THE JOURNAL OF INVESTIGATI VE DERMATOLOGY, 67:8-14. 1976 Vol. 67 , No.1 Copyright © 1976 by The Williams & Wilkins Co. Printed in U.S.A.

GROWTH AND DIFFERENTIATION OF SKIN

B. ALLEN FLAXMAN, M.D., AND PAUL F. A. MADERSON, PH.D. Subsection of Dermatology, Department of Medicine, Brown University and the Miriam Hospital, Providence, Rhode Island, and Department of Biology , Brooklyn College, New York, New York, U. S. A .

During the past 25 years, attention has turned from the morphologic aspects of skin development which are well known, to the mechanisms that control this development. Although our knowledge of these mechanisms is still limited, some avenues of investigation appear promising, e.g., epithelial-mesenchymal interactions, cell communication and other cell surface phenomena, and certain aspects of the proliferative process. In the analysis of what controls growth and differentiation, skin, as the experimental system, has played a major role.

During the quarter century history of the Sym­ it serves as a useful preliminary identifying feature posia on the Biology of Skin, many aspects of skin for taxonomic purposes. Classical 19th century biology have been reviewed but, surprisingly light microscopic studies showed that at their enough, very little attention has been directed earliest stages, ectodermal (epidermal) and so­ toward the embryogenic aspects. Most of the matic mesodermal (dermal) structures were the current symposium concerns specific attributes of same in all vertebrate embryos. Only in the last 25 the mature system, so we shall confine ourselves years have sufficient data become available to mainly to "Growth and Differentiation" during the formulate hypotheses which adequately explain all embryonic period. Our increased knowledge of the aspects of the subsequent diversity of the adult dynamics of integumental development consti­ organ system. tutes an exciting recent chapter. The develop­ Successive divisions of the fertilized egg lead to ment of the vertebrate integument has provided an formation of a 3-layered embryo whose organiza­ especially good opportunity to study the control tion differs somewhat according to species. The mechanisms and their relation to the whole process outermost layer, or ectoderm, forms surface epider­ of embryogenesis. We will concentrate on the few mis and the epithelial component of all skin areas of skin development that have generated, appendages. The middle layer, or mesoderm, gives and will continue to generate, new answers and rise to cells that form mesenchyme (dermis in the questions. adult), whose interaction with ectoderm is signifi­ The sequence of our knowledge of how the cant throughout the entire life of the organism. vertebrate integument develops parallels that of The two germ layer constituents of the verte­ many other organ systems. Extensive morphologic brate integument have quite different potentials analyses by classical light microscopic studies have for development. The mesenchymal cells of the now been confirmed and elaborated by ultrastruc­ somatic mesoderm give rise to dermal fibroblasts tural studies and, to a limited degree, by biochemi­ which secrete the precursors of self-assembling, cal investigations. Recently, as a result of using the extracellular collagen, as well as elastin, glyco­ experimental approach to analyze development, a proteins, and glucosaminoglycans. Dermal blood considerable number of reports document findings vessels and fat cells also derive from the basic in skin that are relevant to development elsewhere mesodermal cell population. The resultant " skele­ in the organism. A fundamental problem of devel­ tal" and "nutrient" functions of the end-products opmental biology is exemplified by the formation of mesenchymal differentiation are ubiquitous of the integument: the genesis of eventual hetero­ among species. Recently we have come to realize geneity in structure and function from initially that, during development, mesoderm ally derived homogeneous embryonic cell populations. In this matrix material undergoes significant changes paper we will emphasize and review how our which are better detected at the biochemical than understanding of this phenomenon has advanced. at the morphologic level (1). The ectodermal cells DEVELOPMENT OF THE VERTEBRATE INTEGUMENT: which form the general body epidermis may even­ GENERAL CHARACTERISTICS tually synthesize predominantly mucoproteins (in Integumentary structure varies so much, not fish) or keratinaceous proteins. Further diversity is only within a species but also between species, that found within certain epidermal appendages such as the sebaceous glands whose main function is Supported in part by Grant CA 11536 from the lipid synthesis. In higher vertebrates, the correla­ National Cancer Institute. tion between the type of intracellular filaments Reprint requests to: Dr. B. A. Flaxman, Miriam Hospital, 164 Summit Avenue, Providence, Rhode Island seen under the electron microscope and the x-ray 02906. diffraction patterns obtained from mature epider- 8 July 1976 GROWTH AND DIFFERENTIATION OF SKIN 9 mal cells l2,3] indicates two distinct