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An In-silico Genomic Survey to Annotate Genes Coding for Early Development-Relevant Signaling Molecules in the Pearl Oyster, Pinctada fucataAuthor(s): Davin H. E. Setiamarga , Keisuke Shimizu , Junpei Kuroda , Kengo Inamura , Kei Sato ,Yukinobu Isowa , Makiko Ishikawa , Reo Maeda , Tomoyuki Nakano , Tomoko Yamakawa , RyoHatori , Akira Ishio , Kayo Kaneko , Kenjiroo Matsumoto , Isao Sarashina , Shinnosuke Teruya , RanZhao , Nori Satoh , Takenori Sasaki , Kenji Matsuno and Kazuyoshi EndoSource: Zoological Science, 30(10):877-888. 2013.Published By: Zoological Society of JapanDOI: http://dx.doi.org/10.2108/zsj.30.877URL: http://www.bioone.org/doi/full/10.2108/zsj.30.877

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2013 Zoological Society of JapanZOOLOGICAL SCIENCE 30: 877–888 (2013)

An In-silico Genomic Survey to Annotate Genes Coding for Early

Development-relevant Signaling Molecules in the

Pearl Oyster, Pinctada fucata

Davin H. E. Setiamarga1*†, Keisuke Shimizu1†, Junpei Kuroda2‡, Kengo Inamura1,

Kei Sato1,3, Yukinobu Isowa1, Makiko Ishikawa1, Reo Maeda2, Tomoyuki Nakano4,

Tomoko Yamakawa2‡, Ryo Hatori2, Akira Ishio2, Kayo Kaneko2, Kenjiroo Matsumoto2,

Isao Sarashina1,3, Shinnosuke Teruya1,3, Ran Zhao1, Nori Satoh5,

Takenori Sasaki1,3, Kenji Matsuno2‡, and Kazuyoshi Endo1

1Department of Earth and Planetary Science, Graduate School of Science, The University of Tokyo,

7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan2Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki,

Noda, Chiba Prefecture 278-8510, Japan3The University Museum, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

4Seto Marine Biological Laboratory, Kyoto University, 459 Shirahama, Nishimuro,

Wakayama Prefecture 649-2211, Japan5Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University,

1919-1 Tancha, Onna, Okinawa 904-0495, Japan

The pearl oyster Pinctada fucata has great potential as a model system for lophotrochozoan devel-

opmental biology research. Pinctada fucata is an important commercial resource, and a significant

body of primary research on this species has emphasized its basic aquaculture biology such as

larval biology and growth, aquaculture, pearl formation and quality improvement, shell formation,

and biomineralization. Recently, a draft genome sequence of this species was published, and many

experimental resources are currently being developed, such as bioinformatics tools, embryo and

larva manipulation methods, gene knockdown technique, etc. In this paper, we report the results

from our genomic survey pertaining to gene families that encode developmental signaling ligands

(Fgf, Hedgehog, PDGF/VEGF, TGFββ, and Wnt families). We found most of the representative genes

of major signaling pathways involved in axial patterning, as well as copies of the signaling molecule

paralogs. Phylogenetic character mapping was used to infer a possible evolutionary scenario of the

signaling molecules in the protostomes, and to reconstruct possible copy numbers of signaling

molecule-coding genes for the ancestral protostome. Our reconstruction suggests that P. fucataretains the ancestral protostome gene complement, providing further justifications for the use of

this taxon as a model organism for developmental genomics research.

Key words: pearl oyster, signaling molecules, ancestral protostome, lophotrochozoans, early development

INTRODUCTION

Current progress in molecular developmental biology

has brought a deep understanding of the molecular under-

pinnings of animal morphogenesis. Although such research

has been performed on only a handful of ecdysozoans (the

fruit fly Drosophila melanogaster and the nematode

Caenorhabditis elegans) and vertebrates (the mouse Mus

musculus, the African clawed frog Xenopus tropicalis, the

chicken Gallus gallus, and the zebrafish Danio rerio), metic-

ulous molecular, genetic, and genomic dissections of the

development of such model systems have provided signifi-

cant information on the role of gene regulations and signal-

ing networks during animal embryonic development. We

now know that in metazoans, seven of about 20 signaling

pathways controlling cellular interactions and differentiations

are involved in the morphogenetic processes during embry-

onic development (Barolo and Posakony, 2002; Pires-da

Silva and Sommer, 2003; Gazave et al., 2009).

Although extrapolative and limited, information from

these model organisms has provided some insight into the

genetic mechanisms of metazoan body plan evolution.

* Corresponding author. Tel. : +81-3-5841-4553;

Fax : +81-3-5841-4555;

E-mail: dsetiamarga@eps.s.u-tokyo.ac.jp† These authors contributed equally.‡ Present address: Department of Biological Science, Osaka Uni-

versity, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan

Supplemental material for this article is available online.

doi:10.2108/zsj.30.877

D. H. E. Setiamarga et al.878

Recent progress in the field of evolutionary developmental

biology (evo-devo), which compares different modes of ani-

mal development and their evolution has revealed that a

basic core of signaling pathways is involved in the morpho-

genesis of all metazoans. Research utilizing non-model

experimental animals such as the acorn worm, amphioxus,

annelids, sea anemones, etc., have confirmed the presence

of at least some of the representative gene members of the

developmental signaling pathways in such animals (e.g.,

Kusserow et al., 2005; Saina et al., 2009; Dray et al., 2010;

Pani et al., 2012; Onai et al., 2012). Until relatively recently,

despite the current popularity of invertebrate evo-devo stud-

ies, developmental genomics research on lophotrochozoans

has remained arguably lacking. This may be due in part to

difficulties in obtaining sufficient numbers of fresh embryos,

lack of basic descriptive information and experimental tools,

and the paucity of genomic resources.

