Frizzled6 Deficiency Disrupts the Differentiation Process of Nail Development Chang-Yi Cui, Joakim Klar, Patrik Georgii-Heming, Anne-Sophie Fröjmark,

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Frizzled6 Deficiency Disrupts the Differentiation Process of Nail Development Chang-Yi Cui, Joakim Klar, Patrik Georgii-Heming, Anne-Sophie Fröjmark, Shahid M. Baig, David Schlessinger, Niklas Dahl Journal of Investigative Dermatology Volume 133, Issue 8, Pages 1990-1997 (August 2013) DOI: 10.1038/jid.2013.84 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression profiling of mouse digit tips during claw development. (a) Schematic representation of early-stage claw development. A primordial claw/nail field is formed on the digit tips in mice at around embryonic day 14.5 (E14.5). Primitive nail matrix is discernible at E16.5, and nail matrix starts to form nail plate at around E18.5. Broken lines indicate excised region for RNA collection. (b) Scatter plots show number of genes differentially expressed in wild-type (WT) and Fzd6−/- digit tips at E14.5, E16.5, and E18.5. KO, knockout. Journal of Investigative Dermatology 2013 133, 1990-1997DOI: (10.1038/jid.2013.84) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Keratin (Krt) and keratin-associated protein (Krtap) genes were significantly affected in Fzd6−/- digit tips. (a) Twenty-nine hard and epithelial krts and five Krtap genes were affected in Fzd6−/- digit tips during claw development, most of them were downregulated (Dn). Up, upregulated. Bolded genes are known genes expressed in nails or known to be responsible for nail abnormalities. Numbers in parentheses represent fold differences. (b) Examples of the time course of expression for four affected hard Krt genes known to be involved in nail formation are shown. Krt86, 34, and 31 were significantly Dn in the knockout (KO) mice at embryonic day 16.5 (E16.5), whereas Krt81 was at E14.5. WT, wild type. *P<0.05. Journal of Investigative Dermatology 2013 133, 1990-1997DOI: (10.1038/jid.2013.84) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunohistochemical staining examples of the hard keratin Krt86, the epithelial keratin Krt6b, and the transglutaminase substrate Ivl in planar sections of digit tips embryonic day 16.5 (E16.5). (a–c) Krt86, Krt6b, and Ivl proteins are expressed in the suprabasal layers of the wild-type (WT) digit tips. (d–f) Only background levels were detected for the corresponding proteins in Fzd6−/- embryos. Hematoxylin (blue color) was used as background staining. Boxes indicate enlarged areas. SB, stratum basale. Journal of Investigative Dermatology 2013 133, 1990-1997DOI: (10.1038/jid.2013.84) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Transglutaminases (Tgms) and their substrates were significantly affected in Fzd6−/- mice. (a) Tgms 1, 3, and 5 were significantly downregulated in Fzd6−/- mice along with their substrates including 12 Lces, 4 S100s, 6 Sprrs, and Tchh, Lor, Csta, Ivl, Flg. Dn, downregulated; Up, upregulated. Bolded gene (Tgm1) is known to be responsible for nail abnormalities. Numbers in parentheses indicate fold differences. (b) Expression levels of Tgm1 and its substrates Lce1h, Sprr1a, and Involucrin were shown. Note a time lag of expression pattern, Tgm1 was significantly downregulated at embryonic day 14.5 (E14.5), Lce1h was at E16.5, Sprr1a was at E14.5 and E16.5, and Involucrin at E16.5. KO, knockout; WT, wild type. *P<0.05. Journal of Investigative Dermatology 2013 133, 1990-1997DOI: (10.1038/jid.2013.84) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Signaling proteins downregulated in the Fzd6−/- mice are shown. Expression patterns of Fzd6, Dkk4, and Bmp2k. Dkk4 was significantly downregulated in the knockout (KO) mice at embryonic day 16.5 (E16.5) and E18.5, but not at E14.5. Like Fzd6, Bmp2k was downregulated at all three time points. WT, wild type. *P<0.05. Journal of Investigative Dermatology 2013 133, 1990-1997DOI: (10.1038/jid.2013.84) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Claw phenotype of K14-Dkk4 transgenic mice. Second digit tips of wild-type (WT) and Dkk4 transgenic mice (TG) are shown. Claw is slightly thinner and the angle between lateral claw fold and horizontal skin stripes is larger in the transgenic mice compared with WT littermates. Journal of Investigative Dermatology 2013 133, 1990-1997DOI: (10.1038/jid.2013.84) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions