Twist2 contributes to skin regeneration and hair follicle formation in mouse fetuses

doi:10.21203/rs.3.rs-3977629/v1

Download PDF

Article

Twist2 contributes to skin regeneration and hair follicle formation in mouse fetuses

https://doi.org/10.21203/rs.3.rs-3977629/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Unlike adult mammalian wounds, early embryonic mouse skin wounds completely regenerate and heal without scars. Analysis of the underlying molecular mechanism will provide insights into scarless wound healing. Twist2 is an important regulator of hair follicle formation and biological patterning; however, whether it plays a role in skin or skin appendage regeneration remains unclear. We aimed to elucidate Twist2 expression and role in fetal wound healing. ICR mouse fetuses were surgically wounded at embryonic days 13 (E13), E15, and E17, and Twist2 expression in tissue samples from these fetuses was evaluated via in situ hybridization, immunohistochemical staining, and reverse transcription-quantitative polymerase chain reaction. Twist2 was upregulated in the dermis of E13 wound margins but downregulated in E15 and E17 wounds. Twist2 knockdown at E13 left visible marks at the wound site, inhibited regeneration, and resulted in defective follicle formation. Twist2-knockdown dermal fibroblasts lacked the ability to undifferentiate. Furthermore, twist2 hetero knockout mice (Twist+/-) formed visible scars, even at E13, where all skin structures, including texture, should regenerate. Thus, Twist2 expression correlated with skin texture formation and hair follicle defects in late mouse embryos. These findings may help develop a therapeutic strategy to reduce scarring and promote hair follicle regeneration.

Biological sciences/Developmental biology/Disease model

Biological sciences/Developmental biology/Morphogenesis

Biological sciences/Developmental biology/Pattern formation

Biological sciences/Cell biology/Mechanisms of disease

Twist2

scar

skin regeneration

hair follicle formation

wound healing

The economic and social consequences of scarring are significant. More than 100 million patients acquire scars each year, some of which cause considerable functional or psychosocial morbidity [1, 2]. Therefore, the development of effective therapies for scarless wound healing has a significant impact and gained much attention.

Wound healing is a complex process that usually occurs in the postnatal period through the formation of scar tissue, and regenerative healing in adult animals is limited to liver and bone [3, 4]. In contrast, the skin wounds of fetuses are known to heal without scars by regenerating normal skin structures, including the recovery of cutaneous appendages and neurovasculature up to a specific developmental stage [5, 6]. Scar tissue is characterized by (1) fibrosis of the dermis, (2) loss of skin texture, (3) loss of skin appendages, and (4) color tone changes. Therefore, to determine whether a wound has regenerated, the morphology of the scar (excluding color tone) should be evaluated from two perspectives: skin texture and dermal structure [7, 8]. In a model of wound healing in mammalian fetuses, an early regenerative healing period is followed by a period in which the wound heals with an extracellular matrix (ECM) that is indistinguishable from intact tissue but fails to regenerate dermal appendages, leaving a visible mark [9]. Then, late in development, the wound changes to an adult-like phenotype with wound healing due to excess collagen in the ECM, loss of dermal appendages, and flattening of the epidermis, classified as a scar. Until now, this change was thought to occur progressively. However, using our proprietary mouse fetal wound model, we found a critical point where complete regeneration without visible scarring occurs until embryonic day 13 (E13) and regeneration of dermal structures occurs until E16, after which it becomes similar to the adult animal type [10, 11]. A deeper understanding of the basic mechanisms of fetal wound healing will allow us to identify therapeutic targets that can minimize scar formation. In particular, observing the factors that change before and after the critical point we discovered may be important in the treatment of post-wound skin regeneration. Skin appendages, such as bird feathers, mammalian hair, and zebrafish scales, have long been considered to be essential interactions between the epidermis and dermis [12]. Several signaling pathways and components, such as Wnt, fibroblast growth factor (FGF), and Sonic hedgehog (SHH), and transcription factors, such as Twist, have been shown to be essential in regulating feather and hair formation and patterning [13–15].

In particular, Twist1 and Twist2 (also called Dermo1) are highly conserved basic helix-loop-helix transcription factors that are structurally conserved, containing basic amino acids flanking two amphipathic alpha-helices separated by an interhelix loop [16]. Twist plays an important role in dorsoventral patterning and mesodermal incision in Drosophila [17, 18]. In chickens, Twist2/Dermo1 is expressed in feather-forming cells, indicating its role in feather formation [19]. In mice, Twist2 knockout showed progressive growth retardation, possibly as a result of increased cytokine signaling, leading to thin skin and sparse distorted hair follicles before death [20]. Thus, while Twist2 signaling is involved in patterning and hair follicle formation, it is unclear whether these paralogs have a primary function in skin texture or skin appendage regeneration. However, since patterning has been reported to be important for epidermal texture and hair follicle formation [21], we hypothesized that Twist2 is involved in skin regeneration after wounding during early development.

