Mammalian skin contains a diverse array of structures – such as hair follicles and nerve endings – that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.
Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale – as seen, for example, in the orderly arrangement of cell types within a single hair follicle – and on a macroscopic scale – as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.
The skin is one of the largest organs in the body, with important functions in somato-sensation, insulation/thermoregulation, and immune defense1. Understanding the molecular and cellular basis of skin development and function has been of longstanding interest because of the fundamental importance of skin as a biological system and its relevance to dermatology. Mammalian skin contains a variety of multicellular structures, including stratified layers of keratinocytes, dermal connective tissue, several types of hair follicles, sebaceous glands, arrector pili muscles, blood vessels, and at least a dozen distinct classes of afferent (sensory) and efferent nerve fibers (Figure 1). Different regions of the body are associated with characteristically different types of skin. In most mammals, nearly the entire body surface is covered with skin that is densely packed with hair follicles. [Humans and naked mole rats constitute exceptions to this pattern.] Hair is missing from the palmar surfaces of the hands and feet, which are also associated with specialized epidermal patterns (dermatoglyphs), exocrine glands, and sensory nerve endings. The cellular and molecular events that control the growth, differentiation, and spatial arrangement of cells within the hair follicle are of special interest as each follicle exhibits, in miniature, many of the central features of organogenesis2. These features include the existence of stem cells and a stem cell niche, precisely choreographed cell migrations, and the assembly of multicellular structures from embryologically distinct components.
This article describes methods for dissecting, fixing, labeling, and imaging mouse skin as an intact two-dimensional sheet, referred to as a “whole mount” or “flat mount” preparation. Since mouse skin is relatively thin, it is possible to image through the full thickness of flattened skin using conventional confocal microscopy. The flat mount approach to imaging mammalian skin is technically advantageous because it bypasses the need for physical sectioning, thereby allowing structures to be reconstructed entirely by optical sectioning. Since nearly the entire skin is processed as a single object, the flat mount approach also facilitates the imaging of multiple regions of the body surface while preserving information about position and orientation relative to the body axes. Finally, structures within the skin are typically present in patterns that are repeated at regular intervals, thus facilitating the collection of images from multiple representatives of a given structure. These characteristics are familiar to neurobiologists who work on the retina, a two-dimensional part of the central nervous system that enjoys analogous advantages for studies of neuronal morphology3.
The flat mount approach described here is of special utility for studying structures that exhibit spatial organization on a relatively large scale within the two-dimensional plane of the skin. One example of large-scale spatial organization is the coordinated polarity of hair follicles and hair follicle-associated structures – Merkel cell clusters, arrector pili muscles, sebaceous glands, and nerve endings4. Hair follicles are oriented at an angle with respect to the plane of the skin, and the component of the follicle vector that lies within the 2-dimensional plane of the skin generally exhibits an orientation with respect to the body axes that is precisely determined for each position on the body. For example, hair follicles on the back point from rostral to caudal and hair on the dorsal surface of the feet point from proximal to distal. Hair follicle orientation is controlled by planar cell polarity signaling (PCP; also called tissue polarity5). This signaling system was discovered in Drosophila where a small set of core PCP genes was found to control the orientation of cuticular hairs and bristles. Three mammalian orthologues of core PCP genes – frizzled homolog 6 (Fzd6, also referred to as Fz6), cadherin EGF LAG seven-pass G-type receptor 1 (Celsr1), and vang-like 2 (Vangl2) – play analogous roles in mammalian skin, coordinating the orientations of hair follicles with the body axes. Studies of Fz6 knockout mice (Fzd6tm1Nat, hereafter referred to as Fz6-/-) show that the primary defect in the absence of PCP signaling is an initial randomization or disorganization of hair follicle orientation, with no effect on the intrinsic structure of the follicles6-8. A second non-PCP system acts later to promote local alignment of nearby follicles, which leads to the production of large-scale hair patterns such as whorls and tufts.
A second example of large-scale spatial organization within the skin is seen in the morphologies of sensory axon arbors. Sensory neurons that innervate the skin have their cell bodies in the dorsal root and trigeminal ganglia. These neurons detect temperature, pain, itch, and various types of mechanical deformations impinging on the skin and hair9. They can be divided into subtypes based on axon diameter and conduction velocity, terminal nerve ending structure, and the patterns of expression of receptors, channels, and other molecules. Because of the high density of innervation within the skin, analyses that involve visualizing all axons (e.g., anti-neurofilament immunostaining) or even all axons of a single class (as seen when a single cell type is marked by expression of a fluorescent reporter) generally reveals a dense superposition of axons that makes it impossible to define the morphology of an individual arbor. To circumvent this problem, we have used extremely sparse genetically-directed labeling to produce dorsal skin samples in which individual well-isolated axon arbors are visualized by expression of a histochemical reporter, human placental alkaline phosphatase10. This approach allows the unambiguous visualization of individual axon arbor morphologies and a definition of somatosensory neuron types based on morphologic criteria.