categories of of cell differentiation can be so altered that constit­ keratinaceous proteins: the hair or alpha-type and uent units of "differentiated" tissues can dedif­ the feather or beta-type l4J. Comparative studies ferentiate to provide a source of new cells. The suggest that the ability to synthesize alpha-keratin elegant experiments of Gurdon [7] have shown that is the most fundamental characteristic of the an adult amphibian can be obtained via appar­ vertebrate epidermis [2] . However, the reptilian ently normal embryonic and larval development lineages associated with the major Mesozoic radia­ which resulted from placing an adult epidermal tions (from which arose modern reptiles and birds) cell nucleus in an enucleated oocyte. These experi­ have an additional faculty, the ability to synthe­ ments indicate that the entire genome is present in size beta-keratin, which is differentially expressed each adult nucleus, that differential gene repres­ in different forms [4]. sion must occur during development, and that the During evolution, the vertebrate integument cytoplasm has important inf1uences on nuclear acquired the ability to form " appendages," which behavior. Apparently, therefore, whatever control have been defined as "localized centers of special­ factors may be demonstrated in embryos or adults, ized epidermal and/or dermal cell proliferation and they ultimately impinge on a fundamental, and differentiation, within an otherwise generalized probably continuous, nuclear- cytoplasmic interac­ integument" [5J. Ontogenetically, they seem to be tion. associated with the developmental fields which are The number of factors that may inf1uence nor­ believed to be associated with the scaled pattern of mal cell behavior continually increases as our the vertebrate integument. They ref1ect the evolu­ knowledge of cell biology is expanded by increas­ tionary predilection of the organ system for selec­ ingly sophisticated experimental tools. We do not tion in various environments. The formation of know with certainty the number and types of these structures involves similar basic mechanisms mechanism(s) that ultimately control cell behav­ and suggests an intimate relation between ecto­ ior. In addition, there probably are different con­ derm and mesoderm. (See review by Serri and trols for different aspects of behavior. These may Cerimele [6 J.) During embryogenesis, down­ be overlapping, e.g. , mesenchyme may regulate growths of ectoderm into mesenchyme provide the both organogenesis and cell differentiation, but epithelial component of the organ. For hair and there may also be other distinctly different control feather, the clear-cut formation of associated mes­ factors which may change with time. enchymal structures is characterized by a close The existence of epithelial-mesenchymal in­ aggregation of cells in relation to the overlying teractions as an empirically demonstrable category ectoderm. The ingrowth of ectoderm and adjacent of controlling factors in all of embryogenesis has condensing mesenchyme forms the basic organ. A been generally accepted for some time [8-10], and second ingrowth of the epithelium of the develop­ investigations in this field have been paramount ing hair follicle into the mesenchyme leads to the for many developmental biologists. The experi­ formation of sebaceous and apocrine glands. Ec­ mental findings for skin have been reviewed by crine sweat glands initially grow as solid cords of several authors lll,12J. The anatomy of develop­ epidermal cells which then develop a lumen by the ment of certain integumentary appendages sug­ formation and coalescence of intracytoplasmic gests both mesenchymal and ectodermal participa­ vacuoles. In this process, the morphologic associa­ tion, but morphology alone tells us little about the tion with the mesenchyme is not as clear as for hair nature of the interactions. . Whatever they may be, and feather. These initial morphologic events are they may not always be the same, or even present followed by the establishment of distinctive pat­ at all, in some situations. It is generally assumed terns of cell differentiation in each appendage. that mesenchymal inf1uences on the ectoderm (or dermis on epidermis) are the more important, but CONTROLS OF GROWTH AND DIFFERENTIATION recent data strongly indicate that the ectodermal The foregoing brief review of the fundamentals potential is significantly autonomous and may of vertebrate integumentary structure indicates even exert the principal effects on the subjacent that from a developmental viewpoint there are at mesenchyme. Thus, in analyzing integumentary least three levels at which " determination" must development, one must determine how far the be analyzed: '( 1) the patterns of macromolecular ectoderm has developed before it begins to interact svnthesis which are eventually expressed by with the mesenchyme. Clearly no interaction can cells-"cell determination" or "cytogenesis"; (2) occur before gastrulation, i.e., before the germ lay­ the general topographic distribution of groups of ers are laid down. In fact, the ectoderm has proba­ cells involved in similar synthetic