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Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Identification of Aldh1a, Cyp26 and RAR orthologs in protostomes pushes back
the retinoic acid genetic machinery in evolutionary time to the bilaterian ancestor
Ricard Albalat a,∗ , Cristian Cañestro b,∗
a
b
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, 645, E-08028 Barcelona, Spain
Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA
a r t i c l e
i n f o
Article history:
Received 8 August 2008
Received in revised form 5 September 2008
Accepted 9 September 2008
Available online xxx
Keywords:
Retinoic acid signaling
Deuterostomes
Invertebrates
Protostomes
Ecdysozoans
Lophotrocozoans
Aldh
Retinaldehyde dehydrogenases
Bilaterian evolution
Comparative genomics
a b s t r a c t
In vertebrates, retinoic acid (RA) is an important morphogenetic signal that controls embryonic development, as well as organ homeostasis in adults. RA action depends on the function of the RA-genetic
machinery, which includes a metabolic module and a signaling module. The metabolic module regulates
the spatiotemporal distribution of RA by the combined action of the RA-synthesizing Aldh1a enzymes,
and the RA-degrading Cyp26 enzymes. The signaling module includes members of the nuclear hormone
receptors family RAR and RXR, and controls the transcriptional state of RA-target genes. RA-signaling has
been described primarily in chordates, but the recent finding of elements of the RA-genetic machinery
in non-chordate deuterostomes has changed our perspective on the evolutionary origin of this morphogenetic signal, challenging previous assumptions that related the invention of the RA-genetic machinery
with the origin of the chordate body plan. To illuminate the evolutionary origin of the RA machinery we
have conducted an extensive survey of Aldh1a, Cyp26 and RAR orthologs in genomic databases of 13 nondeuterostome metazoans. Our results show for the first time the presence of Aldh1a, Cyp26 and RAR in
protostomes, which implies that the components of the RA machinery may be ancient elements of animal
genomes, already present in the last common ancestor of bilaterians. Interestingly, our data also reveal
that independent losses of the RA toolkit have occurred multiple times during animal evolution, which
may have been relevant for the evolution and developmental diversity of the current metazoan lineages.
© 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Retinoic acid (RA), a bioactive derivative of vitamin A, is a signaling molecule that regulates the expression of genes involved
in the development of vertebrate embryos, as well as in numerous
physiological processes in adults (reviewed in [1]). In humans, as in
other vertebrates, the alteration of the RA-signaling system causes
congenital malformations, fertility problems and vision defects,
and can lead to tumorigenesis and neurodegenerative disorders
(reviewed in [2]). For instance, an excess of RA during embryonic development induces homeotic transformations in the axial
patterning and changes the fate of anterior structures into more
posterior ones, a phenotype known as RA-induced posteriorization
[3], which has been hitherto reported only in species of the chordate
Abbreviations: Aldh, aldehyde dehydrogenase; Cyp, cytochrome P450 enzyme;
NR, nuclear hormone receptor; RA, all-trans retinoic acid; RAR, retinoic acid receptor;
RXR, retinoid X receptor; RARE, retinoic acid response element.
∗ Corresponding author.
E-mail addresses: [email protected] (R. Albalat), [email protected]
(C. Cañestro).
phylum. Because the genetic machinery that mediates the RAsignaling had previously been described only in chordates, it was
hypothesized that the invention of the RA machinery (i.e., Aldh1a1,
Cyp26 and RAR) was an evolutionary milestone in the origin of the
chordate body plan (reviewed in [4,5]). Recent extensive surveys
of genomic databases, however, have challenged this hypothesis
after revealing the presence of the key players of the RA machinery
in non-chordate deuterostomes [6–8]. Interestingly, phylogenetic
analysis showed that the RA-genetic machinery diversified substantially in different animal lineages due to recurrent extensive
gene duplications and losses [6]. Such diversification raised the
possibility that interspecific differences in the RA machinery could
be related to morphological and developmental changes in extant
deuterostomes [6].
The RA machinery consists of two basic modules: a metabolic
module and a signaling module. The metabolic module controls
the spatiotemporal levels of RA by the coordinated action of RAproducing and RA-degrading enzymes. This module includes the
retinaldehyde dehydrogenase enzymes (Aldh1a, formerly Raldh)
and the cytochrome P450 enzymes Cyp26 [9]. Vertebrate Aldh1a
enzymes are cytosolic proteins that catalyze the irreversible oxidation of retinal to its carboxylic acid, retinoic acid (reviewed in [10]).