However, intensive studies on development and breed-

ing of some lophotrochozoan species has partially solved

the problem of obtaining fresh samples, while recent devel-

opments in sequencing technology have allowed for the

development of genomics tools. For example, one recent

report on spiralian genome sequences (Simakov et al.,

2013) shed further insights into the phylogeny and evolution

of metazoan at the genomic level. Some lophotrochozoans,

such as the pearl oyster, are cultured species, and therefore

have the potential to be developed as experimental model

systems. Recently, draft genome sequences of the Japanese

pearl oyster Pinctada fucata (Takeuchi et al., 2012) and the

oyster Crassostrea gigas (Zhang et al., 2012) have been

determined. The availability of these genome sequences

helps further development of these aquaculture lophotro-

chozoan species for developmental genomics and genome

evolution studies. In this report, we focus on the Japanese

pearl oyster P. fucata, as a part of the effort to annotate its

recently determined draft genome.

As a commercial species, the Japanese pearl oyster

Pinctada fucata has been cultured for more than 100 years

in Japan (Masaoka and Kobayashi, 2007). Its fertilized eggs

can be obtained constantly for ca. five months, and its con-

trolled fertilization and embryology have been intensely

studied, albeit from the aquaculture perspective (e.g.,

Fujimura et al., 1995). Abundant research on shell formation

and pearl development (e.g., Takeuchi et al., 2008; Fang et

al., 2011; McGinty et al., 2012) has been conducted, provid-

ing much insight into biomineralization and development. P.

fucata. EST libraries from an adult (Kinoshita et al., 2011)

and various developmental stages (Takeuchi et al., 2012)

have also been reported. The availability of aquaculture

information, experimental methods, recently available

genomic resources, and most importantly, copious fresh

embryos make this species ideal for development as a

model system for the investigation of early lophotrochozoan

development.

As one of the initial steps of providing basic information

needed to establish the Japanese pearl oyster as a model

system, we report the result of our genomic survey for early

development-relevant genes in the recently reported full

nuclear genome of the pearl oyster (Takeuchi et al., 2012).

We then focus on the signaling molecule gene families

involved in axial patterning (the FGF, Hedgehog, TGFβ,

VEGF, and Wnt families). Signaling molecules play impor-

tant roles in many morphogenetic events during various

stages of development, such as axis formation, muscle dif-

ferentiation, and nervous system development. These mole-

cules work by diffusing out from a signaling center, producing

a concentration gradient. Also known as morphogens, they

bind to receptors located on receptive surrounding cells,

inducing specific responses that depend upon the concen-

tration of the signaling molecules reaching them. Our results

reported herein indicate that most members of these signal-

ing molecule families are present in protostomes. We then

discuss the implications of our findings for interpretation of

the evolution of protostome signaling molecule genes

involved in their early development.

MATERIALS AND METHODS

Gene model searches and confirmations

We employed two different methods to identify Pinctada fucata

gene homologs. In the first, we obtained amino acid sequences of

the genes of interest from other organisms in GenBank. Retrieved

sequences were used as TBLASTN and BLASTP queries to search

the P. fucata gene models version 1.1 and the genome assembly

version 1.0. This analysis was conducted using the available

Genome Browser (http://marinegenomics.oist.jp/pinctada_fucata).

For the second method, we used the “Pfam domain search” function

available on the P. fucata Genome Browser.

Amino acid sequences of the obtained gene models from both

methods were then subjected to TBLASTN and BLASTP against

the NCBI non-redundant (nr) database for identification confirma-

tion. We also conducted TBLASTN and BLASTP searches against

the P. fucata transcriptome EST database, which is available at the

Genome Browser, to obtain additional confirmation of the gene

models. The EST sequence data were obtained from transcriptome

sequencing of several embryos and adults using Roche 454 Next

Generation Sequencer (Takeuchi et al., 2012).

To illuminate the evolution of the signaling molecule genes in

the protostomes, we browsed the available annotated draft

genomes of two lophotrochozoans (the gastropod Lottia gigantea

(Lg) and the polychaete Capitella teleta (Ct); Simakov et al., 2013),

for orthologous gene sequences.

Phylogenetic analyses of signaling molecule-coding genes

For phylogenetic analyses of signaling molecule genes with

multiple paralogous copies, we used sequences of the gene models

obtained from the P. fucata genome. We used EST sequences

when they showed a longer conserved domain sequence of the

gene than that predicted by gene models. We then used conserved

domain sequences from human (Hs) and fruit fly (Dm) obtained

from GenBank. We also obtained homologous sequences from the

draft genome of the two lophotrochozoans (the polychaete and the

limpet), by doing TBLASTN and BLASTP searches of genome

sequences using various sequence queries from mouse, fruit fly,

and molluscan sequences available in GenBank. We predicted their

conserved protein domains using SMART, and included the domain

sequences in our phylogenetic analyses.

Sequence alignments were conducted using the online version

of PROMALS3D, since this program allows users to input structural

constraints for known domains (Pei et al., 2008). Accordingly, we

first obtained “core” domain alignments from PROSITE (http://

prosite.expasy.org/) and used them as alignment constraints in

PROMALS3D. The obtained alignment was then edited manually by

using Mesquite v2.75 (Maddison and Maddison, 2011) or MEGA v5.0

(Tamura et al., 2011). Afterwards, we used MEGA v5.0 to search

for the best amino acid substitution model of the edited alignments.

