# FGF Signaling Emerged Concomitantly with the Origin of Eumetazoans

Stéphanie Bertrand, Thomas Iwema, Hector Escriva

► To cite this version:

Stéphanie Bertrand, Thomas Iwema, Hector Escriva. FGF Signaling Emerged Concomitantly with the Origin of Eumetazoans. Molecular Biology and Evolution, 2013, 31 (2), pp.310 - 318. 10.1093/mol-bev/mst222. hal-03882514

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# FGF Signaling Emerged Concomitantly with the Origin of Eumetazoans

Stéphanie Bertrand,<sup>*1</sup> Thomas Iwema,<sup>2</sup> and Hector Escriva<sup>1</sup>

## Abstract

Complex metazoan bodies require cell-to-cell communication for development, a process often mediated by signaling molecules binding to specific receptors. Relatively few signaling pathways have been recruited during evolution to build multicellular animals from unicellular zygotes. Of these few signaling pathways, one of particular importance is the receptor tyrosine kinase (RTK) pathway. In metazoans, fibroblast growth factors (FGFs) bind to receptors in the RTK family, but the origin of the FGF gene family has so far remained a mystery. Here we show that extant bona fide FGFs most likely originated from proteins bearing an FGF-like domain that arose in a choanoflagellate/metazoan ancestor. We found orthologous genes closely related to FGF in choanoflagellates as well as in many metazoans such as sponges, acoels, protostomes, or nonvertebrate deuterostomes. We also show that these genes have a common evolutionary history with *Retinitis Pigmentosa 1* (RP1). Even if some metazoan signaling pathways emerged long before multicellularity, we show that FGFs, like their receptors, originated in a eumetazoan ancestor.

## Introduction

Multicellularity has evolved independently several times in the evolutionary history of eukaryotes. Deciphering the order of emergence of individual cell-cell communication pathways in each lineage is crucial for our understanding of how multicellular organisms first appeared from single-celled life forms. In metazoans, only a few types of signaling pathways are known to effect intercellular communication during embryogenesis, namely, the Wingless (Wnt), transforming growth factor-β, receptor tyrosine kinase (RTK), Janus kinase/signal transducers and activators of transcription, nuclear receptor (NR), notch, hedgehog (Hh), G protein coupled receptor, and tumor necrosis factor receptor pathways (Pires-daSilva and Sommer 2003; Malbon 2005; Mathew et al. 2009).

About 20 families of RTKs are known in eumetazoans (D'Aniello et al. 2008), including fibroblast growth factor receptors (FGFRs). FGFs are small proteins, characterized by a conserved domain with a beta-trefoil fold structure, which are generally secreted in the extracellular space. They act as signals between the cells by binding to the extracellular Immunoglobulin-like (Ig-like) domains of FGFRs and are implicated in many developmental processes in bilaterians and cnidarians (Thisse B and Thisse C 2005; Matus et al. 2007). FGFs were first discovered in vertebrates, and 22 genes from eight subfamilies are known in mammals (Itoh and Ornitz 2004; Oulion et al. 2012). Recently, thanks to the sequencing of complete genomes from diverse metazoan species, we know that at least two or three of the FGF subfamilies emerged before the divergence of cnidarians and bilaterians (Oulion et al. 2012). Although genomes from other metazoans such as *Trichoplax* or the sponge *Amphimedon*, and

of opisthokonts closely related to metazoans, were scanned for different signaling molecule genes, no *FGF* homologs were found outside Eumetazoa. The origin of the FGF family of proteins so far remains unknown (Popovici et al. 2005; Oulion et al. 2012).

Using careful searches in publicly available genome and expressed sequence tag (EST) databases, we found genes coding for proteins with domains that are more closely related to FGF than to any other family (FGF-like domains) in all the major metazoan phyla as well as in choanoflagellates. From these data, and considering animal deep evolutionary relationships (Philippe et al. 2009), we propose an evolutionary scenario for the emergence of bona fide FGFs. In this scenario, the first *FGF* gene appeared after duplication of a gene bearing a FGF-like domain in the ancestor of eumetazoans, concomitantly with the appearance of the FGF receptor.

## Results

### Putative FGF Domains Are Found Outside Eumetazoans

To decipher when the FGF family appeared, we first searched nr (all nonredundant GenBank CDS translations + RefSeq Proteins + PDB + SwissProt + PIR + PRF), complete genome, and EST databases for FGF homologs outside eumetazoans. We then used iterative analysis to uncover all the proteins bearing a putative FGF domain. We found two classes of undescribed domains outside the bona fide FGF family that will be referred to as FGF-like. The first class is found in orthologs of vertebrates *RP1/RP1L1* (see later), whereas the second correspond to other genes that we will henceforth name *FGFLs*. We found *FGFL* genes in the choanoflagellate *Monosiga brevicollis* that belongs to the sister group of

<footnote n="1"><sup>1</sup>UPMC Univ Paris 06, CNRS, UMR 7232, BIOM, Observatoire Océanologique de Banyuls sur Mer, Banyuls/Mer, France</footnote>

<footnote n="2"><sup>2</sup>Université de la Réunion, GRI, EA4517, CYROI, Saint-Denis, Reunion Island, France</footnote>

<footnote>*Corresponding author: E-mail: stephanie.bertrand@obs-banyuls.fr.</footnote>

<footnote>Associate editor: David Irwin</footnote>
metazoans and in several sponge species (*Amphimedon queenslandica*, *Oscarella carmela*, *Lubomirskia baicalensis*, *Oopsacas minuta*, *Suberites domuncula*, and *Heterochone calyx*), whereas we did not find bona fide FGFs in these groups. We also found *FGFLs* in the acel *Convoluta pulchra*, in three deuterostomes (*Saccoglossus kowalevskii*, *Strongylocentrotus purpuratus*, and *Branchiostoma floridae*), in several lophotrochozoans (*Alvinella pompejana*, *Capitella teleta*, *Lottia gigantea*, *Mytilus californianus*, and *Lymnaea stagnalis*), and in the spider *Parasteatoda tepidariorum* but failed to find *FGFLs* in any vertebrate species. Although we did not find proteins with FGF-like domains in cnidarians, the two ctenophores *Mnemiopsis leidyi* and *Pleurobrachia pileus* do have at least one gene coding for such a domain. In these newly described FGFL proteins, the FGF-like domain is usually not associated to any other known protein domain, except in a few cases (e.g., one gene in *S. kowalevskii* that codes for a protein with two FGF-like domains, one gene in *B. floridae* encoding a protein with FGF-like, Pox virus and Zinc finger (POZ), and Kelch-motif domains, one gene in *O. carmela* that codes for a protein with galactoside-binding lectin and FGF-like domains, and a ctenophore gene coding for a protein with one DUF3421 and one FGF-like domain; fig. 1 and table 1).

In nonmetazoan eukaryotes, excepting choanoflagellates, we only found a putative FGF domain in *Giardia intestinalis*. This eukaryote belongs to the excavata group and is a unicellular human parasite. The gene coding for a FGF domain contains no intron and we could not find any ortholog outside eumetazoans. We suggest this gene is the result of horizontal gene transfer between a eumetazoan host and the parasite, and therefore we did not include it in further analyses.

Two entries in the Pfam database, from *Dictyostelium discoideum* (Q54DM0) and *Eubacterium ventriosum* (A5Z345), are also wrongly described as members of the FGF family (PF00167). Indeed, if these sequences are used as queries in BlastP, Position-Specific Iterated Blast (PSI-Blast) with five

iterations or in phmmer, no FGF or FGF-like sequences are recovered as significant hits. Moreover, HHpred analysis with these putative FGF domains gives Fascin as the best hit.