activities-"tis­ bly acquired many of its specific epidermal charac­ : ue determination" or "histogenesis"; and (3) the teristics before the mesenchyme enters the picture. c' oordinated organization of groups of cells to form This is certainly true of the capacity for specific 'ecognizable units-"organ determination" or "or­ differentiation into mucus-secreting ciliated cells ,:'anogenesis. " by amphibian ectoderm [13] or keratinized cells by Available data strongly suggest that the expres­ avian ectoderm [14,15]. Initially, ectoderm is prob­ ;ive capacity of an adult cell is not immutably ably totipotential in its reactivity and, under the :-ixed. Indeed, in certain natural situations such as appropriate stimulus, can form all mesodermal or limb regeneration in the salamander, the patterns even some endodermal tissues, at least in amphibi- 10 FLAXMAN AND MADERSON Vol. 67, No.1 ans [13 J. During normal development, this poten­ characteristics as the number of layers, the overall tiality becomes progressively restricted so that thickness, or the extent of keratinization are once a cell embarks on a particular pathway of strongly dependent upon connective tissue influ­ behavior, other pathways are excluded. This "loss ences [17,23 J. However, some epithelia are much of competence" refers to the inability of a cell more resistant than others to the effects of foreign population to respond to exogenous stimuli with dermis (e.g., in mixed grafts); such behavioral the passage of time. For example, when isolated differences have not been adequately explained presumptive neural tube ectoderm is first placed in [23 J. vitro and then recombined with notochord in vivo, We have just alluded to certain problems associ­ the ability of this ectoderm to respond to induction ated with attempts to determine how much control by the underlying notochord lessens in relation to the dermal mesenchyme exerts over basic epider­ the length of time of prior in vitro incubation l13J. mal cytogenesis and histogenesis. However, a The loss of competence appears to be an intrinsic wealth of data indicates that during organogenesis property of the ectodermal tissue. the interactions between the two tissues are com­ Even though the pathways of ectodermal behav­ plex and more obviously interdependent. To argue ior become restricted during development, reversi­ whether one or the other is "more important" on a ble mod ulations of cell differentiation can occur. general basis is futile since the organogenesis of Thus, early chick body ectoderm can become such specialized integumentary derivatives as mucus-secreting when placed on gizzard mesen­ feather, hair, or teeth does not occur without both chyme [14,15 J or treated with vitamin A [16]. the epithelial and mesenchymal components. However, these changes persist only when the Studies of the dynamics of integumentary develop­ appropriate stimulus is applied; when it is re­ ment in chicks have contributed significantly to moved, the epidermis reverts to its original kerati­ our understanding of organogenesis. The adult (or nizing state [17 J. Further modifications of epider­ even newly hatched chick) integument can be mal cell differentiation are also possible, e.g., lipid conveniently divided into three topographic re­ production in a sebaceous gland, and these appear gions: (1) the scaled integument over the lower leg, to be closely associated with specific egan forma­ (2) the regions where feathers, the most character­ tion. Thus, differentiated "sebaceous cells" are istic avian epidermal appendages, are seen, and (3) never seen in the general surface epidermis. Seba­ the regions where various other appendages (claws, ceous cell differentiation can be maintained only beak, wattles, etc.) are found. The scaled leg by a continuously operative, intact stimulus; if it is integument consists of a series of folds, whose outer removed (e.g., after wounding), the sebaceous aspect consists of cells which synthesize beta­ gland cells revert to keratinization as their pre­ keratin and whose inner aspect consists of jux­ ferred pathway of differentiation l18 J. We infer taposed cells which synthesize alpha-keratin. from this that non keratinizing differentiation in Thus, there is a conspicuous horizontal alternation epidermal appendages must represent modula­ of protein synthetic capacities across the entire tions from the basic keratinizing state. epidermal surface [4]. A feather is essentially The histogenesis from the initial ectodermal an "island" of beta-keratin synthesis in a "sea" epithelium of a stratified, squamous keratinizing of alpha-keratin synthesizing cells (interfolicular epithelium, which could be specifically identified epidermis) [24,25]. In other appendages, the as "epidermis," was first thought to depend on epidermal cells synthesize beta-keratin (claws, mesenchymal influences since early in vitro experi­ beak) or alpha (wattles). What factors cause this ments showed that survival and development were heterogeneity in avian integumentary structure? dependent on contact with subjacent mesenchyme. With respect to feather formation, mesenchyme (See review by Flaxman l19 