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Rongqing Zhang - One of the best experts on this subject based on the ideXlab platform.

  • cellular regulation of biomineralization in Pinctada fucata
    2019
    Co-Authors: Rongqing Zhang
    Abstract:

    Because it is widely recognized that the mantle tissue controls nacre precipitation, intensive studies have been conducted to unravel the regulatory mechanism underlying nacre formation. In this chapter, we will focus on the cellular regulation of shell formation in the pearl oyster Pinctada fucata. First, the morphology and proliferation rate in different parts of the mantle tissue have been investigated, and a proliferation hot spot located in the center of the thinnest mantle region was found. However, mantle tissues mainly composed of the mantle edge were used for primary cell culture, due to their high yield of migrated mantle cells. The primary mantle cell culture is used to study how the mantle cells regulate calcium carbonate precipitation. It was found that the physiological functions of the mantle cells were maintained in vitro. High expression of many shell matrix proteins, including ACCBP, Pif80, and nacrein, and high activities of carbonic anhydrase and alkaline phosphatase were detected. Numerous crystals were found inside the cultured cells by polar light microscopy and scanning electron microscopy, and FTIR and XRD analysis demonstrated that these particles were amorphous calcium carbonate (ACC). What’s more, the cultured mantle cells promoted and regulated calcium carbonate precipitation in the culture dishes. These results showed that mantle cells may directly participate in shell formation. In addition, we have studied the function of hemocytes in shell formation. Hemocytes were identified from P. fucata and found to be present in the extrapallial space (EPS). Many components involved in immunity and calcification were identified by proteomics analysis. Poststimulation of lipopolysaccharide and shell damage, most of the tested immune genes and calcification, was upregulated. Moreover, polar light microscopy, scanning electron microscopy, and energy spectrum showed that some hemocytes carried crystals of calcium carbonate, indicating they participated in both immunity and biomineralization. Our studies elucidated the vital roles of mantle cells and hemocytes in regulating shell growth and nacre formation, which would shed light on the improvement of the cultured pearl quality.

  • Molecular Regulation Mechanism of Biomineralization of Pinctada fucata
    Biomineralization Mechanism of the Pearl Oyster Pinctada fucata, 2018
    Co-Authors: Rongqing Zhang
    Abstract:

    To elucidate the mechanism of biomineralization in Pinctada fucata, most of the researchers put their attention on the roles of matrix proteins in shell formation. Our group has identified and characterized many essential matrix proteins in the regulation of deposition of calcium carbonate crystals in both prism and nacre layers, as described in the former chapter. Meanwhile, the regulation of the transcription, translation and expression of matrix proteins and how these regulatory factors mediate the biomineralization have become a hot spot in recent years. In this chapter, we mainly assayed RACE to obtain the sequence of the members of signaling pathways and the transcriptional factors; real time-qPCR to analyze the expression pattern of these factors in different tissues and/or distinct time during shell repair and pearl sac; in situ hybridization to find out the expression location of specific genes in mantle tissue; luciferase assay and electrophoretic mobility assay (EMSA) to clarify the recruitment of transcriptional factors on promoters of matrix protein; yeast two hybridization to explore the interactions between different pathways. We demonstrate the function of NF-κB, TGFβ, Wnt signal pathway, G protein-mediated pathway and several transcriptional factors in mediating the biomineralization in Pinctada fucata. The mechanism of transcriptional regulation can give deep sight to the matrix protein expression pattern which enrich the theory of bionimeralization in Pinctada fucata.

  • The Study on Enzymes Related to Biomineralization of Pinctada fucata
    Biomineralization Mechanism of the Pearl Oyster Pinctada fucata, 2018
    Co-Authors: Rongqing Zhang
    Abstract:

    As an efficient, economical, and clean catalyst, enzymes are expected to have novel features. Efforts have been made to screen new enzymes in marine organisms. In our recent studies, we explored the basic characteristics and functions of alkaline phosphatases, acid phosphatases, carbonic anhydrases, tyrosinase, and a novel astacin-like metalloproteinase in Pinctada fucata.

  • Ecological Study on Biomineralization in Pinctada fucata
    Biomineralization Mechanism of the Pearl Oyster Pinctada fucata, 2018
    Co-Authors: Rongqing Zhang
    Abstract:

