
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

39257814
10.1101/2024.08.30.610562
preprint
1
Article
Decrypting the phylogenetics history of EGF-CFC proteins Cripto and Cryptic
http://orcid.org/0000-0002-0466-896X
Shylo Natalia A. 1
http://orcid.org/0000-0003-2774-3624
Trainor Paul A. 12#
1 Stowers Institute for Medical Research, Kansas City, MO, USA
2 Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Missouri, USA
# Author for correspondence: pat@stowers.org
01 9 2024
2024.08.30.610562https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
nihpp-2024.08.30.610562.pdf
EGF-CFC proteins are obligate coreceptors for Nodal signaling and are thus required for gastrulation and left-right patterning. Species with multiple family members show evidence of specialization. For example, mouse Cripto is required for gastrulation, whereas Cryptic is involved in left-right patterning. However, the members of the family across model organisms have little sequence conservation beyond the EGF-CFC domain, posing challenges for determining their evolutionary history and functional conservation. In this study we outline the evolutionary history of the EGF-CFC family of proteins. We traced the EGF-CFC gene family from a single gene in the deuterostome ancestor through its expansion and functional specialization in tetrapods, and subsequent gene loss and translocation in eutherian mammals. Mouse Cripto and Cryptic, zebrafish Tdgf1, and all three Xenopus EGF-CFC genes (Tdgf1, Tdgf1.2 and Cripto.3) and are all descendants of the ancestral Tdgf1 gene. We propose that subsequent to the family expansion in tetrapods, Tdgf1B (Xenopus Tdgf1.2) acquired specialization in the left-right patterning cascade, and after its translocation in eutherians to a different chromosomal location, Cfc1/Cryptic has maintained that specialization.

Cripto
Cryptic
Tdgf1
EGF-CFC
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pmcINTRODUCTION

Nodal signaling pathway is central to body patterning due to its involvement in gastrulation, anterior-posterior, dorsal-ventral and left-right patterning processes1. EGF-CFC proteins are membrane-anchored and serve as obligate coreceptors for Nodal to assemble the receptor complex, thus making them indispensable for most aspects of Nodal signaling - mesoderm and endoderm formation, as well as anterior-posterior and left-right patterning2,3. The family of EGF-CFC proteins got its name from a conserved domain, composed of an EGF-like motif, and a novel CFC sequence, first identified in mouse Cripto, frog FRL-1, and mouse Cryptic/Cfc14. Zebrafish Tdgf1 is another well-known member of the family5. one-eyed-pinhead (oep) mutation in zebrafish Tdgf1 is frequently used to knock out all Nodal signaling6. Despite high conservation of the EGF-CFC domain, the protein sequence is poorly conserved amongst members of the family. This led to hypotheses that there was no orthologous relationship between family members from different species4,7,8.

The mouse genome possesses two EGF-CFC genes – Cripto and Cryptic, which have specialized functions. Cripto regulates mesoderm formation and anterior-posterior patterning, whereas Cryptic influences left-right patterning9. In contrast, single zebrafish Tdgf1 fulfils both functions of mesoderm formation and left-right patterning10. In humans, Cryptic/CFC1 underwent an additional duplication, resulting in CFC1B and CFC1 genes (Figure 3B) and mutations in both genes have been reported in patients with congenital heart disease and heterotaxy, suggestive of roles in left-right patterning11,12.

The evolutionary history of the EGF-CFC gene family is heretofore unknown, and orthologous relationships between the members of the family remain contentious. It has been suggested that EGF-CFC genes underwent a duplication event after the speciation of vertebrates from chordates, and that the gene corresponding to the Cripto lineage was subsequently lost in non-mammalian species, while being retained and conserved in eutherian mammals2,8.

