Morphogenesis and Malformations of the Skin NICHD/NIADDK Research Workshop

Anne K . Krey, Al an N . M os hell, Delbert H. Dayton, Roger H . Sawyer, and Karen A . H o lbrook N atio nal Institute o f C hild Hca lth and Human Development/ National Institute of Arthri tis. Diabetes. Digest ive and Kidncy Diseases. Bethesda . M aryland . U . S. A.

Developmentall y ca used skin m alform a ti o n s con stitute a spectrum of birth defects , some of w hi ch ca n b e recogni zed prena tall y b y morpholog ic o r bioch emical m ea ns . T h e number of prena tal ly di agnosa ble sk in diseases could be g rea tly expanded with an in creased unders tanding of the molecul ar and cellul a r bases of skin developm ent and the m ech anisms tha t result in th e gene ration of sk in d efects. T he N a tio n a l Ins titute of C hild H ea lth and Human De­ve lopment and the N a tional Institute of Arthriti s, Diabetes,

The sk in is a co mplex o rgan co nsisting of cel ls and tissues deri ved from ectoderm (the epidermis . its ap­pendages, nerve networks), mesoderm (cell s and ex­tracellular matri x of thc dermi s, vascular systems) , and t.he neural crest (melan ocy tes). Each of these ti s­

sues becomes uniqu e in its structure, biochem istry and fun cti on during developm cnt of the ski n. Each also main ta ins cell-specific character istics th at are ex pressed in o ther o rga ns throughout th e body. For example, ep iderma l cells, like all epithelial cell s, contain kerat in pro teins; th e fibro us pro teins present in th e dermis arc also sy nthes ized in tendon , bone, and co rn ea; and all basa l lam­inae, including the dermal-epiderm al junction, have ce rtain struc­tura l proteins and anti gens in common. The skin is access ible, and sa mples m ay be obta ined eas il y fo r cutaneo us studies specif­ica ll y, and for genera l in vest iga ti ons of cell and tiss ue interactions and of important macro molecules th at have a genera l body dis­tributi on.

ell and tissue interacti ons arc d ifficu lt to stud y in adult skin beca use of its stru ctural and biochemi ca l co mplexity. T he de­velop ing sk in provides a situat ion in whi ch stru cture is achieved in a step-w ise fas hion . T hus, co mpli ca ted events such as epithelial­mesenchymal in teractions, patternin g of epidermal :lppenda ges,

Genetics and Tera to logy Branch (AKK . DHD)N IC HD . and Skin D is­eases Branch (AN M)N IA DDK. N ational Institu tes of Hea lth, Bethesda . M aryland. D epartm ent of Bio logy (I<H S). U ni ve rsity of Sou th Ca ro lina, Co lumbia. South C aro lina, and Departm ents of Biological Structure and M edic ine (Dermato logy) (KAH), U ni ve rsity o f Wash ing ton School of Medi cine. Sea ttl e. Washin gton, U.S.A .

Reprint req ues ts to Anne K. Krey, Geneti cs and Teratology Bra nch, NI C HD. 9000 Rockv ill e Pike, Land ow 7C08, Bethesda. MD 20892.

Abbreviations : APUD: amine precursor up take and deca rboxy lation BP: bu ll ous pemphigoid DEJ: der mal-epidermal j un ction EBA: epidermo lysis bu llosa acq uisira EDs: ectoderma l dysp las ias EGA: es tim ated gesta tional age EGF: epidermal growth fa cto r M EL: mouse crythro lcukemic

Diges ti ve and Kidney Diseases, the refore , sponsored a worksh o p that recommended basic bio logic studies co m­bin ed w ith clini ca l investiga ti o n s of n o rm al and abno rmal cutan eous development set forth in this a rticl e. [n vestiga­tions resultin g from these resea rch recommendatio n s a re intended to contribute to the knowledge that s h o uld aid in the prevention of d evelopm entall y ca used sk in d efo rmiti es. J Inv est Dermato l 88:464-473, 1987

and assembly of derm al conn ectivc ti ssue macro molecules may be approached more eas il y. T he pro blems that m ay be studied in developing skin are as varied as the components of skin itself, and requ ire the interest and talents of a d iverse group of inves­tiga tors.

Until the last decade. this divcrsit y was no t reAected in studies of cutaneo us morphogenesis; rather, descripti ve m orphology and experimenta l embryology do minated th e field. T here is at prcsent a heightened interest in cutaneous developmcnt in m an. In part, this may be a conseq uen ce of several new techn o logies and may also be a result of a more wi despread acceptance o f clinical pro­cedures fo r the prenatal diagnosis of inherited skin diso rders. Immunolog ic techniqu es, parti cularl y the development and usc o f monoclonal antibodies, have been important in eva luatin g the state of differenti ati on of normal fetal ski n and in assess ing de­velopmental abnorma liti es in skin from fetuses and new borns with geneti c skin disease. The relatively recent success in cultu ring cpiderm al kerat inocy tes has opened the doo r to studies of normal and abnorm al epidermis in th e sa me m anncr that fibrob last cell culture has allowcd connective tissue biologists to in vest igate the molecul ar basis for inherited co nn ective tissue di so rders so suc­cessfull y. T he usc of the nude mouse as a host fo r transplants of no rm al and abn ormal hum an sk in has provided an opportunity to inves ti ga te the site of ac tion of an abnorm al gene in th e sk in and to condu ct epiderm al-dermal recomb inant studics w ith hu­man tissue in a physio logic env ironment . T he developmcnt of gene probes and the use of these in sequencing and mappi ng genes is at the forefront of resca rch related to genetic diseases of the skin . All of these techn ologies are being used successfully to an­swer questi ons about no rm al and abnormal ad ult skin. T he bi­o logy of no rm al and abno rm al skin durin g development can now be approached with similar techniques.

C utaneous mor phogcnesis and malformation in humans was th e top ic of a March of Dimes (MOD)-spo nso red sympos ium in 1')80 . T his l:lndmark meeting brought inves ti ga to rs togcther to discuss sk in development in man. The m orpho logy of human skin dcvelopment was rev iewed, new resea rch techniques were ex plained , the usc of mutant strains of mi ce th at are applicable to ex perim ental studies of skin development were discussed , and

0022-202X/87/S03.50 Copyrig ht © 1987 by T he Society for Invcstigative Derm ato logy. Inc.

464

VO L. 88. N O.4 APRIL 1987

the underst~nd in g of the basis for specifi c geneti c skin disease was reviewed. Agin g syndromes we re included as the antithes is to development. The meetin g cnded w ith a di scuss ion of the current o pti ons for prenatal dia gnosis of skin dise~ se and a pro­spec tive look at the potential for expandin g this capab ilit y.

The M OD m eetin g helped to set th e sta ge for the worksho p spo nso red by the Nati o nal In stitutes of Health (Nil-I) o n M o r­phogenesis and M alfo rm at io ns of the Ski n, w hi ch was held in Septem ber '1983. C o sponsorship of this meetin g by two NIH In stitutes , the National In stitu te of C hild H ea lth and Hum an Development (N IC HD) and the Nati onal In stitute of Arthriti s, Diabetes, Diges ti ve and Kidne y Diseases (N IADDK), reAected the In stitutes ' overlappin g interests, i.e . , the specific co ncern for skin development by NI C HD, sk in biology b y NIADDK, and fo r abnormalities in dcvelopment o r geneti c skin di sease by bo th units.

This workshop bro ught together basi c and clinical scientists work in g in the area of skin deve lopment, o r in a related area that would prov idc an important perspective o n so m e aspect o f skin morphogenes is . Workshop participants had experti se in CLIt~­neous diffcrentiation; m olecular structure of the sk in and /or skin­related m ac ro m olecul es; geneti c diseases of the skin ; stru cture, fu nction , biochcmistry, and / o r ph ys io logy of the skin; and / o r prenatal dia gnos is . The parti cipants were: Eugene A. Bauer, Rob­ert A. Brigga m an , Frank C h ytil , Benoit de C ro mbrugghe, Bev­erl y A . Dale , Delbert H. Da yton , l<j ell E lgio, Cynthi a J. Fisher, Elain e V . Fuchs, Paul F. Goetin ck, Lowcll A. Go ldsmith , Karen A. H olbrook, Barbara J o hnson- Wint, Stephen I. Katz, Llo yd E. Kin g, Ann e K. Krey, Alfred T. Lane, Alan Moshell, Vin cent M. Ri cca rdi , C lara V. Riddle , Dennis R. Roop , J oe l Rosenbloom , Roger H. Sawyer, Philippe Sengel, Geo rg e Stamatoyannopou los, M alcolm S. Steinberg, Peter M . Stei nert, Marry Ellen Stewart, George P. Stri cklin , Bryan P. Toole, Ju o ni J. Uitto, J ames A. Weston, and Sumner J. Yaffe.

The prog ram of the workshop was necessarily broad , reAectin g the diversity of topi cs relevant to future skin devel o pment and diseases resea rch . Dr. Stamatoya nn opou los, of the University of Washin gton , was invited to be the key note speaker beca use of his el ega nt studi es o n gene reg ulat io n durin g develo pment. His work o n hem oglobin switchin g provides a prototypic model for studies of the developmental contro l o f gene activ ity, a to pi c that is particularly appli ca ble to the skin .

The firs t of two sess ions o f the w o rkshop, entitled "Mor­phology and M acromolecular Expression," concentrated o n th e morphology of the ep id ermi s and its appendages, derma l-ep i­dermal jun ction , and the dermi s; and o n the express ion and reg­ulation of structura l and enzy m~ti c proteins and lipids, in th e respective reg io ns of the skin durin g differentiation in the adult and in development. The topi cs that were discussed in the second workshop sess ion , L'Iltitied "Control Mechani sms," in cluded th e mechanism s o f ti ss uc inte rac tio ns du rin g cutaneous devel o plllent, the role of cell-cell and cell-matri x inter~ct ion s , and the cffects of intrinsic and cxt rin sic regulato ry compo unds (e.g., ho rm o nes, growth factol's, vitamin s, and stero id s) in m od ulatin g sk in de­velopment. Whcreas these were logical categories fo r the purpose of presentation , it was apparent that the resea rch th at needs to be done is goin g to fo cus either o n questions invo lvin g the epider mis , th e dermal-epidermal jun ction, the dermis, o r derm~l-epiderl11al (mesenchymal-epithclial) interactio ns . This report wi ll follow the latter scheme o f o rgani za tion.

THE EPII ER M IS

Structure of the Epidennis During Dcveloprncnt Stud ies of the structure of the ep idcr mis ha ve revcaled the characte risti c features and th e timin g of events in the develo pment and differ­entiation of fetal ep ide rmi s in utero . O ur undcrstandin g of these events in ad ult epid e rmi s has all owed us to look for impo rt~ nt developmenta l markers. The characteri stics th at definc the ep i­dermis at differcnt stages of dcve lop m ent ~ re: stratifi ca tion, ap­pearance of ill1mig rant cells (melanocy tes, Lan gerhans cells, and

N IC HD/ N IADI)K RESEA I(CH WORK SHOP 465

Merkel cell s), chan ges in pcriderm stru cture, initi at io n and de­ve lopment o f epiderm al appendages (pil osebaceous stru cture, cc­crine and apocrine swc~ t g lands, and nail s), and keratini za tion

111· A feature thn is unique ro the deve loping epiderm is is the

periderm , a tran sient , simple epitheliulll first prescnt in cm bry­oni c skin as the superfi cial la yer of the two-Iaycred cpidermal ti ss ue. The perid erm is retained throughout the first two trimes­tcrs o f deve lo pment and undergoes a remarkable scries o f m o r­ph o logic changes in w hi ch Aattened , p:lVemel1t- lik e epithelia l cell s proj ect Iargc , multiple blebs, then reg ress to a Aattened epithelium 12 1. Its la st cells desqu am ate into the amnio ti c Auid at the time cell s of the underl y in g epidermis pro per undcrgo ker~tiniza tion. The am niotic surface o f the periderm is densely covcred with microvi lli , evcn ove r the blebs, indi ca tin g th at this layer presents an en o rm o us amolillt of surfa ce arca to the ~ mnio ti c cav ity-a stru ctural feature that ma y havc fun ctio nal propcrti es in terms of exch ~ n ge between th e fetu s ~ nd the amni o ti c Au id 13 1. Recent studies have shown that th e periderm contain s the keratin po lypep­tides that arc characteristic of sim ple epithelia, and that these are ret~ined throu gho ut gcs tation, cven th oug h rh e und erl y in g epi­derm al la yers at thc sa me tim e prog ress ivel y sy nthesize new ker­ati n species 141. Therc i littl e m o re th~n speculation , however, ~s to the ro le of the periderm , the o ri g in of this tissue, and its rel atio nship to the unde rl y ing epidermis and poss ibl y w ith the amnio n. Ncw m onociona l antibodi es that recogni ze an antigen on peride rm cells shou ld be v~ lu ~ble in sortin g out the identities of thi s la yer as comparcd with other epithelia , and in no tin g other prope rties of thesc unique cell s 151.

