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【摘要】
Epithelial stem cells reside in the hair follicle (HF) bulge region and possess the ability to differentiate into a variety of cutaneous epithelial cells. The evolutionarily conserved Musashi family of RNA-binding proteins is associated with maintenance and/or asymmetric cell division of neural progenitor cells, and a mammalian Musashi protein is expressed in various epithelial stem/progenitor cells, including gut, stomach, and mammary gland. Thus, we hypothesized that Musashi might be expressed in stem cells and early progenitor cells of HF epithelium. Reverse transcriptase-polymerase chain reaction and immunoblotting identified Musashi-1 (Msi-1) and Musashi-2 (Msi-2) mRNA and protein in cultured mouse keratinocytes, but only Msi-1 was identified in human keratinocytes. In mice, immunohistochemical studies showed that Msi-1 and Msi-2 were expressed in the epidermis and HFs from E14.5 until adulthood. In the early anagen phase, Msi-1 and Msi-2 were expressed in the bulge and secondary germ cells and subsequently in inner root sheath (IRS) cells, especially the middle IRS cells, during the late anagen phase. In human skin, Msi-1 was detected in fetal HF cells but not in adult HFs. These observations suggest that Musashi functions not only in the asymmetric division of early progenitor cells but also in the differentiation of IRS cells during HF development and hair cycle progression.
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During skin development, a population of multipotent stem cells gives rise to both the epidermis and its appendages, including hair follicles (HFs). HF morphogenesis is triggered by a series of epithelial-mesenchymal cues, and recent findings obtained from mutant mice have revealed various signaling molecules involved in HF development and hair cycle progression.1 Other studies have reported that HF stem cells lie in the bulge region of HFs.2-5 Cells in this region have a high colony-forming capacity,6,7 are slow cycling, and have a quiescent nature.8 Transplantation studies suggest that these bulge cells possess the ability to differentiate into multiple different types of cutaneous epithelial cells, including the sebaceous gland and even epidermal cells.8-12 Some putative HF stem cell markers have been reported, but none of them have been proven to be definitive markers.
The Musashi family of proteins is an evolutionarily conserved group of RNA-binding proteins, initially identified in Drosophila where they are required for early asymmetric cell divisions in the sensory organ precursor cells.13-15 In mammals, Msi-1 and Msi-2 have been identified in mice,14,16 but only Msi-1 is expressed in humans.17 It has subsequently been demonstrated that Msi-1 and Msi-2 are selectively expressed in neural progenitor cells, including stem cells, and have key roles in the maintenance of the stem cell state and differentiation.14-16,18-20 Moreover, Msi-1 has been shown to be a positive regulator of Notch-signaling through its interaction with m-Numb mRNA.21 Outside the nervous system, Msi-1 is a selective marker for various epithelial stem or early progenitor cells present in intestine,22-24 gastric mucosa,25 and mammary gland,26 among others. Because similar asymmetric divisions are thought to maintain the HF stem cell compartment, we hypothesized that Musashi might be expressed in either HF stem cells or early progenitor cells.
In this report, we have examined the expression pattern of Musashi family proteins during HF development and adult hair cycles in both mice and humans. We found that Msi-1 and Msi-2 were expressed in mouse stem cells in the bulge region. In addition, Msi-1/2 was also expressed in the secondary hair germ, the HF matrix, and the inner root sheath (IRS) cells, at all developmental stages until adulthood. In humans, Msi-1 expression sites were similar to those in mice, although Msi-1 was expressed only in developing skin. These observations suggest that Musashi functions not only in asymmetric stem cell or early progenitor cell division but also in the differentiation of IRS cells during HF development and hair cycle progression.
【关键词】 expression rna-binding follicle development progression
Materials and Methods
Cell Culture
Neonatal human keratinocytes (NHKs) were purchased from Cambrex Bio Science Walkersville, MD. Mouse keratinocytes were obtained from C57BL/6J mouse skin after 3 to 5 hours of dispase enzyme digestion, followed by trypsinization of the separated epidermis. Both human and mouse keratinocyte cells were cultured in defined keratinocyte serum-free medium (Invitrogen, San Diego, CA). Both human and mouse keratinocytes were cultured in low Ca2+ conditions (0.09 mmol/L) to maintain a basal cell-like population of undifferentiated cells. To induce terminal differentiation, CaCl2 was added directly to the culture media at a final concentration of 2 mmol/L. Photographs were taken using a Nikon Coolpix (Nikon, Tokyo, Japan).
Semiquantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted using RNeasy (Qiagen, Chatsworth, CA). cDNA was synthesized by reverse transcription of 1 µg of total RNA, using a cDNA synthesis kit (Invitrogen). The following sets of oligonucleotide primers were used: for mouse Msi-1, 5'-CGAGCTCGACTCCAAAACAAT-3' (sense) and 5'-GGCTTTCTTGCATTCCACCA-3' (anti-sense); mouse Msi-2, 5'-GTCTGCGAACACAGTAGTGGAA-3' (sense) and 5'-GTAGCCTCTGCCATAGGTTGC-3' (anti-sense); human Msi-1, 5'-GGCTTCGTCACTTTCATGGACCAGGCG-3' (sense) and 5'-GGGAACTGGTAGGTGTAA-3' (anti-sense); human keratin 1, 5'-CACTTATTCCGGAGTAACCAG-3' (sense) and 5'-GAATAGGATGAGCTAGTGTAA-3' (anti-sense); mouse glyceraldehyde-3 phosphate-dehydrogenase (mGAPDH), 5'-TTAGCCCCCCTGGCCAAGG-3' (sense) and 5'-CTTACTCCTTGGAGGCCATG-3' (anti-sense); human GAPDH (hGAPDH), 5'-TCATCTCTGCCCCCTCTGCT-3' (sense) and 5'-CGACGCCTGCTTCACCACCT-3' (anti-sense). The PCR amplification for Musashi was performed as follows: 95??C for 10 minutes, 40 cycles of 94??C for 30 seconds, 55??C for 30 seconds, and 72??C for 1 minute, with a final extension step of 72??C for 5 minutes. Keratin 1 and GAPDH were amplified for 25 cycles. PCR products were analyzed by agarose gel electrophoresis.
Immunoblotting
Cell lysates were prepared by homogenization in lysis buffer (50 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate), followed by centrifugation at 15,000 rpm for 5 minutes. Cell lysates (100 µg of protein per lane) were resolved on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels that were then electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA) using a wet transfer apparatus. Membranes were probed with either rat monoclonal anti-Msi-1 antibody (final dilution, 1:500)18 or rabbit polyclonal anti-Msi-2 (final dilution, 1:500)16 antiserum. Proteins were detected with horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch Laboratory, West Grove, PA), and specific bands were visualized by chemiluminescence.
Mice and Tissue Preparation
C57BL/6J mice were purchased from Clea Japan Inc. (Tokyo, Japan). The mice were kept in isolator cages in a barrier facility under a 12-hour light cycle and maintained under specific pathogen-free conditions. Pregnant mice were purchased to get embryos at 14 to 18 days of gestation.
For hair morphogenetic studies, the dorsal skin samples were obtained from untreated newborn mice (male and female) on days 1, 4, 8, 11, 18, and 21 after birth. The samples were processed for immunohistological staining. For hair cycle experiments, 7-week-old female C57BL/6J mice were purchased. After conditioning for 1 week, the anagen phase of the hair growth cycle was induced by gently depilating their dorsal hair shafts using depilatory chemicals. They were sacrificed at 0, 1, 4, and 11 days after depilation for further studies of Msi-1 expression at specific stages of the hair cycle, ie, the anagen phase (1, 4, and 11 days after depilation) and the telogen phase (0 days after depilation).
Human Fetal and Adult Skin Specimens
Human embryonic and fetal skin specimens were obtained from abortuses of 49 to 163 days estimated gestational age (EGA) through the Central Laboratory of Human Embryology at the University of Washington, Seattle, WA, with the approval of the Human Subjects Review Board and in accordance with the United States Department of Health, Education, and Welfare policies. The ages, the autopsy sites, and the numbers of embryos or fetuses included in this study were as follows: 49 to 64 days EGA, scalp (n = 2), trunk (n = 2); 65 to 84 days EGA, scalp (n = 3), trunk (n = 2); 85 to 104 days EGA, scalp (n = 2), trunk (n = 2); 105 to 135 days EGA, scalp (n = 2), trunk (n = 2); and >135 days EGA, scalp (n = 2), trunk (n = 2). Two or three skin specimens from each fetus were used for this study. EGA was determined from maternal histories, fetal measurements (crown-rump and foot length), and comparative histological appearance of the epidermis. Normal adult human skin samples were obtained at skin surgical operations of benign subcutaneous tumors under fully informed consent at Department of Dermatology, Hokkaido University School of Medicine.