    Pinctada fucata inhabits in tropical and subtropical seas, so the sea water conditions have great impacts on its growth and shell formation. To determine the potential effects of global climate change and the water pollution on the oyster growth in the near future, we have conducted a series of studies about the metabolism and shell formation of P. fucata under varied stressors. Various factors controlling biomineralization, such as genes, environmental factors, and ions that ocean acidification (OA), ocean warming (OW), and metal pollution have great effects on the metabolism, biomineralization process, as well as the immune defense, were pointed by our study. It was found that, during short-term exposure, the impact of CO2 and temperature stresses was not manifested in the shell ultrastructure, although it affected the process of biomineralization. If the stressors are present in the long-term, they will have adverse results on the biomineralization of pearls, most likely affecting pearl quality, which results in substantial economic losses for the aquaculture industry. In addition, OA and OW have a great impact on the physiological conditions of hemocytes, including altering pH value of hemolymph and increasing the total hemocyte count, total protein content, and percentage of large hyalinocytes and granulocytes, while decreasing phagocytosis ability. We query the metal accumulations and the corresponding enzymatic responses in the pearl oyster Pinctada fucata after copper exposure. We found that the gills and the digestive gland played different roles associated with distinct copper concentrations and observed the adaptation and recovery of the oysters. Our studies have indicated potential implications for predictions of the effects of environmental changes on pearl aquaculture. Moreover, it also provides theoretical basis for precautions to avoid adverse impact of environmental challenges on the marine pearl aquaculture in the near future.

  • Identification (Characterization) and Function Studies of Matrix Protein from the Oyster Pinctada fucata
    Biomineralization Mechanism of the Pearl Oyster Pinctada fucata, 2018
    Co-Authors: Rongqing Zhang
    Abstract:

    Shell matrix protein (SMP) which is extracted from shell and extrapallial fluid matrix protein (EPFMP) which is extracted from extrapallial fluid have important functions in biomineralization during shell formation of Pinctada fucata. In the past decades, the functions of SMPs and EPFMPs were gradually revealed. In 2015, our group identified 72 unique SMPs in Pinctada fucata by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteins extracted from the shells of P. fucata aligned with a draft genome. In this chapter, we introduced SMPs from nacreous layer (P14, N40, and PfN23), from prismatic layer (KRMP, KRMP-3, and Prisilkin-39), from both prismatic and nacreous layer (MSI7, PfN44, and PfY2), EPFMP and extracellular matrix protein expressed by mantle (EFCBP and Ferritin). For example, P14 plays a crucial role during nacre biomineralization. N40 could stimulate the nucleation of aragonite drastically by serving as a nucleation site. The basic protein PfN23 might be a key accelerator during the regulation of crystal growth in nacre. KRMP protein family plays important roles in the framework formation of prism.

Maoxian He - One of the best experts on this subject based on the ideXlab platform.

  • pfsmad1 5 can interact with pfsmad4 to inhibit pfmsx to regulate shell biomineralization in Pinctada fucata martensii
    Marine Biotechnology, 2020
    Co-Authors: Mi Zhao, Maoxian He
    Abstract:

    The BMP2 signal transduced by SMAD1/5 plays an important role in osteoblast differentiation and bone formation. Shell formation of Pinctada fucata martensii is a typical biomineralization process that is similar to that of teeth/bone formation. However, whether the Pinctada fucata BMP2 (PfBMP2) signal transduced by PfSMAD1/5 occurs in P. f. martensii, how the PfBMP2 signal is transduced by PfSMAD1/5, and how PfSMAD1/5 regulates the biomineralization process in this species and other shellfish are poorly understood. Therefore, injection experiments of recombinant PfBMP2 and inhibitor dorsomorphin revealed that PfSMAD1/5 can transduce PfBMP2 signals. Subcellular localization and bimolecular fluorescence complementation assays indicated that PfSMAD1/5 phosphorylated by PfBMPR1b interacts with PfSMAD4 in the cytoplasm to form a complex, which translocates to the nucleus to transduce PfBMP2 signals. Co-immunoprecipitation and luciferase assays revealed that PfSMAD1/5 may interact with PfMSX to dislodge it from its binding element, resulting in initiation of mantle gene transcription. The in vivo functional assay showed that knockdown of PfMSAD1/5 decreased expression of shell matrix genes and disordered the nacreous layer, and the correlation assay of shell regeneration showed the concomitant expression pattern of PfSMAD1/5 and shell matrix genes. Together, these data showed that PfSMAD1/5 can transduce PfBMP2 signals to regulate shell biomineralization in P. f. martensii, which illustrated conservation of the BMP2-SMAD signal pathway among invertebrates. Particularly, the results suggest that there is only one PfMSX gene, which functions like the Hox gene in vertebrates, that interacts with PfSMAD1/5 in a protein-protein action form and plays the role of transcription repressor.