In most occasions, the top four models suggested were WAG+G,

Signaling Molecule Genes in Pearl Oyster 879

Table 1. Various early developmental genes in Pinctada fucata.

gene namegene model

numberin assembly

gene model ID BLAST best hit (accession + species)

ADAM10 1 pfu_aug1.0_16539.1_68981 XP_003486941, [Bombus impatiens]

ADAM17 1 pfu_aug1.0_16.1_07317 XP_001647994, [Aedes aegypti]

ADMP 2 pfu_aug1.0_192.1_22046.t1 NP_001158394.1, [Saccoglossus kowalevskii]

Aldh1 1 pfu_aug1.0_12252.1_39303 XP_002736990.1, [Saccoglossus kowalevskii]

Aldh2 1 pfu_aug1.0_507.1_36564 ACL79834.1, [Lymnaea stagnalis]

BarH-like 1 1 pfu_aug1.0_13057.1_17948 XP_003446537.1, [Oreochromis niloticus]

BAMBI-like 1 pfu_aug1.0_5683.1_02051.t1 XP_002589511.1, [Branchiostoma floridae]

Brachyury 1 pfu_aug1.0_72.1_00130 ACI97437.1, [Lottia gigantea]

Cyp26-like 1 pfu_aug1.0_8297.1_24368 XP_003223822.1, [Anolis carolinensis]

Deltex 3-like 2 pfu_aug1.0_7873.1_02511 EHB07638.1, [Heterocephalus glaber]

pfu_aug1.0_9498.1_02758 XP_002732882.1, [Saccoglossus kowalevskii]

Engrailed 1 pfu_aug1.0_307148.1_36000 J03066.1, [Homo sapiens]

Forkhead 1 pfu_aug1.0_370.1_29216 AJ507424.1, [Patella vulgata]

Fringe 2 pfu_aug1.0_11483.1_24900 XP_002696180.1, [Bos taurus]

pfu_aug1.0_24112.1_11591 XP_002404236.1, [Ixodes scapularis]

Furin 1 pfu_aug1.0_16592.1_18358 NP_001191568.1, [Aplysia californica]

Gastrulation brain homeobox 1 pfu_aug1.0_2817.1_30257 XM_002609023.1, [Branchiostoma floridae]

Goosecoid 1 pfu_aug1.0_4752.1_16501 AJ507423, [Patella vulgata]

Hex 1 pfu_aug1.0_2973.1_59082 EU196052.1, [Patiria miniata]

Iroquois 1 pfu_aug1.0_17634.1_61730 XM_002400517.1, [Ixodes scapularis]

lhx1/5 1 pfu_aug1.0_5181.1_30887.t1 GU169421.1, [Platynereis dumerilii]

Mad-like 1 pfu_aug1.0_4230.1_16321 CAD90766.1, [Crassostrea gigas]

MAPK 2 pfu_aug1.0_2298.1_58856.t1 AAA83210.1, [Aplysia californica]

pfu_aug1.0_149.1_65261.t1 DAA25284.1, [Bos taurus]

MAPKK 1 pfu_aug1.0_17318.1_69071 NP_001135384.1, [Salmo salar]

Msx 1 pfu_aug1.0_13100.1_39443 BAF91569.1, [Corbicula fluminea]

MyoD 1 pfu_aug1.0_15323.1_39685.t1 AAC47320.1, [Trichinella spiralis]

Netrin-1 1 pfu_aug1.0_1643.1_58611 EF384215.1, [Platynereis dumerilii]

Neuralized 1 pfu_aug1.0_14515.1_61368.t1 XP_001507422, [Ornithorhynchus anatinus]

Nicastrin 1 pfu_aug1.0_10456.1_02919.t1 XP_003214000, [Meleagris gallopavo]

Notch 3 pfu_aug1.0_4087.1_16284 XP_002736950.1, [Saccoglossus kowalevskii]

pfu_aug1.0_812.1_22323 GU076071.1, [Saccoglossus kowalevskii]

pfu_aug1.0_9129.1_09893 AF000634.1, [Lytechinus variegatus]

Numb 1 pfu_aug1.0_88.1_57956 XP_001123108.2, [Apis mellifera]

O-fut1 2 pfu_aug1.0_6710.1_09406 CAH40835.1, [Bombyx mori]

pfu_aug1.0_4592.1_67023 EHJ67995.1, [Danaus plexippus]

Omb-like 1 pfu_aug1.0_294.1_36469 ACN66456.1, [Neanthes arenaceodentata]

Otx 1 pfu_aug1.0_595.1_65534 AF440098.1, [Patella vulgata]

Pax2/5/8 3 pfu_aug1.0_16816.1_54344 NM_001032480.1, [Ciona intestinalis]

pfu_aug1.0_19663.1_61932 XM_001363515.2, [Monodelphis domestica]

pfu_aug1.0_406.1_65408 AJ505023.1, [Platynereis dumerilii]

Pax6 3 pfu_aug1.0_30445.1_26527 AM422131.1, [Sepia officinalis]

pfu_aug1.0_8418.1_67856 NM_001168156.1, [Saccoglossus kowalevskii]

pfu_aug1.0_28617.1_26433 GU224214.1, [Saccoglossus kowalevskii]

Pecanex-like protein 1 1 pfu_aug1.0_3846.1_23422 XP_001368516.1, [Monodelphis domestica]

Pitx 2 pfu_aug1.0_7478.1_31393 ADI48168.1, [Crepidula fornicata]

pfu_aug1.0_7478.1_31392 AAH73479.1, [Xenopus laevis]

Presenilin 1 pfu_aug1.0_9933.1_68120 AAT07667.1, [Aplysia californica]

Retinal homeobox 1 pfu_aug1.0_1605.1_15546 XM_001379867.1, [Monodelphis domestica]

Serrate-like 1 pfu_aug1.0_20597.1_54689 XP_001850921.1, [Culex quinquefasciatus]

Single-minded 1 pfu_aug1.0_1455.1_37019 XP_002413286.1, [Ixodes scapularis]

Six1/2 1 pfu_aug1.0_806.1_22307 NM_001174113.1, [Ovis aries SIX homeobox]

