Morpho-Regulation of Ectodermal Organs
2004; Elsevier BV; Volume: 164; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63197-5
ISSN1525-2191
AutoresMaksim V. Plikus, Wen Pin Wang, Jian Liu, Xia Wang, Ting‐Xin Jiang, Cheng‐Ming Chuong,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoEctodermal organs are composed of keratinocytes organized in different ways during induction, morphogenesis, differentiation, and regenerative stages. We hypothesize that an imbalance of fundamental signaling pathways should affect multiple ectodermal organs in a spatio-temporal-dependent manner. We produced a K14-Noggin transgenic mouse to modulate bone morphogenic protein (BMP) activity and test the extent of this hypothesis. We observed thickened skin epidermis, increased hair density, altered hair types, faster anagen re-entry, and formation of compound vibrissa follicles. The eyelid opening was smaller and ectopic cilia formed at the expense of Meibomian glands. In the distal limb, there were agenesis and hyperpigmentation of claws, interdigital webbing, reduced footpads, and trans-differentiation of sweat glands into hairs. The size of external genitalia increased in both sexes, but they remained fertile. We conclude that modulation of BMP activity can affect the number of ectodermal organs by acting during induction stages, influence the size and shape by acting during morphogenesis stages, change phenotypes by acting during differentiation stages, and facilitate new growth by acting during regeneration stages. Therefore during organogenesis, BMP antagonists can produce a spectrum of phenotypes in a stage-dependent manner by adjusting the level of BMP activity. The distinction between phenotypic variations and pathological changes is discussed. Ectodermal organs are composed of keratinocytes organized in different ways during induction, morphogenesis, differentiation, and regenerative stages. We hypothesize that an imbalance of fundamental signaling pathways should affect multiple ectodermal organs in a spatio-temporal-dependent manner. We produced a K14-Noggin transgenic mouse to modulate bone morphogenic protein (BMP) activity and test the extent of this hypothesis. We observed thickened skin epidermis, increased hair density, altered hair types, faster anagen re-entry, and formation of compound vibrissa follicles. The eyelid opening was smaller and ectopic cilia formed at the expense of Meibomian glands. In the distal limb, there were agenesis and hyperpigmentation of claws, interdigital webbing, reduced footpads, and trans-differentiation of sweat glands into hairs. The size of external genitalia increased in both sexes, but they remained fertile. We conclude that modulation of BMP activity can affect the number of ectodermal organs by acting during induction stages, influence the size and shape by acting during morphogenesis stages, change phenotypes by acting during differentiation stages, and facilitate new growth by acting during regeneration stages. Therefore during organogenesis, BMP antagonists can produce a spectrum of phenotypes in a stage-dependent manner by adjusting the level of BMP activity. The distinction between phenotypic variations and pathological changes is discussed. The integument forms the interface between an organism and its environment. During development and evolution, different types of epithelial organs form on the body surface to allow animals to adapt to different environments. Although these organs, such as hairs, glands, teeth, and so forth, appear to be very different in structure and function, developmental studies suggested that they are all products of epithelial-mesenchymal interactions, with variations overlaid on a common theme.1Chuong C-M Molecular Basis of Epithelial Appendage Morphogenesis. Landes Bioscience, Austin1998Google Scholar Genes involved in human ectodermal dysplasias have recently been cloned. These studies show that a single gene defect can cause abnormalities in several ectodermal organs.2Wisniewski SA Kobielak A Trzeciak WH Kobielak K Recent advances in understanding of the molecular basis of anhidrotic ectodermal dysplasia: discovery of a ligand, ectodysplasin A and its two receptors.J Appl Genet. 2002; 43: 97-107PubMed Google Scholar, 3Headon DJ Overbeek PA Involvement of a novel Tnf receptor homologue in HF induction.Nat Genet. 1999; 22: 370-374Crossref PubMed Scopus (309) Google Scholar, 4Brunner HG Hamel BC Bokhoven HVH P63 gene mutations and human developmental syndromes.Am J Med Genet. 