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Flightless I Expression Enhances Murine Claw Regeneration Following Digit Amputation
Accepted Manuscript Flightless I expression enhances murine claw regeneration following digit amputation Xanthe L. Strudwick, James M. Waters, Allison J. Cowin PII:
S0022-202X(16)32353-3
DOI:
10.1016/j.jid.2016.08.019
Reference:
JID 508
To appear in:
The Journal of Investigative Dermatology
Received Date: 12 April 2016 Revised Date:
5 August 2016
Accepted Date: 5 August 2016
Please cite this article as: Strudwick XL, Waters JM, Cowin AJ, Flightless I expression enhances murine claw regeneration following digit amputation, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2016.08.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Flightless I expression enhances murine claw regeneration following digit amputation
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Xanthe L. Strudwick1, James M. Waters2 & Allison J. Cowin1 Future Industries Institute, University of South Australia, Mawson Lakes, South Australia,
Women’s and Children’s Health Research Institute, North Adelaide, South Australia, Australia.
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Australia;
Correspondence:
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Xanthe Strudwick, Future Industries Institute, University of South Australia, Mawson Lakes Boulevard, Mawson Lakes South Australia, Australia
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Short Title:
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E-mail: [email protected]
Flightless I enhances claw regeneration
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Abstract
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The mammalian digit tip is capable of both reparative and regenerative wound healing dependent upon the level of amputation injury. Removal of the distal third of the terminal phalange results in successful regeneration, whilst a more severe, proximal, amputation heals by tissue repair.
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Flightless I (Flii) is involved in both tissue repair and regeneration. It negatively regulates wound repair but elicits a positive effect in hair follicle regeneration, with Flii overexpression resulting
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in significantly longer hair fibers. Using a model of digit amputation in Flii overexpressing (FIT) mice we investigated Flii in digit regeneration. Both WT and FIT digits regenerated following distal amputation with newly regenerated FIT claws being significantly longer than intact controls. No regeneration was observed in WT mice after severe proximal amputation, however
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FIT mice showed significant regeneration of the missing digit. Using a 3D model of nail formation, connective tissue fibroblasts isolated from the mesenchymal tissue surrounding the WT and FIT digit tips and co-cultured with skin keratinocytes demonstrated aggregate structures
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resembling rudimentary nail buds only when Flii was overexpressed. Moreover, β-Catenin and Cyclin D1 expression was maintained in the FIT regenerating germinal matrix suggesting a
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potential interaction of Flii with Wnt signaling during regeneration.
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Introduction Amputation of the mammalian digit tip may result in complete regeneration depending upon the
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severity of the injury. Removal of the distal half of the terminal phalange results in successful regeneration of all components of tissue, including skeletal structure, whilst a more severe,
proximal amputation, removing more than 2/3 of the terminal phalanx, will illicit no discernible
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regenerative response with healing following that of cutaneous tissue repair (Muneoka et al., 2008; Strudwick and Cowin, 2012; Takeo et al., 2013). The presence of an intact nail bed
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following injury and conservative management of the wound, appears critical for successful regeneration in humans (Mohammad et al., 1999). Studies investigating pathways involved in regeneration versus repair of the digit tip may identify targets for therapeutic development to
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stimulate the regeneration of tissue following injury (Fathke et al., 2006).
Flightless I (Flii) has been implicated in both tissue repair and regeneration. This highly conserved protein is a unique member of the gelsolin family of cytoskeletal remodeling proteins,
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being the only one containing an N-terminal Leucine Rich Repeat domain and is essential for embryonic development (Silacci et al., 2004). Flii has both cytoplasmic and nuclear activities,
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localizing with β-tubulin based structures involved in cell division and within lammellipodia and filopodia associated with migrating cells (Davy et al., 2001; Strudwick and Cowin, 2012). In the nucleus, it is a transcriptional coactivator of the estrogen and thyroid receptors (Lee et al., 2004) and has a differential effect upon β-Catenin dependent transcription and cell cycle progression (Lee and Stallcup, 2006; Seward et al., 2008). Flii is secreted into human plasma and has immunological roles via its interaction with LRRFIP2, Myd88 and TLR4 signaling (Dai et al.,
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2009; Jin et al., 2013; Lei et al., 2012; Ruzehaji et al., 2013). Flii is a negative regulator of wound repair, with decreased Flii expression associated with increased proliferation of dermal and epidermal cells, resulting in smaller, more contracted wounds (Cowin et al., 2007; Jackson et
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al., 2012). A decrease in Flii expression, either endogenously in heterozygous knockout mice, or via the topical application of a neutralizing antibody raised against the LRR domain in murine and porcine wound models results in improved healing (Cowin et al., 2007; Jackson et al., 2012;
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Strudwick and Cowin, 2012). Conversely, overexpression of Flii in the mouse resulted in larger scars, with a slower, impaired wound healing responses (Cowin et al., 2007; Jackson et al., 2012;
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Strudwick and Cowin, 2012), with fibroblast-specific upregulation of Flii giving a similar magnitude of wound healing impairment to non-tissue-specific upregulation (Turner et al., 2015). However, Flii also has a positive effect upon hair follicle regeneration, with Flii overexpression resulting in significantly longer hair fibers in regenerated follicles and reduced
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Flii expression resulting in delayed regeneration (Waters et al., 2011). Recently, Flii has also been shown to play a positive role in cell migration and is required for Rac1 mediated contraction (Marei et al., 2016). It has been suggested that Flii plays a dual role in regulation of
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al., 2016).
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migration depending upon the cellular context and the availability of binding partners (Marei et
These contrasting outcomes indicate a dynamic role for Flii and highlight the need for closer investigation of the difference between regenerative and reparative healing. Utilizing a mouse model to stimulate regeneration or repair in the digit tip, we aimed to investigate the effect of Flii upon tissue regeneration. Understanding the mechanisms involved in mammalian tissue
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regeneration will assist the development of new therapeutic approaches aimed at reducing
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scarring and fibrosis.
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Results
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Murine claw regeneration is observed in Flii over expressing but not WT mice after proximal amputation.
Distal and proximal amputations of the terminal phalange (Supplementary Figure 1a) were
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performed on the middle three digits of the left hind limb of 4 week old wild-type (WT) and Flii overexpressing (FIT) mice and compared with uninjured, intact claws of the right hind limb 8
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weeks after amputation (Figure 1a). Following distal amputation, complete digit regeneration and claw regrowth was observed in both WT and FIT mice 8 weeks after amputation (Figure 1b). FITs additionally showed significantly overgrown claws with measurements revealing that claw lengths were significantly longer than intact controls and were greater than those observed in WT
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mice (Figure 1c). The rate of regeneration following distal amputation was also significantly accelerated in FIT mice (Supplementary Figure 2), with complete regeneration observed by 4 weeks in FIT mice, whilst WT were significantly delayed. Following proximal amputation, no
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regrowth of the amputated tip was observed in WT mice (Figure 1a). However, significant regeneration was observed in FIT mice, although full regeneration was not achieved even after 8
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weeks (Figure 1a, b). The claws of the FIT mice also regenerated to 42% of the length of intact controls while WT claws were less than 15% (Figure 1c). Bone regeneration was assessed by alizarin red whole mount staining (Figure 1a, d) and showed that after the less severe, distal, amputation the terminal phalange of both WT and FIT mice regenerated to a similar length compared to intact digits. Despite the macroscopic observance of digit regeneration in FIT mice
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following proximal amputation, no significant regeneration of the bone was observed in either
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WT or FIT mice.