0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2008.09.017
Please cite this article in press as: R. Albalat, C. Cañestro, Identification of Aldh1a, Cyp26 and RAR orthologs in protostomes pushes back the
retinoic acid genetic machinery in evolutionary time to the bilaterian ancestor, Chem. Biol. Interact. (2008), doi:10.1016/j.cbi.2008.09.017
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CBI-5725; No. of Pages 9
2
ARTICLE IN PRESS
R. Albalat, C. Cañestro / Chemico-Biological Interactions xxx (2008) xxx–xxx
Vertebrates have three Aldh1a paralogs, named Aldh1a1, Aldh1a2
and Aldh1a3, with the exception of rodents that have an additional Adh1a enzyme (Aldh1a4 in rat, and Aldh1a7 in mouse) [11]
and teleosts, which appear to have only two forms (Aldh1a2 and
Aldh1a3) [6,12]. Outside vertebrates, an extensive catalog of Aldh1a
enzymes has recently been identified in cephalochordates, urochordates and hemichordates, showing for the first time the presence
of the Aldh1a family in non-chordate organisms [6]. The Cyp26
hydroxylases of the cytochrome P450 superfamily are endoplasmic reticulum enzymes that transform RA to biologically inactive
metabolites such as 4-hydroxy-RA and 4-oxo-RA. Typically, vertebrates have three Cyp26 enzymes, Cyp26A1, Cyp26B1 and Cyp26C1,
which are responsible for catabolism of RA in embryonic and adult
tissues [13–16]. Outside vertebrates, Cyp26 enzymes have been
identified in cephalochordates, urochordates, hemichordates and
echinoderms [6,17].
The RA-signaling module is responsible for the regulation of
gene expression in a ligand-dependent mode. RA activates heterodimers of a retinoic acid receptor/retinoid X receptor (RAR/RXR),
which bind to retinoic acid response elements (RARE) in the regulatory regions of direct target genes. RAR and RXR are members of
the nuclear hormone receptor (NR) superfamily of ligand-activated
transcription factors [18]. Vertebrates possess three different RAR
(␣, ␤ and ␥) and three RXR (␣, ␤ and ␥) whereas cephalochordates and urochordates have a single representative of each type of
receptor [6–8,17–20]. The only non-chordate RAR so far identified
belongs to the sea urchin Strongylocentrotus purpuratus [6–8,21],
and no RAR orthologs have been reported in any protostome
species. Conversely, orthologs of the RXR, a promiscuous receptor
capable of forming heterodimers with many other NR such as the
thyroid hormone receptor (THR), the vitamin D receptor (VDR) or
the ecdysone receptor (EcR), have been identified in nearly all animals analyzed. Therefore, in contrast to the RAR that specifically
binds RA to mediate gene transcription regulation, the identification of RXR is not diagnostic of the presence of an RA-signaling
system in a given organism.
The discovery of Aldh1a, Cyp26 and RAR homologues in
non-chordate deuterostomes [6] led us to wonder whether the
RA-genetic machinery was an innovation of the deuterostome
lineage or, alternatively, whether it had a deeper evolutionary
origin. To answer this question, in this work we have screened
13 metazoan genomes for Aldh1a, Cyp26 and RAR orthologs as a
diagnostic feature of the presence of a RA-genetic machinery in
non-deuterostome animals. Our results show for the first time
the presence of the three main components of RA machinery in
some protostome lineages. This finding pushes back in evolutionary time our understanding of the origins of the RA machinery and
reveals that it was already present in the last common ancestor of
the bilaterians. Interestingly, independent losses of the RA toolkit
appear to have happened in the two main branches of protostomes
(i.e., lophotrochozoans and ecdysozoans), supporting the possibility that the diversification of the RA-genetic machinery could have
favored the evolution and the developmental diversity of animals.
2. Materials and methods
2.1. Sequence analyses and gene identification
Human ALDH1A, ALDH2, ALDH1L, CYP26A, CYP51, CYP4V2,
RARA, RXRA and THRA, and D. melanogaster Eip75B and EcR protein
sequences were used as starting queries for TBLASTN searches [22]
against public genomic and EST databases of 13 animal species,
including lophotrochozoans such as the annelids Capitella sp.
(coverage 7.9-fold) and Helobdella robusta (coverage 7.9-fold), the
mollusc Lottia gigantea (coverage 8.9-fold); ecdysozoans such as the
platyhelminth Schistosoma mansoni (coverage 7.0-fold), the arthropods Anopheles gambiae (African malaria mosquito) (coverage
10.2-fold), Apis mellifera (honeybee) (coverage 7.5-fold), Drosophila
melanogaster (fruit fly), Tribolium castaneum (red flour beetle)
(coverage 7.3-fold) and Daphnia pulex (common water flea) (coverage 8.7-fold), the nematodes Brugia malayi (coverage 9.0-fold)
and Caenorhabditis elegans; and the cnidarian Nematostella vectensis (starlet sea anemone) (coverage 7.8-fold) and the placozoan
Trichoplax adhaerens (coverage 8.1-fold). Automatically annotated
genes were revised and edited manually to maximize the similarity with available ESTs or with human proteins. Accession numbers
of the genes and database URLs used in this work are provided in
Table 1.
