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Skin and Adnexa of the Laboratory Mouse
CHAPTER
Ski n an d Ad nexa o f th e La b o r a t o r y M ouse | John P Sundberg The Jackson Laboratory, Bar Harbor, ME, USA
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The skin is the largest of the intermediate-sized organs (Goldsmith, 1990). Dermatology, anatomy, and histology textbooks assign simple functions to this organ system which, in reality, is as complicated as any organ in the body. More importantly, it is integrated with every organ of the body, not simply a wrapping to hold things together. The list of functions of the skin are constantly expanding. Table 12.1 is a summary from a recent published debate on this topic (Chuong et al., 2002). Spontaneous and genetically engineered mutations in laboratory mice have changed the basis of our knowledge of the function of the skin and how gene expression in the skin may be a reflection of similar expression in different organs (Sundberg and King, 2000). For example, for a long time there was a general thought that mice without hair (alopecia) have some form of immunodeficiency. This was largely based on the nude mouse. These mutant mice appear to lack hair at the gross level and lack a cell mediated immune system due to failure of the thymus to develop normally. The Laboratory Mouse Copyright 2004 Elsevier ISBN 0- ! 233-6425-6
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In fact, these mice have hair follicles and produce hair fibers but the structures are defective due to the role of the nude gene (Foxnl"") that acts as a transcription factor to down regulate hard keratin production (Mecklenburg et al., 2001). This gene also plays a role in terminal differentiation of keratinocytes at various anatomic sites (Baxter and Brissette, 2002). Hairless (hr), another mutant mouse that has been available for
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over half a century (Gaskoin, 1856) also has a minor abnormality in its immune system (Sprecher et al., 1990). The advent of the severe combined immunodeficiency mutant mice (Prkdc 'cid) with normal pelage and hair cycle changed that limited correlation (Sundberg and Shultz, 1994). We now know that each skin defect can be unique and may or may not be associated with visceral lesions (Sundberg and King, 2000). Numerous mutations have occurred in laboratory mice spontaneously, induced by radiation or various chemical mutagens, or created using transgenesis or targeted mutagenesis (Sundberg, 1994a; Sundberg et al., 1995; Sundberg and King, 1996a, b; 2000; Nakamura et al., 2002a, b; Randall et al., 2003). It is beyond the scope of this chapter to cover these mutant mice. In addition to the reviews provided, much information is available on the Mouse Genome Informatics web site (www.informatics.jax.org). This chapter will provide an overview and references as sources for more specific information on normal anatomy, development, and cycling of the skin and its adnexa. It will also provide information on routine methods to prepare specimens for analysis. General, systematic descriptions of necropsy procedures evaluating all organ systems can be found in another chapter in this book.
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Clinical evaluation The normal mouse is covered with hair. Careful examination reveals that at least two hair types are obvious, as is the case with most domestic mammals. A fine short hair coat covers most of the body (truncal hairs) while long hairs are evident around the head (vibrissae, incorrectly called whiskers by many investigators since there is no anatomical similarity to androgen induced facial hair in humans). If the hair is carefully studied there are many hair types present. Within the pelage hairs there are classically four types: guard hairs are long, straight, thick, and protrude above the level of most hairs; auchene hairs are nearly as long as guard hairs with a gradual bend at the distal end; awl are also straight with a bend
Figure 12.1 Scanning electron micrograph of telogen stage plucked guard (A), awl (B),and zigzag (C) hairs from the dorsal truncal skin of an adult mouse. at the distal end but are short and thin; lastly zigzag hairs are the underhairs that have two bends giving in a 'Z' shape (Figure 12.1). These hair types are best differentiated in plucked samples examined with magnification. Historically many people stuck hairs on double sticky tape to a glass microscope slide. An easier method is to place a few hairs on a glass slide, add a drop of mounting medium, and drop on a cover slip. This forces the hairs to lay in one plane. The hairs can be examined with a microscope, photographed, and a variety of light sources used that can provide diagnostic information (Sundberg and Hogan, 1994; Sundberg et al., 1998). In addition to the pelage or truncal hairs there are many other specialized hair types in the mouse. The tail is covered with very short, broad fibers. Ears have a variety of very short fine fibers (Figure 12.2). Eyes have vibrissae above the eyelids and a network of long hairs protruding from the lid margins called cilia.
Figure 12.2 Subgross photograph of pilosebaceous units (hair follicles with the sebaceous gland at its base) in cleared skin from the ear of an adult mouse.
Figure 12.4 Anagen stage perianal hair. Anus (A),dermal or follicular papilla (DP) within the bulb, sebaceous gland (S), telogen stage truncal hair follicles (T).
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dissection microscope, scanning electron microscopy, or some other means of magnification. Hairs are usually thin and straight with a uniform distribution pattern within a strain. Variations, especially hair loss, can suggest that the mice have a mutant phentype but only after simple diagnostic methods rule out infectious causes or infestations. Ectoparasites remain common in many mouse rooms and will result in alopecia often mistaken the novice for a mutant phenotype (Figure 12.5). Mites are easily diagnosed by placing a piece of hairy skin in a dosed petri dish into a refrigerator then examining it after an hour or so with a hand lens. Mites migrate to the tips looking for another host. They can also be easily identified by histologically if hairs are not shaved during preparation of the skin (Figure 12.5). Other infectious diseases require the assistance of a trained veterinary pathologist for correct diagnosis.
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Vibrissae found around the mouth, eyelids, and near the foot pads on the lower legs (Figure 12.3). Perianal hairs are large thin structures that form a network above the opening of the anus (Figure 12.4). Hairs change around nipples and the base of the ear. These differences often can only be seen using a hand lens,
Tissue collection and preservation for histologic evaluation of the skin Every pathologist has his or her own preference for fixation of tissues. It is always best to work with the pathologist who will be evaluating the tissues before proceeding. Neutral buffered formalin solution is the most universal fixative used. Tissues are often left in formalin for long periods. Due to cross linking of amino groups by the aldehydes, many epitopes are modified making immunohistochemistry difficult or impossible. Fekete's acid alcohol formalin minimizes this problem, especially when tissues are transferred to 70% ethanol after overnight fixation. Commercially available zinc based preservatives are being promoted to
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maintain epitopes, optimize immunohistochemical results while maintaining some degree of the histologic quality that pathologists are used to with paraffin sections. Bouin's solution is popular as a general fixative but it hyalinizes collagen fibers so fine detail of the skin can be difficult to interpret. Use of Bouin's solution requires washing in tap water and transfer to ethanol. Failure to do so results in major artifacts often making the tissue unusable. These and other fixatives are discussed in Chapter 30 on Necropsy Methods which includes formulations for their preparation. Since skin, and especially hair follicles, vary dramatically by location, several locations should be sampled in order to evaluate potential changes. Collection of tissue consistently throughout a study will make specimens comparable. Dorsal skin can be collected over the thorax making sure to label cranial and caudal orientations so the tissue will be trimmed correctly. Ventral skin covering the thorax is also taken. Both dorsal and ventral skin are very similar histologically so they can be placed in separate cassettes when trimmed together with other skin that has distinct histologic features. Vibrissae on the head are collected by removing all the skin on the head as a complete unit. Vibrissae on the muzzle are trimmed as one piece. Eyelids are sectioned from this piece of skin as well to include upper and lower lids. Ear and tail are removed from the body and fixed by immersion. Tail skin can be removed from the bone and muscle or collected together. If the latter is done the bone must be decalcified. Footpads are also collected. Details are provided in Chapter 30 on Necropsy Methods and elsewhere (Sundberg et al., 1998; Relyea et al., 2000a). Nails are collected attached to the feet and digits. Distal limbs can be disarticulated and fixed in tom. If the paw is to be examined, it can be fixed under weight to lay it flat then sectioned horizontally to include all the joints after decalcification. Sagittal sections are the most useful. Digits are processed in toto and serially sectioned lengthwise after decalcification.
