Overexpression of Leap2 impairs Xenopus embryonic development and modulates FGF and activin signals

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Overexpression of Leap2 impairs Xenopus embryonic development and modulates FGF and activin signals Pierre Thiébaud a,b,1 , Bertrand Garbay c,1 , Patrick Auguste a,b , Caroline Le Sénéchal c , Zuzanna Maciejewska a,b , Sandrine Fédou a,b , Xavier Gauthereau a,d , Patricia Costaglioli c,2 , Nadine Thézé a,b,∗,2 a

Univ. Bordeaux, F-33076 Bordeaux, France INSERM U1035, F-33076 Bordeaux, France c Bordeaux INP, BPRVS, EA4135, F-33000 Bordeaux, France d CNRS UMS 3427, F-33076 Bordeaux, France b

a r t i c l e

i n f o

Article history: Received 27 March 2016 Received in revised form 15 June 2016 Accepted 17 June 2016 Available online xxx Keywords: Leap2 Xenopus laevis Embryo FGF Activin Cell migration HUVEC

a b s t r a c t Besides its widely described function in the innate immune response, no other clear physiological function has been attributed so far to the Liver-Expressed-Antimicrobial-Peptide 2 (LEAP2). We used the Xenopus embryo model to investigate potentially new functions for this peptide. We identified the amphibian leap2 gene which is highly related to its mammalian orthologues at both structural and sequence levels. The gene is expressed in the embryo mostly in the endoderm-derived tissues. Accordingly it is induced in pluripotent animal cap cells by FGF, activin or a combination of vegT/␤-catenin. Modulating leap2 expression level by gain-of-function strategy impaired normal embryonic development. When overexpressed in pluripotent embryonic cells derived from blastula animal cap explant, leap2 stimulated FGF while it reduced the activin response. Finally, we demonstrate that LEAP2 blocks FGF-induced migration of HUman Vascular Endothelial Cells (HUVEC). Altogether these findings suggest a model in which LEAP2 could act at the extracellular level as a modulator of FGF and activin signals, thus opening new avenues to explore it in relation with cellular processes such as cell differentiation and migration. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Liver-expressed antimicrobial peptide 2 (LEAP2) was originally identified as a circulating peptide in human blood [1]. It is predominantly expressed in the liver as a 77-amino-acid pre-propeptide and is then processed into a 55-amino-acid propeptide, which is finally converted into the mature LEAP2 peptide of 40 residues. The mature human peptide is cationic (isoelectric point = 9.2) with four positive charges at pH 7.0 and contains two intramolecular disulfide bonds that are cross-linked in a 1–3, 2–4 pattern. Determination of the 3D structure by NMR spectroscopy identified a new fold with a central core stabilized by the two disulfide bonds and a network of hydrogen bonds [2].

∗ Corresponding author. Univ. Bordeaux, INSERM U1035, F-33076 Bordeaux, France. E-mail address: [email protected] (N. Thézé). 1 These authors contributed equally to this work. 2 These authors share senior authorship.

The biological role of LEAP2 is not completely understood. A first report demonstrated its antimicrobial activity against some bacteria and fungi [1]. However, it was later demonstrated that the antibacterial activity of human LEAP2 is moderate, as the Minimum Inhibitory Concentration (MIC) measured in vitro are in the 50–100 ␮M range [2,3]. Moreover, it has been found that the sequence of the mature LEAP2 peptide is highly conserved among mammals and birds, and that orthologues can be found in reptiles and several species of fish [1,4–12]. This high level of conservation is quite unusual for antimicrobial peptides because they usually evolve in each animal species in order to adapt to its specific pathogens. Indeed, LEAP2 induction differs in response to bacterial infection depending on the species considered [7]. In this respect, it closely resembles another human peptide, LEAP1/hepcidin, a small peptide present in human blood that was initially identified as an antimicrobial peptide but which regulates iron homeostasis in mammals [13]. By analogy, it has been proposed that LEAP2 may have an additional biological function to its antibacterial activity. However, it has been recently reported that human LEAP2 does not play any role in cell proliferation and does not exhibit any chemo-

