Abstract
BackgroundMollusca is the second largest phylum in nature. The shell of molluscs is a remarkable example of a natural composite biomaterial. Biomineralization and how it affects mollusks is a popular research topic. The BMP-2 signaling pathway plays a canonical role in biomineralization. SMAD4 is an intracellular transmitter in the BMP signaling pathway in mammals, and some genomic data show SMAD4’s involvment in BMP signaling in invertbrates, but whether SMAD4 plays a conservative role in pearl oyster, Pinctada fucata, still need to be tested.ResultsIn this study, we identified a SMAD4 gene (hereafter designated PfSMAD4) in pearl oyster Pinctada fucata. Bioinformatics analysis of PfSMAD4 showed high identity with its orthologs. PfSMAD4 was located in the cytoplasm in immunofluorescence assays and analyses of PfSMAD4 mRNA in tissues and developmental stages showed high expression in ovaries and D-shaped larvae. An RNA interference experiment, performed by PfSMAD4 double-stranded RNA (dsRNA) injection, demonstrated inhibition not only of nacre growth but also organic sheet formation with a decrease in PfSMAD4 expression. A knockdown experiment using PfBMP2 dsRNA showed decreased PfBMP2 and PfSMAD4 mRNA and irregular crystallization of the nacreous layer using scanning electron microscopy. In co-transfection experiments, PfBMP2-transactivated reporter constructs contained PfSMAD4 promoter sequences.ConclusionsOur results suggest that PfSMAD4 plays a role in biomineralization and can transduce BMP signals in P. fucata. Our data provides important clues about the molecular mechanisms that regulate biomineralization in pearl oyster.
Highlights
Mollusca is the second largest phylum in nature
Homology analysis revealed that the whole PfSMAD4 sequence shared 27.8–77.5 % identity to other known SMAD4 sequences, while the Mad homology 1 (MH1) domain shared 62–93.7 % identity and MH2 domain shared 56.1–96.8 % (Fig. 1)
PfSMAD4 expression in tissues and developmental stages To investigate the expression pattern of PfSMAD4 among various tissues and developmental stages in pearl oyster, Quantitative PCR (qPCR) analysis was performed with gene specific primers
Summary
The shell of molluscs is a remarkable example of a natural composite biomaterial. Biomineralization and how it affects mollusks is a popular research topic. The BMP-2 signaling pathway plays a canonical role in biomineralization. The plain outer surface of pearl oyster shells conceal the lustrous beauty of the mother-of-pearl lining ‘nacre’. It combines a high mechanical strength similar to many ceramics, with elasticity, reducing the brittleness of the shell [1, 2]. Bone morphogenic proteins (BMP) are the largest subgroup in the transforming growth factor-beta (TGF-β) superfamily [6] and play a canonical role in biomineralization [7, 8].
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