2002; 112: 284-290Crossref PubMed Scopus (99) Google Scholar This further substantiates the notion that fundamental molecular pathways are frequently shared in the building of different epithelial organs. The commonly used molecular pathways include bone morphogenic protein (BMP), fibroblast growth factor (FGF), sonic hedgehog (SHH), Wnt, Notch, Eda pathways and so forth.5Chuong C-M Chodankar R Widelitz RB Jiang TX Evo-devo of feathers and scales: building complex epithelial appendages.Curr Opin Dev Genet. 2000; 10: 449-456Crossref PubMed Scopus (111) Google Scholar, 6Fuchs E Merrill BJ Jamora C DasGupta R At the roots of a never-ending cycle.Dev Cell. 2001; 1: 13-25Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar In each pathway there are multiple ligands, receptors, intracellular signaling transducers, and extracellular antagonists. Knowledge of these pathways motivates us to investigate how these molecular activities are translated into tissue morphogenesis. In the context of tissue engineering, such knowledge will also be required to guide epithelial stem cells appropriately to form the tissues/organs desired. Here we selected the BMP pathway for further analysis.7Botchkarev VA Bone morphogenetic proteins and their antagonists in skin and HF biology.J Invest Dermatol. 2003; 120: 36-47Crossref PubMed Scopus (151) Google Scholar BMPs play an important role in many developmental systems. Initially identified for their effects on osteocyte proliferation and differentiation, BMPs were further shown to act as regulators of proliferation, differentiation, apoptosis, cell adhesion, and migration during the development of multiple organs in many organisms studied.8Reddi AH Role of morphogenetic proteins in skeletal tissue engineering and regeneration.Nat Biotechnol. 1998; 16: 247-252Crossref PubMed Scopus (696) Google Scholar, 9Miyazono K Kusanagi K Inoue H Divergence and convergence of TGF-beta/BMP signaling.J Cell Physiol. 2001; 187: 265-276Crossref PubMed Scopus (453) Google Scholar, 10Wozney JM Overview of bone morphogenetic proteins.Spine. 2002; 27: S2-S8Crossref PubMed Scopus (326) Google Scholar Loss-of-function mutations of various components of the BMP pathway lead to severe developmental abnormalities often resulting in early embryonic lethality.11Itoh S Itoh F Goumans MJ Ten Dijke P Signaling of transforming growth factor-beta family members through Smad proteins.Eur J Biochem. 2000; 267: 6954-6967Crossref PubMed Scopus (454) Google Scholar The effect of BMPs on proliferation, differentiation, and apoptosis in different developmental systems is complex. It is, however, concentration-dependent. Low or high dosages of BMPs often result in opposite cell fate decisions: either proliferation or apoptosis.12Piscione TD Phan T Rosenblum ND BMP7 controls collecting tubule cell proliferation and apoptosis via Smad1-dependent and -independent pathways.Am J Physiol. 2001; 280: F19-F33Google Scholar BMP activity in a given tissue depends on the concentration and distribution of BMPs and their antagonists. A number of secreted proteins including Noggin, Follistatin, Chordin, and others antagonize BMP-mediated signaling.13Massague J Chen YG Controlling TGF-beta signaling.Genes Dev. 2000; 14: 627-644PubMed Google Scholar Noggin is the most powerful BMP-2 and BMP-4 antagonist.14Zimmerman LB De Jesus-Escobar JM Harland RM The Spemann organizer signal Noggin binds and inactivates bone morphogenetic protein 4.Cell. 1996; 86: 599-606Abstract Full Text Full Text PDF PubMed Scopus (1327) Google Scholar, 15Piccolo S Agius E Lu B Goodman S Dale L De Robertis EM Cleavage of chordin by xolloid metalloprotease suggests a role for proteolytic processing in the regulation of Spemann organizer activity.Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar Different effects of BMPs are often mediated by distinct BMP receptors (BMPRs). Several models suggest that the proliferative effect of BMPs is mainly mediated via BMP receptor IA (BMPR-IA).16Yamaguchi K Nagai S Ninomiya-Tsuji J Nishita M Tamai K Irie K Ueno N Nishida E Shibuya H Matsumoto K XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway.EMBO J. 1999; 18: 179-187Crossref PubMed Scopus (324) Google Scholar, 17Panchision DM Pickel JM Studer L Lee S-H Turner PA Hazel TG McKay RDG Sequential actions of BMP receptors control neural precursor cell production and fate.Genes Dev. 2001; 15: 2094-2110Crossref PubMed Scopus (285) Google Scholar Apoptotic signaling is mainly mediated through the receptor BMPR-IB.18Kimura N Matsuo R Shibuya H Nakashima K Taga T BMP2-induced apoptosis is mediated by activation of the TAK1–p38 kinase pathway that is negatively regulated by Smad6.J Biol Chem. 2000; 275: 17647-17652Crossref PubMed Scopus (209) Google Scholar, 19Zhang H Bradley A Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development.Development. 