Open Access

Unexpected link between polyketide synthase and calcium carbonate biomineralization

  • Motoki Hojo1, 6,
  • Ai Omi1, 7,
  • Gen Hamanaka2,
  • Kazutoshi Shindo3,
  • Atsuko Shimada1,
  • Mariko Kondo4,
  • Takanori Narita1, 8,
  • Masato Kiyomoto2,
  • Yohei Katsuyama5,
  • Yasuo Ohnishi5,
  • Naoki Irie1 and
  • Hiroyuki Takeda1Email author
Zoological Letters20151:3

DOI: 10.1186/s40851-014-0001-0

Received: 19 September 2014

Accepted: 15 October 2014

Published: 13 January 2015

Abstract

Introduction

Calcium carbonate biominerals participate in diverse physiological functions. Despite intensive studies, little is known about how mineralization is initiated in organisms.

Results

We analyzed the medaka spontaneous mutant, ha, defective in otolith (calcareous ear stone) formation. ha lacks a trigger for otolith mineralization, and the causative gene was found to encode polyketide synthase (pks), a multifunctional enzyme mainly found in bacteria, fungi, and plant. Subsequent experiments demonstrate that the products of medaka PKS, most likely polyketides or their derivatives, act as nucleation facilitators in otolith mineralization. The generality of this novel PKS function is supported by the essential role of echinoderm PKS in calcareous skeleton formation together with the presence of PKSs in a much wider range of animals from coral to vertebrates.

Conclusion

The present study first links PKS to biomineralization and provides a genetic cue for biogeochemistry of carbon and calcium cycles.

Keywords

Biomineralization Calcium carbonate Otolith Polyketide synthase Medaka Spicule

Introduction

Biominerals are produced by living organisms through genetically controlled biological processes, and confer stability, rigidity, defences and functionality to organisms; examples include grass opal (rice; silicate), shell (shellfish; calcium carbonate), and bone/teeth (vertebrate; hydroxyl apatite) [1,2]. The addition of organic components determines organism-specific shape, size, hardness and crystallographic axis orientation of biominerals [3]. Among biominerals, calcium carbonate is the most abundant and is known to play an important role in the global biogeochemical cycles of carbon and calcium through reef building and massive precipitation by calcifying algae in the ocean [4-6], and thus has attracted much attention, together with calcium phosphate mineralization of vertebrate skeleton and teeth.

The biomineral crystallization begins with nucleation through the phase of prenucleation clusters [7,8]. Experimental and modeling studies proposed that nucleation from saturated solution is not a simple event, but is preceded by a phase of amorphous precursors, such as amorphous calcium carbonate (ACC) or amorphous calcium phosphate, depending on the type of biominerals. These precursors then coalesce and rearrange to form nuclei, a core of subsequent crystal growth, a process called ‘nucleation’. Although these processes of biomineralization are well described, the underlying mechanism, particularly regarding the initial nucleation step, remains a mystery.

Otoliths (ear stones) in teleost fish are composed of calcium carbonate and organic materials (0.2–10%) [9], and primarily function in gravity and motion sensing, providing excellent systems for biochemical and genetic analyses of the mechanisms of calcium carbonate biomineralization. Factors involved in initial step of mineralization have not been definitively identified thus far, except for a report that glycogens are always found in the core of nascent otoliths [10]. This is in part due to the lack of genetic studies targeting otolith mineralization. Indeed, while most zebrafish otolith mutants exhibit defects in the number, shape, and position, only a few completely lack otoliths, and in the cases where the causal gene was identified, its function in the context of mineralization remained unclear [11,12].

To gain insights into the crystallization of calcium carbonate in organisms, we analyzed the medaka spontaneous and homozygous viable mutant, ha, defective in otolith formation. Positional cloning identified a mutation in a novel gene encoding polyketide synthase (pks), named OlPKS, specifically expressed in the otic vesicle at the onset of mineralization. We demonstrate that the compound synthesized by OlPKS is secreted in the endolymph and it acts as a nucleation facilitator in otolith mineralization. Furthermore, sea urchin pks-2 expressed in skeletogenic cells was found to be indispensable for the formation of spicules, calcareous larval skeletons, indicating the generality of a novel link between calcium carbonate biomineralization and animal polyketide synthase.

Materials and methods

Fish strains and mapping

The medaka (Oryzias latipes) d-rR strain was used as a wild-type. ha was isolated as a spontaneous mutant by Tomita [13]. ki79 was isolated from a ENU-mediated mutant screening (at the Tokyo Institute of Technology, Japan). Mapping of the ha locus was carried out as previously described [14].

Otolith staining

Alizarin Red staining for visualizing mineralized otoliths was essentially done as described [15]. Embryos were fixed with 4% PFA/2× PBS (phosphate buffered saline) containing 1% sodium hydroxide for three hours at room temperature. The samples were then washed with PBS several times, and stained with 2% Alizarin Red S/1% sodium hydroxide solution overnight. After quickly washing with 0.5% KOH, samples were mounted in 80% glycerol.

Transmitted electron microscopy

Embryos were fixed with 2% PFA and 2% glutaraldehyde buffered in 0.1 M cacodylate buffer and postfixed in 2% OsO4 in 0.1 M cacodylate buffer. Uranyl acetate and lead stain solution were used for contrast enhancement. The specimens were embedded in Quetol-812. Ultrathin sections (70 nm) were analyzed and documented with an electron microscope (JEOL, JEM-1400 Plus).

Whole-mount in situ hybridization

All whole-mount in situ hybridization analyses were performed as previously described [16]. cDNAs isolated by RT-PCR were used as the templates for the probes of marker genes (see Additional file 1).

Immunofluorescence

Immunofluorescence for OMP-1 was performed as previously described Murayama et al. [17], whereas staining for acetylated α-tubulin, OlPKS and PKC ζ were done as previously described by Kamura et al. [18] (see Additional file 1). Polyclonal rabbit OlPKS [amino acids 1520–1711] antibodies were raised by immunization of rabbits with bacterially expressed His-tagged truncated proteins.

Morpholino knockdown and mRNA rescue in medaka embryos

The morpholino antisense oligonucleotide (MOs; Gene Tools LLC) for first-Met of OlPKS was as follows: 5'-AACCACGGCTATTCCGTCCTCCATG-3'. in vitro syntheses of mRNAs were conducted as reported previously [19]. Full length of olpks was isolated from the d-rR strain by RT-PCR. The sequences of primers are described in Additional file 1 (Whole-mount in situ hybridization). For injection of olpks mRNA with active site mutation, ki79 fish was used, because it likely carries a null mutation. Amino acid of the active site in each enzymatic domain was substituted as followed (with nucleotide sequences): KS, 173Cys → Asn (TGC → AAC); AT, 608Ser → Ala (TCC → GCA); DH,914His → Ala (CAC → GCC); KR,1859Tyr → Phe(TAC → TTC); ACP, 2010Ser → Ala(TCC → GCC); Loop (interdomain region; control), 435Thr → Ala(ACC → GCC) [20-22]. Microinjection of MO or mRNAs was carried out as previously described [19].

Genomic research

Animal type I PKS sequences were retrieved by TBLASTN against genome sequences of each species or BLASTP search against nr-protein database using the OlPKS sequence. For each candidates, domain search was done by Pfam sequence search, and amino acid sequences having basic PKS domains KS and AT, and additional domains (e.g., DH, KR, ER, ACP etc.) were defined as an animal type I PKS.

Generation the ha chimeric medaka

Transplantation experiments were performed based on the previous works [23]. [Tg (β-actin:DsRed)] were used as wt-cell donors, whereas ha fish were prepared as hosts. Whether otoliths were formed or not and how many fluorescence cell were contained were confirmed by observations around st. 29 using a confocal fluorescence microscope (Zeiss, LSM710). A transplantation experiment was conducted as a negative control in which [ha −/−; Tg (β-actin:DsRed)] animal was used as the donors (detailed in Additional file 1).

OlPKS expression in A. oryzae and medaka rescue assay

The entire OlPKS ORF was amplified and subcloned into a fungal expression vector, pTAex3. The resulting expression plasmid pTA-olpks was used for transformation of A. oryzae M-2-3 according to the protoplast-PEG method described by Gomi et al. [24]. The mycelia of transformants were extracted with acetone, followed purification using partition between ethyl acetate/H2O two times and the ethyl acetate layer was concentrated to dryness, and partitioned between hexane/90% methanol. The methanol layer was concentrated to dryness to give the material for medaka assay. Each extracts re-dissolved in DMSO was added to the medium in which ha medaka embryos were incubated. (detailed in Additional file 1).

Morpholino knockdown in sea urchin embryos

Microinjection of MOs was carried out as described previously with some modifications [25] (see Additional file 1). MOs (Gene Tools LLC) for first-Met blocking and their five-mispair controls were as follows (small letters in control sequences indicate substituted nucleotides).

hppks-1: 5'-CTGGTTTTATTGCTTCCCATGTTGA-3', hppks-2: 5'-CCCTCCAACTATCTTCCATAACTCA-3', hppks-1 control: 5'- CTGcTaTTATTcCTTCCgATcTTGA -3', hppks-2 control: 5'-CCgTgCAAgTATgTTCgATAACTCA-3'.

Results

The ha embryo fails to mineralize otoliths

Medaka ha is a spontaneous and homo-viable mutant defective in otolith formation [13] (Figure 1A and C). The gross morphology of ha was previously reported as significantly delayed mineralization of otoliths, slightly enlarged otic vesicles (OVs) and malformed semicircular canals [26,27]. We further characterized the ha phenotypes using molecular markers at embryonic stages at which the formation of the OV and otoliths normally takes place (st. 22– 30).
Figure 1

ha embryos fail to mineralize otoliths. (A) DIC images of OVs at st. 30 (dorsal views of the left OV). Grown otoliths are observed in wt OV. Mutant OV contains numerous seeding particles that form a paste-like precipitate (Inset). Red arrows: seeding particles; Asterisks: otoliths. Scale bars: 20 μm. (B) Immunofluorescence of an otolith matrix protein, OMP-1 (dorsal views of the left OV). Anti-Oncorhynchus mykiss (Om) OMP-1 serum is used. In mutant OV, immunoreactive substances cling to the epithelium. Scale bars: 20 μm. (C) Alizarin Red staining for mineralized otolith. Crystal is never observed in mutant OV (dorsal views of the head; white dotted lines show OV). Scale bars: 100 μm. (D) TEM images of the epithelium of the OV at st. 25 when the otolith is forming (the prospective macula region; lateral views). In a wt embryo ‘globules’ coalesce to form the otolith precursor in the posterior end of the OV. In the mutant, by contrast, very fine particles are observed at posterior end of OV (D) and mid-position of the OV (D’). Asterisks: growing otoliths; Black arrows: fine particles; ‘g’:globule; ‘s’:seeding particle. Scale bars: 1 μm. (E) Immunofluorescence of acetylated α-tubulin st. 24 (dorsal views of the left OV). Many short cilia protruded from the epithelium are visible in ha OV as well as wt one. Scale bars: 5 μm.

Otolith formation in medaka proceeds in a way similar to zebrafish [28]; ‘seeding particles’ start to float in the endolymph of the OV (Figure 1A and D) from st. 23 and coalesce into small crystal at st. 24 (Additional file 2K; Upper Left). Otoliths are stereo-microscopically visible as two small crystals at st. 25 and continuously increase in size (Figure 1A and D, and Figure 2D). In zebrafish, essential roles of cilia in otolith formation have been repeatedly shown [28-31]; tethering seeding particles by long kinocilia (5–8 μm) protruded from hair cells in the prospective macula regions and stirring the fluid by shorter motile cilia (1.5–5 μm) lining the entire of the OV epithelium. However, this scenario may not hold true in medaka embryos. Though cilia were found on the OV epithelium of medaka, they are much smaller in size (<1 μm) and their motility was hard to be detected (Figure 1E). Furthermore, probably due to their small size, we failed to identify kinocilia. At least, motile cilia do not contribute to otolith formation in medaka, as the medaka mutant kintoun with paralyzed cilia develops otoliths as normal (Additional file 2K) [32].
Figure 2

ha gene encodes a polyketide synthase. (A) Positional cloning of the ha mutation in linkage group (LG) 20. The number of recombinants at each marker is shown. Sequencing of ha revealed a 9-nucleotide deletion. ORF: open reading frame. (B) Architecture of OlPKS (2051 amino acid-length) predicted by a Pfam search. Each domain is shown by abbreviation. An arrow indicates mutation site of ha, which is located at 279–281 (K, P and S). (C) Whole-mount in situ hybridization with an antisense RNA probe for olpks at otolith forming developmental stages. A representative picture is shown at st. 21 (Upper; dorsal view; dotted line indicates embryonic body). olpks transcripts detected in various stages are shown at high magnification (Lower; dorsal views of left and right OVs). Scale bars: 50 μm. (D) Period of the expression of olpks in the context of otolith growth. Purple area shows the period of olpks expression. Line graphs show the sizes (longest linear dimensions) of otoliths at some developmental stages. Data are the means and standard deviations of measurements taken of at least 7 specimens each. Some observable changes in the OV during otolith development are described with arrows. Red curcle: anterior otolith; Blue triangle: posterior otolith; hpf: hours post fertilization.

In ha mutant embryos, the OV, macula and cilia develop normally (Additional file 2A to D, I and J), and seeding particles are floating in the OV (Figure 1A and Additional files 3 and 4). Furthermore, organic materials such as OMP-1, a major soluble organic matrix protein, are secreted into the endolymph (Figure 1B) (other known organic components, starmaker-like and sparc are normally expressed in the OV (Additional file 2E and F)). Nevertheless, in ha mutants, mineralized stones never form (Figure 1A and C), but instead OMP-1-positive particles precipitate in the endolymph (Figure 1A Inset and B). This was further supported by TEM observation; fine substances (Figure 1D and D′, arrows), probably organic substances supplied by seeding particles, were accumulated around the epithelium in ha (Figure 1D Right and 1D’ Right), instead of growing otoliths (Figure 1D Left and D′ Left, asterisks). In wild-type (wt) OVs, fine particles were compacted into a round ‘globule’ (Figure 1D Left, ‘g’; [10]), and the globules then form otoliths by coalescing near the macula (Figure 1D asterisks; see also Additional file 2G). Taken together, in ha embryos, otolith mineralization is completely inhibited, even though major organic materials are supplied into the endolymph.

