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A dynamic history of gene duplications
and losses characterizes the evolution
of the SPARC family in eumetazoans
rspb.royalsocietypublishing.org
Stephanie Bertrand1, Jaime Fuentealba2, Antoine Aze1,†, Clare Hudson4,
Hitoyoshi Yasuo4, Marcela Torrejon2, Hector Escriva1 and Sylvain Marcellini3
1
CNRS, UMR7232, Université Pierre et Marie Curie Paris 06, Observatoire Océanologique, Banyuls-sur-Mer, France
Department of Biochemistry and Molecular Biology, and 3Laboratory of Development and Evolution,
Department of Cell Biology, Faculty of Biological Sciences, University of Concepción, Concepción, Biobı́o
Region, Chile
4
CNRS, UMR7009, Université Pierre et Marie Curie Paris 06, Laboratoire de Biologie du développement,
Observatoire Océanologique, Villefranche-sur-mer, France
2
Research
Cite this article: Bertrand S, Fuentealba J,
Aze A, Hudson C, Yasuo H, Torrejon M, Escriva
H, Marcellini S. 2013 A dynamic history of
gene duplications and losses characterizes the
evolution of the SPARC family in eumetazoans.
Proc R Soc B 280: 20122963.
http://dx.doi.org/10.1098/rspb.2012.2963
Received: 12 December 2012
Accepted: 31 January 2013
Subject Areas:
evolution, developmental biology, genomics
Keywords:
SPARC gene family, eumetazoans,
ancient duplication, independent losses,
vertebrate evolution
Authors for correspondence:
Stephanie Bertrand
e-mail: [email protected]
Sylvain Marcellini
e-mail: [email protected]
†
Present address: London Research Institute,
Cancer Research UK, Clare Hall Laboratories,
Blanche Lane, South Mimms, Potters Bar,
Hertfordshire EN6 3LD, United Kingdom.
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2012.2963 or
via http://rspb.royalsocietypublishing.org.
The vertebrates share the ability to produce a skeleton made of mineralized
extracellular matrix. However, our understanding of the molecular changes
that accompanied their emergence remains scarce. Here, we describe the
evolutionary history of the SPARC (secreted protein acidic and rich in
cysteine) family, because its vertebrate orthologues are expressed in cartilage, bones and teeth where they have been proposed to bind calcium and
act as extracellular collagen chaperones, and because further duplications
of specific SPARC members produced the small calcium-binding phosphoproteins (SCPP) family that is crucial for skeletal mineralization to occur.
Both phylogeny and synteny conservation analyses reveal that, in the eumetazoan ancestor, a unique ancestral gene duplicated to give rise to SPARC
and SPARCB described here for the first time. Independent losses have
eliminated one of the two paralogues in cnidarians, protostomes and
tetrapods. Hence, only non-tetrapod deuterostomes have conserved both
genes. Remarkably, SPARC and SPARCB paralogues are still linked in the
amphioxus genome. To shed light on the evolution of the SPARC family
members in chordates, we performed a comprehensive analysis of their
embryonic expression patterns in amphioxus, tunicates, teleosts, amphibians
and mammals. Our results show that in the chordate lineage SPARC and
SPARCB family members were recurrently recruited in a variety of unrelated
tissues expressing collagen genes. We propose that one of the earliest steps of
skeletal evolution involved the co-expression of SPARC paralogues with
collagenous proteins.
1. Introduction
Vertebrates appeared more than 500 Ma, and have acquired specialized cell
types able to produce mineralized extracellular matrix found in cartilage,
bone and teeth [1]. Skeletal development relies on a myriad of extracellular proteins that create biochemical conditions compatible with biomineralization [2,3].
Hence, a comprehensive analysis of the evolutionary history of extracellular
proteins expressed by skeletal cells is essential to improve our understanding
of the molecular changes that contributed to the emergence of vertebrates.
For instance, the fibrillar collagen proteins are the principal component of the
vertebrate mineralized matrix [4] and their evolution has been studied in
great details. Fibrillar collagen orthologues are present in sponges, cnidarians
and bilaterians [5– 8], and have been recruited into the vertebrate mineralizing
skeletal matrix to perform a structural role by facilitating the nucleation of
hydroxyapatite crystals [1,9,10]. The SPARC homologues represent another
gene family that is intimately linked to skeletal evolution for a variety of
reasons. First, SPARC proteins bind calcium and act as extracellular collagen
& 2013 The Author(s) Published by the Royal Society. All rights reserved.