Markers of Fetal and Adult Epidennal Differentiation Dif­fe rentiation in the adult ep idcrmi s is eva luated in terms of stru c­tur~1 pro tein s (parti cularl y keratin s and filaggrin), ce ll surfacc anti gens and recepto rs, proteo lytic enzy m es (e.g.,' plasmin ogen activator) and epiderma l lipids th at arc cx pressed.

S (rJ/ ( /lIral Pr()/cillS

K [ IU\ T INS There has been signifi c lI1t prog ress in und ersta ndin g th e stru cture and biochcmistry of ke ratin prote in s in the cpidermis and other epithelia both in v ivo and in vitro. ,enes fo r some of the k e r~tin s in m o use and human cpidermis have been sequ enced, and eDNA probes that c~n h ybridi ze w ith mRNAs from ti ss ue and cultured keratin ocytes have bee n prepared .

The keratin s are a multi gene f.1 mil y ranging in apparent mo­Iecui:1r mass (M,) from 4(),OO() to 68,000 (40-68 K). M ost of th e keratin s are products of distin ct mRNA s 16,71. A few (the 65 and 66 K keratins) are th e result o f pos ttran slatio nal modifi cat io n of th e (,7 K kerat in 181. There is consid erab le ev id encc fo r posttran­slati onal m odifi ca tio n in mammali an epidermis 19, 101. but th e m echanism is not unde rstood. The keratins belo ng to cither an ac idi c (I) or b~ s i c (II ) sub f.1mi ly and arc fo und w ith o th er m em bers o f th eir g roup in th e same ker~ tin interm cdi ate fi lam ent 1111 . The m o lecular o rga ni za tion of keratin s in keratin fil aments has been inferred o n thc basis of amin o ac id sequ cnce ~nd stnl cture of certain keratin genes 11 2 1, and confirm cd b y scannin g transmis­sion electron mi croscope studies 11 3 1. O ur present underst~ nding of kerat in filament exprcss io n by, and the filamcnts' loca li zation in the ep idermis h ~s thus reli ed I~rge l y o n protein chemistry, ce ll­frec tr~nslation o f m RN As fro m tissue , and 111 0no cion:l1 antibody studies f14- 161 ·

Studies usin g cultured keratin ocytes havc :l lso suggested that the v~ riou s kcratins arc produ cts of di st in ct mHNAs 16 1. C ul ture sys tem s ha ve been parti cularl y im port31H in the demonst rati o n of cnvironlll ental effects o n kcratin exp ression and the Ill od ulation o f epidermal differentiation in the ~d ult. ultures of fetal ke rat­in ocytes have no t been used in su ch studies.

Keratin cxpression by epidermal keratin ocy tcs depends upon th e state o f diffcrcn tiation of these cells. In ad ult epidermis, kcr­atins o f in crcasin g M, arc synthesizcd as cells mi g rate from the basal ce ll layer into the stratum g ranul OS UIl1 and stratum co rn eulll .

466 K I ~EY ET AL

The 50 and 58 K keratins expressed by basal keratinocytes, and the 56.6 and 67 K keratins expressed by supra basal cells are con­sidered markers of epidermal st rat ifi cation and keratinization [17J.

A similar seq uence of keratin expression occurs in fetal epi­dermis, with progressive stages of gestation and concom itant epidermal stratification [4). At the time of the embryonic-fetal transition, a la yer of intermediate cells is added between the basal and periderm layers. T hese intermediate cells start syn thesizing the high-M, keratins around 11 to 14 weeks, when hair follicles begin to form and initiate keratinization . Posttranslationally mod­ifi ed keratins are expressed around 24 weeks, after epidermal keratin ization is complete. At the same stage, 40 and 52 K keratins expressed uniquely by the fetal epidermis are lost, probably due to desq uamation of the periderm o r masking by other keratins . The seq uence of keratin expression suggests that new genes are activated at different developmental stages, with perhaps contin­uing expression of the keratin genes from the ea rlier stages. The knowledge of keratin expression by embryonic and fetal epider­mis provides a rational basis for the diagnosis of disorders that involve abnormalities in keratin filament structure and biochem­istry expressed in utero sufficien tl y ea rly to be recognized by prenatal diagnosis based on amniocentesis an dlor fetoscopy.

The normal pattern of kera tin filament expression is altered in certain keratinization (e. g. , some of the ichthyoses) and hyper­proliferative (e. g., psoriasis [1 8]) disorders affecting the epider­mis. A mong these alterations are an absence of expression of some of the tissue-specific keratins, the reexpression of embryonic and feta l keratins, and the expression of "new" keratins. The 48 and 56 K keratins have been described as hyperproliferation markers [191. Such markers need to be studied .

Keratin expression can be modulated by hormones during nor­mal physiologic events (e .g., cyclic changes in vaginal epithe­lium), by addition of exogenous hormones to culture systems (corticosteroids), by various pharmacologic factors (e.g., reti­noids), and by components of the dermis (dermal-epidermal in­teractions) .

The hormonal regulation of keratin expression has been studied using cultured mouse epidermal cells and the hormonally-regu­lated cyclic differentiation of the rat vaginal epithelium . Estradiol admin istered to ovariectomized mice induces differential expres­sion of keratin genes in a temporal seq uence . Twenty-four h after the hormone is administered, the epithel ial cel ls begin to prolif­erate, and genes for the 50, 55, and 60 K keratins are expressed. After 48 h, the epithel ium is stratified and keratinized, and the cells express the higher-M, keratins. cD NA probes have been prepared that hybridize with mRNAs of differentiated mouse epidermal keratins (M, 55, 59, and 67 K) and to mRNAs for low­M, keratins (M, 50, 55, and 60 K) [1 6,17J. The use of similar probes in studying human skin development would be valuable for correlating the expression of keratin genes with the timing of onset of synthes is of the specifi c protein markers.

The influence that hormones (or o ther substances) in the am­niotic fluid or circulating in the fetus might have on keratin expression in the human fetal epidermis has not been investigated to date. It is known that the amniotic fluid is high in steroid hormones (estrogens, etc.) and that fluctuations in the levels of these substances in other body fluids influence, at least, the struc­ture of the epithelia (e.g., the ep ithelium of the third ventricle) they bathe . It is intriguing to consider that there may be a similar influence on the developing sk in, and that hormones might affect the express ion of keratin genes.

Avian systems have been particularly valuable in studies of the pharmacologic contro l of the epithelial differentiation (keratin polypeptide expression) . Most of the efforts in these systems have been concentrated on stud ies of dermal-epidermal interactions, and wi ll be discussed separately below. Other studies have in­volved injections of hydrocortisone and triamcinolone into chick embryos at different stages of development. T he result of this treatment is an inhibition of scale formation, because {3 keratin synthesis is blocked in the steroid-treated embryos [20]. It is not

THE JOURNAL OF INV ESTIGATIV E DERMATOLOGY

known if, or how, fetal skin in humans binds and responds to corticosteroids that may be present in the feta l ci rculation o r in the amniotic fluid. .

T he relationship between vitamin A metabolism and the pro­motion and maintainence of keratinization in the skin has long been recognized. However, the mechanism of action of the vi­tamin A-derived substances (retinoids), their physiologic role, and the application of this knowledge to clinical practice have been rather recent developments. Vitamin A added to kera tino­cyte cultures inhibits the expression ofhigh-M, keratin genes, but permits terminal keratinocyte differentiation if it is deleted from the culture medium [21]. The action of vitamin A (retinol) and retinoic acid may be mediated by two distinct intracellular binding proteins, which have been purified to homogeneity and charac­terized. Both proteins are found in all animals, bind retinoids in a species- but not tissue-specific manner, are expressed at different stages of differentiation, and have been identified in fetal chick skin extracts [22]. They may be the second messenger system th at can activate and rep ress the genome at different stages of differentiation [23]. In spite of the well-known teratogenicity of retinoids for a number of fetal organ systems [24] and their in­fluence on the differentiation of the epidermis, there has been no effort to determine if the intracellular retinol or retinoic acid bind­ing proteins are present in human fetal skin. The presence of these molecules may mediate the retinoids' influence on cutaneous mor­phogenesis.

FILAGGHIN Filaggrin and its precursor, profilaggrin, are pro­teins found in the highly differentiated epidermal layers, granular and cornified. Profilaggrin is a highly phosphorylated, polymeric, basic protein found in keratohyalin g ranules [25] . It is converted by dephosphorylation into tri-, di-, and monomeric units of fi ­laggrin during the granular- cornified cell transition [26]. Filaggrin molecules are broken down to free amino acids, urocanic acid, and pyrrolidone carboxylic acid in the outer la yers of the stratum corneum (27]. Recent studies combining protein biochemistry, immunohistochemistry, and electron microscopy have shown that filaggrin is expressed in keratinocytes offetal epidermis only when there is morphologic evidence for granular and cornfied cells. This occurs around 14 weeks estimated gestational age (EGA) at the site of developing hair follicles, and approximately 10 weeks later in the interfollicular epidermis [4] . Thus, this marker of terminal differentiation of the epidermis appears significan tl y later in development than the high-M, keratins. T his is an un expected finding, because it is known that there is coord inated expression of profilaggrin and the high-M, keratins in vitro [28]. The factors that initiate the express ion of filaggrin are unknown.

DESMOGLEINS AND DESMOPLAK INS Keratinocytes in all layers of the em bryonic epidermis, including the periderm, are con­nected by desmosomes, the protein components of which are important for the integrity of tissue structure. Desmosomal pro­teins may be in volved in some of the aca ntho lytic disorders of I

the skin, such as Hailey-Hailey's disease and Darier's disease. Two groups of proteins have been recognized: those that occur extra­cellularly (desmogleins), and those that are found in the cyto­plasmic environment (desmoplakins). These proteins have been isolated, characterized by M" used as antigens to produce both polyclonal and monoclonal antibodies, and localized in the epi­dermis by immunoelectro n microscopy [29,30] . Three mono­clonal antibodies produced against one of the desmogleins also react with the a-2 type 1 coll agen band, suggesting that there may be a collagen-like region in the desmoglein molecule. Only 2 of the 5 monoclonal antibodies against desmosomal proteins recognize proteins of the hemidesmosome, indicating that these connections to the basal lamina are, in spite of their morphologic sim ilarity to desmosomes, biochemically distinct.