J.) However, an impor­ probably controls the location and perhaps acts as tant advance was made when both Dodson [20 J the primary stimulus for ectodermal participation and Wessells [21] showed that collagen gel, or even in the formation of these appendages [26]. Mes­ a Millipore filter, could sustain epidermal differen­ enchymal influence may last only a short period of tiation from the primary ectodermal layer. The time, after which the epidermis itself is able to main role of mesenchyme in epidermal develop­ regulate mesenchyme. How the ectoderm grows ment seemed, therefore, to be merely to provide an down into the mesenchyme is not known, but attachment site for ectodermal cells; given such an since epidermis can both synthesize and secrete attachment, the ectoderm could then organize collagenase [27,28], this enzyme may function in itself into a fully developed, differentiating epider­ organogenesis. Since transplants of early apical mis since it possessed the requisite information. limb bud mesoderm beneath wing ectoderm pro­ Studies on isolated epidermal cells from adult duced scales and claws [29], mesenchymal con­ human subjects support these conclusions l22]. trol of appendage formation was inferred. Rawles Although the general epidermis appears in many [30] extended these data by an exhaustive series ways tp act autonomously, we cannot assume that of tissue and age chimaeras grown on chorioal­ the only function of the dermis is to provide a lantoic grafts and showed conclusively that feather substratum for germinal cell attachment. Several and beak morphogenesis are also under mesen­ studies have shown that such regional epidermal chymal control. Even more significantly, Rawle's July 1976 GROWTH AND DIFFERENTIATION OF SKIN 11 data emphasized the importance of time in the underwent characteristic morphologic changes; in development of ectodermal and mesenchymal the absence of mesenchyme, no such development ability to interact. took place. Since early microscopic studies had Initially, mesenchyme seems to be paramount in shown that no cells or processes entered through determining the role of the epidermis in appendage the body of the filter, a search was joined for a formation but later the epidermis strongly influ­ "diffusible" chemical factor, the long-sought ences the outcome of the interaction. For instance , "Holy Grail" of primary induction [34J. Suffice it when the dorsal epidermis of 5- to 7 -day-old chicks, to say that the factor has not yet been identified which normally forms feathers is combined with with certainty. It should be pointed out that many tarsometatarsal mesenchyme which promotes inductive stimuli are nonspecific, i.e. , various scale formation, feathers are formed as an outcome different stimuli can cause competent tissue to of the interaction (30]. Epidermis apparently re­ react normally. Thus, the task of identifying a acts to the inductive scale mesenchyme, but previ­ specific stimulus is made more difficult. ous association with dorsal mesenchyme has lim­ This failure to find a diffusible inductive factor ited its response to feather formation. Similarly, in vitro has led to the suggestion that the mac­ when snout epidermis, which forms the large romolecules that form beneath the epithelial com­ vibrissae, is combined with mesenchyme from the ponent may play an inductive role. Both collagen midback of a 13-day-old mouse where smaller and mucopolysaccharides are deposited beneath pelage hairs are located, vibrissa-type follicles the epithelium in vitro [35 J; more specifically, develop [31]. Experiments with adult feather and similar findings have been reported in ectodermal hair have given similar results and suggest strongly epithelium [36J. The origin of these molecu­ that during development the follicular epidermis les-whether from epithelium, mesenchyme, or acquires a propensity, not only for self-organiza­ both-is still being disputed, but it has been tion but for the organization of the associated suggested that they play a role in regulating dermis as well, which is comparable to the ability development. The deposition of collagen may be of interfollicular epidermis to organize itself into involved in morphogenic events since when sali­ the stratified squamous state. vary gland rudiments were treated with collagen­ The danger of generalizing about the sequence of ase they failed to branch and form acini l35 ,37 J. events in early epithelial-mesenchymal interac­ The presence of collagen at specific sites during tions during organogenesis has recently been em­ organ formation may control the important process phasized by Sawyer and his colleagues [32], who of cell movement l38) . In an elegant comparative have produced an elegant series of morphologic study of normal and talpid 3 mutant chick limb bud and experimental studies of normal and "scaleless by scanning and transmission electron microscopy, mutant" leg integument. These studies have Ede, Bellairs, and Bancroft l39] emphasized the shown that it is the epidermis which appears to role of the intact basement membrane in control­ initiate scale differentiation and that whatever ling