## Orthologs of RP1/RP1L1 Bear Two FGF-Like Domains

As previously mentioned, we found FGF-like domains in *FGFL* genes but also in genes coding for multidomain proteins with two doublecortin (DCX) domains followed by two polycystin-1, lipoxygenase, alpha-toxin (PLAT) domains, two FGF-like domains, and several more PLAT domains (fig. 1 and supplementary table S1, Supplementary Material online). Such genes, that we will henceforth call *proRP1s*, are found in many eumetazoan species as well as in the placozoan *Trichoplax adhaerens* and are orthologs of vertebrate *RP1* and *RP1L1*, although these genes have been highly modified during vertebrate evolution. Indeed, in vertebrates, both RP1 and RP1L1 have two DCX domains, but these are followed by a poorly conserved long acidic region, and they lack the FGF-like and PLAT domains. However, in the genomic region of *RP1* of several vertebrates, an alternative transcript coding for a protein with a similar organization as proRP1 is predicted (supplementary table S1, Supplementary Material online). Moreover, in humans, the existence of this predicted transcript is supported by experimental data (supplementary table S2, Supplementary Material online). Besides being supported by phylogeny using the two DCX domain sequences (supplementary fig. S1, Supplementary Material online), the orthology between vertebrate *RP1/RP1L1* genes and the *proRP1* genes found in *Trichoplax* and nonvertebrate metazoans is also supported by synteny conservation (supplementary fig. S2, Supplementary Material online). Indeed, in all the genomes analyzed, this gene is placed next to, or close to, orthologs of the human *C8orf74* gene. We searched for orthologs of *proRP1* outside of placozoans and eumetazoans. In *A. queenslandica*, as in other sponges, we found a unique gene coding for a protein with two DCX domains that is orthologous to vertebrate doublecortin domain-containing protein 2 (DCDC2) genes (supplementary fig. S1,

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Domain organization of a selection of FGF and proRP1 proteins. Domain organizations are presented for FGF1 and FGF2 of the choanoflagellate *Monosiga brevicollis*, for FGFLs of the sponges *Amphimedon queenslandica* and *Oscarella carmela*, of the ctenophore *Mnemiopsis leidyi*, and of the oyster *Crassostrea gigas*, for FGF2 of the cephalochordate *Branchiostoma floridae*, for FGF1 of the hemichordate *Saccoglossus kowalevskii*, and for proRP1 of the placozoan *Trichoplax adhaerens*. FGF-like domain is in blue, galactoside-binding lectin domain in purple, DUF3421 domain in orange, POZ domain in dark purple, Kelch-motif domain in green, DCX domain in pink, and PLAT domain in light blue. Yellow bars correspond to position of phase 0 introns, red to phase 1 introns, and blue to phase 2 introns. For clarity, we neither represent the 17 PLAT domains that are found after the two FGF-like domains of *Trichoplax proRP1* nor the position of introns outside the FGF-like domains.</figcaption></figure>
<table><caption>Table 1. FGFL Gene Descriptions.</caption><thead><tr><th>Clade</th><th>Species</th><th>Name</th><th>Accession Number</th><th>Database</th><th>Position of the FGF-Like Domain</th><th>e-Value (Independent)</th><th>Other Domains</th></tr></thead><tbody><tr><td rowspan="2">Choanoflagellata</td><td rowspan="2">Monosiga brevicollis</td><td>FGFL1</td><td>XP_001745187</td><td>GenBank</td><td>150-283</td><td>1.10e-05</td><td>None</td></tr><tr><td>FGFL2</td><td>XP_001742802</td><td>GenBank</td><td>259-390</td><td>3.70e-08</td><td>TM</td></tr><tr><td rowspan="6">Porifera</td><td rowspan="3">Amphimedon queenslandica<br>Heterochone calyx</td><td>FGFL</td><td>XP_003387084</td><td>GenBank</td><td>17-169</td><td>5.70e-14</td><td>None</td></tr><tr><td>FGFL1</td><td>GO088037.1</td><td>GenBank</td><td>91-215</td><td>7.10e-12</td><td>None</td></tr><tr><td>FGFL2</td><td>GO085879.1</td><td>GenBank</td><td>27-169</td><td>2.10e-12</td><td>None</td></tr><tr><td>Oopsacas minuta</td><td>FGFL</td><td>Contig of AM761341.1,<br>AM761591.1, AM761586.1</td><td>GenBank</td><td>132-265</td><td>1.50e-12</td><td>None</td></tr><tr><td>Oscarella carmela</td><td>FGFL1</td><td>g4231.t1</td><td>Compagen</td><td>143-276</td><td>3.30e-07</td><td>Galactoside-<br>binding lectin</td></tr><tr><td>Suberites domuncula</td><td>FGFL2</td><td>g10575.t1</td><td>Compagen</td><td>2-102</td><td>9.00e-08</td><td>None</td></tr><tr><td rowspan="2">Ctenophora</td><td rowspan="2">Mnemiopsis leidyi<br>Pleurobrachia pileus</td><td>FGFL</td><td>MLRB04679</td><td>NHGRI</td><td>150-280</td><td>2.1e-06</td><td>DUF3421</td></tr><tr><td>FGFL</td><td>CU419264</td><td>Compagen</td><td>148-282</td><td>1.8e-06</td><td>DUF3421</td></tr><tr><td rowspan="5">Deuterostomia</td><td rowspan="3">Branchiostoma floridae<br>Saccoglossus kowalevskii</td><td>FGFL1</td><td>EEN43793</td><td>GenBank</td><td>239-369</td><td>4.40e-06</td><td>None</td></tr><tr><td>FGFL2</td><td>EEN51389</td><td>GenBank</td><td>146-287</td><td>1.90e-11</td><td>POZ-Kelch</td></tr><tr><td>FGFL1</td><td>XP_002738881</td><td>GenBank</td><td>156-299 and<br>334-475</td><td>1.2e-13,<br>1.8e-12</td><td>None</td></tr><tr><td rowspan="2">Strongylocentrotus<br>purpuratus</td><td>FGFL2</td><td>NP_001164695</td><td>GenBank</td><td>50-192</td><td>1.80e-12</td><td>None</td></tr><tr><td>FGFL3</td><td>XP_002740914</td><td>GenBank</td><td>95-215</td><td>0.015</td><td>None</td></tr><tr><td></td><td>FGFL</td><td>XP_003723601</td><td>GenBank</td><td>43-174</td><td>2.60e-05</td><td>None</td></tr><tr><td>Acoelomorpha</td><td>Convoluta pulchra</td><td>FGFL</td><td>EV602615.1</td><td>GenBank</td><td>111-259</td><td>2.60e-13</td><td>None</td></tr><tr><td rowspan="6">Lophotrocozoa</td><td rowspan="2">Alvinella pompejana</td><td>FGFL</td><td>Contig of FP522214.1, FP496044.1,<br>GO191934.1, GO214047.1,<br>GO166221.1</td><td>GenBank</td><td>36-183</td><td>8.20e-06</td><td>None</td></tr><tr><td>FGFL</td><td>209049</td><td>JGI</td><td>113-259</td><td>0.023</td><td>None</td></tr><tr><td>Capitella teleta</td><td>FGFL</td><td>EKC36540</td><td>GenBank</td><td>858-998</td><td>5.4e-22</td><td>None</td></tr><tr><td>Grassostrea gigas</td><td>FGFL</td><td>230861</td><td>JGI</td><td>81-239</td><td>3.10e-05</td><td>TM</td></tr><tr><td>Lottia gigantean</td><td>FGFL</td><td>Contig of ES573811.1, ES291579.1,<br>ES575140.1, ES578045.1</td><td>GenBank</td><td>93-227</td><td>0.018</td><td>None</td></tr><tr><td>Lymmaea stagnalis</td><td>FGFL</td><td>Contig of GE765430.1, GE762820.1,<br>GE762505.1, GE757041.1,<br>GE756172.1, GE757679.1,<br>GE756671.1</td><td>GenBank</td><td>67-226</td><td>4.30e-05</td><td>None</td></tr><tr><td>Mytilus californianus</td><td>FGFL</td><td>Contig of GE765430.1, GE762820.1,<br>GE762505.1, GE757041.1,<br>GE756172.1, GE757679.1,<br>GE756671.1</td><td>GenBank</td><td>67-226</td><td>4.30e-05</td><td>None</td></tr><tr><td>Ecdysozoa</td><td>Parasteatoda tepidariorum</td><td>FGFL</td><td>FY222438</td><td>GenBank</td><td>1-137</td><td>2.9e-05</td><td>None</td></tr></tbody></table>