ha gene encodes a polyketide synthase

Using positional cloning, we narrowed down the ha locus to a 64.7 kb region in linkage group 20, which contains four open reading frames (ORFs 1–4). Sequencing analysis identified a 9-nucleotide deletion (3-amino acid deletion) in ORF 2 that encodes a type I polyketide synthase (PKS) (Figure 2A). We thereafter named this gene Oryzias latipes polyketide synthase (olpks). The olpks transcript is 6153 nt in length, comprises six exons, and encodes a 2051 amino acid protein. PKSs are multifunctional enzymes mainly found in bacteria, fungi and plant, and catalyze the biosynthesis of a diverse group of second metabolite, polyketides, some of which are used for pharmaceuticals with antibiotic and mycotoxic properties [33]. Type I PKS has a set of distinct enzymatic domains that individually catalyze a series of reactions, to produce a final compound. Likewise, OlPKS contains five distinct domains: ketoacyl synthase (KS), acyl transferase (AT), dehydratase (DH), ketoreductase (KR), and acyl carrier protein domain (ACP) (Figure 2B). Essential amino acid sequence motifs for the function of each domain are conserved in OlPKS (Additional file 5B). The vertebrate fatty acid synthase (FAS), essential for all organisms, is thought to be an evolutionary subset of this family [34,35]. Animal FASs and another iterative type I PKSs share a conserved structure that includes KS, AT and ACP domains. FASs contain additional enzymatic domains such as KR, DH, ER and TE, which are present on different PKSs in different combinations. OlPKS possesses the minimal module (KS, AT and ACP) and additional KR and DH domains (Figure 2B and Additional file 5B).

We confirmed that olpks is indeed responsible for the ha phenotype by the following results, (i) phenocopy by injection of an antisense morpholino-oligonucleotide (MO) (Table 1), (ii) identification of a mutation of the olpks locus in another allele, ki79 (Additional file 5A and B) which was isolated from a N-ethyl-N-nitrosourea (ENU)-driven screen (unpublished; screen conducted for medaka mutants with defects in bone or blood development at the Tokyo Institute of Technology, Japan), and (iii) phenotypic rescue by injection of full length olpks mRNA (Table 1). We also confirmed that all domains of OlPKS are indeed required for otolith formation by injecting mRNAs, each of which causes one amino acid substitution at one of the four enzymatic active sites and the essential site of ACP (Additional file 5B and Table 1).
Table 1

Otolith formation in MO or mRNA of OlPKS

Experiment

Nucleltide

Fish

4 otoliths

1-3 otoliths

No otolith

n

Phenocopy

olpks first-Met MO

wt

0%

0%

100%

39

Rescue

(uninjected)

ha

0%

0%

100%

31

olpks mRNA

73%

23%

3%

30

Active site mutation

(uninjected)

ki79

0%

0%

100%

14

olpks-mRNA

65%

35%

0%

43

KS*-mRNA

0%

0%

100%

15

AT*-mRNA

0%

2%

98%

57

DH*-mRNA

0%

0%

100%

18

KR*-mRNA

0%

0%

100%

17

ACP*-mRNA

0%

0%

100%

27

Loop*-mRNA

53%

40%

7%

60

Asterisks indicate one amino acid mutation are introduced.

‘Loop’: interdomain region.

Fully rescued: 4 otoliths in one animal.

Partially rescued: 1–3 otoliths in one animal.

Not rescued: no otolith.

The expression of olpks is transient and exclusively restricted to the OV in developing embryos. The expression initiates at the early somite stage (st. 19) and disappears between 16-somite (st. 24) and 19-somite stage (st. 25), a period when otolith mineralization initiates (Figure 2C and D). The expression becomes restricted to the medial and dorsal region of the vesicle at later stages (Figure 2C and Additional file 5C). The medaka genome has three pks related genes including olpks (Table 2) and we confirmed that the other two are not expressed at embryonic stages and adult tissues (Additional file 5D).
Table 2

Type I PKSs found in animal lineage

Type 1 PKS?

Species name

General name

Gene name

Accession number

Reference genome

Gene ID

Corresponding RNA (Unigene or RNA)?

Domians

 

Homo sapiens

Human

-

-

Homo sapiens GRCh37.p13

-

-

-

 

Bos taurus

Cattle

-

-

Bos taurus UMD3.

-

-

-

 

Canis (lupus) familiaris

Dog

-

-

Canis lupus familiaris CanFam3.1

-

-

-

 

Tursiops truncatus

Dolphin

-

-

Tursiops truncatus turTur1

-

-

-

 

Mus musculus

Mouse

-

-

Mus musculus GRCm38.p1

-

-

-

Monodelphis domestica

Opossum

PREDICTED: phthioceranic/hydroxyphthioceranic acid synthase-like

XP_001375980

MonDom5

LOC100024851

no

KS-AT-DH-KR-ACP

Sarcophilus harrisii

Tasmanian devil

mycocerosic acid synthase-like [Sarcophilus harrisii (Tasmanian devil)]

XP_003771909

Devil_refv7.0

LOC100922065

N.A

KS-AT-DH-KR-ACP

 

Ornithorhynchus anatinus

Platypus

LOC100091954 fatty acid synthase-like [Ornithorhynchus anatinus (platypus)]

-

Ornithorhynchus_anatinus-5.0.1

LOC100091954 (pseudo gene)

N.A

N.A (pseudo gene)

Gallus gallus

Chicken

PREDICTED: phthioceranic/hydroxyphthioceranic acid synthase-like is oformX2 [Gallus gallus].

XP_418588

Gallus_gallus-4.0

LOC420486

Yes (brain; connective, blood)

KS-AT-DH-KR-ACP

Taeniopygia guttata

Zebra finch

PREDICTED: phthioceranic/hydroxyphthioceranic acid synthase-like [Taeniopygia guttata].

XP_002189754

Taeniopygia_guttata-3.2.4

LOC100231542

No

KS-AT-DH-KR-ACP

PREDICTED: Taeniopygia guttata phthioceranic/hydroxyphthioceranic acid synthase-like

XP_002190558

LOC100222288

N.A

KS-AT-DH-KR-ACP

Falco peregrinus

Peregrine falcon

PREDICTED: probable polyketide synthase 1-like [Falco peregrinus].

XP_005234016

F_peregrinus_v1.0

LOC101916009

N.A

KS-AT-DH-KR-ACP

Anolis carolinensis

Green anole

PREDICTED: phthioceranic/hydroxyphthioceranic acid synthase-like [Anolis carolinensis].

XP_003222100

AnoCar2.0

LOC100564455

No

KS*-AT-DH-KR-ACP

PREDICTED: phthioceranic/hydroxyphthioceranic acid synthase-like [Anolis carolinensis].

XP_0032222101

LOC100564655

No

KS-AT-DH-KR-ACP

PREDICTED: phthioceranic/hydroxyphthioceranic acid synthase-like [Anolis carolinensis].

XP_0032222102

LOC100564856

No

KS-AT-DH-KR-ACP

Chelonia mydas

Green sea turtle

Phthioceranic/hydroxyphthioceranic acid synthase [Chelonia mydas].