Scanning electron microscopy of the skin and hair fibers Scanning electron microscopy provides a detailed three dimensional view of structures at various magnifications. X-ray microanalysis can determine the relative elemental content of a specimen which may be useful for evaluation of some mutant mice. Hairs are made up of what used to be called the high sulfur keratins or hard keratins, now called the hair keratins. Changes in sulfur levels can be detected that suggest abnormalities in these hairs. Such is the case with the ichthyosis mutant mouse that has a form of trichothiodystrophy, low sulfur levels in the hair fibers (Figure 12.6) (Itin et al., 1990). Toxic agents, especially heavy metals, can also be identified using this method (Chatt etal., 1990; Takeuchi etal., 1990; Bache etal., 1991). Whole mounts of skin or nails can be easily made by removing tissues at the time of necropsy, spreading soft tissues out on a firm nylon membrane to fix them flat, and placing them in buffered glutaraldehyde using standard methods. Electron microscopists will critically point dry the specimen, coat it with gold, and then examine it with the investigator (Bechtold, 2000). Hairs can be examined in whole mounts or manually removed and examined individually. Adult mouse hair follicles are in telogen for prolonged periods so the hairs can be easily removed manually from lightly anesthetized animals without causing pain but more importantly, damage to fibers is rare since they come out easily. Hairs are placed in a dry vial and processed routinely. Shipments we receive from collaborating laboratories for evaluation are routinely disinfected on the outside surface and are then filled with 70% ethanol and stored for a week or longer before processing due to the common infestation of mites in m a n y research colonies. This approach kills the mites thus avoiding introduction into your colonies.
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microscopy of the skin and hair fibers Transmission electron microscopy can provide a great deal of information but is technically difficult and labor intensive. Tissue is removed during necropsy but should be finely minced into I mm 3 pieces since glutaraldehyde ftxatives do not penetrate tissues deeply. Cacodylate or phosphate buffered glutaraldehyde are commonly used but others are available and described elsewhere. Tissues should be stored refrigerated and embedded soon after collection to minimize artifacts (Bechtold, 2000).
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mouse (A), one with focal alopecia areata (B), and one with diffuse alopecia areata (C).There is a quantifiable increase in heat loss associated with increased hair loss.
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Other methods Many different methods have been developed to evaluate skin. We have tested a thermal imaging device that measures infrared radiated from mice under general anesthesia (Thermogenic Imaging, Billerica, MA). It appeared to be a useful device for determining response to treatment for mutant mice with thick, scaly, neovascularized skin or those with various forms of alopecia (Figure 12.7). Longitudinal studies revealed that thermal changes over time reflected the hair cycle in both mutant and control mice since the hypodermal fat layer, and therefore the insulation value of the skin, varied dramatically through the hair cycle. Transepidermal water loss is an important measurement in mice with abnormalities in the cutaneous water barrier. Mice are first sedated with 100 mg/kg ketamine HCI plus 0.5 mg/kg xylazine intraperitoneally. Dorsal hair is removed with electric clippers and then depilated for 5 min with a chemical agent such as Neet
O (Reckitt and Coleman, Wayne, NJ). Transepidermal water loss is measured 24 h later by placing a Servo Med Evaporimeter EPI probe (Servomed AB, Stockholm, Sweden) on the bald area (Setup and Jemec, 1995; Sundberg et al., 2000). Surface lipids can be collected by dipping euthanized mice into 40 ml of acetone 10 times and drying the acetone under argon gas. The residue is dissolved in toluene and plated in separate lanes on silica gel G chromatographic plates (Merck, Rahway, NJ). The plates are developed to 19 cm in hexane-ether-acetic acid (80:20:1). Following drying of the plate it is sprayed with 50% sulfuric acid (Downing and Stranieri, 1980; Sundberg etal., 2000). Kinetic studies can be easily done if considered at the time of necropsy. Mice can be injected with bromodeoxyuridine (50 ~g/gm body weight) 1 h before necropsy (Smith et al., 2000). A consistent time interval between injection and necropsy is critical since
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it will determine the rates at which this compound is incorporated into DNA currently being synthesized. Unstained sections are processed routinely for immunohistochemistry (Relyea et al., 2000b) and an antibromodeoxyuridine antibody used. Positive cells in 'S' or the DNA synthesis phase of the cell cycle will have nudei that are brown or red depending upon the chromogen and enzyme system used (Relyea et al., 2000b; Smith et al., 2000). An alternative is to use tritiated thymidine. This radionuclide requires special safety precautions, takes 3-6 weeks for development, and can be difficult to interpret so it is less commonly used today. Interpretation is complicated and can depend upon what types of proliferation rates are needed for evaluation of a particular mutant. Standard approaches are described for interfollicular skin such as counting the number of positive nudei per 1000 basal cell nuclei or per linear millimeter of skin, if it lays flat (Leblond etal., 1964; Skerrow and Skerrow, 1985; Kwochka and Rademakers, 1989; Kwochka, 1990; Sundberg et al., 1994a). Some mutant mice have marked proliferation of the infundibulum which requires modifications and special adaptation of counting criteria (Sundberg et al., 1997a). Gene arrays are a relatively new technology that is becoming more widely accessible to investigators. A variety of methods are now available. The critical starting
material is high quality RNA. What tissue to select and how to prepare it are developing and controversial topics as the technology evolves. We have used the entire skin of mice that develop a generalized cutaneous phenotype. The advantage is that an adequate volume can be obtained to provide enough RNA for many experiments. The disadvantage is that hair follicles in various stages are obtained, anatomically discreet areas are mixed and not all areas are affected. Assuming similar anatomical effects are found in age and gender matched controls, the differences in gene expression profiles should represent those related to the disease under investigation. More specific sites can be chosen as the disease is better understood (Carroll et al., 2002). The main advantage of the gene arrays is that complex pathways that can take a great deal of time to analyze using traditional methods can be screened with a small group of animals in a matter of days to generate the data but weeks to months to analyze it. For example, the mouse flaky skin (fin) mutation develops a psoriasiform demaatitis (Sundberg et al., 1994a; Sundberg et al., 1997b; Pelsue et al., 1998). One run with an Affymetrix Gene Chip | determined that this mutant mouse had primarily a Th2 immune response in contrast to the Thl response found in human psoriasis patients (Figure 12.8). This explained variations in
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therapeutic responses between the species (Carroll and Sundberg, unpublished data). Tissue arrays are the next technology with direct application to many research projects. Tissue arrays are build on traditional histology methods whereby paraffin blocks are systematically punched at prescribed sizes and the cores placed into predrilled holes in a new paraffin block (Moch et aL, 2001). Large numbers of tissues from many different organs or different case materials of similar lesions from the same organs can be used. This is providing a tool to specifically evaluate the cells producing proteins from the up and down regulated transcripts detected using gene arrays. Currently custom or predesigned arrays are commercially available. As the technology becomes more accepted, many institutions will probably be adopting it.
