BACKGROUND
the skin is the largest organ in the body and its role in innate immunity as a barrier between the external and internal environment makes it of major importance for the maintenance of homeostasis.
this organ is well supplied with blood vessels and nerve endings that receive tactile and thermal stimuli from the environment  <cit> .
the skin has evolved from a simple respiratory epithelium in the amphioxus  <cit>  to a complex multicellular and multipurpose tissue in vertebrates  <cit> .
the general structure of skin in all vertebrates has been conserved and it is composed of an upper epidermal layer that is an interface with the exterior, an intermediate dermal layer and the basal hypodermal layer.
fish skin differs in several aspects from mammalian skin and the functional divergence between skin in a terrestrial and aquatic environment is presumably underpinned by significant divergence in molecular and cellular processes.
while in human skin the primary physical barrier that confers protection is the stratified epidermis that is composed of dead keratinized cells, in fish the epidermis is composed of metabolically active cells with little keratinization .
goblet cells and club cells produce mucous rich in proteases, mucins, immunoglobulins and antimicrobial peptides  that protect the living epidermis of the fish integument.
the most pronounced difference between the skin in terrestrial and aquatic vertebrates is the presence in fish skin of scales that are mineralized structures of dermal origin, that protect the underlying dermis from abrasion and damage caused by predation  <cit> .
the importance of the skin as a protective barrier and in the maintenance of internal homeostasis means that damage has to be rapidly repaired.
the process of skin repair in vertebrates is complex and involves a cascade of local and systemic responses to restore tissue integrity.
in mammals the outcome of injury to skin is repair and scarring but in amphibians and fish regeneration occurs and the disrupted tissue is replaced by skin with the original tissue architecture  <cit> .
in fish, scale removal provokes a wound and the loss of epidermal cells, scales and the superficial dermis.
the removal of scales damages a key barrier of the innate immune system and consequently provokes an inflammatory response and activation of the processes associated with healing and skin and scale re-growth  <cit> .
fish skin heals rapidly and the wound surface is rapidly covered in mucus and re-epithelialization occurs from the wound margin  <cit> .
skin and scale regeneration in fish involves, re-epithelialization and differentiation of scale-forming cells , production of the external layer matrix , production of the basal-plate matrix  and finally partial mineralization of the basal plate   <cit> .
wound repair and skin regeneration studies are numerous in mammals  <cit>  and amphibians  <cit> , but are much less frequent in the fishes, the largest group of extant vertebrates  <cit>  and the molecular basis of skin repair is generally restricted to single gene studies .
recent studies have used microarrays to assess the response of fish skin to damage or ectoparasites  <cit>  and members of the angiopoietin family are among the differentially expressed genes detected.
in mammals, the angptl family is composed of  <dig> secreted glycoproteins  that regulate a plethora of physiological and pathophysiological processes and in the skin they are involved in tissue repair and cell proliferation  <cit> .
members of this family are characterised by the presence of an amino-terminal coiled-coil domain , a linker region and a carboxyl-terminal fibrinogen-related domain .
the exception is angptl <dig> that is an atypical shorter family member that has lost the fred domain and is only described in mammals  <cit> .
angptls are structurally similar to angiopoietins , an important family of vascular growth factors .
recently it was demonstrated that some of the actions of angptl are mediated by receptors that belong to the immunoglobulin-like superfamily  <cit> .
in humans, angptl <dig> induces keratinocyte migration during wound healing  <cit>  and epidermal differentiation post-healing  <cit> .
in mouse, overexpression of angptl <dig> in skin promotes epidermal hyperplasia and enlargement of dermal lymphatic and blood vessels to favour wound healing  <cit> .
angptl <dig> regulates extracellular matrix  formation  <cit>  and is highly expressed in keratinocytes and is a potent anti-angiogenic factor in the cornea  <cit> .
this protein is also described to inhibit tumour growth in a mouse xenograph model  <cit>  and is required for the regeneration of human hematopoietic stem and progenitor cells   <cit> .
the functional importance of angptl in mammalian skin makes them interesting candidate molecules for skin regeneration in fish.
homologues of several mammalian angptl members have been described in teleosts.
in particular, orthologues of human angptl <dig>  human angptl <dig> and human angptl <dig> have been described respectively, in fin repair  <cit> , in the dermatome  <cit>  and in metabolically modified skin  <cit>  of fish.
the preceding observation together with the reported role of angptl in mammalian skin repair led us to hypothesize that angptl plays a role in skin regeneration in fish.
the existence of multiple members of the angptl family in vertebrates and the deficit of knowledge about this gene family in fish made it necessary to first characterize the evolution of the angptl gene family and gene synteny in order to identify the candidate gene family targeted in this study.
to assist in designation of putative function we identified the motifs in the deduced piscine angptl proteins that have been conserved during evolution.
we then mapped the tissue distribution of gene family members using in silico molecular resources  and confirmed the association of angptl family members with the integument by qpcr in sea bream intact skin and regenerating skin after scale removal.
taking into consideration the role of angptl in tissue repair, cell proliferation and angiogenesis in mammals we correlated the expression patterns of angptl1b, angptl2b, angptl4a, angptl4b and angptl <dig> with the initial phases of piscine skin regeneration to test if the function of the angptl family was conserved during the evolution of the vertebrates.
methods
genome and est database searches
homologues of human angiopoietin-like  family members were procured in  <dig> fish genome assemblies .
using as queries the deduced mature protein sequences of human angptls, ten teleost genomes were explored, nine of which were available from ensembl  <cit> , accessed in may  <dig>  and included: two puffer fishes , stickleback , nile tilapia , medaka , platyfish , atlantic cod , cavefish  and zebrafish  and the sea bass  assessed from the sea bass genome assembly  <cit> .
searches were complemented by mining additional fish genomes at ensembl  <cit> , accessed in may  <dig>  a basal ray-finned fish, the spotted gar , the coelacanth  that is basal to the tetrapod lineage and a jawless fish, the marine lamprey .
the genome of two cartilaginous fishes the elephant shark  and little skate  were also analysed.
to assess angptl gene family evolution, searches were extended to genomes of terrestrial vertebrates and invertebrates .
this included  <dig> terrestrial vertebrates ;  <dig> early deuterostomes ;  <dig> protostomes  and  <dig> early metazoans  were accessed from the ensembl genomes database  <cit> , accessed in may  <dig>  searches for putative angptl-like transcripts for the target invertebrate species were also performed at the ncbi database  <cit>  using the deduced protein of human angptl against the species-specific nucleotide collections .
the identity of all retrieved sequences as angptl family members was confirmed by reverse blast searches against the human ncbi non-redundant protein sequence  <cit>  database.
to aid in the identification of angptl candidates with a functional role in fish skin, the deduced sea bass angptl protein sequences were used to identify angptl transcripts isolated from skin est libraries using a tblastn query against the teleost est collection  <cit>  .
the est sequence hits with e < − <dig> score were retained and their identity was confirmed by reverse blast against the human genome.
microarray probes modified in a sea bream skin/scale regeneration experiment  <cit>  and a transcriptome assembly of sea bass skin  were also analysed for skin angptl candidates.
for the skin expression studies, the angptl family members from the gilthead sea bream  were identified from the species-specific ncbi est database subset  <cit>   and a sea bream transcriptome assembly prepared from multiple tissues  <cit> .
phylogenetic analysis
phylogenetic analysis of fish and other metazoan angptl family members was performed using the deduced mature protein sequences.
two hundred and twenty-six sequences including the angptl <dig> to  <dig> and also angpt  <dig>   <dig> and  <dig> sequences from  <dig> vertebrates including the  <dig> fish species and the cephalochordate representatives were used to construct the phylogenetic trees.
the deduced mature protein sequences were aligned using clustalw   <cit> .
gaps that resulted from the sequence alignment were removed using the aliview v  <dig> .
<dig>  <cit>  and the edited angpt/ angptl protein alignment was submitted to the prottest  <dig>  server  <cit>  to identify the best model to study protein evolution using the akaike information criterion  statistical model  <cit> .
phylogenetic analysis was performed using two approaches: bayesian interference  and maximum likelihood .
the bi tree was built in mrbayes  <dig>   <cit>  using a jtt substitution model   <cit>  and  <dig> .
<dig> generations sampling and probability values to support tree branching.
the ml method was performed with  <dig> bootstrap replicates to test the robustness of the phylogenetic clades in the atgc interface   <cit> .
the ml tree was built with a jtt substitution model with a fixed proportion of invariable sites value  and  <dig> gamma-distributed rate categories .
both bi and ml phylogenetic trees were rooted using the metazoan angpt clade and they had similar branch topologies.
multiple sequence comparisons and analysis
the deduced mature proteins of the fish angptl family were compared with human homologues to identify conserved motifs that have been maintained across vertebrates or that are characteristic of each family cluster identified by phylogenetic analysis.
alignments were performed in clustalw   <cit>  and manually edited using genedoc  <cit>  software that was also used to calculate the percent of sequence identity/similarity between fish, terrestrial vertebrates and cephalochordate homologues.
conserved domains in the fish sequences were identified using smart  <cit>  and uniprot  <cit>  softwares.
the mature protein sequences of the sea bream angptl transcripts were deduced using the expasy translate tool  <cit> .
short-range gene linkage
to further confirm gene identity and to establish an evolutionary model for the metazoan angptl genes, the gene environment of the chromosomes or genome fragments of vertebrate angptl <dig> was isolated to establish if a homologue genome region existed in the cephalochordate.
similarly, the gene environment of mammalian angptl <dig> was characterized to comprehend the absence of this gene in other non-mammalian vertebrates.
the gene environment of the novel non-mammalian angptl <dig> identified in this study was explored to understand its origin and evolution in vertebrates.
short-range gene linkage comparisons included human, chicken  or lizard , coelacanth, spotted gar and elephant shark and also two teleosts the tilapia and the zebrafish.
the vertebrate neighbouring gene environment was retrieved from the genomicus database  <cit>  using the gene environment of human angptl <dig> and angptl <dig> and the spotted gar angptl <dig> as the reference.
the homologue genome regions in elephant shark and lamprey were characterized by querying their specific genome assemblies with the conserved flanking genes identified in teleosts and tetrapods.
the identity of the cephalochordate genes was confirmed by their similarity with the human proteins.
sea bream skin regeneration challenge
manipulation of animals was performed in compliance with international and national ethics guidelines for animal care and experimentation, under a “group-i” license from the portuguese government central veterinary service to ccmar and conducted by a certified investigator .
a stock of adult sea bream of the same age class  were purchased from a commercial supplier  and transferred to ramalhete, the marine station of the centre of marine sciences .
fish were maintained in 1000 l tanks supplied with a continuous flow of aerated sea water at 18–20 °c, ph  <dig> – <dig> , 37 ppt salinity, >80% oxygen saturation and at a density of <5 kg · m− <dig>  fish were fed with a commercial feed  at 2% of the total kg of fish / tank twice daily.
for the skin regeneration challenge, adult sea bream  were divided randomly between five 500 l tanks  supplied with a continuous flow of aerated seawater at 20 ± 2 °c and maintained under the conditions described above.
for the skin regeneration challenge, fish were anesthetised with 2-phenoxyethanol in seawater  and scales were removed from the left flank of the body by gently stroking the skin with forceps to minimise damage to the dermis.
a group of fish  were killed immediately  after scale removal and samples of intact skin  and damaged skin  were snap frozen in liquid nitrogen and subsequently stored at −80 °c for molecular analysis or were fixed in 4% paraformaldehyde  for histology.
in this way, the same fish provided control and regenerating skin samples and they could be directly compared.
to minimize undue stress to the fish the  <dig> tanks represented the different time points of the sample time series after scale removal: 6 h and day  <dig>   <dig>   <dig> and  <dig>  intact and regenerating skin samples  were collected from fish anaesthetised in 2-phenoxyethanol  and were weighed, length measured and a photograph taken.
fish were killed by decapitation and the skin below the dorsal fin on the left  and right hand flank  of the same fish was collected and a portion frozen in liquid nitrogen and the other portion fixed for histological examination.
skin histological and morphometric analysis
intact and regenerating skin samples from sea bream  were fixed in 4% pfa, decalcified overnight in  <dig>  m ethylenediaminetetraacetic acid  and dehydrated in ethanol , saturated in xylene and impregnated and embedded in low melting point paraffin wax .
serial 5 μm sections of skin were mounted on 3-aminopropyltriethoxysilane  coated glass slides, dried overnight at 37 °c, cooled to room temperature and stored until required.
masson’s trichrome staining was used to distinguish between collagen rich and/or mineralized and non-mineralized tissue as previously described  <cit> .
stained sections were analysed using a microscope  coupled to a digital camera  linked to a computer for digital image analysis.
digital images were used to quantify the thickness of the epidermis, basement membrane and dermis as well as the number and diameter  of blood vessels in intact  and regenerating  skin using imagej v <dig> o software  <cit> .
rna extraction and cdna synthesis
total rna was extracted from sea bream intact and regenerating skin using a maxwell®  <dig> mdx instrument  and a maxwell  <dig> total rna purification kit , according to the manufacturer’s instructions.
the quality and integrity of total rna was verified using a nanodrop  <dig> spectrophotometer .
purified total rna  was treated with  <dig>  u dnase  following the manufacturer’s instructions.
dna free total rna  was used for first strand cdna synthesis in a 20 μl reaction volume containing 100 mm random hexamers ,  <dig> u of revertaid™ reverse transcriptase  and  <dig> u of ribolock™ rnase inhibitor .
