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Complex Hair Cycle Domain Patterns and Regenerative Hair Waves in Living Rodents

      Single hair follicles go through regeneration and involution cycles. In a population, hair follicles may affect each other during anagen re-entry, thus forming propagating regenerative hair waves. We review these regenerative hair waves and complex hair cycle domains, which were recently reported in transgenic mice. Two non-invasive methods to track the propagating hair wave in large populations of hair follicles in vivo are described. We also reviewed early accounts of “hair growth patterns” from classical literature. We decipher the “behavior rules” that dictate how dynamic hair waves lead to complex hair cycle domains. In general, a single domain expands when a regenerative hair wave reaches a responsive region and boundaries form when the wave reaches a non-responsive region. As mice age, multiple hair cycle domains form, each with its own regeneration rhythm. Domain patterns can be reset by physiological events such as pregnancy and lactation. Longitudinal sections across domains show arrays of follicles in a continuum of hair cycle stages. Hair cycle domains are different from regional specificity domains. Regenerative hair waves are different from the developmental wave of newly forming hair follicles. The study provides insights into the dynamic states of adult skin and physiological regulation of organ regeneration.

      Introduction

      Understanding the regulation of organ regeneration is of fundamental importance in the age of stem cell biology and regenerative medicine. Hair follicles make a great model for this objective because they molt and regenerate repetitively in the adult as part of their physiological process.
      When we study the regenerative behavior of an organ, most of the time, we focus on the single organ such as the cycling of a single hair follicle. However, there are thousands of hair follicles on the mouse skin, giving another dimension of organ regenerative behavior, one based on population. We became interested in this process when we observed the complex and dynamic hair cycle domains in C.Cg-Msx2tm1Rilm/Mmcd mice (
      • Ma L.
      • Liu J.
      • Wu T.
      • Plikus M.
      • Jiang T.X.
      • Bi Q.
      • et al.
      “Cyclic alopecia” in Msx2 mutants: defects in hair cycling and hair shaft differentiation.
      ). The propagation of a regenerative hair wave in BomTac:NMRI-Foxn1nu, B6.Cg/NTac-Foxn1nu NE9, and B6129-Foxn1tw mice was also observed (
      • Militzer K.
      Hair growth pattern in nude mice.
      ;
      • Suzuki N.
      • Hirata M.
      • Kondo S.
      Traveling stripes on the skin of a mutant mouse.
      ). We also found that this phenomenon and its relationship with systemic hormone levels have been reported in classical literature in mid-twentieth century. We have learned that it is hair wave dynamics that lead to the formation of complex hair cycle domains. Here, we review classical literature and more recent works, extend the study to document the process, and present a simple method that would allow investigators to examine the hair growth pattern on living mice. The study provides insights into the dynamic states of adult skins and physiological regulation of organ regeneration.

      Editor's note

      In this issue the Perspectives series continues with highlights from the 56th Montagna Symposium on the Biology of Skin, which focused on skin appendages, their development, and disease. In this issue, Plikus et al. review the hair cycle of mice and the insight that study of hair can provide insightinto the growth and development of the skin as a whole. In the second article, Huntzicker and co-workers review the reciprocal signaling that occurs among hair follicles, the dermis, and epidermis, and the role of that signaling in hair follicle development and appendage neoplasms. Hair was a long-standing interest of Dr Montagna's, as illustrated by one of his first publications entitled “Relation of hair proliferation to damage induced in the mouse skin” (J Invest Dermatol 19:83, 1952) and his later publication, “Reinnervation of hair follicle end organs and Meissner Corpuscles in skin grafts of Macaques” (J Invest Dermatol 78:210, 1982). This month's Perspectives are a fitting tribute to Dr Montagna's dedication to the science of dermatology and to learning together by “talking about the skin” through the JID and 56 years of Montagna Symposia.
      Russell P. Hall, III
      Deputy Editor