structure. The root sheaths begin to form and differentiate. Stage 5 is the bulbus peg stage with elongation of the inner root sheath and development of the bulge and first sebocytes. Melanin begins to form at this stage in pigmented mice. At stage 6 the follicle begins to extend below the level of the dermis into the hypodermal fat layer. The hair canal can now be identified. In stage 7 the tip of the hair fiber leaves the inner root sheath and enters the hair canal at the level of the infundibulum of the forming sebaceous gland. Stage 8 is the maximum length of the hair follicle where it extends down to the panniculus carnosus muscle and the hair fiber emerges through the epidermis. This process begins in utero and is completed for all follicle types by 5 days post partum when the hair is evident on the skin of most strains of normal mice. The epidermis develops from a single layer into a multilayered structure. In newborn mice it is thick at all anatomic sites and keratinocytes follow a classical differentiation scheme for stratified squamous epithelium (Figures.12.9). Cuboidal basal cells (keratins 5and 14 posmve) are located on the basal lamina (HogenEsch etal.,1999). Above this layer the cells differentiate into the statum spinosum or prickle-cell layer. Here the cells begin to elongate along the axis of the skin and have prominent intercellular bridges (desmosomes) that are evident under high magnification. These spine-like Each hair follicle type starts to develop at different structures are due to artifactual shrinkage of the tissues time points during embryogenesis. Therefore, it is not during preparation. This layer can be identified by the surprising to find clusters of hair follicles in a section presence of keratins 1 and 10. The next layer, the straat different stages of development. The large vibris- tum granulosum, has cells that are flattened along the sae develop earliest and are nearly fully developed by axis of the skin and contain prominent basophilic granbirth. In spite of this, all hair follicles develop in a sim- ules (keratohyalin granules). Two types of granules are ilar anatomic fashion. This developmental scheme is present in the mouse, profilaggrin (P) and loricrin (L) detailed both historically and anatomically elsewhere granules. The larger profllaggrin granules are the blue (Paus etal., 1999) and serves as a guide for the summary structures seen by light microscopy (Presland et al., below. 2000). The most superficial layer, the stratum corneum, The sequential stages of hair follicle development is brightly eosinophilic and consists of compacted, flatbegin with the pregerm stage which is hard to recog- tened keratinocytes. This is the critical portion of the nize histologically but can be defined with various skin that provides a strong aqueous barrier due to the immunohistochemical markers. It consists of a sharply presence of lamellar bodies, small lipid based structures demarcated plaque of basal and suprabasal epidermal only detectable by special staining and transmission keratinocytes. In stage 1, the pregerm develops into an electron microscopy (Elias, 1988). histologically evident epidermal thickening where the The epidermis of a newborn mouse is thick. As the keratinocytes display a vertically polarized orientation mouse ages, within two weeks, the truncal epidermis compared with the more cuboidal appearance of adja- thins to only about two cell layers with the stratum cent basal cells. Concurrently, dermal fibroblasts granulosum and corneum becoming very thin and increase in number immediately below this structure often hard to visualize by light microscopy. Other forming what will become the dermal (follicular) anatomic sites do not change. The tail skin remains papilla. Stages 2-4 produce a column of epidermal thick throughout the mouse's life. The muzzle skin is keratinocytes that develop a cap, invagination of the thinner than at birth but thicker than truncal skin. dermal papilla, and formation of the basic hair follicle Foot pads remain thick.
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This anatomy of the skin and hair follicles are illustrated in Figures 12.4 and 12.9. The top layer of epithelial cells is called the epidermis. This layer differentiates from the cuboidal basal cells in the stratum basale into the polygonal cells of the stratum spinosum, then more flattened cells with fine blue granules in the stratum granulosum, and ultimately into the flat cells that lack a nucleus and become very eosinophilic at the surface in the stratum corneum. The outermost layer of cells separating from the surface are sometimes called the stratum dysjunctum. The hair follicle is a very complicated structure that invaginates into the dermis and hypodermal fat undergoing major changes on a regular basis with the hair cycle (see below). A large gland protrudes from its side that consists of swollen pale cells with fine uniform vacuoles. These are sebaceous glands that produce oils to coat the surface of the skin and hair fiber. The oils can be visualized in frozen sections stained with oil red O, sudan black, or other histologic means to follow how the lipids spread out over the
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surface of the skin in normal compared with mutant mice (Sundberg et al., 1997a). The dermis consists of dense irregular collagenous connective tissue, elastic connective tissue, blood vessels, nerves, smooth muscle (arrector pili muscles that lift hair follicles and fibers; Figure 12.10), and a variety of individual cell types including fibroblasts, mast cells, and small numbers of cells from the immune system. One important feature of skin that is characteristically found in many rodents, especially laboratory mice, is that apocrine sweat glands are not present. Modified apocrine glands, mammary glands are abundant because of the large litter size most mice have (Sundberg et al., 1996). The mouse does not normally have f e t e ridges where the lower aspect of the epidermis forms ridges of cells that extend into the dermis. The dermis between such ridges is commonly called the dermal papillae, a term also used by hair biologists for the specialized fibroblasts that populate the base of an anagen hair follicle called the bulb. Because of this, the fibroblasts within the bulb are also called the follicular papilla. Rete ridge-like structures become prominent when mouse skin heals following ulceration. These changes resemble those found in neoplasms of the epidermis such that the changes are referred to as pseudoepitheliomatous or pseudocarcinomatous hyperplasia. Below the dermis is a layer of fat, the hypodermal fat layer. The thickness of this fat layer changes with the hair cycle being thickest during anagen when follicles need a great deal of energy to produce a hair
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fiber. The panniculus muscle separates the hypodermal fat layer from the adventitia, loose collagenous connective tissue that attaches the skin to the underlying musculature and fat. Mammary glands are found in the fat below this skeletal muscle layer,
limited to the bulb of anagen follicles and hair fibers. Interfollicular epidermis rarely contains pigment and when it does, it is usually only in mutant mice (Sundberg et al., 1994b). If the mice are shaved, irregular pigmented areas will be seen. These are areas containing anagen follicles. If mice are followed daily, these Hair cycle in the m o u s e pigmented patches will migrate caudally. This feature is ~' dramatic in mutant mice such as hairless. These mice The skin undergoes major changes during the first have normal hair 5 days after birth but not subsequent 2 weeks of life (Figure 12.9). Hair follicles continue to hair cycles. Beginning at 2 weeks of age their hair is develop and enter late stage anagen 5 days postpartum shed from head to tail (Sundberg, 1994a). Other hair when hair fibers emerge through the epidermis. The follicle types have different hair cycles. This is why truncal epidermis is relatively thick at birth and thins hairless mice appear to retain vibrissae while being to normal by 2-3 weeks of age. Hair follicles produce completely bald. The length of the hair cycle deterfibers until around 14 days of age over the thorax at mines the length of the hair fiber. This is consistent which time the lower portion undergoes apoptosis. The with why hairless mice have long, persistent vibrissae dermal papilla is retracted by actin filaments (Figure while short pelage hairs are lost. This feature was 12.10) that reside below the isthmus in the resting or demonstrated with angora (Fgf5 g~ mutant mice that telogen stage until the hair cycle is reinitiated. This have a three day prolongation of their truncal hair cycle usually lasts about 3 days in young mice. The follicle and as a result often have long, shaggy hair compared develops into a new anagen stage follicle pushing the with normal littermates (Sundberg et al., 1997c). Numerous genes regulate development and cycling old follicle laterally. The new fiber emerges adjacent to the old one. At some point the old fiber is lost in what of the hair follicles (Hardy, 1992; Pans, 1996, 1998; is now called the exogen stage (Milner et al., 2002). Millar, 1997; Chuong and Noveen, 1999; Stenn and The general features of the different stages of the hair Paus, 2001; Millar, 2002). Classic work done half a cycle are illustrated in Figure 12.9. Development of the century ago detailed changes in the skin and hair follihair follicle embryologically and progression through cles as they cycle, not just the changes in the follicles the hair cycle regularly throughout life have been dis- but also changes in sebaceous gland size and shape as sected anatomically and with molecular and immuno- well as the thickness of the hypodermal fat layer (Chase logical markers to differentiate numerous stages within et al., 1951, 1953; Chase, 1954, 1955; Chase and each major portion of the hair cycle that have been Eaton, 1959; Straile, 1960, 1965, 1969; Straile et al., detailed and reviewed elsewhere (Pans et al., 1999; 1961). Furthermore, hormones cause changes as well Muller-Rover et al., 2001; Millar, 2002). What is com- (Deplewski and Rosenfield, 2000). These are impormonly called the second hair cycle, or the first real hair tant to understand when comparing differences cycle after embryogenesis, has a short anagen stage and between wild type, normal mice, and mutant mice. prolonged telogen stage. The hair cycles in a cranial to Not only should the mice be age and gender matched caudal pattern that can be easily visualized in pigmented in such studies but it is critical to match the stage of the mice. Unlike humans, pigment in the mouse skin is hair cycle as well.