cdna was synthesized by incubating for 10 min at 20 °c, followed by 50 min at 42 °c and 5 min at 72 °c.
the quality of cdna was checked by pcr amplification of rps <dig> with specific primers  using the following cycle: 10 min at 95 °c, followed by 25 cycles of 95 °c for 30 s, 60 °c for 30 s and 72 °c for 30 s and a final 5 min at 72 °c.
the pcr products were run on a 1% agarose gel to confirm amplicon size and the absence of contamination with genomic dna.table  <dig> list of the primers used for gene expression analysis by quantitative real-time pcr in sea bream  skin
angptl1b
angptl2b
angptl4a
angptl4b
angptl7
vegfab
rps18
ß-actin
accession numbers, primer sequence, amplicon length , annealing temperature  and qpcr efficiency  and r <dig> are indicated for each primer pair
abbreviations: f forward, r reverse primer
quantitative expression analysis
the expression of angptl1b, angptl2b, angptl3b, angptl4a, angptl4b, angptl <dig> and angptl9b was confirmed in sea bream skin and the abundance of the amplified transcripts were subsequently characterised in intact and regenerating skin by quantitative real-time pcr.
transcript specific primers were designed using the sea bream sequences as templates  and qpcr was carried out in duplicate 10 μl reactions of 1× ssofast-evagreen supermix  containing cdna  and  <dig> nm of forward and reverse primers.
quantification was performed in a steponeplus thermocycler  using the standard-curve method  and the following program: 30 s at 95 °c, 45 cycles of 5 s at 95 °c and 15 s at 60 °c.
a standard curve was included to permit the initial quantity of target template to be related to amplification cycle.
a final melting curve was carried out between  <dig> and 95 °c and produced a single product dissociation curve for each gene.
relative expression ) was estimated using the geometric mean of two-reference transcripts rps <dig> and ß-actin that did not vary significantly  between the control and regenerating skin samples.
statistical analysis
significant changes in relative transcript expression in intact and regenerating skin during the wound healing process were assessed using a two-way anova followed by a fisher’s least significant difference  post-test using the statplus:mac le v <dig>  <dig> .
relative expression data are presented as mean ± standard error of the mean .
statistical significance was considered at p <  <dig> .
significant differences in intact or regenerating skin at different time points during the experiment are annotated with different letters and significant differences between intact and regenerating skin at the same time point are annotated with an asterisk.
correlation analysis was performed with graphpad prism version  <dig>  for macintosh,  to associate patterns of gene expression with key morphological events  during the initial phases of piscine skin regeneration.
RESULTS
angptls in fish and other metazoans
sequence homologues of human angptl family members were identified in several fish.
a previously undescribed member of this family was identified in fish genomes and also in xenopus, lizard and chicken genomes and was designated angptl <dig>  the new angptl family member was absent from mammalian genomes.
in contrast, orthologues of human angptl <dig> were absent from fish and other non-mammalian vertebrate genomes .
ten teleost fish genomes were analysed and the total angptl gene number retrieved per genome varied from  <dig> to  <dig> depending on retention or not of duplicate gene copies of angptl <dig>  angptl <dig>  angptl <dig> and angptl <dig> and the new angptl <dig> gene identified in this study.
duplicates of human angptl <dig> gene homologues were identified in all teleost genomes analysed but the persistence of paralogues for the other family members was species-dependent and it was not possible in some species to establish if the full complement of genes was present due to the incompleteness of their genome assemblies.fig.
<dig>
angptl gene family members in fish.
the number of predicted genes is indicated.
the chicken and human genes are also indicated to allow comparisons with the fish homologues.
“●“ represents the teleost specific genome duplication .
ni: not identified
in the genome of the spotted gar , which diverged prior to the teleost radiation, a single angptl gene copy was found.
the lobe-finned coelacanth, a fish basal to tetrapods, had a similar gene repertoire to the spotted gar with the exception of the duplicate angptl <dig> genes.
in the cartilaginous fish the elephant shark  and the little skate ,  <dig> and  <dig> angptl genes, respectively were retrieved and orthologues of the teleost angptl <dig>  angptl <dig> and angptl <dig> remain to be identified .
searches in the jawless fish, the marine lamprey , recovered putative angptl <dig> and angptl <dig> genes but the incomplete nature of its genome assembly meant that the existence of other family members was not established .
in the gilthead sea bream  that does not have a sequenced genome,  <dig> angptl transcripts were retrieved but the orthologues of the teleost angptl3a and angptl <dig> were not identified .
terrestrial vertebrates including, the amphibian , the anole lizard , the chicken , the opossum  and the mouse  had a similar gene repertoire to human but in non-mammalian genomes an orthologue of the fish angptl <dig> gene also existed.
orthologues of human angptl <dig> were only identified in mammals and were absent from other vertebrates.
in the cephalochordate  at least  <dig> putative angptl-like genes were identified  indicating that this gene family is ancient and arose prior to the vertebrate radiation.
data mining of other early deuterostome genomes failed to retrieve annotated genes although predicted transcripts orthologous to human angptl <dig> were identified in a urochordate, the sea squirt , in an echinoderm, the sea urchin , and in a hemichordate, the acorn worm .
an orthologue of human angptl <dig>  was also identified in the sea urchin, indicating that different members of the angptl family are present in non-vertebrate genomes.
however, their deduced transcripts are highly divergent in sequence  and length  with the putative human homologues and were not considered for further analysis.
in protostomes sequence hits for proteins related to the vertebrate angptls such as tenascins, ficolins, fibrinogen and others were also obtained but were not explored in this study.
phylogeny of the fish angptls
phylogenetic analysis of the vertebrate and cephalochordate angptl family revealed that the genes shared a common origin and that the family members emerged early during the deuterostome radiation .
according to the tree topology, four main protein clusters that contain distinct members of the angptl family exist: the angptl1-2- <dig> cluster , the angptl3- <dig> cluster , the angptl <dig> cluster  and the angptl7- <dig> cluster .
according to the tree topology, the angptl3- <dig> cluster diverged early after the gene duplication event that gave rise to the ancestral gene from which the angptl1-2- <dig>  angptl <dig> and angptl7- <dig> subsequently emerged.
this suggests that the ancestral angptl gene duplicated prior to the radiation of the vertebrates and that the family members arose from different ancestral genes.fig.
<dig> phylogenetic tree of the fish angptl with the tetrapod and cephalochordate homologues.
tree was constructed with the bayesian interference  built in mrbayes  <dig>  and branch support values  are shown only for the major protein family clades.
the angptl family clusters are boxed with different colours.
the phylogenetic tree is compacted and was generated from the original tree available in additional file 2: figure s <dig>  the tree was rooted using the deuterostome angpt clade  but details are not show in the figure to facilitate interpretation.
for the same reason, the detailed presentation of the teleost and tetrapod members of each family group have been collapsed.
a tree with similar topology was obtained using the maximum-likelihood algorithm and  <dig> bootstrap replicates .
angptl <dig> was not included in the phylogenetic analysis as it is an atypical family member and lacks the fred domain
in teleosts, duplicate copies of angptl <dig>  angptl <dig>  angptl <dig>  angptl <dig> and angptl <dig> arose from the whole genome duplication event reported in this lineage  <cit> .
the teleost angptl gene duplicates are differentiated using the letters a and b and the gene environment of paralogue a shares the greatest conservation with the homologue chromosome regions in human and spotted gar  <cit> .
in other non-teleost fish, single gene family members exist with the exception of angptl <dig> that is duplicated in the coelacanth genome.
the five cephalochordate angptl-like genes clustered with the vertebrate angptls .
four of which group within the vertebrate angptl <dig> clade and three of which  seem to have arisen due to a species-specific gene duplication.
the fifth angptl  sits in the phylogenetic tree prior to the emergence of the vertebrate angptl1-2- <dig> and angptl7- <dig> clades .
the existence of other angptl genes in amphioxus was not established but they are likely to exist and were not identified due to the incompleteness of the genome assembly.
sequence conservation of the fish angptls with human and cephalochordate
amino acid  sequence alignment of the fish angptls with the human orthologues revealed that they are highly conserved and protein domains and sequence motifs are shared by teleost and tetrapod sequences .
this includes an n-terminal coiled-coil domain  and a highly conserved fibrinogen-related domain  in the c-terminal region that is also present in angpt proteins  <cit> .
within the fred motif, four highly conserved cysteine residues predicted to establish two intramolecular disulphide bonds were conserved in human and fish, however their importance in protein structure and function still remain to be established  <cit>  .
human and fish angptl <dig>   <dig> and  <dig> were the most highly conserved members and their deduced mature protein sequence share at least  <dig>   <dig> and 77% aa sequence similarity, respectively .
the deduced sequence of angptl <dig> was also highly conserved and shared 75–78% aa similarity between fish and non-mammalian tetrapods.
the most divergent forms of angptl were angptl <dig> and angptl <dig> that shared a maximum of  <dig> and 55% aa sequence similarity between fish and human, respectively .fig.
<dig> schematic representations of the deduced structure and conserved consensus motifs of the fish and human angptls.
the signal peptide region , the coiled-coil domain  and the highly conserved fibrinogen-related domain  are annotated and to facilitate visualization protein structures were aligned using the fred motif.
the four conserved cysteine residues within the fred motif potentially involved in the establishment of two intramolecular disulphide bonds are represented and indicated by “s-s” in the angptl <dig> protein structure and their positions were obtained from uniprot annotation.
other vertebrate conserved cysteine residues are represented by “c” and predicted n-glycosylation  motifs are annotated with “n”.
across fish, the amino acid residues that regulate the activity of the human angptl <dig>   <cit>  are also conserved.
the figure is not drawn to scale and the percent of amino acid sequence similarity between the fish and the human orthologues is indicated with the exception of angptl <dig> that considers the similarity across fish.
the angptl  <dig> is not represented, as it is only present in mammals.
the complete alignments of the human and fish angptl proteins are available in additional file 4: figure s3a–d.
* the sequence similarity of the coelacanth duplicates was not considered, as their sequence was highly divergent
although overall all the vertebrate angptl family members shared relatively high sequence conservation some specific amino acid sequences that have been linked to protein processing or protein structural configuration/function were also common across fish and human .
this included the three cysteine residues and two glycosylation sites within the ccd of angptl <dig>  angptl <dig> and angptl6; an n-glycosylation site  and a n-glycosylation site  in angptl3; an n-glycosylation site in the ccd of vertebrate angptl <dig> and the amino acids his <dig>  gln <dig> and gln <dig> that are important for the regulation of lipoprotein lipase  in humans and the elevation of plasma triglyceride levels in mice  <cit> ; the vertebrate angptl <dig> contained two conserved cysteine residues that may form an extra disulphide bridge within the fred domain; and three n-glycosylation sites, one within the ccd and two in the fred domains were conserved across the vertebrate angptl <dig>  a unique n-glycosylation site within the ccd was predicted in the deduced protein of angptl <dig> from the teleosts and the spotted gar.
the deduced cephalochordate angptl-like  proteins shared the highest sequence similarity  with vertebrate angptl <dig> and within the deduced lamprey proteins the fred domain was the most highly conserved region .
neighbouring gene analysis
to better understand the evolution of the angptl gene family during the vertebrate radiation, the neighbouring gene environment of mammalian angptl  <dig>  and non-mammalian angptl  <dig>  were compared between fish and tetrapods.
the gene environment of the cephalochordate angptl-like genes that cluster with the vertebrate angptl <dig> clade was also characterised .
in human, the angptl <dig> gene mapped to chromosome  <dig> and orthologues of the flanking genes were found in other vertebrate genomes.
a chromosome region with a similar gene repertoire to that flanking human angptl <dig> was found in the lizard and in the fish  even though the angptl <dig> gene was absent from their genomes.
of the nine genes that flank human angptl <dig>  eight retain linkage in chromosome  <dig> of the lizard and in chromosome lg <dig> of the spotted gar genome suggesting the loss of this gene in these species is potentially a consequence of lineage specific gene deletions .fig.
<dig> comparison of the homologous genome regions harbouring the human angptl <dig> with the fish and lizard.
the gene environment of the human angptl <dig> gene was characterised and was used to identify homologous genes in the anole lizard and several fish: the coelacanth , the spotted gar , the elephant shark  and the marine lamprey  genomes.
horizontal lines represent the chromosome fragments; arrow boxes indicate genes and the arrowhead points in the direction of the predicted gene transcription.
only genes that were conserved across species are represented.
gene names are indicated according to the human annotation and the same colour is used for gene homologues and they are presented according to their order in the chromosome.
the size of the genome fragments analysed and the predicted location of the genes in the chromosomes are indicated in megabase pairs.
gene names and symbols are: coactivator-associated arginine methyltransferase  <dig> , low density lipoprotein receptor , kn motif and ankyrin repeat domains  <dig> , dedicator of cytokinesis  <dig> , member ras oncogene family , transmembrane protein  <dig> , coiled-coil domain containing  <dig> , lipid phosphate phosphatase-related protein type  <dig>  and swim-type zinc finger  <dig> associated protein  <dig>
fig.
<dig> comparison of the spotted gar genome region harbouring angptl <dig> with other fish, chicken and human.
the gene environment of the spotted gar angptl <dig> gene was characterised and was used to identify homologous genome regions in the teleost , the coelacanth , the elephant shark , marine lamprey , chicken and human.
horizontal lines represent the chromosome fragments; arrow boxes indicate genes and the arrowhead points to the orientation of the predicted gene transcription.
only genes that were conserved across species are represented.
gene symbols are indicated and homologue genes are represented by the same colour and they are represented according to their order in the chromosome.
the size of the genome fragments analysed and the location of the gene in the chromosome are indicated in megabase pairs.