NOTE.—The accession number, position of the FGF-like domain, e-value for HMWscan, and position of other domains are given.
Supplementary Material online). We only observed two proteins with both DCX and PLAT domains outside metazoans in the two choanoflagellate species *M. brevicollis* and *Salpingoeca* sp. ATCC 50818. However, the double DCX domain of these choanoflagellate proteins seem more closely related to the DCDC2 proteins (supplementary fig. S1, Supplementary Material online). From these data, we cannot exclude the possibility that in the ancestor of choanoflagellates and metazoans a double DCX domain from which the domains of DCX/DCLK/DCLK2 and RP1/RP1L1 evolved was present and thereafter lost in sponges and choanoflagellates. Although our data suggest that the evolutionary history of the DCX domain-containing proteins is complex, as previously mentioned (Reiner et al. 2006), they support the fact that *proRP1*, coding for DCX, FGF-like, and PLAT domains, appeared in the ancestor of placozoans and eumetazoans.

## Evolutionary and Structural Relationships between FGF and FGF-Like Domains

Although the domains found in bona fide FGFs, proRP1s and FGFLs seem to be evolutionarily related, their sequences are quite divergent. Moreover, the size of the FGF domain is small (~120 amino acids), which makes phylogenetic reconstruction poorly informative. However, we used BI phylogenetic tree reconstruction with all the FGF-like sequences and members of the FGF family. The resulting tree shows that FGF-like

are not bona fide FGFs, and that, as expected, the sequences of the first FGF-like domain of proRP1 group together as do the sequences of the second FGF-like domain of proRP1 (fig. 2A). As these data do not give us information on the evolutionary relationship of the FGF-like domains to other domains than FGF, we analyzed the all-against-all pairwise similarities using clustering by CLuster ANalysis of Sequences (CLANS) (Frickey and Lupas 2004). The FGF domain belongs to the beta-trefoil superfamily (Pfam CL0066) and is closely related to Fascin, FRG1, and DUF569 domains. Members of these three families were included in our analysis together with representatives of the FGF family and with all the FGF-like domains found in this study. We observe in the graph that FRG1 and DUF569 sequences form compact clusters, whereas the Fascin sequences seem to separate into two clusters (fig. 2B). We also observe that FGF-like domain sequences from both proRP1s and FGFLs clearly form a cluster with FGFs.

We also decided to use profile hidden Markov model (HMM) comparisons to test the evolutionary relationships between bona fide FGFs and the newly found FGF-like domains (Eddy 1998). Profile HMMs were built for the first and second FGF-like domains of proRP1 sequences, for all the FGF-like domains of FGFLs and for all FGF-like domains (from both FGFLs and proRP1s). We also undertook the same analysis with all the FGF-like domains of FGFLs excluding protostome sequences, which show several indels, and with the FGF-like domains of protostome FGFLs alone. When all the

A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** FGF-like domains are closely related to FGFs. (A) BI phylogenetic tree of FGFLs, proRP1s, and FGFs using FGF and FGF-like domain sequences. The branches corresponding to FGFs to the first FGF-like domain of proRP1s and to the second FGF-like domain of proRP1s are in green, blue, and purple, respectively. The nodes with a posterior probability value between 0.9 and 1 are labeled with a circle. The scale bar indicates the number of substitutions per site. (B) Cluster map of FGF, FGF-like, and related beta-trefoil families. All the FGF-like domains and representatives of FGF, Fascin, DUF569, and FRG1 families were tested for all-versus-all similarity (ten iterations of PSI-Blast), and clustered by CLANS (Frickey and Lupas 2004). All pairs with similarity threshold $P \le 10^{-25}$ are joined by lines, which are darker and shorter with greater similarity.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Three-dimensional protein models of FGF-like domains. (A) backbone structure of human FGF1 colored using a rainbow scale with N-terminus colored in blue and C-terminus in red. (B) Model of the first FGF-like domain of *T. adhaerens* proRP1. (C) Model of the FGF-like domain present in *M. brevicollis* gene XP_001742802. (D) Model of *A. queenslandica* FGF-like domain. The models are colored using a continuous scale representing amino acid hydrophobicity (Eisenberg et al. 1984), ranging from blue (hydrophobic) to red (hydrophilic). All the models share a similar orientation.</figcaption></figure>

more closely related to FGF than to any other protein family (i.e., *FGFLs*). These genes are present in choanoflagellates and eumetazoans, but from the available data, they seem to be absent from vertebrates and cnidarians. We also show that FGF-like domains are present in multidomain proteins encoded by genes orthologous to vertebrate *RP1* and *RP1L1* (*proRP1*) that are found in placozoans and eumetazoans (see supplementary table S4, Supplementary Material online). Considering present phylogenomic knowledge of animal deep evolutionary relationships (Philippe et al. 2009), we propose a hypothesis for the origin of the FGF family (fig. 5). In this scenario, a gene coding for a protein with a FGF-like domain (*FGFL*) appeared in the ancestor of metazoans and choanoflagellates. Later on, in the ancestor of placozoans and eumetazoans, the gene coding for proRP1, which bears two FGF-like domains, appeared by duplication and domain shuffling. Then, in the ancestor of eumetazoans, the first bona fide FGF emerged after duplication of a *FGFL* gene, and the FGF family thereafter rapidly diversified. An earlier emergence of FGFs followed by secondary losses cannot be excluded, but we searched in many sponge and other opisthokont genomes and transcriptomes and could not find FGFs outside eumetazoans. Even if the monophyly of Porifera is still debated (Worheide et al. 2012), our data strongly suggest that bona fide FGFs only appeared after the divergence of poriferans and the other metazoans.

