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. 2017 Oct 13;13(10):e1007047. doi: 10.1371/journal.pgen.1007047

Fig 6. Alx4 binds a terrestrially conserved element in the Fgf10 locus.

Fig 6

(A) In new born pups, Alx1, Alx4 and Fgf10 were expressed in the lacrimal gland mesenchyme, whereas the FGF response gene Etv5 was expressed in the epithelium. (B) Alx4 was excluded from Pax6-positive epithelial cells and SMA-positive myoepithelial cells, but it was expressed in the neural crest derived mesenchymal cells labeled by Wnt1-Cre induced tdTomato fluorescence. (C) Sequence alignment identified an Alx4 site within an intronic region of Fgf10, which was conserved from human to lizard, but not in species ranging from Xenopus to fish. It resided next to DNase hypersensitivity peaks in NIH3T3 cells. (D) The Alx4 site in the Fgf10 locus matched the Alx consensus sequence. (E) Chromatin immunoprecipitation showed that Alx4 directly bound the Fgf10 intronic site in both lacrimal gland mesenchyme and NIH3T3 cells. Student’s t test: *P<0.01, n = 4; **P<0.001, n = 3. (F) Schematic diagram of mesenchymal cell culture isolated from newborn pups and treatment with Alx siRNA. LGM: lacrimal gland mesenchyme. LGE: lacrimal gland epithelium. (G) Alx4 siRNA significantly down regulated Fgf10 expression in lacrimal mesenchymal cells, whereas additional application of Alx1 siRNA did not lead to further reduction. One Way ANOVA: *P<0.01, **P<0.001, n = 3.