Six4/5 1 pfu_aug1.0_8342.1_45842 EF195741.1, [Branchiostoma floridae]

Snail 1 pfu_aug1.0_5975.1_59906 AAL06240.1, [Patella vulgata]

Sox2 2 pfu_aug1.0_4847.1_59628 DQ917178, [Strongylocentrotus purpuratus]

pfu_aug1.0_802.1_00500 HQ840788, [Hyriopsis schlegelii]

Spalt-like 1 pfu_aug1.0_2082.1_22863 XP_003263028.1, [Nomascus leucogenys]

Sprouty 1 pfu_aug1.0_3628.1_66754 NP_001082238.1, [Xenopus laevis]

Tlx 1 pfu_aug1.0_2408.1_15802 ABQ10643.1, [Platynereis dumerilii]

Vent 1 pfu_aug1.0_9305.1_68025 NM_001168018.1, [Saccoglossus kowalevskii]

Vestigial 1 pfu_aug1.0_4300.1_16336 CBN80870.1, [Dicentrarchus labrax]

D. H. E. Setiamarga et al.880

WAG+G+I, JTT+G, and JTT+I+G. Whenever possible, we used the

best model suggested for subsequent phylogenetic analyses. Max-

imum Likelihood (ML) phylogenetic analyses were conducted using

the online version of RAxML (RAxML Blackbox; Stamatakis et al.,

2008; http://phylobench.vital-it.ch/raxml-bb/), with 100 bootstrap

replications.

Protein domain re-prediction using SMART for signaling mole-

cule genes

Although the Genome Browser provided a domain prediction

for all of its gene models, for further confirmation, we re-predicted

domain structures using the online version of the protein domain

annotation software, SMART (Letunic et al., 2012; Schultz et al.,

1998; http://smart.embl-heidelberg.de/). We based our diagrams of

signaling molecule domain structures on SMART results. In our fig-

ures, we provided diagrams of only our gene models, not of EST

sequences.

RESULTS AND DISCUSSION

Early development-relevant genes in the Pinctada fucata genome

We found and annotated 80 gene models, related to

various early developmental events, in the genome of

Pinctada fucata (Tables 1 and 2). Although we did not

exhaustively search for all currently known early develop-

ment-relevant genes, we did identify representatives of

major signaling pathways involved in early development. As

with all genes identified through wholesale in-silico gene

model predictions and annotations of draft genome

sequences, further experimental analyses through sequence

cloning and functional analyses will be necessary to confirm

their existence and functions. Further quality improvement of

the draft genome and the transcriptome data will also help

future verifications. Nonetheless, our results presented here

provide a starting point for such analyses, and at least at the

current stage, allow preliminary insights into the genomic

reality of development-relevant genes in P. fucata.

Signaling molecule gene families in the Pinctada fucata genome

We obtained gene models for most of the signaling mol-

ecule genes involved in axial patterning, confirming their

presence in the P. fucata genome (Table 2). We next

checked the EST data bank (obtained by transcriptome

Table 2. List of annotated gene models for early development-relevant signaling molecules and their receptors in Pinctada fucata.

Family Category gene namegene model

numberin assembly

gene model ID BLAST best hit (accession + species)

FGF Ligand FGF8/17/18 1 pfu_aug1.0_622.1_36612 NP_001164697.1, [Saccoglossus kowalevskii]

FGF19/21/23 1 pfu_aug1.0_1195.1_58407 XP_003226252.1, [Anolis carolinensis]

Receptor FGFR 1 pfu_aug1.0_1303.1_07891 BAI67805.1, [Idiosepius paradoxus]

Headghog Ligand Hedgehog 1 pfu_aug1.0_424.1_50858 EU394706.1, [Lottia gigantea]

Receptor Patched 1 pfu_aug1.0_5322.1_16629.t1 EFX90375.1, [Daphnia pulex]

PDGF/VEGF Ligand PVF1-like 1 pfu_aug1.0_938.1_58309 XP_002411998.1, [Ixodes scapularis]

Receptor PDGF- and VEGF-receptor 1 pfu_aug1.0_3981.1_16235.t1 BAI67804.1, [Idiosepius paradoxus]

TGFβ Ligand Dpp-BMP2/4 1 pfu_aug1.0_790.1_51081.t1 BAD16731.1, [Pinctada fucata]

BMP3 2 pfu_aug1.0_43625.1_63151.t1 ACF93445.1, [Branchiostoma japonicum]

pfu_aug1.0_102538.1_06302 XP_001494823.2, [Equus caballus]

Gbb-BMP5-8 1 pfu_aug1.0_23580.1_26106 XP_002407531.1, [Ixodes scapularis]

BMP9/10 1 pfu_aug1.0_2637.1_30198 CAD67715.1, [Crassostrea gigas]

Nodal 1 pfu_aug1.0_447.1_29262 ACB42422.1, [Biomphalaria glabrata]

Myostatin 1 pfu_aug1.0_156.1_22035 ABJ09581.2, [Chlamys farreri]

Marverick-like 1 pfu_aug1.0_6051.1_16824 CAD67714.1, [Crassostrea gigas]

Receptor BMPR1 1 pfu_aug1.0_257.1_50753 CAE11917.1, [Crassostrea gigas]

BMPR2 2 pfu_aug1.0_14312.1_32434 XP_001184902.1, [Strongylocentrotus purpuratus]

pfu_aug1.0_27640.1_04591 XP_001184902.1, [Strongylocentrotus purpuratus]

ACVR1 2 pfu_aug1.0_40446.1_70 ADD80738.1, [Pinctada fucata]

pfu_aug1.0_38196.1_0503 ADD80738.1, [Pinctada fucata]

ACVR1B 1 pfu_aug1.0_11.1_50544 CAD20573.1, [Crassostrea gigas]

