Hojo M et al. (2015), Unexpected link between polyketide synthase and...

ECB-ART-50023

Zoological Lett 2015 Jan 13;1:3. doi: 10.1186/s40851-014-0001-0.

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Unexpected link between polyketide synthase and calcium carbonate biomineralization.

Hojo M , Omi A , Hamanaka G , Shindo K , Shimada A , Kondo M , Narita T , Kiyomoto M , Katsuyama Y , Ohnishi Y , Irie N , Takeda H .

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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.

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	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.
	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.
	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.
	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.

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