Detection of Slow-Cycling Cells
To examine the proliferating ability of the skin cells, we labeled neonatal C57BL/6J mice skin with subcutaneous injections of 5-bromo-2-deoxyuridine (BrdU; 50 µg/g body weight) from day 3 of life, twice a day for 3 days. The mice were sacrificed, and skin samples were obtained for further immunolabeling immediately after the BrdU labeling until 2 weeks after the start of the initial labeling to demonstrate the entire labeled cell population and label-retaining cells (slow-cycling cells), respectively. We performed double immunostaining for BrdU and Msi-1 or Msi-2 using the following methods to demonstrate the proliferating cells or label retaining cells and Msi-positive cells in the same tissue sections.
Immunofluorescent Labeling
The dorsal skin samples removed from the mice were immediately frozen in OCT compound (Tissue-Tek; Sakura Finetechnical, Tokyo, Japan) and cut at a thickness of 6 µm. Sections were fixed with 4% paraformaldehyde for 15 minutes at 4??C. The sections were then placed in 0.01 mol/L citrate buffer, pH 6.0, and microwave-treated (500 W) for 10 minutes to facilitate antigen retrieval. Sections were incubated with 10% goat serum in PBS for 1 hour and then incubated with primary antibodies at 4??C overnight: anti-mouse Msi-1 antibody18 (14H-1; final dilution, 1:1000), anti-mouse Msi-2 antibody16 (final dilution, 1:100), anti-BrdU antibody (Roche, Mannheim, Germany; final dilution, 1:100), anti-CD34 antibody (BD Pharmingen, San Diego, CA; final dilution, 1:50), or anti-Ki67 antibody (Novocastra, Newcastle on Tyne, UK; final dilution, 1:250). The sections were incubated with secondary antibodies at room temperature for 1 hour: fluorescein isothiocyanate (FITC)-conjugated anti-rat IgG (Jackson Immunoresearch Laboratory; final dilution, 1:50), FITC-conjugated anti-rabbit-IgG (Jackson Immunoresearch Laboratory; final dilution, 1:50), FITC-conjugated anti-mouse-IgG (Jackson Immunoresearch Laboratory; final dilution, 1:50), or tetramethyl-rhodamine isothiocyanate anti-mouse IgG (Southern Biotechnology Associates, Inc., Birmingham, AL; final dilution, 1:50). The sections were then incubated with 10 µg/ml of propidium iodide at 37??C for 10 minutes for nuclear counterstaining. Sections were observed under an Olympus Fluoview confocal laser-scanning microscope (Olympus, Tokyo, Japan).
Results
Expression of Msi-1 and Msi-2 mRNAs in Mouse and Human Keratinocytes
The RT-PCR-amplified products obtained using Msi-1-specific and Msi-2-specific primers showed clear bands of the predicted sizes. Msi-1 and Msi-2 gene expression was identified in mouse keratinocytes derived from C57BL/6J mice cultured in low Ca2+ medium (0.09 mmol/L), and Msi-1 gene expression was observed in normal human keratinocytes (NHKs) cultured in low Ca2+ medium (0.09 mmol/L) (Figure 1A) . Msi-2 was not identified in human tissues. Thus, using human tissues, we could study only Msi-1 expression. Keratinocytes cultured in low Ca2+ medium (0.09 mmol/L) remain undifferentiated; raising the Ca2+ concentration greater than 2 mmol/L induced expression of differentiation-associated molecules and several morphological changes (Figure 1B) . We found that human Msi-1 message levels increased 2 hours after raising the extracellular calcium concentration to 2 mmol/L, while the GAPDH message expression levels were unaffected (Figure 1B) . In immunoblots of mouse and human cultured keratinocytes, the anti-Msi-1 and anti-Msi-2 antibodies recognized the expected protein bands (Figure 1C) . Msi-1 was detected as a major band of 37 to 40 kd in the human and mouse keratinocyte extracts, and Msi-2 was detected as a major band of 35 to 37 kd in the mouse keratinocyte extract.
Figure 1. Msi-1 and Msi-2 expression in mouse and human keratinocytes. The expression of Msi-1 and Msi-2 was detected in mouse and human keratinocytes. A: RT-PCR analysis of Msi-1 and Msi-2 mRNA expression in mouse and human keratinocytes cultured in low Ca2+ medium (0.09 mmol/L). Expression of mouse Msi-1, Msi-2 (Msi-1 and Msi-2, left), and human Msi-1 (Msi-1, right) was detected in cultured keratinocytes. B: Normal human keratinocytes (NHKs) cultured in low Ca2+ medium remain undifferentiated, and raising the Ca2+ concentration greater than 2 mmol/L induces the expression of the differentiation-associated molecule keratin 1 and morphological changes including stratification. Msi-1 expression increased 2 hours after raising Ca2+ culture levels for human keratinocytes. GAPDH expression is shown as an internal control. Top: Human Msi-1 expression (hMsi-1); middle: human keratin-1 expression (hKeratin 1); bottom: human GAPDH expression (hGAPDH). C: Western blot analysis of Msi-1 and Msi-2 proteins in mouse and human keratinocytes. Msi-1 was expressed in mouse and human keratinocytes (37- to 40-kd bands) and Msi-2 was detected in mouse keratinocytes (35- to 37-kd band).