ACVR2 1 pfu_aug1.0_3667.1_16148 CAR92545.1, [Crassostrea gigas]

Wnt Ligand WntA 1 pfu_aug1.0_964.1_00569 CAD37169.2, [Platynereis dumerilii]

Wnt1 1 pfu_aug1.0_11928.1_60961 CAD37164.2, [Platynereis dumerilii]

Wnt2 1 pfu_aug1.0_9858.1_38969 CAD37171.1, [Patella vulgata]

Wnt4 1 pfu_aug1.0_8897.1_53252 CAD37166.2, [Platynereis dumerilii]

Wnt6 1 pfu_aug1.0_39331.1_62990 ACE79727.1, [Branchiostoma lanceolatum]

Wnt7 1 pfu_aug1.0_76.1_29065 ABD16199.1, [Euprymna scolopes]

Wnt9 2 pfu_aug1.0_189.1_36368 XP_003206944.1, [Meleagris gallopavo]

pfu_aug1.0_189.1_36369 XP_003205766.1, [Meleagris gallopavo]

Wnt10 1 pfu_aug1.0_16143.1_54282 CAD37172.1, [Patella vulgata]

Wnt11 1 pfu_aug1.0_48418.1_70635 ADK38674.2, [Platynereis dumerilii]

Wnt16 1 pfu_aug1.0_5965.1_31091 ADR81924.2, [Platynereis dumerilii]

Receptor Frz-1/2/7 1 pfu_aug1.0_87126.1_42186.t1 NP_001079207.1, [Xenopus laevis]

Frz-4 1 pfu_aug1.0_3031.1_44550.t1 XP_002730495.1, [Saccoglossus kowalevskii]

Frz-5/8 1 pfu_aug1.0_16140.1_03676.t1 ADZ61652.1, [Ptychodera flava]

Signaling Molecule Genes in Pearl Oyster 881

sequencing; Takeuchi et al., 2012), which is available on the

genome browser homepage, to see if the gene models for

the signaling molecules are expressed. We found ESTs for

some, but not all, gene models (Table 3). One possible

explanation for this is that developmental genes are

expressed at relatively low levels compared to housekeep-

ing and metabolic genes, and therefore the transcriptome

sequencing was unable to detect them.

1. FGF

A Pfam domain search for the FGF domain containing

genes on the P. fucata Genome Browser suggested five

prospective gene models. Subsequent confirmations identi-

fied the presence of only two fgf homologs, and the other

three were false hits. TBLASTN, BLASTP, and phylogenetic

analyses confirmed the identities of the two gene models as

fgf8 and fgf19 homologs. We named the gene models

accordingly (Pifuc-fgf8L and Pifuc-fgf19L – the suffix “L”

denotes “Like”) (Table 2; Fig. 1). SMART predicted the pres-

ence of a signal peptide motif and an FGF domain on Pifuc-

Fgf8L, indicating that this is a full-length gene. Pifuc-Fgf19L

lacked the signal peptide domain, suggesting that this gene

model is most likely partial, lacking the 5′ end (Fig. 1A).

However, we detected an EST sequence of this gene in the

transcriptome data, giving further support to the presence of

Pifuc-fgf19L in P. fucata. We also identified a single FGF

receptor (Pifuc-fgfr) gene model (Table 2).

Interestingly, dual copies of the fgf gene are found in

other protostomes too, with one copy being a supported

homolog of fgf8, while the homology of the other copy is

uncertain (e.g., Stathopoulos et al., 2004; Huang and Stern,

2005; Bertrand et al., 2011). Two of the three fgf genes in

the fruit fly, pyramus and thisbe, are fgf8 orthologs, and

they are most likely products of a lineage-specific local

duplication (Stathopoulos et al., 2004). Kadam et al. (2009)

showed that at least in the fruit fly, the two types of FGF

ligands (Pyramus+Thisbe vs. Branchless) are functionally

non-interchangeable. Although the phylogenetic interrela-

tionships among the non-fgf8 genes are unclear, it is possi-

bly a homolog to “the rest” of the fgfs (the non-fgf8s). These

observations suggest the possibility that the two copies of P.

fucata fgf gene models are not unique to this species, but

synapomorphic in the protostomes (Fig. 6). Future studies

involving gene structure and synteny analyses among vari-

ous metazoans and functional analyses may help to reveal

such interrelationships among fgf genes.

2. Hedgehog

We identified one hedgehog gene model (Pifuc-hh) in

the P. fucata genome. Homology of the gene model was

confirmed by TBLASTN, BLASTP and phylogenetic analy-

ses (Fig. 2A, B). The phylogenetic tree shows the affinity of

Pifuc-hh to its Hs, Dm, Lg, and Ct homologs. Protein domain

structure prediction using SMART indicated the presence of

a signal peptide, a Hedge domain, and a Hint (“Hog”)

domain, indicating that the gene model for Pifuc-hh is full-

length (Fig. 2A). We also identified one Hedgehog receptor

gene model (Pifuc-hhR, Table 2). The single gene copy of

Pifuc-hh is consistent with current insights about the copy

numbers of hh in protostomes (Fig. 6).

The presence of a complete hedgehog with both the

Hedge and Hint domains is unsurprising, as it is consistent

with findings from L. gigantea and C. teleta genomes. In

those two lophotrochozoans, we found single copies of

hedgehog with both the Hedge and Hint domains present,

as predicted by SMART. Previously, Matus et al. (2008)

suggested that lophotrochozoan Hedgehog might have lost

its Hedge domain. This was concluded because a genomic

search for hedgehog homologs in C. teleta found only Hint

domain-containing copies. However, the absence of Hedge

domain in C. teleta might have been caused by the quality

of the available draft genome data at that time. Meanwhile

in Ecdysozoa, although the fruit fly has a complete hedgehog

gene containing both the Hedge and the Hint domains, the

nematode C. elegans has apparently lost its Hedge domain

and has duplicated its remaining Hint containing hedgehog

homologs (Aspöck et al., 1999; Hao et al., 2006; Matus et

al., 2008). However, since other nematodes may have a

complete hedgehog with both Hedge and Hint domains

present (Matus et al., 2008), it is most likely that the missing

Hedge in C. elegans was caused by a lineage-specific loss.