This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocol MO11M29 of the Johns Hopkins Medical Institutions. Consult your local Institutional Animal Care and Use Committee guidelines for approved methods of euthanasia. Wear gloves, lab coat, and safety glasses when handling aldehyde fixatives or organic solvents.
1. Preparation of Materials
2. Skin Dissection, Fixation, and Clearing
3. Staining Reactions
4. Imaging and Image Analysis
Brightfield imaging of skin flatmounts can be used to image cutaneous sensory afferents (Figure 3A10) and hair follicle patterns based on melanin pigmentation (Figure 4). Confocal imaging of skin flatmounts can be used to define the geometry of (1) Merkel cell clusters, visualized with anti-cytokeratin-6 or with AM dye uptake (Figures 3I-L), (2) arrector pili muscles, visualized with anti-smooth muscle actin (Figures 3G,H), (3) sebaceous glands, visualized with Oil Red O (Figures 3C-F), and (4) hair follicles, visualized with a Krt17-GFP transgene or by staining with anti-Krt17 antibodies4 (Figures 3E-G,K,L). It is straightforward to manually score the orientations and spatial arrangements of structures in such images by placing vectors of known orientation on the structures using Adobe Photoshop or Illustrator, and then quantifying the resulting vector populations. While this approach suffices for a scale of several hundred vectors, it would be prohibitively tedious on a 10- or 100-fold larger scale.
Figure 1. Schematic of mammalian hairy skin showing the major structures. (Copyright, Terese Winslow.)
Figure 2. Skin dissection and sample handling. A) Dissection tools. B) A P5 dorsal skin pinned to a Sylgard plate. C) A P5 dorsal hind foot skin pinned to a Sylgard plate. D) A dorsal skin mounted between 2 glass gel plates for imaging with a 10X objective and DIC or brightfield illumination.
Figure 3. Skin structures in flat mount views. A,B) A single cutaneous sensory arbor (left) and its traced image (right) from a P21 dorsal skin of a Brn3aCKOAP/+;NFL-CreER/+ mouse exposed to low dose tamoxifen at gestational day 17. This protocol results in Cre-mediated recombination of the reporter in a very small fraction of dorsal root ganglion neurons, and therefore a small fraction of cutaneous sensory arbors are labeled with the human placental alkaline phosphatase (AP) reporter. The labeled axons are visualized with NBT/BCIP histochemistry. Brn3a is equivalently referred to as Pou4f1. C,D) Tail skin stained with Oil Red O to visualize sebaceous glands from WT (left) andFz6-/- (right) mice at P21. The Fz6-/- structures are disorganized. P, proximal; D, distal. E,F) Dorsal foot skin from WT (left) and Fz6-/- (right) mice carrying Krt17-GFP. The skin was harvested at P21 and stained with Oil Red O. The Fz6-/- skin has a hair whorl in its center. P, proximal; D, distal. G,H) Dorsal skin from a WT mouse at P21 showing hair follicles (visualized with a Krt17-GFP transgene and anti-GFP immunostaining; panel G) and arrector pili muscles [visualized with anti-smooth muscle actin (SMA) immunostaining; panel H]. Depth within the confocal Z-stack image is color coded. A, anterior; P, posterior. I-L) A Merkel cell cluster (visualized with cytokeratin8 immunostaining or AM11-43 dye uptake) and its central hair follicle (visualized in panels K and L with Krt17-GFP). Images were obtained from P1 dorsal skin from the indicated genotypes. The Fz6-/- Merkel cell cluster forms a closed circle, whereas the WT Merkel cell cluster is open toward the anterior. A, anterior; P, posterior. Scale bars: A and B, 300 µm; C and D, 500 µm; E and F, 500 µm; G and H, 200 µm; I-L, 50 µm. (Panels A and B are reproduced from eLife and Proc. Natl. Acad. Sci. USA, with permission4,10) Please click here to view a larger version of this figure.
Figure 4. Hair and hair follicles in dorsal skin at P1 and P7 visualized with melanin pigmentation. A,B) P1 skin from WT and Fz6-/- mice. C,D) P7 skin from Fz6-/- mice. Scale bars: A and B, 500 µm; C and D, 1 mm.