The orthology of the proteins was inferred initially by reciprocal best BLASTP searches against human and Drosophila protein
databases [23], and confirmed by phylogenetic reconstructions. For
the Aldh family, in addition to the reciprocal BLASTP approach,
we combined phylogenetic analysis with other types of information with low homoplasy, such as differences in exon–intron
organization and the presence or absence of specific sequence
motifs (e.g., N-terminal signaling sequences that determine the
subcellular localization of the Aldh enzymes), to unambiguously
distinguish between Aldh1a and Aldh2 families [6]. Gene structures were deduced by merging the genomic sequences with
ESTs when available, or by comparison with well characterized Aldh genes described in other species. The iPSORT program
(http://hc.ims.u-tokyo.ac.jp/iPSORT/) was used to predict the subcellular localization of the deduced enzymes [24].
2.2. Phylogenetic analysis
Protein sequence alignments were generated with clustalX [25].
Only the regions rendering unambiguous alignments among paralogs were considered for the phylogenetic analysis: from codon
I40 to I513 of human ALDH1A2 for Aldh alignment; from codon
S74 to E156 and from I236 and S417 of human RARA for nuclear
receptor alignment; and from codon P45 to F490 of human CYP26A
for cytochrome P450 alignment. A neighbor-joining (NJ) phylogenetic tree was constructed and visualized with NJPlot and Unrooted
programs [26]. Confidence in each node was assessed by 1000 bootstrap replicates. Maximum-likelihood (ML) analysis was performed
using the PHYML 2.4.5 program [27] from the MacGDE package, following the JTT model of amino acid substitution [28], estimating the
amino acid frequencies from the data set and taking into account
the among-site rate heterogeneity with four gamma-distributed
categories. The confidence of each node was assessed by 500 bootstrap replicates.
3. Results
3.1. Identification of Aldh1a and Aldh2 in protostomes
To unambiguously distinguish Aldh1a enzymes from the closely
related mitochondrial Aldh2 enzymes, we used a recently proposed
integrative approach that combines phylogenetic reconstructions,
identification of Aldh-family-specific signatures (the exon–intron
organization of the Aldh genes) and the prediction of the subcellular localization of the deduced proteins [6]. Aldh1a genes lack intron
4 but include an extra intron 12b, whereas the opposite – the presence of intron 4 and absence of 12b – is characteristic of Aldh2 genes.
Aldh1a proteins are cytosolic enzymes, whereas Aldh2 localize in
the mitochondria. During our in silico screening, identification of
the Aldh1L genes, the next most closely related Aldh family, was
considered evidence that the complete catalog of Aldh1a and Aldh2
genes present in the databases had been retrieved.
Please cite this article in press as: R. Albalat, C. Cañestro, Identification of Aldh1a, Cyp26 and RAR orthologs in protostomes pushes back the
retinoic acid genetic machinery in evolutionary time to the bilaterian ancestor, Chem. Biol. Interact. (2008), doi:10.1016/j.cbi.2008.09.017
Aldh2
Aldh1L
Cyp26
Cyp51
Cyp4
RAR
THR
Eip75B-Ppar
EcR-Lxr
RXR
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
151890
145966
159056
+
183731
+
228199
+
173561
+
+
+
168520
+
167148
+
62897
+
125155
+
164614
Helobdella robustac
186284
194011
108094
+
212322
150007
95922
129163
nf
157030
+
nf
nf
67463
108893
186353
Lottia gigantead
+
207312
164897
+
157947
+
198550
+
232238
111018
+
205907
+
+
+
142757
+
224598
+
136477
+
170342
+
206562
+
Smp 050390
+
Smp 022960
−
nf
−
nf
−
nf
+
+
−
nf
+
AAR29358
AAR29359
+
AAR30507
−
nf
+
AAD16119
AAD45325
+
XP 313331
XP 319075
XP 392104 + XP 623252
AAF56646
XP 970835
215225
305826
+
XP 313425
+
XM 318614
−
nf
−
nf
+
+
−
nf
+
nf
+
XP 320316
+
XP 320323
+
XP 320944
XP 623084
NP 609285
XP 967960
318586
XP 623798
NP 610107
XP 969916
326816
nf
nf
nf
nf
nf
nf
nf
nf
+
+
+
+
nf
nf
nf
nf
nf
nf
nf
316465
NP 001073579
NP 730321
XP 971362
205205
NP 001091685
NP 724456
NP 001107650
319648
301472
NP 001011634
NP 476781
NP 001107766
219609
−
nfh
nf
+
nfh
NP 503467 NP 498081
+
nfh
NP 502054
−
nf
nf
−
nf
nf
+
+
nf
−
nf
nf
+i
nf
nf
+
XP 001899570
Q9XUK7
+
ABQ28713
nf
+
ABQ28715
nf
Mollusca
Platyhelminthes
Schistosoma mansonie
Ecdysozoa
Arthropoda
Anopheles gambiaef
Apis melliferaf
Drosophila.melanogasterf
Tribolium castaneumf
Daphnia pulexg
Nematoda
Brugia malayif
Caenorhabditis elegansf
Abbreviations: Aldh: Aldehyde dehydrogenase; Cyp: Cytochrome P450 enzyme; RAR: Retinoic acid receptor; THR: Thyroid hormone receptor; Eip75B: Ecdysone-induced protein 75B; Ppar: Peroxisome proliferator activated
receptor; EcR: Ecdysone receptor; Lxr: Liver X receptor; RXR: Retinoid X receptor.