  • PfSMAD1/5 Can Interact with PfSMAD4 to Inhibit PfMSX to Regulate Shell Biomineralization in Pinctada fucata martensii
    Marine Biotechnology, 2020
    Co-Authors: Mi Zhao, Maoxian He
    Abstract:

    The BMP2 signal transduced by SMAD1/5 plays an important role in osteoblast differentiation and bone formation. Shell formation of Pinctada fucata martensii is a typical biomineralization process that is similar to that of teeth/bone formation. However, whether the Pinctada fucata BMP2 (PfBMP2) signal transduced by PfSMAD1/5 occurs in P. f. martensii, how the PfBMP2 signal is transduced by PfSMAD1/5, and how PfSMAD1/5 regulates the biomineralization process in this species and other shellfish are poorly understood. Therefore, injection experiments of recombinant PfBMP2 and inhibitor dorsomorphin revealed that PfSMAD1/5 can transduce PfBMP2 signals. Subcellular localization and bimolecular fluorescence complementation assays indicated that PfSMAD1/5 phosphorylated by PfBMPR1b interacts with PfSMAD4 in the cytoplasm to form a complex, which translocates to the nucleus to transduce PfBMP2 signals. Co-immunoprecipitation and luciferase assays revealed that PfSMAD1/5 may interact with PfMSX to dislodge it from its binding element, resulting in initiation of mantle gene transcription. The in vivo functional assay showed that knockdown of PfMSAD1/5 decreased expression of shell matrix genes and disordered the nacreous layer, and the correlation assay of shell regeneration showed the concomitant expression pattern of PfSMAD1/5 and shell matrix genes. Together, these data showed that PfSMAD1/5 can transduce PfBMP2 signals to regulate shell biomineralization in P. f. martensii, which illustrated conservation of the BMP2-SMAD signal pathway among invertebrates. Particularly, the results suggest that there is only one PfMSX gene, which functions like the Hox gene in vertebrates, that interacts with PfSMAD1/5 in a protein–protein action form and plays the role of transcription repressor.

  • pf dmrt4 a potential factor in sexual development in the pearl oyster Pinctada fucata
    Journal of Oceanology and Limnology, 2018
    Co-Authors: Qi Wang, Maoxian He
    Abstract:

    The mechanisms of sex determination and sex differentiation in the pearl oyster Pinctada fucata are currently poorly understood. We therefore investigated the roles of orthologs of the Dmrt gene family, key players in male gonad differentiation in mammals, in P. fucata sex differentiation and sexual development. Pf-Dmrt4 exhibits features typical of the D mrt family, and displays significant homologies to the DMRT4 cluster. Pf-Dmrt4 mRNA expression in the gonads during a gametogenic cycle, measured by quantitative polymerase chain reaction, was maximal in mature individuals. Pf-Dmrt4 expression, demonstrated by in situ hybridization, was localized in the spermatozoa, spermatids, oocytes and vitellogenic oocytes. Knockdown of Pf-Dmrt4 with double-stranded RNA resulted in decreased mRNA expression levels. And Pf-Dmrt4-dsRNA-injected groups showed spawning-stage male gonads, with ruptured follicles and released spermatozoa. Our results enhance the understanding of sex determination and differentiation in P. fucata and suggest that Pf-Dmrt4 could be involved in male gonadal development, and maintenance of male gonadal function.

  • pfsmad4 plays a role in biomineralization and can transduce bone morphogenetic protein 2 signals in the pearl oyster Pinctada fucata
    BMC Developmental Biology, 2016
    Co-Authors: Mi Zhao, Maoxian He, Xiande Huang, Qi Wang
    Abstract:

    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 involvment in BMP signaling in invertbrates, but whether SMAD4 plays a conservative role in pearl oyster, Pinctada fucata, still need to be tested.

  • analysis of dna methylation in tissues and development stages of pearl oyster Pinctada fucata
    Genes & Genomics, 2015
    Co-Authors: Yaoguo Li, Qin Li, Yunyan Guan, Maoxian He
    Abstract:

    In this study, we developed a methylation-sensitive amplification polymorphism technique to investigate DNA methylation profiles in different tissues and in the early-development stages of the pearl oyster Pinctada fucata (P. fucata). Methylation levels in adductor muscle, digestive gland, axe foot, heart, and gill ranged from 11.71 to 14.71 %, and significant differences (P < 0.05) between methylation levels in different tissues were observed. The DNA methylation levels of sperm, egg cells, two-cell embryos, morula embryos, trochophore larvae and D-shaped larvae were 13.51, 11.80, 12.14, 12.60, 14.65 and 13.18 %, respectively. Development stages of two-cell embryos, morula embryos, trochophore larvae and D-shaped larvae indicated a higher number of identical DNA methylation status loci in the egg, compared to that in the sperm. It is probable that DNA methylation patterns of the progeny are mainly influenced by the egg, while the sperm may become increasingly important during the process of early embryo development. The observed differences in methylation levels in the tissues and the development stages of P. fucata suggest that DNA methylation may act as an epigenetic regulator during tissue differentiation, individual growth, and development.

Norikazu Ueyama - One of the best experts on this subject based on the ideXlab platform.

Mi Zhao - One of the best experts on this subject based on the ideXlab platform.