OTHER PROTEIN MARKERS OF DIFFERENTIATION Involucrin is a protein marker of epidermal differentiation produced in soluble form by cells in the spino us layer, but crosslinked in the granul ar

VOL. ~8. NO. ~ AI' IUL 1987

layer cell s by the calcium-req uirin g ep ide rm al transg lutamin ase [31] . The cross linked protein ~sse mblcs belH:ath the pbsma m em­brane of co rnifi ed ce ll s ~s a thin, elect ro n-den se la ye r that , to ­gethe r w ith the pbsma m embrane, fo rm s the cornitl ed cell en­velope. T he regubtion of in vo lucrin ex pressio n has been exa min ed in keratin ocy te culture . Postmito tic keratinocytes tint either re­ma in in o r have left the basa l I~ye r sy nth es ize invo lu crin 132 1. In vo lu crin is exp ressed in develo pin g rabb it skin onl y after the epidermi s has stratifi ed 133 1· In vo lu crin sy nthesis by human feta l ep idermal cell s ha s not been in ves ti gated to date .

Cell S III:fncc A /lfI~~ L'II S nlld a cceptors: Seve ral anti gens present at the sur f.1ce of ker~tinocy tes appear to mark th e state of kerati ­nocyte differentiati o n. For example, the g lycoco nju gates on ke­ra tin ocyte surfa ces o f different b yers o f the ad ult ep idermis 134 1. and o n keratin ocy tes in vitro 135 1 bind Iectin s diffe rentiall y, co r­respond in g to a loss o f ca rboh ydrate mo ieties as differentiati on proceeds. Furthermore, blood g ro up an tigens arc exp ressed dif­fe rentl y by feta l epide rmi s and o ral mu cosa cells 136 1, but it is not kn own how these an tig Cll s, o r the various Icctin s label the ep ide rmi s durin g prenatal deve lopment in utero.

Va ri o us ce llular rece ptors ma y be essentia l to keratin ocyte dif­fe rentiation. Fo r exa mlle, the ep id erm al g rowth factor (EGF) recepto r has been iso lated and purified. and an ant ibody ha s been prepared to it th at appea rs to bind preferentiall y to the least dif­fe rentiated cell s in the epide rmi s and in ke ratin ocyte ce ll cultures [37-39 1. Localiza tion o f the EG F rece ptor ha s been studi ed in norma l and di seased skin and in cultured keratin ocy tes, and EGF ac tivit y has been determined therei n . EGF exerts its g ro w th­regul at in g effects by binding to the EGF recepto r, w hi ch m ay be the sa me m o lecule as the EGF kina se and m ajo r endogeno us membrane pro tein ; subsequ entl y, it is intern ali zed and rapidl y deg raded. The elcm ent(s) of this regulato ry m echani sm that stim­ulate cell proliferation are not kn o wn , but one signal ma y be the increased ph os phorylation o f tyros in e residues of end ogeneous and exogeno us protein s, w hi ch occurs when EGF binds to its recepto r molecul e. The EGF sys tem in adu lt skin is one of the best-studied m odels o f g rowth and diffe rentiat io n . EGF recepto r distribution has also been studied in feta l rat skin , w here it was fou nd to label basa l epiderma l ce ll s, except fo r the devel o pin g fo llicl es 140 1, but the pos iti on of EG F receptors in develo pin g human skin and the ro le of EGF, o r of similar m orp hogenetic substan ces, in the ontogeny of this tissue ha s no t been in ves ti­ga ted .

Epidef/llnl Lipids: Epidcr J11 al lipid s arc o ften neg lected as markers of differentiati on. alth o ug h the lipi ds sy nth esized by the cell s o f the more hig hl y d iffe renti ated epidermal la yers are as specifi c fo r these ce lls as th e keratin s they possess. The laJ11elbr g ranul es present in spin ous and g ranular cells arc the source of neutral lipid-ri ch lam ell ae that arc relea sed b y exocy tos i ' between the g ranular and co rnified cell layers 14 11 . T hese: sta cks o f lamelbe are th o ug ht to be im porta nt ill regulatin g permeability of the adu lt epid ermi s. Lamellar g ranul es art' recogni zed in the keratin­izin g hair ca nals o f feta l skin at around 15 weeks , and in the interfo lli cular epidermis just at the onset of kerat in izati o n, around 22 to 24 wee ks [42 1. Epidcrm all y deri ved lipid s arc also impo rtant in promoting th e desquamation o f ho rn y ce ll s, an essentia l act ivity if the epide rmis is to be maintain ed in a consta nt thi ckness 1411.

Lipids present on the skin surface are a mi xture of epide rm al lipids and sebaceo us g land- derived sebum . Sec retion of lipid s by the sebaceo us g land is influen ced by circulatin g levels o f and ro­gens, alth o ug h the fatty acid co mpos ition of th ese lipids is influ­enced b y bo th ho rmon es and geneti c fa cto rs. There is m orpho­logic ev iden ce tl13't seb ulll sy nthesi s beg ins in fetal skin at around 15 weeks of ges tation and pro babl y makes signi fica n t contribu­tions to th e li pid-ri ch material o f the verni x caseosa that covers feta l skin , parti cul arly in later stages o f gestation [1/. It is not known if or how th e ve rni x influen ces permeability pro perties of

NIC I ID/ N IAIJDK RESEARCH WOR KSHOP 467

the fetal skin , o r whether the co mpositio n oflipids in the amnio tic fluid could be used as a criterion fo r prenatal diagnosis in so me ichth yoses.

The first repo rted stud ies of feta l cutaneous lipids have shown distinct difTeren ce between epidt'rma l and dcrmallipids , and that even in the first trimester, the epidermis synthesizes so me o f the lipid s that are chara cterist ic of adu lt ti ssue 143J. T here arc insuf­fic ient data at present to co rrel ate lipids in feta l skin with its permeability, althoug h studies with feta l skin in vit ro have dem­onstrated that water transport across hum an feta l skin declines m arked ly when the co mposition o f amnio tic fluid changes and th e fetal epidermi s beg ins to keratinize /441. M o re recent studies of epidermal mo rpho logy by freeze-fracture techniques have sho wn that the pbsm a membranes of fetal epidermal cells have pro perties typical o f epithelia in vo lved in water tran sport durin g the first ha lf o f ges tation 131. The ro le of the epide rl11 allipids in the fetu s ' exchange with the amnio ti c fluid is an important problem that need s in vest igati on .

Epidermal Stem Cells and Regulation of Epidermal Prolifel'ation

Epidcnllnl S tel/l Cells: Heterogeneity in the pro li ferative ca pa­bilities o f basa l epiderma l cells has onl y recentl y been appreciated in work o n rodent and av ian epidermis 145,46J. Studies of adu lt hum an epidermis g rafted to the nude mouse 1471 have revealed a po pulation o f label-reta inin g ce ll s that ma y be eq ui va lent to ce rtain basal cells in adu lt rodent epidermis that have properties of stem cell s: the y cy cle slowly and retain label fo r long perio ds of time. Similar cells have also be:en identitied in the monkey pa lm , w here they are loca ted rather precisel y /481. There arc as yet no ge:neral m o rph o logic o r cy tochemi ca l markers that ca n distin guish stem cells fi'o m o ther basa l cells in adplt epidermis. Pro lifcrati o n o f the epidermis in the human embryo or fetus has been studied in onl y a few inst;1I1 ces, and then limited in scope to a few attempts at label in g pro liferating epiderm al cells in o rgan culture 149 1. There have been no attempts to id entify stem cell s in this rapidl y g row in g ti ssue.

A challen ge fo r the future is to improve o ur understa ndin g of thl: kineti ca ll y diffe rent populations of basal epidermal cell s and to devel o p m arkers that ca n be used to identify a population o f cell s that possess th e pro perties ass igned to stem cclls. Such cell s llIust pla ya role in regubtin g norm al epiderm al stru cture and Ill ay be target cell s in h yperpro liferative disorders. The estab­lishment of em bryoni c and fetal skin and/or kcratinocy tes in o r­ga n and cell cu lture sho uld provide the o pportunity for studies o f epid erlll al ce ll kineti cs, and ste m ce LI s, in devel o pin g skin .

ae.~ l/ln ti" " tlJEpidcrlllnl Prolifcrnrioll : In epidermal as well as o the r ce ll sys telll s, m o re differentiated cell s have been shown to regu late th e aillo un t of ce ll pro life ration by ncga tive feedback . Extracts of epide rmis ca n inhibit pro li fe ratio n of basal keratinocy tes at ce rtain times in the cell cy cle. T his inhibition is prolifcration­dependent, and requires certain cAMP levels to act. An acidic penta peptide w ith the sequen ce pG lu-A sp-Ser-Gl y-Ala has been identified as an e:pidcrma l inhibito r that influ ence:s the cell cycle after co mpletion o f DNA synthesis . This inhibitor stron gly sup­presses mitoses in rat tongue ep ithelium w hen administered in low doses , but has a stimulatory effect at hi gh doscs. A g ranu­locy te inhibito r of onl y five amino acids has a sim ilar inhibitory effect on cells o f the bo ne m arrow [50 1. The mechanism fo r thi s inhibitory effect and th e tiss ue specific ity of the inhibi tors have no t been determ ined as yet.

Model Studies for Probes of Gene Regulation During Epi­dermal Development and Differentiation Significant prog­ress has been made in understandin g protein markers of epidermal differentiation , but the genes that spe:cify form and fun ctio n of the ep idermis have not been adequately investiga ted as yet. Recent advances in ana lyses of mo k cuiar contro ls of develop m ent now

468 K I1EY ET AL

m ake it poss ibi<: to dete rmin e the contributin g fa cto rs th at inAu­ence the deve lo pmental regula t io n o f th e epiderm al genes . Con­tro llin g inAuences have been eicga ntl y stu d ied fo r th e hem og lo bin genes durin g erythro po ies is . Such inves ti ga ti ons of relative con­t ribu tio ns of embryoni c enviro llm ent and intrin sic ce llular co m­mi tm ent to the develo pmental switchin g of hem oglo bin genes are discussed below to pro vide a m odel fo r pro bes into the reg­ul ato ry con tro ls of epide rm al genes durin g deve lo pment o f th e skin .

Ery thropo ies is beg ins in the yo lk sac, but is taken over by the li ver at approxi m ately 5 weeks EG A, and is ultim ately th e ro le of th e bo ne m arrow, at approx im ately 20 weeks. Simultaneously, g lo bin sy n thes is undngoes develo pmental changes that in volve a switch (o r switches) fro m primiti ve to m o re m ature fo rm s of g lob in ex press io n . Acti vity of the () gene in th e embryo is repl aced by ex press io n of th e two ll' genes in fetal and adult life. Genes o f th e {3 locus switch fro m ex pression o f th e € chain in th e embryo to the "Y chain in fe ta l life and to th e adult {3 chain , with onset in the fetus and an in crease in the pe rin atal pe riod . The () to ll' , and € to "Y switches occur by th e 5th week of gesta ti on , w hen he­m ato po ies is changes fro m yo lk sac to li ver, w ith co mpletion by 60-80 days EG A 15·11 . Activa tio n o f th e "Y and {3 genes at the begi nnin g of th e fe tal peri od occur simultaneously, but "Y chains are prod uced at a rate 50 tim es hi g her than {3 chain s at th at stage 152 1. The switch fro m "Y to {3 in th e perin atal peri od is no t cor­related w ith a sw itch in site of hem ato po iesis, but appea rs to correspond to the develo pmental age o f th e fetu s/newbo rn 1531 . Switchin g is de la yed in develo pmentall y reta rd ed fetuses, and is not m odifi ed in sheep fetu ses that have been eith er th yro idecto­mi zed , adrenalecto mized , hypoph ysec to mi zed , o r nephrecto­m ized (to tes t fo r a poss ible influence of ho rm o nes o n the switch) 1541·

Of the two hypo th eses th at ha ve been pro posed to ex pl ain he lllogio bin switchin g durin g ontogeny, o ne assulll es a switch in ste m cell lin eages, e;tch w ith a diffe ren t p rogram o f g lo bin exp ression ; the o the r suggests a hem og lo bin switch w ithin the sa m e ce ll lin eage th at is cont ro ll ed eithe r intrin sica ll y, and/o r ma y be influenced indu cti vely by ce ll -en viro nlll ent interac ti ons. The fi ndi ngs that p rimiti ve and definiti ve ery thro po ieti c ce ll s are no t c\onall y de termined to m ake onl y embryoni c o r definiti ve hem o­g lo bin chain s, and th at fetal and ad ult chain s m ay be coex pressed in o ne cell , argue ag ain st the first (switch in cell lineage) h y­pothes is.