a
Chordate and Ambulacraria sequences are those reported in [6]. Ambulacraria LXR and PPAR correspond to the predicted sea urchin XP 779997 and XP 781750 proteins, respectively.
b
JGI Capitella sp. I v1.0: http://genome.jgi-psf.org/Capca1/Capca1.home.html.
c
JGI H. robusta v1.0: http://genome.jgi-psf.org/Helro1/.
d
JGI L. gigantea v1.0: http://genome.jgi-psf.org/Lotgi1/Lotgi1.home.html.
e
GeneDB S. manosoni: http://www.genedb.org/genedb/smansoni/blast.jsp.
f
NCBI: www.ncbi.nlm.nih,gov.
g
JGI D. pulex v1.0: http://genome.jgi-psf.org/Dappu1/Dappu1.home.html.
h
Three partial sequences similar to Aldh1a, Aldh2 and Aldh1L enzymes can be found in the incomplete B. malayi genome project (TIGR database: http://www.tigr.org/tdb/e2k1/bma1/); accession numbers: BRBFS81TR,
BRQGH23TF and BRNJU76TF.
i
Schmidtea mediterranea THRa and THRb sequences reported in [56].
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Species
BILATERIANS
Deuterostomia
Chordataa (Vertebrata + Urochordata + Cephalochordata)
Ambulacrariaa (Hemichordata + Echinodermata)
Lophotrochozoa
Annelida
Capitella sp.b
R. Albalat, C. Cañestro / Chemico-Biological Interactions xxx (2008) xxx–xxx
Please cite this article in press as: R. Albalat, C. Cañestro, Identification of Aldh1a, Cyp26 and RAR orthologs in protostomes pushes back the
retinoic acid genetic machinery in evolutionary time to the bilaterian ancestor, Chem. Biol. Interact. (2008), doi:10.1016/j.cbi.2008.09.017
Table 1
Phylogenetic distribution of RA-genetic machinery and related sequences.
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Table 2
Invertebrate Aldh1a and Aldh2. Accession numbers and family signatures.
Species
Gene
Accession number
pMTP
I4/I12b
Capitella sp.
CspAldh1a1/2/3a
CspAldh1a1/2/3b
CspAldh1a1/2/3c
CspAldh2
HrAldh1a 1/2/3
HrAldh2
LgAldh1a1/2/3a
LgAldh1a1/2/3b
LgAldh2
Pm˝-crystallin
SmAldh1a1/2/3
SmAldh2
AgAldh1a1/2/3a
AgAldh1a1/2/3b
AgAldh2
AmAldh1a1/2/3
AmAldh2
DmAldh1a1/2/3
DmAldh2
TcAldh1a1/2/3
TcAldh2
DpAldh1a1/2/3a
DpAldh1a1/2/3b
DpAldh2
CeAldh2a
CeAldh2b
NvAldh1a-2
NvAldh1a-2
NvAldh1a-2
TaAldh1a-2
TaAldh1a-2
TaAldh1a-2
151890
145966
159056
183731
186284
194011
207312
164897
157947
AAF73122
Smp 050390
Smp 022960
XP 313331
XP 319075
XP 313425
XP 392104+XP 623252
XP 623084
AAF56646
NP 609285
XP 970835
XP 967960
215225
305826
318586
NP 503467
NP 498081
245626
181421
179476
37388
35686
63774
−
−
−
+
−
+
−
−
+
−
−
−
−
−
+
−
+
−
+
−
+
−
−
+
−
+
−
−
+
−
−
−
−/+
−/+
−/+
+/−
−/+
+/−
−/+
−/+
+/−
−/+
−/+
+/−
na
na
na
−/+
−/−
na
na
−/+
−/−
−/+
−/+
−/−
na
na
−/+
−/+
−/+
+/+
+/+
+/+
Helobdella robusta
Lottia gigantea
Placopecten magellanicus
Schistosoma mansoni
Anopheles gambiae
Apis mellifera
Drosophila melanogaster
Tribolium castaneum
Daphnia pulex
Caenorhabditis elegans
Nematostella vectensis
Trichoplax adhaerens
pMTP: presence (+) or absence (−) of a mitochondrial-targeting peptide based on iPSORT predictions.