In this study, we aimed to determine whether Twist2 plays a role in wound healing using a mouse model. Since fetal mouse wounds epithelialize between 48 and 72 h after wounding [10], we especially focused on wounds 24 h after wounding to observe the effect of Twist2 on the wound healing process. Next, to analyze the function of Twist2, we knocked it down using a siRNA and examined its effect on wound healing. Furthermore, since Twist2 homozygous knockout mice are lethal and heterozygous knockout mice are viable and fertile, we observed the wound healing process in the fetuses of Twist+/- mice [17]. In addition, we investigated the relationship among dermal fibroblast function, hair follicle formation, and Twist2 expression, as previous studies have shown that dermal fibroblasts lose their ability to form hair follicles when cultured in normal two-dimensional (2D) culture, but when cell aggregates are formed in nonadherent culture dishes, they regain their ability to form hair follicles, express undifferentiated markers such as Sox-2 and CD133, and acquire pluripotency [19].

We investigated the effects of twist2 on wound regeneration/repair and hair follicle formation using our own fetal mouse wound healing model and a three-dimensional cell model.

2.1. Twist2 Is Upregulated in the Dermis of E13 Wounds

In situ hybridization was performed to observe the expression of the Twist2 gene during wound healing in embryonic development. The results showed that Twist2 mRNA was specifically expressed in the dermis, and its expression was enhanced as it approached the epidermis. In relation to the wound, Twist2 mRNA expression was high at the wound margin at E13 and tended to decrease as it approached the wound margin at E15 and E17 (Fig. 1a).

In immunostaining, Twist2 protein was specifically expressed in the dermis, and its expression was enhanced as it approached the epidermis. In addition, the expression of Twist2 protein at E15 and E17 decreased around the wound site compared with that at the normal site, whereas that at E13 tended to increase at the wound site compared with that at the normal site (Fig. 1b).

When mRNA was extracted from only dermis of the wound via laser microdissection (LMD), real-time PCR revealed that the expression was significantly increased in the wound compared with that in the normal skin at E13 (P = 0.02). In contrast, the expression at E15 (P = 0.03) and E17 (P = 0.036) was decreased in the wound area compared with that in the normal skin (Fig. 1c). Thus, Twist2 is upregulated in fully regenerating E13 wounds and downregulated in later developmental stages, suggesting its involvement in the E13-type wound healing phenotype.

2.2. Knockdown of Twist2 Inhibits Mouse Fetal Skin Regeneration

To evaluate whether Twist2 is required for fully regenerative E13-type wound healing, we knocked down Twist2 in the skin by injecting siRNA into the amniotic fluid. Mice analyzed 72 h after Twist2 siRNA administration during surgery showed significant knockdown of Twist2 levels in both wounds and normal skin (control, P = 0.027; wound, P = 0.025). In addition, significant knockdown of Twist2 was observed in both the control and normal skin (control, P = 0.027; wound, P = 0.019). Conversely, in the group treated with negative control siRNA, Twist2 was significantly upregulated in the wound (P = 0.03; Fig. 2a). This confirmed the successful knockdown of Twist2 in fetal mouse skin via Twist2 siRNA administration.

In the morphological observation of wounds using a stereo microscope, the wounds were usually completely regenerated even in E13 mice, but treatment with Twist2 siRNA in amniotic fluid prevented skin regeneration, leaving visible marks with no regeneration of the skin structure (Fig. 2b).

Hematoxylin and eosin staining of tissue sections showed that hair follicle regeneration was lost in E13 wounds treated with Twist2 siRNA (Fig. 2c). In the negative control siRNA group, the skin structures, including hair follicles, completely regenerated, indicating that Twist2 is involved in skin regeneration.

2.3. Wounds in twist2 Heterozygous Knockout Mice Do Not Regenerate

The pattern of wound healing in twist2 knockout mouse fetuses was then observed. Crossing of twist 2 heterozygotes yielded homozygous and heterozygous mutant offspring at the predicted Mendelian ratio.

