Abstract
The core molecular mechanisms of dorsal organizer formation during gastrulation are highly conserved within the chordate lineage. One of the key characteristics is that Nodal signaling is required for the organizer-specific gene expression. This feature appears to be ancestral, as evidenced by the presence in the most basally divergent chordate amphioxus. To provide a better understanding of the evolution of organizer-specific gene regulation in chordates, we analyzed the cis-regulatory sequence of amphioxus Chordin in the context of the vertebrate embryo. First, we generated stable zebrafish transgenic lines, and by using light-sheet fluorescent microscopy, characterized in detail the expression pattern of GFP driven by the cis-regulatory sequences of amphioxus Chordin. Next, we performed a 5′deletion analysis and identified an enhancer sufficient to drive the expression of the reporter gene into a chordate dorsal organizer. Finally, we found that the identified enhancer element strongly depends on Nodal signaling, which is consistent with the well-established role of this pathway in the regulation of the expression of dorsal organizer–specific genes across chordates. The enhancer identified in our study may represent a suitable simple system to study the interplay of the evolutionarily conserved regulatory mechanisms operating during early chordate development.
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Acknowledgements
We are grateful to Veronika Noskova for the amphioxus facility maintenance and Dr. Sarka Takacova for proofreading the manuscript. We acknowledge the Light Microscopy Core Facility, IMG CAS, supported by RVO – 68378050-KAV-NPUI, for help with the light-sheet microscopy presented herein.
Funding
This work was supported by the Czech Science Foundation grant 20-25377S.
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I.K. and Z.K. conceived the study. S.M. and Z.K. performed experiments. I.K. wrote the main manuscript text. S.M. prepared Figs. 1, 2, 3, and 4, that were supervised/corrected by I.K.Z.K. prepared Supplementary Figure 1. S.M. prepared all videos. All authors reviewed and edited the manuscript.
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Communicated by Ildiko Somorjai.
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Fig. 5
Supplementary Figure 1 Epigenetic data from B. lanceolatum embryos indicating regions of open chromatin. Region used for reporter gene assays is shown by green line. Blue box indicates the enhancer region. Putative Smad-binding elements are highlighted within the nucleotide sequence of B. floridae enhancer.170 KB
Fig. 6
Supplementary Figure 2 Scheme of the vector used for zebrafish transgenesis27.7 KB
Supplementary video 1 Stable zebrafish transgenic line containing the BfChrd::eGFP (- 1.5kb) construct was imaged with a Zeiss Light Sheet Z.1 microscope. The embryo is in the dorsal view. (AVI 975 KB)
Supplementary video 2 Stable zebrafish transgenic line containing the BfChrd::eGFP (- 1.5kb) construct was imaged with a Zeiss Light Sheet Z.1 microscope. The embryo is in the lateral view. The reporter gene expression starts in the ectoderm and is then much stronger in the involuting mesoderm. (AVI 869 KB)
Supplementary video 3 Stable zebrafish transgenic line containing the BfChrd::eGFP (- 1.5kB/-1.0kb) construct was imaged with a Zeiss Light Sheet Z.1 microscope. The embryo is in the dorsal view. (AVI 1481 KB)
Supplementary video 4 Stable zebrafish transgenic line containing the BfChrd::eGFP (- 1.5kB/-1.0kb) construct was imaged with a Zeiss Light Sheet Z.1 microscope. The embryo is in the lateral view. The reporter gene expression starts in the ectoderm and is then much stronger in the involuting mesoderm. (AVI 2028 KB)
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Machacova, S., Kozmik, Z. & Kozmikova, I. Identification of Nodal-dependent enhancer of amphioxus Chordin sufficient to drive gene expression into the chordate dorsal organizer. Dev Genes Evol 232, 137–145 (2022). https://doi.org/10.1007/s00427-022-00698-z
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DOI: https://doi.org/10.1007/s00427-022-00698-z