With the recent exponential growth of sequenced and annotated genomes, we revisited the evolutionary history of the EGF-CFC family and discovered that the EGF-CFC genes indeed underwent a duplication event as part of a larger gene cassette, after the speciation of vertebrates. However, our analysis shows that one of the two orthologous genes was lost shortly after the duplication event. A more functionally significant event was gene expansion in a tetrapod ancestor that resulted in three tandem copies of Tdgf1 gene (named Tdgf1A-C here). Based on protein conservation we have determined that Tdgf1A was lost in the eutherian ancestor, and that Tdgf1B was translocated to a novel chromosomal position and is now known as Cryptic/Cfc1. Considering that the Xenopus Tdgf1B ortholog Tdgf1.2 is involved in left-right patterning similar to Cryptic/Cfc1, we propose that this specialization occurred in a tetrapod ancestor and has been conserved ever since.

RESULTS

EGF-CFC genes in deuterostome genomes.

The evolutionary history of EGF-CFC protein family has been previously examined in a limited capacity, mostly restricted to the sequence comparison of the family members to determine their evolutionary relationship2,8,13. With vastly more genomes now available, we carried out a thorough survey of the EGF-CFC genes, present in deuterostome genomes, and corroborated their orthologous relationships through analysis of regions of chromosomal homology and sequence conservation.

This survey of the EGF-CFC genes in deuterostomes revealed that all deuterostomes, except for tunicates (ex. Cional intestinalis) have at least one copy of an EGF-CFC gene, with the ancestral gene most frequently named Tdgf1 (teratocarcinoma-derived growth factor 1) (Figure 1). Furthermore, most eutherians have an additional Cryptic/Cfc1 gene, except for the cow genome in which it has been lost, and in humans, which experience a duplication in that region, resulting in two genes - CFC1B and CFC1. We noted additional gene expansions in amphibians and reptiles in the Cripto/Tdgf1 region, with many lineages having 2–3 genes in the same chromosomal region (Figure 1).

Ancestral Tdgf1 gene.

The key to determining homologous relationships between genes is examining them in the context of gene linkage groups across the phylogenetic tree. Although the EGF-CFC family originated in a urbilaterian ancestor2, in this study we limited our analysis to deuterostomes. Using previously published protein sequences2, we began by identifying the Tdgf1 genes in echinoderms, hemichordates and chordates like the Florida lancelet (Supplementary Table 1). Although we noted several rearrangements and recombinations, we also identified clear linkage groups (Figure 2). Most importantly, Tdgf1 is linked to Rtca and Dbt1 in echinoderms, hemichordates and lancelets (Figure 2). Although we were able to locate Rtca, Dbt1 and other genes from the linkage group in Ciona intestinalis, we were unable to identify Tdgf1 in the genome, consistent with prior observations2. The Tdgf1 gene in the craniate sea lamprey was located at a novel chromosomal position, however, our analysis was complicated by the limitations of partial genome assembly14.

EGF-CFC genes in jawed vertebrates.

We then tracked the genomic history of Tdgf1 further into the jawed vertebrate lineage. Although we detected linkage between Lrrc2, Cripto/Tdgf1 and Fam240 in most jawed vertebrates we examined, which clearly established orthologous relationship between these genes (Figure 3A), this linkage group had no apparent similarity with the ancestral Tdgf1 region (Figure 2), and the linkage between Tdgf1, Dbt1 and Rtca was apparently lost.

Interestingly, in jawed vertebrates Dbt1 and Rtca remain linked, but lack an EGF-CFC gene nearby (Supplementary Figure S1A). We noted the similarity between the extended genomic region containing Dbt1 and Rtca and the one containing Cripto/Tdgf1 in jawed vertebrates, most notably the presence of Lrrc family genes in proximity to Tdgf1 and Dbt1 (Lrrc2 and Lrrc39, respectively), indicating that these regions likely arose as the result of a duplication, with subsequent gene loss (Figure 3A, Supplementary Figure S1A). This likely happened in a vertebrate ancestor, since only a single region of homology is present in lancelets (Supplementary Figure S1B), and only single copies of genes from the linkage groups could be identified in Ciona intestinalis (not shown).