I4/I12b: presence (+) or absence (−) of the family-specific introns 4 and 12b in Aldh genes; na, not applicable because gene structure is completely reorganized.
To recognize the protostome Aldh1a, we identified all the putative Aldh1a and Aldh2 genes in the genomic databases of eleven
lophotrochozoan and ecdysozoan species, which covered five different phyla (Tables 1 and 2). Our genomic survey yielded a
total of twenty-five Aldh sequences (Table 1). Phylogenetic analyses revealed that each protostome Aldh grouped either into the
Aldh1a or Aldh2 families. Significantly, we found that at least one
representative of both Aldh families was identified in most of
the species analyzed (Fig. 1). Maximum-likelihood and neighborjoining methods for tree construction yielded the same topology
defining the two families of enzymes. Phylogenetic assignment of
the protostome enzymes was consistent with the Aldh1a and Aldh2
family-specific signatures (Fig. 2) and with the prediction of subcellular localization for the new protostome enzymes (Table 2),
which provided further support for the orthology relationships of
the enzymes. In lophotrochozoans, seven Aldh1a proteins (three
from Capitella sp., one from H. robusta, two from L. gigantea and
one from S. mansoni) were predicted to be cytosolic (Table 2), and
their genes showed the Aldh1a signature (i.e., absence of intron
4 but presence of intron 12b) (Fig. 2). The opposite situation –
i.e., prediction of mitochondrial localization, and the presence of
intron 4 and absence of 12b – was found in four putative Aldh2
sequences in the same species (Table 2 and Fig. 2). Additional
lophotrocozoan Aldh sequences from other species whose genomes
have not been sequenced were included in the analysis. One Aldh
enzyme from Enchytraeus buchholzi (earthworm) (CAA64680) and
the -crystallins of Octopus dofleini (giant octopus) (AAA29392),
Ommastrephes sloani (arrow squid) (AAA29406) and Placopecten
magellanicus (sea scallop) (AAF73122) had been reported to be
evolutionarily related to the Aldh1a-Aldh2 enzymes [29]. Our phylogenetic analysis suggested that these proteins belong to the
cytoplasmic Aldh1a family (Fig. 2). Consistent with this result,
-crystallins lack the mitochondrial-targeting signal ([29] and references therein), and the structure of the sea scallop gene [30]
concurs with the Aldh1a-signature (Fig. 2 and Table 2). Overall,
our analysis revealed the presence of Aldh1a and Aldh2 forms in
lophotrochozoan species.
In ecdysozoans, Aldh exon–intron organizations were more variable than in lophotrocozoans, but some sequences still showed a
gene structure informative for family assignment. One Aldh gene
from A. mellifera, one gene from T. castaneum and two genes from
D. pulex showed the Aldh1a signature (Fig. 2). These genes encode
for proteins predicted to localize in the cytosol (Table 2), and
in phylogenetic reconstructions, grouped together with the other
invertebrate Aldh1a enzymes (Fig. 1). In addition, A. mellifera, T. castaneum and D. pulex genomes contained one Aldh gene that partially
retained the Aldh2 signature (absence of intron 12b), encoded for
enzymes predicted to have a mitochondrial localization, and clustered within the Aldh2 group in the phylogenetic tree. Therefore,
the overall analysis allowed us to convincingly assign each Apis, Tribolium and Daphnia Aldh to either the Aldh1a or the Aldh2 families.