Flii overexpression gives rise to thicker, more curved claws following regeneration.
The appearance of the fully regenerated digit (following distal amputation) was analyzed to
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assess the effect of increased Flii expression upon claw phenotype including claw thickness and curvature as well as the angle of orientation at the tip of the digit (Figure 2a). Overexpression of
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Flii resulted in a reproducible change in the overall appearance of the claws of the regenerated digits. The thickness of the claws at both at the base and as an average overall when measured in 2D planar view were no different between WT and FIT mice (Figure 2bi). The angle between the claw tip and the borderline of the lateral claw fold in FIT mice was 6 degrees less than in WT
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mice, which translates to significantly greater curvature of the claw (Figure 2bii). The orientation of the claw at the tip of the digit was not affected with no difference observed in the angle between the borderlines of the lateral claw fold and the horizontal stripe (Figure 2biii). Thus,
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while the orientation of the claw at the tip of the digit did not change, the claws appeared longer, and were more curved. This subtle but reproducible effect was clearly seen by an overlay of the
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2D trace of the claws (Figure 2c). The effect of Flii expression upon claw phenotype was also assessed in uninjured control digits (Supplementary Figure 3). The claws of FIT mice were significantly longer than WT but no obvious changes to claw thickness, curvature or orientation were observed indicating that phenotypic changes seen in regenerated digits were exacerbated following injury.
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Presence of a germinal matrix is observed in FIT but not WT mice following proximal amputation.
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The germinal matrix within the nail organ supplies the pool of proliferative cells which give rise to the nail organ or the claw in mice (De Berker et al., 2000). The presence of a germinal matrix was confirmed histologically and by staining with Keratin 10 (K10) (Figure 3a). K10 is strongly
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expressed in normal stratified epithelium including that of the digit tip, but is low or absent
within the cells which comprise the nail organ, including the germinal matrix (De Berker et al.,
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2000; Perrin et al., 2004). K10 staining was absent from the region underlying the claw in intact WT and FIT digits and of the regenerated digits following distal amputation. Although the area of the germinal matrix and the K10 negative nail organ region in the intact digits of FIT mice appeared somewhat smaller than WT, this was not statistically significant. WT digits following proximal amputation however, displayed continuous K10 expression around the entire digit tip
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indicating that no nail organ or germinal matrix was present. However, in FIT mice, a distinct germinal matrix was observed following proximal amputation with a similar K10 staining pattern to that observed in intact and distal regenerated digits. A significant increase in the area and
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length of the germinal matrix area was observed after distal amputation in WT but not FIT mice
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(Figure 3b). Whilst no germinal matrix was observed in WT mice following proximal amputation, it was clearly present in FIT mice, albeit significantly smaller in area and length than intact controls.
Flii expression is maintained in the germinal matrix of Flii overexpressing mice following distal and proximal amputation.
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FIT mice have between 1.3 and 1.5 times the normal amount of Flii expressed in the skin, heart and muscle (Thomsen et al., 2011). In response to wounding, a four-fold increase in Flii levels is seen in the skin (Cowin et al., 2007). Flii expression was detected strongly throughout the
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epithelial tissue of the intact digit tip in both WT and FIT mice with further expression apparent within the underlying structures of the dermis (Supplementary Figure 4a). Flii expression in intact digits was not significantly different between WT and FIT mice however epithelial
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expression (Supplementary Figure 4a, b) showed significantly elevated levels of Flii in FIT mice 8 weeks after both distal and proximal amputation. Similar changes in expression are seen in the
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intact back skin compared to 3 days post incisional wounding and in the intact whole digits of these mice using semi-quantitative western analysis (Supplementary Figure 5). In WT mice, a marked decrease in expression was observed, whereas Flii levels in FIT mice were similar to intact digits and significantly higher than WT. Flii expression in the germinal matrix (within
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white boundaries) was comparable in the intact digit between WT and FIT mice (Supplementary Figure 4a, c). Following distal amputation Flii expression was significantly decreased in WT mice, whilst expression was maintained at levels equal to intact controls in FITs. Following
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proximal amputation Flii was expressed within the germinal matrix in FIT mice at levels equal to that of the intact and distally amputated digit. No germinal matrix was observed in WT digits at 8
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weeks and therefore no Flii expression could be measured.
Proliferation is increased in the regenerated germinal matrix of Flii overexpressing mice. Proliferation within the germinal matrix was assessed by immunofluorescent detection of the proliferating cell nuclear antigen (PCNA). Few PCNA positive cells were observed within the
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germinal matrix of WT and FIT intact digits and while an increase in the number of PCNA positive cells was observed in the germinal matrix of FIT mice following distal amputation, this was not statistically significant (Supplementary Figure 4d, e). However, in FIT mice following
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proximal amputation where there is the presence of a germinal matrix, a significant increase in
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proliferation within this area was observed (Supplementary Figure 4d, e).
Claw regeneration is stimulated by Flii expression within the surrounding mesenchyme of
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the digit tip.
Connective Tissue Fibroblasts (CTFs) were isolated from the distal, third (Ph3) and the more proximal, second (Ph2) phalangeal skeletal elements of WT and FIT mice (Supplementary Figure 1b) and cultured with normal human keratinocytes in a 3D model of nail formation
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(Figure 4a). Aggregate structures resembling rudimentary nail buds were induced to form by WT and FIT distal Ph3 CTFs. Keratinocytes cultured with CTFs isolated from the more proximal Ph2 skeletal elements of WT digits however formed a normal looking stratified epidermis.
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Keratinocytes cultured with Ph2 CTFs from FIT digits formed aggregate structures much like those cultured with the Ph3 CTFs. K10 staining (Figure 4a) was strongly detected in the
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normally stratified epidermis of the 3D culture but markedly reduced in aggregate structures formed by keratinocytes cultured with Ph3 CTFs (both WT and FIT), or with the more proximal pool of CTFs which overexpressed Flii (FIT Ph2). Flii expression within the newly formed epidermis or aggregate structures formed by normal human keratinocytes was unchanged, regardless of which pool of CTFs they were cultured with (Figure 4b). Interestingly, βCat was found to be significantly reduced in the normally stratified epidermis formed by keratinocytes
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cultured with the more proximal pool of WT CFTs compared to aggregate structures formed by culture with either distal WT CTFs or both distal and proximal FIT CTFs (Figure 4c). This was
the rudimentary nail like structures fail to form (Figure 4d).
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also associated with greatly reduced expression of CyclinD1 in the WT proximal cultures where
germinal matrix of Flii overexpressing mice.