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tactic activity on monocytes [14]. Finally, a role for LEAP2 during embryonic development cannot be ruled out because the corresponding transcripts were detected in chicken embryos, and at high level in early embryonic stages of the blunt snout bream [6,11]. LEAP2 mutants could be helpful to obtain insights into protein function but no natural mutation has yet been reported and no knock-out mouse for Leap2 is currently available. To circumvent this problem, we decided to turn to a simple vertebrate model that allows efficient gain-of-function and loss-of-function strategies. We investigated leap2 in the amphibian Xenopus laevis. Xenopus has emerged as one of the most tractable models for the study of vertebrate embryogenesis thanks to its numerous advantages such as rapid development and the possibility to overexpress gene products through mRNA injection and a morpholino antisense oligonucleotide-mediated gene knockdown assay [15–17]. Moreover, animal cap explant from blastula embryo is a useful model of pluripotent cells that can be used to investigate signaling pathways that function in normal development [18]. In the present work, we report the structure and expression of the leap2 gene in Xenopus and provide experimental data about its potential function in vertebrate development. Embryos in which leap2 has been overexpressed display developmental defects. We found that in pluripotent animal cap cells, leap2 stimulates the FGF signal while it inhibits activin signaling. Finally, we provide data that sustain a role of human LEAP2 in the down-regulation of FGF2 induced cell migration. Together our data constitute the first description of new functions for LEAP2 in mammals in cell signaling regulation and unveils unexpected physiological role for this peptide. 2. Material and methods 2.1. Ethics statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the European Community. The protocol was approved by the “Comite´ı d’éthique en expérimentation de Bordeaux” N◦ 33011005A. 2.2. Embryonic manipulations Xenopus laevis oocytes and embryos were obtained and staged using standard procedures and cultured in OR2 buffer and 0.1XMMR respectively [19,20]. Stage 36 embryos were dissected on agarose-coated Petri dish with forceps and pieces were directly lysed in TNES buffer before RNA extraction [20]. Stage 45/46 Xenopus embryos guts were dissected out and washed in 0.1XMMR before fixation for 1 h in MEMFA solution and stored in 100% ethanol [21]. 2.3. Cloning and sequence analysis Total RNA was isolated from X. laevis intestine as previously described [22]. 1 ␮g of RNA was reverse-transcribed with the AffinityScript QPCR cDNA Synthesis kit (Agilent Technologies) using a mix of oligo dT and random nonamer primers at a 5:1 ratio (w/w). One ␮l aliquot from the 20 ␮l single-stranded cDNA reaction and the corresponding RT negative control was diluted into 49 ␮l of PCR reaction mix. 30 amplification cycles (30 s at 95 ◦ C, 45 s at 55 ◦ C, and 90 s at 72 ◦ C) were performed using a iCycler (Biorad) and Taq DNA polymerase (New England Biolabs). Primers used for leap2 amplification were based on the EST sequence of leap2 from Xenopus laevis (GenBank: DT081114.1): F 5 - GGGATCCATGTTCCTGCAGCCTGG-3 and R 5 - GGAATTCTTACCTGCACAACTTGG-3 . The underlined sequences represent sites for the restriction enzymes BamHI and EcoRI. The

bold sequence represents the initiation codon. These two primers are located respectively in exon 1 and exon 2 of the Xenopus leap2 gene. After amplification, the PCR product was purified with Nucleospin Gel and PCR clean-up (Macherey-Nagel), ligated into pGEM-T vector (Promega) using Quick Ligase (New England Biolabs), and the plasmid was then electroporated into JM109 E. coli competent cells (Promega). Clones were thereafter selected and sequenced (MilleGen Biotechnologies, Toulouse, France). RACE experiments were performed using the Gene RacerTM kit (Invitrogen) and Xenopus laevis intestine RNA (2.5 ␮g/experiment). PCR products were cloned by using the TOPO TA Cloning Kit (Invitrogen) before sequencing. All experiments were performed according to the manufacturer’s instructions. The leap2 specific primers used for the RACE experiments were: 5 -AGGGGCCTGTCTTTGCGCCCATTAG-3 (3 RACE) and 5 -GCGCAAAGACAGGCCCCTCCAAAAT-3 (5 RACE). Xenopus leap2 sequences were retrieved from GenBank with the alignment search tool tBlastN from the National Center for Biotechnology Information (NCBI). Synteny analysis was performed using Ensembl and metazome and the gene structure was deduced by comparison between genomic and cDNA sequences. 2.4. Constructs, mRNA and injections Xenopus leap2 cDNA was subcloned into BamHI XbaI sites of pCS2 plasmid (pCS2-leap2) (F1 and 5 -GCGGGATCCATGTTCCTGCAGCCTGGG-3 ; R1 5 GCGTCTAGAATTACTAAAAGTCTTCAGCGACCAG-3 ). For microinjection experiments, capped mRNAs were synthesized using the mMessenger mMachine SP6 Kit (Ambion) from Acc65Ilinearized plasmids. For the overexpression study, 4-cell stage embryos were injected with 2.5 ng of leap2 mRNA. To identify the injected side, nlacZ (250 pg) were used as tracer and lacZ expression was revealed with the Red-Gal substrate (Research Organics). For the animal caps assay, 2.5 ng of leap2 mRNA, 500 pg of vegT, 200 pg of ␤-catenin or 1 ng of XFD mRNAs were injected into the animal pole of 2-cell stage embryos. In control experiments, lacZ mRNA was injected at the same dose than leap2 or vegT + ␤catenin mRNAs. Animal cap explants were dissected from stage 8–9 embryos and cultured until the control embryos reached the appropriate stage before RT-qPCR analysis. For growth factor treatment, FGF2 (100 ng/ml final) and activin (50 or 100 ng/ml final) were provided by R&D Systems. For inhibitor treatment, SU5402 (Calbiochem) was dissolved in DMSO (50 mM stock solution) and diluted in 0.1XMBS to 50 ␮M for animal cap treatment. U0126 and PD98059 were dissolved at 20 mM and 100 mM respectively before dilution to 50 ␮M. 2.5. RT-PCR, in situ hybridization Total RNA was isolated from Xenopus laevis embryo and adult tissues as previously described [22]. RNA concentration was determined by spectrophotometry and integrity was assessed by agarose gel electrophoresis. For each sample, 1 ␮g of total RNA was reverse-transcribed with the AffinityScript QPCR cDNA Synthesis kit (Agilent Technologies) using a mix of oligo dT and random nonamer primers at a 5:1 ratio (w/w). To evaluate a possible genomic DNA contamination, an RT negative control was generated for each RNA sample and used as a control in the PCR experiments. PCR was performed as described above with the primer pairs listed in Table S1. 5 ␮l of PCR reactions was analyzed on a 2% agarose gel containing ethidium bromide in TBE buffer and documented with a Gel Documentation System (Biorad). In situ hybridization was carried out as previously reported [23]. Antisense and sense probes were made from pCS2-leap2 linearized plasmid with BamHI (+T7 RNA polymerase) and XbaI (+SP6 RNA polymerase) respec-