However, both regions (Figure 3A and Supplementary Figure S1A) are still markedly different from the relatively small region of homology that contains the ancestral Tdgf1 that we highlighted (Figure 2). An extended analysis of landmark genes in the lancelet, green sea urchin and common starfish, spanning larger chromosomal regions (Supplementary Figure S1B) reveal that the genes present in the two duplicated regions in jawed vertebrates (Figure 3A, Supplementary Figure 1A) are all present on a single chromosome in those deuterostomes, with a clearly identifiable linkage block intercalated with each other (Supplementary Figure S1). Therefore, mouse Cripto, zebrafish Tfgf1, and all three Xenopus EGF-CFC genes (Tdgf1, Tdgf1.2 and Cripto.3) and are all descendants of the ancestral Tdgf1 gene.

This analysis didn’t, however, reveal the origin of the Cryptic/Cfc1 locus. It emerged in eutherian mammals, and gene linkage analysis of the region did not reveal any homology to Cripto/Tdgf1 and the ancestral Tdgf1 regions.

Tdgf1 gene expansion.

We next examined more closely the Tdgf1 genomic region in amphibians and reptiles. Xenopus Frl-1 (Cripto.3) is one of the founding members of the EGF-CFC family and one of three Tdgf1 genes in Xenopus7. Xenopus is the only widely used model organism that has three Tdgf1 genes (Figure 1). We will refer to them as Tdgf1A, Tdgf1B and Tgf1C, corresponding to Xenopus Tdgf1, Tdgf1.2 and Cripto.3, accordingly (Figure 3A). Humans, mice, chickens and zebrafish all contain a single copy of the Cripto/Tdgf1 gene in that genomic locus (Figure 1), so it was reasonable to assume that this gene expansion was unique to Xenopus, similar to the expansion of Nodal genes to over 7 in some frog species15,16.

However, our analysis of amphibian species revealed other lineages with 2 or 3 Tdgf1 genes, like the caecilian Geotrypetes seraphini (2 genes), and common frog (3 genes) (Figure 1, Supplementary Table 1), with Fam240 located between Tdgf1B and Tdgf1C. Notably, we identified many reptilian species also containing 2–3 Tdgf1 genes, e.g. tiger rattlesnake (2 genes), green sea turtle (3 genes), and painted turtle (2 genes) (Figure 1, Supplementary Table 1), some of which also contain one or two Fam240 genes interspersed with Tdgf1 genes (we will refer to these as Fam240_1 and Fam240_2) (Supplementary Figure S2A). Based on the similarity of their positioning in the genomic locus, we wondered if the three Tdgf1 genes and two Fam240 genes in amphibians and reptiles were orthologous. Thus, we compared the sequences of a large number EGF-CFC proteins, uncovering an orthologous relationship between the three Tdgf1 genes in amphibians and reptiles (Figure 4). Likewise, Fam240 genes show clear orthology in the species examined (Supplementary Figure S2B).

The protein clustering revealed patterns of gene inheritance (Figure 4). We found that Tdgf1A is restricted to amphibians and reptiles, whereas Tdgf1C is closely homologous with the eutherian Cripto/Tdgf1 proteins. Notably, Tdgf1B is most closely related to Cryptic/Cfc1 protein, finally revealing its evolutionary origin (Figure 4). These data suggest that Tdgf1B underwent translocation to a new locus in the genome and is now known as Cryptic/Cfc1 (Figure 5). Interestingly, platypus Tdgf1 is most closely related to Tdgf1C, whereas marsupial Tdgf1 protein is more closely related to Tdgf1B. Platypus has retained Tdgf1C, whereas marsupials have retained Tdgf1B, and the eutherian mammals have retained Tdgf1C in the ancestral locus, with a Tdgf1B translocation to a novel genomic locus, where it became known as Cryptic/Cfc1. With additional evidence from two Fam240 genes in the marsupial genomes, and their position in relation to Tdgf1 gene, we propose that the last common eutherian ancestor had two Tdgf1 genes (Tdgf1B and Tdgf1C) and two Fam240 genes.

