The Functional And Molecular Mechanistic Research Of Musashi2 In Skin And Hair Follicle Development

Hair follicle (HF) is the biggest appendage in skin, allowing the higher animals to maintain thermostasis and adapt the atrociously terrestrial environment. Hair follicle stem cells (HFSCs) are the driving source of hair follicle, which are essential for HF function. HFSCs reside in the upper HF named "bulge" and undergoes highly dynamic with periodic activation that drives recurrent hair cycles of growth (anagen), cessation (catagen) and rest (telogen). During the transition of HF from telogen to anagen, HFSCs are activated, which is crucial for a physiological progress of hair cycling. The process is deemed to coordinate by cross-talk of variety of signal pathways. However, it is still not fully understood of the molecular mechanisms in regulating HFSCs. RNA binding proteins have been shown to play important roles in RNA splicing, transfering and translation. Musashi2 (Msi2), an evolutionarily conserved RNA binding protein, has been identified as a critical regulator of adult stem cells and carcinogenesis in varieties of organs, such as neural system, blood, intestine, mammary gland. It has been shown that Msi2 is expressed in HF bulge, secondary hair germ. However, it is remain unknown of Msi2 in regulating hair follicle stem cells and hair cycling. Thus, this study focuses on investigate the function and mechainsms of Msi2 in activation of HFSCs and progression of hair cycle.Using qRT-PCR and IHC, the study demonstrates that Msi2 is extensively expressed in HFSCs and partial differentiated keratinocytes of HF during different hair cycling stages. To further investigate the role of Msi2 in HF, an inducible transgenic mouse model(TRE-MSI2::K14rtTA, DTG), allowing that Mis2 is specifically overexpressed in hair follicle and epidermis upon Dox treatment, and a conditional KO mouse mode (Msi2 flox/flox::K14Cre, cKO), resulting in deletion of Msi2 in hair follicle and epidermis, was generated respectively. With 3 days Dox administration, MSI2 was overexpressed with high level and retards the transition from telogen to anagen during hair cycling at P21 to P24. To initiate a new synchronous hair growth cycle, the depilation assay was performed at P21 with Dox pretreated for 3 days from P18. After 3 days post-depilation, the DTG HFs are delayed to enter anagen. By contrast, deletion of Msi2 significantly promotes the anagen process of the regenerate HF after depilation. BrdU chase experiment suggested that Msi2 induction impaired HFSC activation, which could account for the delayed anagen process. Further, FACS and stem cell marker immunostaining demonstrate that Msi2 maintains quiescent state of HFSCs rather than the number of HFSCs population. In addition, Msi2 also represses hair neogenesis in wound healing.To gain mechanistic insights into the molecular events underlying Msi2 function in hair follicle, transcriptome profiles on ontrol and DTG backskin after 3-day Dox treatment (P21-P24) was performed. The hierarchical clustering of heatmap reveals that a number of genes are significantly altered. With a KEGG pathway annotation, the genes of Sonic hedgehog (Shh) signaling pathway were found with significantly enriched in the profile. The alteration of the Hh pathway was further validated in DTG and cKO mice using qRT-PCR, IHC and western blotting. Based on the reported Msi binding motif sequence, a binding site of Msi2 was identified in 3’-UTR of Shh mRNA. The luciferase assay confirmed Msi2 is able to directly bind to 3’-UTR of Shh mRNA in keratinocytes. Moreover, CLIP-qPCR and in-situ assay further indicated that Msi2 is co-located with Shh mRNA in Mx of anagen hair follicles and reduces the mRNA expression level. Further, RNA stability assay confirmed that Msi2 induction promotes decays of Shh mRNA in vitro. Consistently, the expression of downstream genes of Hh pathway is regulated by Msi2 as well. Furthermore, the delayed external hair regrowth and impaired HF development in DTG mice was able to be rescued by Hh pathway agonist-SAG administration, further supporting the notion that Msi2 directly represses the Shh/Hh pathway. In summary, newly uncovered Msi2-Shh/Hh pathway adds to the growing understanding of the hair growth network and broaden the regulatioal region of Msi, and provides a novel potential therapeutic strategies for skin diseases.