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Fig. 1. Conservation of vertebrate LEAP2 at genomic and protein levels. (A) Conserved synteny regions between human (Hsa), mouse (Mmu), chicken (Gga) and Xenopus tropicalis (Xtr) chromosomal regions containing LEAP2 locus. Genes are represented as colored boxes with the arrow indicating the orientation of the transcription unit. Boxes with the same color correspond to orthologue genes. The drawing is not to scale to avoid complexity. (B) Amino acid sequence comparison between Xenopus tropicalis (Xtr), Xenopus laevis (Xla) and human (Hsa) leap2. The different regions of the proteins are indicated above the sequence. Identical amino acids between amphibian and human sequences are shaded gray. Amino acids italicized in the Xenopus tropicalis sequence are derived from genomic sequences. (C) Schematic representation of human LEAP2 gene structure and comparison with other species. Exons are represented by black boxes, introns (I–II) by a thin line and untranslated regions by white boxes (not on scale). The size of exons and introns for each species is indicated below. The class splice site is indicated below the introns.

tively. Real time RT-PCR was performed, using the SYBR Green qPCR master mix (Promega, Charbonnières-les-Bains, France) and specific primers are listed in Table S1. Real-time PCRs were run on a LightCycler® 480 Thermal Block Cycler (Roche Applied Science). Each sample was measured in duplicate. Gene expressions were normalized relative to ODC housekeeping gene using the comparative Ct method.

2.6. HUVEC culture and migration assay HUman vascular endothelial cells (HUVEC) were cultured in Endothelial Growth Medium with supplement mix. HUVEC cell migration was measured using 24-well-Transwell plates (Becton Dickinson). 5 × 104 cells were seeded in the upper compartment filter (8 ␮m pore size) in 500 ␮l of Endothelial Growth Medium, 0.1% supplement mix. FGF2 (10 ng/ml), FGF2 and human LEAP2 (100 nM, 4405-S from Peptide Institute Inc, Japan, or recombinant homemade) or LEAP2 alone were added in the lower compartment as chemo-attractant and cells were allowed to migrate for 5 h throughout the membranes. Migrated cells in the lower part of the filter were fixed in 4% paraformaldehyde and labeled with DAPI. 10 fields (20X magnification) per filter were photographed and cells were counted using Image J software. Experiments were repeated at least three times and statistical analysis used ANOVA.

3. Results 3.1. Characterization of Xenopus leap2 gene and protein In the search for Xenopus leap2 sequences, we retrieved from Xenbase and GenBank a cDNA clone sequence from adult intestine X. Tropicalis that encodes a 78-amino-acid protein (BC155465). The corresponding gene in Ensembl (Xenopus JGI 4.2, Scaffold 139:173975:177978) is composed of 3 exons and 2 introns. The genomic organization around X. tropicalis leap2 gene, as revealed by synteny analysis, is strongly conserved between vertebrates at both the position and the orientation of flanking genes (Fig. 1A). In human, mouse, chicken and Xenopus genomes, the immediate neighbors of LEAP2 are AFF4, ZCCHC10 and HSPA4 on one hand, and are UQCRQ, GDF9, SHROOM1, ANKRD43, and SEPT8 on the other, with the sense of transcription being totally conserved except for GDF9. In contrast, there is no synteny conservation with fish genome such as fugu, medaka or stickleback. Together, this clearly identifies the leap2 sequence in the genus Xenopus. In a blast search with the X. tropicalis sequence, we identified an EST corresponding to X. laevis leap2 mRNA (AGENCOURT 55718936; GenBank: DT081114.1). The full-length cDNA sequence was established by 5 and 3 RACE experiments and deposited at Genbank (ID: JX987285). Xenopus laevis leap2 cDNA is 450 nucleotides long with a 240 open reading frame encoding an 80-amino-acid protein. X. laevis and X. tropicalis

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Fig. 2. Xenopus laevis leap2 gene expression during development and in adult tissues. (A) RT-PCR analysis of leap2 in embryo from egg to stage (St.) 38. (B) RT-PCR analysis of leap2 in adult tissues. All tissues have been dissected from a single adult except for heart, kidney, lung testis and brain from 5 adults. (C) RT-PCR analysis of leap2 in stage 36 embryonic tissues. Parts of the dissected and analyzed embryo are numbered on the schematic drawing as follows: DA (Dorso Anterior), D (Dorsal), VA (Ventral Anterior) and V (Ventral). Rpl8 gene expression was used as control. (D) leap2 in situ hybridization analysis of stage 45 embryo digestive tract. Sense control probe is shown in the inset of the panel. (E) An enlargement of a single digestive tract in D is shown. Int, intestine; Sto, stomach; Li, liver; Lu, lung; P, pancreas.