Both hetero and homo fetuses survived post-placental surgery, with similar survival rates as wild type (WT). Wound creation and retrieval were possible, but about half of the homo-operated individuals had congestive morphology with lymphangiogenesis imperfecta, and many had severe edema (Fig. 3a). Histologically, the skin morphology was thinner in the homo, and in situ hybridization showed no expression of twist2 in the homo (Fig. 3b). Wounds were created in E13 twist2 knockout mice, and changes in wound healing were examined. Wounds in WT mice regenerated without a visible scar in a normal pattern, whereas those in hetero mice had visible scar and homo mice were difficult to evaluate on the body surface due to edema (Fig. 3c). Observation of the wound tissue showed that all structures, including hair follicles, were regenerated in WT, whereas twist2 knockout mice did not regenerate hair follicles in the wound (Fig. 3d).

2.4. Twist2 Is Upregulated by 3D Culture of Fibroblasts

We previously reported that fetal dermal fibroblasts express undifferentiated markers, such as Sox2 and CD133 when cultured in nonadherent culture dishes and regain follicle-inducing ability once expression is lost [22]. To investigate how Twist2 is involved in the expression of undifferentiated markers and in hair follicle formation, we performed in vitro knockdown of Twist2. We divided E17 fetal dermal fibroblasts into superficial (17S) and deep (17D) fibroblasts in relation to the dermal panniculus carnosus muscle. These cells were cultured in 2D or 3D using nonadherent culture dishes. No morphological changes were observed depending on the site of origin of the cells (Fig. 4a). However, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) showed that Twist2 (17S, P = 0.00056; 17D, P = 0.0014) and the undifferentiated markers Sox2 (17S, P = 0.0000056; 17D, P = 0.026) and CD133 (17S, P = 0.00029; 17D, P = 0.018) were significantly upregulated in the 3D-cultured cells (Fig. 4b). When the siRNA reagent of Twist2 was administered to the 3D culture medium, Twist2 was significantly knocked down (17S, P = 0.0075; 17D, P = 0.011), and at the same time, Sox2 (17S, P = 0.04; 17D, P = 0.0097) and CD133 (17S, P = 0.028; 17D, P = 0.044) were significantly decreased compared to when the negative control siRNA was used (Fig. 4c).

2.5. Knockdown of Twist2 Reduces the Migratory Ability of Dermal Fibroblasts

To further investigate the effect of Twist2 expression on wound healing in terms of cell migration, we performed a scratch assay and found that dermal fibroblasts with Twist2 knocked down had clearly reduced migratory ability (P = 0.0001; Fig. 5a and b). Thus, the expression of Twist2 in terms of cell migration ability also has an effect on wound healing.

Most adult mammalian tissues do not undergo regenerative healing, a process that reproduces functional tissue that is virtually indistinguishable from its original form [23]. Small skin wounds in mice result in the formation of scar tissue or fibrosis, which is composed primarily of ECM components, such as collagen and fibronectin, and lacks new accessory structures, such as hair follicles. This is similar to the wound skin process in humans. However, the mediators that determine the balance between scar formation and regenerative skin wound healing remain unknown. In particular, whether the scar prevents regeneration, whether scar cells inherently lack regenerative capacity, or whether there are simply no regenerative cues is unknown. Thus, there is a strong need to develop novel and efficient strategies for the treatment of this disorder, which requires a comprehensive understanding of the underlying cellular and molecular mechanisms.

We focused on Twist2, one of the factors involved in patterning and follicle formation, using an originally developed mouse embryonic wound healing model and compared it at the time points when wounds switch from regeneration to repair. The results showed that Twist2 was upregulated in the dermis of E13 wounds, which were completely regenerated, and was downregulated in wounds after E15. Knockdown of Twist2 at E13 resulted in wound healing, leaving a visible mark, and inhibited regeneration. Interestingly, follicle formation was not observed in Twist2 knockdown wounds. Similarly, for twist2 knockout mice, wounds did not regenerate at E13 and follicle formation in the wound was also inhibited. To elucidate this mechanism, fibroblasts were made to acquire the ability to undifferentiate via 3D culture and were observed in vitro. The 3D-cultured dermal fibroblasts showed increased expression of undifferentiated markers as well as high expression of Twist2, and knockdown of Twist2 reduced the expression of these markers. In terms of migration capacity, knockdown of twist2 also reduced the migratory capacity of fibroblasts.