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Claw regeneration may be stimulated by maintained βCat/CyclinD1 signaling within the
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Digits were collected from WT and FIT mice at 1, 2 and 4 weeks post distal and proximal amputation. Immunohistochemical analysis revealed that following distal amputation the expression of βCat at 1 week post-amputation was similar within the germinal matrix of both groups of mice (Figure 5a, b). However, following the more proximal amputation a significant
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decrease in expression of βCat was seen in the residual germinal matrix of WT but not FIT mice (Figure 5a, b). The expression of the downstream target of βCat signaling, CyclinD1, which regulates cell progression through the G1 phase and is required for proliferation (Neumeister et
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al., 2003), showed that the number of CyclinD1 positive cells within the germinal matrix following distal amputation (Figure 5c, d) was reduced in FIT digits at 1 week post amputation
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compared to WT but increased to significantly higher levels at 4 weeks post amputation, with a similar number of positive cells as seen at the same time point in WT digits. After proximal amputation (Figure 5e, f) similar levels of CyclinD1 expression were observed in the germinal matrix of WT and FIT mice at 1 week post amputation however where the number of positive cells decreased significantly by 4 weeks post amputation in the WT digits, expression was significantly higher in FIT digits at levels similar to both 1 and 2 weeks post amputation.
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Although, a residual germinal matrix was observed at 1 week post proximal amputation in both WT and FIT mice, the area of the germinal matrix subsequently decreased in size in the WT
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mice whereas it increased in size in Flii overexpressing mice (Supplementary Figure 6).
Discussion
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Tissue regeneration is the ultimate goal following injury and understanding the processes
involved is fundamental to developing new approaches to stimulate regeneration vs repair.
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Mammalian regeneration is currently restricted to certain organs and tissues however the fingertip is capable of regeneration depending on the severity of injury (Takeo et al., 2013). Distal amputation of the fingertip leads to complete regeneration of the bone, skin and nail organ whereas a more severe, proximal amputation will result in healing by repair with no
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regeneration. In our study, proximal amputations in mice overexpressing Flii mice led to up to 42% regeneration of the amputated digit with significant regrowth of the claw. Histologically, a germinal matrix and rudimentary claw could be seen in FIT digits at 8 weeks, whilst none
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appeared in wild-type mice. The germinal matrix is the organ that supplies the pool of keratinocytes which undergo proliferation and differentiation to form the nail or claw of the digit
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tip (De Berker et al., 2000). Few PCNA positive cells were observed within the germinal matrix of the intact digits nor at 8 weeks following distal amputation when regeneration is considered complete (Simkin et al., 2013). The claws in mice overexpressing Flii showed greatly increased proliferation within the germinal matrix and this heightened proliferation may contribute to the continued claw regrowth and may lead to further regeneration of the digit tip beyond the 8 week time frame of this study.
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Not only was there a significant increase in digit tip regeneration and claw regrowth but a change
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in the appearance of the regenerated claws in mice overexpressing Flii was observed post amputation. Newly regenerated claws in FIT mice were significantly elongated compared to than intact control digits and were sharper and more curved. Changes in murine claw phenotype
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have been described in Frizzled 6 (FZD6) deficient mice which exhibit thinner, more pointed claws more vertically orientated at the digit tip (Cui et al., 2013). FZD6 belongs to a family of
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membrane bound receptors through which the canonical, βCat dependent Wnt signaling pathway operates (Schulte, 2010) and FZD6 deficiency leads to a reduction in β-catenin signaling through the canonical pathway. Flii has previously been implicated in the regulation of the canonical Wnt signaling pathway (Lee and Stallcup, 2006). Flii is a negative regulator of the canonical Wnt signaling pathway, inhibiting βCat dependent transcription by disrupting the binding of the
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murine Flightless Associated Protein 1 (FLAP1) or its human homologues Leucine Rich Repeat In FLII Interacting Protein 1/2 (LRRFIP1/2) with βCat (Lee and Stallcup, 2006). Also, Flii interacts with CARM1 and GRIP1 to enhance hormone mediated transcription (Lee et al., 2004).
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These same coactivators are positive transcriptional regulators of the βCat:TCF/LEF complex
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(Lee et al., 2004; Li et al., 2004). In the current study we found that βCat expression was maintained within the germinal matrix of Flii overexpressing mice following proximal amputation and that expression of CyclinD1 in this area continued to be expressed in the regenerating germinal matrix. No expression for βCat or CyclinD1 was observed in the germinal matrix of WT mice which failed to regenerate the claw suggesting that Flii may affect the regulation of the canonical, βCat dependent, Wnt pathway leading to increased regenerative capacity.
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The development of skin and appendages such as hair and nails, or claws, is dependent upon
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interactions between the epidermis and the underlying mesenchyme (Cui et al., 2013). Epithelial cells are stimulated to form into either a normal stratified epidermis or nail-like aggregates via signaling from cells within the mesenchyme surrounding the digit tip in a position specific
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manner (Wu et al., 2013). Connective tissue fibroblasts isolated from the distal terminal or third phalangeal element and cultured with keratinocytes, induce the formation of these nail-like
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aggregates but keratinocytes cultured with a more proximal pool of connective tissue fibroblasts from the second phalangeal element lose this ability to form elemental nail buds and stratify into normal epithelium (Wu et al., 2013). However, when normal human keratinocytes were cultured with connective tissue fibroblasts isolated from the second phalangeal element of mice which overexpress Flii, the epithelial cells were able to form rudimentary nail like structures similar to
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those cultured with fibroblasts isolated from distal, third phalangeal elements. This indicates that the regeneration of the nail organ seen in FIT mice after proximal amputation is likely to be via
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changes in signaling from the mesenchyme due to increased expression of Flii within these cells.
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To be able to drive the body towards a more complete replacement of native tissues, rather than merely repairing a tissue after significant trauma is the goal of many research groups. Utilizing the mammalian digit tip to understand the mechanisms by which tissue is stimulated to regenerate rather than simply repair following amputation may help our understanding of this complex process. These studies suggest that Flii is not just a negative regulator of reparative
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wound healing, but may also be a key regulator of both regenerative and reparative processes
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depending on the cells and tissues involved.
Materials and Methods
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Animal Studies
All mouse strains were congenic on the BALB/c background and BALB/c inbred mice were used
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as wild-type control animals (designated Flii+/+). Flii transgenic strain ((Tg1FLII)2Hdc) incorporating a 17.8 Kb fragment of a human cosmid clone that spans the entire FLII locus, with animals homozygous for the transgene in addition to the endogenous Flii allele designated FliiTg/Tg or FIT. Details of the generation of the FIT mice were described previously, showing
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Digit Amputations
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elevated Flii protein levels in various tissues including skin (Thomsen et al., 2011).
All animal procedures were approved by the WCHN Animal Ethics Committee (AE847/11/2013,
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AE972/2/2017) and carried out in accordance with the Australian code of practice for the care and use of animals for scientific purposes. One third (distal) to greater than two thirds (proximal) of the terminal phalanges of the central three digits of the hind limb of 4 week old wild-type (WT) and Flii overexpressing (FIT) mice were amputated as described previously (Han et al., 2008; Neufeld and Zhao, 1995). Amputation levels were calculated from amputated tip against intact age-matched control digits using whole mount Alizarin Red stain (Han et al., 2008). Briefly, digit tips collected at time of surgery were fixed in 95% ethanol overnight before 15
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incubation with 0.0033% w/v Alizarin Red S (Sigma-Aldrich, Sydney, Australia) in 1.6% Aqueous KOH for two days prior to clearing in 1.6% KOH and Mall’s Solution (20% Glycerin in 1% KOH) to glycerol for visualization. Measurement of terminal phalange and amputated tip
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(MediaCybernetics Inc., Rockville, MD, USA).