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BLASTP with default parameters on the Ensembl database. For all
the sequences that had hits localized in human chromosomes 4, 5
or/and 10, orthology was assessed by phylogenetic reconstruction. The aligned amino acid sequences of the corresponding
gene families were retrieved from Ensembl. Other sequences were
obtained from Genbank and the amphioxus sequences were thereafter added using clustalW [46]. Finally, alignments were manually
corrected in SeaView [40] and ML trees were constructed with
PhyML [45] implemented in SeaView with the WAG þ G þ I
model of protein evolution, and aLRT was computed for
branch support.
Partial cDNAs from Mus musculus SPARC and SPARCL1, Xenopus
tropicalis SPARC, Danio rerio SPARC, SPARCL1 and SPARCB,
Branchiostoma lanceolatum SPARC/SPARCL1 and SPARCB were
cloned by RT-PCR in pGEM-T Easy vector (Promega). Primers
used for each gene are listed in the electronic supplementary material, table S2. The Ciona intestinalis SPARC/SPARCL1
(cilv13k23) and SPARCB (ciad43l22) clones were obtained
from Nori Satoh’s Gene Collection Plates. Dioxigenin-labelled
RNA probes were synthesized using appropriate enzymes according to the manufacturer’s instructions (Roche). Ripe animals of the
Mediterranean amphioxus (B. lanceolatum) were collected in
Argelès-sur-Mer (France), and gametes were obtained by heat
stimulation [47,48]. Fixation and whole-mount in situ hybridization were performed as described in [49], except the
chromogenic reaction which was performed using BM Purple
[50]. Mouse embryos were dissected in PBS, fixed in 4 per cent
paraformaldehyde and whole-mount in situ hybridization was
performed according to Wilkinson [51]. In situ hybridization on
frog embryos was performed according to Harland [52] and
Maldonado-Agurto et al. [53]. In situ hybridization on zebrafish
embryos was performed according to [54]. In situ hybridization
on ascidian embryos was performed according to Hudson &
Yasuo [55] and Wada et al. [56].
2. Material and methods
(a) Phylogenetic analysis
Amino acid sequences of the SPARC and SPOCK families
were obtained from the NCBI, the JGI and the Ensembl databases (see the electronic supplementary material, table S1). For
Oncorhynchus mykiss and Pimephales promelas, EST sequences corresponding to putative SPARCB orthologues were assembled
using CAP3 [38]. The resulting nucleotide sequences were translated in the adequate frame. The amino acid sequences were
all aligned using Clustal Omega [39] and the alignment was
manually corrected using SeaView [40]. Only amino acids
located between the Kazal and the SPARC domains were kept
for the phylogenetic reconstruction. Bayesian inference (BI) tree
was inferred using MRBAYES v. 3.1.2 [41,42], with the model
recommended by ProtTest [43] under the Akaike information
criterion (we used WAG þ G þ I model because LG is not
implemented in MRBAYES), using the CIPRES Science Gateway
[44]. Two independent runs were performed, each with
4 chains. A burn-in of 25 per cent was used and the consensus
tree was calculated for the remaining trees. Maximum likelihood
(ML) analysis was performed using PhyML [45] with the model
recommended by ProtTest (LG þ G þ I) and aLRT support for
branches was computed. The phylogenetic tree obtained using
ML had a topology consistent with the one obtained by BI (see
the electronic supplementary material, figure S1).
(b) Synteny conservation analysis
We searched human orthologues for all the predicted amino acid
sequences found in amphioxus scaffolds 131, 329 and 562 using
3. Results
(a) A new SPARC gene family member
In our search for SPARC and SPARCL1 orthologues in
metazoan genomes, we encountered a third gene that was
not previously described and that we named SPARCB.