DISCUSSION

Here we report on the evolutionary history of the EGF-CFC family of proteins, which act as obligatory co-receptors for Nodal signaling. A newcomer to the field of Nodal-related research may find it difficult to unravel the relationships between the genes in the EGF-CFC family, as it may be not immediately clear what the difference is between Cripto and Cryptic, and how they relate to Cfc1 and Tdgf1 proteins. In addition, which model species have a single EGF-CFC gene, which ones have more, and what may be the difference in their functions, has yet to be succinctly described and summarized. Furthermore, in addition to a collection of historic names and several acceptable aliases, gene names have not been assigned altogether to the identified transcripts in nearly half of the genomes examined in this study, making any work with this gene family particularly challenging.

Previous studies revealed that the EGF-CFC gene first appeared in the ancestor of all bilaterians - an urbilaterian and can be found in deuterostomes and several lophotrochozoans2,17. Interestingly, no EGF-CFC genes could be identified in ecdysozoans, which have also lost the Nodal gene, thus suggesting they co-evolved2. Several studies in deuterostomes such as tunicates, also failed to identify an EGF-CFC transcript, and our analysis in Ciona intestinalis of the genomic regions, linked to Tdgf1 in other animals, has confirmed its absence in the genome (Figure 1, Supplementary Table 1). Ciona intestinalis does, however, have Nodal, and given the requirement for EGF-CFC proteins for proper Nodal signaling, the mechanism for Nodal signal transduction in tunicates begs to be investigated.

Our analysis did however reveal a single EGF-CFC gene (Tdgf1) in echinoderms and hemichordates, as well as the Florida lancelet, linked to Rtca and Dbt1 genes (Figure 2, 5). Subsequently, a large region containing Tdgf1 underwent duplication (Figure 5). Shortly thereafter genes became lost from the two loci, such that Cripto/Tdgf1 gene was lost from the proximity of Rtca and Dbt1 in one duplicated region, and Rtca and Dbt1 were lost from the other region (Figure 5). We have not yet identified a genome where Tdgf1 is present in both duplicated regions, pointing to rapid gene loss from the two regions.

We propose that the duplication occurred in an early vertebrate ancestor, since we don’t find evidence for it in tunicates, but we did detect some duplicated cassettes in the sea lamprey. However, the current state of genome assemblies and annotation for Ciona intestinalis and lampreys make a more precise analysis challenging.

Arguably, the most significant event in the history of EGF-CFC genes occurred in a tetrapod ancestor, when the Tdgf1 gene underwent expansion, resulting in three copies of Tdgf1 in tandem (Figure 5, Supplementary Figure S2A). Subsequent loss of Tdgf1 paralogs in different tetrapod lineages obscured the orthologous relationship between genes, making it difficult to unravel these relationships until now. In eutherian mammals Tdgf1A was entirely lost from the genome, and Tdgf1B underwent a translocation, coupled with chromosomal rearrangements. Genomic regions previously located on separate chromosomes came together, with Tdgf1B landing between them, becoming known as Cryptic/Cfc1 (Figures 3B, 5).

Based on the data obtained in Xenopus, the three Tdgf1 paralogs underwent specialization, with Tdgf1B (Xenopus Tdgf1.2) becoming the exclusive member involved in left-right patterning7. Cryptic/Cfc1 is likewise involved in left-right patterning, suggesting that it has retained its specialization subsequent to the translocation event. This orthologous relationship and conservation of specialization should be further investigated by including analyses from other tetrapod models, in particular reptiles. Reptiles have historically been understudied in developmental biology, but our recent work with veiled chameleons holds a lot of promise for the study of early developmental processes, and roles of orthologous genes in different patterning events18,19.

An important area for this field to focus on in the future, is to analyze the consequences of gene loss and its effects on Nodal signaling, like the complete loss of Tdgf1 in Ciona intestinalis. The function of EGF-CFC genes in monotremes and marsupials is equally interesting. Both have a single copy of the gene, with platypus retaining the Tdgf1C ortholog, and marsupials retaining Tdgf1B in its original genomic location. Hypothetically, in both cases the genes could have re-acquired their generalized functions in mesoderm and endoderm formation, as well as anterior-posterior and left-right patterning2,3. In contrast, we were not able to identify a Cryptic/Cfc1 gene in the cow genome and thus whether cow Cripto/Tdgf1 re-acquired its left-right patterning functions, remains to be determined.