have 90.8% identity at the nucleotide level (Fig. S1). We observed that the X. laevis protein has 2 extra amino acids at its N-terminal end (Methionine-Phenylalanine) but a close examination of the X. tropicalis genomic sequence in the open reading frame revealed a methionine codon and a valine codon upstream of the start codon in the originally described protein (BC155465) (Fig. S1). We thus consider that the two amphibian proteins have 80 amino acids and that they display 50% of identity with their human orthologue (Fig. 1B). The overall organization of the Xenopus leap2 protein is well conserved, with the presence of an N-terminal pre-sequence domain, followed by a pro-sequence and the mature peptide. There are 4 amino acids difference between the two pre-region sequences, two of them being conservative. The pro-region sequence is identical for both amphibians and there are only three amino acids changes over the 40 residues of the mature peptide. While the human and amphibian sequences showed large sequence variation in their preand pro-regions, they displayed a good conservation for the biologically active mature peptide with 67.5% of identity (Fig. 1C). The mature peptides were predicted to have 3 (X. tropicalis) and 5 (X. laevis) positive charges at pH7.0, isoelectric points of 8.97 (X. tropicalis) and 9.58 (X. laevis) and a putative molecular weight of 4.45 kDa for both species. As expected from the sequence conservation, these values are close to those of the human peptide with 4 positive charges at pH7.0, a putative molecular weight of 4.6 kDa and an isoelectric point of 9.37. In the final release of the Xenopus laevis genome (JGIv8.0), we identified a scaffold that contains the leap2 gene whose structure was established by comparing cDNA and genomic sequences. Like other vertebrate genes, X. laevis and X. tropicalis leap2 genes are both composed of three exons and two introns of similar size but with longer introns (Fig. 1C). According to the available sequences, the synteny between X. laevis and X. tropicalis genes is conserved (data not shown).

3.2. Leap2 gene expression in Xenopus laevis Leap2 expression was analyzed by RT-PCR in embryo from different developmental stages (Fig. 2A). No significant transcript level was detected in unfertilized egg nor neurula (stage 15). Leap2 expression started to be detected in tadpole stage embryo (stage 31) and then remains constant. The expression of leap2 was next evaluated in a number of adult tissues (Fig. 2B). Leap2 was expressed in intestine, heart, lung, ovary and kidney. No significant amount of leap2 transcripts was detected in brain, testis or spleen. Our attempt to localize leap2 mRNA in the embryo by whole mount in situ hybridization gave no signal above the background when using antisense versus sense probe (data not shown). Therefore, to obtain more insights into the embryonic expression of leap2, we dissected out stage 36 embryos into four parts that corresponded to Dorsal Anterior (DA), Ventral anterior (VA), Ventral (V) and Dorsal (D) fractions respectively before RNA extraction and RTPCR analysis (Fig. 2C). No leap2 transcripts were detected in DA and D regions that corresponded roughly to brain and somite regions, respectively. A low level of expression was detected in the embryonic VA region that contains the heart and a strong expression was found in the ventral fraction that encompasses the digestive tract of the embryo. To examine leap2 expression more closely in the digestive tract of the embryo, guts were dissected out from 4-day-old embryos (stage 45) and then processed for whole mount in situ hybridization [21]. Leap2 expression was detected in the stomach and intestine but not in the lung or pancreas and it was barely detectable in liver (Fig. 2D, E). Together, these findings indicate that Xenopus leap2 is not expressed maternally and that its major sites of expression in the embryo are the intestine and the stomach, while liver and lung expression is restricted to adult tissues. It should be noted that the only leap2 transcript that is detected in Xenopus laevis tissues corresponds to the correctly spliced mRNA.

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Fig. 3. Leap2 gene regulation in embryonic cells. (A) Animal cap explants from blastula embryos were treated (+) or not (−) with growth factors (GF) before leap2 expression analysis by RT-qPCR. 50 or 100 ng/ml of activin (Act) or 100 ng/ml of FGF2 were used. For comparison, the expression of pdx1 and foxa1 genes was assayed. (B) Embryos were injected with vegT and ␤-catenin mRNAs and explants dissected at the blastula stage before leap2 expression by RT-qPCR analysis. The expression of 3B6 and darmin genes was assayed for comparison. Ctrl, control explants injected with lacZ mRNA. Each value was normalized to the level of ODC expression. (C) Embryos were injected with 2.5 ng of leap2 mRNA in future dorsal or ventral parts of the embryo. One embryo representative of each phenotype is shown. Control embryos injected with 2.5 ng of lacZ mRNA show normal phenotype.

3.3. Leap2 regulation in pluripotent animal cap cells and gain-of-function in embryo Leap2 is expressed in embryonic gut which is mainly constituted by endodermal derivatives. Pluripotent animal cap explants derived from blastula embryo model can be induced to endoderm cells after activin treatment [24]. Therefore, we tested by RT-qPCR whether leap2 was expressed in animal cap cells after activin treatment. Leap2 was not expressed in animal cap control cells and was slightly expressed at 50 ng/ml of activin (Fig. 3A). Its expression level increased with 100 ng/ml of activin (Fig. 3A). Leap2 is also induced by FGF2 (Fig. 3A). In these experiments, pdx1 and foxa1 were expressed in animal cap cells as expected for pancreatic and liver-specific endoderm marker respectively, which are induced in activin-treated animal caps [25,26]. Endoderm differentiation can be obtained in animal cap cells under conditions where the TGF␤ and wnt pathways have been stimulated by the expression of the transcription factors vegT and ␤-catenin respectively [27]. Therefore, we tested whether leap2 was expressed in these conditions. Animal cap cells from uninjected embryos did not express leap2 nor 3B6 and only a low level of darmin, the latter two being endoderm markers (Fig. 3B). However, 3B6, darmin and leap2 were expressed in animal cap explants derived from embryos co-injected with vegT and ␤-catenin mRNA.

This is in agreement with the endoderm differentiation of embryonic cells (Fig. 3B). As a first step towards evaluating the leap2 function during development, we used a gain-of-function strategy based on mRNA overexpression. 2.5 ng of leap2 mRNA were injected into 4-cell stage embryos in either the prospective dorsal or ventral side. When leap2 mRNA was injected on the future dorsal side of the embryo, 30% of it (n = 60) showed a bent phenotype with a curvature of the body axis (Fig. 3C). Embryos where leap2 was overexpressed on the future ventral side had a normal phenotype compared to control embryos injected with lacZ mRNA alone (Fig. 3C 100%, n = 65). These data suggest that the overexpression of leap2 affects normal embryonic development.

