Summary Gla‐rich protein (GRP) is a novel vitamin K‐dependent protein with the highest Gla content of any protein known to date that has been identified in all taxonomic groups of vertebrates (named GRP1) with a paralog in bony fish (named GRP2). In sturgeon, as well as during mouse development, cartilaginous tissues or their precursors are primary sites of GRP expression. In this article, we identify two grp isoforms, grp1 and grp2, encoded by two distinct genes localized in zebrafish chromosomes 25 and 4, respectively. These two genes span, respectively, 6 kb and 9 kb of genomic DNA and are both composed of five exons. Within the coding regions, the overall amino acids identity is 48.6 and 42.0%, respectively, for Grp1 and Grp2 compared to the human GRP. We also have identified the presence of splice variants already previously described in mouse, and corresponding expression levels were determined during embryonic stages and in different adult tissues. The levels of grp expression appear to be inversely correlated, with grp1 being expressed first and remaining high during early development while expression of grp2 appears later and increases in late larval and juvenile stages, having greater prevalence in adult tissues. We conclude that in zebrafish, grp paralogs exhibited distinct patterns of expression suggesting different regulatory pathways for each gene.

Sturgeons are representative of an ancient fish group, and present mainly an internal cartilaginous skeleton, with bone found essentially in the ganoid plaques forming the exogenous skeleton. Because of its archaic genetics, sturgeon represents an important model organism to understand the role of bone and cartilage-related Gla proteins and determine if their molecular mechanisms of action were maintained throughout evolution. Of particular relevance is understanding the regulation, in sturgeon, of those proteins known to be involved in tissue mineralization in mammals, as well as unveiling the function of newly identified calcification-related genes such as the one encoding the recently discovered Gla-rich protein (GRP), thus contributing to understand the poor calcification observed in sturgeon endoskeleton. However, regulation of gene expression and promoter functional analysis of sturgeon cartilage and bone-specific genes has been hampered by lack of suitable in vitro cell systems. We have recently developed the first sturgeon vertebra (VAn2H) and branchial arches (BAAn1F) derived cell cultures, and here we report their inability to mineralize their ECM under mineralizing culture conditions, as detected by von Kossa staining. Although a more extensive characterization of these systems is ongoing, our first data indicate that these cells represent a valuable tool for expression analysis of sturgeon bone and cartilage genes.

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Summary In an effort to provide more information on the function of Grp isoforms in zebrafish, two zebrafish transgenic lines were generated that could conditionally overexpress grp genes. Reported here is the process and strategy used for the generation of these transgenic lines. Overexpression of Grp1 and Grp2 were induced by heat‐shock treatment to investigate their possible roles in the skeleton development. Differences in skeleton development were analysed by alizarin red staining in the larvae. Although no differences in total deformities between wild type and Grp overexpressing larvae were observed, Grp1 and Grp2 overexpressing larvae presented a high percentage of deformities in abdominal and caudal fin vertebra, respectively, with a suggested specific localization of deformities for each isoform.

Summary Gla‐rich protein (GRP, also known as UCMA for upper zone of growth plate and cartilage matrix associated protein), a novel vitamin K‐dependent (VKD) protein, has been identified and isolated from sturgeon by our group and found to have orthologs in nearly all taxonomic groups of vertebrates (named GRP1) and a paralog in bony fish (named GRP2). Additional evidence supporting this hypothesis was recently obtained in zebrafish through analysis of the genomic environment of its 2 grp genes compared to that of the mouse gene. Data confirmed that fish and mammalian GRP genes are true orthologs and that the two zebrafish genes result from ancestral genomic fragment duplication now located in zebrafish chromosomes 4 and 25. In sturgeon, as well as during mouse development, cartilaginous tissues or their precursors are primary sites of GRP expression. Recently, our laboratory has shown that GRP is a circulating protein also expressed and accumulated in some soft tissues and clearly associated with ectopic calcification events. Since this protein has the highest Gla density of any known VKD protein, it was proposed that GRP might be a potent physiological modulator of soft tissue calcification. Contradictory functional studies have recently been published for grp. While in zebrafish recent data indicated a role of Grp‐1 (Ucmaa) during zebrafish skeletal development, another recent genetic study using a knockout strategy failed to identify a relevant function for grp during mouse development. Therefore, further studies are required in order to understand this discrepancy and provide insight into grp function and the molecular players involved in its regulation. In the present work we have taken a computational approach to identify cis‐regulatory transcription factor (TF) binding motifs in both grp1/ucmaa and grp2/ucmab genes from two model fish with compact and well characterized genomes, the fugu (Takifugu rubripes) and the tetraodon (Tetraodon nigroviridis). By comparing promoters from ortholog genes we expected to define conserved transcriptional motifs indicative of regulatory networks affecting these genes. Accordingly, our computational methods identified several TFs, whose binding profiles are available in the TRANSFAC database, which can be important for the regulation of these two genes.