The Aldh nature of the genes from D. melanogaster (2 genes), A. gambiae (3 genes) and C. elegans (2 genes) (Table 1) was inferred from
their position in the evolutionary tree (Fig. 1) and from their predicted subcellular localization (Table 2). The exon–intron structure
of the Aldh genes in such species was poorly conserved and it could
not be used for family assignment. One D. melanogaster and two A.
gambiae proteins clustered with the ecdysozoa Aldh1a forms and
were predicted to be cytoplasmic, while one D. melanogaster, one A.
gambiae and the two C. elegans sequences grouped within the invertebrate Aldh2 and were predicted to be mitochondrial (except C.
elegans Aldh2a; Table 2). Thus, based on the phylogenetic relation-
Please cite this article in press as: R. Albalat, C. Cañestro, Identification of Aldh1a, Cyp26 and RAR orthologs in protostomes pushes back the
retinoic acid genetic machinery in evolutionary time to the bilaterian ancestor, Chem. Biol. Interact. (2008), doi:10.1016/j.cbi.2008.09.017
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5
Fig. 1. Phylogenetic analysis of Aldh1a and Aldh2 families reveals the presence of the Aldh1a in protostomes. The unrooted phylogenetic tree was generated by the neighborjoining (NJ) method based on the clustalX alignment from I40 to I513 of human ALDH1A2. The same tree topology was obtained by the maximum-likelihood (ML) method.
Figures at the nodes are the percentage of bootstrap values supporting the Aldh1a and Aldh2 families, and vertebrate Aldh1a and Aldh2 groups (n = 1000 for NJ, and n = 500
for ML (underlined)). Species abbreviations: Ag, A. gambiae; Am, A. mellifera; Bf, B. floridae; Ce, C. elegans; Csp, Capitella sp.; Dm, D. melanogaster; Dp, D. pulex; Dr, D. rerio; Eb,
E. buchholzi; Gg, G. gallus; Hr, H. robusta; Hs, H. sapiens; Lg, L. gigantea; Mm, M. musculus; Od, O. dofleini; Os, O. sloani; Pm, P. magellanicus; Rn, R. novergicus; Sm, S. mansoni;
Sp. S. purpuratus; Tc, T. castaneum; Tr, T. rubripes; Xt, X. tropicalis.
ships and the predictions of subcellular localization, fly, mosquito
and worm enzymes were classified into Aldh1a and Aldh2 families. In conclusion, our analysis provided robust evidence for the
presence of orthologs to the deuterostome Aldh1a genes in the
two main branches of protostomes, lophotrochozoans and ecdysozoans, and therefore indicated that the origin of Aldh1a predated
the deuterostome–protostome split.
3.2. Identification of Cyp26 enzymes in protostomes
No Cyp26 proteins had been previously identified outside
deuterostomes. To discover whether protostomes have Cyp26
orthologs, we analyzed the same set of protosome genomes used
in the Aldh analysis. In lophotrochozoans, we identified four Cyp26
sequences in the Capitella sp. genome and two in L. gigantea (Table 1
and Fig. 3), while no Cyp26 genes were found in H. robusta or S.
mansoni. Orthologies, initially established by the reciprocal BLAST
method [23], were corroborated by phylogenetic analyses (Fig. 3).
Members of the Cyp51 family (the closest family to Cyp26) and
other cytochrome P450 enzymes such as Cyp4 forms were searched
for and used as outgroups in the phylogenetic analyses. Interestingly, our surveys revealed that none of the seven ecdysozoan
species (A. gambiae, A. melliferea, D. melanogaster, T. castaneum, D.
pulex and C. elegans) had convincing Cyp26 orthologs. Identification
of more distant cytochrome P450 genes in the BLAST searches using
Cyp26 and Cyp51 proteins as starting queries was considered as
evidence that Cyp26 genes have either been lost or their sequences
have diverged beyond recognition in the species analyzed.
3.3. Identification of RAR, RXR and other nuclear hormone
receptors
Heterodimers of RAR and RXR mediate RA-signaling. Because
RXR have been identified in the most basal metazoan phyla, such
as Porifera [31,32] and Cnidaria [33], the presence of RXR orthologs
in most animal species was expected. In accordance with this prediction, RXR orthologs were found in all species analyzed, except in
C. elegans (Table 1), in which the lack of RXR orthologs had already
been reported [34,35].