EMP37033

CheMyd_v1.0

locus_tag: UY3_05838

N.A

KS-AT-DH-KR-ACP

Polyketide synthase Pks N [Chelonia mydas]

EMP24664

locus_tag: UY3_18267

N.A

KS*-AT-DH-KR-ACP

Chrysemys picta

Painted turtle

PREDICTED: uncharecterized protien LOC101936604 [Chrysemys picta bellii]

XP_005291085

Chrysemys_picta_bellii-3.0.1

LOC101936604

N.A

KS-AT*-DH*-KR-ACP

PREDICTED: uncharecterized protien LOC101937174 [Chrysemys picta bellii]

XP_005291087

LOC1019371714

N.A

other-KS-AT*-DH*-KR-ACP

Pelodiscus sinensis

Chinese soft shell turtle

pep: KNOWN_BY_PROJECTION_protein_coding

Scaffold no. JH209275.1

PelSin_1.0

ENSPSIG00000004874

No

KS*-AT-DH-KR-ACP

 

Xenopus (Silurana) tropicalis

Tropical clawed toad

-

-

Xenopus (Silurana) tropicalis build 1 genome database (v4.2 assembly)

-

-

-

Oryzias latipes

Medaka

OIPKS (PREDICTED: phthioceranic/hydroxyphthioceranic acid synthase-like [Oryzias latipes])

XP_004081385

Oryzias latipes ASM31367v1

LOC101169887

No

KS-AT-DH-KR-ACP

OIPKS-2 (PREDICTED: phthioceranic/hydroxyphthioceranic acid synthase type I P ps D-like [Oryzias latipes])

XP_004081384

LOC101169644

No

KS-AT-DH*-KR-ACP

OIPKS-3 (PREDICTED: probable polketide synthase 1-like [Oryzias latipes])

XP_004080917

LOC101170716

No

KS-AT-DH*-KR-ACP

Danio rerio

Zebrafish

Danio rerio wu:fc01d11 (wu: fc01d11),mRNA

XP_682975

Danio rerio Zv9

wu:fc01d11

Yes (muscle)

KS-AT-DH-KR-ACP

si: dkey-61p9.11

NP_001041530

LOC100000781

Yes (kidney)

KS-AT-DH-KR-ACP

Takifugu rubripes

Fugu

phthioceranic/hydroxyphthioceranic acid synthase-like

XP_003968201

FUGU5

LOC101079294

No

KS-AT-DH-KR-ACP

PREDICTED: Takifugu rubripes lovastatin nonaketide synthase-like (LOC101079519), mRNA

XP_003968202

LOC101079519

No

KS-AT-DH-KR-ACP

 

Petromyzon marinus

Lamprey

-

-

Pmarinus_7.0

-

-

-

Branchiostoma Floridae

Lancelet

hypothetical protien BRAFLDRAFT_205831, partial [Branchiostoma floridae].

XP002599684 (=EEN55696)

Branchiostoma floridae v1.0

Gene ID:7231796

N.A.

KS-AT-DH-KR

   

hypothetical protien BRAFLDRAFT_247081 [Branchiostoma floridae]

XP_002591573

 

Gene ID:7219804

N.A.

KS-AT-DH*-KR

   

hypothetical protien BRAFLDRAFT_90481 [Branchiostoma floridae]

XP_002589799

 

Gene ID:7210376

N.A.

KS-AT-DH*-KR-AMP

   

hypothetical protien BRAFLDRAFT_96868 [Branchiostoma floridae]

XP_002598386

 

Gene ID:7254845

N.A.

KS-AT-DH-KR*-TE-C-AMP

   

hypothetical protien BRAFLDRAFT_96863 [Branchiostoma floridae]

XP_002598380

 

Gene ID:7248951

N.A.

KS-AT-DH-KR*-ACP-TE-C-AMP

   

hypothetical protien BRAFLDRAFT_87472 [Branchiostoma floridae]

XP_002589000

 

Gene ID:7246004

N.A.

KS-AT-DH-KR*-ACP-C-AMP

   

hypothetical protien BRAFLDRAFT_125690 [Branchiostoma floridae]

XP_002610053

 

Gene ID:7207083

N.A.

KS*-AT-DH-KR*-ACP*-TE-C

   

hypothetical protien BRAFLDRAFT_91451 [Branchiostoma floridae]

XP_002608071

 

Gene ID:7214024

N.A.

KS-AT-DH-MT-ADH-KR-ACP-α

   

hypothetical protien BRAFLDRAFT_87410 [Branchiostoma floridae]

XP_002605916

 

Gene ID:7243248

N.A.

KS-AT-DH-MT-ADH-KR-ACP

   

hypothetical protien BRAFLDRAFT_89867 [Branchiostoma floridae]

XP_002610100

 

Gene ID:7206066

N.A.

KS-AT-DH-MT-ADH-KR-ACP

hypothetical protien BRAFLDRAFT_87413 [Branchiostoma floridae]

XP_002605913

Gene ID:7246000

N.A.

KS-AT-DH-MT-ADH-KR-ACP

   

hypothetical protien BRAFLDRAFT_125650 [Branchiostoma floridae]

XP_002610103

 

Gene ID: 7207596

N.A.

AMP-KS-AT-DH-MT-ADH-KR-ACP

   

hypothetical protien BRAFLDRAFT_71890 [Branchiostoma floridae]

XP_002613500

 

Gene ID: 7224978

N.A.

KS*-AT-DH

Saccoglossus kowalevskii

Acorn worm

PREDICTED: fatty acids synthase-like

XP_002734101

Skow_1.1

LOC100373061

N.A

KS*-AT-DH-KR-ACP-C-AMP

 

Ciona intestinalis

Ascidian

-

-

Ciona intestinalis KH

-

-

-

Strongy locentrotus purpuratus

Purple sea urchin

LOC588806 probable polyketide synthase 1-like [Strongylocentrotus purpuratus (purple sea urchin)]

XP_793564.2

Spur_3.1

LOC58806

Yes

KS-AT*-DH*-MT-ADH-KR*-ACP

LOC592147 polyketide synthase 2

NP_001239013.1

LOC592147

Yes

KS-AT-DH*-KR-ACP-TE

Acropora digitifera

Coral

aug_v2a.12941.t1 aug_v2a.12941scaf5202:2805-26950(−)

aug_v2a.12941

Adig_1.0

aug_v2a.12941

N.A.

other-KS-AT-DH-KR-ACP*-TE

aug_v2a.16843.t1 aug_v2a.16843scaf8086:11276-29700(−)

aug_v2a.16843

aug_v2a.16843

N.A.

other-KS-AT-DH-KR-TE

aug_v2a.16847.t1 aug_v2a.16847scaf8086:95926-115499(−)

aug_v2a.16847

aug_v2a.16847

N.A.

other-KS-AT-DH-KR-TE

 

Nematostella vectensis

Sea anemone

-

-

Nematostella vectensis v1.0

-

-

-

 

Hydra magnipapillata

Hydra

-

-

Hydra magnipapillataHydra_RP_1.0

-

-

-

 

Amphimedon queenslandica

Sponges

-

-

Amphimedon queenslandicav1.0

-

-

-

Caenorhabditis elegans

Nematode

Protein C41A3.1 [Caenorhabditiselegans]

NP_508923

Caenorhabditis elegansWBcel235

C41A3.1

Yes

KS-KS-DH-ACP-KS-ACP-ACP-KR-ACP-AT-DH-KS-KR-KS-AT-ADH-ACP-C-AMP-ACP-TE

 

Drosophila melanogaster

Fruit fly

-

-

Drosophila melanogasterRelease 5

-

-

-

Asterisks show domains lacking a residue of the active site that is contained in OlPKS.

Sequence sources are mainly NCBI protein database, except dolphin, Chinese soft shell turtle, lamprey, coral, and sea anemone.

KS: ketoacyl synthase, AT: acyl transferase, DH: dehydratase, KR: ketoreductase, ACP: acyl carrier protein domain, TE: thioesterase, AMP: AMP-binding site, MT: methyltransferase, ADH: alcohol dehydrogenase, C: condensation domain.

Based on above results, we conclude that OlPKS is only required for the early step of otolith mineralization and that the mutation in olpks is responsible for the ha phenotype.