In summary, our results strongly suggest that the pearl

oyster P. fucata has retained the protostomian ancestral

condition of a complete single hedgehog gene with both

Hedge and Hint domains (Fig. 6).

Table 3. List of ESTs of Pinctada fucata’s annotated early devel-

opment-relevant signaling molecule gene models.

Family gene name

Number

of RNA

sequence

EST ID

FGF FGF8/17/18 0 –

FGF19/21/23 1 pfudmixc36137g12718

Headghog Hedgehog 0 –

PDGF/VEGF PVF1-like 0 –

TGFβ Dpp-BMP2/4 2 pfudmixc14451g01574

pfudmixc14449g01574

BMP3 1 pfudmixc32706g09287

Gbb-BMP5-8 3 pfudegg-4cellc02598g01138

pfud8-16cellc02367g00970

pfudmixc24496g05056

BMP9/10 0 –

Nodal 1 pfudmixc32568g09149

Myostatin 2 pfudmixc22489g04023

pfudtrochophorec00808g00102

Marverick-like 0 –

Wnt WntA 3 pfudmixc33241g09822

pfudtrochophorec02900g01620

pfudDshapec04488g02500

Wnt1 0 –

Wnt2 2 pfudegg-Dshapec05041g03189

pfudmixc38205g14786

Wnt4 3 pfud8-16cellc02776g01379

pfudDshapec04106g0211

pfudmixc08397g00554

Wnt6 0 –

Wnt7 0 –

Wnt9 0 –

Wnt10 0 –

Wnt11 2 pfudtrochophorec03235g01955

pfudmixc33657g10238

Wnt16 1 pfudmixc35143g11724

D. H. E. Setiamarga et al.882

3. PDGF/VEGF

We found one PDGF/VEGF-related factor gene

model, which we call Pfupvf, in the P. fucata genome.

TBLASTN, BLASTP and phylogenetic analyses con-

firmed that Pifuc-pvf was a homolog of the fruit fly pvf1

(Fig 3). SMART predicted the presence of a PDGF

domain on Pifuc-PVF (Fig. 3A). However, this gene

model does not include a signal peptide, indicating that

it is probably not a full-length sequence. We also iden-

tified a single PDGF/VEGF-related factor receptor gene

model (Table 2).

The fruit fly D. melanogaster has three pvf-like

genes (Cho et al., 2002; Duchek et al., 2001; Heino et

al., 2001; Pietrokovski and Shilo, 2001), while our draft

genome searches of several other insects (e.g., Tribo-

lium castaneum, Acyrthosiphon pisum, and Nasonia vit-

ripennis) found variable pvf gene copy numbers: two in

T. castaneum, one in A. pisum, and one in N. vitripennis.

A genome survey of the nematode C. elegans sug-

gested that the nematode most likely has only one copy

(Popovici et al., 1999; Popovici et al., 2002). Our surveys

Fig. 1. Pifuc-Fgf protein structures and phylogenetic tree. (A) Domain structures of the two Pifuc-Fgf identified in this study. Both gene mod-

els have an Fgf protein domain. However, the signal peptide domain is not present in PfuFgf19-Like gene model, suggesting that this gene

model might be incomplete. (B) Phylogenetic tree of the Fgfs based on the amino acid sequences of their conserved Fgf domains. We included

all Fgf homologs and paralogs from fruit fly (Dm), human (Hs), limpet (Lg), and a polychaete (Ct). The two P. fucata gene models are placed

with their putative homologs, confirming BLAST results. Bootstrap supports below 40% are not shown.

Fig. 2. (A) The protein structures of Pifuc-Hh. Domain structures of

Pifuc-Hh clearly show a complete Hedgehog protein, containing a signal

peptide, a Hedge, and a Hint (or Hog) domains. (B) Molecular phylogeny

of Pifuc-Hh clustered it together with the hedgehog homologs from other

protostomes. Bootstrap supports below 40% are not shown.

Signaling Molecule Genes in Pearl Oyster 883

on the two lophotrochozoan draft genomes also suggest a

variety of copy numbers (two in L. gigantea and three in C.

teleta). This variety of copy numbers suggest that a dynamic

evolutionary events involving local duplication and/or gene

losses have happened to pvf-like genes in the protostomes.

We were unable to confidently deduce the evolutionary

history of the PVDF/VEGF genes through the result of our

phylogenetic analysis, because most branches were not

supported by high bootstrap values. However, despite the

lack of a robust tree, one can infer that duplication and sub-

sequent sub-functionalization events have happened in ver-

tebrates independent from those that occurred in the protos-

tomes, causing the duplicated genes to differentiate into

PDGF and VEGF subfamilies (Holmes and Zachary, 2005).

Our observation here underlines the importance of improved

draft genomes and further molecular studies involving clon-

ing and functional studies, to conclusively elucidate the evo-

lutionary history of this gene family.