      Hair Growth In A Population of Hair Follicles

      A single hair follicle undergoes successive growth cycles. The four major stages of the hair growth cycle are as follows: anagen (the period of active growth), catagen (the period of cessation of the growth and regression), telogen (the period of relative inactivity), and exogen (the event of the old hair fiber shedding;
      • Dry E.
      The coat of the mouse (Mus musculus).
      ;
      • Chase H.B.
      • Rauch H.
      • Smith V.W.
      Critical stages of hair development and pigmentation in the mouse.
      ;
      ;
      • Fuchs E.
      • Merrill B.J.
      • Jamora C.
      • DasGupta R.
      At the roots of a never-ending cycle.
      ;
      • Stenn K.S.
      • Paus R.
      Control of hair follicle cycling.
      ;
      • Milner Y.
      • Sudnik J.
      • Filippi M.
      • Kizoulis M.
      • Kashgarian M.
      • Stenn K.
      Exogen, shedding phase of the hair growth cycle: characterization of a mouse model.
      ). Morphological changes and cellular events associated with the hair growth cycle have been well documented. Signaling pathways operating at different stages of the cycle are the subjects of extensive studies. Multiple signaling pathways have been implicated in the regulation of follicular stem cells’ dynamics, growth activities, and the transition between cycle phases, including bone morphogenetic protein, Wingless-related MMTV integration site, sonic hedgehog, fibroblast growth factor, neurotrophins, transforming growth factor β, hepatocyte growth factor, IFN-γ, retinoids (reviewed by
      • Cotsarelis G.
      Epithelial stem cells: a folliculocentric view.
      ;
      • Plikus M.V.
      • Sundberg J.P.
      • Chuong C.M.
      Mouse skin ectodermal organs.
      ;
      • Fuchs E.
      Scratching the surface of skin development.
      ).
      In a population of hair follicles, hair growth cycles can be mosaic, when neighboring hair follicles proceed through their own cycle stages autonomously. This is observed in vibrissae follicles (
      • Greaves D.K.
      • Hammill M.O.
      • Eddington J.D.
      • Pettipas D.
      • Schreer J.F.
      Growth rate and shedding of vibrissae in the gray seal, Halichoerus grypus: a cautionary note for stable isotope diet analysis.
      ) and pelage hair follicles in the guinea-pig (
      • Chase H.
      Growth of the hair.
      ). At the same time, extraneous stimuli can influence the dynamics of hair cycle progression across the entire animal's skin, synchronizing anagen entry. The effect of systemic stimuli on the hair growth cycle was recognized. For example, estrogens have profound systemic influence on the hair growth cycle. In many animals, including mice, estrogens such as 17β-estradiol inhibit anagen initiation (
      • Fraser A.S.
      • Nay T.
      Growth of the mouse coat II. Effects of sex and pregnancy.
      ). This effect of 17β-estradiol is mediated via estrogen receptors expressed in hair follicles (
      • Ohnemus U.
      • Uenalan M.
      • Conrad F.
      • Handjiski B.
      • Mecklenburg L.
      • Nakamura M.
      • et al.
      Hair cycle control by estrogens: catagen induction via estrogen receptor (ER)-alpha is checked by ER beta signaling.
      ). Rapid and profound anagen induction in female mice can be achieved by gonadectomy (
      • Chanda S.
      • Robinette C.L.
      • Couse J.F.
      • Smart R.C.
      17β-estradiol and ICI-182780 regulate the hair follicle cycle in mice through an estrogen receptor-alpha pathway.
      ). Application of extraneous 17β-estradiol extends telogen. Prolactin is another potent systemic modulator of the hair growth (
      • Pearson A.J.
      • Parry A.L.
      • Ashby M.G.
      • Choy V.J.
      • Wildermoth J.E.
      • Craven A.J.
      Inhibitory effect of increased photoperiod on wool follicle growth.
      ,
      • Pearson A.J.
      • Ashby M.G.
      • Wildermoth J.E.
      • Craven A.J.
      • Nixon A.J.
      Effect of exogenous prolactin on the hair cycle.
      ;
      • Nixon A.J.
      • Ford C.A.
      • Wildermoth J.E.
      • Craven A.J.
      • Pearson A.J.
      Regulation of prolactin receptor expression in ovine skin in relation to circulating prolactin and wool follicle growth status.
      ;
      • Craven A.J.
      • Nixon A.J.
      • Ashby M.G.
      • Ormandy C.J.
      • Blazek K.
      • Wilkins R.J.
      • et al.
      Prolactin delays hair regrowth in mice.
      ). Commonly, in animals with seasonal hair growth an increase in pituitary prolactin during spring induces new anagen (
      • Dicks P.
      The role of prolactin and melatonin in regulating the timing of spring moult in the Cashmere goat.
      ). Prolactin has also been observed to be produced locally and function in regulating the hair cycle in a non-systemic way (
      • Craven A.J.
      • Ormandy C.J.
      • Robertson F.G.
      • Wilkins R.J.
      • Kelly P.A.
      • Nixon A.J.
      • et al.
      Prolactin signaling influences the timing mechanism of the hair follicle: analysis of hair cycles in prolactin receptor knockout mice.
      ;
      • Foitzik K.
      • Krause K.
      • Nixon A.J.
      • Ford C.A.
      • Ohnemus U.
      • Pearson A.J.
      • et al.
      Prolactin and its receptor are expressed in murine hair follicle epithelium, show hair cycle-dependent expression, and induce catagen.
      ).
      Although systemic endocrine factors regulating synchronized hair growth have been elucidated, the mechanism of localized hair cycle coordination within neighboring hair follicles is not yet understood. At the same time, a wealth of morphological evidence shows the existence of such localized coordination of hair cycling. Classic literature contains many experimental accounts of what appears to be patterns or waves of hair growth in mouse, rat, hamster, chinchilla, and rabbit (Figure 1;
      • Mottram J.C.
      Effect of change of coat on the growth of epidermal warts in mice.
      ;
      • Durward A.
      • Rudall K.M.
      Studies on hair growth in the rat.
      ;
      • Whiteley H.J.
      • Ghadially F.N.
      Hair replacement in the domestic rabbit.
      ;
      • Whiteley H.J.
      Studies on hair growth in the rabbit.
      ;
      • Chase H.B.
      • Eaton G.J.
      The growth of hair follicles in waves.
      ). Such waves were defined as “orderly progression in time and space of follicles entering the growth phase, that is, anagen, of their cycles” (
      • Chase H.B.
      • Eaton G.J.
      The growth of hair follicles in waves.
      ). Early observations showed that hair follicles on the trunk of rats cycle in the form of successive waves, spreading from the ventral side of the body to the dorsal over the trunk. This spreading can be visualized as zones of skin, variable in width, with hair follicles in the anagen growth phase (
      • Mottram J.C.
      Effect of change of coat on the growth of epidermal warts in mice.
      ;
      • Durward A.
      • Rudall K.M.
      Studies on hair growth in the rat.
      ). The width of these zones was shown to decrease with the age (
      • Butcher E.O.
      Hair growth on skin transplants in the immature albino rat.
      ;
      • Durward A.
      • Rudall K.M.
      Studies on hair growth in the rat.
      ). It was also mentioned that in the head region and around the limbs the pattern of hair growth is more complicated (
      • Durward A.
      • Rudall K.M.
      Studies on hair growth in the rat.
      ). Others point out that initial wave-like hair cycle patterns breakdown into “islands of growth, especially on the dorsum” (
      • Chase H.B.
      • Eaton G.J.
      The growth of hair follicles in waves.
      ).
      Figure thumbnail gr1
      Figure 1Evidence of hair cycle domain patterns in rodents from classic literature. (a) Identification of hair growth patterns in albino rats based on the appearance of new orange-pigmented hairs upon systemic administration of flavin (
      • Haddow A.
      • Elson L.A.
      • Roe E.M.F.
      • Rudall K.M.
      • Timmis G.M.
      Artificial production of coat colour in the albino rat. Its relation to pattern in the growth of hair.
      ; figure reprinted with permission from Nature Publishing Group). (b) Identification of hair growth patterns upon shaving of pigmented rats (
      • Durward A.
      • Rudall K.M.
      Studies on hair growth in the rat.
      ). (c) Identification of hair growth patterns and speed of anagen spreading waves in pigmented mice upon topical treatment with 8-hydroxyquinoline (
      • Searle C.E.
      The selective depigmenting action of 8-hydroxyquinoline on hair growth in the mouse.
      ).