T he seco nd h ypo th es is , intralin eage switchin g, has been ex­amin ed us in g cell and o rgan cultures and chi ck-qu ail chim eras 155 1 to eva lu ate th e res po nse o f hem ato po ieti c cell s o f di fferent deve lo pmental ages in a deve lo pmental en vironm ent of a g iven age . T ranspl antatio n m ode ls also have been used w hereby fetal stem ce lls arc inj ected in to leth all y irradi ated adult anim als and adult hemato po ieti c ce lls a re injected into a fetus 150,571. T he data demonstrate that changes in the en viro nm ent m ay playa ro le in the hem og lo bin switch , but are no t th e sin g le fac to r co n­tro ll in g the switch . Switchin g mig ht refl ect a change in the ab ili ty of ce ll s to parti cipate in indu cti ve interac ti ons. O th er studi es sug­ges t th at hem ato po ieti c ce lls have an in t rins ic program th at directs th e switch 158 1.

Gene st ructure as well as nucl ea r-cyto plasmi c inte rac ti ons pl ay a ro le in the hem og lo bin switch. This inAu ence is suggested by th e ex tent to w hi ch meth ylati on affects g lo bin ex pressio n in m an 159,(,0 1· It is also in d ica ted b y changes in chro matin o rga ni zat io n of g lo bin genes durin g deve lopm ent, as shown by the genes' sensit iv it y to DNAse I di ges ti o n Inl], and by th e fac t that th e genes' exp ressio n can be regul ated b y cyto pl as mi c pro tein s (trans­actin g c lem ents), w hi ch attach to specifi c seg m ents o f DNA , typ ica ll y th e D NA se I- hypersensitive sites 162].

Studies w ith transgeni c mi ce have dem o nstrated that trans­fected hum an {3 g lo bin genes are ex pressed o nl y in th e e ry thro id cell s (tiss ue-s pecifi c express ion) and onl y in cell s o f the definiti ve erythro id lin eage, not in yolk sac-de ri ved ce lls (s tage-specifi c

TH E JOUI{NA L O F INVESTIGATIVE DEHM AT O LOGY

ex press io n). Transgeni c mi ce containin g human "Y g lo bin g cnes synth es izcd "Y glo bin onl y in e rythro po ieti c cell s o f th e yo lk sac . 163,64 1. T hese studies suggest th at onl y th e appro priate ly staged ce ll s co ntain the tran sactin g eicm ents th at, in additi on to (is-activ e DN A segments participate in the contro l o f the hem oglo bin switch.

So m ati c cell hybrids prepared by fu sion o f m o use cry thro leu­kemic (MEL) and human cell s we re also used to stud y hem og lo­bin ex pression . Fetal ery thro id cell s + M E L cell s syn thes ized fetal "Y glo bin , but with in cre:l sing tim e in culturc ·such cell s even­tu all y synth esized the adul t program o f {3 hemoglo bins. Adult Iy mph o blas ts o r fibrobla sts + M EL cells synthes ized {3 g lobin but no t "Y r65]. The deve lo pmental histo ry apparentl y detcrmines whethe r th e "Y gene will be turn ed o n , but the m o lecular bas is fo r this effect, o r fo r th e "Y to {3 switch, is unknown. N everth eless, th e understandin g th at has alrea d y been gained o f th e develo p­mental regul ati o n o f g lo bin genes, and th e ex pe rim ent:lI m odels used to o btain the data , sho uld be va luable fo r similar gene reg­ulato ry studies o f the skin.

Immigrant Cells in the Epidermis of Developing Skin

Mclall vc ),tes: Melanocy tcs arc neural crest-de rived cell s that are th ought to enter the epide rmis durin g the first trim es ter. The timin g o f this event is based o n the o bserva ti o n o f ce lls w ithin th e embryoni c and earl y fetal epidermi s that possess a no nk er­atin ocy te nu clear m o rph o logy. O th er m arkers o f mel anoc ytes, includin g m elanoso lll es and cell surface anti gens, arc no t present at that tim e o r have no t been studied . M elanoso m es can be rec­ogni zed in th e cy toplas m o f these ce lls after nO days EG A, but the y do no t show Ill o rph o log ic cv iden ce of pi g ment sy nthesis and tran sfer until the second trimester 111 .

Whethe r the neural cres t devel o ps into m cbnocy tes o r o ther progen y (g li al ce ll s) depend s o n en viro nlll ental co nditio ns. Cells des tined to beco me g li al ce ll s differenti ate into such o nly if th ey intera ct in ti ss Li e w ith neuro nal ce ll s; if this inte rac tio n is blocked th ey beg in to ca rr y o ut mebnin synthesis. N eural cres t cells in vitro also sy n thes ize mebnin if they are g row n o n pl as ti c, bu t melJnogcnes is is inhibited if so mite- conditioncd m edium is :!dded to the culture o r if cells arc g ro wn o n a collagen substrate . It i no t kn o wn how the derm al en viro nment mi ght influence neur:!1 cres t ce ll s as th ey mi g rate into the epidermi s and differentiate into melan ocy tes, o r if the dermi s affec ts th e pi g m ent ce ll s o nce thcy arc differentiated . Som e inte rac tio n be tween epiderm al m cian o­cytes and m as t cell s o f the dermis is, howeve r, implicated in the pathogen esis o f neurofi bro m atos is 166 1. T he dcvelo pmental o ri­g in of this diso rd er that manifes ts itse lf cutan eo usly as pi g men­tati on defccts o r neuro fibro mas can now be in ves tiga ted imlllLl­nocytochemicall y w ith th e aim o f improv in g o ur unde rstanding o f th e ontogeni c inAu ences th at yie ld this specifi c progen y o f the neural cres t.

Lallgrrhall s Cells: Langc rh ans ce ll s arc bonc m arrow-deri ved cells of th e epidermi s 167] th at fun cti o n as anti gen-presentin g cells to helper T cell s to m ount an immul1c res po nse. Lik e o ther cells w ith this fun ctio n, the y bea r a number o f cluracteristi c antigens (eg, HLA - DR , T 200, and 5 1(0) and rece pto rs (fo r Fc-l gG, C3b, M g2

' - ac ti vated ATPase) o n the cell surface, and ex prcss a de­termin ant , O KT6, typi ca ll y fo und o n imm ature T cell s 168 1. They arc ca pabl e o f all ogeneic T-ce ll s timul atio n in a mi xcd epidermal cell- ly mph ocy te reactio n and arc a source of th e cy to kin c inter­Icu kin 11 69 1. Based n the o bservati o n o f Lan ge r han s cell g ranules in th e cy to pl as m , Langerh ans cell s ha ve been identifi ed in fe tal epide rmis as ea rly as 10 w eeks. M o re recentl y, epidermal sheets, prepared fro m embryoni c and fet al skin and cxa mined histo­chcmi ca ll y and imllluno hi stochemi ca ll y, have reveal ed a po pu­lati o n o f epide rmal cell s that stain pos iti vely fo r Lan gerh ans cell dete rmin ants (ATPase and HLA-DR) in th e embryoni c ti ssuc and fo r an additi ona l m arker (O KT6) in fe tal ti ss ue after approx i­m atel y l2 weeks 170]. There ex ist no fun cti onal studies of Lang­e rh ans cell s in v itro no r co rrelated studi es to determine whether

VO L. 88, N O . 4 AI'HIL 1987

a po pulation of rccepti ve Iymphocytcs is also present in the dermis at these ea rl y ages .

Merkel Cells: Merkcl cell s in adult skin arc loca ted in thi ck (palmar/ plantar) and hairy po rtio ns, where they fun ction as slow- adapting m echano recc pto rs. Thcy alT typICall y fo und In asse mbli es, associated w ith a sin g le ncurite, called to uch do m es. Dense- co re g ranules th at arc m o rph o logica ll y similar to m ono­amine-containing granules are present in th e cy toplas m of th e Merkel cell , but a specifi c neuro transmitter has no t been identified [71] . Merkel cell s m ay be part of th e ditTu se neuroendocrin e amin e precurso r up ta ke and deca rboxy lati on (APUD) sys tem o f the bod y. T he o ri gin o f M erkel cells is un certain , altho ugh current evidence favo rs an ectodermal derivatio n 172]. Like epithelial cells, they co ntain bundles o f keratin filam ents and fo rm desm osomes with adj acent keratin ocy tes. T hey lack bo th vimentin and neu­rofi lam ents. Merkel cell s have no t been recogni zed in fet al skin until late in th e seco nd trimcs ter, substanti all y later th an o ther immig rant ce lls. This m ay correlatc w ith their m o re loca lized distributio n in skin th an th at o f the o ther immig rant ce lls , o r m ay be a co nsequen ce of not lookin g at skin fro m ea rlier fetu ses w ith the appro pri ate pro bes to recogni ze them .

DE RMAL-EPID E RMAL JUN CTIO N

T he derm al-epiderm al junctio n (DEJ ) is a basem ent membrane, separatin g the epidermis and the dermis, w hich fun cti ons as a zone of interact io n , adh es io n, and as a scmipenetrable ba rri cr between the two ti ssues. It is in vo lved in a number o f hereditary blisterin g diso rd ers (eg, the epiderm o lys is bullosa g ro up of di s­eases) and in ce rtain autoimmune diso rders (eg, bullous pem­phi go id). Stru ctural regio ns of the DEJ (l amin a lu cida, lamin a densa) and di stin ct entities (hemides m oso m es, ancho rin g fil a­ments, and :lI1 cho ring fibril s) have been identifi ed , and num erous biochcmi ca l co nstitu ents (type IV an d VII co llagens, heparan sul­fa te pro teoglyca n, laminin , nidin ogen, entac tin) and anti gens (bullo us pemphigo id IBPI, KF-l , epiderm o lysis bullosa acquisita IEBAL and an cho ring fibril I AFI and AF2 i) have been recognized in each . The absence o f som e o f the anti geni c compo nents (KF-1, AF- l , A F-2) has becn correlated w ith gencti c diseasc.

O ntogenetic studies of the expressio n o f biochemica l and an­tigeni c compo nents o f the devel o pin g D EJ havc sho wn th at : (1 ) ty pe IV coll agen is always present in its lamin a densa; (2) laminin is ex pressed in the lamin a lu cida at all ages, and the BP antlgen appea rs afte r the epidermis is stratified and hemidesmoso m es arc stru cturall y co mplete (a ro und 10 w eeks) 173,741; (3) the EB A antigen is present in the lamin a densa of thi ck skin as ea rl y a s 8 weeks when the ti ssue is strati fie d 175 1; (4) th e KF- I antigen appea l:s in the lamina densa aro und 16 ~eeks [731; and (5) the AFI and AF2 anti gens are ex pressed after 26 w ecks EG A [761, even th o ugh stru ctures w ith the appea ran ce of ancho rin g fibril s are present in first-trimester skin 111. There have been no studies to determine w hether an antibody to ty pe VII co ll agen that rec­ognizes ancho rin g fibril s in ad ult skin will identify pres umpti ve feta l ancho rin g fibril s at ea rlie r stages. It is also no t kn o wn If o r how the differentiatio n o f the epidermi s and the compositio n of the derm al m atri x at va ri o us sta ges o f develo pment determine ex pressio n o f components o f the DEJ . The ontogenetic expression of D EJ s tru ctures and anti gens has been studied in other devel­opment-like systems, such as wo und hea ling , and epibo ly in o r­gan culture [73 1. Studies to answer these ques tIo ns 111 the devel­opin g sk in co uld be condu cted uSlll g cul ture system s and tiss ue recombinant m ethods.

