation of cells expressing human or mouse CFC2, or chick CFC on the right side of Hensen's node randomized heart looping without affecting expression of genes involved in left-right axis formation, including SnR, Nodal, Car, or Pitx2. Application of antisense oligodeoxynucleotides to the midline of Hamburger-Hamilton stage 4-5 embryos also randomized heart looping, but in contrast to the overexpression experiments, antisense oligodeoxynucleotide treatment resulted in bilateral expression of Nodal, Car, Pitx2, and NKX3.2, whereas Lefty1 expression in the midline was transiently lost. Application of the antisense oligodeoxynucleotides to the lateral plate mesoderm abolished Nodal expression. Thus, chick CFC seems to have a dual function in left-right axis formation by maintaining Nodal expression in the lateral plate mesoderm and controlling expression of Lefty1 expression in the midline territory.     

Molecular evolution of the EGF-CFC protein family

The epidermal growth factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) proteins, characterized by the highly conserved EGF and CFC domains, are extracellular membrane associated growth factor-like glycoproteins. These proteins are essential components of the Nodal signaling pathway during early vertebrate embryogenesis. Homologs of the EGF-CFC family have also been implicated in tumorigenesis in humans. Yet, little is known about the mode of molecular evolution in this family. Here we investigate the origin, extent of conservation and evolutionary relationships of EGF-CFC proteins across the metazoa. The results suggest that the first appearance of the EGF-CFC gene occurred in the ancestor of the deuterostomes. Phylogenetic analysis supports the classification of the family into distinct subfamilies that appear to have evolved through lineage-specific duplication and divergence. Site-specific analyses of evolutionary rate shifts between the two major mammalian paralogous subfamilies, Cripto and Cryptic, reveal critical amino acid sites that may account for the observed functional divergence. Furthermore, estimates of functional divergence suggest that rapid change of evolutionary rates at sites located mainly in the CFC domain may contribute towards distinct functional properties of the two paralogs.     

The EGF-CFC gene family in vertebrate development

EGF-CFC genes encode extracellular proteins that play key roles in intercellular signaling pathways during vertebrate embryogenesis. Mutations in zebrafish and mouse EGF-CFC genes lead to defects in germ-layer formation, anterior-posterior axis orientation and left-right axis specification. In addition, members of the EGF-CFC family have been implicated in carcinogenesis. Although formerly regarded as signaling molecules that are distant relatives of epidermal growth factor (EGF), recent findings indicate that EGF-CFC proteins act as essential cofactors for Nodal, a member of the transforming growth factor beta (TGF-beta) family. Here, we review molecular genetic evidence from mouse and zebrafish on biological and biochemical roles of the EGF-CFC family, and discuss differing models for EGF-CFC protein function.     

LRP2 is an auxiliary SHH receptor required to condition the forebrain ventral midline for inductive signals

Sonic hedgehog (SHH) is a regulator of forebrain development that acts through its receptor, patched 1. However, little is known about cellular mechanisms at neurulation, whereby SHH from the prechordal plate governs specification of the rostral diencephalon ventral midline (RDVM), a major forebrain organizer. We identified LRP2, a member of the LDL receptor gene family, as a component of the SHH signaling machinery in the RDVM. LRP2 acts as an apical SHH-binding protein that sequesters SHH in its target field and controls internalization and cellular trafficking of SHH/patched 1 complexes. Lack of LRP2 in mice and in cephalic explants results in failure to respond to SHH, despite functional expression of patched 1 and smoothened, whereas overexpression of LRP2 variants in cells increases SHH signaling capacity. Our data identify a critical role for LRP2 in SHH signaling and reveal the molecular mechanism underlying forebrain anomalies in mice and patients with Lrp2 defects.     