      Observing Hair Waves On Living Mice

      Despite the presence of well-documented accounts of patterned cycling behavior in classic literature, little attention is given to this phenomenon in most of the current studies on the hair cycle. As a standard, hair follicles from the dorsal skin from post-natal day 1 to day 12 are typically used to represent anagen, from day 17 to represent catagen, and from day 21 to represent telogen (
      • Paus R.
      Principles of hair cycle control.
      ;
      • Ma L.
      • Liu J.
      • Wu T.
      • Plikus M.
      • Jiang T.X.
      • Bi Q.
      • et al.
      “Cyclic alopecia” in Msx2 mutants: defects in hair cycling and hair shaft differentiation.
      ). Generally, this is correct because hair follicles on dorsal skin during the first month cycle in a nearly synchronized manner. However, dorsal skin in mature and aging mice soon develops more complicated patterns. It would be critical to account for these changing hair cycle patterns while designing experiments related to hair regeneration and cycling, especially because these patterns can also be modulated by physiological events, such as pregnancy (
      • Johnson E.
      Quantitative studies of hair growth in the albino rat. II. The effect of sex hormones.
      ). Classic studies on patterned cycling behavior are controversial and limited to an ambiguous description of the patterning process. Thus, it is important to clearly establish the presence and dynamics of hair cycle patterns in normal rodents as a baseline. Moreover, comparative studies of the altered patterns of hair cycle domains in mutant mice versus control mice may provide new insights into hair cycle control mechanisms. Analysis of these phenomena will help us understand the requirements for hair follicle regeneration in adults. Furthermore, the continuous distribution of hair follicles at different hair cycle stages can be analyzed in longitudinal sections of a single skin strip to facilitate more precise molecular profiling of hair cycle stages. Finally, it will also encourage the development of new models to explain complex hair pattern formation in living mammals.
      To identify hair cycle stages of follicles in the living mouse, we need to have non-invasive assays that can reveal recognizable traits of these specific stages. Currently, several methods are available for research of this nature. The first method is based on pigmentation; this method was utilized in most of the classic studies. In pigmented mice, such as inbred C57BL/6J mice, melanin production is restricted to the hair follicles and not to interfollicular skin. Melanogenesis starts at anagen IIIa, becomes prominent in anagen IIIb, and continues until catagen (so-called anagen-coupled melanogenesis;
      • Muller-Rover S.
      • Handjiski B.
      • van der Veen C.
      • Eichmuller S.
      • Foitzik K.
      • McKay I.A.
      • et al.
      A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.
      ; Figure 2a and b). Therefore, hair follicles during most of anagen appear gray or black, while hair follicles in telogen have no pigment and the skin becomes pink. When the mouse is covered by hairs (both anagen and telogen club hairs), the pigment difference among hair follicles is concealed. When hairs adjacent the skin surface are clipped, differences in pigmentation of the proximal hair follicles become apparent (
      • Slominski A.
      • Paus R.
      Melanogenesis is coupled to murine anagen: toward new concepts for the role of melanocytes and the regulation of melanogenesis in hair growth.
      ). Patchy regions (domains) of black and pink are revealed, corresponding to areas of hair growth and quiescence. These domains are seen on both the dorsal and ventral sides of the body (Figures 3, 4a and b). They appear because pelage hair follicle populations in the mouse do not cycle independently, but rather show coordinated hair cycle stages within the same domain and discordant stages among adjacent domains. Interestingly, these domains develop into complex patterns from cycle to cycle as age progresses.
      Figure thumbnail gr2
      Figure 2Schematic illustration showing three non-invasive methods used to visualize propagating regenerative wave and hair cycle domains in living animals. (a) Hair follicles are in a continuum of hair cycle stages. (b) Changes of pigmentation. In pigmented mice (such as C57BL/6J), pigment starts to be produced in hair follicles in anagen IIIA and disappears in catagen. However, hair filaments obscure these patterns, and pigmented versus non-pigmented regions can be revealed clearly after hair clipping. (c) Biolabeling with 8-hydroxyquinoline. Upon systemic 8-hydroxyquinoline administration, melanogenesis is ablated in very early anagen hair follicles, resulting in formation of bands of white hairs in pigmented mice. (d) Cyclic alopecia. In mice with cyclic alopecia, hair filaments are shed at a specific hair cycle stage, resulting in hairy and alopecic regions, helping visualization of domains in different stages.
      Figure thumbnail gr3
      Figure 3Propagation of hair regenerative wave in the ventral skin. Hair cycle was followed in (a) the first mouse for 89 days and (b) for 93 days in the second mouse. Propagation of hair regenerative wave forms on both dorsal (
      • Ma L.
      • Liu J.
      • Wu T.
      • Plikus M.
      • Jiang T.X.
      • Bi Q.
      • et al.
      “Cyclic alopecia” in Msx2 mutants: defects in hair cycling and hair shaft differentiation.
      , Plikus et al., in press) and ventral skin.
      Figure thumbnail gr4
      Figure 4Complex patterns of hair cycle domains and topographic sector map of the mouse skin. (a, b) Visualization by cyclic changes of pigmentation. (a) Dorsal and (b) ventral view of wild-type (C57BL/6J) mice. The far left panels: mice before hair clipping. To the right: mice after clipping. Two similar looking black mice can show entirely different patterns of pigmented/non-pigmented skin regions after clipping. (c) Visualization by cyclic alopecia. Two C.Cg-Msx2tm1Rilm/Mmcd mice show distinct hairy/alopecic regions. Far right panels: to help describe the pattern, we propose to use arbitrary topographic sector maps of the mouse skin. There are 12 dorsal and 12 ventral sectors, D, dorsal; L, lateral; M, medial; V, ventral. Parentheses are used to indicate that two domains are connected. For example, the mouse marked with # can be described as (DL1, DL3, DM3; DM4, DR1, DR3). The mouse marked with ^ can be described as (VR2; VM2, VM3, VM4; VL2). It must be emphasized that the sector map is for convenience of description, and its borders do not correspond exactly with boundaries of hair cycle domains. (d, e) Similar hair cycle domain patterns can also be identified in (d) albino Crl:CD1(ICR) and (e) agouti C3H/HeJ strains of mice. In C3H/HeJ agouti mice, the hair fiber pigmentation is yellowish in the distal (eumelanin pigmentation) but black in the more proximal region (pheomelanin pigmentation). This helps us visualize the molting line (flanked by red dots) or the wave front of the hair cycle domain. Bars=(ad) 20 mm; (e) 20 and 2 mm.
