Hiroko Kawakami1,2,3, Austin Johnson1, Yu Fujita4, Avery Swearer1, Naoyuki Wada4, Yasuhiko Kawakami1,2,3. 1. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota. 2. Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota. 3. Developmental Biology Center, University of Minnesota, Minneapolis, Minnesota. 4. Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba, Japan.
Abstract
BACKGROUND: Fgf10 is expressed in various tissues and organs, such as the limb bud, heart, inner ear, and head mesenchyme. Previous studies identified Fgf10 enhancers for the inner ear and heart. However, Fgf10 enhancers for other tissues have not been identified. RESULTS: By using primary culture chick embryo lateral plate mesoderm cells, we compared activities of deletion constructs of the Fgf10 promoter region, cloned into a promoter-less luciferase reporter vector. We identified a 0.34-kb proximal promoter that can activate luciferase expression. Then, we cloned 11 evolutionarily conserved sequences located within or outside of the Fgf10 gene into the 0.34-kb promoter-luciferase vector, and tested their activities in vitro using primary cultured cells. Two sequences showed the highest activities. By using the Tol2 system and electroporation into chick embryos, activities of the 0.34-kb promoter with and without the two sequences were tested in vivo. No activities were detected in limb buds. However, the 0.34-kb promoter exhibited activities in the dorsal midline of the brain, while Fgf10 is detected in broader region in the brain. The two noncoding sequences negatively acted on the 0.34-kb promoter in the brain. CONCLUSIONS: The proximal 0.34-kb promoter has activities to drive expression in restricted areas of the brain. Developmental Dynamics 247:1253-1263, 2018.
BACKGROUND:Fgf10 is expressed in various tissues and organs, such as the limb bud, heart, inner ear, and head mesenchyme. Previous studies identified Fgf10 enhancers for the inner ear and heart. However, Fgf10 enhancers for other tissues have not been identified. RESULTS: By using primary culture chick embryo lateral plate mesoderm cells, we compared activities of deletion constructs of the Fgf10 promoter region, cloned into a promoter-less luciferase reporter vector. We identified a 0.34-kb proximal promoter that can activate luciferase expression. Then, we cloned 11 evolutionarily conserved sequences located within or outside of the Fgf10 gene into the 0.34-kb promoter-luciferase vector, and tested their activities in vitro using primary cultured cells. Two sequences showed the highest activities. By using the Tol2 system and electroporation into chick embryos, activities of the 0.34-kb promoter with and without the two sequences were tested in vivo. No activities were detected in limb buds. However, the 0.34-kb promoter exhibited activities in the dorsal midline of the brain, while Fgf10 is detected in broader region in the brain. The two noncoding sequences negatively acted on the 0.34-kb promoter in the brain. CONCLUSIONS: The proximal 0.34-kb promoter has activities to drive expression in restricted areas of the brain. Developmental Dynamics 247:1253-1263, 2018.
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