Vertebrate SPARCB orthologues are absent from tetrapod
genomes, but were identified in zebrafish (D. rerio) and coelacanth (Latimeria chalumnea) genomes. We also found EST
sequences corresponding to SPARCB genes in two other
teleost species, O. mykiss and P. promelas. SPARCB genes code
for proteins that have the same general domain organization
as SPARC, with a signal peptide followed by a Kazal domain
and a SPARC domain. However, the zebrafish SPARCB does
not possess a large region (typical of vertebrate SPARCL1) separating the signal peptide from the Kazal domain, nor does it
display the acidic region previously described for bilaterian
SPARC and SPARCL1 (not shown and electronic supplementary material, figure S2). In fact, this atypical organization
has previously been described for the Nematostella vectensis
SPARC1–4 proteins [13], which, as we show here, belong to
the SPARCB subgroup. In non-vertebrates, we found
SPARCB orthologues in amphioxus (Branchiostoma floridae),
tunicates (C. intestinalis), ambulacrarians (Strongylocentrotus
purpuratus and Saccoglossus kowalevskii) and cnidarians
(N. vectensis and Hydra magnipapillata), but not in protostomes
(annelids, molluscs, nematodes and arthropods). In order
Proc R Soc B 280: 20122963
(c) Cloning and in situ hybridization
2
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chaperones [11,12]. Second, the SPARC family predates the emergence of vertebrates [13–17]. Third, among osteichthyans (bony
fishes and tetrapods), most species exhibit two paralogues
called SPARC and SPARCL1 that are expressed in skeletal
cells [12,18–26]. Finally, the SPARCL1 paralogue underwent
a series of tandem duplications, thereby giving rise to the
small calcium-binding phosphoproteins (SCPP) gene members
[20,21]. The SCPP members are expressed in cartilage, bone
and teeth and encode highly acidic proteins regulating extracellular matrix biomineralization [18,27–34]. Therefore, while
SPARC and SPARCL1 are neither sufficient in vitro nor
required in vivo for biomineralization to occur [35–37], the
SPARC family has been instrumental in the emergence of
vertebrate genes involved in skeletal tissue mineralization.
Here, we present new data supporting a novel scenario for
the evolutionary history of the SPARC family. Most importantly, both phylogeny and synteny conservation analyses
reveal that an ancient gene duplication in the last common
ancestor of eumetazoans produced SPARC and SPARCB, a
gene first described here. We further show that three independent losses eliminated one of the two paralogues in the
lineage of the cnidarians, protostomes and tetrapods. Consequently, cnidarians exhibit a single gene which is actually
orthologous to SPARCB and not SPARC. In order to shed
light on the ancestral expression pattern of SPARC family
genes, we analysed and compared the embryonic expression pattern of homologues from all major vertebrate and
invertebrate chordate groups. We show that SPARC family
members have independently been recruited to embryonic tissues expressing collagenous proteins, providing novel insights
regarding the earliest molecular changes involved in skeletal
matrix evolution.
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To decipher the evolutionary history of the chordate SPARC/
SPARCL1/SPARCB family, we looked at the genomic
position of SPARC/SPARCL1 and SPARCB genes of species
that possess both genes (i.e. B. floridae, C. intestinalis,
S. kowalevskii, S. purpuratus, L. chalumnea and D. rerio). We
found that they are located on separate contigs, scaffolds or
chromosomes, except in amphioxus. We then analysed the
synteny conservation between amphioxus genomic regions
around SPARC/SPARCL1 and SPARCB, and the human
and zebrafish genomes. As shown in figure 1b, the
amphioxus homologues are located on the scaffold 329 (containing SPARCB) and on the scaffolds 131 and 562
(overlapping at the level of SPARC/SPARCL1). All three scaffolds can be assembled into a single and continuous 2,8 Mb
genomic fragment, showing that the amphioxus SPARC/
SPARCL1 and SPARCB are syntenic (figure 1b). We then
systematically compared all the predicted coding sequences
from this amphioxus region with the human genome at the
Ensembl database website (http://www.ensembl.org/) using
BLASTP. We focused on the best BLAST hits that aligned
to three well-defined paralogous regions ( paralogons) of
the human genome located on chromosomes 4 (containing
SPARCL1), 5 (containing SPARC) and 10 (see figure 1b and
[57]). This approach led us to identify 25 amphioxus genes
(see the electronic supplementary material, table S3), for
which orthology with vertebrate genes was unambiguously confirmed (see the electronic supplementary material,
figure S3 and S4). Therefore, we show that the amphioxus
genomic region containing SPARC/SPARCL1 and SPARCB
harbours many genes whose human orthologues are found
around the human SPARC or SPARCL1 genes or in the
chromosome 10 paralogon. For most of these amphioxus
genes, we were also able to detect conservation of synteny in
zebrafish (figure 1b; electronic supplementary material, figure
S4 and table S3). We found that the zebrafish SPARCB is
located in a region of chromosome 17 which is orthologous
to the aforementioned human chromosome 10 paralogon
(figure 1b). Combined to our phylogenetic analyses, these findings strongly suggest that the SPARC/SPARCL1 and SPARCB
genes were linked in the chordate ancestor, and further confirm
the fact that SPARC and SPARCL1 result from two wholegenome duplication events that occurred in the vertebrate
lineage [22,58].
