MicroRNA-205 promotes hair regeneration by modulating mechanical properties of hair follicle stem cells

Significance In this study, we have determined spatiotemporal compartmentalization of mechanical properties and their associated cell activities within the HF-SC niche during quiescence and activation. The spatial compartmentalization of stiff and quiescent hair follicle stem cells and soft and primed HGs is distinct from the architecture of the epidermis, where the basal, proliferative SC layer is soft and the suprabasal, differentiated layer is stiff. We have identified miR-205, a versatile and potent regulator of the actin cytoskeleton, whose expression modulates actomyosin contractility and the dynamics of cell size changes. Together, this study has implicated the dynamic changes of cell size and subsequent cell cycle reentry as a mechanism to sense and respond to mechanical forces during tissue regeneration.

3 approaching force-indentation curve was fitted with the Hertz (Spherical) model (eq. 1). Constant parameters were chosen to minimize the bias for different samples.
where is the force of the cantilever, is the indentation distance of the cell pressed by the cantilever, is the Young's modulus of the cell layer, is the radius of the spherical indenter, and is the Poisson ratio. The Poisson ratio of cell is normally in the range of 0.3-0.5. We chose = 0.5 in all calculations.

Quantification and 3D F-actin vs YAP analysis
To quantify the immune-staining signals of YAP and cortical F-actin in hair germs, the images were converted to Imaris for further quantification. Briefly, Imaris Surface module was applied to automatically select YAP positive areas in the nucleus in hair germ cells. The mean/median intensity of YAP signals of the selected region was obtained from the statistical tab. Cortical Factin signals were quantified using Line quantification, crossing F-actin bundles, in Fiji software.
The mean peak intensities of 5 line quantifications per cell was calculated.
After quantification of F-actin and YAP signals in wildtype control and miR-205 ind samples, we did normalization of F-actin values and YAP values by dividing all the values with the maximum value of the condition. For wildtype data, we also assigned a phase of the hair cycle (Telogen and Anagen) to each cell depending on their morphology and hair cycle stages. We then defined Telogen Center, t, and Anagen Center, a, by calculating the mean of normalized F-actin value and YAP value for Telogen and Anagen cells in wildtype respectively. We then defined a binary phase score for a cell c as: Binary Phase Score = dist( , ) − dist ( , ) , where the distance is between normalized F-actin and YAP of the cell and two centers.

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Observe that if a cell is closer to the Telogen Center, it will have a positive binary phase score.
Otherwise, if it is closer to the Anagen Center, it will have a negative binary phase score. If a cell is equidistant from both centers, it will have a zero score.
For visualization, we defined a Telogen-Anagen phase landscape by finding binary phase scores for simulated normalized F-actin and YAP value combinations (n=21 2 , where both values in any combination is out of a sequence ranging from 0 to 1, incremented by 0.05) and created a smoothed 3D surface from simulated combinations with

Figure S8
A   (D) Actomyosin forces on α-catenin are weaker in early anagen than that in telogen, indicated by reduced α18 signals. Quantification is showed in the right panel. P values are determined by Student's t-test. (E) miR-205 induction promotes the transition of HG cells from the quiescent telogen state to the activated anagen state, visualized by mathematical modeling and plotting of the F-actin and nuclear YAP score. The color gradient indicates the "distance to centers" score of each sample point. (F) F-actin and nuclear YAP signals, modeled as a distance to centers score, indicate that miR-205 induction promotes the transition of HG cells from telogen to anagen. Note some cells are still classified as telogen cells whereas many are classified as anagen (**p<0.01).
Differential Gene Expression Upon miR205 Induction (4 days) in HFSCs and HG, determined by single-cell RNA-seq Movie S1. Hair germ contraction during telogen.
In the 4-hour intravital imaging, the HG but not the bulge HF-SC compartment contracts by ~30% In the 6-hour intravital imaging, the HG exhibits pulsatile contraction and enlargement activities.
Note the constant position and location of HF-SCs during the same period. All epithelial cells are labeled with H2b-GFP. The green arrowheads track the movement of the bottom of the HG.

Movie S3. Hair germ contraction during telogen.
In the 4-hour intravital imaging, the HG but not the bulge HF-SC compartment contracts by ~30%

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In the 4-hour intravital imaging, the HG enlarges by ~20% in volume in the absence of cell division. Note the HF-SC compartment also enlarges but less than the HG. All epithelial cells are labeled with H2b-GFP. The constant red dotted line outlines the HF-SC and HG compartments.
The yellow dotted line and green arrowhead track the movement of the bottom of the HG.
Movie S5. Hair germ enlargement in an miR-205 induced hair follicle.
In the 4-hour intravital imaging, the HG initially enlarges and then slightly contracts toward the end of the imaging session in the absence of cell division. Note the HF-SC compartment also enlarges slightly. All epithelial cells are labeled with H2b-GFP. The constant red dotted line outlines the HF-SC and HG compartments. The yellow dotted line and green arrowheads track the movement of the bottom of the HG.