Immunofluorescent Identification of Melanocytes in Murine Hair Follicles

Govender, Dheshnie; Davids, Lester M.; Kidson, Susan H.

doi:10.1007/s10735-005-9011-8

Cited by 6 publications

(5 citation statements)

References 16 publications

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“…For immunofluorescence, sections were permeabilized in 0.3%Triton X-100 in PBS for 20 min and blocked for 1 h at room temperature in blocking buffer including 2.5%normal donkey serum, 2.5%normal goat serum(or 5% normal donkey serum alone when goat primary antibodies were used), 0.5% BSA and 0.1% Triton X-100 in PBS. For melanocyte markers, including DCT, TYRP1 and TYR, sections were permeabilized with 0.3% H 2 O 2 in cold methanol for 30 min at-20 °C 36 .MOM Basic kit (Vector Laboratories) was used for blocking when primary antibodies were generated from mouse. Primary antibodies were diluted in blocking buffer and sections were incubated overnight at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

NFIB is a governor of epithelial–melanocyte stem cell behaviour in a shared niche

Chang

Pasolli

Γιαννοπούλου

et al. 2013

Nature

155

151

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Adult stem cells reside in specialized niches where they receive environmental cues to maintain tissue homeostasis. In mammals, the stem cell niche within hair follicles is home to epithelial hair follicle stem cells and melanocyte stem cells, which sustain cyclical bouts of hair regeneration and pigmentation1–4. To generate pigmented hairs, synchrony is achieved such that upon initiation of a new hair cycle, stem cells of each type activate lineage commitment2,5. Dissecting the inter-stem-cell crosstalk governing this intricate coordination has been difficult, because mutations affecting one lineage often affect the other. Here we identify transcription factor NFIB as an unanticipated coordinator of stem cell behaviour. Hair follicle stem-cell-specific conditional targeting of Nfib in mice uncouples stem cell synchrony. Remarkably, this happens not by perturbing hair cycle and follicle architecture, but rather by promoting melanocyte stem cell proliferation and differentiation. The early production of melanin is restricted to melanocyte stem cells at the niche base. Melanocyte stem cells more distant from the dermal papilla are unscathed, thereby preventing hair greying typical of melanocyte stem cell differentiation mutants. Furthermore, we pinpoint KIT-ligand as a dermal papilla signal promoting melanocyte stem cell differentiation. Additionally, through chromatin-immunoprecipitation with high-throughput-sequencing and transcriptional profiling, we identify endothelin 2 (Edn2) as an NFIB target aberrantly activated in NFIB-deficient hair follicle stem cells. Ectopically induced Edn2 recapitulates NFIB-deficient phenotypes in wild-type mice. Conversely, endothelin receptor antagonists and/or KIT blocking antibodies prevent precocious melanocyte stem cell differentiation in the NFIB-deficient niche. Our findings reveal how melanocyte and hair follicle stem cell behaviours maintain reliance upon cooperative factors within the niche, and how this can be uncoupled in injury, stress and disease states.

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Section: Methodsmentioning

confidence: 99%

NFIB is a governor of epithelial–melanocyte stem cell behaviour in a shared niche

Chang

Pasolli

Γιαννοπούλου

et al. 2013

Nature

155

151

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“…Specimens were then left to cool for 5 min in the hot sodium citrate and transferred to room-temperature PBS for an additional 20 min. Deionized water washes were performed again followed by immersion in 3% hydrogen peroxide in methanol for 15 min at room temperature to reduce background (18). Tissue sections on slides were then boxed with a PAP pen followed by a 1% BSA/PBS block for 30 min in a humidified chamber at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Two- and Three-Dimensional Models for Risk Assessment of Radiation-Enhanced Colorectal Tumorigenesis