  • PfSMAD1/5 Can Interact with PfSMAD4 to Inhibit PfMSX to Regulate Shell Biomineralization in Pinctada fucata martensii
    Marine Biotechnology, 2020
    Co-Authors: Mi Zhao, Maoxian He
    Abstract:

    The BMP2 signal transduced by SMAD1/5 plays an important role in osteoblast differentiation and bone formation. Shell formation of Pinctada fucata martensii is a typical biomineralization process that is similar to that of teeth/bone formation. However, whether the Pinctada fucata BMP2 (PfBMP2) signal transduced by PfSMAD1/5 occurs in P. f. martensii, how the PfBMP2 signal is transduced by PfSMAD1/5, and how PfSMAD1/5 regulates the biomineralization process in this species and other shellfish are poorly understood. Therefore, injection experiments of recombinant PfBMP2 and inhibitor dorsomorphin revealed that PfSMAD1/5 can transduce PfBMP2 signals. Subcellular localization and bimolecular fluorescence complementation assays indicated that PfSMAD1/5 phosphorylated by PfBMPR1b interacts with PfSMAD4 in the cytoplasm to form a complex, which translocates to the nucleus to transduce PfBMP2 signals. Co-immunoprecipitation and luciferase assays revealed that PfSMAD1/5 may interact with PfMSX to dislodge it from its binding element, resulting in initiation of mantle gene transcription. The in vivo functional assay showed that knockdown of PfMSAD1/5 decreased expression of shell matrix genes and disordered the nacreous layer, and the correlation assay of shell regeneration showed the concomitant expression pattern of PfSMAD1/5 and shell matrix genes. Together, these data showed that PfSMAD1/5 can transduce PfBMP2 signals to regulate shell biomineralization in P. f. martensii, which illustrated conservation of the BMP2-SMAD signal pathway among invertebrates. Particularly, the results suggest that there is only one PfMSX gene, which functions like the Hox gene in vertebrates, that interacts with PfSMAD1/5 in a protein–protein action form and plays the role of transcription repressor.

  • pfsmad1 5 can interact with pfsmad4 to inhibit pfmsx to regulate shell biomineralization in Pinctada fucata martensii
    Marine Biotechnology, 2020
    Co-Authors: Mi Zhao, Maoxian He
    Abstract:

    The BMP2 signal transduced by SMAD1/5 plays an important role in osteoblast differentiation and bone formation. Shell formation of Pinctada fucata martensii is a typical biomineralization process that is similar to that of teeth/bone formation. However, whether the Pinctada fucata BMP2 (PfBMP2) signal transduced by PfSMAD1/5 occurs in P. f. martensii, how the PfBMP2 signal is transduced by PfSMAD1/5, and how PfSMAD1/5 regulates the biomineralization process in this species and other shellfish are poorly understood. Therefore, injection experiments of recombinant PfBMP2 and inhibitor dorsomorphin revealed that PfSMAD1/5 can transduce PfBMP2 signals. Subcellular localization and bimolecular fluorescence complementation assays indicated that PfSMAD1/5 phosphorylated by PfBMPR1b interacts with PfSMAD4 in the cytoplasm to form a complex, which translocates to the nucleus to transduce PfBMP2 signals. Co-immunoprecipitation and luciferase assays revealed that PfSMAD1/5 may interact with PfMSX to dislodge it from its binding element, resulting in initiation of mantle gene transcription. The in vivo functional assay showed that knockdown of PfMSAD1/5 decreased expression of shell matrix genes and disordered the nacreous layer, and the correlation assay of shell regeneration showed the concomitant expression pattern of PfSMAD1/5 and shell matrix genes. Together, these data showed that PfSMAD1/5 can transduce PfBMP2 signals to regulate shell biomineralization in P. f. martensii, which illustrated conservation of the BMP2-SMAD signal pathway among invertebrates. Particularly, the results suggest that there is only one PfMSX gene, which functions like the Hox gene in vertebrates, that interacts with PfSMAD1/5 in a protein-protein action form and plays the role of transcription repressor.

  • pfsmad4 plays a role in biomineralization and can transduce bone morphogenetic protein 2 signals in the pearl oyster Pinctada fucata
    BMC Developmental Biology, 2016
    Co-Authors: Mi Zhao, Maoxian He, Xiande Huang, Qi Wang
    Abstract:

    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 involvment in BMP signaling in invertbrates, but whether SMAD4 plays a conservative role in pearl oyster, Pinctada fucata, still need to be tested.

  • a homeodomain transcription factor gene pfmsx activates expression of pif gene in the pearl oyster Pinctada fucata
    PLOS ONE, 2014
    Co-Authors: Mi Zhao, Maoxian He, Xiande Huang, Qi Wang
    Abstract:

    We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5′ flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster.

Kazuyuki Takahashi - One of the best experts on this subject based on the ideXlab platform.