## FGF Signaling Pathway Is a Eumetazoan Innovation

Most of the FGFs are extracellular proteins that bind to FGFRs, excepting the fibroblast growth factor homologous factors (FHFs) of vertebrates, also known as FGF11-14, which function as intracellular proteins in an FGFR-independent manner (Goldfarb 2005). FGFRs are members of the RTK superfamily and bear Ig-like domains in their extracellular region. The number of Ig-like loops is variable among eumetazoans; however, the interaction with the ligand only implies two loops that are conserved among the different phyla. If we now consider the origin of the FGF receptor, although sponges and choanoflagellates possess a big repertoire of RTKs with a great variety of domain organisations, a clear Tyrosine Kinase (TK) domain orthologous to the one of FGFRs was only found in the sponge *O. carmela* (Nichols et al. 2006; King et al. 2008; Suga et al. 2012; supplementary fig. S5, Supplementary Material online). This TK domain is not associated to extracellular Ig-like domains, suggesting that it may not be a true FGFR. In the placozoan *Trichoplax*, a FGFR gene was described, coding for three Ig-like loops and an intracellular TK domain (Rebscher et al. 2009). However, phylogenetic analyses using the TK domain does not support orthology with eumetazoan FGFRs. Indeed, although the domain architecture of the *Trichoplax* RTK is similar to the one of FGFR, the TK domain is orthologous to the one of Cad96Ca (supplementary figs. S5 and S6, Supplementary Material online). Altogether, these data suggest that the first FGFR appeared by domain shuffling in the ancestor of eumetazoans, concomitantly with bona fide FGFs and indicates that the origin of the FGF/FGFR couple was an innovation specific to the Eumetazoa.

## Evolution of *RP1* and Possible Implications in Retinitis Pigmentosa

*RP1* codes for a protein produced in photoreceptor cells, which is associated to microtubules of the cilia (Pierce et al. 1999; Liu et al. 2004). A paralog of *RP1* called *RP1L1* has recently been discovered in vertebrates (Conte et al. 2003). Human *RP1* has four exons, with exon 2 and exon 3 coding for two DCX domains, whereas the fourth exon codes for a long poorly conserved region with unknown function. We have shown that in humans the *RP1* genomic region also codes for an alternative transcript that shares the exon 3 with *RP1* and produces a protein with a domain organization close to the one of proRP1. In all the vertebrates for which we found an alternative transcript at the *RP1* locus, the nonconserved C-terminal region of RP1 is encoded by one or few exons located between the exon coding for the second DCX domain and the exons coding for the PLAT and FGF-like domains. We could not find an alternative transcript in any vertebrate *RP1L1* genomic region, and we did not find any exon in nonvertebrate *proRP1* gene coding for a domain similar to the C-terminal region of vertebrates RP1 and RP1L1. Altogether, these data suggest that *RP1* and *RP1L1* are vertebrate innovations that appeared by acquisition of new exon(s) between the DCX-coding exon and the PLAT-coding exons in a *proRP1* gene. This insertion occurred probably
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Evolutionary scenario for the origin of the FGF family. The different clades analyzed and their evolutionary relationships are presented schematically. The boxes on the right represent the different domains that were found in each clade, and the color code is given at the bottom left. The branches of metazoan clades are included in a light gray box, and the branches of eumetazoan clades in a dark gray box. Putative losses are indicated in each branch.</figcaption></figure>

before the two whole genome duplications (i.e., 2R) that took
place at the base of the vertebrate lineage (Dehal and Boore
2005), and the exons coding for PLAT and FGF-like domains
were lost in the *RP1L1* gene after the 2R.

RP1, also known as *oxygen-regulated protein* gene, was first
described in human as a gene in which mutations induce a
degenerative eye disease, retinitis pigmentosa (RP; Pierce et al.
1999). About 40 mutations in human RP1 are known to cause
RP (Audo et al. 2011). Most of them are located in a hotspot region in the fourth exon and result in a truncated protein (Audo et al. 2011). However, mutations in exons 2 and 3 were described in patients showing autosomal recessive RP (Audo et al. 2011). The alternative transcript we found in the *RP1* locus codes for a protein that shares a DCX domain, encoded by exon 3, with RP1. Mutations in *RP1* exon 3 might therefore have consequences on this protein function. These data indicate the need for a careful analysis of the function of the protein produced by the alternative transcript we describe here, to better understand the phenotype of patients with RP associated with mutations in *RP1*.

## Conclusion

Each of the cell-to-cell signaling pathways that were previously thought to be eumetazoan-specific has its own evolutionary story to tell. For example, in the Hh pathway, some actors were already present in the ancestor of choanoflagellates and metazoans, but a bona fide *Hh* gene first appeared after the divergence of sponges through domain shuffling (King et al. 2008; Matus et al. 2008; Srivastava et al. 2010). In the case of Wnt and NR gene families, it has been shown that they are present only in metazoans, having an origin before the divergence of sponges, and showing further diversification in eumetazoans (King et al. 2008; Srivastava et al. 2010). Our data show that the FGF signaling pathway obeys another scenario with an FGF-like domain arising in a choanoflagellate/metazoan ancestor followed by duplications and domain shuffling that gave rise to the first bona fide FGF in an ancestral eumetazoan. The sequence of emergence of these ligands parallels that of their receptors that appeared at the same time by domain shuffling. Although the function of FGFLs is still unknown, our data reveal that the FGF/FGFR couple emergence is probably not linked to the appearance of multicellularity but rather with the increasing complexity of body shapes and developmental processes.

## Materials and Methods

### Search for FGF Homologs

We first searched using BlastP, with FGF sequences of each subfamily as queries, the following databases: nr, Compagen (Hemmrich and Bosch 2008), Ensembl, GeneDB, Broad Institute, Joint Genome Institute (JGI), National Human Genome Research Institute (NHGRI). We also searched using TBlastN the EST database at National Center for Biotechnology Information (NCBI) and the EST data at Compagen. The EST sequences obtained were assembled using CAP3 (Huang and Madan 1999), and the resulting contigs were translated in the appropriate frame. All the amino acid sequences obtained were scanned for putative FGF domains using HMMscan (Finn et al. 2011) with default parameters. Only sequences giving FGF domain as a hit were retained. The portion of each sequence corresponding to the putative FGF domain was then used as a query for new BlastP or TBlastN analysis. We repeated this search until no new sequence with a putative FGF domain could be found. A

summary of the results is presented in supplementary table S4, Supplementary Material online.

### Analysis of FGF-Like Sequences

The putative FGF region of each amino acid sequence was retrieved, and the sequences were used for all-against-all Blast analysis using CLANS (Frickey and Lupas 2004), together with FGF, Fascin, FRG1 and DUF569 sequences. Ten iterations of PSI-Blast and a *P*-value threshold of 10<sup>-25</sup> were used. The same sequences were aligned using Clustal Omega (Sievers et al. 2011) with the help of the profile HMM of the FGF family (Pfam database (Finn et al. 2006)) The alignments were manually corrected in SeaView (Gouy et al. 2010) and were used to search for homology using PRC and HHpred (Soding et al. 2005; Brandt and Heringa 2009). The alignments of the putative FGF sequences with the FGF family profile obtained after PRC analysis were then retrieved for intron position analysis. They were also used to generate profile HMM with HMMbuild at the mobyle portal (Neron et al. 2009), and these profiles were used to generate HMM logos (Schuster-Bockler et al. 2004). We searched for internal repeats in the obtained putative FGF using HHrepID (Biegert and Soding 2008). Putative FGF protein models were generated by homology modeling using modeler 9v9 (Sali and Blundell 1993) based on an alignment with human FGF1, FGF2, FGF4, FGF8, and a synthetic beta-trefoil (1RG8, 1CVS, 1IJT, 2FDB, and 3O4B, respectively).