“●”: teleost specific genome duplication .
gene names and symbols are: kelch-like family member  <dig> , adenylate cyclase  <dig> , dnaj  homolog, subfamily c member  <dig> , efr <dig> , proopiomelanocortin , dna -methyltransferase  <dig> , monooxygenase, dbh-like  <dig>  and sorting nexin  <dig>
fig.
<dig> comparison of the vertebrate and cephalochordate angptl <dig> homologous genome regions.
the vertebrate angptl <dig> conserved gene environment was characterised using the human genome region as reference and was used to identify a homologous region in the amphioxus genome region that house the cephalochordate angptl7-like genes.
horizontal lines represent chromosome fragments and its names and genome fragments are indicated at the right side; coloured block arrows represent genes according to the order in the chromosome and the arrowhead points to the predicted gene transcription.
the location of the gene in the chromosome is indicated below each arrow, in megabase pairs.
“●”: teleost specific genome duplication .
gene names and gene symbols are: tar dna binding protein , mannan-binding lectin serine protease  <dig> , spermidine synthase , exosome component  <dig> , mechanistic target of rapamicin   and ubia prenyltransferase domain containing  <dig>
in the spotted gar, the angptl <dig> gene maps to lg <dig> and in the chicken to chromosome  <dig> and eight genes in linkage were identified.
in the human genome a chromosome region was identified that was homologous to the gene environment flanking the fish angptl <dig> gene even though the gene has been lost from mammalian genomes .
the genes that flank the angptl <dig> gene in fish and in chicken are shared between two human chromosomes  indicating reorganisation of this genome region during the radiation of mammals.
characterisation of the neighbouring gene environment of the duplicate teleost angptl <dig> genes revealed that they map to genome regions that share a similar gene complement confirming that they emerged from the teleost genome tetraploidization.
the gene environment of angptl <dig> in fish  and tetrapods revealed similarity with cephalochordate scaffold_ <dig> that houses amphioxus angptl-like_ii, iii and iv genes and suggests that vertebrate and cephalochordate angptl <dig> shared a common ancestral origin .
the neighbourhood of amphioxus angptl-like_i that maps to scaffold_ <dig> shared no gene linkage with any of the vertebrate chromosomes/scaffolds containing angptl genes and its location may be the result of gene duplication and subsequent translocation.
morphological and morphometric evaluation of sea bream intact and regenerating skin
longitudinal transverse sections of intact and regenerating sea bream skin samples were used to characterize the ontogeny of tissue regeneration after scale removal .
the three typical layers, the epidermis, dermis and hypodermis were observed in histological sections of intact sea bream skin.
the scales sat in individual scale pockets, inserted in the dermis and several layers of mineralized collagen were visible .
the removal of scales damaged the epidermis, dermis and scale pocket and 1 day after scale removal  the torn edges of the ruptured epidermis although still attached to the skin left the dermis and scale pocket exposed directly to the aquatic milieu.
blood vessels were observed in the loose dermis but not in the compact dermis.
fast re-epithelialization of the epidermis occurred and 2 days after scale removal a new epidermis covered the dermis .
a continuous basal layer and basal membrane were observed and formed an interface between the epidermis and the loose dermis.
the scale papilla was also evident in the loose dermis 2 days after scale removal and delineated the location of the future scale pocket and new scale.
establishment of the external barrier was completed 2 days after scale removal  and numerous blood vessels were observed in the loose dermis.
a thin layer of non-mineralized tissue was visible inside the scale pocket 3 days after scale removal and corresponded to the forming scale.
by day  <dig> after scale removal  the structure of the regenerating skin already resembled that of the intact skin, although the mineralized scale was very thin and still did not correspond in thickness or size to the ontogenetic scale.fig.
<dig> morphological evaluation of sea bream intact and regenerating skin  stained with masson’s trichrome.
the posterior region of the scale is orientated to the right.
connective tissue is stained green and mineralized and collagen-rich tissues are stained bright red.
a intact skin before scale removal; b–e regenerating skin at  <dig>   <dig>   <dig> and 4 days after wounding, respectively.
ep: epidermis; dm: demis; dm-l: loose dermis; dm-c: compact dermis; hyd: hypodermis; sc: scale: scp: scale pocket; msc: muscle; bsm: basement membrane; bsl: basal layer; dmp: dermal papilla; mlc: melanocytes; blv: blood vessel
morphometric evaluation of the sea bream skin  revealed the most dramatic changes in the regenerating skin where a marked increase in the thickness of the basement membrane  from 6 h onwards and this was positively correlated  with the progressive increase in the thickness of the epidermis  from 1 day onwards compared to time  <dig>  no changes in the thickness of the dermis were observed during the experiment .
in the intact skin the thickness of the epidermis , basement membrane  and dermis  remained constant throughout the healing period.fig.
<dig> morphometric evaluation of sea bream skin during wound healing after scale removal.
the thickness of the epidermis, basement membrane and dermis during sea bream skin recovery after scale removal is represented.
each value represents the mean ± sem .
statistical significance between groups was assessed using a two-way anova followed by fisher’s least significance difference  post-test.
statistical significances were considered at p <  <dig>  and differences in intact and regenerating skin during the experimental trial are annotated with different letters and comparisons between intact and regenerating skin at the same time point are signalled with an asterisk.
a histological image of the skin layers at the start  and end  of the experimental trial is represented beside each graph in order to illustrate the induced tissue aggression and the respective recovery with time.
ep: epidermis; bsl: basal layer; bsm: basement membrane; dm-l: loose dermis; dm-c: compact dermis; sc: scale; scp: scale pocket; mlc: melanocytes; blc: blood vessel; ext: exterior.
scale bars: 20 μm  and 50 μm
in order to assess the recovery of the vascular system in regenerating skin during the healing process the number and diameter of the blood vessels in the different periods analysed were determined .
an increase in the number of blood vessels was observed at 6 h after wounding in the regenerating skin  compared to time  <dig> .
this was subsequently followed by a decrease in the number of blood vessels until day  <dig> after wounding when similar numbers to that observed at time  <dig> were found, thereafter their number remained relatively constant .
variation in the number of blood vessels was positively correlated with the increase in the thickness of the epidermis  and basement membrane  .
analysis of the blood vessel diameter  revealed that an increase in diameter was progressively observed during the first 24 h post wounding in regenerating skin.
blood vessel diameter was relatively constant in intact skin .
this revealed that the increase in blood vessel number 6 h after wounding in the damaged skin was not the consequence of improved detection due to vasodilation and suggests that new blood vessels were formed.fig.
<dig>
a number of blood vessels and b blood vessel diameter during skin wound healing in sea bream.
each value represents the mean ± sem .
statistical significance between groups was assessed using two-way anova followed by fisher’s least significance difference  post-test.
statistical significance was considered at p <  <dig>  and differences in intact and regenerating skin during the experimental trial are annotated with different letters and comparisons between intact and regenerating skin at the same time point are signalled with an asterisk
expression of angptl family members during sea bream skin regeneration
analysis of angptl transcript distribution in fish by ests in ncbi revealed that they have a widespread tissue distribution .
the results of in silico analysis  indicated that angptl2b, angptl3b, angptl4a, angptl4b, angptl <dig> and angptl9b transcripts are expressed in fish skin .
verification by qpcr using cdna from sea bream skin confirmed the presence of angptl1b, angptl2b, angptl4a, angptl4b and angptl <dig> transcripts but not angptl3b and angptl9b and they were excluded from further analysis.table  <dig> digital expression analysis of angptls transcripts in the teleost skin
symbol
angptl1a
angptl1b
angptl2a
angptl2b
angptl3a
angptl3b
angptl4a
angptl4b
angptl5
angptl6
angptl7
angptl9a
angptl9b
searches were performed against the teleost ncbi database, sea bass skin transcriptome  and sea bream skin scale microarray probes  <cit>  ni: not identified
the abundance of angptl1b, angptl2b, angptl4a, angptl4b and angptl <dig> transcripts was evaluated during sea bream skin regeneration along with vegfab, a mediator of vascular development in zebrafish  <cit>  .
the transcript abundance of angptl1b and angptl2b was significantly increased  at initial stages of skin regeneration compared to the undamaged skin  and subsequently decreased significantly .
the abundance of angptl1b and angptl2b transcripts during the sea bream skin regeneration was correlated .fig.
<dig> relative expression of the sea bream angptl1b, angptl2b, angptl4a, angptl4b, angptl <dig> and vegfab in intact and regenerating skin.
expression levels were obtained by qpcr and each value, that represents the mean ± sem  of the relative expression ), was estimated using the geometric mean of rps <dig> and ß-actin in intact and regenerating skin at time 0 h, 6 h and days  <dig>   <dig>   <dig> and  <dig> after wounding.
statistically significant differences between groups was assessed using two-way anova followed by the fisher’s least significant difference  post-test.
statistical significances were considered at p <  <dig>  and differences in intact and regenerating skin during the experimental trial are annotated with different letters and comparisons between intact and regenerating skin at the same time point are signalled with an asterisk
in contrast, angptl <dig> was significantly down-regulated from 6 h to 4 days  in regenerating skin relative to time  <dig>  the transcription of angptl4a and angptl4b was similar and significantly  down-regulated from 1 day to 4 days in both intact and regenerating skin relative to time  <dig>  the change in transcript abundance over time of angptl4a and angptl4b were highly correlated  and no significant differences in transcript abundance existed between intact and regenerating skin.
the expression of angptl1b  in regenerating skin relative to skin at time  <dig> was significantly increased at 6 h  up until day  <dig> and subsequently decreased at day  <dig> and  <dig> when it was significantly lower  than at the start of the experiment.
in intact skin taken from the undamaged flank of sea bream, angptl1b decreased significantly from day  <dig> ,  <dig>  to  <dig>  relative to the start of the experiment .
pairwise comparisons of angptl1b transcript abundance in intact and regenerating skin at each time point revealed significant up-regulation in regenerating skin at 6 h  and 1 day  after scale removal.
angptl2b  transcripts in regenerating skin were significantly up-regulated  at days  <dig>   <dig> and  <dig> and then strongly and significantly down-regulated at day  <dig>  compared to skin at time  <dig>  in the intact skin angptl <dig> transcripts were significantly decreased at 6 h  and day  <dig>  relative to time  <dig>  in contrast, pairwise comparisons of intact and regenerating skin at each time point revealed that angptl2b transcripts were significantly up-regulated in the regenerating skin relative to intact skin at 6 h  and 1 day  after scale removal .
angptl4a and angptl4b  transcripts had a similar pattern of expression and their abundance decreased progressively after scale removal and were significantly down-regulated  1 day after scale removal in both intact and regenerating skin.
pairwise comparisons of intact and regenerating skin in the same individual at each time point did not reveal any significant differences in angptl4a and angptl4b transcript abundance.
expression of angptl <dig> was variable in both intact and regenerating skin samples over the 4 days of the experiment .
in regenerating skin, angptl <dig> was significantly down-regulated  at 6 h and on days  <dig>   <dig> and  <dig> after scale removal relative to time  <dig>  in intact skin, angptl <dig> transcripts were significantly down-regulated  6 h after the start of the experiment, then significantly up-regulated at day  <dig>  and day  <dig>  relative to 6 h. by day  <dig>  angptl <dig> transcript abundance in intact skin was similar to time  <dig>  comparison of angptl <dig> transcripts in intact and regenerating skin of the same individual at each time point analysed revealed significant down-regulation  of angptl <dig> in regenerating skin  <dig> and 3 days after scale removal.
expression of vegfab in intact and regenerating skin was not significantly different  at any time point analysed.
in intact skin the expression of vegfab transcripts increased progressively and was significantly up-regulated  from day  <dig> onwards relative to skin at time  <dig> .
pairwise comparisons of vegfab transcripts in intact and regenerating skin of the same individual at each time point analysed did not reveal any significant differences.
no correlation between vegfab and angptl expression was found .
discussion
the angptl family is a large group of multifunctional proteins involved in skin regeneration and angiogenesis in mammals.
the present study characterised for the first time the fish angptl gene repertoire and identified the angptl family members that were expressed in the integument of teleost fish and their expression during skin regeneration in the teleost sea bream.
the results reveal that the involvement of angptl family members in skin repair has been conserved during vertebrate evolution.
in fish a new angptl family member was found  and the orthologue of the mammalian angptl <dig> gene was lost.
in teleost angptl genes duplicated as a consequence of the lineage specific genome doubling and some of the paralogues that persisted have a role in skin homeostasis.
orthologues of human angptl <dig> , angptl <dig> , angptl <dig>  and angptl <dig> that play a key role in mammalian angiogenesis, pro-inflammatory response, tissue re-epithelisation/cell proliferation and avascularity, respectively are expressed in sea bream skin.
variation in the abundance of angptl1b, angptl2b and angptl <dig> during sea bream skin regeneration indicates that their role in tissue repair has been conserved .
the role in sea bream skin of the teleost angptl <dig> paralogues that in mammals has a prominent role in skin repair, remains to be established.fig.