mesenchymal cell movement during limb "scale-inducing capabilities" the dermis possesses, development. it acquires from the epidermis. "Scaleless" leg More recently, evidence quite contrary to the epidermis cannot form the initial placodes, but in earlier in vitro findings has been reported. Saxen's appropriate chimaeras, scaleless dermis can par­ group [40,41], which has recently investigated the ticipate in scale formation. Thus, the available classical in vitro model for transfilter induction of data suggest that we still must be wary of any kidney tubule formation, has demonstrated that general conclusions about which comes first during cell processes penetrate through the filter from one early organogenesis, epithelial or mesenchymal side to the other. Cytoplasmic processes of ectoder­ influences. mal epithelium (cornea) can also penetrate a filter The mechanisms by which the epithelium and to contact the underlying mesenchymal tissue [36 J. the mesenchyme interact are not known. The The more recently used filters have been the above discussion suggests that, in some instances Nucleopore type, which appear to differ substan­ at least, mesenchyme passively facilitates the tially from the Millipore filters used in earlier expression of preexisting ectodermal potentials for studies; this discrepancy makes it difficult to ke ratinization and histogenesis. Despite the con­ evaluate the earlier work [42J. However, in a recent centrated research efforts reported in recent sym­ study, cell processes penetrated Millipore filters posia [8,10], the means by which these potentials l43]. Thus direct intercellular contact between are realized are only beginning to be uncovered. tissues could playa significant role in regulating Most experimental data have been obtained from embryonic development. Some recent in vivo stud­ in vitro studies which may not be relevant to ies support this hypothesis [44,45]. normal, in vivo processes. How can cell contact between tissues act as a The experiments of Grobstein [33] and others regulatory factor in development? Recent ad­ established that the interactions occur over a vances in electrophysiologic methods, which dem­ distance of 50 to 100 11 and do not rely upon onstrate electrical communication between cells cell-to-cell contact. In transfilter studies, isolated and organs in vivo and in vitro, may provide the kidney epithelium placed opposite mesenchyme answer. During the early development of chick 12 FLAXMAN AND MADERSON Vol. 67, No.1 embryos, there is a high level of communication may influence subsequent behavior patterns. between organs which decreases and disappears Electrical communication is only one example of during later stages [46]. This communication oc­ how the cell surface may be involved in cell curs by direct cell-to-cell con tact, not by the control. There are other phenomena, including cell passage of current through the extracellular space recognition and adhesion, which are potentially l47]. Since individual plasma membranes have a important for normal development. For example, if high electrical resistance, the flow of current be­ dissociated mixed embryonic skin cells are forced tween cells indicates that preferential, low-resist­ to regain contact, they sort out and form append­ ance connections permit the passage of ions. A host ages and thus show the proper relation between of ultrastructural studies, using special stains and epithelial and mesenchymal components l56,57 J. freeze-cleavage techniques, indicate that cells and The mechanism of recognition is under intense tissues which have these special conductive prop­ study because it may be important for both normal erties have a preponderance of specialized intercel­ development and the abnormal behavior seen in lular junctions that are variously called "nexuses" cancer. or "gap" junctions [48j . In vitro studies of cells One aspect of development that has thus far with such junctions permit direct visual observa­ been overlooked in this discussion is the recent tion of charged fluorescent molecules injected into information on the role of cell proliferation, which one cell passing through to adjacent cells without must be closely interlocked with the events of leakage into the extracellular space l49]. The morphogenesis and the onset of differentiation. upper limit of molecular weight of the dye mole­ One of the effects of mesenchymal-epithelial in­ cules used is in the order of several hundred. teraction may be to promote or delay cell prolifera­ Basically, these morphologic and experimental tion at critical times by some as yet unknown data suggest that small charged molecules can, mechanism. This seems to be the case during the and probably do, pass from one cell to another and early inductive interactions which provide the may, therefore, provide a regulatory mechanism requisite number of cells for organ formation and for behavior. result in feather and hair formation l58]. Some The relevance of the above data to skin biology studies have suggested that a certain amount of lies in the fact that since nexuses have been proliferation is required before the specialized demonstrated