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Development Genes and Evolution

, Volume 228, Issue 2, pp 131–139 | Cite as

Asymmetric pitx2 expression in medaka epithalamus is regulated by nodal signaling through an intronic enhancer

  • Vladimir Soukup
  • Simona Mrstakova
  • Zbynek Kozmik
Original Article
  • 126 Downloads

Abstract

The epithalamic region of fishes shows prominent left-right asymmetries that are executed by nodal signaling upstream of the asymmetry-determining transcription factor pitx2. Previous reports have identified that nodal controls the left-sided pitx2 expression in the lateral plate mesoderm through an enhancer present in the last intron of this gene. However, whether similar regulation occurs also in the case of epithalamic asymmetry is currently unresolved. Here, we address some of the cis-regulatory information that control asymmetric pitx2 expression in epithalamus by presenting a Tg(pitx2:EGFP) 116-17 transgenic medaka model, which expresses enhanced green fluorescent protein (EGFP) under control of an intronic enhancer. We show that this transgene recapitulates epithalamic expression of the endogenous pitx2 and that it responds to nodal signaling inhibition. Further, we identify that three foxh1-binding sites present in this enhancer modulate expression of the transgene and that the second site is absolutely necessary for the left-sided epithalamic expression while the other two sites may have subtler regulative roles. We provide evidence that left-sided epithalamic pitx2 expression is controlled through an enhancer present in the last intron of this gene and that the regulatory logic underlying asymmetric pitx2 expression is shared between epithalamic and lateral plate mesoderm regions.

Keywords

Epithalamus Asymmetry Transgene Enhancer Medaka pitx2 

Notes

Acknowledgements

Our thanks go to Jochen Wittbrodt for providing the p817 vector, to Jindra Lekesova and Veronika Kovacsova for medaka colony care, to Ivana Novakova and Vlada Filimonenko for embryo sectioning, to Peter Fabian for proofreading earlier versions of the manuscript, to Trevor Epp for English correction, and to two anonymous reviewers for their comments. VS is supported by the Czech Science Foundation (GACR 14-20839P) and by institutional support RVO68378050. VS, SM, and ZK acknowledge grant from the Ministry of Education, Youth and Sports of the Czech Republic “Biomodels for health – Centre of Model Organisms” (LO1419) for their support with the imaging presented herein.

Author contribution

VS and ZK conceived and designed the experiments; VS performed the experiments and analyzed the data; SM helped with photography and establishment of the transgenic lines; VS wrote the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interests

The authors declare that they have no competing interest.

Supplementary material

427_2018_611_MOESM1_ESM.tif (2.3 mb)
Supplementary figure 1 Comparison of expression of Tg(pitx2:EGFP) 116–17 and of the endogenous pitx2. Transverse sections through the epithalamic, lens and diencephalic regions of st. 25 embryos. Arrowhead marks expression in the left epithalamus, double arrowhead expression in lens and arrow expression in the ventral diencephalon. (TIFF 2384 kb)
427_2018_611_MOESM2_ESM.docx (14 kb)
Supplementary file . Nucleotide sequence of the cloned promoter and intron 2 of the medaka pitx2 gene with an indication of mutations introduced into the putative foxh1-binding sites. (DOCX 13 kb)
427_2018_611_MOESM3_ESM.xlsx (9 kb)
Supplementary table 1 List of primers used in this study. (XLSX 9 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Molecular Genetics of the Czech Academy of SciencesPragueCzech Republic
  2. 2.Department of Zoology, Faculty of ScienceCharles University in PraguePragueCzech Republic

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