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1997MicrosResTechnique

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Molecular Histology in Skin Appendage MorphogenesisRANDALL B. WIDELITZ,1TING-XIN JIANG,1ALEXANDER NOVEEN,1SHEREEA. TING-BERRETH,1ERIC YIN,1HAN-SUNG JUNG,2AND CHENG-MING CHUONG1*1Pathology Department School of Medicine, University of Southern California, HMR 204, 2011 Zonal Avenue, Los Angeles, CA 900332Department of Anatomy and Developmental Biology, University College and Middlesex School of Medicine, University CollegeLondon, Windeyer Building, Cleveland Street, London W 1P 6DB UKKEY WORDS hair; feather; development; evolution; homeobox genes; adhesion molecules;growth factors; signaling moleculesABSTRACT Classical histological studies have demonstrated the cellular organization of skinappendages and helped us appreciate the intricate structures and function of skin appendages. Atthis juncture, questions can be directed to determine how these cellular organizations are achieved.How do cells rearrange themselves to form the complex cyto-architecture of skin appendages? Whatare the molecular bases of the morphogenesis and histogenesis of skin appendages? Recently, manynew molecules expressed in a spatial and temporal specific manner during the formation of skinappendages were identified by molecular biological approaches. In this review, novel moleculartechniques that are useful in skin appendage research are discussed. The distribution of exemplarymolecules from different categories including growth factors, intracellular signaling molecules,homeobox genes, adhesion molecules, and extracellular matrix molecules are summarized in adiagram using feather and hair as models. We hope that these results will serve as the ground workfor completing the molecular mapping of skin appendages which will refine and re-define ourunderstanding of the developmental process beyond relying on morphological criteria. We also hopethat the listed protocols will help those who are interested in this venture. This new molecularhistology of skin appendages is the foundation for forming new hypotheses on how molecules aremechanistically involved in skin appendage development and for designing experiments to testthem. This may also lead to the modulation of healing and regeneration processes in futuretreatment modalities. Microsc. Res. Tech. 38:00–00, 1997.r1997 Wiley-Liss, Inc.INTRODUCTIONHistology, which demonstrates the cellular organiza-tion of tissues, has helped us to appreciate the struc-tures and function of skin appendages. Skin append-ages derive from interactions of the epidermis anddermis within the skin. Elaboration of defined domainsof the skin leads to the formation of new skin append-ages with different functions. In general, there are twocategories of skin appendages, characterized by eitherprotrusion out of or invagination into the body surface.The skin appendages that protrude out of the bodysurface are hair, feather, scale, nail, claw, etc. Theyprovide a variety of functions to individuals, rangingfrom environmental protection to ritual mating dis-plays. The skin appendages that invaginate are thesebaceous gland, sweat gland, mammary gland, etc.They provide specialized physiological functions toindividuals, ranging from environmental adaptation tochild rearing.How are the complex cyto-architecture of these skinappendages achieved? Recent progress in molecularbiology has allowed us to identify many new moleculeswhich are important in cell interactions and embryonicdevelopment. These molecules can be categorized asgrowth factors, homeobox genes, cell adhesion mol-ecules, extracellular matrix molecules, etc (Chuong,1993; Chuong et al., 1993; Chuong et al., 1996). Usingimmunocytochemistry, in situ hybridization and othernovel molecular biology techniques, it is now possible tostudy the spatial and temporal distribution of thesemolecules during the morphogenesis of skin append-ages. The objective of this article is to introduce newtechniques used in the molecular histology of skinappendagesandto summarizerecentfindings infeatherand hair development as useful references. These find-ings will set the ground work for us to explore themechanism(s) underlying the processes of develop-ment, cycling, and regeneration in skin appendagemorphogenesis. We also discuss the fundamental impli-cations that these molecular studies have for develop-ment and evolution of skin appendages.MATERIALS AND METHODSMolecular Histology Methods for SkinAppendage ResearchIdentifying mRNA ExpressionWhole mount in situ hybridization with non radioac-tive probe. This method permits a three dimensionaloverview of the distribution of specific mRNAs. This*Correspondence to: Cheng-Ming Chuong, Pathology Department School ofMedicine, University of Southern California, HMR 204, 2011 Zonal Ave., LosAngeles, CA90033.Contract grant sponsor: NIH; contract grant sponsor: NSF; contract grantsponsor: CTR; contract grant sponsor: Wright Foundation; contract grant spon-sor: The Norris Cancer Center Breast Cancer Research Project; contract grantsponsor: NorrisCancer Center Postdoctoral Supplement Fund.Received 17 February 1995;Accepted inrevised form 19 May 1995MICROSCOPY RESEARCH AND TECHNIQUE 38:452–465 (1997)r1997 WILEY-LISS, INC.replaces the tedious work of three dimensional recon-struction from section staining using an image analysisprogram.Furthermore, insitu hybridizationprobes caneasily be generated using PCR technology (Erlich,1989). In addition, it is easy for us who study skin toobtain a piece of appropriately sized skin (eg., up to10 3 5 3 1 mm) for this purpose. We think this tech-niquewill play amajorrole in themolecular mapping ofskin development in the future.In our laboratory, we use a protocol based on Sasakiand Hogan (1993). Briefly, chicken embryo skins dis-sected in RNase free phosphate-buffered saline (PBS)are fixed in 4% paraformaldehyde for 2 hours or at 4°Covernight. Tissues are dehydrated, rehydrated,bleached, and treated with proteinase K. Samples thenare subjected to secondary fixation in 0.2% glutaralde-hyde in 4% paraformaldehyde/PBS before hybridiza-tion at 70°C overnight in buffer (50% formamide,5 3 SSC, 1% SDS, 50 µg/ml heparin, 50 µg/ml tRNA)containing 2 µg/ml digoxigenin-labeled riboprobes. Af-ter washing and RNase A treatment (50 µg/ml) thetissues are incubated with anti-digoxigenin Fab8 conju-gated to alkaline phosphatase (Boehringer Mannheim).Alkaline phosphatase is detected with 4.5 µl/ml NBTwith 3.5 µl/ml BCIP (Promega) following standardprotocols.Samplesaredehydrated,rehydrated,


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