Flightless I Expression Enhances Murine Claw Regeneration Following Digit Amputation  Xanthe L. Strudwick, James M. Waters, Allison J. Cowin  Journal of Investigative Dermatology  Volume 137, Issue 1, Pages 228-236 (January 2017) DOI: 10.1016/j.jid.2016.08.019 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Claw regeneration is enhanced by flightless I (Flii) overexpression in murine digits. (a) Effect of Flii expression on regeneration was assessed macroscopically (macro) and by alizarin red staining of the bone 8 weeks after either distal or proximal amputation in wild-type (WT) and Flii overexpressing (FIT) mice. The approximate level of amputation is shown by a black dashed line. Original magnification ×2.5. Black scale bars represent 600 μm. (b) Macroscopic regeneration assessed qualitatively by five independent assessors using a modified Leichardt scale comparing amputated digits with associated intact control digit on the opposite hind limb. The scale ranges from 0 to 5, where 0 equals no regeneration visible, 1 is grossly undergrown, 3 is equal to intact control, and 5 is grossly overgrown. The solid line represents normal intact control digits. (c) Claw regeneration was assessed by the measurement of the length of the claw from the borderline of the lateral claw fold to the claw tip on macroscopic images and expressed as percent of the length of the associated intact control digit on the opposite hind limb. Mean ± SEM. **P < 0.01, ***P < 0.005. The solid line represents length normal intact control claws. (d) Bone regeneration was assessed by the measurement of the length of the terminal phalange after alizarin red staining and expressed as percent of the length of the associated intact control digit on the opposite hind limb. Mean ± SEM. **P < 0.01, ***P < 0.005. The solid line represents the length of normal intact control terminal phalange. SEM, standard error of the mean. Journal of Investigative Dermatology 2017 137, 228-236DOI: (10.1016/j.jid.2016.08.019) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Flii overexpression results in thinner, more curved claws after regeneration of the digit tip. The effect of Flii expression on the regenerated claw appearance was assessed in WT and FIT mice 8 weeks after distal amputation. (a) The thickness of the claw was determined both as an average along the claw and at the base of the claw between the borderline of the lateral claw fold and the proximal claw fold. The overall curve of the claw was taken as the angle between the borderline of the lateral claw fold and the tip of the claw. The orientation of the claw at the tip of digit tip was calculated as the angle between the horizontal stripe and the borderline of the lateral claw fold. Images included are for the demonstration of the technique only. (b) Measurements of claw thickness in WT and FIT mice were expressed as average and base thickness in μm. The angles of the claw curvature and orientation to the digit tip were expressed graphically in degrees. Mean ± SEM. *P < 0.05. (c) Overall claw appearance was demonstrated by overlaying two-dimensional traces of claw thickness, curve angle, and orientation angle taken from representative macroscopic images of WT and FIT claws. Original magnification ×2.5. Black scale bars represent 600 μm. Flii, flightless I; FIT, Flii overexpressing; SEM, standard error of the mean; WT, wild type. Journal of Investigative Dermatology 2017 137, 228-236DOI: (10.1016/j.jid.2016.08.019) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Presence of a germinal matrix is observed in FIT but not WT mice after proximal amputation. (a) Four-micrometer sagittal sections taken from paraffin-embedded digits collected 8 weeks after distal or proximal amputation and the associated intact control digits of WT and FIT mice. The presence of a germinal matrix was confirmed histologically (H&E) and by the absence of keratin 10 (K10) immunofluorescence (pseudo-stained red). Original magnification ×4. Arrowheads denote the region of the nail organ with absent K10 staining. The germinal matrix (gm) shown bounded by white in the inset ×10 magnification images. All scale bars represent 200 μm. Measurements of the regenerated germinal matrix were expressed as a percent of the area (b) and length (c) of the associated intact control digit. Mean ± SEM. *P < 0.05. FIT, Flii overexpressing; H&E, hematoxylin and eosin; SEM, standard error of the mean; WT, wild type. Journal of Investigative Dermatology 2017 137, 228-236DOI: (10.1016/j.jid.2016.08.019) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Claw regeneration appears to be stimulated by flightless I expression within the surrounding mesenchyme of the digit tip. (a) Normal human keratinocytes were cultured with “distal” or “proximal” connective tissue fibroblasts for 21 days at the air-liquid interface on de-epidermalized dermis. Four-micrometer sections were assessed histologically (H&E) and by immunofluorescent detection for the expression of keratin 10 (K10, pseudo-stained green), Flightless I (Flii, pseudo-stained green), βCat (pseudo-stained orange), or cyclin D1 (pseudo-stained orange). The junction between the dermis (d) and epidermis (e) is marked by a dashed line. Rudimentary nail formation is marked by the presence of aggregate structures (as) within the neo-epidermis. White arrows indicate positive cyclin D1 expression within the nucleus. Original magnification ×20. All scale bars represent 50 μm. Flii (b) and βCat (c) expression was measured quantitatively as the optical density (OD) of fluorescent images in the epidermis. Mean ± SEM. (d) The expression level of cyclin D1 was measured quantitatively as the number of cyclin D1 positive (+ve) cells per 100 μm. Mean ± SEM. *P < 0.05, **P < 0.01. βCat, β-catenin; FIT, Flii overexpressing; H&E, hematoxylin and eosin; SEM, standard error of the mean; WT, wild type. Journal of Investigative Dermatology 2017 137, 228-236DOI: (10.1016/j.jid.2016.08.019) Copyright © 2016 The Authors Terms and Conditions

Figure 5 βCat and cyclin D1 expression is maintained in the germinal matrix of Flii overexpressing mice after distal and proximal amputation. (a) Immunofluorescent detection of βCat (pseudo-stained orange) within the germinal matrix (gm) shown bounded by white separated from the underlying nail dermis (nd) on 4-μm sagittal sections taken from paraffin-embedded digits of WT and FIT mice collected 1 week after distal or proximal amputation. Original magnification ×20. All scale bars represent 20 μm. (b) βCat expression was measured quantitatively as the optical density (OD) of fluorescent images within the germinal matrix of distal and proximal amputated digits from WT and FIT mice. Mean ± SEM. *P < 0.05, ***P < 0.005. Immunofluorescent detection of cyclin D1 (pseudo-stained yellow) within the germinal matrix (gm) bounded by white separated from the nail dermis (nd) on 4-μm sagittal sections taken from paraffin-embedded digits of WT and FIT mice collected 1, 2, and 4 weeks after amputation at the distal (c) or proximal (e) level. White arrows indicate positive cyclin D1 expression within the nucleus. Composite ×20 magnification images. All scale bars represent 20 μm. The expression level of cyclin D1 was measured quantitatively as the number of cyclin D1 positive (+ve) cells per 100 μm within the volar epithelium after distal (Dist) (d) or proximal (Prox) (f) amputation. Mean ± SEM. *P < 0.05, ***P < 0.005. βCat, β-catenin; FIT, Flii overexpressing; Flii, flightless I; SEM, standard error of the mean; WT, wild type. Journal of Investigative Dermatology 2017 137, 228-236DOI: (10.1016/j.jid.2016.08.019) Copyright © 2016 The Authors Terms and Conditions