T he use o f man y of th e antibo di es to DEJ compo nents has been very impo rtant in specifi c diagnosis o f genetic diseases invo lvin g the D EJ in pos tnatal ski n [76J. Antibodi es have recentl y also been used in studies of frozen sectio ns of fetal skill o bta1l1ed by fetos­copy. M o re and im pro ved data underl yin g this antibod y use are, howeve r, needed to a ll o w the co ntinued and ex panded use of th e

N ICH D/ N IADDK RESEA RCH WO RKSHOP 469

antibody proced ure for rapid diagnoscs o f abno rm alities in feta l as we ll as adult skin .

T H E DE RMI S

Collagen in the Adult and Fetal Dermis Fo rm and fun cti on o f the derm is are determin cd by its ex tracellul ar m atri x, the con­stitu en ts of w hich arc also fo un d in o ther conn ective ti ss ues througho ut the body. O ne of th e principal m atri x compo nents, collagen, is represented by a fa mil y of at least 10 genetlcall y distin ct pro teins th at occur in ti ss ue-specific patterns 111 the ad ult and arise durin g develo pment in a defmed tempo ral sequence. The interstitial co llagens (types I, III , and V) for m thc fi brIllar part o f th e m at ri x of th e dermis. Ty pe VI coll agen assembles in beaded fi lam ents adj acent to the coll agen fibers; type IV IS a componellt o f the basa l lamin ae not only at th e DEJ bu t also around vessels, nerves, and beneath epider mal appendages; and ty pe VII coll agen is a co mponent of th e anchorin g fi b rils. T he biosyn thes is of collagens w ithin the cells of the dermIS, 1I1c1udll1g the va ri o us enzy m e- ca talyzed steps, arc well understood . T he events impo rtant in the secretio n of co ll agen m o lecules in to thc ex tracellular m atri x and their asse mbl y and stabili zation as co l­lagen fi bers arc also reasonabl y wcll understood. Severa l human coll agen genes have been cloned and iso lated, as well as partiall y seq uenced , and th eir chro m oso m al loca tio n dete rmin ed. .

E rrors at va rio us levels of collagen biosymh esis have been Iden­tified in fibrob las ts cultured fro m paticnts w ith inherited con­nective ti ss ue diso rders. Reco mbin ant D NA techn ology has en­abled in ves tigato rs to define these erro rs at the gene level 177].

Insight into the develo pmem al reg ulatio n of co ll agen biosy n­thesis Ius been o btained th ro ug h studies o f th e collagen genes ' regulato ry mechanisms usin g cultured fibroblas t syste ms trans­fo rm ed by viruses, o r m o usc embryos inj ected w ith .v irus. A nim al systems, such as Drosophila and Cacl/or/labdilis elega l/ s, have pro­vided info rm atio n abo ut the tempo ral ex pression o f the co ll agen genes [781·

U sing a chi ck fibro blast system , it w as fo und that cellul ar t rans­fo rm ati on w ith RN A o r D N A virus ca used a n13 rked red uction in type I collagen sy nthes is. Injecti o n of a plas mi d that com ained the pro m o ter region of thc cl oned chick 0' 2 type I coll agen gen.e linked to a bacterial gene, into m o use cell s (where th e plas mid IS stably integrated into the geno m e), fo llowed by viral transfo r-_ matio n of the m o use cells, allo w ed redu ctio n of th e synth eSIS ot the bac teri al m essage to th e sa m c extent as th at of m o use coll agcn ImRNA . This suggests the sa m e regul ati on of the co llagcn pro­m oter after reintro ductio n in to m o use cells as o f the end ogeneous co ll agen pro mo ter. Such studies sho uld prov ide a systcm fo r th e determin atio n of w hi ch pro m o ter sequences m ay be Im po rtant fo r the regul atio n of co ll agen gencs durin g develo pment of the sk in .

T he ty pc of collagen synth esized in fe tal kin are the sa m e as th ose in the adult and th ey are distributed in th e sa m e pattern 17<) ]. In fetal skin , however, co llagens III and V arc slig htl y m o re abundant th an in the adult [80 1. HI gher Icvels of enzym es related to posttranslati onal m odifica ti on of collagen I and III have been no ted in the fetus, but m echanisms that regulate prenata l collagen gene ex pression b y fe tal fib ro blas ts have no t been explo red . T he interactions o f co llagens with o th er n1.a trix m o lecules, and ce ll s of th e dermis and the epidermi s in develo ping skin arc also un ex­plo red .

Collagenase Expression in DeVelopment Collagenase is in­volved in the initial stages o f coll agen deg rada tio n associated with res tructurin g of the m atri x in no rm al tissue m aintenance, path o­logic condi tions in volvin g tiss ue inflammation (eg, arthritis) and as part of m o rph ogeneti c processes such as wound repaIr and develo pment. A stru cturall y and enzy m atica ll y abno rm al co lla­genase m o lecule is the bas is fo r the severe inherited blistering diso rder, recessive dys tro phic epiderm ol ysis bullosa 181]. Col­la genase is prod uced by fibrob la sts in the dermis. bu t IllUSt be

470 K I(EY ET AL

reg ula ted st rin gentl y so that anomalous patte rn s of co llagen breakdow n o r accuillul at io n do not occur.

Co ll agenase ex press io n by fibro blasts cultured frolll hum an embryoni c and feta l ski n in creases w ith prog ress ive gestat io nal age. T he am o unt of ca talyt icall y acti ve pro tein is substantiall y lower (4-10 times) in feta l fibro blasts th an th at synthesized by adult fibrob lasts, altho ug h the am o unt of trans latable co lla genase IllH.NA is o nl y 2-3 tim es less. T hese data sugges t that there is regula tion of coll agenase sy nthesis by th e fetal ce ll s at the lev els of t ranscrip tion and tra nslatio n [82]. Feta l and adult fib ro bla sts showed similar levels o f synth es is of co llagenase inhibitory pro­te in /83 /. Hi g h leve ls of th e inhibito r, howe ver, are present in amni otic Auid and alT produ ced by fetal lun g fibrobla sts. Puri­ficatio n of so m e fetal co ll agenases has revea led that they have low specifi c act ivities, and thus th ere ma y be stru ctural alid /or ca talyti c differences between these enzy m es and those o f th e adult, sug­gest in g the possibility o f synth esis o f a feta l ty pe of colla gen ase [82]. It is no t clear w hether specifi c populati o ns of fibrobla sts in fetal skin prod uce co ll agenase o r if a ll have th e sa m e po tenti al. In the ad ult , co ll agena se produ ct io n appea rs to be ma ximi zed by ce ll s nca r the derm al-ep id erm al junction /84 /. This m ay sugges t an interd epend en ce of epid erm al and derm al ce lls in the regulatio n of co lla genase sy nthes is that sho ul d be invest iga ted in adu lt and feta l skin.

Ep iderm al cells frol11 feta l and ad ult rabbit skin , cocultured w ith derma l fibro bl asts, s timul ate co ll agenase sy nthesis several fo ld throug h the prod uctio n of a cy tok ine. Prod uctio n of this co lla genase sti mulati ng substa nce is ce ll density- and Caz·' -de­pendent. Feta l ep i·derma l cell s arc very effective in th e produ cti o n of the cytok in e. T h e state o f di fferenti atio n o f the epiderm al cells th at produce this m ate rial has not been exa min ed ri goro usly, althoug h the p resence of C az , sugges ts that m o re-d iffe rentiated epiden1131 ce ll s m ay be in volved /85 /. Co ll agenase synthes is in feta l ski n Illa y be correl ated w ith in vas ivc and m o rph ogenetic events such as th e form at ion of epidermal append ages and th e estab li shm cnt of nerve- vascular netwo rks. Purifi catio n o f the ef­fecto r substa nce that s timul ates coll agena se sy nthesis , and efforts to identi fy the prod uctio n of this m ateri al o r o f a similar actin g co mpo un d fro m other derma l ce ll s sho ul d receive consid era t io n.

Elastin Expression in Development E las tin , th e seco nd ma­j o r m at rix co mpo nent of the dermis, is the elect ro n-lu cent portio n that acco unts for up to 90% of th e m ature elas tin fib er typica ll y presen t in th e deep ret icul ar dermis of adu lt skin ; the remain in g protein is that of the mi crofi bril. Mi crofi bril s are readil y apparent in less mature elas ti c networks in th e m o re superfI cial derm is. T he en tire elas tin network is co ntinu o us and im portant in the elasti c recoil of the de rmi s after defo rmatio n.

E last in appears to be a late gene p rodu ct in man y feta l ti ss ues , includin g th e de rmis. In hum an feta l sk in , clastic fib ers are not ev ident by m o rpho logic criteri a until the last few weeks of the second t rim ester /86 /. It is no t clea r w hether the soluble elastin precurso r, tropoelastin , ca n be detected b iochemi call y o r im­mun olog ica ll y in the ti ssue at ea rlier stages. Mi cro fibrils arc al­read y recogni zed in embryoni c sk in , but there is no ev idence that th ese morpho logic entities are biochemica ll y th e sa m e as the elas­tin Illi crofi brils. T he contro l of elas tin mHNA in develo pment appea rs to be largely govern ed by the am o unt of elastin mRNA transcribed /87 \. E lastin cDNA and geno mi c clones have been o btain ed, and the loca ti o n of th e elas tin gene o n hum an clHO-1l1 0SO lll e 2 has been determin ed /88]. U se of these probes m ay prove va lu ab le for further s tudi es of th e reg ul ation o f ela stin sy nth esis in deve lo pin g ti ss ues .

Glycosaminoglycans, Proteoglycans, and Glycoproteins in Development Proteog lyca ns for m a ge l-like milieu that inAu­ences the assembl y and orga ni za tion o f co ll agen and elastin fib ers in the derm al m atri x. T hey prov id e ri g id ity and co mpress ibilit y to the dermis, and affect , li ke other m atrix m o lecules, cell dif­ferentiation in bo th th e adult and fetal ti ss ue. Proteoglycans arc fo un d in the deve lop in g derm is as h ya luro ni c ac id-stabili zed pro-

T H E JOURNAL OF INVESTIGATIVE DERMATOLOG Y

teog lycan aggregates with m o no m eric uni ts that m ay exhibit het­erogeneity in co re pro tein co mpo nents. The develop m ental reg- · ulatio n of proteog lyca n sy nth es is needs to be in vcst iga ted with rega rd to the m an y steps that contri bute to th e concerted assembly of pro teog lyca n const itu ent g lycosa min oglyca ns and co re pro­tein s, the hitter of w hi ch could be approached usin g antibo d y an d cD NA techniques .

O th er matri x co m ponents in vo lved in va riou s aspects of cell behav io r in the dermis are g lycop ro tein s and s pecifIC g lycos­amin oglyca ns, w hi ch affect the cells' attachm ent to the extra cel­lul ar matri x, as we ll as th eir mi g rat io n , pro liferat io n , and differ­enti atio n. An exa mple for such a ro le is that of the predom in ant g lycosa min oglyca n, hyaluro ni c acid , in th e embr yoni c dermis /89 /. H ya luronic ac id participates in the fo rmatio n of peri cellula r coats and subst ra tes fo r mig ratin g m esenchymal ce lls that prevent cellul ar interacti o n o r ad hes io n prerequisite fo r dermal diffe ren­tiatio n. Decreases in h yaluro ni c ac id levels co rrelate with an o nset o r in crease in sul f.l ted proteoglyca n bi osy nthesis at a stage w hen there is also in creased acculllula t io n o f co llagen :lI1d elast in fibers, and restri ction in ce ll m o bility occurs r90 1. T hc fun ctio n of h y­aluro ni c ac id and the m od ulati o n of hya luro ni c acid-cell inter­ac ti o ns in the developmenta l regubti o n of dermal cell behavio r need to be in ves ti ga tcd fo r a better understandin g of thi s matri x co mpon cn t's con tributi ons to the morphogenesis and differen­ti atio n of th e skin.