      Although less distinctive than the method described above, hair clipping can be used to reveal hair cycle patterns in non-pigmented strains of mice, such as albino Crl:CD1(ICR) mice. This reveals white (anagen) patches on otherwise pink (telogen) skin (Figure 4d). Additionally, hair cycling can be monitored by observing regrowth of white hairs in albino animals after artificial coloring of the coat (
      • Whiteley H.J.
      • Ghadially F.N.
      Hair replacement in the domestic rabbit.
      ), or by the appearance of new orange-pigmented hairs upon intraperitoneal administration of flavin 9-phenyl-5:6-benzoisoalloxazine dissolved in oil (Figure 1a;
      • Haddow A.
      • Elson L.A.
      • Roe E.M.F.
      • Rudall K.M.
      • Timmis G.M.
      Artificial production of coat colour in the albino rat. Its relation to pattern in the growth of hair.
      ). In mice with an agouti coat, such as C3H/HeJ mice, hair clipping allows distinction not only between anagen and telogen but also between early and late anagen stages. This is possible due to the fact that in agouti mice hair follicles switch from pheomelanin to eumelanin production during anagen (
      • Bultman S.J.
      • Michaud E.J.
      • Woychik R.P.
      Molecular characterization of the mouse agouti locus.
      ). Because the switch between alternative pigments occurs at the same anagen stage in all hair follicles, a black band of anagen hairs enables visualization and monitoring of the timing of the anagen spreading wave (Figure 4e). It is also possible to monitor speed of the anagen spreading wave by artificially ablating melanogenesis in pigmented mice with 8-hydroxyquinoline and measuring width of bands of white hairs (Figure 1c). Upon application, 8-hydroxyquinoline inhibits melanogenesis (via unknown mechanism) only in the very early anagen hair follicles and for the rest of their hair cycle (
      • Searle C.E.
      The selective depigmenting action of 8-hydroxyquinoline on hair growth in the mouse.
      ). Late anagen hair follicles are not affected. By modulating frequency of 8-hydroxyquinoline treatment, one can lengthen or shorten a time window during which melanogenesis is ablated at the front of the continuously spreading anagen wave.
      The second method is based on the recently documented “cyclic alopecia” phenotype described by
      • Ma L.
      • Liu J.
      • Wu T.
      • Plikus M.
      • Jiang T.X.
      • Bi Q.
      • et al.
      “Cyclic alopecia” in Msx2 mutants: defects in hair cycling and hair shaft differentiation.
      . This phenotype is characterized by multiple hair growth domains giving the skin an appearance of patterned patchiness. Basic requirements for the cyclic alopecia phenotype are (1) that hair follicles continuously undergo regenerative cycling and (2) that hair shafts dislodge from the follicle at a particular stage of the hair cycle, making affected skin regions alopecic until hair follicles regenerate during the next anagen (Figure 2a and c). During recent years, increasing numbers of transgenic mice displaying cyclic alopecia phenotype have been reported. Thus far, the cyclic alopecia phenotype has been documented in C.Cg-Msx2tm1Rilm/Mmcd mice with defective club hair formation (
      • Ma L.
      • Liu J.
      • Wu T.
      • Plikus M.
      • Jiang T.X.
      • Bi Q.
      • et al.
      “Cyclic alopecia” in Msx2 mutants: defects in hair cycling and hair shaft differentiation.
      ;
      • Plikus M.
      • Chuong C.M.
      Making waves with hairs.
      ), in B6;129X1-Dsg3tm1Stan/J mice with defective desmosomal attachment of telogen hair to the outer root sheath (
      • Koch P.J.
      • Mahoney M.G.
      • Cotsarelis G.
      • Rothenberger K.
      • Lavker R.M.
      • Stanley J.R.
      Desmoglein 3 anchors telogen hair in the follicle.
      ), in BomTac:NMRI-Foxn1nu, B6.Cg/NTac-Foxn1nu NE9, and B6129-Foxn1tw mice with the defect in Foxn1 gene (
      • Militzer K.
      Hair growth pattern in nude mice.
      ;
      • Suzuki N.
      • Hirata M.
      • Kondo S.
      Traveling stripes on the skin of a mutant mouse.
      ), in transgenic mice overexpressing gain-of-function allele of Notch1 under the involucrin promoter (B6C3-Tg(IVL-Notch1)IC;
      • Uyttendaele H.
      • Panteleyev A.A.
      • de Berker D.
      • Tobin D.T.
      • Christiano A.M.
      Activation of Notch1 in the hair follicle leads to cell-fate switch and Mohawk alopecia.
      ), in B6-Dsc1tm1Dga (
      • Chidgey M.
      • Brakebusch C.
      • Gustafsson E.
      • Cruchley A.
      • Hail C.
      • Kirk S.
      • et al.
      Mice lacking desmocollin 1 show epidermal fragility accompanied by barrier defects and abnormal differentiation.
      ) and in Ppp3r1tm1Grc/Ppp3r1tm1Grc; +/KRT5-cre mice (
      • Mammucari C.
      • Tommasi di Vignano A.
      • Sharov A.A.
      • Neilson J.
      • Havrda M.C.
      • Roop D.R.
      • et al.
      Integration of Notch 1 and calcineurin/NFAT signaling pathways in keratinocyte growth and differentiation control.
      ).
      Analysis of hair cycle patterns can be made more informative by overlapping an anagen hair pigmentation pattern with a static hair distribution pattern. Because each pelage hair follicle is associated with a sebaceous gland (forming what is known as pilo-sebaceous unit), its position on the skin surface can be marked by Oil red fat staining. According to this method, staining of skin is performed prior to its removal from a killed mouse; this is performed to prevent background staining of subcutaneous fat. The shaved skin of the killed mouse is then soaked with propylene glycol, followed by incubation with standard Oil red dye. Upon completion of the staining (after 30–40 minutes) and washing, the position of individual pilo-sebaceous units is clearly visualized as bright red dots on a pale pink skin. Skin can be then removed and inverted, which allows more precise and informative evaluation of anagen spreading wave dynamics over the static hair pattern (Figure 5a).
      Figure thumbnail gr5
      Figure 5Cellular basis of a hair cycle domain. (a) Whole mount and (b) longitudinal sections through the hair cycle domain. The region with the most advanced anagen hair follicles (anagen VI in this case) probably started the hair cycle earliest. Adjacent hair follicles to the left are seen in gradient stages from anagen V to I. This represents a spreading wave of hair cycle activation. A boundary of the hair cycle domains forms when a skin region does not respond to the spreading wave and remains in telogen. The blank arrow is used to point to the direction of the spreading waves. Apparently, the spreading can occur at a different rate (ranging from 1 to 12.5 mm day−1, n=24), and the mechanism remains to be investigated. Bars= (a, b) 1 mm and 200 μm.