<dig> summary of angptl gene expression during skin regeneration in sea bream.
the x-axis indicates time after wounding  and the y-axis relative expression of angptl1b, angptl2b and angptl <dig>  colours represent the approximate timing of angiogenesis and the re-epithelialization processes that overlap during the initial 4 days of wound healing in sea bream skin illustrated according to our morphometric evaluations , in arbitrary units
angptl members in fish
homologues of the mammalian angptls exist in fish.
in the teleosts they have duplicated and phylogenetic analysis and gene synteny confirmed that this was the result of the lineage specific genome duplication  <cit> .
some of the gene duplicates have persisted and the ests retrieved for the angptl paralogues and their divergent in silico distribution in skin suggests that after genome duplication functional specialization occurred and that they acquired a range of different physiological functions.
angptl genes were found from lamprey  to coelacanth  to mammals.
the angptl family members from fishes shared highly conserved sequence and structural motifs with the human homologues and some of the residues of functional importance in the human protein have also been maintained  <cit> .
the deduced proteins in fish and human share two highly conserved and characteristic signature motifs of this family: the n-terminal coiled-coil domain , which likely contributes to protein oligomerization, and the c-terminal fibrinogen-like domain  that in the related angpt protein family is a receptor-binding domain  <cit> .
a conserved n-terminus signal peptide in fish angptls suggests that they are secreted and the identification of conserved potential n-glycosylation consensus sites in the predicted proteins suggests that like the mammalian orthologues they are glycosylated proteins and may function as pleotropic endocrine/autocrine factors  <cit> .
in common with the human angptls, the fish members possess four conserved cysteine residues within the c-terminus fred motif that potentially form two intramolecular disulphide bonds.
however, the importance of the four highly conserved cysteine residues in the protein structure remains to be discovered as alanine replacement studies with human angptl <dig> failed to reveal functional modifications  <cit> .
angpt proteins possess a further two conserved cysteine residues compared to angptl and the resulting conformational difference between the two sets of proteins is proposed to be the basis of receptor selectivity for the two protein families  <cit> .
a number of changes have occurred during the evolution of the angptl family and a new vertebrate member, angptl <dig> was identified in the present study in non-mammalian vertebrates such as fish, the xenopus, anole lizard and chicken.
gene synteny analysis suggests that the loss of angptl <dig> from mammals was probably the result of chromosome rearrangements that occurred early in their radiation.
similarly, orthologues of the mammalian angptl <dig> gene were absent from the sequenced genomes of fish and other non-mammalian vertebrates.
angptl <dig> is an atypical family member as it lacks the fred domain, the glycosylation sites and the amino acids forming the intramolecular disulphide bonds but it has overlapping functions with angptl <dig> and  <dig> and inhibits lipoprotein lipase activity  <cit> .
the absence of an angptl <dig> gene in non-mammalian vertebrate genomes but the existence of a conserved gene environment across the vertebrates suggests that gene loss may have been due to lineage-specific deletions.
in mammals, the fred is essential for the angiogenic activity of the angptl protein while the ccd domain seems to be more important for other physiological functions, including regulation of lipid metabolism by inhibition of lipoprotein lipase  <cit> .
the functional importance of angptl proteins and their duplicates in fish physiology is unknown but given their high sequence homology with mammalian orthologues we hypothesized that their function might be conserved.
in fact, in the zebrafish, the only teleost where members of this family have previously been described, the orthologues of human angptl <dig>   <dig> and  <dig> are ubiquitously expressed and have a similar tissue distribution to human.
the zebrafish angptl <dig> and  <dig> share a conserved role in vascular development with the human orthologues  <cit> .
recently, angptls were found to activate immune inhibitory receptors of the leukocyte immunoglobulin -like family, a group of innate immune receptors, that are expressed on immune cells and involved in the control of inflammatory responses and cytotoxicity  <cit> .
studies of human angptl <dig> and its receptor  indicate that neither the ccd nor the fred binds to lilrb <dig>  the authors suggest that ligand-receptor interactions occur via the receptors immunoglobulin domain and that angptl <dig> protein multimerization is essential for downstream signalling  <cit> .
receptors for the fish angptl are currently unknown, although potential immunoglobulin-like receptor transcripts have been described in fish and it will be important to establish if they have a similar role to those found in mammals  <cit> .
angptl emerged early and evolved via gene duplications and deletions in the vertebrate radiation
in vertebrates, angptl evolved via gene duplication and deletion events and they are proposed to have shared a common evolutionary origin with angpt, with which they share sequence and structure similarities.
four main angptl vertebrate protein clusters  emerged from duplications of the ancestral angptl prior to the vertebrate radiation and the family members expanded during the early vertebrate genome doublings and segmental genome duplications .
the teleosts are by far the most successful and diverse group of vertebrates with at least  <dig>  species identified  <cit> .
this success has been linked to the teleost specific genome duplication that is suggested to have provided the raw material for the evolutionary adaptations and innovation that are a characteristic of this group  <cit> .
the phylogenetic analysis indicates that the angptl genes also duplicated early in the teleost radiation and this was followed by gene loss so that in extant teleost genomes only a few paralogues persisted  <cit> .
in the coelacanth, species-specific gene duplication affected angptl <dig> and the duplicates map in tandem to the same genome fragment  and clustering in the phylogenetic tree suggests that their sequence has diverged considerably from that in other vertebrates.
the identification of only two angptls  in the lamprey and the non-identification of angptl <dig>   <dig> and  <dig> in the cartilaginous fish genomes may be a consequence of their incomplete genome assemblies or linked to their unique physiological adaptations to an aquatic environment and associated gene deletions after their divergence from the common vertebrate ancestor.
members of the angptl family are suggested to have emerged prior to the vertebrate radiation and a sea squirt  gene homologue is equally similar to all the vertebrate members  <cit> .
we have also found other putative angptl-like genes in the genomes of several other early deuterostome species but their similarity with the vertebrate angptl family was very low.
the exception was in the cephalochordate  genome where  <dig> angptl-like genes were found.
the cephalochordate angptl-like deduced protein cluster revealed that four of the genes were orthologues of the vertebrate angptl <dig>  furthermore, the conserved gene environment flanking the cephalochordate angptl-like genes and the vertebrate angptl <dig> suggests that the ancestral angptl <dig> gene emerged prior to the vertebrate radiation.
a putative angptl1-2-6-like gene also seems to exist in the amphioxus and others genes of this family may potentially exist and their presence in the gene repertoire of the cephalochordate or in other early deuterostomes suggests this is an ancient gene family and their expansion in vertebrates may be a functional innovation linked to increased complexity of organisms and their physiology.
angptls in sea bream skin wound healing
in mammals’ skin wound healing is a complex and highly coordinated sequence of events.
this process is triggered by blood clotting and is followed by inflammation, vascularization, formation of granular tissue and tissue remodelling  <cit> .
in fish the sequence of events is different, re-epithelialization of damaged skin initiates immediately after wounding and the few studies of skin healing in fish reveals that this is independent of the inflammatory signals released by the blood clot  <cit> .
in all vertebrates during healing new blood vessels are formed from pre-existing ones in the dermis  <cit>  and in mammals’ new blood vessels are observed 3 days after wounding  <cit> .
in teleosts this process is initiated much earlier and in zebrafish skin new blood vessels are observed 1 day after damage to the epidermis and dermis  <cit> .
similarly, in our sea bream skin regeneration model new blood vessels were observed 6 h after scale removal and did not correlate with a peak of vegfab , an established vertebrate blood vessel maker, which reached maximal expression 1 day after wounding.
in teleosts duplicate vegfa genes have been described, vegfaa and vegfab, and both can bind the kdra and kdrb  in vitro, participating in vascular development  <cit> .
the involvement of the vegfaa paralogue in our sea bream skin healing model remains to be established and in the future characterisation of this and of other angiogenic factors  in tissue recovery will be of interest.
the expression in mammalian and teleost skins of angptls, their link with integument repair and specifically their co-ordinated appearance with key steps of the skin repair program in the sea bream suggests that they may share a conserved role in vertebrate skin regeneration.
in sea bream angptl1b, angptl2b, angptl4a, angptl4b and angptl <dig> are expressed in skin and their variable pattern of expression during wound healing and the differences in regenerating and intact skins 48 h post-wounding suggests they have acquired distinct roles in tissue re-epithelialization and angiogenesis.
in zebrafish and mice angptl <dig> and angptl <dig> are suggested to stimulate angiogenesis and to promote endothelial cell apoptosis for the initiation of vascular development  <cit> .
in mice, angptl <dig> was also found to regulate the pro-inflammatory response and it activates resident murine peritoneal monocytes and macrophages  <cit> .
we did not study markers of inflammation, however the increased expression of angptl1b and angptl2b was correlated with the number and diameter of blood vessel, respectively, during the early stages of skin regeneration in sea bream and suggests they also have a role in angiogenesis .
in human angptl <dig> is an anti-angiogenic factor and in vitro studies using cornea keratocyte cells demonstrated that this gene may be responsible for maintaining tissue avascularity in the eye  <cit> .
the decreased expression of angptl <dig> during sea bream skin regeneration and its negative correlation with blood vessel number suggests it may have a similar role to that in mammals .
in mammals, angptl <dig> regulates skin re-epithelialization, its expression is increased during this process in mice  <cit>  and in angptl4-knockout mice there was decreased expression of genes involved in epidermal differentiation and cell proliferation in skin  <cit> .
in murine ischemic tissues, angptl <dig> bound to the ecm inhibits endothelial cell motility, sprouting and formation of new blood vessels  <cit> .
the down-regulation of the duplicate angptl <dig> genes during sea bream skin regeneration and their negative correlation with the thickness of epidermis and basement membrane  suggests they are unlikely to be involved in re-epithelialization.
nonetheless, the time-frame of angptl1b and angptl2b up-regulation suggests they may be involved in this process .
the responsiveness of angptl family members in the intact skin of fish with a damaged flank was notable.
in human and mammalian experimental models a systemic effect of local skin injuries, such as burns or diabetic wounds is well described in relation to cardiovascular and metabolic parameters  <cit> .
the results obtained for expression of angptl1b, 2b and  <dig> in the intact skin of fish with a damaged flank suggests that it is the combination of both local and systemic effects that contribute to skin recovery; it will be important in the future to establish which cells are actively producing these proteins in skin and their role in teleost skin repair and homeostasis.
CONCLUSIONS
the present study characterizes the homologues of the human angptls in fish and identifies the candidate members that are involved in teleost skin regeneration.
in fish, amphibian and chicken a novel member of this family  was found but this gene is absent from mammals.
homologues of human angptl <dig> are not found in fish and they are only present in mammalian genomes.
in the teleosts the angptl family expanded and some gene paralogues acquired a role in the skin.
in the sea bream, angptl1b, angptl2b, angptl4a, angptl4b and angptl <dig> transcripts are present in skin but only angptl1b, angptl2b and angptl <dig> are modified in response to damage and are presumably involved in skin repair.
the change in abundance of angptl1b and angptl2b transcripts correlates with the timing of re-epithelialization and angiogenesis detected by histology.
in contrast, angptl <dig> is down-regulated and negatively correlated with angiogenesis suggesting that in common with mammals it is an anti-angiogenic factor .
overall, the results indicate that angptl family members are involved in sea bream skin homeostasis and repair.
additional files
additional file 1: table s <dig>  accession numbers of the fish, tetrapod and cephalochordate angptl genes and sea bream transcripts.
* very incomplete sequence and not included in the phylogenetic tree; ni-not identified.
additional file 2: figure s <dig>  expanded phylogenetic tree of the fish and other metazoan angptl family members generated using bayesian interference .
details are available from fig.
<dig>  accession numbers of the sequences used are given in the additional file 1: table s <dig> and additional file 9: table s <dig>
additional file 3: figure s <dig>  phylogenetic tree of the fish and other metazoan angptl family members constructed with the maximum-likelihood  algorithm.
analysis was performed in atgc  using the deduced amino acid sequence and a fixed value for the proportion of invariable sites  <dig> ,  <dig> gamma-distributed rate categories  and  <dig> bootstrap replicates according to prottest.
tree was rooted using the metazoan angpt clade .
accession numbers are given in the additional file 1: table s <dig> and additional file 9: table s <dig>
additional file 4: figure s <dig>  sequence alignments of the human, spotted gar, zebrafish, sea bass and sea bream angptls.
sequences were compared according to the clustering of the phylogenetic tree .
a angptl1-2-6; b angptl3-4; c angptl <dig> and d angptl7- <dig>  conserved amino acids in the sequence alignment are shaded; dark grey represents 80% conservation and black 100% conservation.
in the human sequences the signal peptide is underlined and in bold and the coiled-coil domain  are in bold and coloured blue.
the conserved fibrinogen-related domain  in human and in fish is boxed in red and the four conserved cysteine residues within this motif that are involved in two intramolecular disulphide bonds are highlighted in yellow and the predicted glycosylation  motifs are highlighted in red.
additional file 5: table s <dig>  percent of amino acid sequence identity/similarity of the fish angptl family members with the human orthologues.
additional file 6: figure s <dig>  sequence alignment of the deduced cephalochordate angptl-like  <dig> protein with the human and spotted gar angptl <dig>  conserved amino acids in the sequence alignment are shaded; dark grey represents 80% conservation and black 100% conservation.
the coiled-coil domain  is coloured in blue for the human sequence.
the conserved fibrinogen-related domain  is boxed and the four conserved cysteine residues within this motif that are potentially involved in the establishment of two intramolecular disulphide bonds of the vertebrate proteins are highlighted in yellow.
additional file 7: table s <dig>  correlation analysis of gene expression profile and changes in skin morphology during the initial phases of piscine skin regeneration .
a, skin parameters during sea bream skin regeneration ; b, gene expression and c, gene expression and skin parameters.