Mastery of the dissection methods described above requires only patience, a steady hand, and a few good dissection tools. The dorsal skin dissection is relatively easy, but the tail and foot skin dissections – especially at early postnatal ages – are more challenging. At early prenatal ages (e.g., before E15), the skin is difficult to remove without tearing it. Conveniently, for many studies of growth and patterning of skin structures in mice, the events of interest occur postnatally, as seen for example in studies of the growth of arrector pili muscles19.
Imaging deep into the skin in a flat mount configuration is challenging because skin is a relatively refractile tissue. This challenge becomes greater as the skin matures due to the differentiation and increased thickness of its constituent layers. One partial solution to this problem is to separate the dermis and the epidermis and analyze the epidermis as a separate structure (“epidermal whole mounts”). This approach is useful for visualizing hair follicles and their associated structures, especially for mouse tail skin. However, obtaining high-quality epidermal whole mounts from mouse dorsal skin is difficult, presumably due to (1) the high density of hair follicles that extend deep into the dermis and (2) the thinness of the interfollicular epidermis13.
In our experience, BBBA treatment is a highly effective method for rendering skin optically transparent while preserving immunofluorescent signals without the need of separating the epidermal and dermal layers. Benzyl-benzoate has been used to clear tissues for decades20 and it is widely used to clear frog and fish embryos. However, BBBA has the disadvantage that it denatures GFP and other fluorescent proteins, it dissolves Oil Red O, and it can contaminate equipment (e.g., microscopes). It would be of interest to systematically compare BBBA treatment of skin with other clarification methods such as SeeDB21, ClearT22, Scale23, 3DISCO24, and CLARITY25, some of which are compatible with fluorescent proteins. If BBBA treated samples are to be imaged using a dissecting microscope, a conventional light/fluorescent microscope, or a confocal microscope, the samples should be placed in a glass dish or between two large glass plates to minimize BBBA contamination.
We have not discussed data analysis in any detail, as this is more idiosyncratic to the biological question under investigation. However, as noted in the Introduction, the presence of many nearly identical structures such as hair follicles, Merkel cell clusters, and nerve endings, and the completeness with which these structures can be imaged in flat mounts provides an opportunity to measure structural or morphologic parameters in a statistically rigorous manner, as seen for example in recent studies of cutaneous sensory axon morphologies10 and of hair follicles and follicle-associated structures in wild type and Fz6 mutant mice4,8. With the development of more sophisticated image analysis tools, it may be possible to automate or semi-automate some of the analyses that, at present, are performed manually, thereby facilitating wider use of skin flat mount imaging as a method for investigating the principles underlying multicellular organization.
The authors have nothing to disclose.
The authors thank Dr. Amir Rattner for helpful comments on the manuscript. Supported by the Howard Hughes Medical Institute.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
5-bromo-4-chloro-indolyl phosphate (BCIP) | Roche | 11383221001 | |
AM1-43 | Biotium | 70024 | |
AM4-65 | Biotium | 70039 | |
Benzyl alcohol | Sigma | 402834 | |
Benzyl benzoate | Sigma | B-6630 | |
Confocal microscope | Zeiss | LSM700 | |
Cy3-alpha smooth muscle actin antibody | Sigma | C6198 | 1:400 |
Cytokeratin-8 | Developmental Studies Hybridoma Bank | TROMA-I-c | 1:500 |
Dissecting microscope | |||
Dissection tools | Fine Science Tools | scissors and forceps | |
Electric razor | |||
Fluoromount G | EM Sciences | 17984-25 | |
Formalin | Sigma | HT501320 | |
Glass dishes | Pyrex | 6 cm and 10 cm diameter | |
Glass plates | Amersham Biosciences | SE202P-10 | 10 cm x 8 cm x 1 mm |
Hair remover | Nair | ||
Horizontal rotating platform | Hoefer | PR250 Orbital shaker | |
Insect pins | Fine Science Tools | 26002-20 | |
Ketamine/xylazine | Sigma | K113 | |
Nitroblue tetrazolium (NBT) | Roche | 11383213001 | |
Oil Red O | Sigma | O0625 | |
Paraformaldehyde | Sigma | P6148 | |
Razor Blades | VWR | 55411-055 | |
Secondary antibodies | Invitrogen | Alexa-dye conjugated | |
Sylgard-184 | Fisher Scientific | NC9020938 | |
Tissue culture plastic dishes | 10 cm diameter | ||
Tissue culture plates | 6- and 12-well |