Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Abstract

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6,11 , we can achieve robust gain-or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models. Video LinkThe video component of this article can be found at https://www.jove.com/video/4344/ Protocol 1. Prepare 0 to 2 days Old Newborn Mice for Skin Dissection 1. Euthanize mouse pups using a CO 2 chamber for at least 20 min. Leave pups on ice up to an hour until dissection. P0-P2 mice are highly recommended as cells prepared from older mice have reduced progenitor capacity that results in lower graft yields. Prepare one dish of 70% ethanol (for step 1.3) and three dishes of wash solution (for step 3) when ready to perform skin dissection. Thaw Dispase Solution and leave it at 4 °C. Cervical dislocate pups after CO 2 overexposure to ensure euthanasia. 2. Place euthanized pups in a culture dish on ice and transfer to a sterile flow hood. 3. Wash pups by briefly immersing in 70% ethanol and place onto sterile culture dish. Dissect Mouse Skin1. Cut off each limb and tail at the base of the torso with sterile scissors. 2. Grasp the body firmly between a pair of curved forceps and make an incision along the dorsal skin from head to tail using a scalpel without penetrating the underlying fascia. 3. Carefully peel the skin away from the midline of the mouse. 4. Grasp the exposed mouse firmly with the long side of the curved forceps and insert another pair of curved forceps underneath the skin at the posterior end of the mouse and gently pull skin over hips toward the ventral half of the mouse. 5. Carefully peel skin completely off the mouse in one smooth motion and discard the carcass. Wash Skin and Incubate with Dispase Solution1. Place the skin in the first dish of wash solution with dermis-side down. Spread skin out and agitate with forceps. Leave skin in the first dish of wash solution while dissecting the next skin.

Actin polymerization controls cilia-mediated signaling

“…Cell culture, drug treatments, and quantitative PCR Primary mDCs were isolated as previously described (Woo et al, 2013). In brief, skin was dissected from 1-to 3-d-old pups and incubated in 2.5 mg/ml dispase in HBSS overnight at 4°C.…”

Dermal papilla cell‐secreted biglycan regulates hair follicle phase transit and regeneration by activating Wnt/β‐catenin

“…Actually, it is well known that the classical Wnt/β‐catenin signalling pathway is essential for HF formation 20–22 . In rodent DP cells, the activation of the Wnt/β‐catenin signalling pathway can prolong HF‐inducing ability 40 .…”

Unlocking the vital role of host cells in hair follicle reconstruction by semi-permeable capsules

“…The cell suspension was centrifuged at 230 g for 5 min, then the cell pellet was resuspended in DMEM. The epidermis was digested in 0.25% trypsin-EDTA (Gibco, Grand Island, NY, USA) at 37°C for 10 min to obtain freshly isolated epidermal cells, as previously reported [ 15 ]. The preparation of cells from GFP newborn mice is the same as previously described.…”