      Deciphering Principles of Hair Follicle Population Behavior

      Hair cycle patterns differ at varying stages of an animals’ life. Generally, patterns increase in complexity with age. It is important to know the degree of pattern complexity in mice of different ages to select animals of the appropriate hair cycle stage best suited for the experimental intent.
      • Chase H.B.
      • Eaton G.J.
      The growth of hair follicles in waves.
      describe the following stages of hair cycle pattern progression in mice based on their analysis of 230 mice for at least the first five hair cycles:
      • (1)
        The first hair cycle “occurs as the follicles develop from the epidermis and the order of development is from the dorsum toward the venter (abdomen) and extremities. This is scarcely to be considered a wave.”
      • (2)
        The first post-natal hair cycle “proceeds generally as a wave from the venter dorsally and posteriorly.”
      • (3)
        The second post-natal hair cycle “proceeds generally from the anterior venter posteriorly, then from the posterior venter anteriorly and dorsally, and then anteriorly on the dorsum after the two posterior lateral portions coalesce.”
      • (4)
        The third post-natal hair cycle “essentially duplicates previous on the venter, but on the dorsum there is a new center of activity on the saddle that proceeds posteriorly.” From the second post-natal hair cycle “on, the dorsal spread from the venter stops at the lateral line, except usually in the posterior region.”
      • (5)
        By the forth post-natal hair cycle “the waves are weak in males and, instead of waves, there are islands of growth, especially on the dorsum. In females this breakdown of waves generally occurs by fifth postnatal hair cycle.”
      • Chase H.B.
      • Eaton G.J.
      The growth of hair follicles in waves.
      further conclude that although the above described pattern of progression is the most common, there are many strain- and gender-related deviations from the patterning process. Patterns can be “more asymmetrical in some individuals than others. Around the head and legs there are even more extensive individual variations.”
      To validate and enrich these classic observations further, we followed the temporal changes of hair cycle patterns in C57BL/6J mice with clipped hairs (n>10). Mice were observed every 48 hours. For comparison, we have also followed the changes of the hair patches in mutant C.Cg-Msx2tm1Rilm/Mmcd mice with cyclic alopecia by observing the same mouse every 24 or 48 hours for a period ranging from 3 to 12 months (n>10). C.Cg-Msx2tm1Rilm/Mmcd mice showed domain pattern changes parallel with those of C57BL/6J mice, although the “rhythm” of the changes in mutant mice was faster. In general, hair cycle domain patterns undergo an age-dependent progression toward increasing complexity (that is, hair follicles cycle in smaller and non-synchronized groups). Our long-term study helped us to enrich previously described pattern progression (
      • Chase H.B.
      • Eaton G.J.
      The growth of hair follicles in waves.
      ).
      • (1)
        Synchronous growth all over the body: During the first hair cycle, hair follicles in the dorsal skin appear to cycle synchronously. As a convention, hair follicles in the dorsal skin from post-natal day 1 to approximately day 12 are used to represent anagen and then they gradually enter telogen after day 12. At approximately post-natal day 25, they start to enter the first true post-natal anagen lasting until around day 40. Dorsal skin from these ages is typically used in hair cycle-related research of transgenic mice (
        • Paus R.
        Principles of hair cycle control.
        ;
        • Oro A.E.
        • Higgins K.
        Hair cycle regulation of Hedgehog signal reception.
        ).
      • (2)
        Formation of a hair cycle spreading waves (Figure 6b, day 0): In agreement with
        • Chase H.B.
        • Eaton G.J.
        The growth of hair follicles in waves.
        , hair follicles start to form gradient anagen spreading waves.
        Figure thumbnail gr6
        Figure 6Some basic rules beneath the complexity of dynamic hair cycle domain patterns. (a) General locations of hair cycle domains can persist over the course of 83 days (about four hair cycles) as shown in the C.Cg-Msx2tm1Rilm/Mmcd mouse here. On the other hand, the boundaries of domains are not precise and shift from cycle to cycle (green arrows). Domain patterns undergo continuous dynamic changes. (b) Increasing complexity of hair cycle pattern in C.Cg-Msx2tm1Rilm/Mmcd mice over the course of 115 days. Big domains break into multiple smaller domains. (c) A typical dorsal hair cycle pattern composed of one central and two symmetric lateral domains in a C.Cg-Msx2tm1Rilmsol;Mmcd mouse. (d) Lack of the central scalp domain and presence of two lateral domains in the interauricular area of the C.Cg-Msx2tm1Rilm/Mmcd mouse. Bars=(a, b) 10 mm; (c, d) 5 mm.
      • (3)
        Formation of horizontal sectors due to the interruption of the hair cycle spreading wave: At this stage (variable number of cycles, >3), the dorsal skin forms 2–4 segment-like discrete domains.
      • (4)