In order to explore the developmental evolution of the SPARC
family members in the chordate lineage, we undertook the
analysis of their expression during embryogenesis using
whole-mount in situ hybridization in species belonging to the
three chordate phyla. Below, we describe the expression of
the SPARC/SPARCL1 gene in amphioxus and tunicates, as
well as its two vertebrate orthologues SPARC and SPARCL1.
We subsequently describe the expression of the SPARCB
members found in tunicates, amphioxus and zebrafish.
In amphioxus, SPARC/SPARCL1 expression is first detected at the early neurula stage in the paraxial mesoderm
(figure 2a; electronic supplementary material, figure S5). At
the mid-neurula stage, the expression increases while remaining restricted to the forming somites (figure 2b). In the larva,
SPARC/SPARCL1 is expressed in the ventral and paraxial
mesoderm and in the notochord (figure 2c). In ascidians,
ubiquitous transcripts of SPARC/SPARCL1 were detected
during early cleavage stages (not shown). Specific expression
begins at late neurula/early tailbud stage in the notochord
(figure 2d). At early tailbud stage, lower levels of transcripts
were also detected in the central nervous system and anteriormost epidermis, as well as a strong expression in the notochord
(figure 2e). In later tailbud stages, expression was detected
specifically in the notochord (figure 2f ).
We next examined the expression patterns of the
osteichthyan SPARC and SPARCL1 paralogues. In zebrafish,
SPARC is first expressed in the otic placode at the beginning
of somitogenesis (not shown). The expression in the otic vesicle persists until at least 72 hpf (figure 2g–j). At 18 hpf,
expression is also detected in the notochord and in the
somites (figure 2g). At 24 hpf, SPARC is expressed in
the same territories as well as in the epidermis (figure 2h).
The epidermal expression persists until 72 hpf (figure 2j ). In
48 hpf zebrafish embryos, SPARC expression is detected in
the epidermis, the posterior notochord, the somites, the otic
vesicle and the apical ectodermal ridge of the pectoral fin
(figure 2i). The 72 hpf embryos display a similar SPARC
expression, except for the notochord where it is no longer
detected (figure 2j ). This expression pattern is in agreement
with previously published data [59]. Regarding amphibians,
our results in X. tropicalis are consistent with the previously
published expression patterns of SPARC in Xenopus laevis
embryos [60]. The X. tropicalis SPARC gene is specifically
expressed in the notochord and neural plate of stage 21 neurula (figure 2k). In stage 26 embryos, SPARC is detected in the
notochord, the presomitic mesoderm and the anterior region
(figure 2l ). In stage 31 tadpoles, transcripts are fading in
the presomitic mesoderm and are detected at the level
of the notochord, otic vesicle, lens, branchial arches and midbrain (figure 2m). In mouse, SPARC is strongly expressed
in extraembryonic tissues at stages E7.5–E8.5 (figure 2n,o).
At E8.5, expression is also detected in the brain (figure 2o).
From E9.5 onward, SPARC is mainly expressed in the
epidermis as well as in the vascular system (figure 2p,q).
At E11.5, the expression is also detected in the heart of the
embryo (figure 2p,r).
As SPARCL1 has been lost in amphibians [20,21], we
focused on the D. rerio and M. musculus orthologues. In zebrafish, restricted SPARCL1 expression is first detected in
24 hpf stage embryos in the lens, the brain and at a lower
3
Proc R Soc B 280: 20122963
(b) Synteny conservation analysis
(c) Developmental expression of the chordate
SPARC family genes
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to unambiguously assess orthology relationships between
SPARC, SPARCL1 and SPARCB, we performed phylogenetic
reconstruction, using SPOCK family sequences as an outgroup.
The phylogenetic tree shows that teleost, coelacanth and nonvertebrate deuterostome SPARCB proteins are orthologous and
group with the cnidarian sequences (figure 1a and electronic
supplementary material, figure S1). In agreement with published
literature, the vertebrate SPARC and SPARCL1 paralogues
cluster as a monophyletic clade (figure 1a and [22]). Our results
also reveal that the genomes of tunicates, amphioxus, ambulacrarians and protostomes harbour a unique gene (referred to as
SPARC/SPARCL1 hereafter), which is co-orthologue to the vertebrate SPARC and SPARCL1 genes (figure 1a and electronic
supplementary material, figure S1). Remarkably, no SPARC/
SPARCL1 orthologues are present in cnidarians (figure 1a and
electronic supplementary material, figure S1).