OlPKS produces lipophilic substances secreted into the endolymph

We hypothesized that, similar to other PKSs, OlPKS synthesizes polyketide-related small compounds in the OV epithelium, which are then secreted into the endolymph for the initial step of otolith mineralization.

To test this idea, we first examined subcellular localization of OlPKS. Immunostaining revealed that the medial wall of the OV exclusively expresses OlPKS at st. 23, which is highly localized at the apical of epithelial cells (Figure 3A Left) (a region for antigen is described in Additional file 5B). Double staining with an antibody to PKC ζ (Figure 3A Center), an apical membrane marker, demonstrated that the distribution of OlPKS enzyme partially overlaps with that of PKC ζ but OlPKS signal is detected closer to the lumen (Figure 3A Right). Thus, substances synthesized at the apical surface can be directly secreted to the endolymph, in a way similar to ‘membrane-localized’ PKS observed in bacterial cells [36,37].
Figure 3

OlPKS produces lipophilic substances secreted into the endolymph. (A) Intracellular localization of OlPKS in the OV epithelium (dorsal views of the left OV). Anti-OlPKS antibody detects the OlPKS protein at the apical region of the epithelial cells. Co-immunostaining with a membrane marker, PKC ζ, shows it localizes near the apical membrane. Yellow dotted line: OV. Scale bar: 10 μm. (B) Schematic procedure of the chimeric experiment. (C) Some images of the chimeric OVs in live embryos. [wt → ha] shows wt cells expressing DsRed are transplanted to an ha embryo. [ha → ha] is a negative control experiment. Yellow dotted lines: OVs. Scale bars: 10 μm. (D) Schematic representation of the heterologous expression system using A. oryzae. (E) Summary of the bioassay in the heterologous expression experiment. Numbers of ha embryos treated by the extract of olpks transfromant or that of empty vector toransformant are shown (Upper table). Grades of recovery of the mineralization: four otoliths (two per OV, fully rescued), 1–3 otoliths (at least one otolith per embryo, partially rescued) and no otolith (not rescued). Representative picture of treated embryo in each category is shown with a picture of wt embryo (Lower). Scale bar: 100 μm.

Next, we performed a chimera experiment in which DsRed-expressing wt cells was transplanted into mutant blastula, and examined for otolith formation when wt donor cells colonized mutant OVs (Figure 3B). Remarkably, irrespective of their number and localization within the OV, wt cells effectively restored otolith formation at the appropriate time and location, the macula region, in ha embryos. Only a few cells, located at any region of the vesicle, were found to be sufficient (Figure 3C and Additional file 6A and B).

Finally, we adopted a heterologous expression system to characterize substances synthesized by OlPKS. Since large-scale expression of PKSs has been established in Aspergillus oryzae [24], we introduced the olpks cDNA into A. oryzae, expecting that exogenous PKS (i.e., OlPKS) could work using endogenous substrates such as acetyl-CoA and malonyl-CoA in fungal cells like their own PKSs (Figure 3D). OlPKS expression in transformed fungi was confirmed by western-blotting (Additional file 6C). Since polyketide-derivatives exhibit moderate hydrophobic nature, we extracted cultivated mycelia with acetone, followed by purification using partition between ethyl acetate/H2O and subsequent purification. We then attempted a simple in vivo assay in which ha mutant embryos were cultured with an aliquot of the extracts (Figure 3D). Remarkably, these extracts restored otolith mineralization in ha embryos, while no such rescue was observed with control extracts (Figure 3E). Taken together, these data demonstrate that it is not OlPKS but ethyl-acetate extractable substances synthesized by OlPKS and secreted into the endolymph that nucleate otolith mineralization.

Broad distribution and conserved roles of polyketide synthases in animals

Animal pks genes were rarely explored, except for two echinoderm pks-1 and pks-2 (isolated from Strongylocentrotus purpuratus) [38,39], and the presence of fish, bird, and nematode pks genes were reported by previous phylogenetic analyses [40-42]. We performed thorough database searches for animal pks genes against current updated genome databases. Identified pks gene candidates were then assessed for a constitution of domains in each predicted amino acid sequence. These searches revealed a remarkably broad distribution of pks genes from Cnidaria to Bilateria, including coral (Acropora digitifera), C. elelgans and reptiles/birds (Figure 4A and Table 2). Usually, 1–3 pks genes are present in each genome, except for the lancelet genome that contains 13 genes. Most vertebrate PKSs have five domains, which are similar to OlPKS (e.g., KS, AT, DH, KR and ACP; Table 2). Zebrafish pks, drpks (wu:fc01d11), expresses in the otic vesicle (Additional file 7B). By contrast, other animal PKSs are not similar to olpks and have versatile domains, especially C. elegans PKS contains 21 domains in the polypeptide (predicted by Pfam search) [42]. The phylogenetic tree constructed using the sequence of KS domain (Additional file 7A) shows that animal PKSs are phylogenetically distinct from animal FASs and rather they are close to microbial PKS genes (Additional file 7A).
Figure 4

Broad distribution and conserved roles of PKSs in animals. (A) Distribution of the pks genes found by the BLAST searches in the schematic phylogenetic tree of animal kingdom. Red font shows the presence of type I pks gene(s) in the species. Except for fly, frog and mammal, most intensively studied models, pks genes could be overlooked due to incomplete genome information. (B) Whole-mount in situ hybridization of H. pulcherrimus with probes for hppks-1 (Upper Panel) and hppks-2 (Lower Panel). hppks-1 was first detected at the mesenchyme blastula stage in the precursors of the secondary mesenchyme cells (SMCs) at the vegetal pole, and the expression persisted until the prism stage, in the SMCs and then in the ectoderm. The expression was no longer observed in pluteus larvae. hppks-2 expression initiates in PMC precursors at the blastula stage and disappear by late gastrula just after spicule formation starting (mid-gastrulation). (C) Representative results of the MO knockdown experiments in H. pulcherrimus. Images were taken at two stages (24 h and 48 h). Arrows indicate pigment cells. HpPKS-2 first Met MO-injected or its control MO-injected embryos were also observed by a dark-field microscope for visualizing the spicules. Each MO was injected at a concentration of 200 μM. ‘CMO’: Control MO, Scale bars: 50 μm.

We hypothesized that like medaka PKSs, some of the other animal PKSs participate in biomineralization, more specifically calcium carbonate mineralization. To test this idea, we focused on echinoderm pks-2 because its reported expression is in primary mesenchyme cells (PMCs) that give rise to spicules, larval skeletons made of calcium carbonate [38,43]. We first confirmed the PMC-specific expression of pks-2 in our experimental system, Hemicentrotus pulcherrimus (hppks-2). Importantly, the expression of sea urchin pks-2 disappears around late gastrula stage just after PMCs begin to form spicules (Figure 4B Lower Panel). We then examined the function of HpPKS-2 by injecting HpPKS-2 MO. As shown in Figure 4C, HpPKS-2 morphants exhibited severe defects in spicule formation while control MO-injected embryos appeared normal (Figure 4C and Additional file 7C). Another echinoderm pks gene, hppks-1 was found to contribute to pigmentation as previously reported [39] (Figure 4B Upper Panel and C, and Additional file 7C). We thus conclude that echinoderm pks-2 plays a critical role in the formation of calcareous skeletal elements in larva.