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bone length were taken from whole mount photographs, using ImagePro Plus 5.1 software
Macroscopic Assessment
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All samples were assessed macroscopically for regenerative success 8 weeks post amputation from images taken at the time of collection of the digits. Images were scored by five independent assessors for macroscopic evidence of regeneration using a modified Leichardt scale on a scale of 0-5 comparing amputated digits with associated intact control digits from the opposite hind
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limb of the same animal (n=36/genotype). Further information on the Leichardt scale assessment
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can be found in Supplementary information.
Semi Quantitative Western Analysis, Histological, Alizarin Red and Immunofluorescent
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Staining
Flii protein expression in intact and wounded back skin and in whole intact digits of WT and FIT mice was analyzed as described in supplementary information. Samples were collected at 1, 2, 4 and 8 weeks post amputation for histological assessment, alizarin red and immunofluorescence as described in supplementary information.
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Connective Tissue Fibroblast Isolation and Nail Formation Model Position specific connective tissue fibroblasts were isolated from hind limb digits of 4-6 week
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old male and female WT and FIT mice according to previously description (Wu et al., 2013) and described in Supplementary information. Isolated P2 or P3 WT and FIT CTFs were cultured with normal human neonatal keratinocytes (Invitrogen, Waverley, NSW, Australia) to form a 3D model of nail formation in triplicate based upon previously described methods (MacNeil et al.,
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2011; Wu et al., 2013). Briefly, 1 × 105 passage 3-5 fibroblasts and 3 × 105 passage 1-3
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keratinocytes, each in 250 mL of 10% Green’s medium were seeded onto de-epidermalized human dermis (European Skin Bank, Beverwijk, Netherlands) fitted with a 1cm internal diameter chamfered metal (medical-grade stainless steel). After 48 hrs with two media changes the steel ring is removed and skin constructs raised on stainless steel grids and allowed to grow in 10% Green’s for 21 days at the air–liquid interface prior to processing for H&E or
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Statistical Analysis
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immunofluorescence staining.
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Statistical differences were determined using the 2-tailed Student's t-test. A p value of less than 0.05 was considered significant.
Conflict of Interest The authors state no conflict of interest.
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Thomsen N, Chappell A, Ali RG, Jones T, Adams DH, Matthaei KI, et al. Mouse strains for the ubiquitous or conditional overexpression of the Flii gene. Genesis 2011;49:681-688.
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Turner CT, Waters JM, Jackson JE, Arkell RM, Cowin AJ. Fibroblast-specific upregulation of Flightless I impairs wound healing. Exp Dermatol 2015;24:692-697. Waters JM, Lindo JE, Arkell RM, Cowin AJ. Regeneration of hair follicles is modulated by flightless I (Flii) in a rodent vibrissa model. J Invest Dermatol 2011;131:838-847. Wu Y, Wang K, Karapetyan A, Fernando WA, Simkin J, Han M, et al. Connective tissue fibroblast properties are position-dependent during mouse digit tip regeneration. PLoS One
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2013;8:e54764.
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Figure Legends Figure 1: Claw regeneration is enhanced by Flightless I (Flii) overexpression in murine digits. (a) Effect of Flii expression upon regeneration was assessed macroscopically (macro) and
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by alizarin red staining of the bone 8 weeks after either distal or proximal amputation in wildtype (WT) and Flii overexpressing (FIT) mice. Approximate level of amputation is shown by black dashed line. Original magnification 2.5X. Black scale bars represent 600µm. (b)
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Macroscopic regeneration assessed qualitatively by 5 independent assessors using a modified Leichardt scale comparing amputated digits to associated intact control digit on the opposite hind
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limb. The scale ranges from 0-5, where 0 equals no regeneration visible, 1 is grossly undergrown, 3 is equal to intact control and 5 is grossly overgrown. Solid line represents normal intact control digits. (c) Claw regeneration was assessed by measurement of the length of the claw from the borderline of the lateral claw fold to the claw tip on macroscopic images and
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expressed as % of the length of the associated intact control digit on the opposite hind limb. Mean +/- SEM. **= p <0.01, ***= p <0.005. Solid line represents length normal intact control claws. (d) Bone regeneration was assessed by measurement of the length of the terminal
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phalange following alizarin red staining and expressed as % of the length of the associated intact control digit on the opposite hind limb. Mean +/- SEM. **= p <0.01, ***= p <0.005. Solid line
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represents length of normal intact control terminal phalange.
Figure 2: Flii overexpression results in thinner, more curved claws following regeneration of the digit tip. The effect of Flii expression upon 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 as well as at the base of the claw between the 21
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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
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stripe and the borderline of the lateral claw fold. Images included are for demonstration of
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
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tip were expressed graphically in degrees. Mean +/- SEM. *= p <0.05. (c) Overall claw
appearance was demonstrated by overlaying 2D traces of claw thickness, curve angle and
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orientation angle take from representative macroscopic images of WT and FIT claws. Original magnification 2.5X. Black scale bars represent 600µm.
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Figure 3: Presence of a germinal matrix is observed in FIT but not WT mice following proximal amputation. (a) 4µm 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
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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 4X.
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Arrowheads denote region of the nail organ with absent K10 staining. The germinal matrix (gm) shown bounded by white in inset 10X magnification images. All scale bars represent 200µm. (b) Measurement of the regenerated germinal matrix were expressed as a percent of the area and length of the associated intact control digit. Mean +/- SEM. *= p <0.05.
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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 are air-liquid interface on de-
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epidermalized dermis. 4µm 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 CyclinD1 (pseudo
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stained orange). Junction between the dermis (d) and epidermis (e) is marked by dashed line. Rudimentary nail formation is marked by the presence of aggregate structures (as) within the
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neo-epidermis. White arrows indicate positive CyclinD1 expression within the nucleus. Original magnification 20X. All scale bars represent 50µm. 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 CyclinD1 was measured quantitatively as the number of CyclinD1
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positive (+ve) cells per 100µm. Mean +/- SEM. *= p <0.05, **=p<0.01, ***= p <0.005.
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Figure 5: βCat and CyclinD1 expression is maintained in the germinal matrix of Flii overexpressing mice following distal and proximal amputation. (a) Immunofluorescent
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detection of βCat (pseudo stained orange) within the germinal matrix (gm) shown bounded by white separated from the underlying nail dermis (nd) on 4µm sagittal sections taken from paraffin embedded digits of WT and FIT mice collected 1 week after distal or proximal amputation. Original magnification of images is 20X. All scale bars represent 20µm. (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 CyclinD1 (pseudo stained 23
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yellow) within the germinal matrix (gm) bounded by white separated from the nail dermis (nd) on 4µm sagittal sections taken from paraffin embedded digits of WT and FIT mice collected 1, 2 and 4 weeks post amputation at the distal (c) or proximal (e) level. White arrows indicate
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positive CyclinD1 expression within the nucleus. Composite 20X magnification images. All scale bars represent 20µm. The expression level of CyclinD1 was measured quantitatively as the number of CyclinD1 positive (+ve) cells per 100µm within the volar epithelium following distal
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(Dist) (d) or proximal (Prox) (f) amputation. Mean +/- SEM. *= p <0.05, ***= p <0.005.
