Regulation of Six1 expression by evolutionarily conserved enhancers in tetrapods.

Shigeru Sato, Keiko Ikeda, Go Shioi, Kazuki Nakao, Hiroshi Yajima, Kiyoshi Kawakami

Division of Biology, Center for Molecular Medicine, Jichi Medical University, Yakushiji, Shimotsuke, Tochigi 329-0498, Japan.

Developmental biology 2012 Aug 1

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The Six1 homeobox gene plays critical roles in vertebrate organogenesis. Mice deficient for Six1 show severe defects in organs such as skeletal muscle, kidney, thymus, sensory organs and ganglia derived from cranial placodes, and mutations in human SIX1 cause branchio-oto-renal syndrome, an autosomal dominant developmental disorder characterized by hearing loss and branchial defects. The present study was designed to identify enhancers responsible for the dynamic expression pattern of Six1 during mouse embryogenesis. The results showed distinct enhancer activities of seven conserved non-coding sequences (CNSs) retained in tetrapod Six1 loci. The activities were detected in all cranial placodes (excluding the lens placode), dorsal root ganglia, somites, nephrogenic cord, notochord and cranial mesoderm. The major Six1-expression domains during development were covered by the sum of activities of these enhancers, together with the previously identified enhancer for the pre-placodal region and foregut endoderm. Thus, the eight CNSs identified in a series of our study represent major evolutionarily conserved enhancers responsible for the expression of Six1 in tetrapods. The results also confirmed that chick electroporation is a robust means to decipher regulatory information stored in vertebrate genomes. Mutational analysis of the most conserved placode-specific enhancer, Six1-21, indicated that the enhancer integrates a variety of inputs from Sox, Pax, Fox, Six, Wnt/Lef1 and basic helix-loop-helix proteins. Positive autoregulation of Six1 is achieved through the regulation of Six protein-binding sites. The identified Six1 enhancers provide valuable tools to understand the mechanism of Six1 regulation and to manipulate gene expression in the developing embryo, particularly in the sensory organs. Copyright © 2012 Elsevier Inc. All rights reserved.

Citation

Shigeru Sato, Keiko Ikeda, Go Shioi, Kazuki Nakao, Hiroshi Yajima, Kiyoshi Kawakami. Regulation of Six1 expression by evolutionarily conserved enhancers in tetrapods. Developmental biology. 2012 Aug 1;368(1):95-108