There have been few reports on the mechanism and role of Twist2 in skin wound healing. However, standard WNT signaling is known to be required for Twist2 expression in dermal fibroblasts during the embryonic stage when hair follicles are formed [13, 24]. The WNT signaling pathway is an evolutionarily conserved intercellular communication system that is important for cell proliferation and differentiation in both development and tissue homeostasis [25]. In hair follicle morphogenesis, active WNT signaling in dermal fibroblasts via β-catenin and WNT co-transcription factors (Lef1, Tcf1, Tcf4, etc.) is a prerequisite for dermal papilla layer commitment [26, 27]. In addition, recent studies have shown that neonatal papillary fibroblasts still exhibit regenerative capacity supported by active WNT signaling at birth, even after fetal hair follicle morphogenesis is complete. Additionally, after birth, chromatin remodeling by Twist2 follows the fibroblast maturation pathway, spontaneous intracellular changes, resulting in loss of regenerative capacity mediated by inactivation of the WNT signaling pathway [28]. It has been suggested that Twist2 expression decreases during postnatal maturation and that a major role of Twist2 is lineage-specific inactivation of WNT signaling, which occurs in a time-dependent manner after birth. Furthermore, transient loss of Twist2 expression in fibroblasts is thought to be necessary for maintaining high WNT signaling activity after completion of dermal papillae differentiation [29]. The present study demonstrates that Twist2 is involved in complete skin regeneration and that its downregulation induces switch to healing that leaves an adult-type visible mark without hair follicle formation, which is consistent with the above mechanisms involved and the altered nature of dermal fibroblasts at E13 onwards.

In terms of cell migration, fibroblasts in the dermis with Twist2 knocked down also had reduced migratory capacity. In relation to re-epithelialization, dermal recovery occurs through fibroblast migration and proliferation, and the response of fibroblasts during wound healing determines the outcome of tissue repair. In response to wounding, macrophages and fibroblasts secrete growth factors that result in further fibroblast migration and proliferation. Recently, transplantation and lineage tracing have identified two distinct dermal lineages that give rise to the upper and lower dermis, respectively [30]. Following wounding, both cell types are mobilized to the injured area; however, one population forms the lower dermis, while the other that gives rise to the upper dermis is mobilized during re-epithelialization and provides the environment for new follicle formation in the wounded area. In our results, we found an altered expression of Twist2 in cells of the upper dermis and reduced cell migratory capacity upon knockdown. Thus, the findings suggest that in wounds at E15 and later stages, reduced Twist2 expression in fibroblasts in the upper dermis leads to reduced migration and impaired follicle formation.

Despite these findings, a limitation of this study is the lack of studies on signaling downstream of Twist2 and target cells in the investigation of wound healing during development. Considering that the reticular dermis and subcutaneous fat also showed significant expansion in the postnatal period, it is possible that inactivation of the WNT signaling pathway by Twist2 is required for the postnatal differentiation of both upper and lower fibroblast lineage cells, and examination of the behavior of these cells in null mice is also needed [31]. Whether this phenomenon is applicable to mammals other than mice also needs to be confirmed.

The findings of the present study show that Twist2 is correlated with non-scarring skin regeneration and hair follicle formation during development. If this can be mimicked in the human skin and wound tissue, it may be useful for the development of scarless wound-healing and regenerative medicine.

4.1. Ethical Consideration

The research protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Keio University School of Medicine (approval number: 20170914). All experiments were conducted in accordance with the institutional guidelines for animal experiments at Keio University. This study is reported in accordance with the Animal Research: Reporting of In Vivo Experiments on Animals (ARRIVE) guidelines.

4.2. Mouse

Jcl:ICR mice were obtained from Sankyo Laboratory Services, Inc. (Osaka, Japan). B6.129X1-Twist2^{tm1.1(cre)Dor/J} mice were purchased from The Jackson Laboratories (#008712, Bar Harbor, ME, USA). C57BL/6J was used as the WT. Heterozygous crosses were made and maintained. Phenotypes of the fetuses were confirmed after genotyping was performed after fetal surgery and wound tissue was recovered.

4.3. Fetal Wounding Procedure

Eight-week-old female ICR mice were used. Vaginal plugs were checked twice daily. When a plug was observed, the fetus was designated E0 and was wounded at E13, E15, and E17. Surgeries were performed on five pregnant mice per time point. Pregnant mice were anesthetized with 3% isoflurane, and the abdominal wall was incised to expose the uterus. Using an operating microscope, the myometrium and amniotic and yolk sacs were incised. Then, using surgical microscissors, a full-layer incision approximately 2 mm in length was made in the lateral thoracic region of the fetus at E13. After wounding, the amnion and yolk sac were sutured with 9 − 0 nylon, while the myometrium was left open and unsutured. In particular, the fetus was returned to the abdominal cavity with the amnion and yolk sac covered but the myometrium uncovered, and the abdomen was closed; at E15 and E17, after fetal wounding, the myometrium was sutured with 9 − 0 nylon, the uterus was returned to the abdominal cavity, and the abdomen was closed. Then, just before the closure of both wounds, a uterine relaxant, ritodrine hydrochloride (FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan; 1 µg/g BW), was administered intraperitoneally. The peritoneum and skin were then sutured with 5 − 0 nylon thread. Maternal mice were euthanized via cervical dislocation, and fetuses were harvested 24 h after wounding. At each time point, wounds were made in at least four fetuses. Fetal skin was harvested and fixed in 4% paraformaldehyde for 24 h, and the fixed tissue was embedded in paraffin and stained.