between adult epidermal cells in aspects of cytodifferentiation can begin. These vivo l50] and in vitro l51] they probably exist in so-called "quantal" mitoses, after which cell­ embryonic tissues as well. Electrical communica­ specific macromolecules are synthesized, are tion has been demonstrated in adult amphibian thought to be important during the differentiation epidermis l52 j and in human epidermal basal cells of many organs [59,60]. Much more needs to be [53,54j. Further studies of the structure and func­ learned about whether such phenomena are opera­ tion of nexuses in embryonic integumentary tissues tive during skin development. For example, does are urgently needed to explain many aspects of the synthesis of lipid by the sebaceous gland after early differentiation. Cell communication could mitosis have anything to do with the proliferative playa role in the morphogenetic movements that events that precede it? According to Stern, Day­ take place during the formation of skin append­ ton, and Duecy [61], the onset of keratinization in ages and in the histogenesis of surface epidermis. fetal mouse epidermis is associated with decreased During early embryogenesis, ectoderm and meso­ proliferative activity, but before these observations derm could interact by establishing low resis­ can be fully evaluated, we need to know such tance junctions between the different cell types. details as the length of the cell cycle and the size of At any rate, morphologic and physiologic evi­ the proliferative pool. dence along this line should be sought. If commu­ Another important factor in proliferation that nication supplies the initial inductive stimuli, it may influence subsequent differentiation pertains may also be responsible for the already demon­ to the length of time a postmitotic cell remains in strated capacity of epidermal cells for later self­ the germinal layer before being released to move regulation during organogenesis and histogenesis. toward the body surface. Earlier studies of the Differentiation may be regulated, at least partially, kinetics of mammalian epidermis, where only in the same way; some studies oflizard skin in vitro alpha-keratin is found, have suggested a random suggest that this is so [55]. Thus, intercellular pattern of cell movement out of the basal layer. communication may be a comprehensive mech­ This, however, does not seem to be the case in the anism whereby the interactions between different epidermis of squamate reptiles, in which the dura­ tissues and between cells during development are tion of stay in the germinal layer is linked to the effected. What these communicating molecules imbsequent synthesis of alpha- or beta-keratin by are and how they act is still a mystery. the differentiating cells [62]. Thus, the pattern of Currently, there is intense interest in the prop­ formation and release of cells from the basal layer erties of the cell surface and their relation to is not necessarily an entirely random phenomenon, countless aspects of behavior. In a sense, the cell but the forces that restrain the cells from leaving surface acts for the cell in its "social" interactions the basal layer are still completely unknown. much as the skin acts for the whole organism. Abnormal keratinization, such as that sometimes Stimuli that impinge on the outer surface of either found in parts of a plaque of psoriasis, may be July 1976 GROWTH AND DIFFERENTIATION OF SKIN 13 related more directly to the length of time spent in Scott, JH Vaughan. New York, McGraw-Hill, the basal layer (during which a cell may receive 1971, pp 49-54 critical messages from the dermis or perhaps from 7. Gurdon JB: The Control of Gene Expression in Animal Development. Cambridge, Harvard Uni­ other epidermal cells) than to the proposed short­ versity Press, 1974 ening of the cell cycle or transit time. The same 8. Fleischmajer R, Billingham RE: J~~J>ithelial­ reasoning may apply to the sebaceous gland where Mesenchymal Interactions. Baltimore, Williams & abnormal differentiation (keratinization) is seen in Wilkins, 1968 acne. 9. Maderson PFA: Embryonic tissue interactions as the basis for morphological changes in evolution. American Zoologist 15:315-327, 1975 CONCLUSIONS 10. Slav kin HC, Greulich RC: Extracellular Matrix Influ­ In general, our understanding of the growth and ences on Gene Expression. New York, Academic, 1975 differentiation of skin has kept pace with that for 1l. Billingham RE, Silvers WK: The origin and conser­ other organ systems during the past 25 years. vation of epidermal specificities. N Engl J Med Because of the relatively insoluble nature of kera­ 268:477-480, 539-545, 1963 tin, however, biochemical advances have lagged 12. Wessells NK: Differentiation of epidermis and epi­ dermal derivatives. N Engl J Med 277:21-33, 1967 somewhat. Experimental techniques used to un­ 13. Holtfreter J: Mesenchyme and epithelia in inductive ravel the mysteries of other organ systems have and morphogenetic processes, Epithelial-Mes­ been and are still being applied to the study of enchymal Interactions. Edited by R Fleisch­ skin. This organ has emerged as an experimental majer, RE Billingham. Baltimore, Williams & Wilkins, 1968, pp 1-30 system widely used by scientists with special 14. McLoughlin CB: The importance of mesenchymal training in dermatology who are looking for special factors in the differentiation of chick epidermis. 