E PID E H.MAL- DERMAL INTE H.A CTIO N S

E piderm al-derma l in te ractio ns arc importa nt in the regul atio n of epid crm al differenti atio n (keratin iza ti o n), m o rph ogenes is (a ppen­dage fo rmati o n), patte rn formati o n (epiderma l th ickness), and in ce rtai n activ ities o f th e dermis. T he las t consequence of these interac tio ns is the leas t we ll docum ented.

T issue interact io ns arc beli eved to inAu en ce gene exp ression durin g o rga nogenesis, but the m echanis m s arc not well under­stood. T he interactio n of th e epidermi s and dermis durin g de­velop m ent has alread y been alluded to, e.g., th e ep iderm al cy­to kin e stimulatio n o f derm al fibro blast produ cti o n o f co lla genase. Other cl ea r exa m ples of derm al-epiderm al interactio ns ha ve been revea led by hetero typic and hcterospecific epidermal-dermal re­co mbination expe rim ents usin g devel o pin g skin. In such studies, it has been shown that the dermis is responsible for determining w hether the skin w ill be glabro us o r bea r appendagcs, and w hat type o r appcndage wi ll form and in w hat distribution [91 ]. The m olecul ar mechanism for this inAu encc o n the deve lo pin g epi­dermi s is no t known. H owever, m o rph o logic studies and im­mun o histochemi cal labelin g of anti gcns and matrix co mpo nents in th e dermis have sugges ted a distributio n of derm al constituents that is differen t at the site o f appendage formati o n (dccreased co llagCll I and JlI , in creased fibron cctin and sul fatcd g lycosa mi­nog lycans) than it is bcnea th thc interappendagea l epidermis [92/. These o bserva tion s sugges t that th ese m atrix co mpo nents ma y be at least part o f the m essage for epidermal m o rphogenesis. Other co mpo nents (type IV co lbgen, laminin , and hepara n-sul­fate proteog lyca n) remain evenl y di st ributed in th e dermis thro ugho ut develo pment. T he m echanism by w hi ch the epider­mis is apparentl y able to sense the mi cro heterogeneo us texture of its de rmal substrate and respo nd to it w ith the appropr iate appendages, rema ins to be studied.

T he ro le of the dermis in reg ulati o n o f epidermal pro lifera ti o n and ke rati niza tion and the pattern and specificity of keratin bio­synth es is has been in vest igated usin g th e chi ck as an animal m odel /93 /. It was o bserved that av ian epiderm al cells sy n thes ize a and f3 keratin s in a scale-specifi c pattern , but that th e regulati on o f these pattern s res ides in the dermis. Derm al ti ss ue fro m each of the vario us scale types, reco mbined w ith the simple epithe lium of the chick cho ri o n and g rafted o n to the chi ck cho rioa llantoic m elll brane, directs the cho rio ni c epithelium to differenti ate and to syn thes ize keratin s accordin g to th e scale type fro m w hi ch the derm al ti ssue wa s o btained. T he studies de m o nstrate the derm al

VOL. 88. NO.4 AI' Il lL 1')87

ca pability to d irect the <.:pidermi s' histogen<.:sis and k<.:ratini zati o n

1941· Studies of the sca le less chi ck mutant (sr/sr) have d<.:m o ns trated

the role of the epider mis in the epiderma l-derm al ti ss ue int<.:ra c­tions. Scal e less <.:mbryos do n ot form the epidern131 placod<.:s of scales; however, r<.: ciprocal epidermal-dermal recombination <.:x­perill1ents us in g nor m al and sca leless skill have show n that mutant dermis attai ns its ab ili ty to dir<.:ct <.:p ide rmal his togencsis thro ug h seq ucntia l interactions w ith a nOrlllJl epid ermi s 194-97 1.

These kinds of studies ma y provide di rection fo r experiments that coul d be do ne w ith hum an skin to beg in to stud y disorders (e . g . , the ectodermal dysplasias 1 EDs]) that arc thou g ht to res ult fro m abnor mal epitheli al-m esen chy m al intera ction s durin g de­ve lopmen t. The EDs arc a Iarg<.:, genetica ll y and cl inica ll y het­erogeneous g ro up of d iso rde rs characterized b y abno rmaliti<.:s o f tecth , ha ir, swea t g lands , and nai ls-a ll append ag<.:s o f th<.: epi­dermis 198 1. The kind s o f reco mbin ant srudi<.:s that ha ve b<.:<.:n done in the chi ck and m o use could b<.: don<.: with hum an f<.:tal skin to beg in to und<.:rs tand the condi tion s in the epidermis and dermis that are present at the time of append age formati o n . With su ch data, it m ay be poss ible to h ypothesize how and w hen durin g development t hese disorders could be m anifest, and what direc­ti on o ne mi g ht take to look fo r the prinury gene defect.

T he hum an skin g raft to the nude m o us<.: (as w ho le skin o r in tiss uc reco mbinant expe rim ents) ha s bem parti cularly help ful in id entifyin g w hethe r the g<.: net ic defect in an inh<.:rited diso rder of the skin is primary o r secondary , and if p rim ary , w h<.:t her it is ex pressed in the epidermis o r der mis . This technique ha s been success ful, fo r example, ill the dem o nstration of the gene re­sponsible for lamellar ichth yos is that causes ab nOrlll aliti es in the epide rmi s 19() I. U se of thi s syste m , however, is predi cted o n havin g app ro priate markers to recogni ze the abnorma lity in the tiss ue eith e r by m o rph o logic or biochemica l techn iques.

C LINI C AL RELEVAN CE OF UNDERSTANDING FETAL SK IN DEVELOPM ENT

Our und ersta ndin g o f the no rm al events in cutaneous m orpho­genes is allows us to predi ct w hi ch inherited disorders of the skin mig ht be expressed in ute ro , and at w hi ch prenatal ages they occur. In s ig ht into both the n o rmal and abno rm al devel o pment is essential for considerations of prenatal dia gn osis and , ulti­mately , in utero therapy. As prenatal dia g nos is for these di so rde rs is ca rri ed out, we extend o ur und erstanding of the natural histo ry o f disease into the prenatal period . At present, S0 111e of the lim­itations in bein g able to m ake a dia gnosis p renata ll y are the lack of understandin g of th e disorders at the gene level , the appa rem absen ce of specific biochemi ca l and / o r m o rphol ogic m ark ers for thc diseases in ad ult skin and , thus, the lack of probes that mig ht be used to recognize th e abnormalities in utero .

In spite of these limitat io ns, there has been co ns id erab le su ccess in reco gnizin g several of the more severe inherited di so rd ers o f the skin in fe tu ses that a re a ffec ted . Some o f these diso rders ca n be dia g nosed o n the basis of a known en zy matic defec t using alllniot ic fluid (e.g., X-linked recess ive ichth yos is) , o r cultured amnioti c fluid ce lls (e. g., Ref., ulll 's diseasc) \1 001. Others , fo r w hi ch a biochemica l d efect has not yet been recogni zed , can be diag nosed in t he adu lt by :1 s tru c tural abnorma lity in the tissuc (eg, bullous co n genita l ichth yos ifo rm e ry throderma). Under­stand in g the s tructural , bio che mi cal , and anti geni c pro perti es of the skin at relevant stages in development provides a basi s fo r predictin g whether an ab no rmality cou ld be expected to be ex­pressed in utero . Successfu l prenatal d iagn os is, based o n recog­nition of stru ctural markers , has becn poss ible for a number o f disorders o f keratini za tion , of th e de rm al-epide rmal junction , and o f pi g ment ce ll abno rm alities 195 1. Attempts arc also bein g made to sea rch for s tructural markers in amnioti c fluid ce lls, o r for variations in populations o f ce lls in the amnio ti c fluid , whi ch ma y be indicative of the disorder. The goa l is to be able to use thc amnioti c fluid and its ce lls for an accurate dia g nosis, thus to elim­inate the m o re invasive and less ava il able procedure offetoscopy.

NIC H D/ N IA()D K I(ESEARCH WO RK SHO P 471

Ultimatel y, o ne wo uld ho pe to de termine the m o lecular basis fo r the disorders and to prepare probes th at could recogni ze the ab­no rmal gene in sa mples of chorionic vi lli . W hen this is possible, thc timin g of prenata l dia g nosis wi ll be m oved back fro m 19-2 1 weeks (fetoscopy) o r 14- 16 weeks (a mni ocente is) to 8-1 0 weeks (cho ri o ni c villi samp lin g).

CO N C LU SIONS AND RECO MMENDATI O N

The wo rksho p empha sized that the ques ti o ns we arc askin g about skin develop m ent arc no t unique to this o rgan , o r to man , but arc general prob lem s in cell and m o lecul ar bio logy th at perta in to all of developmcm as thcy arc pro blems of cell-ce ll imerJC ti o ns, cell-matrix interaction s, ti ss ue imera cti on s, and m o lecubr rl' g u­b tion. Three general areas fo r future resea rch o n cutan cous dc­vclopmem were rcco mm ended: ( I) characterizatio n of var io us events in cutaneOllS m o rphogenesis; (2) regulati o n of m o rph o­gencsis b y imrin sic an d extrin sic con tro ls; and (3) :lppli ca tion of the studies o n clltan eo us m o rph ogencsis to problems of clini ca l impo rt:!n ce. M os t o f the specifi c suggestio ns for research have been in co rpo rated into appropriate sec tio ns in the tex t. T o ac­co mplish m an y o f the suggested goals, it wi ll be necessa ry to develo p ce ll and o rg an culture sys tem s o f fetal sk in :! nd ti ss ues. These ma y then be used for studi es o f th e reg ulatio n of gen <.: express io n :!nd to defi ne m<.:chanism s b y whi ch ce ll s respond to ex tracellubr cues. DN A pro bes fo r s truc tural gencs of the epi­dermi s and dermis wi ll need to be prepared that ca n be used bo th ex perim entall y and c lini c:!lI y , for example in prenatal dia g nosis and carrie r tes tin g for genetic disea ses. Anin131 m od els sho uld be idemificd and used for ex perim<.:ntal and deve lop m enta l s tudies that arc no t poss ible w ith th<.: human tiss lle .