4.4. Immunohistochemistry

Paraffin-embedded specimens were sliced into 7 µm-thick sections and mounted on glass slides. After drying overnight at room temperature (15–25°C) to allow the specimens to adhere to the slides, the paraffin was dissolved in a slide heater (ThermoBrite; Leica Biosystems, Nussloch, Germany) at 65°C for 30 min immediately before use. The slides were then deparaffinized by changing xylene twice at room temperature (5 min each). The slides were transferred twice to 100% ethanol (3 min each), once to 95%, 70%, and 50% ethanol (3 min each), and rehydrated at room temperature. After antigen activation by heating, the samples were incubated with 2% goat serum in phosphate-buffered saline (PBS) for 30 min at room temperature to block nonspecific binding sites. The cells were then incubated with anti-Twist2 antibody (ab66031; Abcam, Cambridge, UK; diluted 1:100 in PBS) overnight at 4°C. After washing three times with PBS, the cells were incubated with an anti-mouse HRPO-labeled rabbit IgG reagent ImmPRESS (Vector Laboratories Inc., CA, USA; diluted 1:500 in PBS) for 1 h at room temperature. Signals were amplified by the avidin-biotinylated peroxidase complex method using the VECTASTAIN ABC kit (Vector Laboratories), and the sections were incubated in 20 mg/dL 3,3'-diaminobenzidine solution (FUJIFILM Wako Pure Chemical Co.) for 1–3 min. The sections were then washed once with running tap water for 5 min before counterstaining the nuclei for 6 s at room temperature with Gill’s hematoxylin solution (Merck Millipore, Billerica, MA, USA). Finally, the sections were washed with tap water for 5 min, dehydrated four times with ethanol (95%, 95%, 100%, and 100%; 5 min each), washed three times with xylene, and sealed with Mount Quick Sealant (Takara Bio, Shiga., Japan). The slides were observed under an integrated stereo microscope (BZ-X800; KEYENCE, Osaka, Japan). The experiment was repeated three times.

4.5. In Situ Hybridization

In situ hybridization analysis was performed using the QuantiGene ViewRNA ISH Tissue Assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, paraffin sections were dried at 60°C for 60 min, and paraffin was removed using Histo Clear (National Diagnostics, Atlanta, GA, USA) and 100% ethanol with an ImmEdge Pen (Vector Laboratories Inc.). After washing with PBS twice, the tissue was fixed in 10% neutral-buffered formalin solution for 5 min and washed again with PBS. The target probe was diluted 50-fold in probe set diluent QF solution warmed to 40°C and incubated at 40°C for 3 h. After washing with wash buffer three times, the preamplifier solution was incubated at 40°C for 25 min. The preamplifier solution was washed with wash buffer three times and then incubated at 40°C for 15 min. The AP enhancer solution was decanted, and Fast Red Tablet was dissolved in Naphthol buffer and incubated at room temperature for 5 min. After decanting the AP enhancer solution, Fast Red Tablet was dissolved in Naphthol buffer and incubated at 40°C for 30 min. After washing with PBS twice, nuclear staining was performed with Gill’s hematoxylin solution, and the cells were washed with water three times. The probe used was Twist2 (VB1-14748-VT).

4.6. LMD, RNA Isolation, and Reverse Transcription

LMD was performed using a PALM MicroBeam (Carl Zeiss, Germany). The manufacturer’s recommended slides and collection tubes (AdhesiveCap 500 opaque; Carl Zeiss) were set up, and the tissue was carefully cut after adjusting the aperture and intensity using a 20× magnification objective lens. The tube caps were filled with Buffer RLT (RNeasy Micro Kit; Qiagen, Germany) with β-mercaptoethanol to allow separation of intact RNA. Total RNA was extracted from cells or skin tissue using a monophasic solution of phenol and guanidine isothiocyanate (ISOGEN; Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. Total RNA was mixed with a random primer, reverse transcriptase, and dNTP mixture (Takara Bio). Reverse transcription was performed on a T100TM thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 25°C for 5 min, 55°C for 10 min, and 80°C for 10 min to heat-inactivate the reverse transcriptase and produce cDNA.