1. systems to answer fundamental problems in biol­ The differentiation in culture of the isolated epi­ ogy. The advances we make in controlling the dermis of the chick and its response to excess vitamin A. J Embryol Exp Morphol 9:370-384, growth and differentiation of skin will probably 1961 parallel those in other organ systems. Some ques­ 15. McLoughlin CB: Mesenchymal influences on epithe­ tions will probably be more easily answered with lial differentiation. Symp Soc Exp BioI 17:359-388, skin as the experimental system whereas other 1963 16. Fell HB, Mellanby E: Metaplasia produced in cul­ questions will require other systems. An under­ ture of chick ectoderm by high vitamin A. J standing of the pathogenesis of many common skin Physiol (Lond) 119:470-488, 1953 diseases such as acne, psoriasis, baldness, etc. may 17. McLouglin CB: Interaction of epidermis with various well be attained only when an understanding of the types of foreign mesenchyme, Epithelial-Mes­ enchymal Interactions. Edited by R Fleisch­ controls that operate in normal skin development maj€r, RE Billingham. Baltimore, Williams & is acquired. Most of the easy questions in biology Wilkins, 1968, pp 244-251 appear to have been solved. Those remaining are 18. Strauss JS, Kligman AM: Pathologic patterns of the incredibly difficult, and progress is limited by the sebaceous gland. J Invest DermatoI30:51-61, 1958 19. Flaxman BA: Principles of skin development, Der­ available research tools and the innate human matology in General Medicine. Edited by TB capacity to comprehend or devise new experi­ Fitzpatrick, KA Arndt, WH Clark Jr, AZ Eisen, ments. It is our hope that scientists with an EJ Van Scott, JH Vaughan. New York, McGraw­ interest in skin development will continue to be in Hill, 1971, pp 54-58 20. Dodson JW: The differentiation of epidermis. 1. The the forefront of those who are working to push back interrelationships of epidermis and dermis in em­ the frontiers of biology. bryonic chicken skin. J Embryol Exp Morphol 17:83-105, 1967 21. Wessells NK: Substrate and nutrient effects upon epidermal basal cell orientation and proliferation. REFERENCES Proc Natl Acad Sci USA 52:252-259, 1964 1. Mechanic GL: The existence of chemically distinct 22. Flaxman BA: Replication and differentiation in vitro soluble collagens in pre-natal and post-natal bo­ of epidermal cells from normal human skin and vine skin and tendon, Extracellular Matrix Inf1u­ from benign (psoriasis) and malignant (basal cell ences on Gene Expression. Edited by HC Slavkin, carcinoma) hyperplasia. In Vitro 8:237-256, 1972 RC Greulich. New York Academic, 1975, pp 23. Billingham RE, Silvers WK: Studies on the conser­ 347-354 vation of epidermal specificities of skin and certain 2. Flaxman BA: Cell differentiation and its control in mucosas in adult mammals. J Exp Med the vertebrate epidermis. American Zoologist 125:429-446, 1967 12: 13-25, 1972 24. Maderson PF A: On how an archosaurian scale might 3. Maderson PFA, Flaxman BA, Roth SI, Szabo G: have given rise to an avian feather. American Ultrastructural contributions to the identification Naturalist 106:424-428, 1972 of cell types in the lizard epidermal generation. J 25. Regal P: The evolutionary origin of feathers. Q Rev Morphol 136:191-210, 1972 BioI 50:35-66 4. Baden HP, Maderson PFA: The morphological and 26. Sengel P: Differenciation in vitro des germes plu­ biochemical identification of fibrous proteins in maires de l'embryon de poulet. C R Soc BioI the amniote epidermis. J Exp Zool 174:225-232, (Paris) 150:2057-2059, 1956 1970 27. Eisen AZ, Gross J: The role of epithelium and .. 5. Maderson PFA: When? why? and how?: some mesenchyme in the production of a collagenolytic speculations on the evolution of the vertebrate enzyme and a hyaluronidase in the American integument. American Zoologist 12:159-171, 1972 tadpole. Dev BioI 12:408-418, 1965 6. Serri, F, Cerimele D: Embryology of the skin, Derma­ 28. Eisen AZ: Human skin collagenase: localization and tology in General Medicine. Edited by TB Fitzpa­ distribution in normal human skin. J Invest Der­ trick, KA Arndt, WH Clark Jr, AZ Eisen, EJ Van matol 52:442-448, 1969 14 FLAXMAN AND MADERSON Vol. 67, No.1