Role of the EGF-CFC gene cripto in cell differentiation and embryo development

The EGF-CFC proteins have been recently recognized as a novel family of extracellular factors required during early vertebrate development. Cripto is the founder member of the EGF-CFC family initially related to the epidermal growth factor (EGF); its expression is increased in human colon, gastric, pancreatic and lung carcinoma and in different types of both mouse and human breast carcinomas. Genetic studies in the mouse have established an essential role of cripto in the formation and correct positioning of the anterior-posterior axis. Furthermore, the absence of cripto results in a defective precardiac mesoderm, unable to differentiate into functional cardiomyocytes. Although mouse and human Cripto have been shown to activate the ras/raf/MAP kinase pathway in mammary epithelial cell lines, genetic evidence in Zebrafish has been provided for a relationship between the EGF-CFC proteins and Nodal, a member of the transforming growth factor family. Here we review the biological role of cripto in development and differentiation, pointing out recent findings on the biochemical interactions of Cripto, Nodal and Activin-like receptors.     

Cripto: a tumor growth factor and more

Cripto, a growth factor with an EGF-like domain, and the first member of the EGF-CFC family of genes to be sequenced and characterized, contributes to deregulated growth of cancer cells. A role for Cripto in tumor development has been described in the human and the mouse. Members of the EGF-CFC family are found only in vertebrates: CFC proteins in zebrafish, Xenopus, chick, mouse and human have been characterized and indicate some common general functions in development. Cripto expression was first found in human and mouse embryonal carcinoma cells and male teratocarcinomas, and was demonstrated to be over-expressed in breast, cervical, ovarian, gastric, lung, colon, and pancreatic carcinomas in contrast to normal tissues where Cripto expression was invariably low or absent. Cripto may play a role in mammary tumorigenesis, since in vitro, Cripto induces mammary cell proliferation, reduces apoptosis, increases cell migration, and inhibits milk protein expression. This prediction is strengthened by observations of Cripto expression in 80% of human and mouse mammary tumors. At least three important roles for Cripto in development have created considerable interest, and each activity may be distinct in its mechanism of receptor signaling. One role is in the patterning of the anterior-posterior axis of the early embryo, a second is a crucial role in the development of the heart, and a third is in potentiating branching morphogenesis and modulating differentiation in the developing mammary gland. Whether these properties are functions of different forms of Cripto, different Cripto receptors or the distinct domains within this 15-38 kDa glycoprotein are examined here, but much remains to be revealed about this evolutionarily conserved gene product. Since all Cripto receptors have not yet been determined with certainty, future possible uses as therapeutic targets remain to be developed. Cripto is released or shed from expressing cells and may serve as an accessible marker gene in the early to mid-progressive stages of breast and other cancers. Meanwhile some speculations on possible receptor complexes for Cripto signaling in mammary cells are offered here as a spur to further discoveries.     

Structure-function analysis of the EGF-CFC family member Cripto identifies residues essential for nodal signalling.

Cripto is the founding member of the family of EGF-CFC genes, a class of extracellular factors essential for early vertebrate development. In this study we show that injection of Cripto recombinant protein in mid to late zebrafish Maternal-Zygotic one-eyed pinhead (MZoep) blastulae was able to fully rescue the mutant phenotype, thus providing the first direct evidence that Cripto activity can be added extracellularly to recover oep-encoded function in zebrafish early embryos. Moreover, 15 point mutations and two deletion mutants were generated to assess in vivo their functional relevance by comparing the ability of cripto wild-type and mutant RNAs to rescue the zebrafish MZoep mutant. From this study we concluded that the EGF-CFC domain is sufficient for Cripto biological activity and identified ten point mutations with a functional defective phenotype, two of which, located in the EGF-like domain, correspond to loss-of-function mutations. Finally, we have developed a three-dimensional structural model of Cripto protein and used it as a guide to predict amino acid residues potentially implicated in protein-protein interaction.     