1996; 122: 2977-2986Crossref PubMed Google Scholar BMP signaling is used in skin and skin appendages development. In the presence of BMP-4, ectodermal cells choose an epidermal over a neural fate early in gastrulation.20Wilson PA Hemmati-Brivanlou A Induction of epidermis and inhibition of neural fate by Bmp-4.Nature. 1995; 376: 331-333Crossref PubMed Scopus (644) Google Scholar Later in skin development, distinct spatial distributions of different BMPs and BMPRs are seen. BMP-6 and BMP-7 are mainly expressed in the epidermis, with BMP-7 present in the basal and BMP-6 in suprabasal layers.21Lyons KM Pelton RW Hogan BL Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development.Genes Dev. 1989; 3: 1657-1668Crossref PubMed Scopus (399) Google Scholar, 22Wall NA Blessing M Wright CV Hogan BL Biosynthesis and in vivo localization of the decapentaplegic-Vg-related protein, DVR-6 (bone morphogenetic protein-6).J Cell Biol. 1993; 120: 493-502Crossref PubMed Scopus (113) Google Scholar, 23Takahashi H Ikeda T Transcripts for two members of the transforming growth factor-beta superfamily BMP-3 and BMP-7 are expressed in developing rat embryos.Dev Dyn. 1996; 207: 439-449Crossref PubMed Scopus (70) Google Scholar Also, BMPR-IA is expressed in the basal layer, whereas BMPR-IB is in the suprabasal layer. Based on several lines of evidence it was proposed that BMPR-IA mediates proliferation effects and BMPR-IB mediates differentiation effects of BMPs in epidermis.17Panchision DM Pickel JM Studer L Lee S-H Turner PA Hazel TG McKay RDG Sequential actions of BMP receptors control neural precursor cell production and fate.Genes Dev. 2001; 15: 2094-2110Crossref PubMed Scopus (285) Google Scholar Unlike BMP-6 and BMP-7, BMP-2 and BMP-4 are expressed during hair follicle (HF) organogenesis. BMP-4 is expressed transiently in the mesenchymal condensations just before HF formation. Therefore, this may be part of the initial dermal signals inducing follicular germ formation. BMP-2, however, is expressed in the epidermal placode, and in more advanced follicles it is found in the matrix and precortex cells.21Lyons KM Pelton RW Hogan BL Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development.Genes Dev. 1989; 3: 1657-1668Crossref PubMed Scopus (399) Google Scholar, 24Bitgood MJ McMahon AP Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo.Dev Biol. 1995; 172: 126-138Crossref PubMed Scopus (1174) Google Scholar, 25Botchkarev VA Botchkareva NV Roth W Noggin is a mesenchymally-derived stimulator of HF induction.Nat Cell Biol. 1999; 1: 158-164Crossref PubMed Scopus (331) Google Scholar At the time of HF induction, BMP signaling inhibits induction whereas Noggin signaling stimulates induction of HFs.25Botchkarev VA Botchkareva NV Roth W Noggin is a mesenchymally-derived stimulator of HF induction.Nat Cell Biol. 1999; 1: 158-164Crossref PubMed Scopus (331) Google Scholar, 26Noramly S Morgan BA BMPs mediate lateral inhibition at successive stages in feather tract development.Development. 1998; 125: 3775-3787PubMed Google Scholar Importantly, induction of secondary (nontylotrich) HFs, but not primary (tylotrich) HFs is affected by BMPs/Noggin.27Botchkarev VA Botchkareva NV Sharov AA Funa K Huber O Gilchrest BA Modulation of BMP signaling by Noggin is required for induction of the secondary (nontylotrich) HFs.J Invest Dermatol. 2002; 118: 3-10Crossref PubMed Scopus (117) Google Scholar The role of Noggin during HF induction was addressed in the Noggin knockout mouse model.25Botchkarev VA Botchkareva NV Roth W Noggin is a mesenchymally-derived stimulator of HF induction.Nat Cell Biol. 1999; 1: 158-164Crossref PubMed Scopus (331) Google Scholar Data were provided using Noggin knockout skin grafts because homozygous Noggin knockout mice die prematurely. It suggests that Noggin is important for secondary HF induction. In this model, secondary HFs failed to form. Induction of primary HFs was not affected, yet their growth was further arrested because of long-term BMP excess. Similar to this, transgenic mice engineered to overexpress BMP-4 in the outer root sheath under the control of the bovine cytokeratin IV promoter had a