Expression of Msi-1 Protein during Hair Morphogenesis in Mouse Skin
At E14.5, weak Msi-1 expression was observed throughout the entire, as yet, unkeratinized epidermis and the hair placode (Figure 2, A, D, and G) . In addition, Msi-1 was expressed in the dermal cells surrounding the hair placode and in the upper dermis. At E15.5, the inner cells of the hair germ showed strong expression of Msi-1 protein compared with the outermost cells of the hair germ and the dermal cells (Figure 2, B, E, and H) . At E18.5, Msi-1 was highly expressed throughout the entire bulbous hair peg and interfollicular epidermal basal layer (Figure 2, C, F, and I) .
Figure 2. Msi-1 was expressed in mice embryonic skin. C57BL/6 mouse back skin samples, at the following developmental stages (E14.5, E15.5, and E18.5), were processed for immunohistological analysis of Msi-1. Sections were subjected to H&E staining (ACC) or immunofluorescence staining with the anti-Msi-1 antibody (DCF). DCF are confocal images in which Msi-1 fluorescence (green, FITC) and nuclear stain (red, propidium iodide) were merged. Msi-1 was expressed in the undifferentiated epidermis and in the dermal cells at E14.5. Msi-1 was expressed in hair germ and bulbous hair peg at each developmental stage. GCI: Schematic representation of immunoreactivity patterns of Msi-1 during murine embryonic epidermal development. Green shows Msi-1 expression in both the nucleus and cytoplasm. A, D, and G, at E14.5; B, E, and H, at E15.5; C, F, and I, at E18.5. Scale bars, 50 µm.
Msi-1 Is Expressed in the Restricted Areas of the HF IRS during the First Hair Cycle (Neogenesis) in Neonatal Mice
During the anagen phase of HF neogenesis in 1-day-old mice, Msi-1 was expressed throughout the entire developing HF and within the basal cells of the interfollicular epidermis (Figure 3, A and G) . This expression pattern was similar to that in E18.5 skin (Figure 2, F and I) . In 4-day-old mice, during the anagen phase, Msi-1-positive cells were seen in the outermost layer of IRS cells in the middle of the HF, just below the IRS start of keratinization (Figure 3 , B (arrowhead) and H). We tentatively termed these cells "middle IRS cells" (MICs). The HF matrix cells were also Msi-1-positive (Figure 3 , B (arrow) and H). From the age of 8 days (11 and 18 days old), during the late anagen to early catagen phase, only MICs showed strong Msi-1 immunoreactivity (Figure 3, CCE and ICK) . These MICs expressed Msi-1 both in the cytoplasm and in the nucleus. The number of Msi-1-positive MICs in the early catagen HF was greater than those in the anagen HF (Figure 3, E and K) . Outer root sheath cells were completely devoid of Msi-1 expression throughout the anagen to catagen phase during the first hair cycle. Msi-1-positive MICs disappeared during the transition phase, telogen to early anagen, at 21 days (Figure 3, F and L) . At this stage, Msi-1 was expressed in the nucleus of the bulge, the secondary germ, and the epidermal cells.
Figure 3. Msi-1 expression in mouse neonatal skin. Dorsal skin samples from C57BL/6 newborn mice on days 1, 4, 8, 11, 18, and 21 after birth were examined. Msi-1 was expressed in matrix and middle IRS cells (MICs) at an early anagen phase, and at late anagen to early catagen phases Msi-1 was found only in MICs. At the transition from telogen to early anagen phase, Msi-1 was expressed in bulge cells. A and G: One-day-old (anagen); B and H: 4-day-old (anagen); C and I: 8-day-old (anagen); D and J: 11-day-old (anagen); E and K: 18-day-old (early catagen); F and L: 21-day-old (during the transition from telogen to anagen state) mice. GCL: Schematic representation of Msi-1 immunoreactivity patterns during murine neonatal skin. Green shows Msi-1 expression in nucleus and cytoplasm. Yellow shows Msi-1 expression only in nucleus. SG, sebaceous gland; Bu, bulge; DP, dermal papilla; MIC, middle IRS cells. Scale bar, 50 µm.