In contrast, RAR homologs had hitherto been described only
in deuterostomes [6–8]. To determine whether or not nondeuterostomes possess RAR orthologs, we surveyed the genomic
databases of the eleven protostome species analyzed in this
work. We found, for the first time, putative RAR genes outside deuterostomes: in the annelid Capitella sp. and in the
mollusc L. gigantea (Table 1). The orthology between deuterostome and protostome RAR was initially based on the reciprocal
BLAST method [23] and was robustly supported by phylogenetic
analyses (Fig. 3). Members of the thyroid hormone receptors
(THR), the closest related receptors to the RAR family, were also
identified in Capitella sp. and L. gigantea genomes (Table 1),
further supporting the RAR nature of the newly identified
genes. Additional invertebrate members of the subfamily I of
nuclear receptors such as the ecdysone-induced proteins 75B
(Eip75B), which are related to vertebrate peroxisome proliferator activated receptors (PPAR), and the ecdysone receptors (EcR),
which are closely related to the vertebrate liver X receptors
Please cite this article in press as: R. Albalat, C. Cañestro, Identification of Aldh1a, Cyp26 and RAR orthologs in protostomes pushes back the
retinoic acid genetic machinery in evolutionary time to the bilaterian ancestor, Chem. Biol. Interact. (2008), doi:10.1016/j.cbi.2008.09.017
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Fig. 2. Comparison of the gene structure of Aldh1a and Aldh2 families across bilaterians reveals the presence of family-specific intron positions, and validates the use of the
exon–intron organization as a homoplastic feature for family assignments [6]. Chordate gene structures are represented by H. sapiens ALDH1A1, urochordate C. intestinalis
Aldh1a1/2/3a and cephalochordate B. floridae Aldh1a1/2/3e genes. For clarity, intron nomenclature (1–12, 12b and 13) is as in [6]. The positions of the introns 4 and 12b, which
define the Aldh-family signatures, are indicated by black arrowheads on a gray background; conserved intron positions are indicated with white arrowheads; lineage-specific
intron positions are indicated with gray arrowheads (not numbered).
Fig. 3. Phylogenetic analysis of Cyp26 and RAR. Neighbor-joining (NJ) and maximum-likelihood (ML) methods yielded the same tree topologies. Figures at the nodes are
the percentage of bootstrap values supporting each node (n = 1000 for NJ, and n = 500 for ML, in italics; poorly supported nodes <50% were collapsed). The tree of the Cyp
enzymes was rooted with the Cyp4v2 sequences, and the tree of the RAR, with the RXR sequences. Capitella sp. and L. gigantea Cyp26 and RAR sequences clearly grouped
with deuterostome Cyp26 and RAR proteins, supporting their orthology to the deuterostome proteins.
Please cite this article in press as: R. Albalat, C. Cañestro, Identification of Aldh1a, Cyp26 and RAR orthologs in protostomes pushes back the
retinoic acid genetic machinery in evolutionary time to the bilaterian ancestor, Chem. Biol. Interact. (2008), doi:10.1016/j.cbi.2008.09.017
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Fig. 4. The phylogenetic distribution of the RA-genetic machinery (black boxes) in protostomes and deuterostomes indicates that it was already present in the bilaterian
ancestor. The lack of RA-genetic machinery (empty box) and the presence of closely related genes (grey boxes) are also depicted. The family identity of cnidarian and placozoan
Aldh proteins was not conclusive (half filled boxes).
(LXR) [36], were also identified in most of the phyla analyzed
(Table 1).
4. Discussion
4.1. Origin of the RA-genetic machinery
The identification of Aldh1a, Cyp26 and RAR orthologs in nonchordate deuterostomes [6] pushed back in the evolutionary ladder
the origin of the RA-genetic machinery and challenged the relationship between the invention of this machinery and chordate-specific
innovations in the body plan. To further investigate the evolutionary origin of the RA machinery, we analyzed a catalogue of eleven
non-deuterostome genomes, searching for Aldh1a, Cyp26 and RAR
orthologs as diagnostic features of the RA-genetic machinery in the
protostome lineage. Our finding of clear orthologs of the Aldh1a,
Cyp26 and RAR families in several protostome species (Table 1)
pushes back once again the origin of the RA-genetic machinery
and implies that these genes already existed in the last common
ancestor of bilaterians (Fig. 4).
In an attempt to narrow down the origin of the RA machinery
during animal evolution, we explored available genomic databases
of non-bilaterian organisms such as the cnidarian N. vectensis and
placozoan T. adhaerens. Three putative Aldh1a-Aldh2 sequences
were identified in the N. vectensis genome (Table 2), but no Cyp26
or RAR orthologs were recognized in the database. In T. adhaerens,
three Aldh1a-Aldh2 (Table 2) and one Cyp26 (ID 60776) sequences
were found, but no RAR orthologs were identified. Although neither
of the two non-bilaterian animals analyzed in this work have a full
RA toolkit, the taxonomic diversity is still too narrow to draw firm
conclusions about whether a complete RA-genetic machinery could
exist in stem metazoans. We also analyzed the genomic database
of the choanoflagellate Monosiga brevicollis, the closest unicellular
relative of animals [37,38]. The absence of any convincing Aldh1a,
Cyp26 and RAR genes in M. brevicollis suggested that this organism
lacks the RA-genetic machinery (Fig. 4), which is consistent with the
absence of members of the NR superfamily in choanoflagellates [39]
and in any other non-metazoan organism [19,40]. Overall, our findings suggest that the RA-genetic machinery might be confined to
metazoans, although a definite answer must await larger databases
and broader phylogenetic samplings.