        Formation of synchronous lateral domains (Figure 6b, day 30 and 5c, day 54): As more hair cycles pass (variable number of cycles, >4), different horizontal segments start to split into one central and two lateral domains that cycle independently. Interestingly, the two lateral domains usually mirror each other in shape and cycling phases, resulting in a bilaterally symmetric appearance. The central domain is positioned along the midline and cycles independently from its lateral counterparts. Occasionally, it can be in phase with the two lateral domains of the segment anterior or posterior to it, thus forming a V-shaped or inverted V-shaped zone.
      • (5)
        Formation of asynchronous lateral domains (Figure 3b, day 93 and 5b, day 115): As the mouse ages, initially synchronous lateral domains within the same segment can become asynchronous.
      • (6)
        Further asynchrony of hair cycle domains: Furthermore, each of the lateral domains may further break into two daughter domains. Thus, the hair cycle asynchrony becomes even greater, and the initial symmetry of the lateral domains can get lost.
      • (7)
        Hair cycle domains can be altered by systemic physiological conditions such as pregnancy, and local conditions such as skin trauma: For example, pregnancy and prolonged lactation in adult female mice can reset complex hair cycle domain patterns (
        • Johnson E.
        Quantitative studies of hair growth in the albino rat. III. The role of the adrenal glands.
        ,
        • Johnson E.
        Quantitative studies of hair growth in the albino rat. II. The effect of sex hormones.
        ). This phenomenon may arise due to the effect of estrogen and/or prolactin levels that are elevated during these physiological states. Both of these hormones are implicated in direct inhibition of anagen induction of anagen initiation in telogen hair follicles (
        • Oh H.S.
        • Smart R.C.
        An estrogen receptor pathway regulates the telogen–anagen hair follicle transition and influences epidermal cell proliferation.
        ;
        • Pearson A.J.
        • Ashby M.G.
        • Wildermoth J.E.
        • Craven A.J.
        • Nixon A.J.
        Effect of exogenous prolactin on the hair cycle.
        ;
        • Craven A.J.
        • Ormandy C.J.
        • Robertson F.G.
        • Wilkins R.J.
        • Kelly P.A.
        • Nixon A.J.
        • et al.
        Prolactin signaling influences the timing mechanism of the hair follicle: analysis of hair cycles in prolactin receptor knockout mice.
        ,
        • Craven A.J.
        • Nixon A.J.
        • Ashby M.G.
        • Ormandy C.J.
        • Blazek K.
        • Wilkins R.J.
        • et al.
        Prolactin delays hair regrowth in mice.
        ;
        • Foitzik K.
        • Krause K.
        • Nixon A.J.
        • Ford C.A.
        • Ohnemus U.
        • Pearson A.J.
        • et al.
        Prolactin and its receptor are expressed in murine hair follicle epithelium, show hair cycle-dependent expression, and induce catagen.
        ). Resetting occurs following the prolonged telogen associated with pregnancy and lactation (most prominent after repetitive pregnancies). In the post-lactation period, there is simultaneous anagen re-entry of most of the hair follicles across the skin regardless of the pre-pregnancy patterning. As a result, most of the hair follicles become synchronized, reminiscent to the status in the first post-natal cycle. Subsequently, new complex hair cycle domain patterns can re-form, unless new pregnancies occur. These data suggest that hair follicles in a hair cycle domain are not permanently “changed” in their structures or fixed in their “memory.” Under certain conditions, they can be reset by responding to appropriate signals or can switch between different domains. Skin trauma can induce new anagen in telogen hair follicles surrounding the wound (
        • Argyris T.S.
        The effect of wounds on adjacent growing or resting hair follicles in mice.
        ). Recently, it has been shown that trauma-induced anagen is caused by Map3k5-dependent infiltration and activation of macrophages in the peri-wound area (
        • Osaka N.
        • Takahashi T.
        • Murakami S.
        • Matsuzawa A.
        • Noguchi T.
        • Fujiwara T.
        • et al.
        ASK1-dependent recruitment and activation of macrophages induce hair growth in skin wounds.
        ). Such trauma-caused centers of new hair growth can propagate anagen induction throughout the adjacent non-wounded skin changing hair cycle domain patterns. Thus, it is important to differentiate trauma-caused and spontaneous anagen induction centers. Hair growth stimulation by wounding can occur after variable intervals and all the way up to 28 days (
        • Argyris T.S.
        • Argyris B.F.
        Factors affecting the stimulation of hair growth during wound healing.
        ), when initial wounding, especially if small, might not be very noticeable. Skin trauma in mice can originate from scratching and fighting. Thus, for long-term pattern monitoring experiments it is advisable to keep each mouse separately.
      • (8)
        Rate of hair wave propagation can vary: While hair follicles from different mice exhibit similar hair cycle domain patterns, the rates (or rhythms) of hair cycles can be different due to the different lengths of anagen and telogen stages in transgenic mice. For example, hair cycle domain patterns in C.Cg-Msx2tm1Rilm/Mmcd mice show similar spatial distributions parallel to that of C57BL/6J mice, but the domains cycle with a faster rhythm (that is, the total time to complete one full hair cycle is shorter). As a result, C.Cg-Msx2tm1Rilm/Mmcd mice develop complex hair cycle domain patterns at a younger age.