Msi-1 Is Expressed in the Bulge and Secondary Hair Germ Cells during Early Anagen and in the MIC and Matrix Cells during the Late Anagen Phase in Adult Mice
In the depilation-induced adult hair cycle, at day 0 immediately after depilation, in the telogen phase, Msi-1-positive cells were not seen in the HF, although Msi-1 was expressed in the interfollicular epidermal basal cell nuclei (Figure 4, A, D, and J) . During the early steps of telogen-anagen transition, at days 1 to 4 after depilation, Msi-1 was expressed in the bulge region and secondary germ cells (Figure 4, B, C, E, F, K, L) . At that time, the bulge cells expressed CD34, a putative HF stem cell marker10-12 (Figure 4G) , and Msi-1-positive cells were also positive for the proliferation marker Ki67 antigen (Figure 4H) . During the late anagen phase, 11 days after depilation, MICs were Msi-1-positive, similar to the Msi-1-positive MICs seen during the anagen phase of HF development (Figure 4, I and M) . In addition, the anagen HF expressed Msi-1 protein in the hair matrix cells (Figure 4, I and M) . In these cells, Msi-1 was expressed both in the cytoplasm and in the nuclei. Throughout the depilation induced adult hair cycle, we found Msi-1-positive nuclei in the interfollicular epidermal basal cells (Figure 4, DCF and ICM) .
Figure 4. Msi-1 expression in the adult hair cycle of mice. Dorsal skin samples from 8-week-old C57BL/6 mice were harvested on days 0, 1, 4, and 11 after anagen induction. Sections were H&E stained (ACC) or processed for immunofluorescence staining with the anti-Msi-1 antibody (DCF and I), anti-CD34 antibody (G) or anti-Ki67 antibody (H). D and J: Msi-1 was not detected in HF at telogen. E, F, K, and L: After anagen induction, Msi-1 was expressed in the bulge and the secondary germ cells at early anagen. I and M: At late anagen, Msi-1 was expressed in MICs and the matrix cells, although the bulge cells were completely devoid of Msi-1 expression. A, D, and J: Day 0 after depilation (telogen); B, E, and K: 1 day after depilation (anagen); C, FCH, and L: 4 days after depilation (anagen); I and M: 11 days after depilation (anagen). G: Expression of CD34 in mice HF 4 days after depilation. H: Expression of Ki67 in mice HFs 4 days after depilation. JCM: Schematic representation of immunoreactivity patterns of Msi-1 during murine adult hair cycle. Green shows Msi-1 expression in nucleus and cytoplasm. Yellow shows Msi-1 expression only in nucleus. SG, sebaceous gland; Bu, bulge; DP, dermal papilla; MIC, middle IRS cells. Scale bar, 50 µm.
Msi-2 Is Expressed throughout the Entire Hair Germ, the Progenitor Cells of the IRS (preIRS) in the Developing HF, and the Entire HF IRS from the Neonatal Until Adult Stages of the Hair Cycle in Mice
We next observed the distribution of Msi-2 protein, another member of the Musashi RNA-binding protein family. We failed to find Msi-2 expression in E14.5 mouse skin (Figure 5, A, D, and G) . However, at E15.5, Msi-2 was expressed throughout the entire hair germ and in the superficial layers of the epidermis (Figure 5, B, E, and H) . In E18.5 and 1-day-old neonatal mouse skin, Msi-2 was expressed in the IRS cone cells, the progenitor cells for the IRS (preIRS) (Figure 5, C, F, and I ; Figure 6, A and G ). After birth, at 4 days and later (8, 11, and 18 days), Msi-2 was expressed in the IRS layer cells including the Msi-1-positive MICs (Figure 6, BCE and HCK) . At 21-days, Msi-2 was expressed in the inner bulge region cells (Figure 6, F and L) . Throughout the neonatal hair cycle, Msi-2 was expressed in the superficial layers of the epidermis (Figure 6, ACL) . Similar to Msi-1, however, Msi-2 was not seen in the HF during the telogen phase (Figure 7, A, D, and H) . In the mouse adult hair cycle, Msi-2 was expressed in the bulge, to a lesser extent in hair germ cells during the early anagen phase (Figure 7, B, E, I, C, F, and J) , and in the IRS layer cells including MICs during the late anagen stage (Figure 7, G and K) .