3.4. Leap2 stimulates FGF while blocking activin signals FGF and activin, two widely used growth factors, elicit distinct phenotypic effects on animal caps that can be easily monitored by simple observation. FGF2 is known to induce mesoderm of ventral type while activin induces mesoderm of dorsal type [28]. Thus, while FGF-treated explants have a pear-like shape activing-treated explants show a typical elongation [29]. Because leap2 displays a signal peptide and has the potential to act outside the cell as an extracellular signaling molecule, we wondered whether it could

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Fig. 4. Leap2 overexpession in pluripotent embryonic cells stimulates FGF2 signal but reduces activin signal. (A) Embryos were injected (+leap2) or not (-leap2) with 2.5 ng of leap2 mRNA and animal cap explants were treated with 100 ng/ml of FGF2 or 50 ng/ml of activin or were untreated (Ctrl). Explant morphology was scored 5 h or 18 h after addition of growth factors. (B) Animal cap explants from embryos injected (+leap2) or not (-leap2) with 2.5 ng of leap2 mRNA were treated with 100 ng/ml of FGF2 and FGF signaling was blocked by injecting 1 ng of the dominant negative FGF receptor (XFD) mRNA or by incubating with SU5402 or PD98059. The phenotype of explants was scored 5 h after addition of FGF2. Untreated animal caps injected with lacZ mRNA were used as control (Ctrl). (C) Animal cap explants displayed in A (5 h for FGF and 18 h for activin) were analyzed for pnp, xbra or chordin expression by RT-qPCR. (D) Proposed model for the action of leap2 on FGF and activin signaling. RTK, Receptor Tyrosine Kinase, RTK-P; Phosphorylated RTK; MEK, MAPK Kinase; ERK, MAP Kinase; RSTK, Receptor Serine/Threonine Kinase. Two possible levels of action of leap2 on activin signaling are depicted (?).

act on FGF or activin pathways. Leap2 mRNA was injected into the animal pole of 2-cell stage embryos and animal cap explants were taken from blastula stage embryos, treated with FGF or activin and scored for morphological changes relative to untreated control explants. Animal caps injected with leap2 mRNA remained round and show no difference with animal cap controls (Fig. 4A). Animal caps treated with FGF2 for 5 h displayed a typical pear-like shape and, in the presence of leap2, displayed a more pronounced deformation and elongate significantly (Fig. 4A). Animal caps treated with activin and analyzed after 5 h of culture displayed a slight deformation compared to untreated control explants. When the caps derived from embryos were injected with leap2 mRNA, they did not show any such deformation. When analyzed after 18 h of culture, activin-treated animal caps displayed the typical elongated form. However, in the presence of leap2, this elongation was less pronounced. This indicates that overexpression of leap2 reduced activin-triggered elongation of animal caps. Together these data suggest that leap2 overexpression enhances FGF signals while it down-regulates activin signals respectively. Expression of XFD, a dominant negative receptor of FGF (FGFR1), has been shown to block FGF signals in the embryo [30]. XFD forms a heterodimer with the endogenous receptor but has its cytoplasmic domain deleted which impairs signaling. Animal caps where XFD had been expressed did not show any response to leap2 stimulation after FGF treatment. Similarly when caps were cultured in the presence of SU5402 and PD98059, two potent FGF inhibitors, they did not show any elongation stimulation with leap2 (Fig. 4B). Together these results suggest that leap2 functions as an agonist of FGF and can potentially act at the extra-cellular level. To further assess the effects of leap2 on FGF and activin signaling, we analyzed the expression of selected target genes by RT-qPCR. Pnp (purine phosphorylase) is known to be induced by FGF while chordin is a dorsal gene induced by activin and xbra is a pan-mesodermic factor induced by both FGF and activin [31–33].

Fig. 5. LEAP2 blocks FGF2 induced migration of HUman Vascular Endothelial Cells. HUman Vascular Endothelial Cells were treated or not (Ctrl) with FGF2 in the presence (+LEAP2) or not of human LEAP2 and tested for their migration as described in Material and Methods. Results are expressed as cell number per field. Two preparations of LEAP2 (A, B) were tested.

Pnp expression was induced in animal caps treated with FGF2 and was slightly increased in caps that overexpressed leap2 (Fig. 4C). In our conditions, pnp expression was stimulated by activin and slightly decreased in the presence of leap2 (Fig. 4C). Xbra expression showed no difference in FGF-treated animal caps in the presence or not of leap2 (Fig. 4C). In contrast, activin-induced xbra expression was clearly decreased in the presence of leap2 (Fig. 4C). Similarly, activin-induced expression of chordin was down-regulated when animal caps overexpressed leap2 (Fig. 4C). These results confirm