REFE RE N CES

I. Ho lbrook K A. HolT MS: tru cturc or thc dcvelopill g l!lllllall Clll ­bryollic alld fcta l skill . Selll DcrlllalOl 3: 185-202. 1')84

2. Holbrook KA, O dlalld GF: Fille stru cture of devclopillg hlllllall ep ider lllis: light. scallllill g and tral1S llli ss ion electron Ill ic rosco py of the peridcnll . J In vcst Dcrlll ato l 65: 16-38, 1')75

3. It iddle C V: Illtralllelllbranous respoll sc to cAMP in fetal epider lll is. Cell T issue I ~e s 2-1 1 :687-689. 1')85

4. Dale BA. Holbrook KA , KimbaIl JI1.. Ho tTM S. SUIl T -T: Expression or epiderllla l keratins and fi laggrin durin g l!lllnan fera l skin de­velopmcnt. J cll Bioi 10 1: 1257- 1269. 1')85

5. Lane AT, Nq~i M Golds lll ith LA : IdL'lltifi c nioll or hUIll :lIl rl,tal peridcnn with a ;nonoc!ollal anribody (abs tr). J IlI vcst Derlll ato l 84 :2')8, 1')85

6. Fuchs E. Gree ll H: Multiple keratins of cultured hUlllan epidl'l'lll al cells arc translatcd (ro lll diflCrenr ml1.NA Illolecuics. Cell 17:573-5H2. 1')7')

7. Moll It Franke WW. Schiller DL: Thc ca talog of hUlllan cyroker­ati ns: patterns o f expression in norlll al epithelia . tUlllors Jnd cul­turcd ccll s. ell 3 1: 11 -24. 1982

S. SlIn TT. Eichner 11. , Nelson WG. T sell g SCG, Weiss I1. A, Jarvinen MD. Woodcock- Mitchell ]: Keratin c1assl's: 1ll 0iccul.,r m:lrk ers fo r difrerelH typcs o f epithelia l difti.:rcllti :nion. J In vcs t DCrIll:ltol 8 1: 10')-1 15. 1')83

'). Stci llert PM , Adler WW: Pos tsYlltheti c 1l1Od ifi ca tions of Illalllm alian cpidermal :tl pha-ker:Hin . Biochemistry I H:S66S-5(,6,). 1')7')

10. [lowdcll PE. Q uinbn itA , \3reitkreutz D. Fusenig NE: Proteolyti c Illodifi ca tion of ac idic :lIld ba sic kerati ns during tl'l'lllinal dilli.: r­cnti:Hion of mouse and hUIll Jn epidermis. Eur J [liochclll 142: 29-36. 1984

II . Sun T-T, Eichner R. Scherlll er D. Cooper D. Nelson WG. Weiss I1.A : C lass ifi cation. cx press ion. and possibk IIl cch:lIlislllS o f evo­lu ti on of III a 111 Ill ali all epithelia l kera tins: a unifyi ll g 1l1 0de l. in Call­cer Cell . vo l. I. Ed itcd by A Levine. W Topp, GN VandeWoudl'. JD Watson. New York. Co ld Spring Harbo r Laboratory. 1')84. pp 169-176

12. Steincrt PM , Parry DAD. Itecoosin EL, Idler WW , teven AC. Trus I3L. l1.oop Dlt Th e cOlllpkte eDNA and deduced alllin o acid sequen ce of a type II Illouse epider lllal keratin o f 60.000 Da:

472 KREY ET AL

analys is of sequ ence differences between type I and II keratins. Proc Nat! Acad Sci USA 81:5709-57 13. 1984

13. Aebi U. G la va ri s EC, E icher R: T he hierarchic o rde r o f intermediate filament stru cture and assembl y. Proc 43rd Annual M eeting o f the E lectro n Microsco pi c Society of America 1985. pp 722-725

14. Woodcock-M itchellJ , Eichner R. Nelson WG, Sun T-T: Immuno­loca li za tion of ke rati n polypeptides in hum an epidermis usin g monoclonal antibod ies. ) Cell Bi o i 95:580-588. 1982

IS. Roop D I<. Haw ley-Nelson p. C heng C K, Yuspa SH : Ex pression o f keratin genes in mouse epiderm is and no rm:l l and malignantl y transfo rm ed epiderm al cells in culture. ) In vest Dermatol 81: 144-- 149. 1983

16. Roop Dn. H awley-Nelson P, C heng C K. Yuspa SH : Keratin gene ex press ion in m ouse epidermis and cultured epiderm al ce ll s. Proc Natl Acad Sci USA 80:71 6-720. 1983

17. Nelson WG, Sun T-T: The 50- an d 58-Kda lton kerati n classes as molecu lar marke rs fo r stratifi ed squamo us epithelia: cell cu lture studies. ) C ell Bio i 97:244--25 1. 1983

18. Steinert PM . Peck G L. Idler WW: Structural changes of epidermal alpha-ke ratin in d isorders o f keratini za tion, in Biochemistry of N o rm al and Abno rma l Epidermal D iffe rentiation. Edited by I Bernstein. M Seij i. T o kyo, University of T okyo Press. 1980, pp 39 1- 406

19. Weiss RA. Eichner R, Sun T -T: M onoclonal ant ibody analys is o f keratin express ion in epiderm al d iseases: A 48- and 56-Kdalto n kerati n as mo lecular markers for hype rp ro li ferative keratin ocy tes. J Cell Bioi 98 : 1397-1406, 1984

20. Fisher Cj, O'Guin WM. Sawyer RH : Altered keratin biosynthesis fo llows in hibition of scalc m orphogensis by h ydrocortisone. Dev Bio i 106:45=-52, 1984

2 1. Fuchs E. G reen H : Regulation of te rminal differentiat ion of cultured human kcratinocytes by vitamin A. Cell 25 :617-625, 198 1

22. Su ni BP, Banerjee C K: Purifi cation and properti es o f retinoic acid binding protein from chi ck embryo skin . Biochem )1 73:643-649, 1978

23. C hytil F, O ng DE: Cellul ar re tinoid-binding proteins, in The Ret­inoids. vo l. II. Ed ited by MB SpOI'l1, AB Roberts. DS Good man. N ew Yo rk . Academi c Press , 1984. pp 89- 123

24 . Benke PJ: T he iso treti noin teratogen synd rome. JAMA 25 1:3267-3269. 1984

25. Lonsdale-Eccles ) 0 . Haugen JA , Da le BA: A phospho rylated ke­ratohya lin-deri ved precurso r of ep idermal stratum corneum basic prote in . ) Bio i C hem 255:2235-2238. 1980

26. Resi ng KA . Walsh KA. Dale BA: Identi fi cation o f two intermediates duri ng process ing of profilaggrin in neonata l mouse epidermis. ) Cell Bio i 99: 1372- 1378, 1984

27. Scott R. H arding C R. Barrett ) G: Histidine-ri ch protein of the ke­ratohya lin g ranules: source o f the free am ino acids, uroca nic acid and pyrro lidonc ca rboxylic acid in st ratum co rneum . Biochim Biophys Acta 7 19: 11 0- 11 7. 1982

28. Flcckman p. Haydock P, B lo mquist C, Da le I3A : Profi lagg rin and the 67 Kd kerati n arc coordinately ex pressed in cultured hum an epiderma l ke ratinocy tcs (abst r). ) C ell Bioi 99:315. 1984

29. Gorbsky G. Steinberg M S: Iso lation of the interce llular g lycoprotcins o f des moso mes. ) C ell Bio i 90:243-248, 1981

30. Giudice GJ . Cohen SM, Patel NH, Steinberg MS: Im munolog ic co mpari son of desmoso mal components from several bovine tis­sues. J Cell Biochem 26:35-45 . 1984

3 1. Th atcher SM, Hice RH: Keratinocy te-specific transglutam inase of cultured human epiderm al ce ll s: rel ation to cross-linked envelope fo rm ation and te rminal differentiati on. Cell 40:685-695 , 1985

32. Watt FM : Selected migration of termin all y differenti atin g cells fro m the basa l laye r of cultured human epidermis. J C ell Bio i 98: 16-2 1. 1984

33. Banks-Schlegel SP, G reen H : Studies on the develo pment of the definitive cell type of embryoni c epidermis lIsing the cross- li nked enve lope as a differentiation m arker. Dev Bioi 74:275-285. 1980

34. N em anic MK, Whitehead J S, Elias PM: Alterations in membrane suga rs durin g ep iderm al differenti ation: visualiza tion w ith lectins and the role of g lycosidases. J Histochem C ytochem 3 1 :887-897, 1983

35. Brabec RK . Peters B P, Bernstein IA . Gra y RH, Goldstein I) : Dif-

TH E JO URNAL OF INV ESTIGATIVE DERMATO LOGY

ferc ntia l lecti n binding to cellular membranes in the epidermis of the newbo rn rat . Proc Natl Acad Sci U SA 77:477-479. 1980

36. Vedto fte P, Dabels teen E. H ako mo ri S-I, Young WW : C hanges in the express ion of blood group ca rbohydrates du ri ng ora l mu cosa develop ment in hum an fetuses. Differe ntiatio n 27 :22 1-228, 1984

37 . Green MR, l3usketter DA, Couchm an JR , Rees D A: D istribution and number of epidermal g rowth f.1cto r recepto rs in skin is re lated to ep ithelial ce ll g rowth . Dev B ioi IOU:506-S I2. 1983

38. O' Keefe E. l3attin T. Pa yne R: Ep iderm al g row th fac to r recepto r in culturcd human epiderm al cdls: direct demo nstratio n in cul ­tured cell s. J In vest Dermato l 7tl :482-487. 1982

3') . N anney LB , M agid M , Stoschek C M. Kin g LE, Jr : Comparison of epider mal g rowth fa ctor binding and receptor d istribution in nor­mal hum an epidermis and epidermal appendages . J In vest Der­mata l R3:385-393, 1984

40. G reen MH, Couchman JR: D istributio n of epidermal g rowth facto r receptors in ra t skin d uring embryonic develo pmcnt: ev idence for a specific inductive interaction between a mesenchyme and an epithelium . Biochem Soc Trans 12:243-244, 1984

41. Elias PM : Epiderm al lipids, barrier fun ction. and desquamation . ) In vest Derm atol 80 (suppl):44-49, 1983

42. O dland GF, H olbrook KA : T he lamellar g ranu les of epidennis. C ur r Probl Derm ato l 9:29-49. 198 1

43. Wi lliams ML. Hincenbergs M , Holbrook KA: Developmental changes ill Illnn an fetal skin lipids (a bstr). C lin I ~es 33:695, 1985

44. Parmley T H . Seeds AE: Fetal skin perm ea bility to isotopic water (TH O) in ea rl y pregnancy. Am) O bstet Gynecol 108: 128-131. 1940

45 . C hri stopher SE, La urence EI3 : Kineti cs and stru ctural o rgani za tion of the cpidermis. C urr Probl Derm ato l 6:87- 106. 1976

46. Bickenbach JR: Identi fica tion and behav io r o f label-retainin g cells in o ralnlli cosa and skill . ) Dellt Res fiO(c): 161 1- 1620, 1981

47. Bri gga m an RA . Kelly T: Contiuous th ymidine labelin g studies of norm al hum an skin g rowth on nude mice: measurem ent o f cycling basa l ce ll s (a bstr). J Ill vest Derlll ato l 78:359. 1982

48. Lavker RM , Sun T -T: Heterogeneity in basal keratinocy tes: 1I10 r­phologica l and fun cti onal correlatio ns. Science 18: 1239- 124 1. 1982

49. Stern 113: T he uptake o f tritiated th ymidine by hum an feta l ep ider­mis. ) In vest Dermatol 63:268-272. 1974

50. Pauk ovits WI<. Laerum OD: Iso lation and synthes is of a hemo­regulatory peptide. Z Naturfo rsch 37c: 1297-1300, 1982

51. H echt F, M otulsk y AG. Lemire HJ . Shepa rd TE: Predominance of hemoglobin gower I in ea rl y hum an embryonic develo pm ent. Science 152:91-92, 1966

52. C ivid alli G, N athan DG. Kan YW , Sant:lma rin a R, Fri go lctto F: Relation o f beta to gamm a synth esis durin g the first trim es ter: an app roach to prenatal diagnos is of tha lassemia. Pediatr I<es 8:553-560, 1974

53. Bard H: Pos tnatal fetal and ad ul t hemoglobin synthes is in earl y prc­term newborn infants. J C lin In ves t 52: 1789- 1795. 1973

54 . Wood WG , N as h J. Wea therall DJ . Robi nson ) S. H arri son FA : T he sheep as an animal m odel fo r the sw itch fro m feta l to adult hem o­globi ns. in Cellular and M o lecular Hegulation of Hemoglo bin Switching. Edited by G Stamatoyannopoulos. A W N ienhius. New York, G runc and Stratton, 1979, pp 153- 167

55. Le Do uarin N: O ntogeny of hematopoiet ic o rgans studied in av ian embryo interspecific chimeras, in Differentiation in N o rm al and N eoplasti c Hematopoietic Cell s. Edited by 13 C larkson. PA M arks, ) T ill. N ew Yo rk , Cold Sprin g H arbo r Labo rato ry. 1978. pp 5-3 1.