Figure 5. Msi-2 was expressed in mouse embryonic skin. C57BL/6 mouse back skin samples at the following developmental stages (E14.5, E15.5, and E18.5) were processed for immunohistological analysis of Msi-2. Sections were stained for H&E (ACC) or indirect immunofluorescence staining with the anti-Msi-2 antibody (DCF), in which Msi-2 fluorescence (green, FITC) and nuclear stain (red, propidium iodide) were merged. Msi-2 was not detected in undifferentiated epidermis at E14.5. At E15.5 and after, Msi-2 was expressed in the hair germ and the bulbous hair peg at developmental stages. A, D, and G, at E14.5; B, E, and H, at E15.5; C, F, and I, at E18.5. GCI: Schematic representation of immunoreactivity patterns of Msi-2 during murine embryonic epidermal development. Green color shows Msi-2 expression in both the nucleus and cytoplasm. Scale bars, 50 µm.
Figure 6. Msi-2 expression in the interfollicular epidermis and HF of neonatal mice. Dorsal skin samples from C57BL/6 newborn mice were harvested on days 1, 4, 8, 11, 18, and 21 after birth. A and G: Msi-2 was expressed in IRS cone (preIRS cells) at 1 day. BCE and HCK: From anagen to early catagen phases, Msi-2 was expressed in IRS cells and MICs. F and L: The transition from telogen to early anagen phase, Msi-2 was expressed in the bulge cells. A and G, 1-day-old (anagen); B and H, 4-day-old (anagen); C and I, 8-day-old (anagen); D and J, 11-day-old (anagen); E and K, 18-day-old (early catagen); F and L, 21-day-old mice (transition from telogen to anagen state). GCL: Schematic representation of Msi-2 immunoreactivity patterns in murine neonatal skin. Green indicates Msi-2 expression in nucleus and cytoplasm. SG, sebaceous gland; Bu, bulge; DP, dermal papilla; MIC, middle IRS cells. Scale bar, 50 µm.
Figure 7. Msi-2 expression during the mouse adult hair cycle. Dorsal skin samples from 8-week-old C57BL/6 mice were harvested on days 0, 1, 4, and 11 after anagen induction. Sections were subjected to H&E staining (ACC) or immunofluorescence staining with the anti-Msi-2 antibody (DCG). D and H: Msi-2 was not detected at telogen phase. E, F, I, and J: After anagen induction, Msi-2 was expressed in the bulge and the secondary germ cells. G and K: At late anagen, Msi-2 was expressed in IRS cells and MICs, although bulge cells were completely devoid of Msi-1 expression. A, D, and H, day 0 after depilation (telogen); B, E, and I, 1 day after depilation (anagen); C, F, and J, 4 days after depilation (anagen); G and K, 11 days after depilation (anagen). HCK: Schematic representation of Msi-2 immunoreactivity patterns during the murine adult hair cycle. Green shows Msi-2 expression in nucleus and cytoplasm. SG, sebaceous gland; Bu, bulge; DP, dermal papilla; MIC, middle IRS cells. Scale bar, 50 µm.
Expression of Msi-1 Is Seen in the Developing Human HF but Not in the Adult Human HF
During the early human HF morphogenesis (65 to 84 days EGA), Msi-1 protein was expressed throughout the entire hair germ, in the cells forming dermal cell condensation, and in the basal layer of the interfollicular epidermis (Figure 8, A and F) . During the early hair peg to bulbous hair peg transition (85 to 134 days EGA), Msi-1 was expressed throughout the entire hair peg and bulbous hair peg cells and in dermal cells surrounding the hair peg and bulbous hair peg (Figure 8, B, C, G, and H) . The outermost cells of the hair peg and bulbous hair peg only weakly expressed Msi-1 compared with the inner cells. In lanugo HFs (>135 days EGA), Msi-1 expression was seen in the IRS and matrix (Figure 8, E and I) . We failed to find any signal in the bulge region (Figure 8, D and I) . We could also not detect Msi-1 in any part of the adult HFs; however, we detected Msi-1 mRNA in NHK derived from human adult skin (data not shown).
Figure 8. Msi-1 expression during human skin development. Human embryonic and fetal skin specimens were obtained from abortuses of 49 to 163 days EGA and subjected to indirect immunofluorescence analysis. Msi-1 was expressed in the hair germ (A, F), peg (B, G), and bulbous peg (C, H). In the lanugo HF (D, E, I), Msi-1 was expressed only in matrix cells, and the bulge cells were devoid of Msi-1 expression. F, G, H: Throughout human skin development, Msi-1 was expressed in the dermal cells surrounding developing HFs. A and F, hair germ cells (65 to 84 days EGA); B and G, hair peg cells (85 to 104 days EGA); C and H, early bulbous peg (108 days EGA); D and I, bulge cells of the lanugo HF (>135 days EGA); E and I, matrix cells of the lanugo HF (>135 days EGA). FCI: Schematic representation of the Msi-1 immunoreactivity pattern during human skin development. Green highlights Msi-1 expression in the nucleus and cytoplasm. SG, sebaceous gland; Bu, bulge; DP, dermal papilla. Scale bar, 50 µm.