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the stimulating effects of leap2 on FGF signaling and its inhibitory effects on activin signaling (Fig. 4D). 3.5. LEAP2 impairs FGF-induced human cell migration Our findings clearly suggest that leap2 can modulate FGF signaling in embryonic cells. Therefore, we wondered whether this could be extended to human cells. In other words, could LEAP2 have some effect on FGF-stimulated human cells? To address this question, we used HUman Vascular Endothelial Cells (HUVEC) which are known to respond to FGF2 stimulation by both increased proliferation and migration [34]. LEAP2 is expressed at a low level in HUVEC cells (data not shown) as in several other human cell lines (see http://www.proteinatlas.org/ENSG00000164406-LEAP2/cell). In a first series of experiments and despite several attempts, we did not observe any significant effect of LEAP2 on FGF-induced proliferation of HUVEC cells (data not shown). In contrast, we repeatedly found that FGF-treated cells migrated less in the presence of human LEAP2 in the culture medium (Fig. 5). These findings suggest that LEAP2 can modulate FGF signaling in human cells and at least the migration of endothelial cells. 4. Discussion In this paper, we report the identification of the leap gene in Xenopus and its expression during development and in adult tissues. The modulation of leap2 expression by gain of function impairs the normal development of the embryo. More importantly, overexpression of leap2 in pluripotent embryonic cells enhances the FGF response while reducing the activin response. Finally, human LEAP2 stimulates the migration of human cells in culture. This is therefore the first report of an unexpected function in leap2 that is related to cell signaling in animals. Our work completes previous studies on leap2 expression in chicken and fish embryo and stimulates interest for further functions in developmental organism models [6,11]. Like its vertebrate counterparts, the Xenopus leap2 encodes a mature peptide of 40 amino acids that has all the hallmarks of the leap2 family. It is generated from an 80-residue precursor that is matured in a pro-sequence, which is then cleaved to produce a native peptide that contains four cysteines allowing the formation of two intracellular disulfide bridges. The identity of the Xenopus leap2 gene has been confirmed by both synteny and gene structure which are highly conserved with its mammalian orthologues. Although the amphibian gene is structurally identical to its mammalian orthologues, its introns are unexpectedly longer since introns I and II are 10 and 5-fold larger than their mammalian counterparts, respectively. This is contrary to what has been observed for the evolution of intron size and what we found for the MRAS or SMMHC genes where the introns were much larger in mammalian genes compared to amphibian genes [22,35,36]. Leap2 is expressed in the Xenopus laevis embryo according to a strict temporal program and mRNA starts to accumulate from stage 27 (one day after fertilization) at the beginning of organogenesis. In catfish, leap2 has been found to be expressed quite late in development (eleven days after fertilization), unlike the situation in grass carp where the leap2 transcript level declines from the 16-cell stage embryo, suggesting a maternal expression [4,5]. In the embryo, leap2 transcripts are mainly found in the digestive tract while it expression is more widespread in adult tissues, as is the case in most fish [5,7,9,11]. In view of its major expression in the endodermis, leap2 is induced in pluripotent animal caps that are differentiated in the endodermis. It is also induced by activin and FGF. We did not detect any of the intron 1-containing variants that have been identified in some human tissues and cell lines

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[1,14]. These observations indicate that leap2 genes from different species are differentially regulated. Most studies that have addressed the functions of LEAP2 have focused on its antimicrobial activity but very few have addressed other physiological properties [14,37,38]. Our work based on gainof-function approach clearly establishes that modifying the level of leap2 expression has profound effects on the normal development of the embryo. Embryos overexpressing leap2 have more or less pronounced axis defects, depending on where leap2 expression has been disturbed. In all cases, those disruptions affect the late but not early phases of embryonic development. This could be interpreted in the light of its late endogenous embryonic expression. In a first attempt to unravel the mechanisms that were disturbed by leap2 in our experiments, we used an animal cap assay since the morphologic changes that it reveals provide important information. Indeed, FGF-treated animal caps have a pear shape and contain mesothelium and mesenchymal tissues, while activintreated explants contain muscle tissues and have an elongated form that is reminiscent of the convergent extension movements that occur during gastrulation [29]. Since leap2 is not expressed maternally, there is no interference with the endogenous protein and the animal cap assay will reveal direct effect of leap2 on the tested signaling pathways. We showed that leap2 enhanced the FGF response and induced a transient elongation of animal cap explants. The use of FGF inhibitors confirmed that leap2 specifically affected the FGF pathway. More importantly, experiments with a dominant negative FGF receptor indicated that leap2 may act at the extracellular level. These findings indicate that the biological effects of leap2 require FGF signaling. We hypothesize that leap2 potentiates the FGF response by increasing FGF-FGFR affinity promoting receptor dimerization, as has been shown for heparan sulfate [39]. Although Leap2 shows no homology with the FGF ligand, it could constitute a new ligand for the FGF receptor, as has been found with FRL1 and FRL2 [40]. Nevertheless, leap2 might also act at an intracellular level by an unknown mechanism. Animal caps treated with activin elongate extensively and mimic the convergent extension of axial mesodermal cells in development [41]. Leap2 partially inhibits this elongation, indicating a negative effect on activin signaling. This was confirmed by the reduced expression of chordin in animal caps. We do not yet have any evidence whether the activin-reduced signaling by leap2 is direct or not and whether it occurs in the extracellular or intracellular space as a consequence of FGF blocking. Indeed it is known that activin-mediated mesoderm induction requires FGF [42]. Although our data suggest that leap2 interferes with FGF signaling pathways in embryonic cells, it is tempting to speculate whether this property is conserved in human cells. FGF are thought to be involved in various cellular processes including apoptosis, cell adhesion, migration, differentiation and proliferation, not only in the embryo but also in numerous cell types and in complex regulated systems such as angiogenesis [43,44]. Since the transient elongation of FGF treated animal cap explants is related to cell migration that occurs during gastrulation movements, we turned to the model of HUman Vascular Endothelial Cells (HUVEC) in which FGF response can be easily monitored [29,34]. Moreover, there is no Xenopus cell line available for in vitro migration study and the model of cell migration (Neural crest cell for instance) in the embryo does not respond to FGF. Our data indicate that FGF-dependent endothelial cell migration is down-regulated in the presence of LEAP2 while it has no effect on cell proliferation. It has also be observed that LEAP2 has no effect on hepatocyte cell proliferation [14]. Our findings reveal that LEAP2 can regulate FGF signaling in human cells.