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S0022-202X(16)32353-3
DOI:
10.1016/j.jid.2016.08.019
Reference:
JID 508
To appear in:
The Journal of Investigative Dermatology
Received Date: 12 April 2016 Revised Date:
5 August 2016
Accepted Date: 5 August 2016
Please cite this article as: Strudwick XL, Waters JM, Cowin AJ, Flightless I expression enhances murine claw regeneration following digit amputation, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2016.08.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Flightless I expression enhances murine claw regeneration following digit amputation
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Xanthe L. Strudwick1, James M. Waters2 & Allison J. Cowin1 Future Industries Institute, University of South Australia, Mawson Lakes, South Australia,
Women’s and Children’s Health Research Institute, North Adelaide, South Australia, Australia.
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Australia;
Correspondence:
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Xanthe Strudwick, Future Industries Institute, University of South Australia, Mawson Lakes Boulevard, Mawson Lakes South Australia, Australia
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Short Title:
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E-mail: [email protected]
Flightless I enhances claw regeneration
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Abstract
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The mammalian digit tip is capable of both reparative and regenerative wound healing dependent upon the level of amputation injury. Removal of the distal third of the terminal phalange results in successful regeneration, whilst a more severe, proximal, amputation heals by tissue repair.
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Flightless I (Flii) is involved in both tissue repair and regeneration. It negatively regulates wound repair but elicits a positive effect in hair follicle regeneration, with Flii overexpression resulting
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in significantly longer hair fibers. Using a model of digit amputation in Flii overexpressing (FIT) mice we investigated Flii in digit regeneration. Both WT and FIT digits regenerated following distal amputation with newly regenerated FIT claws being significantly longer than intact controls. No regeneration was observed in WT mice after severe proximal amputation, however
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FIT mice showed significant regeneration of the missing digit. Using a 3D model of nail formation, connective tissue fibroblasts isolated from the mesenchymal tissue surrounding the WT and FIT digit tips and co-cultured with skin keratinocytes demonstrated aggregate structures
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resembling rudimentary nail buds only when Flii was overexpressed. Moreover, β-Catenin and Cyclin D1 expression was maintained in the FIT regenerating germinal matrix suggesting a
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potential interaction of Flii with Wnt signaling during regeneration.
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Introduction Amputation of the mammalian digit tip may result in complete regeneration depending upon the
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severity of the injury. Removal of the distal half of the terminal phalange results in successful regeneration of all components of tissue, including skeletal structure, whilst a more severe,
proximal amputation, removing more than 2/3 of the terminal phalanx, will illicit no discernible
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regenerative response with healing following that of cutaneous tissue repair (Muneoka et al., 2008; Strudwick and Cowin, 2012; Takeo et al., 2013). The presence of an intact nail bed
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following injury and conservative management of the wound, appears critical for successful regeneration in humans (Mohammad et al., 1999). Studies investigating pathways involved in regeneration versus repair of the digit tip may identify targets for therapeutic development to
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stimulate the regeneration of tissue following injury (Fathke et al., 2006).
Flightless I (Flii) has been implicated in both tissue repair and regeneration. This highly conserved protein is a unique member of the gelsolin family of cytoskeletal remodeling proteins,
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being the only one containing an N-terminal Leucine Rich Repeat domain and is essential for embryonic development (Silacci et al., 2004). Flii has both cytoplasmic and nuclear activities,
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localizing with β-tubulin based structures involved in cell division and within lammellipodia and filopodia associated with migrating cells (Davy et al., 2001; Strudwick and Cowin, 2012). In the nucleus, it is a transcriptional coactivator of the estrogen and thyroid receptors (Lee et al., 2004) and has a differential effect upon β-Catenin dependent transcription and cell cycle progression (Lee and Stallcup, 2006; Seward et al., 2008). Flii is secreted into human plasma and has immunological roles via its interaction with LRRFIP2, Myd88 and TLR4 signaling (Dai et al.,
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2009; Jin et al., 2013; Lei et al., 2012; Ruzehaji et al., 2013). Flii is a negative regulator of wound repair, with decreased Flii expression associated with increased proliferation of dermal and epidermal cells, resulting in smaller, more contracted wounds (Cowin et al., 2007; Jackson et
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al., 2012). A decrease in Flii expression, either endogenously in heterozygous knockout mice, or via the topical application of a neutralizing antibody raised against the LRR domain in murine and porcine wound models results in improved healing (Cowin et al., 2007; Jackson et al., 2012;
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Strudwick and Cowin, 2012). Conversely, overexpression of Flii in the mouse resulted in larger scars, with a slower, impaired wound healing responses (Cowin et al., 2007; Jackson et al., 2012;
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Strudwick and Cowin, 2012), with fibroblast-specific upregulation of Flii giving a similar magnitude of wound healing impairment to non-tissue-specific upregulation (Turner et al., 2015). However, Flii also has a positive effect upon hair follicle regeneration, with Flii overexpression resulting in significantly longer hair fibers in regenerated follicles and reduced
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Flii expression resulting in delayed regeneration (Waters et al., 2011). Recently, Flii has also been shown to play a positive role in cell migration and is required for Rac1 mediated contraction (Marei et al., 2016). It has been suggested that Flii plays a dual role in regulation of
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al., 2016).
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migration depending upon the cellular context and the availability of binding partners (Marei et
These contrasting outcomes indicate a dynamic role for Flii and highlight the need for closer investigation of the difference between regenerative and reparative healing. Utilizing a mouse model to stimulate regeneration or repair in the digit tip, we aimed to investigate the effect of Flii upon tissue regeneration. Understanding the mechanisms involved in mammalian tissue
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regeneration will assist the development of new therapeutic approaches aimed at reducing
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scarring and fibrosis.
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Results
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Murine claw regeneration is observed in Flii over expressing but not WT mice after proximal amputation.
Distal and proximal amputations of the terminal phalange (Supplementary Figure 1a) were
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performed on the middle three digits of the left hind limb of 4 week old wild-type (WT) and Flii overexpressing (FIT) mice and compared with uninjured, intact claws of the right hind limb 8
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weeks after amputation (Figure 1a). Following distal amputation, complete digit regeneration and claw regrowth was observed in both WT and FIT mice 8 weeks after amputation (Figure 1b). FITs additionally showed significantly overgrown claws with measurements revealing that claw lengths were significantly longer than intact controls and were greater than those observed in WT
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mice (Figure 1c). The rate of regeneration following distal amputation was also significantly accelerated in FIT mice (Supplementary Figure 2), with complete regeneration observed by 4 weeks in FIT mice, whilst WT were significantly delayed. Following proximal amputation, no
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regrowth of the amputated tip was observed in WT mice (Figure 1a). However, significant regeneration was observed in FIT mice, although full regeneration was not achieved even after 8
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weeks (Figure 1a, b). The claws of the FIT mice also regenerated to 42% of the length of intact controls while WT claws were less than 15% (Figure 1c). Bone regeneration was assessed by alizarin red whole mount staining (Figure 1a, d) and showed that after the less severe, distal, amputation the terminal phalange of both WT and FIT mice regenerated to a similar length compared to intact digits. Despite the macroscopic observance of digit regeneration in FIT mice
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following proximal amputation, no significant regeneration of the bone was observed in either
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WT or FIT mice.