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(a)
0.99
(b)
amphioxus scaffolds
(i)
SPARCB
SPARC/SPARCL1
131
(iii) Chr. 1
Chr. 4
SPARCL1
Chr. 7
Chr. 5
SPARC
Chr. 14
329
562
zebrafish 2R paralogons
human 2R paralogons
(ii)
Chr .10
SPOCK
1
SPARCB
1
SPARC/SPARCL1
(protostomes)
1
Chr. 17
Chr. 12
SPARCL1
SPARC
SPARCB
Figure 1. Paralogy and orthology relationships of the SPARC gene family members in eumetazoans. (a) Bayesian inference phylogenetic tree of the SPARC family.
SPOCK family was used as outgroup. Posterior probabilities are shown on each node. Scale bar indicates the number of substitutions per site. See the electronic
supplementary material, table S1 for accession numbers and name abbreviations. (b) Synteny conservation among vertebrate and amphioxus SPARC chromosomal
regions. The amphioxus SPARCB and SPARC/SPARCL1 genes are located on three overlapping genomic contigs numbered 329, 562 and 131 (i). This amphioxus region
is co-orthologous to regions of the human (ii) chromosomes 4 (containing SPARCL1), 5 (containing SPARC) and 10, as well as regions of the zebrafish (iii) chromosomes 1 (containing SPARCL1), 7, 14 (containing SPARC), 17 (containing SPARCB) and 12.
level in the notochord (figure 2s). The lens and notochord
expression persists until at least 72 hpf (figure 2t,u).
Additional expression in the brain floorplate is observed at
48 and 72 hpf (figure 2t,u). At this later stage, SPARCL1 is
also expressed in the oral cavity (figure 2u). In E7.5 mouse
embryos, SPARCL1 is specifically expressed in the node
Proc R Soc B 280: 20122963
1
1
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1 SPARCL1_TAKRU
SPARCL1_ORYLA
SPARCL1_DANRE
SPARCL1B_LATCH
1
SPARCL1A_LATCH
1
SPARCL1_MUSMU
0.99
1
SPARCL1_HOMSA
1
SPARCL1_MELGA
1 SPARCL1_GALGA
0.94
0.51SPARCL1_COTJA
SPARC1_PETMA
0.87
1
SPARC2_PETMA
SPARC/SPARCL1_BRAFL
0.93
SPARC/SPARCL1_CIOIN
SPARC/SPARCL1_SACKO
0.95
SPARC/SPARCL1_STRPU
SPARC/SPARCL1_DAPPU
SPARC/SPARCL1_BOMMO
0.66
SPARC/SPARCL1_PEDHU
0.99
0.84
SPARC/SPARCL1_SCHGR
0.75
1 SPARC/SPARCL1_CAMFL
SPARC/SPARCL1_APIME
1
1
0.93
SPARC/SPARCL1_DROME
SPARC/SPARCL1_TRICA
0.95
SPARC/SPARCL1_IXOSC
SPARC/SPARCL1_LOTGI
1
SPARC/SPARCL1_HALDI
SPARC/SPARCL1_ASCSU
1
1
SPARC/SPARCL1_BRUMA
0.6
SPARC/SPARCL1_HETBA
1
SPARC/SPARCL1_CAEEL
1
SPARC/SPARCL1_TRISP
0.98
SPARC/SPARCL1_PHRLA
1
SPARC/SPARCL1_CAPCA
SPARCB_DANRE
1 SPARCB_PIMPR
1
1
SPARCB_ONCMY
SPARCB_LATCH
0.31
SPARCB_SACKO
0.98
1
SPARCB_BRAFL
SPARCB_STRPU
0.32
SPARCB_CIOIN
SPARC1_NEMVE
0.5
SPARC2_NEMVE
1
SPARC4_NEMVE