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5. Conclusion These data suggest the new finding that leap2 can modulate the FGF and activin pathways by acting very likely in the extracellular space. This does not call into question the previously established antimicrobial activity of leap2 but reveals a new physiological role. This opens up new avenues for integrating LEAP2 into the regulation of cellular processes in animals that are awaiting analysis. Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgments This work was supported by the Centre National de la Recherche Scientifique (CNRS), The INSERM, the SFR Transbiomed, Bordeaux INP and the University of Bordeaux. The RT-qPCR analysis was performed on the Real-time PCR Platform CNRS UMS 3427/INSERM US05/FR TBMC Bordeaux University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.peptides.2016. 06.008. References [1] A. Krause, R. Sillard, B. Kleemeier, E. Kluver, E. Maronde, J.R. Conejo-Garcia, W.G. Forssmann, P. Schulz-Knappe, M.C. Nehls, F. Wattler, S. Wattler, K. Adermann, Isolation and biochemical characterization of LEAP-2, a novel blood peptide expressed in the liver, Protein Sci. 12 (2003) 143–152. [2] S.T. Henriques, C.C. Tan, D.J. Craik, R.J. Clark, Structural and functional analysis of human liver-expressed antimicrobial peptide 2, Chembiochem 11 (2010) 2148–2157. [3] A. Hocquellet, B. Odaert, C. Cabanne, A. Noubhani, W. Dieryck, G. Joucla, C. Le Senechal, M. Milenkov, S. Chaignepain, J.M. Schmitter, S. Claverol, X. Santarelli, E.J. Dufourc, M. Bonneu, B. Garbay, P. Costaglioli, Structure-activity relationship of human liver-expressed antimicrobial peptide 2, Peptides 31 (2010) 58–66. [4] B. Bao, E. Peatman, P. Xu, P. Li, H. Zeng, C. He, Z. Liu, The catfish liver-expressed antimicrobial peptide 2 (LEAP-2) gene is expressed in a wide range of tissues and developmentally regulated, Mol. Immunol. 43 (2006) 367–377. [5] F. Liu, J.L. Li, G.H. Yue, J.J. Fu, Z.F. Zhou, Molecular cloning and expression analysis of the liver-expressed antimicrobial peptide 2 (LEAP-2) gene in grass carp, Vet. Immunol. Immunopathol. 133 (2010) 133–143. [6] G. Michailidis, Expression of chicken LEAP-2 in the reproductive organs and embryos and in response to Salmonella enterica infection, Vet. Res. Commun. 34 (2010) 459–471. [7] H.X. Li, X.J. Lu, C.H. Li, J. Chen, Molecular characterization of the liver-expressed antimicrobial peptide 2 (LEAP-2) in a teleost fish, Plecoglossus altivelis: antimicrobial activity and molecular mechanism, Mol. Immunol. 65 (2015) 406–415. [8] G. Yang, H. Guo, H. Li, S. Shan, X. Zhang, J.H. Rombout, L. An, Molecular characterization of LEAP-2 cDNA in common carp (Cyprinus carpio L.) and the differential expression upon a Vibrio anguillarum stimulus; indications for a significant immune role in skin, Fish Shellfish Immunol. 37 (2014) 22–29. [9] T. Liu, Y. Gao, R. Wang, T. Xu, Characterization, evolution and functional analysis of the liver-expressed antimicrobial peptide 2 (LEAP-2) gene in miiuy croaker, Fish Shellfish Immunol. 41 (2014) 191–199. [10] H.X. Li, X.J. Lu, C.H. Li, J. Chen, Molecular characterization and functional analysis of two distinct liver-expressed antimicrobial peptide 2 (LEAP-2) genes in large yellow croaker (Larimichthys crocea), Fish Shellfish Immunol. 38 (2014) 330–339. [11] T. Liang, W. Ji, G.R. Zhang, K.J. Wei, K. Feng, W.M. Wang, G.W. Zou, Molecular cloning and expression analysis of liver-expressed antimicrobial peptide 1 (LEAP-1) and LEAP-2 genes in the blunt snout bream (Megalobrama amblycephala), Fish Shellfish Immunol. 35 (2013) 553–563. [12] Y.A. Zhang, J. Zou, C.I. Chang, C.J. Secombes, Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout, Vet. Immunol. Immunopathol. 101 (2004) 259–269. [13] T. Ganz, Molecular control of iron transport, J. Am. Soc. Nephrol. 18 (2007) 394–400. [14] A. Howard, C. Townes, P. Milona, C.J. Nile, G. Michailidis, J. Hall, Expression and functional analyses of liver expressed antimicrobial peptide-2 (LEAP-2) variant forms in human tissues, Cell. Immunol. 261 (2010) 128–133.