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The mouse has nails or claws on each digit, just like most other mammals including humans. The name claw suggests these structures are different than human nails which may be why little attention has been paid to them. In fact, anatomically they are very similar at the gross and histologic levels. Human nails are dorsoventrally flattened to form a plate while rodent nails are laterally flattened. These changes, not restricted to humans since similar refinements are found in many nonhuman primates, are associated with the function of the nails in primates as a refined tool associated with manual dexterity rather than as a weapon or digging tool. Sagital sections illustrate that mice have a nail matrix, nail plate, nail bed, hyponychium, and other structures (Figure 12.11) identical to but smaller than the human nail. To veterinarians this is not at all surprising since all mammals have nails or claws that are variations on this general theme. Nails can be extremely difficult to prepare and interpret histologically. However, changes in mutant mice can be dramatic when these structures are magnified with a dissection microscope or by scanning electron microscopy (Sundberg and King, 2000).
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cycle that will not be discussed here. Other glands found at specific anatomic sites are modified sebaceous glands, a type of holocrine gland. These include preputial and clitoral glands around the genitals, meibomian glands in the eyelid, and cerruminous glands within the outer ear. All are large glands with a structure similar to that found in the sebaceous glands associated with hair follicles. The major difference is that each has a stratified squamous epithelial lined duct that empties directly onto the structure where it is located (Sundberg and King, 2000). Hair follicles are specialized and have sebaceous glands associated with them that vary in size. The most notable are the perianal hairs which have large sebaceous glands. Salivary, lacrimal, and harderian glands are very different and are described in chapters dealing with the organs they are associated with.
Skin and adnexal mutant phenotypes It is beyond the scope of this chapter to describe or even list all mutant mice with skin and/or hair/nail phenotypes. As a general starting point we have grouped phenotypes into 10 classes (Table 12.2). Detailed lists, descriptions, references, and illustrations are published elsewhere (Sundberg, 1994a; Sundberg and King, 1996a, b, 2000; Nakamura et al., 2002a, b; Randall et al., 2003).
Mammary glands are specialized forms of apocrine sweat glands with a complex developmental and lactation
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Figure 12.11 Normal sagital section of the nail from an adult mouse (A). Hyponechium (H), nail plate (NP), nail bed (B), proximal nail fold (PNF), matrix (M), phalanx 1 (P1), phalanx 2 (P2), sesamoid bone (S),eccrine gland (EG),foot pad (FP). High magnification of a sesamoid bone under P2.
TABLE 12.2: General categories of mutant mouse cutaneous phenotypes Hair and skin color (pigmentation) Eccrine gland defects Sebaceous gland defects Primary scarring disorders Hair follicle cycling disorders Structural defects of hair fibers Hair texture abnormalities Missing hair fiber and follicle types Noninflammatory (ichthyosiform and keratodermas) skin diseases Inflammatory (psoriasiform and proliferative) skin diseases Papillomatous skin diseases Cutaneous carcinogenesis Bullous and acantholytic skin diseases Structural and growth defects of the nails
References Bache, C.A., Lisk, D.J., Scarlett, J.M. and Carbone, L.G. (1991).J. Toxicol. Environ. Health, 34, 423--431. Baxter, R.M. and Brissette, J.L. (2002). J. Invest. Dermatol. 118, 303-309. Bechtold, L.S. (2000). In Systematic Characterization of Mouse Mutations (eds J.P. Sundberg and D. Boggess), pp. 121-129. CRC Press, Boca Raton FL. Carroll, J., McElwee, K.J., King, L.E.J., Byrne, M.C. and Sundberg, J.P. (2002). J. Invest. Dermatol. 119, 392-402. Chase, H.B. (1954). Physiol. Rev. 34, 113-126. Chase, H.B. (1955).J. Soc. Cosmetic Chem. 6, 9-14. Chase, H.B. and Eaton, G.J. (1959). Ann. NYAcad. Sci. 83, 365-368. Chase, H.B., Montagna, W. and Malone, J.D. (1953). Anat. Rec. 116, 75-82. Chase, H.B., Rauch, H. and Smith, V.W. (1951). Physiol. Zool. 24, 1-10. Chatt, A., Holzbecher, J. and Katz, S.A. (1990). Biol. Trace E/em. Res. 26-27, 513-519. Chuong, C.-M. and Noveen, A. (1999). J. Invest. Dermatol. Symp. Proc. 4, 307-311. Chuong, C.M., Nickoloff, B.J., Elias, P.M., Goldsmith, L.A., Macher, E., Maderson, P.A., Sundberg, J.P., Tagami, H., Plonka, P.M., Thestrup-Pederson, K., Bernard, B.A., Schroder, J.M., Dotto, P., Chang, C.M., Williams, M.L., Feingold, K.R., King, L.E. Kligman, A.M., Rees, J.L. and Christophers, E. (2002). Exp. Dermatol. 11, 159-187.
Deplewski, D. and Rosenfield, R.L. (2000). Endocrine Rev. 21,363-392. Downing, D.T. and Stranieri, A.M. (1980). J. Chromatogr. 192, 208-211. Elias, EM. (1988). Drug Develop. Res. 13, 97-105. Gaskoin, J.S. (1856). Proc. Zoo. Soc. London 24, 38-40. Goldsmith, L.A. (1990). Arch. Dernmtol., 126, 301-302. Hardy, M.H. (1992). Trends Genet. 8, 55-61. HogenEsch, H., Boggess, D. and Sundberg, J.P. (1999). Pathobiology 67, 45-50. Itin, P.H., Sundberg, J.P., Dunstan, R.W. and Pittelkow, M.R. (1990).J. Invest. Dermatol. 94, 537. Kwochka, K.W. (1990). Am.J. Vet. Res. 51, 1570-1573. Kwochka, K.W. and Rademakers, A.M. (1989). Am. J. Vet. Res. 50, 587-591. Leblond, C.P., Greulich, R.C. and Pereira, J.P.M. (1964). In Advances in Biology of the Skin (eds W. Montagna and R.E. Billingham), pp. 39-67. Pergamon Press, New York. Mecklenburg, L., Nakamura, M., Sundberg, J.P. and Paus, R. (2001). Exp. MoL Pathol. 71, 171-178. Millar, S. (1997). In Cytoskeletal-membrane Interactions and Signal Transduction (eds P. Cowin and M.W. Klymkowsky), pp. 87-102. Landes Bioscience,Austin, TX. Millar, S.E. (2002).J. Invest. Dermatol. 118, 216-225. Milner, Y., Sudnik, J., Filippi, M., Kizoulis, M., Kashgarian, M. and Stenn, K. (2002). J. Invest. DermatoL 119, 639-644. Moch, H., Kononen, J., KaUioniemi, O.-P. and Sauter, G. (2001). Adv. Anat. Pathol. 8, 14-20. Muller-Rover, S., Handjiski, B., vanderVeen, C., Eichmuller, S., Foitzik, K., McKay, I.A., Stenn, K.S. and Paus, R. (2001). J. Invest. DermatoL 117, 3-15. Nakamura, M., Sundberg, J.P. and Pans, R. (2002a). Exp. Dermatol. 10, 369-390. Nakamura, M., Tobin, D.J., Richards-Smith, B., Sundberg, J.P. and Paus, R. (2002b). J. Dermatol. Sci. 28, 1-33. Paus, R. (1996). Cu~ Opin. DermatoL 3, 248-258. Paus, R. (1998).J. Dermatol. 25, 793-802. Paus, R., Muller-Rover, S., vanderVeen, C., Maurer, M., Eichmiller, S., Ling, G., Hofmann, U., Foitzik, K., Mecklenburg, L. and Handjiski, B. (1999). J. Invest. DermatoL 113, 523-532. Pelsue, S.C., Schweitzer, P.A., Schweitzer, I.B., Christianson, S.W., Gott, B., Sundberg, J.P., Beamer, W.G. and Shultz, L.D. (1998). Eur. J. Immunol. 28, 1379-1388. Presland, R.B., Boggess, D., Lewis, S.P., Hull, C., Fleckman, P. and Sundberg, J.P. (2000). J. Invest. Dermatol., 115, 1072-1081. Randall, V.A., Sundberg, J.P. and Philpott, M.P. (2003) J. Invest. DermatoL Sym. Proc. 8, 39-45. Relyea, M.J., Miller, J., Boggess, D. and Sundberg, J.P. (2000a). In Systematic approach to evaluation of mouse mutations (eds J.P. Sundberg and D. Boggess), pp. 57-90. CRC Press, Boca Raton, FL.
