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. 2021 Jan 19;12:439. doi: 10.1038/s41467-020-20714-y

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Schematic of transgenic reporter construct containing LacZ, a minimal promoter and the putative enhancer sequence of interest. b Table of constructs used for transgenesis, the CE64 subunits included and the number of embryos displaying reporter expression for each construct (i.e. 3/4 means that out of a total of 4 transgenic embryos, 3 display positive reporter expression). c Photomicrographs of representative embryos stained for LacZ activity. Reporter expression in the MHB is only detected in wt Tg(CE64) embryos. Note the staining that is present in the anterior hindbrain of some of the Tg(DEL-B) embryos. Tg(DEL-C) and Tg(64-B) do not manifest any reproducible reporter expression. d In situ hybridization of control, DEL-B and DEL-C deletion embryos at e8.25 with riboprobe against Fgf8. Expression is undetectable in both enhancer subunit deletions (n = 4). Blue box indicates LacZ reporter gene in (a). In all panels, light green, green and dark green boxes indicate 64-A, 64-B, and 64-C subunits, respectively. Scale bar is 1 mm in (c) and 100 μm in (d).