PfSMAD4 plays a role in biomineralization and can transduce bone morphogenetic protein-2 signals in the pearl oyster Pinctada fucata.
BACKGROUND
Mollusca 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 involvement in BMP signaling in invertebrates, but whether SMAD4 plays a conservative role in pearl oyster, Pinctada fucata, still need to be tested.


RESULTS
In 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.


CONCLUSIONS
Our 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.
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China.|University of Chinese Academy of Sciences, Beijing, 100049, China.
Mi
M
Zhao
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China.
Yu
Y
Shi
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China. hmx@scsio.ac.cn.
Maoxian
M
He
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China.
Xiande
X
Huang
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China.|University of Chinese Academy of Sciences, Beijing, 100049, China.
Qi
Q
Wang
D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism*; D002113:Calcification, Physiologic; D003593:Cytoplasm / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D060734:Nacre / Q000378:metabolism; D010802:Phylogeny; D049911:Pinctada / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D021381:Protein Transport; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D015398:Signal Transduction; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D055396:Bone Morphogenetic Protein 2; D060734:Nacre; D012333:RNA, Messenger; D051901:Smad4 Protein
BMP signaling pathway; Biomineralization; Pinctada fucata; SMAD4