4.2. Divergent patterns of conservation of the RA machinery
during protostome evolution
Our genomic survey reveals that protostome species have
retained the components of RA machinery (i.e., Aldh1a, Cyp26,
RAR orthologs) to different extents. Some species, principally from
lophotrochozoa, have preserved most of the elements while other
species, especially from ecdysozoa, have lost nearly all the components. The cases of the lophotrochozoans Capitella sp. and L.
gigantea are particularly interesting, because the finding of complete RA-gene toolkits in their genomes provides for the first time
a mechanistic framework for the presence of a classic RA-signaling
system in protostomes, and raises the possibility of functional
conservation of the RA-signaling system between chordate and
non-chordate species. In this regard, it has been reported that in
the lophotrochozoan Lymnaea stagnalis (great pond snail), RA acts
as a trophic factor and a chemotactic molecule [41] and affects eye
formation [42]. Similarly, RA induces neurogenesis and neurite differentiation, and participates in eye morphogenesis in vertebrates
[43–46].
RA machinery has been lost or modified beyond recognition
in many metazoan species. First, none out of seven ecdysozoan
species possess Cyp26 and RAR orthologs; second, Cyp26 and RAR
genes have also been lost in the lophotrochozoans H. robusta (phylum Annelida) and S. mansoni (phylum Platyhelminthes); and third,
Aldh1a, Cyp26 and RAR genes have been lost during the evolution of
the chordate Oikopleura dioca [6]. The phylogenetic distribution of
these species implies that such losses have occurred independently
during animal evolution. Moreover, the absence of the RA toolkit
implies that RA does not play the classical morphogenetic role in
these animals. This prediction has been experimentally tested in O.
dioca, in which it has been shown the lack of the classical role for RA
in anterior-posterior axial patterning during embryo development
[47,48].
It is worth mentioning that although some protostome species
lack Cyp26 and RAR genes, most of them retain Aldh1a orthologs. In
vertebrates, in addition to the classical RAR-mediated RA-signaling,
RA participates in other functions by the direct binding to other proteins (e.g., protein kinase C alpha) [49,50] for which a source of RA
might be required. Moreover, Nowickyj et al. have recently shown
that in vitro, RXR from the primitive insect Locusta migratoria binds
9-cis-RA and all-trans-RA with high affinity, and that embryonic
Please cite this article in press as: R. Albalat, C. Cañestro, Identification of Aldh1a, Cyp26 and RAR orthologs in protostomes pushes back the
retinoic acid genetic machinery in evolutionary time to the bilaterian ancestor, Chem. Biol. Interact. (2008), doi:10.1016/j.cbi.2008.09.017
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extracts contain significant amounts of endogenous 9-cis- and alltrans-RA [51]. Therefore, although we do not yet know whether RA
plays similar non-RAR mediated roles in other invertebrate species,
this possibility needs to be considered when analyzing the functionality of the Aldh1a enzymes in protostomes. Moreover, it has
to be remembered that orthology does not necessarily imply functional conservation, and that Aldh1a enzymes could have acquired
different activities other than RA synthesis [52]. In this regard,
the -crystallins of molluscs are paradigmatic examples because
although they belong to the Aldh1a family, they lack retinaldehyde
dehydrogenase activity, do not bind NAD or NADP cofactors, and
have been recruited to be preferentially expressed in the invertebrate eye lens [30,53–55]. Therefore, the biochemical activities of
the different protostome Aldh1a identified here, and whether or not
they are involved in RA metabolism, deserve further investigation.
In summary, our data provide robust evidence that the RAgenetic machinery is an old evolutionary device that arose before
the divergence of the major animal phyla, and reveal that the RAgenetic machinery has been differently preserved, lost, recruited
and modified during animal evolution to befit the diverse physiological and developmental requirements of different organisms.
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
Conflict of interest
[22]
The authors declare that there are no conflicts of interest.
[23]
Acknowledgements
This material is based on work supported by Universitat de
Barcelona Grant MC064448 to RA and NSF Grant IOB-0719577 to
CC.
In agreement with our conlusions, Campo-Paysaa and colleagues reviewed the RA signalling in several invertebrate groups,
reaching to similar inferences on the ancient origin of the RA
machinery: F. Campo-Paysaa, F. Marlétaz, V. Laudet, M. Schubert.
Retinoic acid signaling in development: Tissue-specific functions
and evolutionary origins. Genesis (in press).
[24]
[25]
[26]
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retinoic acid genetic machinery in evolutionary time to the bilaterian ancestor, Chem. Biol. Interact. (2008), doi:10.1016/j.cbi.2008.09.017