Flii overexpression gives rise to thicker, more curved claws following regeneration.
The appearance of the fully regenerated digit (following distal amputation) was analyzed to
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assess the effect of increased Flii expression upon claw phenotype including claw thickness and curvature as well as the angle of orientation at the tip of the digit (Figure 2a). Overexpression of
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Flii resulted in a reproducible change in the overall appearance of the claws of the regenerated digits. The thickness of the claws at both at the base and as an average overall when measured in 2D planar view were no different between WT and FIT mice (Figure 2bi). The angle between the claw tip and the borderline of the lateral claw fold in FIT mice was 6 degrees less than in WT
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mice, which translates to significantly greater curvature of the claw (Figure 2bii). The orientation of the claw at the tip of the digit was not affected with no difference observed in the angle between the borderlines of the lateral claw fold and the horizontal stripe (Figure 2biii). Thus,
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while the orientation of the claw at the tip of the digit did not change, the claws appeared longer, and were more curved. This subtle but reproducible effect was clearly seen by an overlay of the
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2D trace of the claws (Figure 2c). The effect of Flii expression upon claw phenotype was also assessed in uninjured control digits (Supplementary Figure 3). The claws of FIT mice were significantly longer than WT but no obvious changes to claw thickness, curvature or orientation were observed indicating that phenotypic changes seen in regenerated digits were exacerbated following injury.
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Presence of a germinal matrix is observed in FIT but not WT mice following proximal amputation.
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The germinal matrix within the nail organ supplies the pool of proliferative cells which give rise to the nail organ or the claw in mice (De Berker et al., 2000). The presence of a germinal matrix was confirmed histologically and by staining with Keratin 10 (K10) (Figure 3a). K10 is strongly
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expressed in normal stratified epithelium including that of the digit tip, but is low or absent
within the cells which comprise the nail organ, including the germinal matrix (De Berker et al.,
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2000; Perrin et al., 2004). K10 staining was absent from the region underlying the claw in intact WT and FIT digits and of the regenerated digits following distal amputation. Although the area of the germinal matrix and the K10 negative nail organ region in the intact digits of FIT mice appeared somewhat smaller than WT, this was not statistically significant. WT digits following proximal amputation however, displayed continuous K10 expression around the entire digit tip
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indicating that no nail organ or germinal matrix was present. However, in FIT mice, a distinct germinal matrix was observed following proximal amputation with a similar K10 staining pattern to that observed in intact and distal regenerated digits. A significant increase in the area and
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length of the germinal matrix area was observed after distal amputation in WT but not FIT mice
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(Figure 3b). Whilst no germinal matrix was observed in WT mice following proximal amputation, it was clearly present in FIT mice, albeit significantly smaller in area and length than intact controls.
Flii expression is maintained in the germinal matrix of Flii overexpressing mice following distal and proximal amputation.
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FIT mice have between 1.3 and 1.5 times the normal amount of Flii expressed in the skin, heart and muscle (Thomsen et al., 2011). In response to wounding, a four-fold increase in Flii levels is seen in the skin (Cowin et al., 2007). Flii expression was detected strongly throughout the
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epithelial tissue of the intact digit tip in both WT and FIT mice with further expression apparent within the underlying structures of the dermis (Supplementary Figure 4a). Flii expression in intact digits was not significantly different between WT and FIT mice however epithelial
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expression (Supplementary Figure 4a, b) showed significantly elevated levels of Flii in FIT mice 8 weeks after both distal and proximal amputation. Similar changes in expression are seen in the
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intact back skin compared to 3 days post incisional wounding and in the intact whole digits of these mice using semi-quantitative western analysis (Supplementary Figure 5). In WT mice, a marked decrease in expression was observed, whereas Flii levels in FIT mice were similar to intact digits and significantly higher than WT. Flii expression in the germinal matrix (within
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white boundaries) was comparable in the intact digit between WT and FIT mice (Supplementary Figure 4a, c). Following distal amputation Flii expression was significantly decreased in WT mice, whilst expression was maintained at levels equal to intact controls in FITs. Following
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proximal amputation Flii was expressed within the germinal matrix in FIT mice at levels equal to that of the intact and distally amputated digit. No germinal matrix was observed in WT digits at 8
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weeks and therefore no Flii expression could be measured.
Proliferation is increased in the regenerated germinal matrix of Flii overexpressing mice. Proliferation within the germinal matrix was assessed by immunofluorescent detection of the proliferating cell nuclear antigen (PCNA). Few PCNA positive cells were observed within the
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germinal matrix of WT and FIT intact digits and while an increase in the number of PCNA positive cells was observed in the germinal matrix of FIT mice following distal amputation, this was not statistically significant (Supplementary Figure 4d, e). However, in FIT mice following
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proximal amputation where there is the presence of a germinal matrix, a significant increase in
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proliferation within this area was observed (Supplementary Figure 4d, e).
Claw regeneration is stimulated by Flii expression within the surrounding mesenchyme of
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the digit tip.
Connective Tissue Fibroblasts (CTFs) were isolated from the distal, third (Ph3) and the more proximal, second (Ph2) phalangeal skeletal elements of WT and FIT mice (Supplementary Figure 1b) and cultured with normal human keratinocytes in a 3D model of nail formation
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(Figure 4a). Aggregate structures resembling rudimentary nail buds were induced to form by WT and FIT distal Ph3 CTFs. Keratinocytes cultured with CTFs isolated from the more proximal Ph2 skeletal elements of WT digits however formed a normal looking stratified epidermis.
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Keratinocytes cultured with Ph2 CTFs from FIT digits formed aggregate structures much like those cultured with the Ph3 CTFs. K10 staining (Figure 4a) was strongly detected in the
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normally stratified epidermis of the 3D culture but markedly reduced in aggregate structures formed by keratinocytes cultured with Ph3 CTFs (both WT and FIT), or with the more proximal pool of CTFs which overexpressed Flii (FIT Ph2). Flii expression within the newly formed epidermis or aggregate structures formed by normal human keratinocytes was unchanged, regardless of which pool of CTFs they were cultured with (Figure 4b). Interestingly, βCat was found to be significantly reduced in the normally stratified epidermis formed by keratinocytes
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cultured with the more proximal pool of WT CFTs compared to aggregate structures formed by culture with either distal WT CTFs or both distal and proximal FIT CTFs (Figure 4c). This was
the rudimentary nail like structures fail to form (Figure 4d).
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also associated with greatly reduced expression of CyclinD1 in the WT proximal cultures where
germinal matrix of Flii overexpressing mice.