### Phylogenetic Analysis

DCX domains amino acid sequences from Dictyostelium, M. brevicollis, A. queenslandica, T. adhaerens, and bilaterian proteins bearing two DCX domains were aligned using ClustalW (Thompson et al. 2002) and manually reviewed in SeaView (Gouy et al. 2010). Bayesian inference (BI) trees were inferred using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003), with the model recommended by ProtTest 1.4 (Abascal et al. 2005) under the Akaike information criterion (WAG + Γ + I), at the CIPRES Science Gateway V. 3.1. Two independent runs were performed, each with four chains and 1 million generations. A burn-in of 25% was used, and a 50 majority-rule consensus tree was calculated for the remaining trees. For the FGF and FGF-like phylogenetic analysis, the alignment given by PRC was used in the same way to infer trees by BI with the (WAG + Γ) model.

## Supplementary Material

Supplementary figures S1–S6 and tables S1–S4 are available at *Molecular Biology and Evolution* online (http://www.mbe.oxfordjournals.org/).

## Acknowledgments

This work was supported by Agence Nationale de la Recherche (ANR-2010-BLAN-1716 01 and ANR-2010-BLAN-1234 02) to H.E. and by CPER-FEDER GRI phase IV to T.I. We thank Nigel Grimsley and Josh Coulcher for careful reading of the manuscript.
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**Supplementary Information**

**Supplementary Figure Legends**

**Figure S1.** **BI phylogenetic tree of proteins containing two DCX domains.** Sequences from *Dictyostelium* were used as an outgroup. Posterior probabilities are shown on each node. The scale bar indicates the number of substitutions per site. The proRP1 sequences group with RP1 and RP1L1 from vertebrates. *Trichoplax* proRP1 is highlighted in pink. The choanoflagellates (yellow) and *A. queenslandica* (orange) sequences group with vertebrates DCDC2. Species name abbreviations are as follows: *AMPQ: Amphimedon queenslandica*, *APIM: Apis mellifera*, *BRAF: Branchiostoma floridae*, *CAEE: Caenorhabditis elegans*, *CAPT: Capitella teleta*, *CIOI: Ciona intestinalis*, *DANRE: Danio rerio*, *DAPP: Daphnia pulex*, *DICD: Dictyostelium discoideum*, *DICF: D. fasciculatum*, *DICP: D. purpureum*, *DROM: Drosophila melanogaster*, *GALG: Gallus gallus*, *HOMS: Homo sapiens*, *IXOS: Ixodes scapularis*, *LOTG: Lottia gigantea*, *MONB: Monosiga brevicollis*, *MUSM: Mus musculus*, *NEMV: Nematostella vectensis*, *SALS: Salpingoeca sp.*, *STRP: Strongylocentrotus purpuratus*, *TETN: Tetraodon nigroviridis*, *XENL: Xenopus laevis*. When sequence name is not described, the accession number is given.

**Figure S2.** **Synteny conservation analysis.** Schematic representation of the genomic regions surrounding *RP1*, *RP1L1* and *proRP1* genes in human, mouse, zebrafinch, *Xenopus*, zebrafish, lamprey, amphioxus, *Capitella* and *Trichoplax*. Data are from Ensembl and JGI databases. Orthologous genes are represented by arrows of the same colour, the others by grey arrows. The number of the chromosome or scaffold is indicated, as well as the position of the genomic segment that is schematised.
**Figure S3. Intron/exon structure conservation.** Positions and phase of the introns in the FGF and FGF-like domains. Alignments of FGF family representatives with FGF-like domains of FGFLs (A), of FGFLs from protostomes (B), and proRP1 (C and D) as obtained from PRC software are presented. Sequences names of FGFs are in blue, and the others in yellow. The positions of introns are highlighted in orange where phase is 1 and in red where phase is 0.

**Figure S4. Three dimensional protein models of FGF-like domains.** A. Backbone structure of human FGF1 coloured using a rainbow scale with N-terminus coloured in blue and C-terminus in red. B. Model of the second FGF-like domain of *T. adhaerens* proRP1. C. Model of the FGF-like domain present in *Monosiga brevicollis* gene XP_001742802. D. Model of *Lottia gigantea* FGF-like domain. These models are coloured by the mean of a continuous scale representative of the amino acid hydrophobicity using Eisenberg hydrophobicity scale (Eisenberg et al. 1984) ranging from blue (hydrophobic) to red (hydrophilic). The models all share a similar orientation.

**Figure S5. Phylogenetic analysis of the TK families closely related to FGFR.** Bayesian phylogenetic tree of FGFR, RET, PDGFR/VEGFR, Cad96Ca, TIE and Torso genes from several metazoan species using the TK domain sequences, estimated under the RtRev+I+Γ model (2 MrBayes runs of 1 million generations each; 25% generation burn-in; 4 chains per run). The sequence XP_001749064 from *Monosiga brevicollis* was used as an outgroup (see (Suga et al. 2012)). *Oscarella carmela* FGFR and *Trichoplax adhaerens* putative FGFR are framed in black. Species name abbreviations are as follows: *AFLO*: *Apis florea*, *AQUE*: *Amphimedon queenslandica*, *BFLO*: *Branchiostoma floridae*, *CGIG*: *Crassostrea gigas*,
CTEL: *Capitella teleta*, CINT: *Ciona intestinalis*, CQUI: *Culex quinquefasciatus*, DRER: *Danio rerio*, DMEL: *Drosophila melanogaster*, HMAP: *Hydra magnipapillata*, HSAP: *Homo sapiens*, HROR: *Halocynthia roretzi*, HVUL: *Hydra vulgaris*, LGIG: *Lottia gigantea*, MBRE: *Monosiga brevicollis*, MLEY: *Mnemiopsis leidyi*, MMUS: *Mus musculus*, NVEC: *Nematostella vectensis*, OCAR: *Oscarella carmela*, PLIV: *Paracentrotus lividus*, SKOW: *Saccoglossus kowalevskii*, SPUR: *Strongylocentrotus purpuratus*, TADH: *Trichoplax adhaerens*, TCAS: *Tribolium castaneum*, TNIG: *Tetraodon nigroviridis*, XTRO: *Xenopus tropicalis*.

**Figure S6. Phylogenetic analysis of the FGFR and RET families.** Bayesian phylogenetic tree of FGFR and RET genes from several metazoan species using the TK domain sequences, estimated under the RtRev+I+Γ model (2 MrBayes runs of 1 million generations each; 25% generation burn-in; 4 chains per run). *Oscarella carmela* FGFR is framed in black. Species name abbreviations are as in Fig. S5.

**Table S1.** ProRP1 homologues in metazoans. For each species, information on database, accession number, genomic position (when available), name and domain composition is presented.

**Table S2.** Accession numbers of cDNAs from human RP1 gene coding for a protein with the same domain organization as *Trichoplax* proRP1.

**Table S3.** Profile HMM analysis of the different FGF-like domains.
**Table S4.** Summary of species we searched for FGF and FGF-like. For each species the database used and the presence (green) or absence (red) of FGF domain, and of FGF-like domains from FGFLs or proRP1 is indicated. We did not show all the species for which none of these domains could be found.

**References**

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Suga H, Dacre M, de Mendoza A, Shalchian-Tabrizi K, Manning G, Ruiz-Trillo I. 2012. Genomic survey of premetazoans shows deep conservation of cytoplasmic tyrosine kinases and multiple radiations of receptor tyrosine kinases. Sci Signal 5: ra35.
<figure><img src="image_7.png" /><figcaption>Figure S1</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure S2</figcaption></figure>

Human

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

Mouse

<figure><img src="image_11.png" /></figure>
<figure><img src="image_12.png" /><figcaption>Zebra Finch</figcaption></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>Xenopus</figcaption></figure>

<figure><img src="image_15.png" /></figure>
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>
<figure><img src="image_20.png" /></figure>

Scaffold GL478245

Lamprey

<figure><img src="image_21.png" /></figure>

Scaffold GL485622
<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /><figcaption>Amphioxus</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>Capitella teleta</figcaption></figure>
<figure><img src="image_25.png" /></figure>

Trichoplax adhaerens

Scaffold7
A

<figure><img src="image_26.png" /></figure>

B

<figure><img src="image_27.png" /></figure>

C

<figure><img src="image_28.png" /></figure>

D

<figure><img src="image_29.png" /></figure>

FGF-like

FGF-like
protostomes

First FGF-like
of proRP1

Second FGF-like
of proRP1
<figure><img src="image_30.png" /><figcaption>Figure S4</figcaption></figure>

A

<figure><img src="image_31.png" /></figure>

B

<figure><img src="image_32.png" /></figure>

C

<figure><img src="image_33.png" /></figure>

D
<figure><img src="image_34.png" /><figcaption>Figure S5</figcaption></figure>
<figure><img src="image_35.png" /><figcaption>Figure S6</figcaption></figure>