Fucosylation of Cripto is required for its ability to facilitate nodal signaling

O-linked fucose modification is rare and has been shown to occur almost exclusively within epidermal growth factor (EGF)-like modules. We have found that the EGF-CFC family member human Cripto-1 (CR) is modified with fucose and through a combination of peptide mapping, mass spectrometry, and sequence analysis localized the site of attachment to Thr-88. The identification of a fucose modification on human CR within its EGF-like domain and the presence of a consensus fucosylation site within all EGF-CFC family members suggest that this is a biologically important modification in CR, which functionally distinguishes it from the EGF ligands that bind the type 1 erbB growth factor receptors. A single CR point mutation, Thr-88 --> Ala, results in a form of the protein that is not fucosylated and has substantially weaker activity in cell-based CR/Nodal signaling assays, indicating that fucosylation is functionally important for CR to facilitate Nodal signaling.     

Lefty blocks a subset of TGFbeta signals by antagonizing EGF-CFC coreceptors

Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFbeta signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFbeta signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFbeta agonists and antagonists and suggest that subtle sequence variations in TGFbeta signals result in greater ligand diversity.     

Membrane-anchorage of Cripto protein by glycosylphosphatidylinositol and its distribution during early mouse development

cripto is the original member of the family of EGF-CFC genes, recently recognized as novel extracellular factors essential for vertebrate development. During the early stages of mouse gastrulation, cripto mRNA is detected in mesodermal cells; later, cripto mRNA is detected only in the truncus arteriosus of the developing heart. Here we describe the in vivo distribution of Cripto protein throughout mouse embryo development and show that cripto mRNA and protein colocalize. By means of immunofluorescence analysis and biochemical characterization, we show that Cripto is a membrane-bound protein anchored to the lipid bilayer by a glycosylphosphatidylinositol (GPI) moiety. We suggest that presentation of Cripto on the cell surface via a GPI-linkage is important in determining the spatial specificity of cell–cell interactions that play a critical role in the early patterning of the embryo.     

Lefty Blocks a Subset of TGFβ Signals by Antagonizing EGF-CFC Coreceptors

Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFβ signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFβ signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFβ agonists and antagonists and suggest that subtle sequence variations in TGFβ signals result in greater ligand diversity.     

Lefty Blocks a Subset of TGFβ Signals by Antagonizing EGF-CFC Coreceptors

Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFβ signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFβ signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFβ agonists and antagonists and suggest that subtle sequence variations in TGFβ signals result in greater ligand diversity.     

XCR2, one of three Xenopus EGF-CFC genes, has a distinct role in the regulation of left-right patterning

Members of the EGF-CFC family facilitate signaling by a subset of TGFbeta superfamily ligands that includes the nodal-related factors and GDF1/VG1. Studies in mouse, zebrafish, and chick point to an essential role for EGF-CFC proteins in the action of nodal/GDF1 signals in the early establishment of the mesendoderm and later visceral left-right patterning. Antisense knockdown of the only known frog EGF-CFC factor (FRL1), however, has argued against an essential role for this factor in nodal/GDF1 signaling. To address this apparent paradox, we have identified two additional Xenopus EGF-CFC family members. The three Xenopus EGF-CFC factors show distinct patterns of expression. We have examined the role of XCR2, the only Xenopus EGF-CFC factor expressed in post-gastrula embryos, in embryogenesis. Antisense morpholino oligonucleotide-mediated depletion of XCR2 disrupts left-right asymmetry of the heart and gut. Although XCR2 is expressed bilaterally at neurula stage, XCR2 is required on the left side, but not the right side, for normal left-right patterning. Left-side expression of XNR1 in the lateral plate mesoderm depends on XCR2, whereas posterior bilateral expression of XNR1 does not, suggesting that distinct mechanisms maintain XNR1 expression in different regions of neurula-tailbud embryos. Ectopic XCR2 on the right side initiates premature right-side expression of XNR1 and XATV, and can reverse visceral patterning. This activity of XCR2 depends on its co-receptor function. These observations indicate that XCR2 has a crucial limiting role in maintaining a bistable asymmetry in nodal family signaling across the left-right axis.     