Overall, CFC-EGF proteins have been historically reported to have little sequence conservation between species, beyond the CFC-EGF domain, which remains relatively well conserved. The reasons behind this evolutionary flexibility currently remain unknown.

We observed frequent rearrangements and recombinations in regions proximal to EGF-CFC genes, in addition to several duplication and translocation events. The human genome is a good example of the outcomes of these processes, since it contains tandemly duplicated CFC1 and CFC1B genes in the CRYPTIC/CFC1 locus, and several pseudogenes, including the CRIPTO3 pseudogene on the X chromosome, which resembles CRIPTO (Figure 1B), and contains part of the CRIPTO promoter region20. The eutherian locus, containing Cripto/Tdgf1 has little resemblance to the ancestral Tdgf1 region due to numerous recombination events, coupled with gene loss. Future analysis should determine whether regions containing EGF-CFC genes represent conserved recombination hotspots across different species, and whether proximal transposable elements may explain gene translocations.

In this study we have outlined the evolutionary history of the EGF-CFC family of proteins. We have traced their evolution in deuterostomes from a single gene through their expansion in tetrapods, then specialization, gene loss and translocation in eutherian mammals. EGF-CFC proteins are critical co-factors for Nodal and are thus involved in many processes in early development and pattern formation, and the co-evolution of EGF-CFC and Nodal proteins is remarkable.

METHODS

Species phylogenetic tree

We used phyloT (v2 Database information: phyloT database version: 2023.2; NCBI taxonomy nodes: 3 905 559; NCBI GenBank ACCs: 1 007 637 254; RefSeq protein IDs 133 123 611; Uniprot protein IDs/ACCS 332 457 571; Genome Taxonomy Database release 214) (https://phylot.biobyte.de/) to generate the species phylogenetic tree, based on the NCBI taxonomy. Individual species of interest were searched in the NCBI taxonomy to add to the “NCBI tree elements” section. The following tree and file options were selected: internal nodes – expanded; polytomy – yes; file format – Newick; ignore errors – yes. We selected to visualize the tree in iTOL (Interactive tree of life v 6.9)21. The tree nodes were rotated to position mammals at the top of the page. Individual scientific names were replaced with common names. For visualization purposes the labels were aligned right, and the scaling factor was set to 0.1 horizontal.

Gene and protein identification

EGF-CFC genes were first identified using the NCBI Orthologs function22. The identity of the genes of interest was ascertained using the neighboring genes in the syntenic block, in comparison to other species. Additionally, we identified some EGF-CFC proteins using BLASTP program, targeting specific species of interest23. Lamprey genes and homologous regions were examined using https://simrbase.stowers.org/14. Lastly, some of the proteins used in this study had been previously identified by Ravisankar et al.8 and Truchado-Garcia et al.2

Protein phylogenetic tree

Individual sequences for Tdgf1/Cripto and Cryptic/Cfc1 homologs were used to determine the relationship of protein sequences. To generate the Tdgf1/Cfc1 phylogenetic tree, we used the https://ngphylogeny.fr/ web interface, selecting “A la Carte” to generate a custom workflow24. We selected MAFT for multiple alignment, with “linsi” option selected25. We selected trimAI for alignment curation, with 0 gap threshold26. For tree inference we selected FastTree with 1,000 bootstrap replicates27–29. “Booster Tree with [id avg transfer distances depth] as branch labels” was selected as most representative of both protein relationships and phylogenetic relationships of species of origin. The phylogenetic tree was visualized using iTOL (Interactive tree of life v 6.9)21. The scaling factor was set to 0.2 horizontal.

To generate the Fam240 phylogenetic tree, we inputted select protein sequences into Clustal Omega Multiple Sequence Alignment (MSA) program30. Phylogenetic tree was exported into iTOL (Interactive tree of life v 6.9) for visualization. The scaling factor was set to 0.2 horizontal.

