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CALCIUM, THE BACKBONE OF FISH CULTURE: IMPORTANCE IN SKELETAL FORMATION, REPRODUCTION AND
NORMAL PHYSIOLOGY
Q5RS-2001-02904 Coordinator: Centre of Marine Sciences (CCMAR), University of Algarve, Faro, Portugal Prof. Deborah M. Power: e-mail: <[email protected]> Viveiro Vilanova - Associated contracter to UALG: Viveiros Vilanova, SA, 7645, Portugal Ms Joana Amaral email: <[email protected]> UNIMAN-Contractor: School of Biological Sciences, University of Manchester, UK Prof. Richard Balment tel: e-mail: <[email protected]> KUN- Contractor: University of Nijmegen, The NetherlandsProf. Gert Flik: e-mail: <[email protected]> 5) MRC- Contractor: HGMP Resource Centre, Wellcome Genome Campus, Cambridge, UK. Dr Melody Clark: email: <[email protected]>
Objectives FISHCAL
• To identify the relative importance of dietary, endogenous and environmental sources of calcium during critical phases of development, growth and reproduction.
• Determine experimentally the roles of PTHrPin whole animal calcium homeostasis.
• Study the molecular mechanisms that underpin bone formation in fish and the way in which PTHrP regulates this process.
• Target species – sea bream (Sparus auratus).
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Benefits and Beneficiaries:
• Multitude of known roles for PTHrP indevelopment, bone formation and immunesystem.
• Insight into the basis of skeletal developmentand how this can go wrong so as to providesome solutions for the problem of highincidence of developmental abnormalities atthe early stages in sea bream.
• Candidate gene approach.•Identification of PTH/PTHrP promoters.• Identification of PTHrP responsive genes.• Gene expression profiles during endochondrial calcification.
Genomics input:
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Candidate gene approach: Comparativeanalysis
THE JAPANESE PUFFERFISHTHE JAPANESE PUFFERFISH((FuguFugu rubripesrubripes))
SeabreamSeabreamSparusSparus auratusauratus
TilapiaTilapiaO. O. mossambicusmossambicus
Example of approach - PTHrP
DNA sequencesFugu Cosmidlibrary screening
DNA extractiona) Probeb) Shot-gun cloning
FuguFugu PTHrPPTHrPgenesgenes
Fugu databasehttp://Fugu.hgmp.mrc.ac.
uk/Pint Assembly Program(Weston, HGMP, unpublished)
d) BLAST against SPTR database
e) Putative PTHrP cosmids
c) Vector clipping and quality checked
PCR walks, PCR walks, SacISacI libraries, libraries, further sequence scansfurther sequence scans
Fugu probe /PCR screenof Sparus cDNA libraries
Physiology
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Parathyroid hormone
Parathyroid hormone-related protein
5 PTH/PTHrPgenes isolated
Canário et al., 2006 Febs Lets
Microarray
1. Production of Differentially Enriched Library from PTHrP and Control larval heads and sequencing of clones
2.Production of Microarray• 2000 clones from libraries selected for spotting onarray slide.• 100 known positive and negative clones spotted to allow verification of data.
3. Experiments Larval tissue treated with PTHrP (dose/time course).
(quadruple screen, dye swap)
4. Analysis of Microarray• Imagene 5.1 used outline and verify spots.• Genespring used to analyse data and establish which clones are up or down regulated (4-fold cut-off).
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Array profile
Positive clones
ControlsLanding lights
0
0.5
1
1.5
2
2.5
1 2 3 4 5 6
0
500
1000
1500
2000
2500
3000
3500
1 2 3 4 5 6
0
500
1000
1500
2000
2500
3000
1 2 3 4 5 6
OSN/actin Col1/actin
DPN/actin
0
1000
2000
3000
4000
5000
6000
7000
1 2 3 4 5 6
Col5/actin
1. Control2. E2
3. Calcitonin4. Vitamin D
5. PTHrP (10nM)6. PTHrP (1000nM)
Candidate gene expression in scales - influence of hormones
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Molecular Tools Generated• Systems for production of recombinant sea bream
proteins.• Recombinant sea bream proteins and specific
antisera.• Quantitative RT-PCR for a range of endocrine
hormones and receptors. • Subtractive larval head cartilage cDNA library and
a Microarray. • Identification of candidate genes.• Specific radioimmunoassays for parathyroid
hormone (PTH) and parathyroid hormone related protein (PTHrP), thyroid hormones and steroids.
• Physiological studies and phenotype.
PublicationsAnjos, L., Rotllant, J., Guerreiro, P.M., Hang, X., Canario, A.V.M. Balment, R. and Power, D.M.
(2005) Production and characterisation of recombinant sea bream (Sparus aurata) parathyroid hormone-related protein (PTHrP) General and Comparative Endocrinology in press
Rotllant, J., Redruello, B., Guerreiro, P.M., Fernandes, H., Canario, A.V.M. and Power D.M. (2005) Calcium mobilization from fish scales is mediated by parathyroid hormone related protein via the parathyroid hormone type 1 receptor. Regulatory Peptides in press
Rotllant, J., Guerreiro, P.M., Redruello, B., Fernandes, H., Apolónia, L., Anjos, L., Canario, A.V.M. and Power, D.M. (2005) Ligand binding and signalling pathways of PTH receptors in seabream (Sparus auratus) enterocytes. Cell and Tissue Research in press
Redruello, B., Estêvão, M.D., Rotllant, J., Guerreiro, P.M., Anjos, L.I., Canário A.V.M. and Power, D.M. (2005) Isolation and Characterization of Piscine Osteonectin and Downregulation of its expression by Parathyroid Hormone-Related Protein. J. Bone and Mineral Research no prelo
Estêvão, M.D., Redruello, B., Canario A.V.M. and Power, D.M. (2005) Ontogeny of osteonectinexpression in embryos and larvae of sea bream (Sparus auratus). General and Comparative Endocrinology 142, 155-162
Rotllant, J., Guerreiro, P.M., Anjos, L., Redruello, B., Canario, A.V.M., and Power, D.M. (2005) Stimulation of cortisol release by the N-terminus of teleost parathyroid hormone-related protein: characterization of ligand determinants for receptor signalling in interrenal cells in vitro. Endocrinology 146, 71-76.
Guerreiro, P.M., Fuentes, J., Flik, G., Rotllant, J., Power, D.M. and Canario, A.V.M. (2003) Water calcium concentration modifies whole-body calcium uptake in sea bream larvae during short-term adaptation to altered salinities. Journal of Experimental Biology 207, 645-653