4. TGFββThe TGFβ superfamily consists of two subfamilies, the

BMP and the Activin/TGFβ subfamilies. A typical TGFβligand has two conserved domains, the TGFβ propeptide and

TGFβ-like domains (Fig. 4A). Previous molluscan studies

have identified three TGFβ ligand-coding genes (Nederbragt

et al., 2002; Grande and Patel, 2009; Kin et al., 2009). How-

ever, those previous studies suffered from the lack of

genomic data. Accordingly, we conducted a genomic survey

of TGFβ superfamily ligands in P. fucata to look for their

presence in the genome. We annotated five gene models as

homologs of BMP subfamily ligands (dpp-bmp2/4, bmp3,

gbb-bmp5-8, bmp9/10, and nodal), an Activin/TGFβ sub-

family ligand (myostatin), and maverick. The homology of

each copy was checked with TBLASTN, BLASTP, and phy-

logenetic analysis. The resulting phylogenetic tree (Fig. 4B)

confidently placed each copy with its tentative homologous

genes from other bilaterians, with reasonable bootstrap sup-

ports.

We did not find any gene model or EST for the other

three TGFβ family ligands (i.e., activin, tgfβ, and lefty) in the

currently available predicted gene models and the transcrip-

tome data. Our survey on the genome data of L. gigantea

and C. teleta also failed to find any gene model for tgfβ and

lefty. Previous studies have suggested that the absence of

tgfβ and lefty is probably synapomorphic to protostomes

(e.g., Van der Zee et al., 2008).

Our genomic survey also failed to identify any gene

model for activin in L. gigantea and C. teleta. Although

Grande and Patel (2009) reported the presence of an activin

homolog in L. gigantea, they did not detect its expression in

the developing embryo, leaving its function unclear, while its

affinity to other activin homologs was poorly supported. In

our phylogenetic analyses, the reported Lg activin grouped

with fruit fly’s activin-like protein with low support (Fig. 4B),

Fig. 3. (A) A comparison of Pifuc-Pvf protein with other VEGF and PDGF genes. PDGF/VEGF was thought to have later diverged into PDGF

and VEGF in vertebrates. Pifuc-Pvf identified in this report lacks a signal peptide, suggesting that it is an incomplete gene model. (B) Molecular

phylogenetic analyses of Pifuc-Pvf included PDGF and VEGF sequences from fruit fly (Dm), human (Hs), limpet (Lg), and a polychaete (Ct).

Bootstrap supports below 40% are not shown.

D. H. E. Setiamarga et al.884

Fig. 4. The protein structure of TGFβ superfamily ligands and their phylogenetic tree. (A) We identified a complete Pifuc-Dpp sequence with

all expected domains present. However, other gene models of TGFβ ligands are most likely partial sequences lacking one or more domains of

the signal peptide, the TGFβ propeptide, or the TGFβ family-like. Rectangles with broken lines indicate partial predicted domains. (B) Phyloge-

netic tree of TGFβ superfamily ligand genes. We included homologs from fruit fly (Dm), human (Hs), limpet (Lg), and a polychaete (Ct). The two

subfamilies, i.e., the BMP and Activin/TGFβ subfamilies, were divided into two monophyletic clades. The eight gene models of P. fucata are

placed within the respective clades of homologous genes, which is in accordance with the BLAST results. Bootstrap supports below 40% are

not shown.

Signaling Molecule Genes in Pearl Oyster 885

Fig. 5. A domain structure diagram and a phylogenetic tree of all Wnt gene models identified from P. fucata genome. We identified 10 Wnts

through a Pfam domain search, although most of the sequences found are partial. (A) Domain structures of all Wnt proteins identified in this

research. Some are very short sequences and thus possibly partial. (B) Wnt sequences from a squid (Es), fruit fly (Dm), human (Hs), limpet

(Lg), and a polychaete (Ct). Nine copies are properly placed together with their homologs with acceptable bootstrap support, confirming BLAST

results. Bootstrap supports below 40% are not shown.

D. H. E. Setiamarga et al.886

but not with other activins. We also identified five gene

models for TGFβ receptors (bmp receptor type I, type II,

activin receptor type I, IB and type II) in the P. fucata

genome (Table 2, Supplementary Fig. S1). Since Myostatin

and probably other BMP ligands also bind to Activin recep-

tors (Huminiecki et al., 2009), the presence of these recep-

tors does not necessarily suggest the presence of activin.

With our current data, we are unable to confidently conclude

the presence of activin in mollusks, or lophotrochozoans.

Therefore, future genomic and molecular analyses will be

needed to conclusively show if activin is really present or

absent in P. fucata and other mollusks. However, since the

presence of two activin homologs has also been reported in

the fruit fly (Zhu et al., 2008), we can deduce that the pres-

ence of activin is probably ancestral to protostomes (Fig. 6).

5. Wnt

A Pfam domain search found 13 wnt candidate gene

models. Further analyses confirmed the presence of ten

PifucWnts gene models (wntA, 1, 2, 4, 6, 7, 9, 10, 11, 16)

(Table 2, Fig. 5). Most of the Pifuc-wnt sequences obtained

were short and probably partial, with Wnt11 as the shortest

(68 amino acids) (Fig. 5A). Phylogenetic analysis confidently

placed most of the gene models with their respective

human, fruit fly, squid (Euprymna scolopes), limpet, and

polychaete homologs, a result that is consistent with the ini-

tial BLASTP and TBLASTN results. We also found three

gene models for Wnt receptors, Frizzled, in the P. fucata

genome (Table 2, Supplementary Fig. S2).