4.7. RT-qPCR

RT-qPCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). A total of 40 cycles were performed, and the fluorescence of each sample was measured at the end of each cycle. The PCR reaction used was as follows: 95°C for 3 s (denaturation) and 60°C for 30 s (annealing and extension). In the subsequent melting curve analysis phase, the temperature was increased from 60°C to 95°C and fluorescence was measured continuously. Twist2 (Mm00492147_m1), Sox2 (Mm03053810_s1), and prom1 (CD133) (Mm00477115_m1) were used as primers (all from Thermo Fisher Scientific). The PCR master mix (cat. 4352042; Applied Biosystems, Foster City, CA, USA) was used according to the manufacturer’s instructions, and ACTB (Mm02619580_g1) was used as a control gene for normalization according to the manufacturer’s instructions. Gene expression levels at normal sites were used as baseline, and fold-change values were determined using the 2-ΔΔCt method.

4.8. siRNA Experiment for Mouse Fetuses

E13 mice underwent normal fetal surgery, and siRNA reagents were injected into the amniotic fluid. The concentration of the siRNA reagent Twist2 (160520 and 61236; Thermo Fisher Scientific) used was 1.5 mg/mL. Complex buffer (1377501; Thermo Fisher Scientific) and siRNA were mixed in equal amounts, and invivofectamine (1377501; Thermo Fisher Scientific) times the amount of siRNA was mixed to make the reagent. Negative siRNA (Silencer™ Negative Control No. 1 siRNA; Thermo Fisher Scientific) was used as a negative control. The prepared reagents were then incubated at 50°C for 30 min, placed in a Float-A-Lyzer dialyzer, and dialyzed with 1 L of PBS (pH 7.4). The final concentration of siRNA preparation reagent (approximately 0.35 mg/mL) was administered in approximately 40 µL amniotic fluid per fetal mouse. Tissues were then harvested 72 h later, and the recovered tissues were embedded in paraffin. Seven micrometer-thick slides were prepared, and hematoxylin and eosin-stained tissues were observed.

4.9. 2D and 3D Culture of Fetal Mouse Dermal Fibroblasts

E17 fetal mouse skin was divided under a microscope into the superficial (S) and deep (D) layer of the dermis. Fibroblasts were established from these tissues, and those at passage 5 were used as mesenchymal cells. They were cultured as previously described [22] in two-dimensional culture using a regular adherent culture dish (Nunclon Delta; Thermo Fisher Scientific) and in three-dimensional culture using a dish that was made non-adherent by coating it with a 1% agarose gel. Next, 10% fetal bovine serum and Dulbecco’s modified Eagle medium with 1% penicillin-streptomycin were used for each culture. Cells were grown at 37°C and 5% CO₂, and the medium was changed twice a week. Three-week-old cultures were then incubated with Lipofectamine 2000 (11668-019; Life Technologies, Invitrogen, Germany) and then transfected with Twist2 siRNA (160520 and 61236, SilencerTM siRNA; Thermo Fisher Scientific) or negative control siRNA (4390843, SilencerTM Select Negative Control No. 1 siRNA; Thermo Fisher Scientific) via injection into the culture medium. After 72 h, RNA was collected from the cells using ISOGEN (Nippon Gene), and specific gene knockdown was evaluated via RT-qPCR.

4.10. Scratch Assay

Knockdown of Twist2 was performed in the same manner on fibroblasts in the D dermis layer used in 3D culture. The cells were treated with 10 µg/mL mitomycin C (NACALAI TESQUE, INC., Kyoto, Japan) for 3 h at 37°C to exclude proliferative effects prior to the assay. The cells were grown to confluence on a plastic dish and scratched using a 500 µm pipette tip. The cells were grown in Dulbecco’s modified Eagle medium at 37°C, 5% CO₂, and 95% relative humidity for up to 24 h. Migration of cells from the edges of the scratches was visualized under a microscope (BZ-X800; KEYENCE).

4.11. Statistical Analysis

Mann–Whitney U test was performed using Statistica software version 9.0 (StatSoft, Tulsa, OK) to determine the significance of differences in gene expression. The results of descriptive statistics are presented as the mean ± standard deviation. Statistical significance was set at P < 0.05. Each experiment was performed in triplicate.

Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Author Contribution

Conceptualization, K.T., A.S. and K.K.; data curation, K.T., A.S., N.A-H. and K.K.; formal analysis, K.K. and A.S.; funding acquisition, K.K.; investigation, K.K., A.S., S.S. and K.K.; methodology, K.K.; project administration, K.O. and K.K.; resources, N.A-H. And K.K.; software, K.K. and A.S.; supervision, K.K.; validation, K.T. and K.K.; visualization, K.T. and A.S.; writing–original draft preparation, K.T.; writing–review and editing, K.K.