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Claw regeneration may be stimulated by maintained βCat/CyclinD1 signaling within the
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Digits were collected from WT and FIT mice at 1, 2 and 4 weeks post distal and proximal amputation. Immunohistochemical analysis revealed that following distal amputation the expression of βCat at 1 week post-amputation was similar within the germinal matrix of both groups of mice (Figure 5a, b). However, following the more proximal amputation a significant
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decrease in expression of βCat was seen in the residual germinal matrix of WT but not FIT mice (Figure 5a, b). The expression of the downstream target of βCat signaling, CyclinD1, which regulates cell progression through the G1 phase and is required for proliferation (Neumeister et
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al., 2003), showed that the number of CyclinD1 positive cells within the germinal matrix following distal amputation (Figure 5c, d) was reduced in FIT digits at 1 week post amputation
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compared to WT but increased to significantly higher levels at 4 weeks post amputation, with a similar number of positive cells as seen at the same time point in WT digits. After proximal amputation (Figure 5e, f) similar levels of CyclinD1 expression were observed in the germinal matrix of WT and FIT mice at 1 week post amputation however where the number of positive cells decreased significantly by 4 weeks post amputation in the WT digits, expression was significantly higher in FIT digits at levels similar to both 1 and 2 weeks post amputation.
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Although, a residual germinal matrix was observed at 1 week post proximal amputation in both WT and FIT mice, the area of the germinal matrix subsequently decreased in size in the WT
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mice whereas it increased in size in Flii overexpressing mice (Supplementary Figure 6).
Discussion
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involved is fundamental to developing new approaches to stimulate regeneration vs repair.
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Mammalian regeneration is currently restricted to certain organs and tissues however the fingertip is capable of regeneration depending on the severity of injury (Takeo et al., 2013). Distal amputation of the fingertip leads to complete regeneration of the bone, skin and nail organ whereas a more severe, proximal amputation will result in healing by repair with no
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regeneration. In our study, proximal amputations in mice overexpressing Flii mice led to up to 42% regeneration of the amputated digit with significant regrowth of the claw. Histologically, a germinal matrix and rudimentary claw could be seen in FIT digits at 8 weeks, whilst none
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appeared in wild-type mice. The germinal matrix is the organ that supplies the pool of keratinocytes which undergo proliferation and differentiation to form the nail or claw of the digit
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tip (De Berker et al., 2000). Few PCNA positive cells were observed within the germinal matrix of the intact digits nor at 8 weeks following distal amputation when regeneration is considered complete (Simkin et al., 2013). The claws in mice overexpressing Flii showed greatly increased proliferation within the germinal matrix and this heightened proliferation may contribute to the continued claw regrowth and may lead to further regeneration of the digit tip beyond the 8 week time frame of this study.
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Not only was there a significant increase in digit tip regeneration and claw regrowth but a change
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in the appearance of the regenerated claws in mice overexpressing Flii was observed post amputation. Newly regenerated claws in FIT mice were significantly elongated compared to than intact control digits and were sharper and more curved. Changes in murine claw phenotype
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have been described in Frizzled 6 (FZD6) deficient mice which exhibit thinner, more pointed claws more vertically orientated at the digit tip (Cui et al., 2013). FZD6 belongs to a family of
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membrane bound receptors through which the canonical, βCat dependent Wnt signaling pathway operates (Schulte, 2010) and FZD6 deficiency leads to a reduction in β-catenin signaling through the canonical pathway. Flii has previously been implicated in the regulation of the canonical Wnt signaling pathway (Lee and Stallcup, 2006). Flii is a negative regulator of the canonical Wnt signaling pathway, inhibiting βCat dependent transcription by disrupting the binding of the
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murine Flightless Associated Protein 1 (FLAP1) or its human homologues Leucine Rich Repeat In FLII Interacting Protein 1/2 (LRRFIP1/2) with βCat (Lee and Stallcup, 2006). Also, Flii interacts with CARM1 and GRIP1 to enhance hormone mediated transcription (Lee et al., 2004).
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These same coactivators are positive transcriptional regulators of the βCat:TCF/LEF complex
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(Lee et al., 2004; Li et al., 2004). In the current study we found that βCat expression was maintained within the germinal matrix of Flii overexpressing mice following proximal amputation and that expression of CyclinD1 in this area continued to be expressed in the regenerating germinal matrix. No expression for βCat or CyclinD1 was observed in the germinal matrix of WT mice which failed to regenerate the claw suggesting that Flii may affect the regulation of the canonical, βCat dependent, Wnt pathway leading to increased regenerative capacity.
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The development of skin and appendages such as hair and nails, or claws, is dependent upon
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interactions between the epidermis and the underlying mesenchyme (Cui et al., 2013). Epithelial cells are stimulated to form into either a normal stratified epidermis or nail-like aggregates via signaling from cells within the mesenchyme surrounding the digit tip in a position specific
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manner (Wu et al., 2013). Connective tissue fibroblasts isolated from the distal terminal or third phalangeal element and cultured with keratinocytes, induce the formation of these nail-like
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aggregates but keratinocytes cultured with a more proximal pool of connective tissue fibroblasts from the second phalangeal element lose this ability to form elemental nail buds and stratify into normal epithelium (Wu et al., 2013). However, when normal human keratinocytes were cultured with connective tissue fibroblasts isolated from the second phalangeal element of mice which overexpress Flii, the epithelial cells were able to form rudimentary nail like structures similar to
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those cultured with fibroblasts isolated from distal, third phalangeal elements. This indicates that the regeneration of the nail organ seen in FIT mice after proximal amputation is likely to be via
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changes in signaling from the mesenchyme due to increased expression of Flii within these cells.
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To be able to drive the body towards a more complete replacement of native tissues, rather than merely repairing a tissue after significant trauma is the goal of many research groups. Utilizing the mammalian digit tip to understand the mechanisms by which tissue is stimulated to regenerate rather than simply repair following amputation may help our understanding of this complex process. These studies suggest that Flii is not just a negative regulator of reparative
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wound healing, but may also be a key regulator of both regenerative and reparative processes
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depending on the cells and tissues involved.
Materials and Methods
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Animal Studies
All mouse strains were congenic on the BALB/c background and BALB/c inbred mice were used
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as wild-type control animals (designated Flii+/+). Flii transgenic strain ((Tg1FLII)2Hdc) incorporating a 17.8 Kb fragment of a human cosmid clone that spans the entire FLII locus, with animals homozygous for the transgene in addition to the endogenous Flii allele designated FliiTg/Tg or FIT. Details of the generation of the FIT mice were described previously, showing
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Digit Amputations
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elevated Flii protein levels in various tissues including skin (Thomsen et al., 2011).
All animal procedures were approved by the WCHN Animal Ethics Committee (AE847/11/2013,
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AE972/2/2017) and carried out in accordance with the Australian code of practice for the care and use of animals for scientific purposes. One third (distal) to greater than two thirds (proximal) of the terminal phalanges of the central three digits of the hind limb of 4 week old wild-type (WT) and Flii overexpressing (FIT) mice were amputated as described previously (Han et al., 2008; Neufeld and Zhao, 1995). Amputation levels were calculated from amputated tip against intact age-matched control digits using whole mount Alizarin Red stain (Han et al., 2008). Briefly, digit tips collected at time of surgery were fixed in 95% ethanol overnight before 15
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incubation with 0.0033% w/v Alizarin Red S (Sigma-Aldrich, Sydney, Australia) in 1.6% Aqueous KOH for two days prior to clearing in 1.6% KOH and Mall’s Solution (20% Glycerin in 1% KOH) to glycerol for visualization. Measurement of terminal phalange and amputated tip
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(MediaCybernetics Inc., Rockville, MD, USA).