56. Bun ch C, Wood WG. Weathera ll DJ . Robinson JS, Corp MJ: H ae­moglo bin synthesis by feta l e ry th ro id cells ill an adu lt environ­ment. Br J Haemato l 49:325-336, 198 1

57. Papa yann opoulou T H . N aka mo to B, Agos tine lli F. M anna M, Lu­ca relli G, Stamatoyanllopoulos G: Feta l to ad ult hemopoieti c cell transplantation in man: insights into hem oglo bin switching. Blood 67:99- 104. 1986

58. Stamatoyannopoulos G, Papayanno po ulou T H . B rice M. Kerrachi S. N akamoto B . Lim G, Farquhar M: C ell bio logy o f hemoglo bin switching I. The swi tch fro m fetal to ad ul t hemoglo bin for m ation during ontogeny, in Hemoglobin in Development and Differen­ti ation. Ed ited by G StamatoyannopOlilos . A W Nienhuis. N ew York . A.n . Liss, 198 1. pp 287-305

VO L. 88. N O.4 APHIL 1'.187

59. Van der Ploeg LHT, Flavel RA : DNA Ill ethylatio n in the hUlllan ga mma-dclta-bcta-g lobin locus in erythro id and no nerythro id tis­sucs. C ell 19:947-958. 1980

60. M av ilio F, G iampao lo A, C are A, Mig lia ccio G, Ca landrini M, Russo G, Pag li ardi G L. M asrrobe rard ino G, M arinucci M, Peschlc C : M olecular mechanism s of hum an hemoglo bin switching : se­lecti ve undermeth ylation and ex pression o f g lobin genes in cm­bryonic. feta l and adu lt e rythro blasts. Proc Nat l Acad Sci USA 80:6907 -69 11 . 1983

61. G ro udi nc M . Ko hw i-Shigematsu T, Gelin as I~ . Stamatoyannopou­lo us G. I'apa yannopoulou TH: Human fe tal to adult hemoglobin switchin g : changes in chro m atin structure of the beta-g lobin locus. Proc N ad Acad Sci USA 80:755 1-7555. 1983

62. Weintraub H , Weisbrod S, Larscn A, G roudine M: C hanges in g lo­bin chro m at in stru cture during red ce ll differentiat ion in chick embryos, in O rganization and Ex press ion of G lobin Gencs. Editcd by G Stamatoya nn opoulos. A W Nienhuis. N ew York , A. R. Liss . 198 1, pp 175- 190

63. M ag ram j. C hada K, Cos tantini F: Develo pmental reg ulation of a cloned adu lt beta-g lobin gene in transgenic mice. N ature 3 15: 338-340, 1985

64. Cos tantini F, Radice G , Mag ram J , Stamatoyannopoulos G. Papay­annopoulo u T H , C hada K: Develop mental regulation of human glo bin gencs in transgenic mice. New York . Cold Sprin g H arbor Sy mpos ia on Q uantitative B io logy 50:36 1-370, 1985

65. Wi lling M C, Nienhu is A W, Anderson WF: Selec tive acti vat ion of hum an bcta- but no t ga l1lm a-globin gene in human fibroblast mo use erythro lcukacmia cc ll hybrids. N ature 277 :534-538. 1979

66. Riccardi V M : C utancous man ifestation of ncuro fibromatosis: cel­lu lar inte ractio ns, pig mentatio n and mast ccll s, in Mo rphogenesis and M alfo rmations o f the Skin , Birth Defects: O rig inal Article Series, vo l X VII . Edited by RJ B landau . N ew Yo rk , A. I~ . Liss, 198 1. pp 129- 146

67. Katz SI, Tamaki K. Sachs DH: E pidermal Langerhans cell s arc de­ri ved from ce ll s o ri ginatin g in bone marrow. Nature 282:324-326, 1979

68 . Murph y GF: Cell membrane g lycopro teins and Lan gerh ans cells. HUI1l Patho l 16: 103- 11 2, 1985

69. Sauder D N , Din arello C A, M orhenn VB : Langerhans cell produc­tio n of inter leukin- 1. J In vest Denm to l 82:605-607, 1984

70. Fos ter C A, Ho lb rook KA , Farr AG : O ntogeny o f Lan g her hans ce ll s in human embryonic and feta l skin: express ion o f HLA-DR and OKT-6 determ inants. J Invest Dcrm ato l 86:240-243, 1986

71. H artschuh W, Weihe E, Buchler M , Hellll staedtcr V, Feurle GE, Fo rss mann W G,: M etenkephalin- li ke immuno reacti vity in Merkel Cell s. Cell Tissue Res 20 1 :343-348, 1979

72. M o ll R, M o ll I, Frankc WW: Identifi ca tion of Merkel cell s in hum an skin by specific cy to kerat in antibodics: changes in cel l density and distributio n in feta l and ad ult plantal epidermis. Differentiation 28: 136- 154, 1984

73. Finc J-D , Sm ith L T, Ho lbrook K A, Katz SI: The appeara nce of four bascment membrane zone antigens in develo ping hum an feta l skin . J In vest Dcrnutol 83:66-69, 1984

74. Lane AT, Helm KF, Goldsmith LA: Id entifi c~t i o n o f bullous pe J1l­phigoid , pemphigus, lam inin , and ancho rin g fibril an tigens in hu­man fctal skin . J In ves t D crmatol 84:27-30, 1985

75 . Q ueen LL, Pall er AS , Woodley DT, Gemmon WH , Briggam an I~A: O rgan specifi c and ph ylogeneti c distribution of ep idermolysis bul­losa acqu isita ant igen (abstr) . J In vest Dermatol 82:432, 1984

76. Fine J D, I3reathn ach SM , I-lintncr 1-1, Katz SI: KI:- I mOlloclo ll al antibod y defin es a specific basement m embrane antigenic defect in d yst rophic fo rms o f epiderm o lys is bullosa. J In vest Dcrmato l 82:35-38. 1984

77 . Prockop DJ , Ki virikk o KI: Heritablc diseascs o f co lla gen . N EngJ J M ed 3 11 :376-386. 1984

78 . Kramer JM , Cox G N , Hirsh D : Comparisons of the complete se­qucnces of two co llagen genes from Caeno rhabditis e1 cga ns. Cell 30:599-606, 1982

N I HD/ NIADDK ItESEA ltCH WORKS H OP 473

79. Smi th L T , Holbrook K A. Madri J S: Collagen types I, III :lIld V in human embryoni c and feta l ski n. Am J Anat 175:507-522, 1986

tlO. Eps tein EH. Jr: \alpha l(III ) \J Hum an ski n co llagen: release by pepsin digcstion and preponderance in fe tal li fe. J Bio i C heJ1l 249:3225-323 1. 1974

8 1. J3 auer EA: Rccessive dys trophic epidermolysis bu llosa: evidence for an altered co ll agenase in tibroblast cultures. Proc N at! Acad Sci USA 7ol :4646-4650, 1977

82. Ba ller EA. Kronberg A. Stri cklin G P, Smith LT. Ho lbrook KA: Age-related changes in co llagenase express io n in cultured cmbry­oni c and feta l human skin fibrobla sts . Exp ell Rcs 16 1:484-ol94. 19t15

83. Wclgus H G. Stri cklin G P: Human skin fib roblast co llagenase inhib­ito r . o mparati vc studies in human connecti ve ti ssucs, se rulll and amniotic Auid. J Bioi C helll 258: 12259- 12264, 1983

84. Eisen AZ: HUl11 an skin co llagcnasc: relatio nship to thc pathogencs is of ep ider lllo lysis bu llos:! dystrophica. J In vest Derlll atol 52:H9-453. 1969

tl5. Johnson-Wi nt B: Rcgulation of connective tissue co llagenase pro­duction : stilllulators from ad ult :lIld feta l epiderm al ce lls. J Cell Bio i 98:90-96. 1984

86. Deutsch T A. Esterl y NH: Elasti c (,bns in fetal de rmi s. J In ves t Dermato l (,5:320-323. 1975

87. J3urnett W, Finnigan-l3unick A, Yoo n K. Rosenb loo m J: Ana lys is of elastin genc ex press io n in the developing chick ao rta usmg cloned elastin cD NA. J !:lio l C hcm 257: 1569- 1572. 19'2

88. Em anuel 13 , Ca nnizza ro L. O rnstein-Goldstein N . Indik Z. Yoo n K, M ay M, O li ver L, 130yd C. Rosenbloom J: C hrolllosom al loca liza tion of the humall elastin gene. AJ1lJ Hum Genet 37:873-8tl2, 1985

89. Hreen M . Weinste in H G. Jo hnson RL. Vcis A, M arshall I~T: Acid g lycosa minoglycans in hum an skin durin g feta l develop ment and adu lt li fe . Biochim Biophys Act:1 20 1 :54-60, 1970

90. V~rm~ RS, Varma R: Glycosa minoglycans and proteoglyca ns o f the ski n. in Glycosa minoglycans and I'ro teog l yc~ n s in Ph ysio logica l ~nd Pa tho l og i c~ ll'rocesses of!:lody Sys tems. Edited by RS V~rma . H Va rm a. Basel . Ka rger, 1982. pp 15 1- 164

9 I. Sengel 1' : Mo rphogenes is of Skin . Develop lll en t~ 1 and Ccll Biology Seri es. Ed ited by M Abercrombi . D I~ N cwth , JB Torrey. C am­bridge, Ca m bridge U niversity Press, 1976

92. Scnge! 1': Ro le of extraccllular matri x in the development of skin and cutancous appendages . in Dcvelopmcntal M echanisms: Nor­mal and Abnormal. Ed ited by J W Las h, L Saxen. New York, Alan It . Liss, 1985. pp 123-136

93. Sawyer RI-I: T he ro le of epithelial- mcscnchym al interactions in reg­ubtin g gene ex press ion durin g avian sca le m orphogenes is, in Ep­ithelial-Mesenchymallnteractio ll s ill Development, Edited by RH Sawyer. JF Fa llon. New York , Praeger Pub lishers 1983, pp 11 5- 146

94. Sawyer 111-1. O'Guin WM. Knapp LW: Avia n Sca le devel opmen t. X. Dermal induction of ti ssue-specific kera tins in ex trae lll bryonic ectoderm . I ev Bio i 10 1:8- 18, 19S4

95. Sawyer RI-I : Avian sc~ 1c devel0pJ1lent: effect of the scakless gene on mo rphogenes is ~nd histogcnesis. Dev Bioi 68: 1-1 5. 1979

96. McAleese SI<, Sawyer RH : Avian Sc~k Develop ment. IX. Scale fo rmatio n by sca le less (,else) epidermis under the inAuence of no r­mal sca le dennis. Dev Bioi 89:493-502, 1982

97. Dhouai ll y D , Sawyer RH : Avian sca le develo pl11ent . X I. In itia l ap­pearance of rhe dermal defect in sca leless skin . Dev B io i 105: 343-350, 1984

98. Freire-M aia N . Pinhiero M : Ectodermal Dysplas ias: A C linica l and Geneti c Stud y, Ncw York , Abn R. Liss, 1984

99. !3riggaman RA . Wheeler CEo Jr: L~niclbr ichthyosis: long-term graft studies on congenitall y athymic nu de mi ce. J Ill ves t Dcrl11ato l 67: 567-572. 1976

100. Ho lbrook KA: Prcna tal d iagnos is of geneti c sk in disea;c. Se lll Der­lII atol 3: 155-264, 1984