Abstract
The transcription factor 12 (tcf12) is a basic Helix-Loop-Helix protein (bHLH) of the E-protein family, proven to play an important role in developmental processes like neurogenesis, mesoderm formation, and cranial vault development. In humans, mutations in TCF12 lead to craniosynostosis, a congenital birth disorder characterized by the premature fusion of one or several of the cranial sutures. Current research has been primarily focused on functional studies of TCF12, hence the cellular expression profile of this gene during embryonic development and early stages of ossification remains poorly understood. Here we present the establishment and detailed analysis of two transgenic tcf12:EGFP fluorescent zebrafish (Danio rerio) reporter lines. Using these transgenic lines, we analyzed the general spatiotemporal expression pattern of tcf12 during different developmental stages and put emphasis on skeletal development and cranial suture patterning. We identified robust tcf12 promoter-driven EGFP expression in the central nervous system (CNS), the heart, the pronephros, and the somites of zebrafish embryos. Additionally, expression was observed inside the muscles and bones of the viscerocranium in juvenile and adult fish. During cranial vault development, the transgenic fish show a high amount of tcf12 expressing cells at the growth fronts of the ossifying frontal and parietal bones and inside the emerging cranial sutures. Subsequently, we tested the transcriptional activity of three evolutionary conserved non-coding elements (CNEs) located in the tcf12 locus by transient transgenic assays and compared their in vivo activity to the expression pattern determined in the transgenic tcf12:EGFP lines. We could validate two of them as tcf12 enhancer elements driving specific gene expression in the CNS during embryogenesis. Our newly established transgenic lines enhance the understanding of tcf12 gene regulation and open up the possibilities for further functional investigation of these novel tcf12 enhancer elements in zebrafish.
Highlights
TCF12, called HEB or HTF4, is a member of the basic Helix-Loop-Helix protein (bHLH) protein family, widely expressed in many vertebrate tissues and cell lines
We recombined the coding elements (CNEs) into the zebrafish Enhancer Detector (ZED) vector, which contains Tol2 sites, a minimal promoter linked to GFP and a cardiac-actin:RFP cassette that serves as control for transgenesis efficiency
The transgenic line showed localized, strong enhanced green fluorescent protein (EGFP) expression in single neurons of the central nervous system, e.g. in neurons of the midbrain, the midbrain-hindbrain boundary, the hindbrain, and the neural tube. These findings indicate that the tcf12:EGFP transgenic zebrafish show equivalent expression patterns to the endogenous tcf12 mRNA expression determined via in-situ hybridization and allow high resolution analyses during development
Summary
TCF12, called HEB or HTF4, is a member of the bHLH protein family, widely expressed in many vertebrate tissues and cell lines. Cranial sutures are bands of non-ossified mesenchymal tissue that separate the calvarial bone plates during vertebrate skull development. Cis-acting enhancer elements within the human TCF12 locus have been reported and investigated in transgenic mouse models [22] These experiments hint to a potential tissue specific regulation of TCF12 by these elements during development and foreshadow regions harboring essential transcription factor binding sites. Human-rodent comparisons, by contrast, are limited due to a comparatively short evolutionary divergence time which comes along with a high overall similarity even in nonfunctional genomic regions [22] To validate such regulatory elements in zebrafish we compared the tcf expression pattern characterized by our newly established transgenic lines to the transcriptional activity of three different TCF12 CNEs in vivo. To investigate the CNE activity during embryogenesis we used the ZED vector, in which CNE sequences combined with a minimal promoter drive fluorescence reporter genes [26]
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