Unexpected link between polyketide synthase and calcium carbonate biomineralization

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. haembryos 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 ?m. (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 ?m. (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 ?m. (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 ?m. (E) Immunofluorescence of acetylated ?-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 ?m.

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 ?m) protruded from hair cells
in the prospective macula regions and stirring the fluid by shorter motile cilia (1.5–5 ?m)
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 ?m) 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. hagene 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 ?m. (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

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

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 ? (Figure 3A Center), an apical membrane marker, demonstrated that the distribution of OlPKS enzyme partially
overlaps with that of PKC ? 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 ?, shows it localizes near the
apical membrane. Yellow dotted line: OV. Scale bar: 10 ?m. (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 ?m. (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 ?m.

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 oryzae24], 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 ?M. ‘CMO’: Control MO, Scale bars: 50 ?m.

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.