FGFR

RET
<table><caption>Table S1. PreRP1 homologs in metazoans. For each species, information on database, accession number, genomic position (when available), name and domain composition is presented.</caption><thead><tr><th>Clade</th><th>Species</th><th>Common name</th><th>Accession number gene</th><th>Name</th><th>Chromosome/Position</th><th>Accession number of the protein sequence</th><th>Database</th><th>Domain organization</th></tr></thead><tbody><tr><td rowspan="10">Vertebrate</td><td>Ailuropoda melanoleuca</td><td>Panda</td><td>ENSAMEG0000011683<br>Ame, R014220</td><td>RPI</td><td>Scaffold GL193312.1: 429,449-648,070<br>Scaffold GL193312.1: 428,867-438,146</td><td>ENSAMEP0000012305<br>Ame, R014220</td><td>Ensembl</td><td>DCX/PLAT/2/FGF/2/PLAT*2</td></tr><tr><td>Anoia carolinensis</td><td>Anole licard</td><td>ENSAMEG0000016125<br>ENSACAG0000020939</td><td>RPI1</td><td>Scaffold GL193323.1: 1,759,204-1,773,255<br>Scaffold GL343244.1: 866,883-952,777</td><td>ENSAMEP0000017019<br>ENSACAP0000017892</td><td>Ensembl</td><td>DCX/PLAT/2/FGF/2/PLAT*2<br>PLAT*2</td></tr><tr><td>Bos taurus</td><td>Cow</td><td>ENSACAG0000027747<br>ENSACAG0000028027</td><td>RPI1L</td><td>Scaffold GL343244.1: 866,883-952,777<br>Chromosome 4: 41,026,363-41,042,736</td><td>ENSACAP0000019388<br>ENSACAP0000019459</td><td>Ensembl</td><td>DCX/PLAT/2/FGF/2<br>PLAT/2/FGF/2</td></tr><tr><td>Danio rerio</td><td>Zebrafish</td><td>ENSARG0000077687<br>ENSARG0000086360<br>ENSARG0000089458</td><td>RPI1 (1/2)<br>RPI (2/2)<br>RPI1L</td><td>Chromosome 14: 24,019-278,241,085<br>Chromosome 14: 23,990-193,239,938<br>Chromosome 8: 8,508,960-8,518,765</td><td>ENSBTAP0000055952<br>ENSBTAP0000014879<br>ENSBTAP0000026846</td><td>Ensembl</td><td>DCX/PLAT/2/FGF/2<br>PLAT/2/FGF/2</td></tr><tr><td>Homo sapiens</td><td>Human</td><td>ENSARG0000014237<br>ENSARG0000183638</td><td>RPI</td><td>Chromosome 2: 30,447,718-30,448,930<br>Chromosome 2: 55,28,627-55,543,394</td><td>ENSSDARP0000114695<br>ENSSDARP0000105907<br>ENSSDARP0000105051</td><td>Ensembl</td><td>DCX/PLAT/2/FGF/2<br>PLAT/2/FGF/2</td></tr><tr><td>Lattimeria chalumnae</td><td>Cow</td><td>ENSARG0000014602<br>ENSARG0000014370</td><td>RPI1</td><td>Chromosome 17: 5,562,212-5,583,829<br>si-dkey/33/22-2,301</td><td>ENSARG00000107244<br>ENSP00000220676</td><td>Ensembl</td><td>DCX/PLAT/2/FGF/2<br>PLAT/2/FGF/2</td></tr><tr><td>Meleagris gallopavo</td><td>Turkey</td><td>ENSARG0000010962<br>ENSARGAG0000014193</td><td>RPI1L</td><td>Chromosome 8: 10,463,859-10,512,612<br>Scaffold JH126724.1: 1,058,502-1,068,932<br>Scaffold JH126724.1: 1,013,189-1,031,086</td><td>ENSP0000017193<br>ENSACAP0000016569<br>ENSACAP0000016310</td><td>Ensembl</td><td>DCX/PLAT/2/FGF/2/PLAT*2<br>PLAT/2/FGF/2</td></tr><tr><td>Otolemur garnettii</td><td>Bushbaby</td><td>ENSARGAG0000003108<br>ENSARGAG0000032424</td><td>RPI1L</td><td>Scaffold JH127442.1: 203,546-225,488<br>Chromosome 3: 60,524,541-60,580,542<br>Chromosome 2: 112,567,137-112,590,099</td><td>ENSMLGAP0000011421<br>ENSMLGAP0000015014<br>Scaffold GL873590.1: 3,835,751-5,620,468</td><td>Ensembl</td><td>DCX/PLAT/2/FGF/2/PLAT*2<br>PLAT/2/FGF/2</td></tr><tr><td>Petromyzon marinus</td><td>Lamprey</td><td>ENSPMAG0000008246<br>ENSPMAG0000008812<br>ENSPMAG000001989</td><td>RPI (1/1)<br>RPI (2/2)</td><td>Scaffold GL873593.1: 1,226,470-1,234,835<br>Scaffold GL480696: 25,583-35,017<br>Scaffold GL478245: 10,970-15,566<br>Scaffold GL485622: 7,659-8,531</td><td>ENSOGAP0000020959<br>ENSPMAP0000000981<br>ENSPMAP00000009707<br>ENSPMAP0000002175</td><td>Ensembl</td><td>DCX/PLAT/2/FGF/2/PLAT*2<br>PLAT/2/FGF/2</td></tr><tr><td>Sarcophaga hartrici</td><td>Tasmanian devil</td><td>ENSSHA0000010365<br>ENSSHA0000011536</td><td>RPI</td><td>Scaffold GL841258.1: 388,974-684,603<br>Scaffold GL841258.1: 673,771-676,698</td><td>ENSSHAP0000011380<br>ENSSHAP0000012092</td><td>Ensembl</td><td>DCX/PLAT/2/FGF/2<br>unknown</td></tr><tr><td>Taeniopygia guttata</td><td>Zebra finch</td><td>ENSSHA0000011254<br>ENSTGU0000011040<br>ENSTGU0000011038<br>ENSTGU0000013316</td><td>RPI1L<br>XP.002196882.1<br>RPI</td><td>Scaffold GL834715.1: 823,401-837,998<br>Chromosome 2: 116,600-349,116,603,949<br>Chromosome 2: 116,575,268-116,583,331<br>Chromosome 3: 111,558,335-111,558,985<br>Scaffold GL172719.1: 2,249,715-2,259,635</td><td>ENSSHAP0000013165<br>XP.002196882<br>ENSTGU0000011377<br>ENSTGU0000013370<br>ENSXETP00000055758<br>ENSXETP0000017755</td><td>Ensembl</td><td>DCX/PLAT/2/FGF/2/PLAT*7<br>DCX/PLAT/2/FGF/2/PLAT*2<br>DCX/PLAT/2/FGF/2<br>PLAT/2/FGF/2</td></tr><tr><td>Xenopus tropicalis</td><td>Xenopus</td><td>ENSXETG0000008106<br>ENSXETG0000008105</td><td>RPI</td><td>Scaffold GL172719.1: 2,205,249-2,235,868<br>Chromosome 7: 4,537,369-4,543,878</td><td>ENSXETP0000017755<br>XP.002196882<br>ENSXETP0000017755</td><td>Ensembl</td><td>DCX/PLAT/2/FGF/2/PLAT*12<br>DCX/PLAT/2/FGF/2/PLAT*12<br>DCX/PLAT/2/FGF/2/PLAT*17<br>DCX/PLAT/2/FGF/2/PLAT*17</td></tr><tr><td>Tunicate</td><td>Clonema intestinalis</td><td>Sen squirti</td><td>LOC100175334<br>tgeneb2 pg scaffold_15000072<br>tgeneb2 pg scaffold_216000019</td><td>PREDICTED similar to Lipoxygenase homology domain-containing protein 1</td><td>Scaffold 13: 16,12142-17,21597<br>Scaffold 216: 52,0591-588,067</td><td>JBGI<br>JBGI</td><td>DCX/PLAT/2/FGF/2/PLAT*17<br>DCX/PLAT/2/FGF/2/PLAT*17</td></tr><tr><td>Cephalopodae</td><td>Branchiostoma floridae</td><td>Anphixalus</td><td></td><td></td><td>Conig. NW_003118940.1<br>Conig. NW_003118940.1</td><td>hmm203004<br>hmm205004</td><td>NCBI<br>NCBI</td><td>DCX/PLAT/2/FGF/2/PLAT*17<br>DCX/PLAT/2/FGF/2/PLAT*11</td></tr><tr><td rowspan="3">Hemichordate</td><td>Saccoglossus kowalevskii</td><td>Acorn worm</td><td></td><td></td><td>Chromosome 1: 5,443,881-5,446,858<br>Chromosome 1: 5,436,801-5,443,029</td><td>F27C1.13<br>F27C1.11</td><td>Ensembl<br>Ensembl</td><td>DCX/PLAT/2/FGF/2/PLAT*3<br>DCX/PLAT/2/FGF/2/PLAT*5<br>DCX/PLAT/2/FGF/2/PLAT*3</td></tr><tr><td>Eclyzozoa</td><td>Carnorhabditis elegans</td><td>Carnorhabditis elegans</td><td>F27C1.13<br>F27C1.11</td><td>putative PLAT/LH2 domain protein<br>Doublecotin family protein</td><td>XP.003372868<br>XP.001898608</td><td>Genbank<br>Genbank</td><td>DCX/PLAT/2/FGF/2/PLAT*4<br>DCX/PLAT/2/FGF/2/PLAT*4</td></tr><tr><td>C. elegans</td><td>Trichinella spiralis<br>Brugia malayi</td><td>Trichinella spiralis<br>Brugia malayi</td><td>Tsp_10427<br>Bm1_35805</td><td>hypothetical protein CAEBREN_29075</td><td>ECG Conig.112.471446.480436<br>ECG 46.162827-183842</td><td>Genbank/Wormbase</td><td>DCX/PLAT/2/FGF/2/PLAT*24<br>DCX/PLAT/2/FGF/2/PLAT*24<br>DCX/PLAT/2/FGF/2/PLAT*24</td></tr><tr><td>Lophotrochozoa</td><td>Capitella telea</td><td>Capitella telea</td><td>adExt_tgeneb1 pg_C_460016</td><td></td><td>supercontig pathogen EMU_scaffold_007728: 9834586-9852616</td><td>EnW_000656500<br>EKC38590</td><td>GeneDB<br>Genbank</td><td>DCX/PLAT/2/FGF/2/PLAT*24<br>DCX/PLAT/2/FGF/2/PLAT*24<br>DCX/PLAT/2/FGF/2/PLAT*24</td></tr><tr><td>Acnel</td><td>Convolita pulchra</td><td>Convolita pulchra</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Placozoa</td><td>Trichoplax adhaerens</td><td>Trichoplax adhaerens</td><td>tgenebA2 pg C. scaffold_7000174</td><td></td><td>scaffold_7: 1644952-1666314</td><td>57956</td><td>JBGI</td><td>DCX/PLAT/2/FGF/2/PLAT*16</td></tr></tbody></table>
<table><caption>**Table S2.** Accession numbers of cDNAs from human RP1 gene coding for a protein with the same domain organization as *Trichoplax* proRP1.</caption><thead><tr><td>cDNA Accession number</td><td>Protein Accession Number</td><td>Domains</td><td>Matching ESTs</td></tr></thead><tbody><tr><td>AK316511</td><td>BAH14882</td><td>DCX-PLAT*2-FGFlike*2-PLAT*2</td><td>DB449676, DN831136, DN930509, DN930508, DV080509, BM689829, DC399661, DB227032, DB230079, DB222023, DB227084, DB232529, BX279907, CR749012, DB222477, DB236513, DB233491, DB222294</td></tr><tr><td>AK304443</td><td>BAG65264</td><td>DCX-PLAT*2-FGFlike*2-PLAT*2</td><td>DB449676, DN831136, DN930509, DN930508, DV080509, DC399661, DC402231, DC419041</td></tr><tr><td>AK302348</td><td>BAG63675</td><td>DCX-PLAT*2-FGF</td><td>DB449676, DN831136, DN930509, DN930508, DC402231, DC399661</td></tr><tr><td>AK098176</td><td>BAG53587</td><td>PLAT*2</td><td>DN831136, DN930509, DN930508, DB348563, DB205048</td></tr></tbody></table>
