Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene
Zebrafish (Danio rerio) is an excellent model organism for the study of vertebrate development including skeletogenesis. Studies of mammalian cartilage formation were greatly advanced through the use of a cartilage specific regulatory element of the Collagen type II alpha 1 (Col2a1) gene. In an effort to isolate such an element in zebrafish, we compared the expression of two col2a1 homologues and found that expression of col2a1b, a previously uncharacterized zebrafish homologue, only partially overlaps with col2a1a. We focused our analysis on col2a1a, as it is expressed in both the stacked chondrocytes and the perichondrium. By comparing the genomic sequence surrounding the predicted transcriptional start site of col2a1a among several species of teleosts we identified a small highly conserved sequence (R2) located 1.7 kb upstream of the presumptive transcriptional initiation site. Interestingly, neither the sequence nor location of this element is conserved between teleost and mammalian Col2a1. We generated transient and stable transgenic lines with just the R2 element or the entire 1.7 kb fragment 5′ of the transcriptional initiation site. The identified regulatory elements enable the tracking of cellular development in various tissues by driving robust reporter expression in craniofacial cartilage, ear, notochord, floor plate, hypochord and fins in a pattern similar to the expression of endogenous col2a1a. Using a reporter gene driven by the R2 regulatory element, we analyzed the morphogenesis of the notochord sheath cells as they withdraw from the stack of initially uniform cells and encase the inflating vacuolated notochord cells. Finally, we show that like endogenous col2a1a, craniofacial expression of these reporter constructs depends on Sox9a transcription factor activity. At the same time, notochord expression is maintained after Sox9a knockdown, suggesting that other factors can activate expression through the identified regulatory element in this tissue.
The emerging role of Wnt/PCP signaling in organ formation
Over the last two decades the zebrafish has been an excellent model organism to study vertebrate development. Mutant analysis combined with gene knockdown and other manipulations revealed an essential role of Wnt signaling, independent of B-catenin, during development. Especially well characterized is the function of Wnt/planar cell polarity (PCP) signaling in the regulation of gastrulation movements and neurulation, described in other reviews within this special issue. Here, we set out to highlight some of the new and exciting research that is being carried out in zebrafish to elucidate the role that Wnt/PCP signaling plays in the formation of specific organs, including the lateral line, craniofacial development, and regeneration. We also summarized the emerging connection of the Wnt/PCP pathway with primary cilia function, an essential organelle in several organ activities.