The presence of multiple wnt genes in P. fucata is con-

sistent with our current knowledge of ancient wnt duplica-

tions, as most wnt genes and their receptors apparently

already existed in ancestral cnidarians (Kusserow et al.,

2005). In this study, we did not find any copy of wnt3 in the

P. fucata genome, or any EST sequence in the transcrip-

tome data. Previous studies have also indicated the

absence of wnt3 in the protostomes, suggesting that the

absence of the wnt3 homolog is possibly a synapomorphic

character of the protostomes, in agreement with previous

studies (Schubert et al., 2000; Kusserow et al., 2005; Cho

et al., 2010; Janssen et al., 2010). We also failed to detect

any gene model for wnt5 gene model in the present draft

genome of P. fucata. However, wnt5 is present in other

mollusks surveyed in our present study on wnt genes (E.

scolopes and L. gigantea). We also detected a wnt5 EST

sequence in the transcriptome data. From these observa-

tions, we can confidently deduce that wnt5 is present in P.

fucata, and its absence is most likely an artifact caused by

the current quality of the P. fucata draft genome. Our result

also suggests that wnt8 is absent in P. fucata, a conclusion

supported by its absence in both the draft genome and tran-

scriptome data. Meanwhile, our L. gigantea draft genome

search also indicated its absence, although we found the

gene in the draft genome of C. teleta. These results suggest

that wnt8 was probably lost in the molluscan lineage, in

agreement with previous studies (Kusserow et al., 2005;

Cho et al., 2011).

It should be noted that originally, Pfam domain search

Fig. 6. Reconstructions of gene copy numbers of the ancestral lophotrochozoans and ancestral protostome. We assumed that the greatest

common factor in which all gene copies present in any extant protostome must have had at least one copy in the ancestors, and if multiple cop-

ies are present in only one species, we assumed that it was duplicated locally. We also put the information of deuterostome genes into consid-

eration. For the ancestral deuterostome, we refer to findings from the basal chordate amphioxus and other vertebrates. Question marks

indicate uncertainties or lack of information; while cross marks on the ancestral protostome indicate possible absence.

Signaling Molecule Genes in Pearl Oyster 887

found two wnt9 gene models (pfu_aug1.0_189.1_3639 and

pfu_aug1.0_189.1_3638), which were located in tandem on

the same scaffold. BLAST searches allied both genes to the

same wnt9a homolog in turkey (Meleagris gallopavo).

When both “genes” were subjected to SMART protein

domain searches, they were shown to be composed of only

partial Wnt domains. We retrieved the portion of the scaffold

sequence where the two gene models are located, and a

quick inspection of that sequence revealed that the

sequence of the region in-between the two original gene

models were undetermined (denoted by a series of N).

Therefore for our phylogenetic analysis, we simply concate-

nated the two gene models and treated them as a single

sequence.

When we plot the number of existing copies of wnt

genes in mollusks, annelids, insects, and nematodes, mol-

lusks and annelids show an almost complete number of wnt

copies, compared to model ecdysozoans (Fig. 6; Guder et

al., 2006; Cho et al., 2011). This suggests that mollusks

retain the ancient number of wnt genes, close to that pre-

dicted for the ancestral protostome. Future functional analy-

ses will be needed to see if both the copy number and the

gene functions are also conserved.

Insights into the evolution of signaling molecule genes

in protostomes

In this study, we conducted a general survey for genes

related to axis formation in early embryonic development.

We then focused our survey on signaling ligand-coding gene

families, namely FGF, Hedgehog, PDGF/VEGF, TGFβ, and

Wnt.

Our results allow us to predict the possible ancestral

condition of the gene copy numbers in protostomes, and

possible evolutionary processes that account for them. In

Fig. 6, we present a phylogenetic character mapping for the

presence/absence of FGF, Hedgehog, Wnt, TGFβ, and

PDGF/VEGF gene family members on the protostome tree.

The results suggested that the ancestral protostome had two

copies of fgf, a single copy of hedgehog, five copies of bmp-

related genes, one copy of activin, one copy of maverick,

and 12 copies of wnt genes. Figure 6 indicates that genomes

of D. melanogaster and C. elegans are possibly highly

derived because of extensive local duplications and lineage

specific losses. It appears that mollusks retain most gene

copies, probably indicating that molluscan genomes are

closer to that of the ancestral protostome.

Future expectations

The presence of a series of long N in various scaffolds

and the relatively short length of some scaffolds in the cur-

rent version of P. fucata draft genome may have been the

cause of our inability to identify complete sequences for

many gene models. Current transcriptome sequencing

method used to generate the currently available transcrip-

tome data may not be sufficiently sensitive to detect genes

expressed at very low levels, hence our inability to obtain

EST data for some signaling molecule genes. Therefore,

future work involving gene cloning and functional analyses

will be needed to confirm the in-silico results presented

here, while further refinements of P. fucata draft genome

and the transcriptome data will allow for better gene predic-

tions and synteny analyses to check gene homologies and

evolutionary histories conclusively (e.g., Bertrand et al.,

2010; Hui et al., 2012). For example, better sequence qual-

ity on longer scaffolds may enable us to see if the two wnt9

copies are actually sequences from a single gene, or if they

originated from two paralogous copies of wnt9 obtained

through a local tandem duplication event. Improved draft

genome and transcriptome data might also allow us to con-

fidently deduce the homology of lophotrochozoan and meta-

zoan non-fgf8 genes, and help us to elucidate the evolution

of the PDGF/VEGF gene family.

ACKNOWLEDGMENTS

For gene model annotations, three Genome Jamboree events

were held in 2011–2012 in Japan. These events were partially funded

by JAMBIO (Japanese Association for Marine Biology). The authors

would like to sincerely thank Takeshi Takeuchi, Takeshi Kawashima,

Ryo Koyanagi (OIST), and Hiroshi Wada (Univ. Tsukuba) for their

invaluable advice and supports. We are indebted to Christopher

Mah (Smithsonian National Muse. Nat. Hist.), Noah Ben-Aderet

(Scripps Inst. Ocean.) and Neil Aschliman (St. Ambrose Univ.) for

helping us to improve the English of our manuscript. The manu-

script’s English was then further improved by a professional editor.

We also thank two anonymous reviewers for their invaluable advice

to improve the manuscript.

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(Received October 23, 2012 / Accepted March 23, 2013)