FRL-1, a member of the EGF-CFC family,is essential for neural differentiation in Xenopus early development

Recent studies indicate an essential role for the EGF-CFC family in vertebrate development, particularly in the regulation of nodal signaling. Biochemical evidence suggests that EGF-CFC genes can also activate certain cellular responses independently of nodal signaling. Here, we show that FRL-1, a Xenopus EGF-CFC gene, suppresses BMP signaling to regulate an early step in neural induction. Overexpression of FRL-1 in animal caps induced the early neural markers zic3, soxD and Xngnr-1, but not the pan-mesodermal marker Xbra or the dorsal mesodermal marker chordin. Furthermore, overexpression of FRL-1 suppressed the expression of the BMP-responsive genes, Xvent-1 and Xmsx-1, which are expressed in animal caps and induced by overexpressed BMP-4. Conversely, loss of function analysis using morpholino-antisense oligonucleotides against FRL-1 (FRL-1MO) showed that FRL-1 is required for neural development. FRL-1MO-injected embryos lacked neural structures but contained mesodermal tissue. It was suggested previously that expression of early neural genes that mark the start of neuralization is activated in the presumptive neuroectoderm of gastrulae. FRL-1MO also inhibited the expression of these genes in dorsal ectoderm, but did not affect the expression of chordin, which acts as a neural inducer from dorsal mesoderm. FRL-1MO also inhibited the expression of neural markers that were induced by chordin in animal caps, suggesting that FRL-1 enables the response to neural inducing signals in ectoderm. Furthermore, we showed that the activation of mitogen-activated protein kinase by FRL-1 is required for neural induction and BMP inhibition. Together, these results suggest that FRL-1 is essential in the establishment of the neural induction response.     

The CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto. Functional and structural insights through disulfide structure analysis.

The disulfide structure of the CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto protein was determined by a combination of enzymatic and chemical fragmentation, followed by chromatographic separation of the fragments, and characterization by mass spectrometry and N-terminal sequencing. These studies showed that Cys115 forms a disulfide bond with Cys133, Cys128 with Cys149, and Cys131 with Cys140. Protein database searching and molecular modeling revealed that the pattern of disulfide linkages in the CFC domain of Cripto is the same as that in PARS intercerebralis major Peptide C (PMP-C), a serine protease inhibitor, and that the EGF-CFC domains of Cripto are predicted to be structurally homologous to the EGF-VWFC domains of the C-terminal extracellular portions of Jagged 1 and Jagged 2. Biochemical studies of the interactions of ALK4 with the CFC domain of Cripto by fluorescence-activated cell sorter analysis indicate that the CFC domain binds to ALK4 independent of the EGF domain. A molecular model of the CFC domain of Cripto was constructed based on the nuclear magnetic resonance structure of PMP-C. This model reveals a hydrophobic patch in the domain opposite to the presumed ALK4 binding site. This hydrophobic patch may be functionally important for the formation of intra or intermolecular complexes.     

Cripto-1-induced increase in vimentin expression is associated with enhanced migration of human Caski cervical carcinoma cells

Cripto-1 (CR-1), a member of the EGF-CFC peptide family, plays an essential role during mesoderm formation in vertebrates as well as in cancer development. Using cDNA gene expression array, Western blot, and indirect immunofluorescence, an increase in vimentin expression was demonstrated in CR-1-transfected human Caski cervical carcinoma cells compared to control vector-transfected cells. In parental Caski cells, recombinant CR-1 induced a dose-dependent increase of vimentin protein expression within 24 h. Since vimentin expression has been demonstrated to correlate with a more aggressive phenotype in human cervical cancer, the migration capacity of CR-1-transfected or CR-1-treated Caski cells was studied in the Boyden chamber assay. Compared to the vector-transfected or untreated Caski cells, CR-1-transfected cells or cells treated with recombinant CR-1 exhibit enhanced migration, both through collagen- and through gelatin-coated membranes. Additionally, CR-1 can function as a chemoattractant for Caski cells. These findings are of biological significance since CR-1 is overexpressed in several types of human carcinomas. The present data demonstrate that CR-1 can increase vimentin expression and modulate migration in human cervical carcinoma cells.