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bone length were taken from whole mount photographs, using ImagePro Plus 5.1 software
Macroscopic Assessment
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All samples were assessed macroscopically for regenerative success 8 weeks post amputation from images taken at the time of collection of the digits. Images were scored by five independent assessors for macroscopic evidence of regeneration using a modified Leichardt scale on a scale of 0-5 comparing amputated digits with associated intact control digits from the opposite hind
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limb of the same animal (n=36/genotype). Further information on the Leichardt scale assessment
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can be found in Supplementary information.
Semi Quantitative Western Analysis, Histological, Alizarin Red and Immunofluorescent
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Staining
Flii protein expression in intact and wounded back skin and in whole intact digits of WT and FIT mice was analyzed as described in supplementary information. Samples were collected at 1, 2, 4 and 8 weeks post amputation for histological assessment, alizarin red and immunofluorescence as described in supplementary information.
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Connective Tissue Fibroblast Isolation and Nail Formation Model Position specific connective tissue fibroblasts were isolated from hind limb digits of 4-6 week
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old male and female WT and FIT mice according to previously description (Wu et al., 2013) and described in Supplementary information. Isolated P2 or P3 WT and FIT CTFs were cultured with normal human neonatal keratinocytes (Invitrogen, Waverley, NSW, Australia) to form a 3D model of nail formation in triplicate based upon previously described methods (MacNeil et al.,
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2011; Wu et al., 2013). Briefly, 1 × 105 passage 3-5 fibroblasts and 3 × 105 passage 1-3
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keratinocytes, each in 250 mL of 10% Green’s medium were seeded onto de-epidermalized human dermis (European Skin Bank, Beverwijk, Netherlands) fitted with a 1cm internal diameter chamfered metal (medical-grade stainless steel). After 48 hrs with two media changes the steel ring is removed and skin constructs raised on stainless steel grids and allowed to grow in 10% Green’s for 21 days at the air–liquid interface prior to processing for H&E or
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Statistical Analysis
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immunofluorescence staining.
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Statistical differences were determined using the 2-tailed Student's t-test. A p value of less than 0.05 was considered significant.
Conflict of Interest The authors state no conflict of interest.
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Turner CT, Waters JM, Jackson JE, Arkell RM, Cowin AJ. Fibroblast-specific upregulation of Flightless I impairs wound healing. Exp Dermatol 2015;24:692-697. Waters JM, Lindo JE, Arkell RM, Cowin AJ. Regeneration of hair follicles is modulated by flightless I (Flii) in a rodent vibrissa model. J Invest Dermatol 2011;131:838-847. Wu Y, Wang K, Karapetyan A, Fernando WA, Simkin J, Han M, et al. Connective tissue fibroblast properties are position-dependent during mouse digit tip regeneration. PLoS One
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Figure Legends Figure 1: Claw regeneration is enhanced by Flightless I (Flii) overexpression in murine digits. (a) Effect of Flii expression upon regeneration was assessed macroscopically (macro) and
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by alizarin red staining of the bone 8 weeks after either distal or proximal amputation in wildtype (WT) and Flii overexpressing (FIT) mice. Approximate level of amputation is shown by black dashed line. Original magnification 2.5X. Black scale bars represent 600µm. (b)
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Macroscopic regeneration assessed qualitatively by 5 independent assessors using a modified Leichardt scale comparing amputated digits to associated intact control digit on the opposite hind
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limb. The scale ranges from 0-5, where 0 equals no regeneration visible, 1 is grossly undergrown, 3 is equal to intact control and 5 is grossly overgrown. Solid line represents normal intact control digits. (c) Claw regeneration was assessed by measurement of the length of the claw from the borderline of the lateral claw fold to the claw tip on macroscopic images and
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expressed as % of the length of the associated intact control digit on the opposite hind limb. Mean +/- SEM. **= p <0.01, ***= p <0.005. Solid line represents length normal intact control claws. (d) Bone regeneration was assessed by measurement of the length of the terminal
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phalange following alizarin red staining and expressed as % of the length of the associated intact control digit on the opposite hind limb. Mean +/- SEM. **= p <0.01, ***= p <0.005. Solid line
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represents length of normal intact control terminal phalange.
Figure 2: Flii overexpression results in thinner, more curved claws following regeneration of the digit tip. The effect of Flii expression upon 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 as well as at the base of the claw between the 21
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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
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stripe and the borderline of the lateral claw fold. Images included are for demonstration of
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
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tip were expressed graphically in degrees. Mean +/- SEM. *= p <0.05. (c) Overall claw
appearance was demonstrated by overlaying 2D traces of claw thickness, curve angle and
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orientation angle take from representative macroscopic images of WT and FIT claws. Original magnification 2.5X. Black scale bars represent 600µm.
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Figure 3: Presence of a germinal matrix is observed in FIT but not WT mice following proximal amputation. (a) 4µm 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
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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 4X.
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Arrowheads denote region of the nail organ with absent K10 staining. The germinal matrix (gm) shown bounded by white in inset 10X magnification images. All scale bars represent 200µm. (b) Measurement of the regenerated germinal matrix were expressed as a percent of the area and length of the associated intact control digit. Mean +/- SEM. *= p <0.05.
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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 are air-liquid interface on de-
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epidermalized dermis. 4µm 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 CyclinD1 (pseudo
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stained orange). Junction between the dermis (d) and epidermis (e) is marked by dashed line. Rudimentary nail formation is marked by the presence of aggregate structures (as) within the
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neo-epidermis. White arrows indicate positive CyclinD1 expression within the nucleus. Original magnification 20X. All scale bars represent 50µm. 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 CyclinD1 was measured quantitatively as the number of CyclinD1
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positive (+ve) cells per 100µm. Mean +/- SEM. *= p <0.05, **=p<0.01, ***= p <0.005.
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Figure 5: βCat and CyclinD1 expression is maintained in the germinal matrix of Flii overexpressing mice following distal and proximal amputation. (a) Immunofluorescent
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detection of βCat (pseudo stained orange) within the germinal matrix (gm) shown bounded by white separated from the underlying nail dermis (nd) on 4µm sagittal sections taken from paraffin embedded digits of WT and FIT mice collected 1 week after distal or proximal amputation. Original magnification of images is 20X. All scale bars represent 20µm. (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 CyclinD1 (pseudo stained 23
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yellow) within the germinal matrix (gm) bounded by white separated from the nail dermis (nd) on 4µm sagittal sections taken from paraffin embedded digits of WT and FIT mice collected 1, 2 and 4 weeks post amputation at the distal (c) or proximal (e) level. White arrows indicate
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positive CyclinD1 expression within the nucleus. Composite 20X magnification images. All scale bars represent 20µm. The expression level of CyclinD1 was measured quantitatively as the number of CyclinD1 positive (+ve) cells per 100µm within the volar epithelium following distal
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(Dist) (d) or proximal (Prox) (f) amputation. Mean +/- SEM. *= p <0.05, ***= p <0.005.
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