      Topographic Map of Hair Cycle Domains

      As a result nearly all domains cycle at their own pace, introducing a great degree of complexity and creating an appearance of randomly distributed hair patches, which become what we call “hair cycle domain patterns.” While the above represents the general trend in more than 25 mice we have observed, it should be noted that variations of this progression are also common. To help describe these patterns, we propose a topographic sector map. We have assigned two longitudinal borders that split the dorsal skin into three large longitudinal sectors. Three horizontal borders were assigned to further divide them into a total of 12 sectors. Similar methods were used for the ventral skin (Figure 4). The proposed mapping includes 12 dorsal sectors, 12 ventral sectors, and 6 cephalic sectors. The sector map helps us describe the observed hair cycle patterns at any given point in their development. However, the sector borders are arbitrarily assigned. While close, they do not coincide exactly with domain boundaries.
      While hair cycle domain patterns undergo progressive temporal changes, their configuration is relatively stable and can be tracked from one cycle to another over the course of an extended period of time (at least 83 days; Figure 6a). However, the precise shape and size of a single hair cycle domain is not identical over the course of several growth cycles. The domain boundaries can shift from one cycle to the next, suggesting that these boundaries are not static. Indeed, we did not find any anatomical structures corresponding to domain boundaries. In fact, one particular hair follicle can belong to domain “A” in one cycle and to domain “B” in the following cycle.