29 . Saunders JW: Inductive specificity in the origin of lium and mesenchyme in the developing chick integumentary derivatives in the fowl, A Sympo­ embryo, Epithelial-Mesenchymal Interactions. sium on the Chemical Basis of Development. Edited by R Fleischmajer, RE Billingham. Bal­ Edited by WD McElroy, B Glass. Baltimore, Johns timore, Williams & Wilkins, 1968, pp 31-55 Hopkins Press, 1958, pp 239-253 46. Sheridan JD: Electrophysiological evidence for low­ 30. Rawles ME: Tissue interactions in the morphogene­ resistance intercellular junctions in the early chick sis of a feather, Biology of the Skin and Hair embryo. J Cell BioI 37:650-659, 1968 Growth. Edited by AG Lyne, BF Short. Sydney, 47. Furshpan EJ, Potter DD: Low-resistance junctions Angus and Robertson, 1965, pp 105-128 between cells in embryo and tissue culture, Cur­ 31. Kolla~ EJ: An in vitro study of hair and vibrissae rent Topics in Developmental Biology, vol 3. development in embryonic mouse skin. J Invest Edited by AA Moscona, A Monroy. New York, Dermatol 46:254- 262, 1966 Academic, 1968, pp 95-127 32. Sawyer RH: Avian scale development. V. Ultrastruc­ 48. McNutt NS, Weinstein RS: Membrane ultrastruc­ ture of the chorionic epithelium induced by ante­ ture at mammalian intercellular junctions. Prog rior shank dermis from the scaleless mutant. J Exp Biophys Mol BioI 26:47-101, 1973 Zool 191: 133-139, 1975 49. Sheridan JD: Dye movement and low-resistance 33. Grobstein C: The problem of the chemical nature of junctions between reaggregated embryonic cells. embryonic inducers, Cell Differentiation. A CIBA Dev BioI 26:627-636, 1971 Foundation Symposium. Edited by AVS DeRewk, 50. Hashimoto K: Intracellular space of the human J Knight. Boston, Little, Brown, 1967, pp 131-136 epidermis as demonstrated with lanthanum. J 34. Saxen L, Toivonen S: Primary Embryonic Induction. Invest Dermatol 57:17-31, 1971 London, Logos Press, 1962 51. Cavoto FV, Flaxman BA: Communication between 35. Grobstein C: Developmental significance of interface normal human epidermal cells in vitro. J Invest materials in epithelial-mesenchymal interaction, Dermatol 59 :370-374, 1972 Epithelial- Mesenchymal Interactions. Edited by 52 . Loewenstein WR, Penn RD: Intercellular communi­ R Fleischmajer, RE Billingham. Baltimore, Wil­ cation and tissue growth. II. Tissue regeneration. J liams & Wilkins, 1968, pp 173-176 Cell BioI 33:235-242, 1967 36. Meier S, Hay ED: Control of corneal differentiation 53. Flaxman BA, Cavoto FV: Low resistance junctions in in vitro by extracellular matrix, Extracellular epithelial outgrowths from normal and cancerous Matrix Influences on Gene Expression. Edited by epidermis in vitro. J Cell BioI 58:219-223, 1973 HC Slavkin, RC Greulich. New York, Academic, 54. VanHeukelom JS, Slaaf DW, Van Der Leun JC: Cell 1975, pp 185-196 communication in the basal cells of the human 37 . Grobstein C, Cohen J: Collagenase: effect on the epidermis. Biophys J 12: 1266-1284, 1972 morphogenesis of embryonic salivary epithelium in 55. Flaxman BA, Maderson PFA, Szabo G, Roth SI: vitro. Science 150:626-628, 1965 Control of cell differentiation in lizard epidermis in 38 . Stuart ES, Garber B, Moscona AA: An analysis of vitro. Dev BioI 18:354-374, 1968 feather germ formation in the embryo and in vitro, 56. Moscona AA, Garber AA: Reconstruction of skin in normal development and in skin treated with from single cells and integumental differentiation hydrocortisone. J Exp Zool 174:97- 118, 1972 in cell aggregates, Epithelial-Mesenchymal In­ 39 . Ede DA, Bellairs R, Bancroft M: A scanning electron teractions. Edited by R Fleischmajer, RE Billing­ microscope study of the early limb-bud in normal ham. Baltimore, Williams & Wilkins, 1968, pp and talpid 3 mutant chick embryos. J Embryol Exp 230-243 Morphol 31 :761-785, 1974 57. Moscona AA: Cell recognition, histotypic adhesion 40. Wartiovaara J , Nordling S, Lehtonen E, Saxen L: and enzyme induction in embryonic cells, Extra­ Transfilter induction of kidney tubules: correlation cellular Matrix Influences on Gene Expression. with cytoplasmic penetration into Nucleopore fil­ Edited by HC Slavkin, RC Greulich. New York, ters. J Embryol Exp Morphol 31:667-682, 1974 Academic, 1975, pp 55-67 41. Saxen L: Transmission and spread of kidney tubule 58 . Wessells NK, Roessner KD: Nonproliferation in induction, Extracellular Matrix Influences on dermal condensations of mouse vibrissae and pel­ Gene Expression. Edited by HC Slavkin, RC lage hairs. Dev BioI 12:419-433, 1965 Greulich. New York, Academic, 1975, pp 523-529 59. Bischoff R, Holtzer H: Mitosis and the processes of 42. Grobstein C: Developmental role of intercellular differentiation of myogenic cells in vitro. J Cell matrix: retrospective and prospective, Extracellu­ BioI 41:188-200, 1969 lar Matrix Influences on Gene Expression. Edited 60. Wessells NK: Problems in the analysis of determina­ by HC Slavkin, RC Greulich. New York, Aca­ tion, mitosis and differentiation, Epithelial­ demic, 1975, pp 9-16 Mesenchymal Interactions. Edited by R Fleisch­ 43. Lehtonen E, Wartiovaara J, Nordling S, Saxen L: majer, RE Billingham. Baltimore, Williams & demonstration of cytoplasmic processes in Mil­ Wilkins, 1968, pp 132-151 lipore filters permitting kidney tubule induction. J 61. Stern IB, Dayton L, Duecy J: The uptake of tritiated Embryol Exp Morphol 33:187-203, 1975 thymidine by the dorsal epidermis of the fetal and 44. Cutler LS, Chaudhry AP: Intercellular contacts at newborn rat. Anat Rec 170:225-234, 1971 the epithelial-mesenchymal interface during the 62 . Flaxman BA, Maderson PF A: Relationship between prenatal development of the rat submandibular patterns of cell migration from the germinal layer gland. Dev BioI 33:229-240, 1973 and changing patterns of differentiation in the 45. Hay ED: Organization and fine structure of epithe- lizard epidermis. J Exp Zool 183:209-216, 1973