Abstract
Besides the active Wnt signalling itself, the extracellular inhibition by Dkk1 is important for various embryonic developmental processes, such as optic vesicle differentiation and facial outgrowth. Although a feedback crosstalk of the active Wnt/β-catenin signaling and Dkk1 regulation has been suggested, the control of Dkk1 transcription by the Tcf/Lef1 mediated Wnt signalling and its connection to additional signalling factors has not been elucidated in vivo. Here, we used a combination of transgenic mouse approaches and biochemical analyses to unravel the direct Dkk1 transcriptional regulation via Tcf/Lefs. By using site directed mutagenesis, we tested several conserved Tcf/Lef1 binding sites within Dkk1 conserved non-coding elements (CNEs) and found that these are required for tissue specific reporter expression. In addition a conserved Msx1/2 binding site is required for retinal reporter expression and Msx2 but not Msx1 binds its conserved binding site within CNE195 in the optic cups. Within craniofacial expression domains, Lef1 interferes with Dkk1 directly via two conserved Tcf/Lef1 binding sites in the craniofacial enhancer CNE114, both of which are required for the general craniofacial Dkk1 reporter activation. Furthermore, these Tcf/Lef1 sites are commonly bound in the whisker hair bud mesenchyme but specifically Tcf/Lef1 (no. 2) is required for mandibular activation and repression of maxillar Dkk1 activation. Lastly, we tested the Tcf/Lef1 binding capacities of the Dkk1 promoter and found that although Lef1 binds the Dkk1 promoter, these sites are not sufficient for tissue specific Dkk1 activation. Together, we here present the importance of conserved Tcf/Lef1 and Msx1/2 sites that are required for differential Dkk1 transcriptional reporter activation in vivo. This requirement directly correlates with Lef1 and Msx1/2 interaction with these genomic loci.
Highlights
During embryonic development, various processes, such as head induction or limb outgrowth are driven by a defined modulation of the active Wnt signalling via the extracellular inhibitor Dkk1 [1,2,3,4,5]
Two of the conserved sites are localized within the Dkk1 craniofacial regulatory enhancer CNE114 and one in the optic cup enhancer CNE195 [34]
We found that Dkk1 expression is overlapping with the anterior Lef1 expression domain in the maxillary region of the 1st brachial arch (Fig. 1C–F) and the frontonasal mass at E10.5 (Fig. 1E+F), the overall Lef1 expression was much broader in the maxillary domain
Summary
Various processes, such as head induction or limb outgrowth are driven by a defined modulation of the active Wnt signalling via the extracellular inhibitor Dkk1 [1,2,3,4,5]. In addition to the relevance of the WntDkk crosstalk during early head induction processes, both, Wnt genes and Dkk are dynamically expressed in various head derivates, such as the retina [6,7,8] and meso- or ectodermal domains of the 1st branchial arch or craniofacial tissues [9,10,11,12,13,14] These partially overlapping expression patterns strongly suggest a regulatory feedback correlation between the active Wnt signalling and Dkk expression. Several studies have indicated that e.g. the outgrowth of craniofacial derivates or hair follicle formation involves a Wnt-Dkk crosstalk [15], [16] These data indicate that Wnt and Dkk protein levels have to be regulated in a well defined and dynamic fashion to maintain a normal tissue homeostasis. Unravelling a potential Wnt-Dkk feedback mechanism in vivo would in addition improve the knowledge about Dkk associated disease formation
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