represented in the tables are the correlation value  and the p-value .
correlations are highlighted in red.
additional file 8: table s <dig>  list of the teleost angptl ests and their origin.
data was retrieved from the ncbi database using sea bass angptl members as the query.
ests retrieved were those present in skin or from teleost fins and bony structures covered with skin.
additional file 9: table s <dig>  accession numbers of the fish, tetrapod and cephalochordate angpt genes and transcripts.
ni-not identified.
abbreviations
aaamino acid
ampantimicrobial peptide
angptangiopoietin
angptlangiopoietin-like
ccdcoiled-coil domain
ecmextracellular matrix
fgfr1fibroblast growth factor receptor 1
fredfibrinogen-related domain
igimmunoglobulin
kdrtype iii receptor tyrosine kinase receptor
lpllipoprotein lipase
qpcrquantitative real-time polymerase chain reaction
tgfßtransforming growth factor beta
vegfaavascular endothelial growth factor aa
vegfabvascular endothelial growth factor ab

 the skin is the largest organ in the body and its role in innate immunity as a barrier between the external and internal environment makes it of major importance for the maintenance of homeostasis. this organ is well supplied with blood vessels and nerve endings that receive tactile and thermal stimuli from the environment. the skin has evolved from a simple respiratory epithelium in the amphioxus to a complex multicellular and multipurpose tissue in vertebrates. the general structure of skin in all vertebrates has been conserved and it is composed of an upper epidermal layer that is an interface with the exterior, an intermediate dermal layer and the basal hypodermal layer. fish skin differs in several aspects from mammalian skin and the functional divergence between skin in a terrestrial and aquatic environment is presumably underpinned by significant divergence in molecular and cellular processes. while in human skin the primary physical barrier that confers protection is the stratified epidermis that is composed of dead keratinized cells, in fish the epidermis is composed of metabolically active cells with little keratinization. goblet cells and club cells produce mucous rich in proteases, mucins, immunoglobulins and antimicrobial peptides that protect the living epidermis of the fish integument. the most pronounced difference between the skin in terrestrial and aquatic vertebrates is the presence in fish skin of scales that are mineralized structures of dermal origin, that protect the underlying dermis from abrasion and damage caused by predation. the importance of the skin as a protective barrier and in the maintenance of internal homeostasis means that damage has to be rapidly repaired. the process of skin repair in vertebrates is complex and involves a cascade of local and systemic responses to restore tissue integrity. in mammals the outcome of injury to skin is repair and scarring but in amphibians and fish regeneration occurs and the disrupted tissue is replaced by skin with the original tissue architecture. in fish, scale removal provokes a wound and the loss of epidermal cells, scales and the superficial dermis. the removal of scales damages a key barrier of the innate immune system and consequently provokes an inflammatory response and activation of the processes associated with healing and skin and scale re-growth. fish skin heals rapidly and the wound surface is rapidly covered in mucus and re-epithelialization occurs from the wound margin. skin and scale regeneration in fish involves, re-epithelialization and differentiation of scale-forming cells, production of the external layer matrix, production of the basal-plate matrix and finally partial mineralization of the basal plate. wound repair and skin regeneration studies are numerous in mammals and amphibians, but are much less frequent in the fishes, the largest group of extant vertebrates and the molecular basis of skin repair is generally restricted to single gene studies. recent studies have used microarrays to assess the response of fish skin to damage or ectoparasites and members of the angiopoietin family are among the differentially expressed genes detected. in mammals, the angptl family is composed of secreted glycoproteins that regulate a plethora of physiological and pathophysiological processes and in the skin they are involved in tissue repair and cell proliferation. members of this family are characterised by the presence of an amino-terminal coiled-coil domain, a linker region and a carboxyl-terminal fibrinogen-related domain. the exception is angptl that is an atypical shorter family member that has lost the fred domain and is only described in mammals. angptls are structurally similar to angiopoietins, an important family of vascular growth factors. recently it was demonstrated that some of the actions of angptl are mediated by receptors that belong to the immunoglobulin-like superfamily. in humans, angptl induces keratinocyte migration during wound healing and epidermal differentiation post-healing. in mouse, overexpression of angptl in skin promotes epidermal hyperplasia and enlargement of dermal lymphatic and blood vessels to favour wound healing. angptl regulates extracellular matrix formation and is highly expressed in keratinocytes and is a potent anti-angiogenic factor in the cornea. this protein is also described to inhibit tumour growth in a mouse xenograph model and is required for the regeneration of human hematopoietic stem and progenitor cells. the functional importance of angptl in mammalian skin makes them interesting candidate molecules for skin regeneration in fish. homologues of several mammalian angptl members have been described in teleosts. in particular, orthologues of human angptl human angptl and human angptl have been described respectively, in fin repair, in the dermatome and in metabolically modified skin of fish. the preceding observation together with the reported role of angptl in mammalian skin repair led us to hypothesize that angptl plays a role in skin regeneration in fish. the existence of multiple members of the angptl family in vertebrates and the deficit of knowledge about this gene family in fish made it necessary to first characterize the evolution of the angptl gene family and gene synteny in order to identify the candidate gene family targeted in this study. to assist in designation of putative function we identified the motifs in the deduced piscine angptl proteins that have been conserved during evolution. we then mapped the tissue distribution of gene family members using in silico molecular resources and confirmed the association of angptl family members with the integument by qpcr in sea bream intact skin and regenerating skin after scale removal. taking into consideration the role of angptl in tissue repair, cell proliferation and angiogenesis in mammals we correlated the expression patterns of angptl1b, angptl2b, angptl4a, angptl4b and angptl with the initial phases of piscine skin regeneration to test if the function of the angptl family was conserved during the evolution of the vertebrates. methods genome and est database searches homologues of human angiopoietin-like family members were procured in fish genome assemblies. using as queries the deduced mature protein sequences of human angptls, ten teleost genomes were explored, nine of which were available from ensembl, accessed in may and included: two puffer fishes, stickleback, nile tilapia, medaka, platyfish, atlantic cod, cavefish and zebrafish and the sea bass assessed from the sea bass genome assembly. searches were complemented by mining additional fish genomes at ensembl, accessed in may a basal ray-finned fish, the spotted gar, the coelacanth that is basal to the tetrapod lineage and a jawless fish, the marine lamprey. the genome of two cartilaginous fishes the elephant shark and little skate were also analysed. to assess angptl gene family evolution, searches were extended to genomes of terrestrial vertebrates and invertebrates. this included terrestrial vertebrates; early deuterostomes; protostomes and early metazoans were accessed from the ensembl genomes database, accessed in may searches for putative angptl-like transcripts for the target invertebrate species were also performed at the ncbi database using the deduced protein of human angptl against the species-specific nucleotide collections. the identity of all retrieved sequences as angptl family members was confirmed by reverse blast searches against the human ncbi non-redundant protein sequence database. to aid in the identification of angptl candidates with a functional role in fish skin, the deduced sea bass angptl protein sequences were used to identify angptl transcripts isolated from skin est libraries using a tblastn query against the teleost est collection. the est sequence hits with e < − score were retained and their identity was confirmed by reverse blast against the human genome. microarray probes modified in a sea bream skin/scale regeneration experiment and a transcriptome assembly of sea bass skin were also analysed for skin angptl candidates. for the skin expression studies, the angptl family members from the gilthead sea bream were identified from the species-specific ncbi est database subset and a sea bream transcriptome assembly prepared from multiple tissues. phylogenetic analysis phylogenetic analysis of fish and other metazoan angptl family members was performed using the deduced mature protein sequences. two hundred and twenty-six sequences including the angptl to and also angpt and sequences from vertebrates including the fish species and the cephalochordate representatives were used to construct the phylogenetic trees. the deduced mature protein sequences were aligned using clustalw. gaps that resulted from the sequence alignment were removed using the aliview v. and the edited angpt/ angptl protein alignment was submitted to the prottest server to identify the best model to study protein evolution using the akaike information criterion statistical model. phylogenetic analysis was performed using two approaches: bayesian interference and maximum likelihood. the bi tree was built in mrbayes using a jtt substitution model and. generations sampling and probability values to support tree branching. the ml method was performed with bootstrap replicates to test the robustness of the phylogenetic clades in the atgc interface. the ml tree was built with a jtt substitution model with a fixed proportion of invariable sites value and gamma-distributed rate categories. both bi and ml phylogenetic trees were rooted using the metazoan angpt clade and they had similar branch topologies. multiple sequence comparisons and analysis the deduced mature proteins of the fish angptl family were compared with human homologues to identify conserved motifs that have been maintained across vertebrates or that are characteristic of each family cluster identified by phylogenetic analysis. alignments were performed in clustalw and manually edited using genedoc software that was also used to calculate the percent of sequence identity/similarity between fish, terrestrial vertebrates and cephalochordate homologues. conserved domains in the fish sequences were identified using smart and uniprot softwares. the mature protein sequences of the sea bream angptl transcripts were deduced using the expasy translate tool. short-range gene linkage to further confirm gene identity and to establish an evolutionary model for the metazoan angptl genes, the gene environment of the chromosomes or genome fragments of vertebrate angptl was isolated to establish if a homologue genome region existed in the cephalochordate. similarly, the gene environment of mammalian angptl was characterized to comprehend the absence of this gene in other non-mammalian vertebrates. the gene environment of the novel non-mammalian angptl identified in this study was explored to understand its origin and evolution in vertebrates. short-range gene linkage comparisons included human, chicken or lizard, coelacanth, spotted gar and elephant shark and also two teleosts the tilapia and the zebrafish. the vertebrate neighbouring gene environment was retrieved from the genomicus database using the gene environment of human angptl and angptl and the spotted gar angptl as the reference. the homologue genome regions in elephant shark and lamprey were characterized by querying their specific genome assemblies with the conserved flanking genes identified in teleosts and tetrapods. the identity of the cephalochordate genes was confirmed by their similarity with the human proteins. sea bream skin regeneration challenge manipulation of animals was performed in compliance with international and national ethics guidelines for animal care and experimentation, under a “group-i” license from the portuguese government central veterinary service to ccmar and conducted by a certified investigator. a stock of adult sea bream of the same age class were purchased from a commercial supplier and transferred to ramalhete, the marine station of the centre of marine sciences. fish were maintained in 1000 l tanks supplied with a continuous flow of aerated sea water at 18–20 °c, ph  –, 37 ppt salinity, >80% oxygen saturation and at a density of <5 kg · m− fish were fed with a commercial feed at 2% of the total kg of fish / tank twice daily. for the skin regeneration challenge, adult sea bream were divided randomly between five 500 l tanks supplied with a continuous flow of aerated seawater at 20 ± 2 °c and maintained under the conditions described above. for the skin regeneration challenge, fish were anesthetised with 2-phenoxyethanol in seawater and scales were removed from the left flank of the body by gently stroking the skin with forceps to minimise damage to the dermis. a group of fish were killed immediately after scale removal and samples of intact skin and damaged skin were snap frozen in liquid nitrogen and subsequently stored at −80 °c for molecular analysis or were fixed in 4% paraformaldehyde for histology. in this way, the same fish provided control and regenerating skin samples and they could be directly compared. to minimize undue stress to the fish the tanks represented the different time points of the sample time series after scale removal: 6 h and day and intact and regenerating skin samples were collected from fish anaesthetised in 2-phenoxyethanol and were weighed, length measured and a photograph taken. fish were killed by decapitation and the skin below the dorsal fin on the left and right hand flank of the same fish was collected and a portion frozen in liquid nitrogen and the other portion fixed for histological examination. skin histological and morphometric analysis intact and regenerating skin samples from sea bream were fixed in 4% pfa, decalcified overnight in  m ethylenediaminetetraacetic acid and dehydrated in ethanol, saturated in xylene and impregnated and embedded in low melting point paraffin wax. serial 5 μm sections of skin were mounted on 3-aminopropyltriethoxysilane coated glass slides, dried overnight at 37 °c, cooled to room temperature and stored until required. masson’s trichrome staining was used to distinguish between collagen rich and/or mineralized and non-mineralized tissue as previously described. stained sections were analysed using a microscope coupled to a digital camera linked to a computer for digital image analysis. digital images were used to quantify the thickness of the epidermis, basement membrane and dermis as well as the number and diameter of blood vessels in intact and regenerating skin using imagej v o software. rna extraction and cdna synthesis total rna was extracted from sea bream intact and regenerating skin using a maxwell® mdx instrument and a maxwell total rna purification kit, according to the manufacturer’s instructions. the quality and integrity of total rna was verified using a nanodrop spectrophotometer. purified total rna was treated with u dnase following the manufacturer’s instructions. dna free total rna was used for first strand cdna synthesis in a 20 μl reaction volume containing 100 mm random hexamers, u of revertaid™ reverse transcriptase and u of ribolock™ rnase inhibitor. cdna was synthesized by incubating for 10 min at 20 °c, followed by 50 min at 42 °c and 5 min at 72 °c. the quality of cdna was checked by pcr amplification of rps with specific primers using the following cycle: 10 min at 95 °c, followed by 25 cycles of 95 °c for 30 s, 60 °c for 30 s and 72 °c for 30 s and a final 5 min at 72 °c. the pcr products were run on a 1% agarose gel to confirm amplicon size and the absence of contamination with genomic dna.table list of the primers used for gene expression analysis by quantitative real-time pcr in sea bream skin angptl1b angptl2b angptl4a angptl4b angptl7 vegfab rps18 ß-actin accession numbers, primer sequence, amplicon length, annealing temperature and qpcr efficiency and r are indicated for each primer pair abbreviations: f forward, r reverse primer quantitative expression analysis the expression of angptl1b, angptl2b, angptl3b, angptl4a, angptl4b, angptl and angptl9b was confirmed in sea bream skin and the abundance of the amplified transcripts were subsequently characterised in intact and regenerating skin by quantitative real-time pcr. transcript specific primers were designed using the sea bream sequences as templates and qpcr was carried out in duplicate 10 μl reactions of 1× ssofast-evagreen supermix containing cdna and nm of forward and reverse primers. quantification was performed in a steponeplus thermocycler using the standard-curve method and the following program: 30 s at 95 °c, 45 cycles of 5 s at 95 °c and 15 s at 60 °c. a standard curve was included to permit the initial quantity of target template to be related to amplification cycle. a final melting curve was carried out between and 95 °c and produced a single product dissociation curve for each gene. relative expression ) was estimated using the geometric mean of two-reference transcripts rps and ß-actin that did not vary significantly between the control and regenerating skin samples. statistical analysis significant changes in relative transcript expression in intact and regenerating skin during the wound healing process were assessed using a two-way anova followed by a fisher’s least significant difference post-test using the statplus:mac le v. relative expression data are presented as mean ± standard error of the mean. statistical significance was considered at p < . significant differences in intact or regenerating skin at different time points during the experiment are annotated with different letters and significant differences between intact and regenerating skin at the same time point are annotated with an asterisk. correlation analysis was performed with graphpad prism version for macintosh, to associate patterns of gene expression with key morphological events during the initial phases of piscine skin regeneration.  