Differential expression of two tropoelastin genes in zebrafish
Introduction
Elastin is the polymeric extracellular matrix protein that provides the properties of extensibility and elastic recoil to large arteries, lung parenchyma, elastic ligaments, elastic cartilages, skin and other elastic tissues of all vertebrates with the exception of the agnathans. Elastin is synthesized as a monomeric protein called tropoelastin, but is rapidly assembled into a polymer in the extracellular matrix where it is stabilized by covalent crosslinks involving lysine resides. For a review of the general biochemistry of elastin see Mithieux and Weiss (2005).
All mammalian and avian tropoelastins sequenced to date have a characteristic and predominant alternating domain arrangement that is thought to be important both for the process of assembly of tropoelastin monomers into a crosslinked polymer and for the elastomeric properties of the polymeric form of the protein (Mithieux and Weiss, 2005). Recently we have identified, sequenced and characterized genes for tropoelastin in zebrafish (Danio rerio) and frog (Xenopus tropicalis) (Chung et al., in press). While the absolute sequence homology both among these teleost and amphibian tropoelastin genes and with their mammalian and avian counterparts is limited, all of these tropoelastins share the characteristic alternating domain arrangements and lysine-containing crosslinking motifs of this family of proteins. An unusual feature of the zebrafish was the presence of two significantly different tropoelastin genes, both of which were expressed (Genbank accession numbers DQ523562 and DQ523563) (Chung et al., in press). To date, this has been the only reported example of a species containing multiple tropoelastin genes.
The presence of two distinct genes for tropoelastin in the zebrafish raised the question as to whether the products of these genes might be differentially expressed, perhaps reflecting differences in functional properties of elastins in different tissues. Indeed, recent investigations using polypeptides modeled after tropoelastin sequences and domain structures have suggested that sequence variations, such as those present among species or, in the case of the zebrafish, between the two tropoelastin genes can strongly influence the physical properties of the protein (Bellingham et al., 2001, Miao et al., 2003, Miao et al., 2005). Physical properties that are affected include both the propensity for self-assembly of tropoelastin, which involves a process of temperature-induced phase separation known as coacervation, as well as the mechanical properties of the elastin polymer itself. In the present study we compare the temporal and spatial expression of these two tropoelastin genes and the tissue localization of their respective gene products in developing and adult zebrafish.
Section snippets
Temporal and spatial expression of two tropoelastin genes in zebrafish
We had previously demonstrated by Northern analysis that both zebrafish tropoelastin genes were expressed in 4 day post-fertilization (dpf) stage embryos (Chung et al., in press). However, while the eln2 transcript could be detected using preparations of total RNA, detection of the eln1 transcript required enrichment of polyA mRNA. This suggested that the two genes might differ in their levels of expression during development.
Differential expression levels for these two tropoelastin genes over
Discussion
Identification of two genes coding for expressed tropoelastin proteins in the zebrafish has provided the first example of a species with multiple genes for tropoelastin. We have suggested (Chung et al., in press) that this pair of genes likely arose not from a single gene duplication, but as part of the genome duplication event that has been reported to have taken place in the bony fish line leading to the teleosts approximately 300 million years ago, after their divergence from the line
Zebrafish
Embryos from the zebrafish AB wild type strain were collected from natural spawnings and staged according to Kimmel et al. (1995). Adult zebrafish of the longfin strain were a gift of Dr. P. Drapeau at the Montreal General Hospital.
Real-time quantitative RT-PCR
Total RNA from zebrafish embryos at various stages of development was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. First-strand cDNA was synthesized from 2 μg of total RNA using SuperScript III reverse transcriptase
Acknowledgements
Support for this work was provided by the Heart and Stroke Foundation of Ontario (FWK), University of Toronto Start-Up Fund and NSERC Discovery Grant (AEEB), and Canadian Institutes of Health Research (ECD). ECD is a Canada Research Chair and MM was a recipient of a Heart and Stroke Foundation of Canada Fellowship. We thank Dr. Ian Scott for useful discussions.
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