0.9
0.87
SPARC3_NEMVE
SPARC3_HYDMA
SPOCK1_DANRE
1
1 SPOCK1_MELGA
SPOCK1_GALGA
SPOCK3_XENLA
0.87
1
0.89 SPOCK3_ANOCA
SPOCK3_MELGA
0.74 SPOCK3_MUSMU
1
0.99SPOCK3_HOMSA
SPOCK3_DANRE
1
1
SPOCK3_TETNI
SPOCK2_XENLA
0.99 SPOCK2_ANOCA
0.98
0.92
SPOCK2_RATNO
1 SPOCK2_HOMSA
1
SPOCK2_TETNI
1
0.92
SPOCK2_DANRE
SPOCK_SACKO
SPOCK_BRAFL
SPOCK_TRICA
0.82
SPOCK_ACYPI
0.58
SPOCK_DROME
1
1
SPOCK_ANOGA
1
SPOCK_APIME
SPOCK_IXOSC
SPOCK_NEMVE
SPOCK_HYDMA
0.49
0.3
SPARC/SPARCL1
(deuterostomes)
0.97
4
SPARC_SPAAU
1 0.43 SPARC_TAKRU
SPARC_GADMO
0.880.97SPARC_ORYLA
SPARC_DANRE
0.470.87SPARC_SALSA
SPARC_LATCH
SPARC_XENLA
0.97 1
SPARC_RANCA
0.62 SPARC_HOMSA
1 SPARC_MUSMU
1
1
SPARC_COTJA
0.98
SPARC_GALGA
0.98SPARC_ANOCA
1 SPARC_GINCI
SPARC_SCYCA
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(a)
(c)
(d)
(i)
( j)
(l)
(m)
(o)
(p)
(q)
SPARC
X. tropicalis
(n)
(r)
M. musculus
(s)
(t)
(u)
D. rerio
(w)
(x)
(y)
(z)
M. musculus
(e')
(c')
( f ')
(g')
(h')
SPARCB
(d')
(b')
B. lanceolatum C. intestinalis
(a')
SPARCL1
(v)
Figure 2. Comparative analysis of gene expression patterns of chordate SPARC homologues. (a – f ) SPARC/SPARCL1 in situ hybridizations, amphioxus embryos are at
(a) the early neurula, (b) mid – late neurula and (c) larvae stages. (d ) Ascidian embryos are at the late neurula, (e) tadpole and (f ) larvae stages. (g– r) SPARC in
situ hybridizations, zebrafish embryos are at stages (g) 18 hpf, (h) 24 hpf, and (i,j ) 72 hpf. Amphibian embryos are at stages (k) 21, (l ) 27 and (m) 31. Mouse
embryos are at stages (n) E7.5, (o) E8.5, ( p) E11.5, (q,r) E10.5. (s– z) SPARCL1 in situ hybridizations, zebrafish embryos are at stages (s) 24 hpf, (t) 48 hpf and
(u) 72 hpf. Mouse embryos are at stages (v) E7.5, (w) E8.5, (x) E9.5, ( y) E10.5 and (z) E11.5. (a0 – h0 ) SPARCB in situ hybridizations, amphioxus embryos are at the
(a0 ) mid – late neurula, late (b0 ) neurula and (c0 ) larvae stages. Ascidian embryos are at the early (d0 ) gastrula, (e0 ) neurula, ( f0 ) early tailbud and (g0 ,h0 ) late
tailbud stages. Embryos are oriented with their anterior and dorsal sides leftward and upward, respectively, except for (a,d,k,e0 ) (dorsal view); (n– r,v – z) (anterior
upward, dorsal towards the left); and (d0 ) (vegetal pole view), (h0 ) (ventral views). For mouse embryos, the images were inverted and the right side of the embryos
is shown. A summary of the phylogenetic relationships of these genes is indicated on the right. Description of the different expression patterns can be found in the
main text.