angptls in fish and other metazoans sequence homologues of human angptl family members were identified in several fish. a previously undescribed member of this family was identified in fish genomes and also in xenopus, lizard and chicken genomes and was designated angptl the new angptl family member was absent from mammalian genomes. in contrast, orthologues of human angptl were absent from fish and other non-mammalian vertebrate genomes. ten teleost fish genomes were analysed and the total angptl gene number retrieved per genome varied from to depending on retention or not of duplicate gene copies of angptl angptl angptl and angptl and the new angptl gene identified in this study. duplicates of human angptl gene homologues were identified in all teleost genomes analysed but the persistence of paralogues for the other family members was species-dependent and it was not possible in some species to establish if the full complement of genes was present due to the incompleteness of their genome assemblies.fig. angptl gene family members in fish. the number of predicted genes is indicated. the chicken and human genes are also indicated to allow comparisons with the fish homologues. “●“ represents the teleost specific genome duplication. ni: not identified in the genome of the spotted gar, which diverged prior to the teleost radiation, a single angptl gene copy was found. the lobe-finned coelacanth, a fish basal to tetrapods, had a similar gene repertoire to the spotted gar with the exception of the duplicate angptl genes. in the cartilaginous fish the elephant shark and the little skate, and angptl genes, respectively were retrieved and orthologues of the teleost angptl angptl and angptl remain to be identified. searches in the jawless fish, the marine lamprey, recovered putative angptl and angptl genes but the incomplete nature of its genome assembly meant that the existence of other family members was not established. in the gilthead sea bream that does not have a sequenced genome, angptl transcripts were retrieved but the orthologues of the teleost angptl3a and angptl were not identified. terrestrial vertebrates including, the amphibian, the anole lizard, the chicken, the opossum and the mouse had a similar gene repertoire to human but in non-mammalian genomes an orthologue of the fish angptl gene also existed. orthologues of human angptl were only identified in mammals and were absent from other vertebrates. in the cephalochordate at least putative angptl-like genes were identified indicating that this gene family is ancient and arose prior to the vertebrate radiation. data mining of other early deuterostome genomes failed to retrieve annotated genes although predicted transcripts orthologous to human angptl were identified in a urochordate, the sea squirt, in an echinoderm, the sea urchin, and in a hemichordate, the acorn worm. an orthologue of human angptl was also identified in the sea urchin, indicating that different members of the angptl family are present in non-vertebrate genomes. however, their deduced transcripts are highly divergent in sequence and length with the putative human homologues and were not considered for further analysis. in protostomes sequence hits for proteins related to the vertebrate angptls such as tenascins, ficolins, fibrinogen and others were also obtained but were not explored in this study. phylogeny of the fish angptls phylogenetic analysis of the vertebrate and cephalochordate angptl family revealed that the genes shared a common origin and that the family members emerged early during the deuterostome radiation. according to the tree topology, four main protein clusters that contain distinct members of the angptl family exist: the angptl1-2- cluster, the angptl3- cluster, the angptl cluster and the angptl7- cluster. according to the tree topology, the angptl3- cluster diverged early after the gene duplication event that gave rise to the ancestral gene from which the angptl1-2- angptl and angptl7- subsequently emerged. this suggests that the ancestral angptl gene duplicated prior to the radiation of the vertebrates and that the family members arose from different ancestral genes.fig. phylogenetic tree of the fish angptl with the tetrapod and cephalochordate homologues. tree was constructed with the bayesian interference built in mrbayes and branch support values are shown only for the major protein family clades. the angptl family clusters are boxed with different colours. the phylogenetic tree is compacted and was generated from the original tree available in additional file 2: figure s the tree was rooted using the deuterostome angpt clade but details are not show in the figure to facilitate interpretation. for the same reason, the detailed presentation of the teleost and tetrapod members of each family group have been collapsed. a tree with similar topology was obtained using the maximum-likelihood algorithm and bootstrap replicates. angptl was not included in the phylogenetic analysis as it is an atypical family member and lacks the fred domain in teleosts, duplicate copies of angptl angptl angptl angptl and angptl arose from the whole genome duplication event reported in this lineage. the teleost angptl gene duplicates are differentiated using the letters a and b and the gene environment of paralogue a shares the greatest conservation with the homologue chromosome regions in human and spotted gar. in other non-teleost fish, single gene family members exist with the exception of angptl that is duplicated in the coelacanth genome. the five cephalochordate angptl-like genes clustered with the vertebrate angptls. four of which group within the vertebrate angptl clade and three of which seem to have arisen due to a species-specific gene duplication. the fifth angptl sits in the phylogenetic tree prior to the emergence of the vertebrate angptl1-2- and angptl7- clades. the existence of other angptl genes in amphioxus was not established but they are likely to exist and were not identified due to the incompleteness of the genome assembly. sequence conservation of the fish angptls with human and cephalochordate amino acid sequence alignment of the fish angptls with the human orthologues revealed that they are highly conserved and protein domains and sequence motifs are shared by teleost and tetrapod sequences. this includes an n-terminal coiled-coil domain and a highly conserved fibrinogen-related domain in the c-terminal region that is also present in angpt proteins. within the fred motif, four highly conserved cysteine residues predicted to establish two intramolecular disulphide bonds were conserved in human and fish, however their importance in protein structure and function still remain to be established. human and fish angptl and were the most highly conserved members and their deduced mature protein sequence share at least and 77% aa sequence similarity, respectively. the deduced sequence of angptl was also highly conserved and shared 75–78% aa similarity between fish and non-mammalian tetrapods. the most divergent forms of angptl were angptl and angptl that shared a maximum of and 55% aa sequence similarity between fish and human, respectively.fig. schematic representations of the deduced structure and conserved consensus motifs of the fish and human angptls. the signal peptide region, the coiled-coil domain and the highly conserved fibrinogen-related domain are annotated and to facilitate visualization protein structures were aligned using the fred motif. the four conserved cysteine residues within the fred motif potentially involved in the establishment of two intramolecular disulphide bonds are represented and indicated by “s-s” in the angptl protein structure and their positions were obtained from uniprot annotation. other vertebrate conserved cysteine residues are represented by “c” and predicted n-glycosylation motifs are annotated with “n”. across fish, the amino acid residues that regulate the activity of the human angptl are also conserved. the figure is not drawn to scale and the percent of amino acid sequence similarity between the fish and the human orthologues is indicated with the exception of angptl that considers the similarity across fish. the angptl is not represented, as it is only present in mammals. the complete alignments of the human and fish angptl proteins are available in additional file 4: figure s3a–d. * the sequence similarity of the coelacanth duplicates was not considered, as their sequence was highly divergent although overall all the vertebrate angptl family members shared relatively high sequence conservation some specific amino acid sequences that have been linked to protein processing or protein structural configuration/function were also common across fish and human. this included the three cysteine residues and two glycosylation sites within the ccd of angptl angptl and angptl6; an n-glycosylation site and a n-glycosylation site in angptl3; an n-glycosylation site in the ccd of vertebrate angptl and the amino acids his gln and gln that are important for the regulation of lipoprotein lipase in humans and the elevation of plasma triglyceride levels in mice; the vertebrate angptl contained two conserved cysteine residues that may form an extra disulphide bridge within the fred domain; and three n-glycosylation sites, one within the ccd and two in the fred domains were conserved across the vertebrate angptl a unique n-glycosylation site within the ccd was predicted in the deduced protein of angptl from the teleosts and the spotted gar. the deduced cephalochordate angptl-like proteins shared the highest sequence similarity with vertebrate angptl and within the deduced lamprey proteins the fred domain was the most highly conserved region. neighbouring gene analysis to better understand the evolution of the angptl gene family during the vertebrate radiation, the neighbouring gene environment of mammalian angptl and non-mammalian angptl were compared between fish and tetrapods. the gene environment of the cephalochordate angptl-like genes that cluster with the vertebrate angptl clade was also characterised. in human, the angptl gene mapped to chromosome and orthologues of the flanking genes were found in other vertebrate genomes. a chromosome region with a similar gene repertoire to that flanking human angptl was found in the lizard and in the fish even though the angptl gene was absent from their genomes. of the nine genes that flank human angptl eight retain linkage in chromosome of the lizard and in chromosome lg of the spotted gar genome suggesting the loss of this gene in these species is potentially a consequence of lineage specific gene deletions.fig. comparison of the homologous genome regions harbouring the human angptl with the fish and lizard. the gene environment of the human angptl gene was characterised and was used to identify homologous genes in the anole lizard and several fish: the coelacanth, the spotted gar, the elephant shark and the marine lamprey genomes. horizontal lines represent the chromosome fragments; arrow boxes indicate genes and the arrowhead points in the direction of the predicted gene transcription. only genes that were conserved across species are represented. gene names are indicated according to the human annotation and the same colour is used for gene homologues and they are presented according to their order in the chromosome. the size of the genome fragments analysed and the predicted location of the genes in the chromosomes are indicated in megabase pairs. gene names and symbols are: coactivator-associated arginine methyltransferase, low density lipoprotein receptor, kn motif and ankyrin repeat domains, dedicator of cytokinesis, member ras oncogene family, transmembrane protein, coiled-coil domain containing, lipid phosphate phosphatase-related protein type and swim-type zinc finger associated protein fig. comparison of the spotted gar genome region harbouring angptl with other fish, chicken and human. the gene environment of the spotted gar angptl gene was characterised and was used to identify homologous genome regions in the teleost, the coelacanth, the elephant shark, marine lamprey, chicken and human. horizontal lines represent the chromosome fragments; arrow boxes indicate genes and the arrowhead points to the orientation of the predicted gene transcription. only genes that were conserved across species are represented. gene symbols are indicated and homologue genes are represented by the same colour and they are represented according to their order in the chromosome. the size of the genome fragments analysed and the location of the gene in the chromosome are indicated in megabase pairs. “●”: teleost specific genome duplication. gene names and symbols are: kelch-like family member, adenylate cyclase, dnaj homolog, subfamily c member, efr, proopiomelanocortin, dna -methyltransferase, monooxygenase, dbh-like and sorting nexin fig. comparison of the vertebrate and cephalochordate angptl homologous genome regions. the vertebrate angptl conserved gene environment was characterised using the human genome region as reference and was used to identify a homologous region in the amphioxus genome region that house the cephalochordate angptl7-like genes. horizontal lines represent chromosome fragments and its names and genome fragments are indicated at the right side; coloured block arrows represent genes according to the order in the chromosome and the arrowhead points to the predicted gene transcription. the location of the gene in the chromosome is indicated below each arrow, in megabase pairs. “●”: teleost specific genome duplication. gene names and gene symbols are: tar dna binding protein, mannan-binding lectin serine protease, spermidine synthase, exosome component, mechanistic target of rapamicin and ubia prenyltransferase domain containing in the spotted gar, the angptl gene maps to lg and in the chicken to chromosome and eight genes in linkage were identified. in the human genome a chromosome region was identified that was homologous to the gene environment flanking the fish angptl gene even though the gene has been lost from mammalian genomes. the genes that flank the angptl gene in fish and in chicken are shared between two human chromosomes indicating reorganisation of this genome region during the radiation of mammals. characterisation of the neighbouring gene environment of the duplicate teleost angptl genes revealed that they map to genome regions that share a similar gene complement confirming that they emerged from the teleost genome tetraploidization. the gene environment of angptl in fish and tetrapods revealed similarity with cephalochordate scaffold_ that houses amphioxus angptl-like_ii, iii and iv genes and suggests that vertebrate and cephalochordate angptl shared a common ancestral origin. the neighbourhood of amphioxus angptl-like_i that maps to scaffold_ shared no gene linkage with any of the vertebrate chromosomes/scaffolds containing angptl genes and its location may be the result of gene duplication and subsequent translocation. morphological and morphometric evaluation of sea bream intact and regenerating skin longitudinal transverse sections of intact and regenerating sea bream skin samples were used to