Characterization of cis-regulatory elements for Fgf10 expression in the chick embryo

Hiroko Kawakami, Austin Johnson, Yu Fujita, Avery Swearer, Naoyuki Wada, Yasuhiko Kawakami

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)1253-1263
Number of pages11
JournalDevelopmental Dynamics
Volume247
Issue number12
DOIs
StatePublished - Dec 2018

Bibliographical note

Funding Information:
Additional supporting information may be found in the online version of this article. Grant sponsor: National Institute of Arthritis, Musculoskeletal and Skin Diseases of the National Institutes of Health; Grant number: R01AR064195; Grant sponsor: Japan Society for the Promotion of Science; Grant number: KAKENHI, 17K08498. Abbreviations: EGFP, enhanced green fluorescent protein; FGF, fibroblast growth factor; HH, Hamburger-Hamilton; LPM, lateral plate mesoderm; qRT-PCR, qunatitative reverse transcription-polymerase chain reaction; TSS, transcription start site; UTR, untranslated region.

Funding Information:
We thank Katherine Chen for editorial assistance, Drs. Naoko Koyano-Nakagawa and Michael O’Connor for sharing their equipment, and Dr. Yoshiko Takahashi for the pCAGGS-Trans-posase construct. A.S. was supported by Carleton College’s Kolenkow-Reitz Fellowship. Y.K. was supported by the National Institute of Arthritis, Musculoskeletal and Skin Diseases and N.W. was supported by the Japan Society for the Promotion of Science KAKENHI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no conflicts to disclose.

Publisher Copyright:
© 2018 Wiley Periodicals, Inc.

Keywords

  • Fgf10
  • chick embryos
  • enhancer
  • in vivo electroporation
  • primary culture

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