Proc R Soc B 280: 20122963
D. rerio
(k)
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(h)
(f)
SPARC/SPARCL1
(g)
C. intestinalis
B. lanceolatum
(b)
5
(e)
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(a) A new evolutionary scenario for the SPARC
gene family
Both phylogenetic reconstructions and synteny conservation
data reveal a surprisingly rich history of gene duplications
and losses for the eumetazoan SPARC family, and the unsuspected existence of the SPARCB paralogy group. Based on
our results, we propose the following scenario for the evolution
of the SPARC gene family in eumetazoans (figure 3). A SPARC
gene was present in the ancestor of eumetazoans and was
duplicated before the divergence between cnidarians and
bilaterians, giving rise to the SPARC/SPARCL1 and SPARCB
paralogues. The SPARC/SPARCL1 gene was thereafter lost
in cnidarians, whereas the SPARCB gene was lost in the ancestor of all protostomes. The fact that SPARC/SPARCL1 and
SPARCB orthologues are present in the genomes of zebrafish,
ambulacrarians, amphioxus and ascidians shows that both
genes were retained in the deuterostome ancestor. In the vertebrate lineage, two whole-genome duplication events [58]
gave rise to SPARC and SPARCL1, and to two SPARCB paralogues. In tetrapods, however, both SPARCB paralogues
were lost. Moreover, only SPARC was retained in amphibians
[20–22]. In teleosts, an additional genome duplication
occurred was followed by extensive gene losses [61], resulting
in the retention of one SPARC, one SPARCL1 and one SPARCB
gene. Finally, the paucity of SPARCB sequences in teleost databases suggests that this gene was recurrently eliminated in this
lineage. Because the zebrafish SPARCB gene is only weakly
expressed in the lens, a tissue in which SPARC and SPARCL1
(b) Shared and derived expression patterns of
SPARC homologues
Several conserved gene expression domains are evident in
chordates for SPARC/SPARCL1 orthologues. First, a notochordal expression of SPARC/SPARCL1 orthologues is evident in
amphioxus, tunicates, teleosts and amphibians (figure 2c,d–f,
g,k–m,s) suggesting that this feature was already present
in the last common ancestor of chordates. Second, the
amphioxus SPARC/SPARCL1, the zebrafish and amphibian
SPARC, and the mouse SPARCL1 orthologues are expressed
in the paraxial mesoderm (figure 2a–c,g–h,k–m,w–z).
Hence, the paraxial mesoderm-specific expression also represents a synapomorphy of chordates, but has been lost in
ascidians concomitantly with the acquisition of a highly
derived mode of embryogenesis. The fact that different vertebrate paralogues are expressed in this region (i.e. SPARCL1
in mouse and SPARC in zebrafish and amphibian) suggests
independent losses of regulatory enhancers, as proposed by
the duplication–degeneration–complementation model [62].
Third, we find that several tissues display a conserved
expression of SPARC/SPARCL1 homologues, such as the
brain (figure 2e,k–m,o), the otic vesicle (figure 2g–j,m,y) and
the lens (figure 2i–j,m,s–t). Finally, some expression territories
of SPARC or SPARCL1 are lineage-specific and include the
teleost apical ectodermal ridge (figure 2i,j), the mouse extraembryonic tissue, vasculature, epidermis and heart (figure 2n–r)
and the mouse node (figure 2v).
The expression patterns of the SPARCB orthologues in
amphioxus, tunicates and zebrafish greatly differ from each
other (figure 2a0 –h0 ; electronic supplementary material,
figure S5), suggesting that SPARCB function evolved faster
than its SPARC/SPARCL1 paralogue. Below, we will discuss
how these rapid changes of SPARCB expression patterns,
and particularly its recruitment in the amphioxus notochord
and the tunicate palps, might be informative regarding the
evolution of the vertebrate skeleton.
(c) Is the origin of the vertebrate skeleton linked to
the co-option of genes involved in collagenous
matrix formation?
Our results strengthen the idea that the convergent coexpression of SPARC family members with collagen genes
is a recurrent phenomenon in bilaterian evolution [22,63].
In the fruitfly, the SPARC/SPARCL1 orthologue functionally
interacts with collagen-IV to stabilize the basal lamina of
6
Proc R Soc B 280: 20122963
4. Discussion
transcripts are abundant (figure 2i,j,t,u; electronic supplementary material, figure S5), it is tempting to propose that
functional redundancy might have facilitated the loss of
SPARCB in most other teleosts. Our synteny conservation
analysis between amphioxus and osteichthyans shows that
SPARC/SPARCL1 and SPARCB were linked in the genome of
the ancestral chordate. No synteny conservation could be
found in the other non-chordate deuterostomes that have
both SPARC/SPARCL1 and SPARCB genes, which could
be due to genomic rearrangements or to incomplete genome
assembly. We, therefore, propose that SPARC/SPARCL1 and
SPARCB arose from a tandem duplication that occurred in
the last common ancestor of eumetazoans.
rspb.royalsocietypublishing.org
(figure 2v). At E8.5, it is detected in the brain, the tailbud and
the somites (figure 2w). At E9.5, the expression is strong in
the dorsal part of the somites and in the tailbud (figure 2x).
SPARCL1 is also expressed in the brain, mainly in the hindbrain, and in the branchial arches at this stage (figure 2x).
At E10.5, expression is still observed in somites, branchial
arches and brain, as well as in the vascular system and in
the otic placode (figure 2y). The same holds true for embryos
at E11.5, with an additional expression in the forelimb
buds (figure 2z).