characterize the ontogeny of tissue regeneration after scale removal. the three typical layers, the epidermis, dermis and hypodermis were observed in histological sections of intact sea bream skin. the scales sat in individual scale pockets, inserted in the dermis and several layers of mineralized collagen were visible. the removal of scales damaged the epidermis, dermis and scale pocket and 1 day after scale removal the torn edges of the ruptured epidermis although still attached to the skin left the dermis and scale pocket exposed directly to the aquatic milieu. blood vessels were observed in the loose dermis but not in the compact dermis. fast re-epithelialization of the epidermis occurred and 2 days after scale removal a new epidermis covered the dermis. a continuous basal layer and basal membrane were observed and formed an interface between the epidermis and the loose dermis. the scale papilla was also evident in the loose dermis 2 days after scale removal and delineated the location of the future scale pocket and new scale. establishment of the external barrier was completed 2 days after scale removal and numerous blood vessels were observed in the loose dermis. a thin layer of non-mineralized tissue was visible inside the scale pocket 3 days after scale removal and corresponded to the forming scale. by day after scale removal the structure of the regenerating skin already resembled that of the intact skin, although the mineralized scale was very thin and still did not correspond in thickness or size to the ontogenetic scale.fig. morphological evaluation of sea bream intact and regenerating skin stained with masson’s trichrome. the posterior region of the scale is orientated to the right. connective tissue is stained green and mineralized and collagen-rich tissues are stained bright red. a intact skin before scale removal; b–e regenerating skin at and 4 days after wounding, respectively. ep: epidermis; dm: demis; dm-l: loose dermis; dm-c: compact dermis; hyd: hypodermis; sc: scale: scp: scale pocket; msc: muscle; bsm: basement membrane; bsl: basal layer; dmp: dermal papilla; mlc: melanocytes; blv: blood vessel morphometric evaluation of the sea bream skin revealed the most dramatic changes in the regenerating skin where a marked increase in the thickness of the basement membrane from 6 h onwards and this was positively correlated with the progressive increase in the thickness of the epidermis from 1 day onwards compared to time no changes in the thickness of the dermis were observed during the experiment. in the intact skin the thickness of the epidermis, basement membrane and dermis remained constant throughout the healing period.fig. morphometric evaluation of sea bream skin during wound healing after scale removal. the thickness of the epidermis, basement membrane and dermis during sea bream skin recovery after scale removal is represented. each value represents the mean ± sem. statistical significance between groups was assessed using a two-way anova followed by fisher’s least significance difference post-test. statistical significances were considered at p <  and differences in intact and regenerating skin during the experimental trial are annotated with different letters and comparisons between intact and regenerating skin at the same time point are signalled with an asterisk. a histological image of the skin layers at the start and end of the experimental trial is represented beside each graph in order to illustrate the induced tissue aggression and the respective recovery with time. ep: epidermis; bsl: basal layer; bsm: basement membrane; dm-l: loose dermis; dm-c: compact dermis; sc: scale; scp: scale pocket; mlc: melanocytes; blc: blood vessel; ext: exterior. scale bars: 20 μm and 50 μm in order to assess the recovery of the vascular system in regenerating skin during the healing process the number and diameter of the blood vessels in the different periods analysed were determined. an increase in the number of blood vessels was observed at 6 h after wounding in the regenerating skin compared to time. this was subsequently followed by a decrease in the number of blood vessels until day after wounding when similar numbers to that observed at time were found, thereafter their number remained relatively constant. variation in the number of blood vessels was positively correlated with the increase in the thickness of the epidermis and basement membrane. analysis of the blood vessel diameter revealed that an increase in diameter was progressively observed during the first 24 h post wounding in regenerating skin. blood vessel diameter was relatively constant in intact skin. this revealed that the increase in blood vessel number 6 h after wounding in the damaged skin was not the consequence of improved detection due to vasodilation and suggests that new blood vessels were formed.fig. a number of blood vessels and b blood vessel diameter during skin wound healing in sea bream. each value represents the mean ± sem. statistical significance between groups was assessed using two-way anova followed by fisher’s least significance difference post-test. statistical significance was considered at p <  and differences in intact and regenerating skin during the experimental trial are annotated with different letters and comparisons between intact and regenerating skin at the same time point are signalled with an asterisk expression of angptl family members during sea bream skin regeneration analysis of angptl transcript distribution in fish by ests in ncbi revealed that they have a widespread tissue distribution. the results of in silico analysis indicated that angptl2b, angptl3b, angptl4a, angptl4b, angptl and angptl9b transcripts are expressed in fish skin. verification by qpcr using cdna from sea bream skin confirmed the presence of angptl1b, angptl2b, angptl4a, angptl4b and angptl transcripts but not angptl3b and angptl9b and they were excluded from further analysis.table digital expression analysis of angptls transcripts in the teleost skin symbol angptl1a angptl1b angptl2a angptl2b angptl3a angptl3b angptl4a angptl4b angptl5 angptl6 angptl7 angptl9a angptl9b searches were performed against the teleost ncbi database, sea bass skin transcriptome and sea bream skin scale microarray probes ni: not identified the abundance of angptl1b, angptl2b, angptl4a, angptl4b and angptl transcripts was evaluated during sea bream skin regeneration along with vegfab, a mediator of vascular development in zebrafish. the transcript abundance of angptl1b and angptl2b was significantly increased at initial stages of skin regeneration compared to the undamaged skin and subsequently decreased significantly. the abundance of angptl1b and angptl2b transcripts during the sea bream skin regeneration was correlated.fig. relative expression of the sea bream angptl1b, angptl2b, angptl4a, angptl4b, angptl and vegfab in intact and regenerating skin. expression levels were obtained by qpcr and each value, that represents the mean ± sem of the relative expression ), was estimated using the geometric mean of rps and ß-actin in intact and regenerating skin at time 0 h, 6 h and days and after wounding. statistically significant differences between groups was assessed using two-way anova followed by the fisher’s least significant difference post-test. statistical significances were considered at p <  and differences in intact and regenerating skin during the experimental trial are annotated with different letters and comparisons between intact and regenerating skin at the same time point are signalled with an asterisk in contrast, angptl was significantly down-regulated from 6 h to 4 days in regenerating skin relative to time the transcription of angptl4a and angptl4b was similar and significantly down-regulated from 1 day to 4 days in both intact and regenerating skin relative to time the change in transcript abundance over time of angptl4a and angptl4b were highly correlated and no significant differences in transcript abundance existed between intact and regenerating skin. the expression of angptl1b in regenerating skin relative to skin at time was significantly increased at 6 h up until day and subsequently decreased at day and when it was significantly lower than at the start of the experiment. in intact skin taken from the undamaged flank of sea bream, angptl1b decreased significantly from day, to relative to the start of the experiment. pairwise comparisons of angptl1b transcript abundance in intact and regenerating skin at each time point revealed significant up-regulation in regenerating skin at 6 h and 1 day after scale removal. angptl2b transcripts in regenerating skin were significantly up-regulated at days and and then strongly and significantly down-regulated at day compared to skin at time in the intact skin angptl transcripts were significantly decreased at 6 h and day relative to time in contrast, pairwise comparisons of intact and regenerating skin at each time point revealed that angptl2b transcripts were significantly up-regulated in the regenerating skin relative to intact skin at 6 h and 1 day after scale removal. angptl4a and angptl4b transcripts had a similar pattern of expression and their abundance decreased progressively after scale removal and were significantly down-regulated 1 day after scale removal in both intact and regenerating skin. pairwise comparisons of intact and regenerating skin in the same individual at each time point did not reveal any significant differences in angptl4a and angptl4b transcript abundance. expression of angptl was variable in both intact and regenerating skin samples over the 4 days of the experiment. in regenerating skin, angptl was significantly down-regulated at 6 h and on days and after scale removal relative to time in intact skin, angptl transcripts were significantly down-regulated 6 h after the start of the experiment, then significantly up-regulated at day and day relative to 6 h. by day angptl transcript abundance in intact skin was similar to time comparison of angptl transcripts in intact and regenerating skin of the same individual at each time point analysed revealed significant down-regulation of angptl in regenerating skin and 3 days after scale removal. expression of vegfab in intact and regenerating skin was not significantly different at any time point analysed. in intact skin the expression of vegfab transcripts increased progressively and was significantly up-regulated from day onwards relative to skin at time. pairwise comparisons of vegfab transcripts in intact and regenerating skin of the same individual at each time point analysed did not reveal any significant differences. no correlation between vegfab and angptl expression was found. discussion the angptl family is a large group of multifunctional proteins involved in skin regeneration and angiogenesis in mammals. the present study characterised for the first time the fish angptl gene repertoire and identified the angptl family members that were expressed in the integument of teleost fish and their expression during skin regeneration in the teleost sea bream. the results reveal that the involvement of angptl family members in skin repair has been conserved during vertebrate evolution. in fish a new angptl family member was found and the orthologue of the mammalian angptl gene was lost. in teleost angptl genes duplicated as a consequence of the lineage specific genome doubling and some of the paralogues that persisted have a role in skin homeostasis. orthologues of human angptl, angptl, angptl and angptl that play a key role in mammalian angiogenesis, pro-inflammatory response, tissue re-epithelisation/cell proliferation and avascularity, respectively are expressed in sea bream skin. variation in the abundance of angptl1b, angptl2b and angptl during sea bream skin regeneration indicates that their role in tissue repair has been conserved. the role in sea bream skin of the teleost angptl paralogues that in mammals has a prominent role in skin repair, remains to be established.fig. summary of angptl gene expression during skin regeneration in sea bream. the x-axis indicates time after wounding and the y-axis relative expression of angptl1b, angptl2b and angptl colours represent the approximate timing of angiogenesis and the re-epithelialization processes that overlap during the initial 4 days of wound healing in sea bream skin illustrated according to our morphometric evaluations, in arbitrary units angptl members in fish homologues of the mammalian angptls exist in fish. in the teleosts they have duplicated and phylogenetic analysis and gene synteny confirmed that this was the result of the lineage specific genome duplication. some of the gene duplicates have persisted and the ests retrieved for the angptl paralogues and their divergent in silico distribution in skin suggests that after genome duplication functional specialization occurred and that they acquired a range of different physiological functions. angptl genes were found from lamprey to coelacanth to mammals. the angptl family members from fishes shared highly conserved sequence and structural motifs with the human homologues and some of the residues of functional importance in the human protein have also been maintained. the deduced proteins in fish and human share two highly conserved and characteristic signature motifs of this family: the n-terminal coiled-coil domain, which likely contributes to protein oligomerization, and the c-terminal fibrinogen-like domain that in the related angpt protein family is a receptor-binding domain. a conserved n-terminus signal peptide in fish angptls suggests that they are secreted and the identification of conserved potential n-glycosylation consensus sites in the predicted proteins suggests that like the mammalian orthologues they are glycosylated proteins and may function as pleotropic endocrine/autocrine factors. in common with the human angptls, the fish members possess four conserved cysteine residues within the c-terminus fred motif that potentially form two intramolecular disulphide bonds. however, the importance of the four highly conserved cysteine residues in the protein structure remains to be discovered as alanine replacement studies with human angptl failed to reveal functional modifications. angpt proteins possess a further two conserved cysteine residues compared to angptl and the resulting conformational difference between the two sets of proteins is proposed to be the basis of receptor selectivity for the two protein families. a number of changes have occurred during the evolution of the angptl family and a new vertebrate member, angptl was identified in the present study in non-mammalian vertebrates such as fish, the xenopus, anole lizard and chicken. gene synteny analysis suggests that the loss of angptl from mammals was probably the result of chromosome rearrangements that occurred early in their radiation. similarly, orthologues of the mammalian angptl gene were absent from the sequenced genomes of fish and other non-mammalian vertebrates. angptl is an atypical family member as it lacks the fred domain, the glycosylation sites and the amino acids forming the intramolecular disulphide bonds but it has overlapping functions with angptl and and inhibits lipoprotein lipase activity. the absence of an angptl gene in non-mammalian vertebrate genomes but the existence of a conserved gene environment across the vertebrates suggests that gene loss may have been due to lineage-specific deletions. in mammals, the fred is essential for the angiogenic activity of the angptl protein while the ccd domain seems to be more important for other physiological functions, including regulation of lipid metabolism by inhibition of lipoprotein lipase. the functional importance of angptl proteins and their duplicates in fish physiology is unknown but given their high sequence homology with mammalian orthologues we hypothesized that their function might be conserved. in fact, in the zebrafish, the only teleost where members of this family have previously been described, the orthologues of human angptl and are ubiquitously expressed and have a similar tissue distribution to human. the zebrafish angptl and share a conserved role in vascular development with the human orthologues. recently, angptls were found to activate immune inhibitory receptors of the leukocyte immunoglobulin -like family, a group of innate immune receptors, that are expressed on immune cells and involved in the control of inflammatory responses and cytotoxicity. studies of human angptl and its receptor indicate that neither the ccd nor the fred binds to lilrb the authors suggest that ligand-receptor interactions occur via the receptors immunoglobulin domain and that angptl protein multimerization is essential for downstream signalling. receptors for the fish angptl are currently unknown, although potential immunoglobulin-like receptor transcripts have been described in fish and it will be important to establish if they have a similar role to those found in mammals. angptl emerged early and evolved via gene duplications and deletions