Supplementary Material

Supplement 1

ACKNOWLEDGEMENTS

We would like to thank members of the Trainor lab for their thoughtful comments and discussion. This work was supported by the Stowers Institute for Medical Research (P.A.T) and a K99 (HD114881) from the National Institute for Child Health and Human Development (N.A.S).

Figure 1 | Phylogenetic distribution of EGF-CFC proteins.

The phylogenetic relationship of deuterostome clades evaluated in this study, showing presence or absence of Crypto/Tdgf1 and Cryptic/Cfc1 homologs in their genomes. The numbers in the squares indicate additional expansion in that region and the number of genes. Orange indicates the Cripto/Tdgf1 gene is present in jawed vertebrates, whereas red is indicative of the ancestral gene. Crypto/Cfc1 is demarcated with a blue colored square.

Figure 2 | Structure of the chromosomal regions containing the ancestral EGF-CFC gene Tdgf1 in deuterostomes.

The ancestral Tdgf1 gene is demarcated by a red triangle. Expanded chromosomal regions and select landmark genes for lancelet, green sea urchin and common starfish are in figure S1B. Dbt and Rtca are linked to Tdgf1 on the ancestral deuterostome chromosome and are highlighted in gray. Vertical tildes designate the location of an ancestral chromosomal recombination events. A square bracket represents the edge of a genomic scaffold. Dashed lines between two sections indicate that the genes are on distant parts of the same chromosome/scaffold. Chromosomes and scaffold numbers are as indicated. Only conserved landmark genes are pictured. Additional species-specific genes are present throughout, and not depicted. Pictured are key model organisms, and additional species, which aid in revealing conserved regions of homology.

Figure 3 | Structure of the chromosomal regions containing EGF-CFC gene family members in jawed vertebrates.

A. The Cripto/Tdgf1 present in jawed vertebrates is demarcated by orange triangles. This gene underwent an expansion in tetrapods. We termed the resulting three genes Tdgf1A, Tdgf1B and Tgf1C in order from Lrrc2. Duplicated Fam240 genes were labeled Fam240_1 and Fam240_2 in order from Lrrc2. B. Structure of the chromosomal regions containing Cryptic/Cfc1 genes, demarcated by blue triangles. Only conserved landmark genes are pictured. Additional species-specific genes are present throughout, and not depicted. Pictured are key model organisms, and additional species, which aid in revealing conserved regions of homology. Vertical tildes designate the location of an ancestral chromosomal recombination events. A square bracket represents the edge of a genomic scaffold. Dashed lines between two sections indicate that the genes are on separate distant parts of the same chromosome. Chromosomes and scaffold numbers are as indicated.

Figure 4 | Phylogenetic relationship of EGF-CFC proteins.

The phylogenetic relationship of EGF-CFC protein sequences in deuterostomes. Cryptic/Cfc1 in eutherian mammals is highlighted in yellow text. Tdgf1A proteins are in red, Tgf1B sequences are in orange, and Tdgf1C are in blue.

Figure 5 | Model of the EGF-CFC genomic regions evolution.

Dbt and Rtca genomic cassette, adjacent to the ancestral Tdgf1 gene, underwent an inversion in a chordate ancestor. In a vertebrate ancestor the entire genomic region underwent duplication, followed by gene loss in both regions. We also note acquisition of Fam240 gene, proximal to Tdgf1, somewhere in the fish lineage. In a tetrapod ancestor, the Tfgf1 and Fam240 genes underwent an expansion event, resulting in three copies of Tdgf1 (named here A-C, counting from Lrrc2 gene) and two copies of Fam240 (named here 1 and 2, counting from the Lrrc2 gene). Subsequently in the eutherian ancestor the ancestral region including Tdgf1A was lost, and Tdgf1B underwent translocation to a new genomic site, becoming known as Cryptic/Cfc1. The ancestral Tdgf1 gene is demarcated by a red triangle, whereas Cripto/Tdgf1 is demarcated in orange and Cryptic/Cfc1 is light blue.

Figure editing

Figures were constructed and edited using Adobe Illustrator.
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