The onset of the amphioxus SPARCB expression occurs at
the mid–late neurula stage, specifically in the differentiating
notochord (figure 2a0 ; electronic supplementary material,
figure S5). At the late neurula stage, before mouth opening,
the expression persists in the notochord and appears in the
pharyngeal endoderm (figure 2b0 ). In amphioxus larvae,
SPARCB is expressed in the anterior-most part of the pharyngeal endoderm (oral cavity) and in the notochord (figure 2c0 ).
In ascidians, SPARCB expression is first detected at the early
gastrula stage in the endoderm precursors (figure 2d0 ).
During neural plate stages, transcripts can be detected
in the a-line-derived part of the neural plate (figure 2e0 ).
At early tailbud stages, expression is detected in the posterior
epidermis at the tip of the tail (figure 2f 0 ). Finally, during
later tailbud stages expression is seen in the three palp precursors (figure 2g0 ). Expression is stronger, and begins
earlier, in the ventral most palp precursor (figure 2h0 ). In zebrafish, only a weak SPARCB expression could be detected in
the lens between 24 and 48 hpf (see the electronic supplementary material, figure S5).
Downloaded from http://rspb.royalsocietypublishing.org/ on June 17, 2017
13
ancestral SPARC gene
SPARC/SPARCL1 gene
SPARCB gene
SPARC gene
Osteichtyan
ancestor
4
5 H. sapiens
10
SPARCL1 gene
SPARCB orthologue
SPARCB orthologue
gene loss
X. laevis
whole-genome duplicaton
Sarcopterygian
ancestor
L. chalumnea
Deuterostome
ancestor
B. floridae
Tandem duplicaton
Bilaterian
ancestor
S. kowalevskii
S. purpuratus
Eumetazoan
ancestor
Protostome
ancestor
D. melanogaster
L. gigantea
Cnidarian
ancestor
H. magnipapillata
Figure 3. Evolutionary scenario of the SPARC gene family in eumetazoans. The diagram depicts a phylogenetic tree of metazoan species whose SPARC homologues
were analysed in this study. Whole-genome duplications are indicated at the base of each branch in which they were inferred to have occurred. We have considered
here a scenario with no gene loss before these duplications and we have indicated the gene losses that probably occurred between these duplications and the
ancestor of osteichtyans or teleosts. On the right the presence of SPARC family members with their syntenic relationships in different eumetazoan species is shown.
embryonic epithelia [11,15,16]. The ascidian and amphioxus
SPARC/SPARCL1 orthologues and the osteichthyan SPARC
genes are expressed in the notochord, a structure expressing
a variety of fibrilar and non-fibrilar collagens [7,8,64]. It
has been shown that fibrilar collagens from clade A, B and
C were independently recruited to the notochord in the
chordate ancestor [7]. Remarkably, such a convergent
phenomenon also characterizes the SPARC family members,
as demonstrated by the notochordal expression shared by the
amphioxus SPARCB and its chordate SPARC/SPARCL1 paralogues (figure 2c,f,k,s,b 0 ). Finally, in developing ascidian
palps, the SPARCB gene is co-expressed with the fibrilar
collagen gene CiFCol4 (this study and [7]). Altogether, these
data suggest a close functional relationship between SPARC
family members and collagenous proteins, which is supported by the presence of a well-conserved collagen-binding
site in SPARC, SPARCL1 and SPARCB proteins (see the electronic supplementary material, figure S2). We, therefore,
propose that one of the earliest steps of skeletal evolution
involved the co-expression of SPARC/SPARCL1/SPARCB
and collagenous proteins in an available pool of embryonic,
multipotent, mesenchymal cells. Clearly, future directions
in the field of skeletal evolution will involve the comparison
of regulatory network architectures from unrelated chordate
tissues that nevertheless have experienced a selective pressure
to convergently acquire a SPARC-dependent collagenous
extracellular matrix.
This work was financially supported by a FONDECYT grant to S.M.
(no. 1110756); the Agence Nationale de la Recherche grants nos.
ANR-2010-BLAN-1716 01 and ANR-2010-BLAN-1234 02 to H.E.;
and a joint ECOS-CONICYT grant to S.M. and H.E. (no. C09B01).
The laboratory of C.H. and H.Y. was supported by the Centre
National de la Recherche Scientifique, the Université Pierre et
Marie Curie and the Agence Nationale de la Recherche (ANR-09BLAN-0013-01). The authors acknowledge Laure Bernard and
Vincent Laudet for kindly providing zebrafish embryos.
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