Musashi-Positive MICs Are Distinct from Slow-Cycling Cells in the HF of Neonatal Mice
Double-immunofluorescence labeling for BrdU and Msi-1 or Msi-2 indicated that most HF epithelial cells and interfollicular epidermal keratinocytes, including MICs, were positive for BrdU just after the labeling (Figure 9, A and C) . After the 2-week chase experiment, the Msi-1- or Msi-2-positive MICs failed to show significant BrdU labeling (Figure 9, B and D ; arrowheads), indicating that the Musashi-positive MICs were not slow-cycling.
Figure 9. Musashi-positive HF epithelial cells in neonatal mice are not slow-cycling cells. To detect slow-cycling cells (putative stem cells), BrdU (50 µg/g body weight) was injected into neonatal C57BL/6J mice from day 3 after birth, twice a day for 3 days. Musashi-positive MICs were also BrdU-positive (A, C; arrowheads) immediately after BrdU injection, although after a 2-week chase MICs (B, D; arrowheads) were not BrdU-positive. Cells retaining the label after 2 weeks (B, D; arrows) were identified as slow-cycling cells. Neither Msi-1-positive cells (B, arrowheads) nor Msi-2-positive cells (D, arrowheads) were slow-cycling cells. A and C, 0 day after labeling; B and D, 2 weeks after labeling. Green (FITC), Msi-1 (A, B), or Msi-2 (C, D); red (tetramethyl-rhodamine isothiocyanate), BrdU; blue (TOPRO), nuclear staining. Asterisk indicates nonspecific staining. Scale bar, 50 µm.
Discussion
The Musashi family of genes encodes RNA-binding proteins that are associated with asymmetric division and/or maintenance of neural progenitor cells.14-16,18 Msi-1 and Msi-2 are selectively expressed in neural progenitor cells, including putative stem cells, where they have key roles in the maintenance of the stem cell state and differentiation. Furthermore, the mammalian Musashi protein is also expressed in various epithelial stem/progenitor cells, including gut, stomach, and mammary gland.22-26 Therefore, we examined whether Musashi family proteins might be expressed in the HF bulge region, which is the niche thought to harbor the HF epithelial stem cell population.9
In the present study, we have demonstrated for the first time that the Musashi family proteins are expressed in the epidermis and the HF of both mouse and human skin. In mice, Msi-1 and Msi-2 mRNA and proteins were expressed in cultured keratinocytes. Furthermore, immunohistochemical studies showed that Msi-1 and Msi-2 were expressed in the epidermis and HF from the earliest stages of fetal development until adulthood. In the transition from telogen to anagen phase, Msi-1 and Msi-2 were both expressed in the bulge region, a putative site rich in stem cells, and in the secondary germ cells, comprising early progenitor cells derived from the putative bulge stem cells. Moreover, during anagen phase, Msi-1 was also expressed in the matrix cells. The cells lie in the bulge region and are normally quiescent, but they are activated during early anagen and rapidly give rise to cycling transit-amplifying (TA) cells.8,27 Thus, cells forming the secondary hair germ and the matrix are thought to be early progenitors of HF stem cells. Considering these facts, these Msi-positive cells may represent progenitor cells, or HF stem cells, supporting previously reported models.14-16,18,22,23 In humans, Msi-1 mRNA and protein were detected in cultured keratinocytes, and Msi-1 protein was expressed in developing HFs, although we failed to detect this immunoreactivity in human adult skin.
In addition, Msi-1 was expressed in the outermost cell layers of the IRS in the mid-follicle, just below the IRS keratinization point (MIC), and Msi-2 was expressed throughout the entire IRS including MICs. MICs were confirmed not to contain BrdU label after 2 weeks of chase and are therefore not label-retaining cells. We thought that these MICs might lie in the Huxley layer of the IRS based on their location within the HF. Moreover, during HF development, Msi-2 was expressed in the cells that lie in the IRS cone that are the progenitor cells for the IRS. This localization pattern suggested that Musashi family proteins play some roles in IRS formation. RT-PCR analysis of the mRNA levels in mouse and human keratinocytes suggested that Musashi may work in both undifferentiated and differentiated keratinocytes. Considering these findings, Musashi family proteins are thought to have at least two roles in HF, ie, one associated with asymmetric division of the stem cell or early progenitor cell population and the other functioning during the differentiation of IRS cells.