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Relyea, M.J., Sundberg, J.P. and Ward, J.M. (2000b). In SystematicApproach to Evaluation of Mouse Mutations (eds J.P. Sundberg and D. Boggess), pp. 131-144. CRC Press, Boca Raton, FL. Serup, J. and Jemec,. G.B.E. (1995). Handbook on NoninvasiveMethods and the Skin, CRC Press, Boca Raton, FL. Skerrow, D. and Skerrow, C.J. (eds) (1985). Methods in Skin Research. John Wiley & Sons Ltd, Chinchester. Smith, R.S., Martin, G. and Boggess, D. (2000). In SystematicApproach to Evaluation of Mouse Mutations (eds J.P. Sundberg and D. Boggess), pp. 111-119. CRC Press, Boca Raton, FL. Sprecher, E., Becker, Y., Kraal, G., Hall, E. and Shultz, L.D. (1990). Arch. Dermatol., 282, 188-193. Stenn, K.S. and Pans, R. (2001). Physiol. R~., 81,449-494. Straile, W.E. (1960). Am.J. Anat., 106, 133-148. Straile, W.E. (1965). In Biology of Skin and Hair Growth (eds A.G. Lyne and B.E Short), pp. 35-37. Angus & Robertson, Sydney. Straile, W.E. (1969). Adv. Biol. Skin, 9, 369-390. Straile, W.E., Chase, H.B. and Arsenault, C. (1961). J. Exp. Zool. 148, 205-216. Sundberg, J., X, M. and Boggess, D. (1998). In Cutaneous Appendages (ed. M. Chuong), pp. 421-435. Molecular Biology Intelligence Unit I, Landes Company, Austin, TX. Sundberg, J.P. (1994a). In Handbook of Mouse Mutations with Skin and Hair Abnormalities: Animal Models and Biomedical Tools (ed. J.P. Sundberg), pp. 291-312. CRC Press, Boca Raton. Sundberg, J.P. (1994b). Handbook of Mouse Mutations with Skin and Hair Abnormalities. Animal Models and Biomedical Tools. CRC Press, Inc, Boca Raton, FL. Sundberg, J.P. and Hogan, M.E. (1994). In Handbook of Mouse Mutations with Skin and Hair Abnormalities: Animal Models and Biomedical Tools (ed. J.P. Sundberg), CRC Press, Boca Raton, FL. Sundberg, J.P. and King, L.E. (1996a). J. Invest. Dermatol. 106, 368-379.
Sundberg, J.P. and King, L.E.J. (1996b). Dermatol. Clin. 14, 619-632. Sundberg, J.P. and King, L.E. (2000). In Pathology of Genetically EngineeredMice (eds J.M. Ward, J.F. Mahler, R.R. Maronpot and J.P. Sundberg), pp. 181-213. Iowa State University Press, Ames. Sundberg, J.P. and Shultz, L.D. (1994). In Handbook of Mouse Mutations with Skin and Hair Abnormalities (ed. J.P. Sundberg), pp. 423-429. CRC Press, Boca Raton, FL. Sundberg, J.P., Dunstan, R.W., Roop, D.R. and Beamer, W.G. (1994a).J. Invest. Dermatol. 102, 781-788. Sundberg, J.P., Dunstan, R.W., Roop, D.R. and Beamer, W.G. (1994b).J. Invest. DermatoL 102, 781-788. Sundberg, J.P., Orlow, S.J., Sweet, H.O. and Beamer, W.G. (1994c). In Handbook of Mouse Mutations with Skin and HairAbnormalities:Animal Modelsand BiomedscalTools(ed. J.P. Sundberg), pp. 159-164. CRC Press, Boca Raton, FL. Sundberg, J.P., HogenEsch, H. and King, L.E. (1995). Mouse Modelsfir ScalySkin Diseases.CRC Press, Inc, Boca Raton. Sundberg, J.P., Hogan, M.E. and King, L.E. (1996). In Pathobiology of the Aging Mouse, Vol. 2 (eds U. Mohr, D.L. Dungworth, C.C. Capen, W.W. Carlton, J.P. St~adberg, and J.M. Ward), pp. 303-323. ILSI Press, Washington, DC. Sundberg, J.P., Boggess, D., Hogan, M.E., Sundberg, B.A., Rourk, M.H., Harris, B., Johnson, K., Dunstan, R.W. and Davisson, M.T. (1997a). Am. J. Pathol., 151,293-310. Sundberg, J.P., France, M., Boggess, D., Sundberg, B.A., Jenson, A.B., Beamer, W.G. and Shultz, L.D. (1997b). Pathobiology 65, 271-286. Sundberg, J.P., Rourk, M., Boggess, D., Hogan, M.E., Sundberg, B.A. and Bertolino, A. (19970. Vet. Pathol. 34, 171-179. Sundberg, J.P., Boggess, D., Sundberg, B.A., Eilersten, K., Parimoo, S., Filippi, M. and Stenn, K. (2000). Am. J. Pathol. 156, 2067-2075. Takeuchi, T., Nakano, Y., Aoki, A., Ohmori, S. and Kasuya, M. (1990). Biol. TraceElem. Res. 26-27, 263-268.
Ski n an d Ad nexa o f th e La b o r a t o r y M ouse | John P Sundberg The Jackson Laboratory, Bar Harbor, ME, USA
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The skin is the largest of the intermediate-sized organs (Goldsmith, 1990). Dermatology, anatomy, and histology textbooks assign simple functions to this organ system which, in reality, is as complicated as any organ in the body. More importantly, it is integrated with every organ of the body, not simply a wrapping to hold things together. The list of functions of the skin are constantly expanding. Table 12.1 is a summary from a recent published debate on this topic (Chuong et al., 2002). Spontaneous and genetically engineered mutations in laboratory mice have changed the basis of our knowledge of the function of the skin and how gene expression in the skin may be a reflection of similar expression in different organs (Sundberg and King, 2000). For example, for a long time there was a general thought that mice without hair (alopecia) have some form of immunodeficiency. This was largely based on the nude mouse. These mutant mice appear to lack hair at the gross level and lack a cell mediated immune system due to failure of the thymus to develop normally. The Laboratory Mouse Copyright 2004 Elsevier ISBN 0- ! 233-6425-6
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In fact, these mice have hair follicles and produce hair fibers but the structures are defective due to the role of the nude gene (Foxnl"") that acts as a transcription factor to down regulate hard keratin production (Mecklenburg et al., 2001). This gene also plays a role in terminal differentiation of keratinocytes at various anatomic sites (Baxter and Brissette, 2002). Hairless (hr), another mutant mouse that has been available for
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over half a century (Gaskoin, 1856) also has a minor abnormality in its immune system (Sprecher et al., 1990). The advent of the severe combined immunodeficiency mutant mice (Prkdc 'cid) with normal pelage and hair cycle changed that limited correlation (Sundberg and Shultz, 1994). We now know that each skin defect can be unique and may or may not be associated with visceral lesions (Sundberg and King, 2000). Numerous mutations have occurred in laboratory mice spontaneously, induced by radiation or various chemical mutagens, or created using transgenesis or targeted mutagenesis (Sundberg, 1994a; Sundberg et al., 1995; Sundberg and King, 1996a, b; 2000; Nakamura et al., 2002a, b; Randall et al., 2003). It is beyond the scope of this chapter to cover these mutant mice. In addition to the reviews provided, much information is available on the Mouse Genome Informatics web site (www.informatics.jax.org). This chapter will provide an overview and references as sources for more specific information on normal anatomy, development, and cycling of the skin and its adnexa. It will also provide information on routine methods to prepare specimens for analysis. General, systematic descriptions of necropsy procedures evaluating all organ systems can be found in another chapter in this book.