<table><caption>**Table S3.** Profile HMM analysis of the different FGF-like domains.</caption><thead><tr><th rowspan="2"></th><th colspan="5">Hhpred against CDD database best hit</th><th colspan="3">PRC against CDD database best hit</th></tr><tr><th>Accession number</th><th>Name</th><th>Prob</th><th>E-value</th><th>P-value</th><th>Accession Number</th><th>Name</th><th>E-value</th></tr></thead><tbody><tr><td>proRP1 first FGF-like domain</td><td>smart00442</td><td>FGF</td><td>100.0</td><td>2.0E-45</td><td>4.4E-50</td><td>PF00167</td><td>FGF</td><td>2.6e-14</td></tr><tr><td>proRP1 second FGF-like domain</td><td>smart00442</td><td>FGF</td><td>100.0</td><td>3.5E-40</td><td>7.8E-45</td><td>PF00167</td><td>FGF</td><td>1.4e-07</td></tr><tr><td>FGFL FGF-like all</td><td>smart00442</td><td>FGF</td><td>100.0</td><td>1.8E-39</td><td>5.7E-44</td><td>PF00167</td><td>FGF</td><td>5.4e-20</td></tr><tr><td>FGFL FGF-like protostomes</td><td>KOG3885</td><td>FGF</td><td>100.0</td><td>4.4E-46</td><td>9.9E-51</td><td>PF00167</td><td>FGF</td><td>2.2e-06</td></tr><tr><td>FGFL FGF-like deuterostomes</td><td>smart00442</td><td>FGF</td><td>100.0</td><td>2.7E-42</td><td>6.0E-47</td><td>PF00167</td><td>FGF</td><td>6.3e-17</td></tr><tr><td>FGFL FGF-like all except protostomes</td><td>smart00442</td><td>FGF</td><td>100.0</td><td>4.9E-42</td><td>1.1E-46</td><td>PF00167</td><td>FGF</td><td>2.2e-22</td></tr><tr><td>FGFL FGF-like non bilaterians</td><td>smart00442</td><td>FGF</td><td>100.0</td><td>2.4E-46</td><td>5.4E-51</td><td>PF00167</td><td>FGF</td><td>1.3e-19</td></tr><tr><td>All FGF-like</td><td>smart00442</td><td>FGF</td><td>100.0</td><td>2.3E-35</td><td>5.1E-40</td><td>PF00167</td><td>FGF</td><td>7.5e-23</td></tr></tbody></table>
<table><caption>Table S4. Summary of species we searched for FGF and FGF-like. For each species the database used and the presence (green) or absence (red) of FGF domain, and of FGF-like domains from FGFLs or proRP1 is indicated. We did not show all the species for which none of these domains could be found.</caption><thead><tr><th></th><th>Species</th><th>FGF</th><th>FGF-like in proRP1</th><th>FGF-like in FGFLs</th><th>Data</th><th>Database</th><th>Webpage link</th></tr></thead><tbody><tr><td rowspan="20">Metazoans</td><td rowspan="10">Vertebrates</td><td>Ailuropoda melanoleuca</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Anolis carolinensis</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Bos taurus</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Danio rerio</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Gallus gallus</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Homo sapiens</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Latimeria chalumnae</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Meleagris gallopavo</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Mus musculus</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Oryzias latipes</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Otolemur garnettii</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Petromyzon marinus</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Rattus norvegicus</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Sarcophilus harrisii</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Taeniopygia guttata</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Takifugu rubripes</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Tetraodon nigroviridis</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Xenopus tropicalis</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td rowspan="2">Tunicates</td><td>Ciona intestinalis</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Ciona savignyi</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ensembl.org/index.html</td></tr><tr><td>Cephalochordates</td><td>Branchiostoma floridae</td><td></td><td></td><td></td><td>Genome</td><td>JGI</td><td>http://genome.jgi-psf.org/</td></tr><tr><td>Hemichordates</td><td>Saccoglossus kowalevskii</td><td></td><td></td><td></td><td>Genome</td><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/</td></tr><tr><td>Echinoderms</td><td>Strongylocentrotus purpuratus</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://metazoa.ensembl.org/index.html</td></tr><tr><td rowspan="7">Arthropods</td><td>Anopheles gambiae</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://metazoa.ensembl.org/index.html</td></tr><tr><td>Apis mellifera</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://metazoa.ensembl.org/index.html</td></tr><tr><td>Bombyx mori</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://metazoa.ensembl.org/index.html</td></tr><tr><td>Daphnia pulex</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://metazoa.ensembl.org/index.html</td></tr><tr><td>Drosophila melanogaster</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://metazoa.ensembl.org/index.html</td></tr><tr><td>Ixodes scapularis</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://metazoa.ensembl.org/index.html</td></tr><tr><td>Parasteatoda tepidariorum</td><td></td><td></td><td></td><td>EST</td><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/</td></tr><tr><td rowspan="2">Nematods</td><td>Brugia malayi</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://metazoa.ensembl.org/index.html</td></tr><tr><td>Trichinella spiralis</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://metazoa.ensembl.org/index.html</td></tr><tr><td rowspan="2">Annelids</td><td>Alvinella pompejana</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://metazoa.ensembl.org/index.html</td></tr><tr><td>Capitella telea</td><td></td><td></td><td></td><td>EST</td><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/</td></tr><tr><td rowspan="3">Mollusks</td><td>Crassostrea gigas</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://www.ncbi.nlm.nih.gov/</td></tr><tr><td>Lottia gigantea</td><td></td><td></td><td></td><td>EST</td><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/</td></tr><tr><td>Lymnaea stagnalis</td><td></td><td></td><td></td><td>EST</td><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/</td></tr><tr><td rowspan="2">Trematods</td><td>Mytilus californianus</td><td></td><td></td><td></td><td>EST</td><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/</td></tr><tr><td>Echinococcus multilocularis</td><td></td><td></td><td></td><td>Genome</td><td>GeneDB</td><td>http://www.genedb.org/Homepage</td></tr><tr><td rowspan="2">Acoelomorph</td><td>Convoluta pulchra</td><td></td><td></td><td></td><td>Genome</td><td>GeneDB</td><td>http://www.genedb.org/Homepage</td></tr><tr><td>Cnidarians</td><td>Acropora millepora</td><td></td><td></td><td></td><td>Genome</td><td>GeneDB</td><td>http://www.genedb.org/Homepage</td></tr><tr><td rowspan="2">Ctenophores</td><td>Acropora hemisphaerica</td><td></td><td></td><td></td><td>EST</td><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/</td></tr><tr><td>Nematostella vectensis</td><td></td><td></td><td></td><td>EST</td><td>Compagen</td><td>http://compagen.zoologie.uni-kiel.de/</td></tr><tr><td rowspan="2">Placozoans</td><td>Hydra magnipapillata</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://metazoa.ensembl.org/index.html</td></tr><tr><td>Trichoplax adhaerens</td><td></td><td></td><td></td><td>EST</td><td>Compagen</td><td>http://compagen.zoologie.uni-kiel.de/</td></tr><tr><td rowspan="5">Poriferans</td><td>Amphimedon queenslandica</td><td></td><td></td><td></td><td>Genome</td><td>Ensembl</td><td>http://metazoa.ensembl.org/index.html</td></tr><tr><td>Heterochore calyx</td><td></td><td></td><td></td><td>EST</td><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/</td></tr><tr><td>Oopsacas minuta</td><td></td><td></td><td></td><td>EST</td><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/</td></tr><tr><td>Oscarella carmela</td><td></td><td></td><td></td><td>Genome</td><td>Compagen</td><td>http://compagen.zoologie.uni-kiel.de/</td></tr><tr><td>Suberites domuncula</td><td></td><td></td><td></td><td>EST</td><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/</td></tr><tr><td rowspan="5">Opisthokonts Non Metazoan</td><td>Choanofflagellates</td><td>Monosiga brevicollis</td><td></td><td></td><td></td><td>Genome</td><td>Broad Institute</td><td>http://www.broadinstitute.org</td></tr><tr><td>Capsaspora</td><td>Capaspora owczarzaki ATCC 30864</td><td></td><td></td><td></td><td>Genome</td><td>Broad Institute</td><td>http://www.broadinstitute.org</td></tr><tr><td>Ichthyosporans</td><td>Sphaeroformata arctica JP610</td><td></td><td></td><td></td><td>Genome</td><td>Broad Institute</td><td>http://www.broadinstitute.org</td></tr><tr><td>Fungi</td><td>Allomyces macrognus ATCC 38327</td><td></td><td></td><td></td><td>Genome</td><td>Broad Institute</td><td>http://www.broadinstitute.org</td></tr><tr><td>Amoebozoans</td><td>Dictyostelium discoideum</td><td></td><td></td><td></td><td>Genome</td><td>Dictybase</td><td>http://dictybase.org/</td></tr></tbody></table>