Acknowledgments:

The authors thank the laboratory members for their technical assistance with experiments. This research was funded by JSPS KAKENHI, grant number JP 20K18422.

Data availability

The data presented in this study are available on request from the corresponding author.

Brown, B.C. et al. The patient-reported impact of scars measure: Development and validation. Plast. Reconstr. Surg. 125, 1439–1449 (2020). doi:10.1097/PRS.0b013e3181d4fd89.
Bayat, A., McGrouther, D.A. & Ferguson, M.W. Skin Scarring. BMJ 326, 88–92 (2003). doi:10.1136/bmj.326.7380.88.
Patel, A., Aslam, R., Jamil, M., Ansari, A. & Khan, S. The effects of growth factors and cytokines on hepatic regeneration: A systematic review. Cureus 14, e24539 (2022). doi:10.7759/cureus.24539.
Tsukagoshi, Y. & Matsushita, Y. Bone Regeneration: a message from clinical medicine and basic science. Clin. Anat. (2022). doi:10.1002/ca.23917.
Longaker, M.T. et al. Studies in fetal wound healing, vi. second and early third trimester fetal wounds demonstrate rapid collagen deposition without scar formation. J. Pediatr. Surg. 25, 63–68 (1990); discussion 68. doi:10.1016/s0022-3468(05)80165-4.
Gurtner, G.C., Werner, S., Barrandon, Y. & Longaker, M.T. Wound repair and regeneration. Nature 453, 314–321 (2008). doi:10.1038/nature07039.
Walmsley, G.G. et al. A mouse fetal skin model of scarless wound repair. J. Vis. Exp. 95, 52297 (2015). doi: 10.3791/52297,Pubmed:25650841.
Ishii, T. et al. Skin wound healing of the adult newt, Cynops pyrrhogaster: A unique re-epithelialization and scarless model. Biomedicines 9, 1892 (2021). doi: 10.3390/biomedicines9121892,Pubmed:34944708.
Lo, D.D., Zimmermann, A.S., Nauta, A., Longaker, M.T. & Lorenz, H.P. Scarless fetal skin wound healing update. Birth Defects Res. C Embryo Today 96, 237–247. doi:10.1002/bdrc.21018.
Kishi, K., Okabe, K., Shimizu, R. & Kubota, Y. Fetal skin possesses the ability to regenerate completely: Complete regeneration of skin. Keio J. Med. 61, 101–108 (2012). doi:10.2302/kjm.2011-0002-ir.
Takaya, K. et al. Actin cable formation and epidermis-dermis positional relationship during complete skin regeneration. Sci. Rep. 12, 15913 (2022). doi: 10.1038/s41598-022-18175-y.
Dhouailly, D. Dermo-epidermal interactions between birds and mammals: Differentiation of cutaneous appendages. J. Embryol. Exp. Morphol. 30, 587–603 (1973). doi:10.1242/dev.30.3.587.
Takaya, K. et al. Role of Wnt signaling in mouse fetal skin wound healing. Biomedicines 10, 1536 (2022). doi: 10.3390/biomedicines10071536.
Takaya, K., Aramaki-Hattori, N., Sakai, S., Okabe, K. & Kishi, K. Effect of Sonic Hedgehog on the regeneration of epidermal texture patterns. Biomedicines 10, 3099 (2022). doi: 10.3390/biomedicines10123099.
Takaya, K. et al. Fibroblast growth factor 7 suppresses fibrosis and promotes epithelialization during wound healing in mouse fetuses. Int. J. Mol. Sci. 23, 7087 (2022). doi: 10.3390/ijms23137087.
Jacob, T. et al. Zebrafish twist2/dermo1 regulates scale shape and scale organization during skin development and regeneration. Cells Dev. 166, 203684 (2021). doi:10.1016/j.cdev.2021.203684.
Thisse, B., el Messal, M. & Perrin-Schmitt, F. The twist gene: Isolation of a Drosophila zygotic gene necessary for the establishment of dorsoventral pattern. Nucleic Acids Res. 15, 3439–3453 (1987). doi:10.1093/nar/15.8.3439.
Wieschaus, E., Nüsslein-Volhard, C. & Jürgens, G. Mutations affecting the pattern of the larval cuticle in drosophila melanogaster. Wilehm Roux. Arch. Dev. Biol. 193, 296–307 (1984). doi:10.1007/BF00848158.
Scaal, M., Füchtbauer, E.M. & Brand-Saberi, B. cDermo-1 expression indicates a role in Avian skin development. Anat. Embryol (Berl). 203, 1–7 (2001). doi:10.1007/pl00008244.
Šošić, D., Richardson, J.A., Yu, K., Ornitz, D.M. & Olson, E.N. Twist regulates cytokine gene expression through a negative feedback loop that represses NF-kappaB activity. Cell 112, 169–180 (2003). doi:10.1016/s0092-8674(03)00002-3.
Glover, J.D. et al. Hierarchical patterning modes orchestrate hair follicle morphogenesis. PLoS Biol. 15, e2002117 (2017).
Shimizu, R. et al. Sphere formation restores and confers hair-inducing capacity in cultured mesenchymal cells. Exp. Dermatol. 20, 679–681 (2011). doi:10.1111/j.1600-0625.2011.01281.x.
Takeo, M., Lee, W. & Ito, M. Wound healing and skin regeneration. Cold Spring Harb. Perspect. Med. 5, a023267 (2015). doi:10.1101/cshperspect.a023267.
Budnick, I. et al. Defining the identity of mouse embryonic dermal fibroblasts. Genesis 54, 415–430 (2016). doi:10.1002/dvg.22952.
Chen, D., Jarrell, A., Guo, C., Lang, R. & Atit, R. Dermal β-catenin activity in response to epidermal Wnt ligands is required for fibroblast proliferation and hair follicle initiation. Development 139, 1522–1533 (2012). doi:10.1242/dev.076463.
Phan, Q.M. et al. Lef1 expression in fibroblasts maintains developmental potential in adult skin to regenerate wounds. eLife 9, e60066 (2020). doi:10.7554/eLife.60066.
Mok, K.W. et al. Dermal condensate niche fate specification occurs prior to formation and is placode progenitor dependent. Dev. Cell. 48, 32–48.e5 (2019). doi:10.1016/j.devcel.2018.11.034.
Kim, J.Y. et al. Twist2-driven chromatin remodeling governs the postnatal maturation of dermal fibroblasts. Cell Rep. 39, 110821 (2022). doi:10.1016/j.celrep.2022.110821.
Guerrero-Juarez, C.F. et al. Single-cell analysis reveals fibroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds. Nat. Commun. 10, 650 (2019). doi:10.1038/s41467-018-08247-x.
Driskell, R.R. et al. Distinct fibroblast lineages determine dermal architecture in skin development and repair. Nature 504, 277–281 (2013). doi:10.1038/nature12783.
Tukel, T. et al. Homozygous nonsense mutations in TWIST2 cause setleis syndrome. Am. J. Hum. Genet. 87, 289–296 (2010). doi:10.1016/j.ajhg.2010.07.009.