Discussion

We here show that olpks encoded by the ha locus is essential for the early step of otolith biomineralization in medaka. In ha mutant embryos, mineralized stones never form, but instead OMP-1-positive particles precipitate in the endolymph. Our TEM observation confirmed that crystal growth is never initiated in ha OV despite the accumulation of otolith materials (Figure 1D), suggesting that the product of OlPKS participates in the initial step, in particular, nucleation of mineralization. The expression pattern of olpks supports its early function; the expression is transient and disappears by the onset of otolith formation (Figure 2D). This pattern of expression contrasts sharply with that of other essential genes identified so far. For instance, otolin-1 and omp-1 expression begins at st. 25 and persists into adulthood as these proteins continue to be deposited on growing otoliths throughout life [44,45]. Furthermore, the expression of medaka starmaker (called starmaker-like), a key regulator of the crystal lattice of calcium carbonates [46], is detected at st. 21 and is again maintained at later stages [47]. Intriguingly, ha fry develop an otolith-like stone (Additional file 2H), and some adult ha fish finally have otolith-like stones, albeit with abnormal shape and size [48]. This does not occur in zebrafish ‘no-otolith’-type mutants such as backstroke and keinstein [49]. These facts further suggest that the endolymph of ha only lacks a trigger. In ha, continuous supply of otolith materials without crystallization could lead to abnormal development of the inner ear and irregular stone formation at later stages. Together with these phenotypic and expression analyses, we conclude that the product of OlPKS functions as a nucleation trigger (facilitator).

Despite our efforts to identify the product of OlPKS, it remains elusive; we failed to detect any specific peak of the synthesized compound in HPLC analysis using extracts of A. oryzae transformed with olpks, probably due to low production of the compound. However, the active substance could have amphiphilic nature like other polyketide (e.g., phenolic lipids [50]) because it was re-solved in medaka culture medium and penetrates into the otic vesicle of the embryo in the rescue experiment (Figure 3D). Given its amphiphilic nature, the product of OlPKS could serve as a bridge between ACC and organic components such as soluble matrix protein (e.g., OMP-1) and/or insoluble scaffold matrices (e.g., Otolin-1)(the possible functions of these organic compounds in biomineralization were proposed by Nagasawa [3]). Supporting this idea, in studies of High-resolution Cryo-TEM imaging, monolayers of fatty acids (arachidic acid or stearic acid) that are also amphiphilic in nature, have been used to artificially induce crystal nucleation [7,51]. Further investigation using cell-free systems [52,53] will be necessary to address at which step of biomineralization PKS products actually work.

Otolith formation shares many features with the formation of vertebrate bones consisting of calcium phosphate (hydroxyapatite) minerals, the best characterized calcification process to date [54]. Intriguingly, in bone formation, phospholipid such as phosphatidylserine (PS) has been implicated in an inducer of hydroxyapatite mineralization and most likely acts as a nucleation facilitator [52,55,56]. Since PS is a small substance with amphiphilic nature, the product of OlPKS could be a functional counterpart in calcium carbonate mineralization, interacting with calcium ions, ACC and matrix proteins.

The phylogenetic analysis based on the KS domain demonstrates the topology in which animal PKSs are separated from the animal FAS clade (Additional file 7A). Unlike the fas gene, the distribution of pks genes in animals is irregular; they are not found in some animal groups, for examples, fly, frog, and mammal, which represent some of the most intensively studied model animals (Figure 4A). These facts complicate the evolutionary origin of animal pks genes, while a single origin of animal fas has been repeatedly supported. Horizontal gene transfer, gene duplications and gene losses could have occurred during evolution, as proposed in bacteria/fungi PKSs [57-59]. Among those animal pks genes, sea urchin pks-2 could be functionally equivalent to olpks because of its early and transient expression in the calcifying cell lineage and its knockdown phenotype, although the domain architecture of sea urchin PKS-2 differs to some degree from that of OlPKS, being KS-AT-DH-ER-KR-ACP-TE and KS-AT-DH-KR-ACP, respectively [38].

Given the conserved role of medaka and sea urchin PKSs, calcium carbonate biomineralization could generally require the products of PKSs for nucleation. PKSs could participate in the production of coral skeletons and calcification of algae in the ocean. For instance, Emiliania huxleyi [58,60] has some pks genes (e.g., fgeneshEH_pg.50__74, protein ID103465, Joint Genome Institute (http://genome.jgi-psf.org/pages/search-for-genes.jsf?organism=Emihu1)). Intriguingly, the presence of PKS in Cnidaria appears to be associated with biomineraliztion; coral (A. digitifera) has a couple of pks genes, whereas Hydra magnipapillata and Nematostella vectensis do not. Furthermore, pks genes of bird and reptile might be involved in production of egg shells. In frogs and mammals, however, pks genes were no longer needed and lost during evolution, although their inner ears still contain calcium carbonate biominerals. This inconsistency may be explained by the fact that their biominerals do not form large stones, but instead small grains called otoconia. However, further intense search in those animals which appear to have lost pks genes will definitely be needed.

Conclusions

The present study addresses the function of the vertebrate pks gene and demonstrates its vital role in calcium carbonate biomineralization. Further functional analyses of newly identified PKSs will uncover a long-overlooked world of polyketides and their derivatives in animals. Our finding also provides genetic and molecular cues for the geochemical study of global carbon and calcium cycles.

Accession numbers

pks genes reported herein have been deposited in GenBank with accession numbers as follow: olpks [AB923905], hppks-1 [AB923906] and hppks-2 [AB923907].

Declarations

Acknowledgements

We thank Dr. Hiromichi Nagasawa for providing anti- O. mykiss OMP-1 serum; Dr. Joachim Wittbrodt for providing the cDNA of pax2; Dr. Akira Kudo for providing the ki79 fish; Yasuko Ozawa for excellent fish care; Sayaka Tayama for her contribution to the chimeric experiment. Members of the Yasuo Ohnishi laboratory, past and present, are acknowledged, particularly Adeline Muliandi, Takayuki Hayashi and Takuya Makino.

This work was supported in part by Exploratory Research ("Houga Kenkyu") Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology.

Authors’ Affiliations

(1)
Department of Biological Sciences, Graduate School of Science, University of Tokyo
(2)
Tateyama Marine Laboratory, Marine and Coastal Research Center, Ochanomizu University
(3)
Department of Food and Nutrition, Japan Women’s University
(4)
Misaki Marine Biological Station, Graduate School of Science, University of Tokyo
(5)
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo
(6)
Present address: Department of Pharmaceutical and Environmental Sciences, Tokyo Metropolitan Institute of Public Health
(7)
Present address: Division of Molecular Pathology, Research Institute for Biomedical Sciences, Tokyo University of Science
(8)
Present address: Laboratory of Veterinary Biochemistry, Nihon University College of Bioresource Sciences