In this study, we showed several differences between mouse and human Musashi expression in the skin. The discrepancy in the expression pattern of Musashi family proteins has also been observed in other tissues.23 Although the exact reasons remain unclear, we believe this Msi-1 and Msi-2 expression discrepancy between mouse and human keratinocytes is mainly due to the fact that only Msi-1 has been identified in humans although both Msi-1 and Msi-2 have been identified in mice. Thus, Msi-1 may serve concurrently as Msi-2 in certain functions in humans.
An additional reason for the discrepancy in Msi-1 and Msi-2 expression is that the manner in which HF development and hair cycle progression takes place are quite different between the mouse and human. In mice, Msi-1 and Msi-2 were expressed in the bulge and secondary hair germ cells that are thought to be putative stem and early progenitor cells. However, we failed to detect Musashi expression in the corresponding regions in the human HF. This discrepancy may be related to differences in the hair cycles: in mice the hair cycle is synchronized whereas in humans it is not synchronized and is more random.
Both Msi-1 and Msi-2 are RNA-binding proteins characterized by two RNP-type RNA recognition motifs (RRMs) that show a remarkable similarity to each other, both in their primary structures and their RNA-binding specificities.14-16 Msi-1 and Msi-2 are known to be predominantly co-expressed in proliferating embryonic pluripotent neural precursors,14-16 and disruption of either the Msi-1 or Msi-2 genes alone shows no effect on the number or self-renewal activity of the central nervous system stem cells.20 Considering these facts, Msi-1 and Msi-2 may be able to compensate for the loss of the other in mice.
Several molecules are suggested to be regulated at the translational level by Musashi family proteins in the epidermis and HF. Msi-1 is a positive regulator of Notch-signaling through its interaction with m-Numb mRNA.21 Notch-1, a large transmembrane receptor, is expressed in the developing or differentiating epidermis and HF.28,29 Notch-1 is also known to initiate epidermal terminal differentiation and regulate HF differentiation, especially in IRS cell lineages.30,31 In our study, we showed that Msi-1 was expressed in the epidermis, the entire hair germ, and inner cells of the hair peg of mice and humans during HF development. This distribution of Msi-1 during HF development is similar to that of Notch-1.28,29 Notch-1 is expressed in the inner cells of the embryonic placode and the follicle bulb cells where it is thought to determine cell fate.28,29 Moreover, Msi-1 was expressed in matrix cells during anagen phase in which Notch-1 was also expressed in the anagen HF. Considering these expression patterns, regulators of the Notch signaling pathway, including m-Numb, are potential targets for Musashi proteins in the HF, although Msi-1 and Msi-2 may work in different ways from those proposed for the other tissues.
Previous studies that identified the RNA sequences targeted by Msi-1 showed that Msi-1 could bind poly(G) and poly(U) RNA in vitro14 and could also bind to UG-rich sequences using SELEX.21 Msi-1 may also bind gene sequences other than m-Numb to promote HF morphogenesis and control the hair cycle. Indeed, several molecules that contribute to development and progression of HF, including ß-catenin, fibroblast growth factor-5, transforming growth factor-ß2, BMP-2, Sonic Hedgehog, and others,1 have this UG-rich consensus sequence. Further studies are needed to identify Musashi mRNA targets in HFs.
In this study, Msi-1 protein expression was mostly cytoplasmic. However, in some instances, nuclear staining was seen in interfollicular epidermal and HF cells. The reasons for these altered expression patterns, with both cytoplasmic and nuclear distributions, remain unclear, although similar variations in Msi-1 subcellular localization have been reported in other tissues.23 Msi-1 localizes to the nucleus in epithelial cells, and Msi-1 possesses a potential phosphorylation site for cdc-2 kinase, a cyclin-dependent kinase that can bind cyclin B and regulate mitosis. Thus, Msi-1 may be posttranslationally modified in the nucleus during specific phases of the cell cycle.
In summary, Msi-1 and Msi-2 proteins were expressed in the HF and epidermis from the earliest developmental stage examined until adulthood in mice whereas only Msi-1 was expressed in the developing human HF. Msi-1 and Msi-2 expression was restricted to the bulge, secondary hair germ, and IRS cells, including MIC. These results suggest that Msi-1 and Msi-2 play important roles in HF development and progression of the hair cycle by regulating asymmetric cell division and the differentiation of IRS cells.
Acknowledgements
We thank Mrs. Megumi Sato and Ms. Kaori Sakai for their technical assistance and Dr. James R. McMillan for his critical reading of this manuscript.
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作者单位：From the Department of Dermatology,* Hokkaido University Graduate School of Medicine, Sapporo; and the Department of Physiology, Keio University School of Medicine, Tokyo, Japan