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Clinical evaluation The normal mouse is covered with hair. Careful examination reveals that at least two hair types are obvious, as is the case with most domestic mammals. A fine short hair coat covers most of the body (truncal hairs) while long hairs are evident around the head (vibrissae, incorrectly called whiskers by many investigators since there is no anatomical similarity to androgen induced facial hair in humans). If the hair is carefully studied there are many hair types present. Within the pelage hairs there are classically four types: guard hairs are long, straight, thick, and protrude above the level of most hairs; auchene hairs are nearly as long as guard hairs with a gradual bend at the distal end; awl are also straight with a bend
Figure 12.1 Scanning electron micrograph of telogen stage plucked guard (A), awl (B),and zigzag (C) hairs from the dorsal truncal skin of an adult mouse. at the distal end but are short and thin; lastly zigzag hairs are the underhairs that have two bends giving in a 'Z' shape (Figure 12.1). These hair types are best differentiated in plucked samples examined with magnification. Historically many people stuck hairs on double sticky tape to a glass microscope slide. An easier method is to place a few hairs on a glass slide, add a drop of mounting medium, and drop on a cover slip. This forces the hairs to lay in one plane. The hairs can be examined with a microscope, photographed, and a variety of light sources used that can provide diagnostic information (Sundberg and Hogan, 1994; Sundberg et al., 1998). In addition to the pelage or truncal hairs there are many other specialized hair types in the mouse. The tail is covered with very short, broad fibers. Ears have a variety of very short fine fibers (Figure 12.2). Eyes have vibrissae above the eyelids and a network of long hairs protruding from the lid margins called cilia.
Figure 12.2 Subgross photograph of pilosebaceous units (hair follicles with the sebaceous gland at its base) in cleared skin from the ear of an adult mouse.
Figure 12.4 Anagen stage perianal hair. Anus (A),dermal or follicular papilla (DP) within the bulb, sebaceous gland (S), telogen stage truncal hair follicles (T).
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dissection microscope, scanning electron microscopy, or some other means of magnification. Hairs are usually thin and straight with a uniform distribution pattern within a strain. Variations, especially hair loss, can suggest that the mice have a mutant phentype but only after simple diagnostic methods rule out infectious causes or infestations. Ectoparasites remain common in many mouse rooms and will result in alopecia often mistaken the novice for a mutant phenotype (Figure 12.5). Mites are easily diagnosed by placing a piece of hairy skin in a dosed petri dish into a refrigerator then examining it after an hour or so with a hand lens. Mites migrate to the tips looking for another host. They can also be easily identified by histologically if hairs are not shaved during preparation of the skin (Figure 12.5). Other infectious diseases require the assistance of a trained veterinary pathologist for correct diagnosis.
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Vibrissae found around the mouth, eyelids, and near the foot pads on the lower legs (Figure 12.3). Perianal hairs are large thin structures that form a network above the opening of the anus (Figure 12.4). Hairs change around nipples and the base of the ear. These differences often can only be seen using a hand lens,
Tissue collection and preservation for histologic evaluation of the skin Every pathologist has his or her own preference for fixation of tissues. It is always best to work with the pathologist who will be evaluating the tissues before proceeding. Neutral buffered formalin solution is the most universal fixative used. Tissues are often left in formalin for long periods. Due to cross linking of amino groups by the aldehydes, many epitopes are modified making immunohistochemistry difficult or impossible. Fekete's acid alcohol formalin minimizes this problem, especially when tissues are transferred to 70% ethanol after overnight fixation. Commercially available zinc based preservatives are being promoted to
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maintain epitopes, optimize immunohistochemical results while maintaining some degree of the histologic quality that pathologists are used to with paraffin sections. Bouin's solution is popular as a general fixative but it hyalinizes collagen fibers so fine detail of the skin can be difficult to interpret. Use of Bouin's solution requires washing in tap water and transfer to ethanol. Failure to do so results in major artifacts often making the tissue unusable. These and other fixatives are discussed in Chapter 30 on Necropsy Methods which includes formulations for their preparation. Since skin, and especially hair follicles, vary dramatically by location, several locations should be sampled in order to evaluate potential changes. Collection of tissue consistently throughout a study will make specimens comparable. Dorsal skin can be collected over the thorax making sure to label cranial and caudal orientations so the tissue will be trimmed correctly. Ventral skin covering the thorax is also taken. Both dorsal and ventral skin are very similar histologically so they can be placed in separate cassettes when trimmed together with other skin that has distinct histologic features. Vibrissae on the head are collected by removing all the skin on the head as a complete unit. Vibrissae on the muzzle are trimmed as one piece. Eyelids are sectioned from this piece of skin as well to include upper and lower lids. Ear and tail are removed from the body and fixed by immersion. Tail skin can be removed from the bone and muscle or collected together. If the latter is done the bone must be decalcified. Footpads are also collected. Details are provided in Chapter 30 on Necropsy Methods and elsewhere (Sundberg et al., 1998; Relyea et al., 2000a). Nails are collected attached to the feet and digits. Distal limbs can be disarticulated and fixed in tom. If the paw is to be examined, it can be fixed under weight to lay it flat then sectioned horizontally to include all the joints after decalcification. Sagittal sections are the most useful. Digits are processed in toto and serially sectioned lengthwise after decalcification.
Scanning electron microscopy of the skin and hair fibers Scanning electron microscopy provides a detailed three dimensional view of structures at various magnifications. X-ray microanalysis can determine the relative elemental content of a specimen which may be useful for evaluation of some mutant mice. Hairs are made up of what used to be called the high sulfur keratins or hard keratins, now called the hair keratins. Changes in sulfur levels can be detected that suggest abnormalities in these hairs. Such is the case with the ichthyosis mutant mouse that has a form of trichothiodystrophy, low sulfur levels in the hair fibers (Figure 12.6) (Itin et al., 1990). Toxic agents, especially heavy metals, can also be identified using this method (Chatt etal., 1990; Takeuchi etal., 1990; Bache etal., 1991). Whole mounts of skin or nails can be easily made by removing tissues at the time of necropsy, spreading soft tissues out on a firm nylon membrane to fix them flat, and placing them in buffered glutaraldehyde using standard methods. Electron microscopists will critically point dry the specimen, coat it with gold, and then examine it with the investigator (Bechtold, 2000). Hairs can be examined in whole mounts or manually removed and examined individually. Adult mouse hair follicles are in telogen for prolonged periods so the hairs can be easily removed manually from lightly anesthetized animals without causing pain but more importantly, damage to fibers is rare since they come out easily. Hairs are placed in a dry vial and processed routinely. Shipments we receive from collaborating laboratories for evaluation are routinely disinfected on the outside surface and are then filled with 70% ethanol and stored for a week or longer before processing due to the common infestation of mites in m a n y research colonies. This approach kills the mites thus avoiding introduction into your colonies.
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microscopy of the skin and hair fibers Transmission electron microscopy can provide a great deal of information but is technically difficult and labor intensive. Tissue is removed during necropsy but should be finely minced into I mm 3 pieces since glutaraldehyde ftxatives do not penetrate tissues deeply. Cacodylate or phosphate buffered glutaraldehyde are commonly used but others are available and described elsewhere. Tissues should be stored refrigerated and embedded soon after collection to minimize artifacts (Bechtold, 2000).