References

  1. Addadi L, Weiner S: Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization. Proc Natl Acad Sci U S A 1985, 82:4110–4114.View ArticlePubMed CentralPubMedGoogle Scholar
  2. Weiner S, Hood L: Soluble protein of the organic matrix of mollusk shells: a potential template for shell formation. Science 1975, 190:987–989.View ArticlePubMedGoogle Scholar
  3. Nagasawa H: The molecular mechanism of calcification in aquatic organisms. Biosci Biotechnol Biochem 2013, 77:1991–1996.View ArticlePubMedGoogle Scholar
  4. Lowenstam HA, Weiner S: On Biomineralization. New York: Oxford University Press; 1989.Google Scholar
  5. Westbroek P, Brown CW, Van Bleijswijk J, Brownlee C, Brummer GJ, Conte M, Egge J, Fernandez E, Jordan R, Knappertsbusch M, Stefels J, Veldhuis M, Van der Wal P, Young, J: A model system approach to biological climate forcing. The example of Emiliania huxleyi. Glob Planet Change 1993, 8:27–46.View ArticleGoogle Scholar
  6. Van CP: Biomineralization and Global Biogeochemical Cycles. Rev Mineral Geochem 2003, 54:357–381.View ArticleGoogle Scholar
  7. Dey A, Bomans PH, Muller FA, Will J, Frederik PM, de With G, Sommerdijk NA: The role of prenucleation clusters in surface-induced calcium phosphate crystallization. Nat Mater 2010, 9:1010–1014.View ArticlePubMedGoogle Scholar
  8. Gebauer D, Volkel A, Colfen H: Stable prenucleation calcium carbonate clusters. Science 2008, 322:1819–1822.View ArticlePubMedGoogle Scholar
  9. Campana SE: Chemistry and composition of fish otoliths: pathway, mechanisms and applications. Mar Ecol Prog Ser 1999, 188:263–297.View ArticleGoogle Scholar
  10. Pisam M, Jammet C, Laurent D: First steps of otolith formation of the zebrafish: role of glycogen? Cell Tissue Res 2002, 310:163–168.View ArticlePubMedGoogle Scholar
  11. Whitfield TT, Granato M, van Eeden FJ, Schach U, Brand M, Furutani-Seiki M, Haffter P, Hammerschmidt M, Heisenberg CP, Jiang YJ, Kane, DA, Kelsh RN, Mullins MC, Odenthal J, Nusslein-Volhard C: Mutations affecting development of the zebrafish inner ear and lateral line. Dev 1996, 123:241–254.Google Scholar
  12. Schibler A, Malicki J: A screen for genetic defects of the zebrafish ear. Mech Dev 2007, 124:592–604.View ArticlePubMedGoogle Scholar
  13. Tomita H: Strains And Mutants. In Biology of the Medaka (Egami, N ed University of Tokyo Press). 1990:111–127.Google Scholar
  14. Kimura T, Jindo T, Narita T, Naruse K, Kobayashi D, Shin IT, Kitagawa T, Sakaguchi T, Mitani H, Shima A, Kohara Y, Takeda H: Large-scale isolation of ESTs from medaka embryos and its application to medaka developmental genetics. Mech Dev 2004, 121:915–932.View ArticlePubMedGoogle Scholar
  15. Ohtsuka M, Kikuchi N, Yokoi H, Kinoshita M, Wakamatsu Y, Ozato K, Takeda H, Inoko H, Kimura M: Possible roles of zic1 and zic4, identified within the medaka Double anal fin (Da) locus, in dorsoventral patterning of the trunk-tail region (related to phenotypes of the Da mutant). Mech Dev 2004, 121:873–882.View ArticlePubMedGoogle Scholar
  16. Hojo M, Takashima S, Kobayashi D, Sumeragi A, Shimada A, Tsukahara T, Yokoi H, Narita T, Jindo T, Kage T, Kitagawa T, Kimura T, Sekimizu K, Miyake A, Setiamarga D, Murakami R, Tsuda S, Ooki S, Kakihara K, Naruse K, Takeda H: Right-elevated expression of charon is regulated by fluid flow in medaka Kupffer's vesicle. Develop Growth Differ 2007, 49:395–405.View ArticleGoogle Scholar
  17. Murayama E, Herbomel P, Kawakami A, Takeda H, Nagasawa H: Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae. Mech Dev 2005, 122:791–803.View ArticlePubMedGoogle Scholar
  18. Kamura K, Kobayashi D, Uehara Y, Koshida S, Iijima N, Kudo A, Yokoyama T, Takeda H: Pkd1l1 complexes with Pkd2 on motile cilia and functions to establish the left-right axis. Dev 2011, 138:1121–1129.View ArticleGoogle Scholar
  19. Yokoi H, Shimada A, Carl M, Takashima S, Kobayashi D, Narita T, Jindo T, Kimura T, Kitagawa T, Kage T, Sawada A, Naruse K, Asakawa S, Shimizu N, Mitani H, Shima A, Tsutsumi M, Hori H, Wittbrodt J, Saga Y, Ishikawa Y, Araki K, Takeda H: Mutant analyses reveal different functions of fgfr1 in medaka and zebrafish despite conserved ligand-receptor relationships. Dev Biol 2007, 304:326–337.View ArticlePubMedGoogle Scholar
  20. Witkowski A, Joshi A, Smith S: Fatty acid synthase: in vitro complementation of inactive mutants. Biochemistry 1996, 35:10569–10575.View ArticlePubMedGoogle Scholar
  21. Smith S, Witkowski A, Joshi AK: Structural and functional organization of the animal fatty acid synthase. Prog Lipid Res 2003, 42:289–317.View ArticlePubMedGoogle Scholar
  22. Reid R, Piagentini M, Rodriguez E, Ashley G, Viswanathan N, Carney J, Santi DV, Hutchinson CR, McDaniel R: A model of structure and catalysis for ketoreductase domains in modular polyketide synthases. Biochemistry 2003, 42:72–79.View ArticlePubMedGoogle Scholar
  23. Shimada A, Yabusaki M, Niwa H, Yokoi H, Hatta K, Kobayashi D, Takeda H: Maternal-zygotic medaka mutants for fgfr1 reveal its essential role in the migration of the axial mesoderm but not the lateral mesoderm. Dev 2008, 135:281–290.View ArticleGoogle Scholar
  24. Gomi K, Iimura Y, Hara S: Integrative Transformation of Aspergillus oryzae with a Plasmid Containing the Aspergillus nidulans argB Gene. Agric Biol Chem 1987, 51:2549–2555.View ArticleGoogle Scholar
  25. Katow H, Katow T, Abe K, Ooka S, Kiyomoto M, Hamanaka G: Mesomere-derived glutamate decarboxylase-expressing blastocoelar mesenchyme cells of sea urchin larvae. Biology open 2014, 3:94–102.View ArticlePubMed CentralPubMedGoogle Scholar
  26. Ijiri K: Vestibular and visual contribution to fish behavior under microgravity. Adv Space Res 2000, 25:1997–2006.View ArticlePubMedGoogle Scholar
  27. Mizuno R, Ijiri K: Otolith formation in a mutant medaka with a deficiency in gravity-sensing. Adv Space Res 2003, 32:1513–1520.View ArticlePubMedGoogle Scholar
  28. Riley BB, Zhu C, Janetopoulos C, Aufderheide KJ: A critical period of ear development controlled by distinct populations of ciliated cells in the zebrafish. Dev Biol 1997, 191:191–201.View ArticlePubMedGoogle Scholar
  29. Colantonio JR, Vermot J, Wu D, Langenbacher AD, Fraser S, Chen JN, Hill KL: The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear. Nature 2009, 457:205–209.View ArticlePubMedGoogle Scholar
  30. Wu D, Freund JB, Fraser SE, Vermot J: Mechanistic basis of otolith formation during teleost inner ear development. Dev Cell 2011, 20:271–278.View ArticlePubMedGoogle Scholar
