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. 2018 Aug 16;8:12240. doi: 10.1038/s41598-018-30801-2

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© The Author(s) 2018

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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Akirin2 expression in the developing limb bud epithelium. (a) RT-PCR using two different primer sets that generate amplicons across multiple exons. Akirin2 is expressed in the limb at E10.5 and E14. (b) In wild type E10.5 cryosections, strong Akirin2 immunoreactivity is seen in the somites (denoted by *) and in the forelimb bud (FL), including expression in the AER (arrows). (c) At E12.5, Akirin2 immunostaining is lost in most cells of the ectoderm overlying the interdigital tissue of the knockout forelimb compared with control; as expected, mesodermal expression is unaffected as Cre is limited to the ectoderm. (d) Expression of a tdTom Cre reporter allele in the developing limb at E11.5 in both control and mutants, counterstained with the nuclear marker Sytox Green, shows epithelium-restricted Cre activity. (e) High magnification image of the tdTom reporter showing widespread, but patchy, expression in the ventral ectoderm of E10.5 control forelimb (arrows indicate tdTom-negative cells). Key: A, anterior; Di, distal; D, dorsal; ec, ectoderm; FL, forelimb; me, mesoderm; P, posterior; Pr, proximal; V, ventral. Scale bar: 100 μm in b; 50 μm in c; 200 μm in d; 50 μm in e.