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Other methods Many different methods have been developed to evaluate skin. We have tested a thermal imaging device that measures infrared radiated from mice under general anesthesia (Thermogenic Imaging, Billerica, MA). It appeared to be a useful device for determining response to treatment for mutant mice with thick, scaly, neovascularized skin or those with various forms of alopecia (Figure 12.7). Longitudinal studies revealed that thermal changes over time reflected the hair cycle in both mutant and control mice since the hypodermal fat layer, and therefore the insulation value of the skin, varied dramatically through the hair cycle. Transepidermal water loss is an important measurement in mice with abnormalities in the cutaneous water barrier. Mice are first sedated with 100 mg/kg ketamine HCI plus 0.5 mg/kg xylazine intraperitoneally. Dorsal hair is removed with electric clippers and then depilated for 5 min with a chemical agent such as Neet
O (Reckitt and Coleman, Wayne, NJ). Transepidermal water loss is measured 24 h later by placing a Servo Med Evaporimeter EPI probe (Servomed AB, Stockholm, Sweden) on the bald area (Setup and Jemec, 1995; Sundberg et al., 2000). Surface lipids can be collected by dipping euthanized mice into 40 ml of acetone 10 times and drying the acetone under argon gas. The residue is dissolved in toluene and plated in separate lanes on silica gel G chromatographic plates (Merck, Rahway, NJ). The plates are developed to 19 cm in hexane-ether-acetic acid (80:20:1). Following drying of the plate it is sprayed with 50% sulfuric acid (Downing and Stranieri, 1980; Sundberg etal., 2000). Kinetic studies can be easily done if considered at the time of necropsy. Mice can be injected with bromodeoxyuridine (50 ~g/gm body weight) 1 h before necropsy (Smith et al., 2000). A consistent time interval between injection and necropsy is critical since
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it will determine the rates at which this compound is incorporated into DNA currently being synthesized. Unstained sections are processed routinely for immunohistochemistry (Relyea et al., 2000b) and an antibromodeoxyuridine antibody used. Positive cells in 'S' or the DNA synthesis phase of the cell cycle will have nudei that are brown or red depending upon the chromogen and enzyme system used (Relyea et al., 2000b; Smith et al., 2000). An alternative is to use tritiated thymidine. This radionuclide requires special safety precautions, takes 3-6 weeks for development, and can be difficult to interpret so it is less commonly used today. Interpretation is complicated and can depend upon what types of proliferation rates are needed for evaluation of a particular mutant. Standard approaches are described for interfollicular skin such as counting the number of positive nudei per 1000 basal cell nuclei or per linear millimeter of skin, if it lays flat (Leblond etal., 1964; Skerrow and Skerrow, 1985; Kwochka and Rademakers, 1989; Kwochka, 1990; Sundberg et al., 1994a). Some mutant mice have marked proliferation of the infundibulum which requires modifications and special adaptation of counting criteria (Sundberg et al., 1997a). Gene arrays are a relatively new technology that is becoming more widely accessible to investigators. A variety of methods are now available. The critical starting
material is high quality RNA. What tissue to select and how to prepare it are developing and controversial topics as the technology evolves. We have used the entire skin of mice that develop a generalized cutaneous phenotype. The advantage is that an adequate volume can be obtained to provide enough RNA for many experiments. The disadvantage is that hair follicles in various stages are obtained, anatomically discreet areas are mixed and not all areas are affected. Assuming similar anatomical effects are found in age and gender matched controls, the differences in gene expression profiles should represent those related to the disease under investigation. More specific sites can be chosen as the disease is better understood (Carroll et al., 2002). The main advantage of the gene arrays is that complex pathways that can take a great deal of time to analyze using traditional methods can be screened with a small group of animals in a matter of days to generate the data but weeks to months to analyze it. For example, the mouse flaky skin (fin) mutation develops a psoriasiform demaatitis (Sundberg et al., 1994a; Sundberg et al., 1997b; Pelsue et al., 1998). One run with an Affymetrix Gene Chip | determined that this mutant mouse had primarily a Th2 immune response in contrast to the Thl response found in human psoriasis patients (Figure 12.8). This explained variations in
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F i g u r e 12.8 Keratin6 is abnormallyexpressedin the suprabasilarepidermis of the flaky skin (fsn) mutant mouse in a pattern similarto that of a human psoriasisbiopsy.Geneexpressionprofilesfrom these tissues revealeda different immunological mechanismunderlying clinically similarphenotypes (black bars representfsn/fsn mice or psoriatic human skin,gray bars represent +/+ normal littermate mice or normal human skin R N A ) .
therapeutic responses between the species (Carroll and Sundberg, unpublished data). Tissue arrays are the next technology with direct application to many research projects. Tissue arrays are build on traditional histology methods whereby paraffin blocks are systematically punched at prescribed sizes and the cores placed into predrilled holes in a new paraffin block (Moch et aL, 2001). Large numbers of tissues from many different organs or different case materials of similar lesions from the same organs can be used. This is providing a tool to specifically evaluate the cells producing proteins from the up and down regulated transcripts detected using gene arrays. Currently custom or predesigned arrays are commercially available. As the technology becomes more accepted, many institutions will probably be adopting it.
structure. The root sheaths begin to form and differentiate. Stage 5 is the bulbus peg stage with elongation of the inner root sheath and development of the bulge and first sebocytes. Melanin begins to form at this stage in pigmented mice. At stage 6 the follicle begins to extend below the level of the dermis into the hypodermal fat layer. The hair canal can now be identified. In stage 7 the tip of the hair fiber leaves the inner root sheath and enters the hair canal at the level of the infundibulum of the forming sebaceous gland. Stage 8 is the maximum length of the hair follicle where it extends down to the panniculus carnosus muscle and the hair fiber emerges through the epidermis. This process begins in utero and is completed for all follicle types by 5 days post partum when the hair is evident on the skin of most strains of normal mice. The epidermis develops from a single layer into a multilayered structure. In newborn mice it is thick at all anatomic sites and keratinocytes follow a classical differentiation scheme for stratified squamous epithelium (Figures.12.9). Cuboidal basal cells (keratins 5and 14 posmve) are located on the basal lamina (HogenEsch etal.,1999). Above this layer the cells differentiate into the statum spinosum or prickle-cell layer. Here the cells begin to elongate along the axis of the skin and have prominent intercellular bridges (desmosomes) that are evident under high magnification. These spine-like Each hair follicle type starts to develop at different structures are due to artifactual shrinkage of the tissues time points during embryogenesis. Therefore, it is not during preparation. This layer can be identified by the surprising to find clusters of hair follicles in a section presence of keratins 1 and 10. The next layer, the straat different stages of development. The large vibris- tum granulosum, has cells that are flattened along the sae develop earliest and are nearly fully developed by axis of the skin and contain prominent basophilic granbirth. In spite of this, all hair follicles develop in a sim- ules (keratohyalin granules). Two types of granules are ilar anatomic fashion. This developmental scheme is present in the mouse, profilaggrin (P) and loricrin (L) detailed both historically and anatomically elsewhere granules. The larger profllaggrin granules are the blue (Paus etal., 1999) and serves as a guide for the summary structures seen by light microscopy (Presland et al., below. 2000). The most superficial layer, the stratum corneum, The sequential stages of hair follicle development is brightly eosinophilic and consists of compacted, flatbegin with the pregerm stage which is hard to recog- tened keratinocytes. This is the critical portion of the nize histologically but can be defined with various skin that provides a strong aqueous barrier due to the immunohistochemical markers. It consists of a sharply presence of lamellar bodies, small lipid based structures demarcated plaque of basal and suprabasal epidermal only detectable by special staining and transmission keratinocytes. In stage 1, the pregerm develops into an electron microscopy (Elias, 1988). histologically evident epidermal thickening where the The epidermis of a newborn mouse is thick. As the keratinocytes display a vertically polarized orientation mouse ages, within two weeks, the truncal epidermis compared with the more cuboidal appearance of adja- thins to only about two cell layers with the stratum cent basal cells. Concurrently, dermal fibroblasts granulosum and corneum becoming very thin and increase in number immediately below this structure often hard to visualize by light microscopy. Other forming what will become the dermal (follicular) anatomic sites do not change. The tail skin remains papilla. Stages 2-4 produce a column of epidermal thick throughout the mouse's life. The muzzle skin is keratinocytes that develop a cap, invagination of the thinner than at birth but thicker than truncal skin. dermal papilla, and formation of the basic hair follicle Foot pads remain thick.