in the vertebrate radiation in vertebrates, angptl evolved via gene duplication and deletion events and they are proposed to have shared a common evolutionary origin with angpt, with which they share sequence and structure similarities. four main angptl vertebrate protein clusters emerged from duplications of the ancestral angptl prior to the vertebrate radiation and the family members expanded during the early vertebrate genome doublings and segmental genome duplications. the teleosts are by far the most successful and diverse group of vertebrates with at least species identified. this success has been linked to the teleost specific genome duplication that is suggested to have provided the raw material for the evolutionary adaptations and innovation that are a characteristic of this group. the phylogenetic analysis indicates that the angptl genes also duplicated early in the teleost radiation and this was followed by gene loss so that in extant teleost genomes only a few paralogues persisted. in the coelacanth, species-specific gene duplication affected angptl and the duplicates map in tandem to the same genome fragment and clustering in the phylogenetic tree suggests that their sequence has diverged considerably from that in other vertebrates. the identification of only two angptls in the lamprey and the non-identification of angptl and in the cartilaginous fish genomes may be a consequence of their incomplete genome assemblies or linked to their unique physiological adaptations to an aquatic environment and associated gene deletions after their divergence from the common vertebrate ancestor. members of the angptl family are suggested to have emerged prior to the vertebrate radiation and a sea squirt gene homologue is equally similar to all the vertebrate members. we have also found other putative angptl-like genes in the genomes of several other early deuterostome species but their similarity with the vertebrate angptl family was very low. the exception was in the cephalochordate genome where angptl-like genes were found. the cephalochordate angptl-like deduced protein cluster revealed that four of the genes were orthologues of the vertebrate angptl furthermore, the conserved gene environment flanking the cephalochordate angptl-like genes and the vertebrate angptl suggests that the ancestral angptl gene emerged prior to the vertebrate radiation. a putative angptl1-2-6-like gene also seems to exist in the amphioxus and others genes of this family may potentially exist and their presence in the gene repertoire of the cephalochordate or in other early deuterostomes suggests this is an ancient gene family and their expansion in vertebrates may be a functional innovation linked to increased complexity of organisms and their physiology. angptls in sea bream skin wound healing in mammals’ skin wound healing is a complex and highly coordinated sequence of events. this process is triggered by blood clotting and is followed by inflammation, vascularization, formation of granular tissue and tissue remodelling. in fish the sequence of events is different, re-epithelialization of damaged skin initiates immediately after wounding and the few studies of skin healing in fish reveals that this is independent of the inflammatory signals released by the blood clot. in all vertebrates during healing new blood vessels are formed from pre-existing ones in the dermis and in mammals’ new blood vessels are observed 3 days after wounding. in teleosts this process is initiated much earlier and in zebrafish skin new blood vessels are observed 1 day after damage to the epidermis and dermis. similarly, in our sea bream skin regeneration model new blood vessels were observed 6 h after scale removal and did not correlate with a peak of vegfab, an established vertebrate blood vessel maker, which reached maximal expression 1 day after wounding. in teleosts duplicate vegfa genes have been described, vegfaa and vegfab, and both can bind the kdra and kdrb in vitro, participating in vascular development. the involvement of the vegfaa paralogue in our sea bream skin healing model remains to be established and in the future characterisation of this and of other angiogenic factors in tissue recovery will be of interest. the expression in mammalian and teleost skins of angptls, their link with integument repair and specifically their co-ordinated appearance with key steps of the skin repair program in the sea bream suggests that they may share a conserved role in vertebrate skin regeneration. in sea bream angptl1b, angptl2b, angptl4a, angptl4b and angptl are expressed in skin and their variable pattern of expression during wound healing and the differences in regenerating and intact skins 48 h post-wounding suggests they have acquired distinct roles in tissue re-epithelialization and angiogenesis. in zebrafish and mice angptl and angptl are suggested to stimulate angiogenesis and to promote endothelial cell apoptosis for the initiation of vascular development. in mice, angptl was also found to regulate the pro-inflammatory response and it activates resident murine peritoneal monocytes and macrophages. we did not study markers of inflammation, however the increased expression of angptl1b and angptl2b was correlated with the number and diameter of blood vessel, respectively, during the early stages of skin regeneration in sea bream and suggests they also have a role in angiogenesis. in human angptl is an anti-angiogenic factor and in vitro studies using cornea keratocyte cells demonstrated that this gene may be responsible for maintaining tissue avascularity in the eye. the decreased expression of angptl during sea bream skin regeneration and its negative correlation with blood vessel number suggests it may have a similar role to that in mammals. in mammals, angptl regulates skin re-epithelialization, its expression is increased during this process in mice and in angptl4-knockout mice there was decreased expression of genes involved in epidermal differentiation and cell proliferation in skin. in murine ischemic tissues, angptl bound to the ecm inhibits endothelial cell motility, sprouting and formation of new blood vessels. the down-regulation of the duplicate angptl genes during sea bream skin regeneration and their negative correlation with the thickness of epidermis and basement membrane suggests they are unlikely to be involved in re-epithelialization. nonetheless, the time-frame of angptl1b and angptl2b up-regulation suggests they may be involved in this process. the responsiveness of angptl family members in the intact skin of fish with a damaged flank was notable. in human and mammalian experimental models a systemic effect of local skin injuries, such as burns or diabetic wounds is well described in relation to cardiovascular and metabolic parameters. the results obtained for expression of angptl1b, 2b and in the intact skin of fish with a damaged flank suggests that it is the combination of both local and systemic effects that contribute to skin recovery; it will be important in the future to establish which cells are actively producing these proteins in skin and their role in teleost skin repair and homeostasis.  the present study characterizes the homologues of the human angptls in fish and identifies the candidate members that are involved in teleost skin regeneration. in fish, amphibian and chicken a novel member of this family was found but this gene is absent from mammals. homologues of human angptl are not found in fish and they are only present in mammalian genomes. in the teleosts the angptl family expanded and some gene paralogues acquired a role in the skin. in the sea bream, angptl1b, angptl2b, angptl4a, angptl4b and angptl transcripts are present in skin but only angptl1b, angptl2b and angptl are modified in response to damage and are presumably involved in skin repair. the change in abundance of angptl1b and angptl2b transcripts correlates with the timing of re-epithelialization and angiogenesis detected by histology. in contrast, angptl is down-regulated and negatively correlated with angiogenesis suggesting that in common with mammals it is an anti-angiogenic factor. overall, the results indicate that angptl family members are involved in sea bream skin homeostasis and repair. additional files additional file 1: table s accession numbers of the fish, tetrapod and cephalochordate angptl genes and sea bream transcripts. * very incomplete sequence and not included in the phylogenetic tree; ni-not identified. additional file 2: figure s expanded phylogenetic tree of the fish and other metazoan angptl family members generated using bayesian interference. details are available from fig.  accession numbers of the sequences used are given in the additional file 1: table s and additional file 9: table s additional file 3: figure s phylogenetic tree of the fish and other metazoan angptl family members constructed with the maximum-likelihood algorithm. analysis was performed in atgc using the deduced amino acid sequence and a fixed value for the proportion of invariable sites, gamma-distributed rate categories and bootstrap replicates according to prottest. tree was rooted using the metazoan angpt clade. accession numbers are given in the additional file 1: table s and additional file 9: table s additional file 4: figure s sequence alignments of the human, spotted gar, zebrafish, sea bass and sea bream angptls. sequences were compared according to the clustering of the phylogenetic tree. a angptl1-2-6; b angptl3-4; c angptl and d angptl7- conserved amino acids in the sequence alignment are shaded; dark grey represents 80% conservation and black 100% conservation. in the human sequences the signal peptide is underlined and in bold and the coiled-coil domain are in bold and coloured blue. the conserved fibrinogen-related domain in human and in fish is boxed in red and the four conserved cysteine residues within this motif that are involved in two intramolecular disulphide bonds are highlighted in yellow and the predicted glycosylation motifs are highlighted in red. additional file 5: table s percent of amino acid sequence identity/similarity of the fish angptl family members with the human orthologues. additional file 6: figure s sequence alignment of the deduced cephalochordate angptl-like protein with the human and spotted gar angptl conserved amino acids in the sequence alignment are shaded; dark grey represents 80% conservation and black 100% conservation. the coiled-coil domain is coloured in blue for the human sequence. the conserved fibrinogen-related domain is boxed and the four conserved cysteine residues within this motif that are potentially involved in the establishment of two intramolecular disulphide bonds of the vertebrate proteins are highlighted in yellow. additional file 7: table s correlation analysis of gene expression profile and changes in skin morphology during the initial phases of piscine skin regeneration. a, skin parameters during sea bream skin regeneration; b, gene expression and c, gene expression and skin parameters. represented in the tables are the correlation value and the p-value. correlations are highlighted in red. additional file 8: table s list of the teleost angptl ests and their origin. data was retrieved from the ncbi database using sea bass angptl members as the query. ests retrieved were those present in skin or from teleost fins and bony structures covered with skin. additional file 9: table s accession numbers of the fish, tetrapod and cephalochordate angpt genes and transcripts. ni-not identified. abbreviations aaamino acid ampantimicrobial peptide angptangiopoietin angptlangiopoietin-like ccdcoiled-coil domain ecmextracellular matrix fgfr1fibroblast growth factor receptor 1 fredfibrinogen-related domain igimmunoglobulin kdrtype iii receptor tyrosine kinase receptor lpllipoprotein lipase qpcrquantitative real-time polymerase chain reaction tgfßtransforming growth factor beta vegfaavascular endothelial growth factor aa vegfabvascular endothelial growth factor ab 
in sea bream, changes in the expression of angptl1b, angptl2b and angptl were correlated with the main phases of skin regeneration, indicating the involvement of angptl family members in skin regeneration has been conserved in the vertebrates. in the teleost fishes a gene family expansion occurred but all the deduced angptl proteins retained conserved sequence and structure motifs with the human homologues. exploration of the fish angptl family in skin sheds new light on the understanding of the molecular basis of skin regeneration an issue of importance for disease control in aquaculture. during the initial stages of skin regeneration angptl1b and angptl2b transcripts were significantly more abundant than in intact skin and angptl transcripts were down-regulated throughout the 4-days of skin regeneration that was studied. no difference in angptl4a and angptl4b transcript abundance was detected during regeneration or between regenerating and intact skin. the skin in vertebrates is a protective barrier and damage is rapidly repaired to re-establish barrier function and maintain internal homeostasis. in sea bream skin angptl1b, angptl2b, angptl4a, angptl4b and angptl transcripts were successfully amplified and they were differentially expressed during skin regeneration. in the first 2 days of skin regeneration, re-establishment of the physical barrier and an increase in the number of blood vessels was observed. angiopoietin-like proteinsevolutionexpressionskin regenerationteleosthttp://dx.doi.org/ /501100001871fundação para a ciência e a tecnologiasfrh/bd/81625/2011uid/multi/04326/2013costa rita a. http://dx.doi.org/http://dx.doi.org/ /501100001871fundação para a ciência e a tecnologiaccmar/multi/04326/2013http://dx.doi.org/http://dx.doi.org/ /501100004963seventh framework programmefp 222719issue-copyright-statement© the author 2017 homologues of human angptl1- were identified in fish, although angptl was absent and a totally new family member designated angptl was identified in fish and other non-mammalian vertebrates. the angptl gene family has expanded in teleost genomes. the present study characterizes for the first time the homologues of human angptls in fish and identifies the candidates that share a conserved role in skin repair using a regenerating teleost skin model over a 4-day healing period. the angiopoietin-like proteins are a family of eight secreted glycoproteins with an important role in skin repair and angiogenesis in humans.
 the skin in vertebrates is a protective barrier and damage is rapidly repaired to re-establish barrier function and maintain internal homeostasis. the angiopoietin-like proteins are a family of eight secreted glycoproteins with an important role in skin repair and angiogenesis in humans. in other vertebrates their existence and role in skin remains largely unstudied. the present study characterizes for the first time the homologues of human angptls in fish and identifies the candidates that share a conserved role in skin repair using a regenerating teleost skin model over a 4-day healing period.  homologues of human angptl1- were identified in fish, although angptl was absent and a totally new family member designated angptl was identified in fish and other non-mammalian vertebrates. in the teleost fishes a gene family expansion occurred but all the deduced angptl proteins retained conserved sequence and structure motifs with the human homologues. in sea bream skin angptl1b, angptl2b, angptl4a, angptl4b and angptl transcripts were successfully amplified and they were differentially expressed during skin regeneration. in the first 2 days of skin regeneration, re-establishment of the physical barrier and an increase in the number of blood vessels was observed. during the initial stages of skin regeneration angptl1b and angptl2b transcripts were significantly more abundant than in intact skin and angptl transcripts were down-regulated throughout the 4-days of skin regeneration that was studied. no difference in angptl4a and angptl4b transcript abundance was detected during regeneration or between regenerating and intact skin.  the angptl gene family has expanded in teleost genomes. in sea bream, changes in the expression of angptl1b, angptl2b and angptl were correlated with the main phases of skin regeneration, indicating the involvement of angptl family members in skin regeneration has been conserved in the vertebrates. exploration of the fish angptl family in skin sheds new light on the understanding of the molecular basis of skin regeneration an issue of importance for disease control in aquaculture. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords angiopoietin-like proteinsevolutionexpressionskin regenerationteleosthttp://dx.doi.org/ /501100001871fundação para a ciência e a tecnologiasfrh/bd/81625/2011uid/multi/04326/2013costa rita a. http://dx.doi.org/http://dx.doi.org/ /501100001871fundação para a ciência e a tecnologiaccmar/multi/04326/2013http://dx.doi.org/http://dx.doi.org/ /501100004963seventh framework programmefp 222719issue-copyright-statement© the author 2017 