Cloning, Expression, and the Functional Analyses of Regulatory Elements of Zebrafish Six6 Genes During Craniofacial Development
Date Issued
2008
Date
2008
Author(s)
Tsai, Min-Nan
Abstract
Six6 gene belongs to the Six gene family, of which the members were identified in mouse and other vertebrates by homologous screening with Drosophila sine oculis (so) gene as a probe. It has been shown that sine oculis plays an import role during Drosophila eye development. In contrast, only vertebrate six3 and six6 are expressed in the optic primodium and are implicated in eye development. Besides, these two genes also participate in brain development, suggesting that they are crucial for neuronal differentiation.he aim of this study is to search the zebrafish six6 orthologue and to analyze the expression and its tissue-specific gene regulation. In the first attempt, NCBI data-base search did not result in the identification of zebrafish six6 gene. However, two zebrafish six6 homologues (zgc: 110344 and zgc: 63871) were presented in the UCSC bio-informatics web site using human SIX6 gene as a reference. It has been well known that six4, six1 and six6 form a genetic linkage as six4-six1-six6 arrangement. The two putative zebrafish six6 homologues were designated as six6.1 and six6.2 according to the presence of the genetic linkage in genome and alignment of amino acid sequences. he 3’UTR cDNAs of the genes were isolated by PCR for anti-sense RNA probe preparation. In situ hybridization results revealed that zebrafish six6.1 and six6.2 transcripts are expressed in ventral diencephalon, hypothalamus and presumptive adenohypophysis. In addition, six6.1 transcripts are located in the neural retina. n regard to the gene regulation, the upstream promoter fragments of the two genes were cloned by PCR amplification and were analyzed in zebrafish embryo with GFP reporter gene by transgenic assay. The results showed that the reporter is expressed in similar patterns as those of endogenous mRNA, suggesting that the respective upstream promoter regions harbor the regulatory elements for six6.1 and six6.2. In parallel, two conserved DNA regions are located in the upstream regions of the two genes by Dot-Matrix analyses. The transgenic assays suggested that the proper cis-regulatory elements for the two genes reside in these conserved locations.
Subjects
promoter
zebrafish
cis-regulatory element
evolution
genetic linkage
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