No competing interests reported.

Download PDF

Editorial decision: Revision requested
16 Apr, 2024
Reviews received at journal
16 Apr, 2024
Reviewers agreed at journal
08 Apr, 2024
Reviews received at journal
05 Apr, 2024
Reviewers agreed at journal
25 Mar, 2024
Reviewers invited by journal
10 Mar, 2024
Editor assigned by journal
10 Mar, 2024
Editor invited by journal
07 Mar, 2024
Submission checks completed at journal
07 Mar, 2024
First submitted to journal
21 Feb, 2024

You are reading this latest preprint version

Twist2 contributes to skin regeneration and hair follicle formation in mouse fetuses

Status:

Version 1

Abstract

Figures

1. Introduction

2. Results

2.1. Twist2 Is Upregulated in the Dermis of E13 Wounds

2.2. Knockdown of Twist2 Inhibits Mouse Fetal Skin Regeneration

2.3. Wounds in twist2 Heterozygous Knockout Mice Do Not Regenerate

2.4. Twist2 Is Upregulated by 3D Culture of Fibroblasts

2.5. Knockdown of Twist2 Reduces the Migratory Ability of Dermal Fibroblasts

3. Discussion

4. Methods

4.1. Ethical Consideration

4.2. Mouse

4.3. Fetal Wounding Procedure

4.4. Immunohistochemistry

4.5. In Situ Hybridization

4.6. LMD, RNA Isolation, and Reverse Transcription

4.7. RT-qPCR

4.8. siRNA Experiment for Mouse Fetuses

4.9. 2D and 3D Culture of Fetal Mouse Dermal Fibroblasts

4.10. Scratch Assay

4.11. Statistical Analysis

Declarations

Conflicts of Interest:

Author Contribution

Acknowledgments:

Data availability

References

Additional Declarations

Status:

Version 1