Identification of a Promoter Element within the Zebrafish colXα1 Gene Responsive to Runx2 Isoforms Osf2/Cbfa1 and til-1 but not to pebp2αA2

Abstract

Type X collagen is a short chain collagen specifically expressed by hypertrophic chondrocytes during endochondral ossification. We report here the functional analysis of the zebrafish (Danio rerio) collagen Xalpha1 gene (colXalpha1) promoter with the identification of a region responsive to two isoforms of the runt domain transcription factor runx2. Furthermore, we provide evidence for the presence of dual promoter usage in zebrafish, a finding that should be important to further understanding of the regulation of its restricted tissue distribution and spatial-temporal expression during early development. The zebrafish colXalpha1 gene structure is comparable to that recently identified by comparative genomics in takifugu and shows homology with corresponding mammalian genes, indicating that its general architecture has been maintained throughout vertebrate evolution. Our data suggest that, as in mammals, runx2 plays a role in the development of the osteogenic lineage, supporting zebrafish as a model for studies of bone and cartilage development.

Establishment of a Bone-Specific col10a1:GFP Transgenic Zebrafish

“…As reported previously (Simoes et al, 2006), we found that the col10a1 promoter contains 2 Runx2 consensus binding sequences (PuACCPu-CA/TGTGGT) upstream of the predicted transcriptional start site (TSS, -812 and -66 positions) and an additional binding site at the +23 position from the TSS (Fig. 2).…”

“…Previous studies have suggested that the col10a1 promoter of vertebrates may contain several components that direct its tissue-specific gene expression, such as the proximal basal regulatory region, and negative and positive regulatory elements (Higashikawa et al, 2009;Li et al, 2011;Simoes et al, 2006;Zheng et al, 2003). Our study also supports the existence of different regulatory elements within the zebrafish col10a1 promoter at least for the osteoblast-specific program.…”

“…Additionally, a negative element present in the -1.2-kb region may act when no positive signal is delivered, which could be a general mechanism for suppressing col10a1 expression in most cell types. Consistent with this possibility, the ~1.1-kb col10a1 promoter (similar to our ~1.3-kb construct) showed only a small amount of transactivation of the luciferase reporter in the Xenopus A6 kidney epithelial cell line (Higashikawa et al, 2009;Li et al, 2011;Simoes et al, 2006;Zheng et al, 2003). Therefore, tissue-specific col10a1 expression in vertebrates may depend on the activity of different elements within the promoter.…”

“…2): the first construct contained the immediate proximal promoter containing 2 Runx2-binding sites at -66 and +23; the second construct included an ~1.3-kb promoter sequence containing 3 binding sites at -812, -66, and +23 bp; and the third construct harbored an ~2.2-kb region containing the same Runx2-binding sites as those present in the second construct and an additional upstream ~900-bp fragment. It has been suggested that the col10a1 promoter contains positive and negative elements for its gene expression in vitro (Simoes et al, 2006); therefore, these constructs were used to test whether different parts of the col10a1 promoter are necessary to render promoter activity in the bone primordia.…”

UDP xylose synthase 1 is required for morphogenesis and histogenesis of the craniofacial skeleton

“…Type X collagen (Col10a1) is a marker of both osteoblasts and maturing chondrocytes in teleosts (Avaron et al, 2006; Clement et al, 2008; Simoes et al, 2006). In wild types, whole-mount in situ hybridization at 5 dpf showed that col10a1 is expressed in osteoblasts of both the endochondral (e.g., ceratohyal, hyomandibular) and intramembranous (e.g., opercle, dentary) skeletons (Fig.…”