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This anatomy of the skin and hair follicles are illustrated in Figures 12.4 and 12.9. The top layer of epithelial cells is called the epidermis. This layer differentiates from the cuboidal basal cells in the stratum basale into the polygonal cells of the stratum spinosum, then more flattened cells with fine blue granules in the stratum granulosum, and ultimately into the flat cells that lack a nucleus and become very eosinophilic at the surface in the stratum corneum. The outermost layer of cells separating from the surface are sometimes called the stratum dysjunctum. The hair follicle is a very complicated structure that invaginates into the dermis and hypodermal fat undergoing major changes on a regular basis with the hair cycle (see below). A large gland protrudes from its side that consists of swollen pale cells with fine uniform vacuoles. These are sebaceous glands that produce oils to coat the surface of the skin and hair fiber. The oils can be visualized in frozen sections stained with oil red O, sudan black, or other histologic means to follow how the lipids spread out over the
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surface of the skin in normal compared with mutant mice (Sundberg et al., 1997a). The dermis consists of dense irregular collagenous connective tissue, elastic connective tissue, blood vessels, nerves, smooth muscle (arrector pili muscles that lift hair follicles and fibers; Figure 12.10), and a variety of individual cell types including fibroblasts, mast cells, and small numbers of cells from the immune system. One important feature of skin that is characteristically found in many rodents, especially laboratory mice, is that apocrine sweat glands are not present. Modified apocrine glands, mammary glands are abundant because of the large litter size most mice have (Sundberg et al., 1996). The mouse does not normally have f e t e ridges where the lower aspect of the epidermis forms ridges of cells that extend into the dermis. The dermis between such ridges is commonly called the dermal papillae, a term also used by hair biologists for the specialized fibroblasts that populate the base of an anagen hair follicle called the bulb. Because of this, the fibroblasts within the bulb are also called the follicular papilla. Rete ridge-like structures become prominent when mouse skin heals following ulceration. These changes resemble those found in neoplasms of the epidermis such that the changes are referred to as pseudoepitheliomatous or pseudocarcinomatous hyperplasia. Below the dermis is a layer of fat, the hypodermal fat layer. The thickness of this fat layer changes with the hair cycle being thickest during anagen when follicles need a great deal of energy to produce a hair
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fiber. The panniculus muscle separates the hypodermal fat layer from the adventitia, loose collagenous connective tissue that attaches the skin to the underlying musculature and fat. Mammary glands are found in the fat below this skeletal muscle layer,
limited to the bulb of anagen follicles and hair fibers. Interfollicular epidermis rarely contains pigment and when it does, it is usually only in mutant mice (Sundberg et al., 1994b). If the mice are shaved, irregular pigmented areas will be seen. These are areas containing anagen follicles. If mice are followed daily, these Hair cycle in the m o u s e pigmented patches will migrate caudally. This feature is ~' dramatic in mutant mice such as hairless. These mice The skin undergoes major changes during the first have normal hair 5 days after birth but not subsequent 2 weeks of life (Figure 12.9). Hair follicles continue to hair cycles. Beginning at 2 weeks of age their hair is develop and enter late stage anagen 5 days postpartum shed from head to tail (Sundberg, 1994a). Other hair when hair fibers emerge through the epidermis. The follicle types have different hair cycles. This is why truncal epidermis is relatively thick at birth and thins hairless mice appear to retain vibrissae while being to normal by 2-3 weeks of age. Hair follicles produce completely bald. The length of the hair cycle deterfibers until around 14 days of age over the thorax at mines the length of the hair fiber. This is consistent which time the lower portion undergoes apoptosis. The with why hairless mice have long, persistent vibrissae dermal papilla is retracted by actin filaments (Figure while short pelage hairs are lost. This feature was 12.10) that reside below the isthmus in the resting or demonstrated with angora (Fgf5 g~ mutant mice that telogen stage until the hair cycle is reinitiated. This have a three day prolongation of their truncal hair cycle usually lasts about 3 days in young mice. The follicle and as a result often have long, shaggy hair compared develops into a new anagen stage follicle pushing the with normal littermates (Sundberg et al., 1997c). Numerous genes regulate development and cycling old follicle laterally. The new fiber emerges adjacent to the old one. At some point the old fiber is lost in what of the hair follicles (Hardy, 1992; Pans, 1996, 1998; is now called the exogen stage (Milner et al., 2002). Millar, 1997; Chuong and Noveen, 1999; Stenn and The general features of the different stages of the hair Paus, 2001; Millar, 2002). Classic work done half a cycle are illustrated in Figure 12.9. Development of the century ago detailed changes in the skin and hair follihair follicle embryologically and progression through cles as they cycle, not just the changes in the follicles the hair cycle regularly throughout life have been dis- but also changes in sebaceous gland size and shape as sected anatomically and with molecular and immuno- well as the thickness of the hypodermal fat layer (Chase logical markers to differentiate numerous stages within et al., 1951, 1953; Chase, 1954, 1955; Chase and each major portion of the hair cycle that have been Eaton, 1959; Straile, 1960, 1965, 1969; Straile et al., detailed and reviewed elsewhere (Pans et al., 1999; 1961). Furthermore, hormones cause changes as well Muller-Rover et al., 2001; Millar, 2002). What is com- (Deplewski and Rosenfield, 2000). These are impormonly called the second hair cycle, or the first real hair tant to understand when comparing differences cycle after embryogenesis, has a short anagen stage and between wild type, normal mice, and mutant mice. prolonged telogen stage. The hair cycles in a cranial to Not only should the mice be age and gender matched caudal pattern that can be easily visualized in pigmented in such studies but it is critical to match the stage of the mice. Unlike humans, pigment in the mouse skin is hair cycle as well.
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The mouse has nails or claws on each digit, just like most other mammals including humans. The name claw suggests these structures are different than human nails which may be why little attention has been paid to them. In fact, anatomically they are very similar at the gross and histologic levels. Human nails are dorsoventrally flattened to form a plate while rodent nails are laterally flattened. These changes, not restricted to humans since similar refinements are found in many nonhuman primates, are associated with the function of the nails in primates as a refined tool associated with manual dexterity rather than as a weapon or digging tool. Sagital sections illustrate that mice have a nail matrix, nail plate, nail bed, hyponychium, and other structures (Figure 12.11) identical to but smaller than the human nail. To veterinarians this is not at all surprising since all mammals have nails or claws that are variations on this general theme. Nails can be extremely difficult to prepare and interpret histologically. However, changes in mutant mice can be dramatic when these structures are magnified with a dissection microscope or by scanning electron microscopy (Sundberg and King, 2000).
Other specialized glands
cycle that will not be discussed here. Other glands found at specific anatomic sites are modified sebaceous glands, a type of holocrine gland. These include preputial and clitoral glands around the genitals, meibomian glands in the eyelid, and cerruminous glands within the outer ear. All are large glands with a structure similar to that found in the sebaceous glands associated with hair follicles. The major difference is that each has a stratified squamous epithelial lined duct that empties directly onto the structure where it is located (Sundberg and King, 2000). Hair follicles are specialized and have sebaceous glands associated with them that vary in size. The most notable are the perianal hairs which have large sebaceous glands. Salivary, lacrimal, and harderian glands are very different and are described in chapters dealing with the organs they are associated with.
Skin and adnexal mutant phenotypes It is beyond the scope of this chapter to describe or even list all mutant mice with skin and/or hair/nail phenotypes. As a general starting point we have grouped phenotypes into 10 classes (Table 12.2). Detailed lists, descriptions, references, and illustrations are published elsewhere (Sundberg, 1994a; Sundberg and King, 1996a, b, 2000; Nakamura et al., 2002a, b; Randall et al., 2003).
Mammary glands are specialized forms of apocrine sweat glands with a complex developmental and lactation
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Figure 12.11 Normal sagital section of the nail from an adult mouse (A). Hyponechium (H), nail plate (NP), nail bed (B), proximal nail fold (PNF), matrix (M), phalanx 1 (P1), phalanx 2 (P2), sesamoid bone (S),eccrine gland (EG),foot pad (FP). High magnification of a sesamoid bone under P2.
TABLE 12.2: General categories of mutant mouse cutaneous phenotypes Hair and skin color (pigmentation) Eccrine gland defects Sebaceous gland defects Primary scarring disorders Hair follicle cycling disorders Structural defects of hair fibers Hair texture abnormalities Missing hair fiber and follicle types Noninflammatory (ichthyosiform and keratodermas) skin diseases Inflammatory (psoriasiform and proliferative) skin diseases Papillomatous skin diseases Cutaneous carcinogenesis Bullous and acantholytic skin diseases Structural and growth defects of the nails
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