[15] J.B. Gurdon, N. Hopwood, The introduction of Xenopus laevis into developmental biology: of empire, pregnancy testing and ribosomal genes, Int. J. Dev. Biol. 44 (2000) 43–50. [16] R.M. Harland, R.M. Grainger, Xenopus research: metamorphosed by genetics and genomics, Trends Genet. 27 (2011) 507–515. [17] J. Heasman, Morpholino oligos: making sense of antisense? Dev. Biol. 243 (2002) 209–214. [18] K. Okabayashi, M. Asashima, Tissue generation from amphibian animal caps, Curr. Opin. Genet. Dev. 13 (2003) 502–507. [19] P.D. Nieuwkoop, J. Faber (Eds.), Normal Table of Xenopus Laevis (Daudin), 2nd ed., Garland, Amsterdam, 1975. [20] H.L. Sive, R.M. Grainger, R.M. Harland, Early Development of Xenopus Laevis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2000. [21] A.D. Chalmers, J.M. Slack, Development of the gut in Xenopus laevis, Dev. Dyn. 212 (1998) 509–521. [22] W. Barillot, K. Treguer, C. Faucheux, S. Fedou, N. Theze, P. Thiebaud, Induction and modulation of smooth muscle differentiation in Xenopus embryonic cells, Dev. Dyn. 237 (2008) 3373–3386. [23] R.M. Harland, In situ hybridization: an improved whole-mount method for Xenopus embryos, Methods Cell Biol. 36 (1991) 685–695. [24] C. Hudson, D. Clements, R.V. Friday, D. Stott, H.R. Woodland, Xsox17alpha and −beta mediate endoderm formation in Xenopus, Cell 91 (1997) 397–405. [25] S. Knochel, J. Lef, J. Clement, B. Klocke, S. Hille, M. Koster, W. Knochel, Activin A induced expression of a fork head related gene in posterior chordamesoderm (notochord) of Xenopus laevis embryos, Mech. Dev. 38 (1992) 157–165. [26] G.L. Henry, I.H. Brivanlou, D.S. Kessler, A. Hemmati-Brivanlou, D.A. Melton, TGF-beta signals and a pattern in Xenopus laevis endodermal development, Development 122 (1996) 1007–1015. [27] Y. Chen, K. Jurgens, T. Hollemann, M. Claussen, G. Ramadori, T. Pieler, Cell-autonomous and signal-dependent expression of liver and intestine marker genes in pluripotent precursor cells from Xenopus embryos, Mech. Dev. 120 (2003) 277–288. [28] J.C. Smith, Mesoderm-inducing factors and mesodermal patterning, Curr. Opin. Cell Biol. 7 (1995) 856–861. [29] J.C. Smith, J. Cooke, J.B. Green, G. Howes, K. Symes, Inducing factors and the control of mesodermal pattern in Xenopus laevis, Development 107 (Suppl) (1989) 149–159. [30] E. Amaya, T.J. Musci, M.W. Kirschner, Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos, Cell 66 (1991) 257–270. [31] P.A. Branney, L. Faas, S.E. Steane, M.E. Pownall, H.V. Isaacs, Characterisation of the fibroblast growth factor dependent transcriptome in early development, PLoS One 4 (2009) e4951. [32] Y. Sasai, B. Lu, H. Steinbeisser, D. Geissert, L.K. Gont, E.M. De Robertis, Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes, Cell 79 (1994) 779–790. [33] J.C. Smith, B.M. Price, J.B. Green, D. Weigel, B.G. Herrmann, Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction, Cell 67 (1991) 79–87. [34] C. Dos Santos, C. Blanc, R. Elahouel, M. Prescott, G. Carpentier, A. Ori, J. Courty, Y. Hamma-Kourbali, D.G. Fernig, J. Delbe, Proliferation and migration activities of fibroblast growth factor-2 in endothelial cells are modulated by its direct interaction with heparin affin regulatory peptide, Biochimie 107 (Pt B) (2014) 350–357. [35] Q. Zhang, S.V. Edwards, The evolution of intron size in amniotes: a role for powered flight? Genome Biol. Evol. 4 (2012) 1033–1043. [36] M.E. Mathieu, C. Faucheux, C. Saucourt, F. Soulet, X. Gauthereau, S. Fedou, M. Trouillas, N. Theze, P. Thiebaud, H. Boeuf, MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate, Development 140 (2013) 3311–3322. [37] Y. Sang, B. Ramanathan, J.E. Minton, C.R. Ross, F. Blecha, Porcine liver-expressed antimicrobial peptides, hepcidin and LEAP-2: cloning and induction by bacterial infection, Dev. Comp. Immunol. 30 (2006) 357–366. [38] C.L. Townes, G. Michailidis, C.J. Nile, J. Hall, Induction of cationic chicken liver-expressed antimicrobial peptide 2 in response to Salmonella enterica infection, Infect. Immun. 72 (2004) 6987–6993. [39] D.M. Ornitz, FGFs, heparan sulfate and FGFRs: complex interactions essential for development, Bioessays 22 (2000) 108–112. [40] N. Kinoshita, J. Minshull, M.W. Kirschner, The identification of two novel ligands of the FGF receptor by a yeast screening method and their activity in Xenopus development, Cell 83 (1995) 621–630. [41] K.M. Kwan, M.W. Kirschner, Xbra functions as a switch between cell migration and convergent extension in the Xenopus gastrula, Development 130 (2003) 1961–1972. [42] R.A. Cornell, D. Kimelman, Activin-mediated mesoderm induction requires FGF, Development 120 (1994) 453–462. [43] P. Auguste, S. Javerzat, A. Bikfalvi, Regulation of vascular development by fibroblast growth factors, Cell Tissue Res. 314 (2003) 157–166. [44] R.T. Bottcher, C. Niehrs, Fibroblast growth factor signaling during early vertebrate development, Encocr. Rev. 26 (2005) 63–77.

Please cite this article in press as: P. Thiébaud, et al., Overexpression of Leap2 impairs Xenopus embryonic development and modulates FGF and activin signals, Peptides (2016), http://dx.doi.org/10.1016/j.peptides.2016.06.008