      Cellular Basis of The Hair Cycle Domain

      A skin specimen containing a hair growth pattern is in and of itself very informative. Longitudinal sections spanning the entire length of an anagen spreading wave show a continuous array of hair follicles in sequential hair cycle stages (Figure 5b;
      • Suzuki N.
      • Hirata M.
      • Kondo S.
      Traveling stripes on the skin of a mutant mouse.
      ). Such histological preparations can be used for temporal molecular profiling of hair cycle stages and can enable identification of transient signaling events within any signaling pathway.
      The hair cycle domains discussed above represent the transient status of hair cycle stages, and not permanent anatomical differences. There are also regional differences between various parts of the skin, known as regional specificity. Regional specificity implies that different skin regions such as the scalp, beard, eyebrows, face, lips, palms, nails, mammary glands, sweat glands, and so on have different characteristics. Epidermal precursors (or stem cells) are initially multipotent and competent of forming all the above structures. During development, special domains of the dermis send specific messages to the epidermis. Through a series of epithelial–mesenchymal interactions, these different skin domains with special structures and functions gradually emerge. The integument diversifies to endow different functions to different parts of the skin (
      • Chuong C.M.
      • Dhouailly D.
      • Gilmore S.
      • Forest L.
      • Shelley W.B.
      • Stenn K.S.
      • et al.
      What is the biological basis of pattern formation of skin lesions?.
      ;
      • Widelitz R.B.
      • Baker R.E.
      • Plikus M.
      • Lin C.M.
      • Maini P.K.
      • Paus R.
      • et al.
      Distinct mechanisms underlie pattern formation in the skin and skin appendages.
      ). Conversely, hair cycle domain boundaries are transient and can be reset by local or systemic hormone events.
      The regenerative hair wave discussed above should also be distinguished from the developmental wave. During skin development, hair primordia form as the result of mechanisms that may involve activators and inhibitors (
      • Nagorcka B.N.
      • Mooney J.R.
      The role of a reaction–diffusion system in the initiation of primary hair follicles.
      ;
      • Maini P.K.
      • Baker R.E.
      • Chuong C.M.
      Developmental biology. The Turing model comes of molecular age.
      ;
      • Sick S.
      • Reinker S.
      • Timmer J.
      • Schlake T.
      WNT and DKK determine hair follicle spacing through a reaction–diffusion mechanism.
      ). The new hair primordia are laid out in a temporal order, suggesting that the formation of new hair primordia may also be facilitated by the adjacent newly formed primordia. In the B6.129P2-Fzd6tm1Nat mice, hair primordia distribute in the shape of multiple whorls (
      • Guo N.
      • Hawkins C.
      • Nathans J.
      Frizzled6 controls hair patterning in mice.
      ;
      • Wang Y.
      • Badea T.
      • Nathans J.
      Order from disorder: self-organization in mammalian hair patterning.
      ), suggesting the involvement of the WNT pathway in this process. In humans, this phenomenon is most clearly manifested in the occipital hair whorl patterns (
      • Plikus M.
      • Chuong C.M.
      Making waves with hairs.
      ). However, once a follicle is born and the patterns are set in development, these arrangements cannot be changed. On the other hand, the regenerative hair wave we have discussed here involves hair follicles with different hair cycle statuses and is transitory.

      Conclusion

      We hope that the experimental evidence reviewed here will facilitate further recognition of the complexity of the hair cycle in the field of hair research. It can be used as a guide for the planning of hair cycle experiments in adult animals. Simple, yet powerful techniques of monitoring and studying hair cycle patterns, such as whole mount pilo-sebaceous units staining with Oil red, can be easily adopted into any experimental design. We also want to advocate the use of transverse sections through hair cycle domains to capture all stages of hair cycle instead of just one. This method is much more advantageous over having to collect multiple skin samples from different discrete hair cycle stages. Our method allows to observe the continuum of hair cycle stages and not to overlook brief events, such as transient expression of signaling molecules. Furthermore, researchers performing hair cycle studies in transgenic mice should recognize that hair growth pattern dynamics can be dramatically altered in mutant animals.
      In the future, it will be most interesting to study the interactive behavior of a population of organs. Here, we have observed that the regenerative behavior of thousands of hair follicles is coordinated. We are interested in computer simulation models that can be used to describe these complex wave patterns. We would like to learn how physicochemical principles can be applied to explain this regenerative behavior. What are the molecules that mediate wave propagation? What are the molecular pathways that receive or do not receive such propagation signals, leading to the complex wave patterns? How do the principles described here help elucidate patterning behaviors in the biological and non-biological world? How do wave patterns behave in different animals? How do the wave patterns changed by systemic physiology help animals adapt to the external environment, either as an individual during its lifespan or as a species during evolutionary adaptation? These are challenging questions waiting to be answered.

      Conflict of Interest

      The authors state no conflict of interest.

      NOTE ADDED IN PROOF

      During review of this manuscript, a paper appeared in the press. We have been studying the molecular basis of the phenomena described here. During review of this manuscript, a paper appeared in the press. It described that bone morphogenetic protein plays an important role in the formation of this wave. Plikus MV, Mayer JA, de la Cruz D, Baker RE, Maini PK, Maxson R et al. Cyclic dermal BMP signaling regulates stem cell activation during hair regeneration. Nature (in press).

      ACKNOWLEDGMENTS

      This work was supported by grants from NIAMS (CMC). We thank Dr John Sundberg, Dr Ralf Paus, Dr Vladimir Botchkarev, Dr Kurt Stenn, Dr Angela Christiano, Dr Valerie Randall, Dr George Cotsarelis, Dr Sarah Millar, and Dr Anthony Oro for their helpful discussions and insights.

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