An Evolutionarily Conserved Mesodermal Enhancer in Vertebrate Zic3

Yuri S Odaka,Jun Aruga,Takahide Tohmonda,Atsushi Toyoda

doi:10.1038/s41598-018-33235-y

Abstract

Zic3 encodes a zinc finger protein essential for the development of meso-ectodermal tissues. In mammals, Zic3 has important roles in the development of neural tube, axial skeletons, left-right body axis, and in maintaining pluripotency of ES cells. Here we characterized cis-regulatory elements required for Zic3 expression. Enhancer activities of human-chicken-conserved noncoding sequences around Zic1 and Zic3 were screened using chick whole-embryo electroporation. We identified enhancers for meso-ectodermal tissues. Among them, a mesodermal enhancer (Zic3-ME) in distant 3′ flanking showed robust enhancement of reporter gene expression in the mesodermal tissue of chicken and mouse embryos, and was required for mesodermal Zic3 expression in mice. Zic3-ME minimal core region is included in the DNase hypersensitive region of ES cells, mesoderm, and neural progenitors, and was bound by T (Brachyury), Eomes, Lef1, Nanog, Oct4, and Zic2. Zic3-ME is derived from an ancestral sequence shared with a sequence encoding a mitochondrial enzyme. These results indicate that Zic3-ME is an integrated cis-regulatory element essential for the proper expression of Zic3 in vertebrates, serving as a hub for a gene regulatory network including Zic3.

Highlights

Zic[3] is required for the maintenance of pluripotency in embryonic stem (ES) cells[6], neuroectoderm and mesoderm differentiation, and determination of left-right axis of the internal organs[7]
To understand the mechanism underlying the regulation of Zic gene expression, we analyzed the cis-regulatory elements required for the expression of vertebrate Zic[1] and Zic[3], and characterized a developmentally critical Zic[3] mesodermal enhancer in terms of its activity in chicken and mouse embryos, potential trans-acting factors, and its evolutionary history
At HH7 (Fig. 1C,I), Zic[1] and Zic[3] expression in the neural folds showed anterior-posterior profiles similar to those in the HH6 stage, but the signal was enhanced in the lateral region of the neural plate as seen in the cross sections (Fig. 1a–c,g–i)

Summary

Introduction

Zic[3] is required for the maintenance of pluripotency in embryonic stem (ES) cells[6], neuroectoderm and mesoderm differentiation, and determination of left-right axis of the internal organs[7]. Several studies have addressed the role of cis-regulatory elements, along with the associated signaling and transcription factors (TFs), in the regulation of Zic gene expression Examples of these cis-regulatory elements include- (1) The 5′ flanking region of mouse Zic[1] that controls the dorsal spinal cord expression[27]; (2) The BMP inhibitor-responsive promoter region in Xenopus Zic128. To understand the mechanism underlying the regulation of Zic gene expression, we analyzed the cis-regulatory elements required for the expression of vertebrate Zic[1] and Zic[3], and characterized a developmentally critical Zic[3] mesodermal enhancer in terms of its activity in chicken and mouse embryos, potential trans-acting factors, and its evolutionary history

Methods

Results

Conclusion