Research Article: On the traces of tcf12: Investigation of the gene expression pattern during development and cranial suture patterning in zebrafish (Danio rerio)

Date Published: June 12, 2019

Publisher: Public Library of Science

Author(s): Rabea Blümel, Miriam Zink, Eva Klopocki, Daniel Liedtke, Chi Zhang.


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.

Partial Text

TCF12, also called HEB or HTF4, is a member of the bHLH protein family, widely expressed in many vertebrate tissues and cell lines. TCF12 can form homodimers and heterodimers with other bHLH proteins to regulate the transcription of various target genes during different developmental processes, like mesodermal and hematopoietic specification as well as T cell development [1–3]. In rodent models, Tcf12 has been shown to be involved in neurogenesis and loss of Tcf12 leads to a reduction in brain size [4–6]. Recent studies assigned an essential role for Tcf12 in osteogenic differentiation of bone marrow stem cells via BMP and Erk1/2 signaling pathways [7]. A number of mutations affecting TCF12 have been identified in patients with coronal craniosynostosis type 3 (OMIM: #615314), prompting that TCF12 plays a key role in coronal suture development and patency [8, 9]. Cranial sutures are bands of non-ossified mesenchymal tissue that separate the calvarial bone plates during vertebrate skull development. They are characterized as highly proliferative zones of osteogenic differentiation and bone formation. Under normal conditions, only the metopic suture fuses during early childhood in humans whereas the other sutures are open up to an age of 40 years [10]. The patency of the sutures during childhood is essential to enable normal skull growth in compliance with the developing brain. Depending on the affected suture that is fused, craniosynostosis patients can exhibit severe skull malformations, compensatory bone growth, facial asymmetries, and raised intracranial pressure [11]. Most cases of craniosynostosis are treated by surgery of affected newborns at an age between 8 and 15 months to reduce intracranial pressure and for correction of the cranial deformity [12]. A cure or even a pharmacological treatment is not available, as the molecular reasons for the suture fusions are heterogeneous and often still unknown [13].

In this study, we present the establishment of a novel transgenic model for observing the changing expression pattern of tcf12 during zebrafish development and cranial suture morphogenesis. In addition, we tested the transcriptional activity of three non-coding elements within the tcf12 locus and compared their activity to the transgene expression of tcf12:EGFP fish.