2009

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Astronauts may be at an increased risk for developing colorectal cancer after a prolonged interplanetary mission given the potential for greater carcinogenic effects of radiation to the colon. In addition, with an increase in age, there is a greater incidence of premalignant colon adenomas with age. In the present study, we have compared the effects of radiation on human colon epithelial cells in two-dimensional (2D) monolayer culture, in three-dimensional (3D) culture, and in intact human colon tissue biopsies. Immortalized colon epithelial cells were irradiated at the NASA Space Radiation Laboratory (NSRL) with either 1 Gy 1 GeV/nucleon 56 Fe particles or 1 Gy 1 GeV/nucleon protons and were stained at various times to assess DNA damage and repair responses. The results show more persisting damage at 24 h with iron-particle radiation compared to protons. Similar results were seen in 3D colon epithelial cell cultures in which 56 Fe-particle-irradiated specimens show more persisting damage at 24 h than those irradiated with low-LET γ rays. We compared these results to those obtained from human colon tissue biopsies irradiated with 1 Gy γ rays or 1 Gy 1 GeV 56 Fe particles. Observations of radiation-induced DNA damage and repair in γ-irradiated specimens revealed more pronounced early DNA damage responses in the epithelial cell compartment compared to the stromal cell compartment. After low-LET irradiation, the damage foci mostly disappeared at 24 h. Antibodies to more than one type of DNA repair factor display this pattern of DNA damage, and staining of nonirradiated cells with non-phosphorylated DNA-PKcs shows a predominance of epithelial staining over stromal cells. Biopsy specimens irradiated with high-LET radiations also show a pattern of predominance of the DNA damage response in the highly proliferative epithelial cell compartment. Persistent unrepaired DNA damage in colon epithelial cells and the differing repair responses between the epithelial and mesenchymal compartments in tissues may enhance tumorigenesis by both stem cell transformation and alterations in the radiation-induced permissive tissue microenvironment that may potentiate cancer progression.

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“…The hair of C57BL/6J mice grows cyclically, and the hair cycle is divided into the growth, regression, and resting phases [34]. Melanocytes in the skin are distributed to the hair follicles [35]. Synthesis, secretion, and darkening of melanin occur during hair growth [36].…”

Section: Discussionmentioning

confidence: 99%

Effects of Baicalin on Alopecia and the Associated Mechanism

Chen

Fan

et al. 2022

BioMed Research International

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The aim of the present study was to explore the potential pharmacological mechanism of baicalin by combining network pharmacology prediction and the experimental verification of alopecia. Networks of baicalin-associated targets and alopecia-related genes were constructed using the STRING database. Potential targets and pathways associated with the therapeutic efficacy of baicalin were identified via enrichment analysis using Cytoscape and the database for annotation, visualization and integrated discovery (Metascape). The back hair of C57BL/6J mice was removed with depilatory cream to verify the therapeutic effect of baicalin. Human hair dermal papilla cells (HHDPCs) were used to explore the mechanism of action of baicalin. Network pharmacology analysis revealed that the potential targets of baicalin mainly include protein serine/threonine kinase, Src protein, epidermal growth factor receptor, and insulin-like growth factor 1 (IGF1), which were indicated to mediate neutrophil degranulation and regulation of cell-cell adhesion, vesicle lumen, cytoplasmic vesicle, membrane raft, and endopeptidase activity. Multiple pathways were identified, such as proteoglycans in cancer, PI3K/AKT, and forkhead box O signaling pathways. Following baicalin treatment for the experimental mice, the coverage, length, and weight of the hair increased in a baicalin dose-dependent manner. Moreover, the histological evaluation showed that the number of hair follicles increased after baicalin treatment and melanin formation were pronounced. In addition, baicalin induced an increase in the phosphorylated p-AKT, p-glycogen synthase kinase-3β, p-PI3K, TGF-β1, and vascular endothelial growth factor levels. Furthermore, the activation levels of key protein p-AKT were increased. Baicalin induced the proliferation of HHDPCs in vitro and significantly upregulated p-AKT, IGF1, and alkaline phosphatase. In conclusion, the present study revealed that the pharmacological mechanisms of baicalin in alopecia therapy were associated with the proliferation of DPCs, the activation of the AKT pathway, and the transmission of downstream signals, indicating that baicalin is a potential drug candidate for the clinical treatment of hair loss.

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Immunofluorescent Identification of Melanocytes in Murine Hair Follicles

Cited by 6 publications

References 16 publications

NFIB is a governor of epithelial–melanocyte stem cell behaviour in a shared niche

NFIB is a governor of epithelial–melanocyte stem cell behaviour in a shared niche

Two- and Three-Dimensional Models for Risk Assessment of Radiation-Enhanced Colorectal Tumorigenesis

Effects of Baicalin on Alopecia and the Associated Mechanism

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