Expression of biomineralisation genes in tissues and cultured cells of the abalone Haliotis tuberculata
- 234 Downloads
Mollusc shell biomineralisation involves a variety of organic macromolecules (matrix proteins and enzymes) that control calcium carbonate (CaCO3) deposition, growth of crystals, the selection of polymorph, and the microstructure of the shell. Since the mantle and the hemocytes play an important role in the control of shell formation, primary cell cultures have been developed to study the expression of three biomineralisation genes recently identified in the abalone Haliotis tuberculata: a matrix protein, Lustrin A, and two carbonic anhydrase enzymes. Mantle cells and hemocytes were successfully maintained in primary cultures and were evaluated for their viability and proliferation over time using a semi-automated assay (XTT). PCR and densitometric analysis were used to semi-quantify the gene expression and compare the level of expression in native tissues and cultured cells. The results demonstrated that the three genes of interest were being expressed in abalone tissues, with expression highest in the mantle and much lower in the hemocytes and the gills. Biomineralisation genes were also expressed significantly in mantle cells, confirming that primary cultures of target tissues are suitable models for in vitro investigation of matrix protein secretion.
KeywordsBiomineralisation Gene expression Primary culture Haliotis tuberculata
We thank S. Huchette from the farm France-Haliotis (Plouguerneau, France) for providing abalones Haliotis tuberculata. This work was financed in part by the ATM programme ‘Biomineralisation’ of the MNHN funded by the Ministère délégué à l’Enseignement supérieur et à la Recherche. M. O’Neill was supported by an Erasmus fellowship from the European Community.
- Auzoux S, Domart-Coulon I, Doumenc D (1993) Gill cell cultures of the butterfish clam Ruditapes decussatus. J Mar Biotechnol 1:79–81Google Scholar
- Auzoux-Bordenave S, Domart-Coulon I (2010) Marine invertebrate cell cultures as tools for biomineralization studies. Journal des Sciences Halieutique et Aquatique 2:42–47Google Scholar
- Bédouet L, Marie A, Berland S, Marie B, Auzoux-Bordenave S, Marin F, Milet C (2012) Proteomic strategy for identifying mollusc shell proteins using mild chemical degradation and trypsin digestion of insoluble organic shell matrix: a pilot study on Haliotis tuberculata. Mar Biotechnol 14:446–458. doi: 10.1007/s10126-011-9425-0 CrossRefGoogle Scholar
- Chomczynski P, Sacchi N (1987) Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159Google Scholar
- Dauphin Y, Cuif J-P, Mutvei H, Denis A (1989) Mineralogy, chemistry and ultrastructure of the external shell-layer in ten species of Haliotis with reference to Haliotis tuberculata (Mollusca: Archaeogastropoda). Bull Geol Inst University Uppsala 15:7–38Google Scholar
- Farcy E, Serpentini A, Fiévet B, Lebel J-M (2007) Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata: transcriptional induction in response to thermal stress in hemocyte primary culture. Comp Biochem Physiol B: Biochem Mol Biol 146:540–550. doi: 10.1016/j.cbpb.2006.12.006 CrossRefGoogle Scholar
- Gaume B, Denis F, Van Wormhoudt A, Huchette S, Jackson DJ, Auzoux-Bordenave S (2013) Characterization and expression of Lustrin A biomineralizing gene during shell formation of the European abalone Haliotis tuberculata, in preparationGoogle Scholar
- Kleinschuster SJ, Parent J, Walker CW, Farley CA (1996) A cardiac cell line from Mya arenaria (Linnaeus, 1759). J Shellfish Res 15:695–707Google Scholar
- Le Roy N, Marie B, Gaume B, Guichard N, Delgado S, Zanella-Cléon I, Becchi M, Auzoux-Bordenave S, Sire J-Y, Marin F (2012) Identification of two carbonic anhydrases in the mantle of the European abalone Haliotis tuberculata (Gastropoda, Haliotidae): phylogenetic implications. J Exp Zool B Mol Dev Evol 318B:353–367CrossRefGoogle Scholar
- Machii A, Wada K (1989) Some marine invertebrates tissue culture. In: Mitsuhashi J (ed) Invertebrate cell system applications, vol 2. CRC Press, Boca Raton, pp 225–233Google Scholar
- Nakahara H, Bevelander G, Kakei M (1982) Electron microscopic and amino acid studies on the outer and inner shell layers of Haliotis rufescens. Venus (Japan. J Malacol) 41:33–46Google Scholar