  31. Stooke-Vaughan GA, Huang P, Hammond KL, Schier AF, Whitfield TT: The role of hair cells, cilia and ciliary motility in otolith formation in the zebrafish otic vesicle. Dev 2012, 139:1777–1787.View ArticleGoogle Scholar
  32. Omran H, Kobayashi D, Olbrich H, Tsukahara T, Loges NT, Hagiwara H, Zhang Q, Leblond G, O'Toole E, Hara C, Mizuno H, Kawano H, Fliegauf M, Yagi T, Koshida S, Miyawaki A, Zentgraf H, Seithe H, Reinhardt R, Watanabe Y, Kamiya R, Mitchell DR, Takeda H: Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins. Nature 2008, 456:611–616.View ArticlePubMed CentralPubMedGoogle Scholar
  33. Nikolouli K, Mossialos D: Bioactive compounds synthesized by non-ribosomal peptide synthetases and type-I polyketide synthases discovered through genome-mining and metagenomics. Biotechnol Lett 2012, 34:1393–1403.View ArticlePubMedGoogle Scholar
  34. Staunton J, Weissman KJ: Polyketide biosynthesis: a millennium review. Nat Prod Rep 2001, 18:380–416.View ArticlePubMedGoogle Scholar
  35. Smith S, Tsai SC: The type I fatty acid and polyketide synthases: a tale of two megasynthases. Nat Prod Rep 2007, 24:1041–1072.View ArticlePubMed CentralPubMedGoogle Scholar
  36. Straight PD, Fischbach MA, Walsh CT, Rudner DZ, Kolter R: A singular enzymatic megacomplex from Bacillus subtilis. Proc Natl Acad Sci U S A 2007, 104:305–310.View ArticlePubMed CentralPubMedGoogle Scholar
  37. Jain M, Cox JS: Interaction between polyketide synthase and transporter suggests coupled synthesis and export of virulence lipid in M. tuberculosis. PLoS Pathog 2005, 1:12–19.View ArticleGoogle Scholar
  38. Beeble A, Calestani C: Expression pattern of polyketide synthase-2 during sea urchin development. Gene Expr Patterns 2012, 12:7–10.View ArticlePubMed CentralPubMedGoogle Scholar
  39. Calestani C, Rast JP, Davidson EH: Isolation of pigment cell specific genes in the sea urchin embryo by differential macroarray screening. Dev 2003, 130:4587–4596.View ArticleGoogle Scholar
  40. Castoe TA, Stephens T, Noonan BP, Calestani C: A novel group of type I polyketide synthases (PKS) in animals and the complex phylogenomics of PKSs. Gene 2007, 392:47–58.View ArticlePubMedGoogle Scholar
  41. Leibundgut M, Maier T, Jenni S, Ban N: The multienzyme architecture of eukaryotic fatty acid synthases. Curr Opin Struct Biol 2008, 18:714–725.View ArticlePubMedGoogle Scholar
  42. O'Brien RV, Davis RW, Khosla C, Hillenmeyer ME: Computational identification and analysis of orphan assembly-line polyketide synthases. J Antibiot (Tokyo) 2014, 67:89–97.View ArticleGoogle Scholar
  43. Rafiq K, Cheers MS, Ettensohn CA: The genomic regulatory control of skeletal morphogenesis in the sea urchin. Dev 2012, 139:579–590.View ArticleGoogle Scholar
  44. Nemoto Y, Chatani M, Inohaya K, Hiraki Y, Kudo A: Expression of marker genes during otolith development in medaka. Gene Expr Patterns 2008, 8:92–95.View ArticlePubMedGoogle Scholar
  45. Murayama E, Takagi Y, Nagasawa H: Immunohistochemical localization of two otolith matrix proteins in the otolith and inner ear of the rainbow trout, Oncorhynchus mykiss: comparative aspects between the adult inner ear and embryonic otocysts. Histochem Cell Biol 2004, 121:155–166.View ArticlePubMedGoogle Scholar
  46. Sollner C, Burghammer M, Busch-Nentwich E, Berger J, Schwarz H, Riekel C, Nicolson T: Control of crystal size and lattice formation by starmaker in otolith biomineralization. Science 2003, 302:282–286.View ArticlePubMedGoogle Scholar
  47. Bajoghli B, Ramialison M, Aghaallaei N, Czerny T, Wittbrodt J: Identification of starmaker-like in medaka as a putative target gene of Pax2 in the otic vesicle. Dev Dyn 2009, 238:2860–2866.View ArticlePubMedGoogle Scholar
  48. Noro S, Yamamoto N, Ishikawa Y, Ito H, Ijiri K: Studies on the morphology of the inner ear and semicircular canal endorgan projections of ha, a medaka behavior mutant. Fish Biol J Medaka 2007, 11:31–41.Google Scholar
  49. Sollner C, Schwarz H, Geisler R, Nicolson T: Mutated otopetrin 1 affects the genesis of otoliths and the localization of Starmaker in zebrafish. Dev Genes Evol 2004, 214:582–590.View ArticlePubMedGoogle Scholar
  50. Kozubek A, Tyman JH: Resorcinolic Lipids, the Natural Non-isoprenoid Phenolic Amphiphiles and Their Biological Activity. Chem Rev 1999, 99:1–26.View ArticlePubMedGoogle Scholar
  51. Pouget EM, Bomans PH, Goos JA, Frederik PM, de With G, Sommerdijk NA: The initial stages of template-controlled CaCO3 formation revealed by cryo-TEM. Science 2009, 323:1455–1458.View ArticlePubMedGoogle Scholar
  52. Boskey AL: Hydroxyapatite formation in a dynamic collagen gel system: effects of type i collagen, lipids, and proteoglycans. J Phys Chem 1989, 93:1628–1633.View ArticleGoogle Scholar
  53. Tohse H, Saruwatari K, Kogure T, Nagasawa H, Takagi Y: Control of polymorphism and morphology of calcium carbonate crystals by a matrix protein aggregate in fish otoliths. Cryst Growth Des 2009, 9:4897–4901.View ArticleGoogle Scholar
  54. Hughes I, Thalmann I, Thalmann R, Ornitz DM: Mixing model systems: using zebrafish and mouse inner ear mutants and other organ systems to unravel the mystery of otoconial development. Brain Res 2006, 1091:58–74.View ArticlePubMed CentralPubMedGoogle Scholar
  55. Wu LN, Genge BR, Dunkelberger DG, LeGeros RZ, Concannon B, Wuthier RE: Physicochemical characterization of the nucleational core of matrix vesicles. J Biol Chem 1997, 272:4404–4411.View ArticlePubMedGoogle Scholar
  56. Satsangi A, Satsangi N, Glover R, Satsangi RK, Ong JL: Osteoblast response to phospholipid modified titanium surface. Biomaterials 2003, 24:4585–4589.View ArticlePubMedGoogle Scholar
  57. Jenke-Kodama H, Dittmann E: Evolution of metabolic diversity: insights from microbial polyketide synthases. Phytochemistry 2009, 70:1858–1866.View ArticlePubMedGoogle Scholar
  58. John U, Beszteri B, Derelle E, Van de Peer Y, Read B, Moreau H, Cembella A: Novel Insights into Evolution of Protistan Polyketide Synthases through Phylogenomic Analysis. Protist 2008, 159:21–30.View ArticlePubMedGoogle Scholar
  59. Foerstner KU, Doerks T, Creevey CJ, Doerks A, Bork P: A computational screen for type I polyketide synthases in metagenomics shotgun data. PLoS One 2008, 3:e3515.View ArticlePubMed CentralPubMedGoogle Scholar
  60. Jones BM, Edwards RJ, Skipp PJ, O'Connor CD, Iglesias-Rodriguez MD: Shotgun proteomic analysis of Emiliania huxleyi, a marine phytoplankton species of major biogeochemical importance. Mar Biotechnol 2011, 13:496–504.View ArticlePubMedGoogle Scholar

Copyright

© Hojo et al.; licensee BioMed Central. 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Advertisement