complete deficiency of hair growth after the first hair cycle and, therefore, were progressively balding.28Blessing M Nanney LB King LE Jones CM Hogan BL Transgenic mice as a model to study the role of TGF-beta-related molecules in HFs.Genes Dev. 1993; 7: 204-215Crossref PubMed Scopus (157) Google Scholar It seems that during development, Noggin prevents interactions between BMPR-IA and BMPs produced by the mesenchyme and placode. In support of this idea, Noggin treatment increases the hair placodes and accelerates HF morphogenesis in embryonic skin organ culture.25Botchkarev VA Botchkareva NV Roth W Noggin is a mesenchymally-derived stimulator of HF induction.Nat Cell Biol. 1999; 1: 158-164Crossref PubMed Scopus (331) Google Scholar Noggin is also required for HF growth during postnatal life. Normally, in adult HFs, Noggin activity is localized to the HF bulb. Noggin, produced by the dermal papilla, supports proliferation in the lower hair matrix.25Botchkarev VA Botchkareva NV Roth W Noggin is a mesenchymally-derived stimulator of HF induction.Nat Cell Biol. 1999; 1: 158-164Crossref PubMed Scopus (331) Google Scholar Overexpression of Noggin in proliferating hair matrix cells and differentiating hair precursor cells under the proximal Mxs2 promoter leads to the disruption of differentiation in epithelial cells controlled here, in part, by BMPs.29Kulessa H Turk G Hogan BL Inhibition of Bmp signaling affects growth and differentiation in the anagen HF.EMBO J. 2000; 19: 6664-6674Crossref PubMed Scopus (160) Google Scholar Another important pathway in hair morphogenesis is Wnt/β-catenin signaling and its up-regulation leads to an induction of excessive numbers of HFs.30Zhou P Byrne C Jacobs J Fuchs E Lymphoid enhancer factor 1 directs HF patterning and epithelial cell fate.Genes Dev. 1995; 9: 700-713Crossref PubMed Scopus (293) Google Scholar, 31Gat U DasGupta R Degenstein L Fuchs E De novo HF morphogenesis and hair tumors in mice expressing a truncated β-catenin in skin.Cell. 1998; 95: 605-614Abstract Full Text Full Text PDF PubMed Scopus (960) Google Scholar Disruption of the Wnt/β-catenin pathway in skin leads to an arrest of HF development.32Huelsken J Vogel R Erdmann B Cotsarelis G Birchmeier W Beta-catenin controls HF morphogenesis and stem cell differentiation in the skin.Cell. 2001; 105: 533-545Abstract Full Text Full Text PDF PubMed Scopus (1099) Google Scholar, 33Andl T Reddy ST Gaddapara T Millar SE WNT signals are required for the initiation of HF development.Dev Cell. 2002; 2: 643-653Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar Inhibition of BMP activity is shown to produce Lef-1 required for the activation of β-catenin/Lef-1 transcriptional complex.25Botchkarev VA Botchkareva NV Roth W Noggin is a mesenchymally-derived stimulator of HF induction.Nat Cell Biol. 1999; 1: 158-164Crossref PubMed Scopus (331) Google Scholar, 34Jamora C DasGupta R Kocieniewski P Fuchs E Links between signal transduction, transcription and adhesion in epithelial bud development.Nature. 2003; 422: 317-322Crossref PubMed Scopus (484) Google Scholar Although the roles of Noggin in HF formation have been studied, its role in other skin appendages remains mostly unknown. To address these questions we created a transgenic mouse model in which ectopic Noggin expression was directed by the K14 promoter. The K14-Noggin mice study showed that Noggin mediates disruption of normal BMP signaling during development, causing multiple abnormalities in a variety of ectodermal organs. Hyperplasia of pelage HFs occurred, ectopic HFs formed on the ventral side of the paw, supernumerary cilia formed in eyelids, and compound vibrissa follicles arose. Claws and footpads failed to form normally. There were also defects in organs that we do not normally consider as skin appendages. For example, we found an increase in the sizes of external genitalia and defects in eyelid opening. Here, we will describe an array of abnormalities and discuss the roles of BMP activity in pathogenesis. Mice were generated in the Norris Cancer Center transgenic mouse facility at the University of Southern California. To generate transgenic mice, human K14 promoter-chicken Noggin-poly A inserts were purified and microinjected into the male pronucleus of fertilized egg of C57BL/6J × CBA/J mice followed by reimplantation of injected eggs into pseudopregnant C57BL/6J × CBA/J females. The purification and microinjection of DNA were performed as described.36Liu YH Ma L Wu LY Luo W Kundu R Sangiorgi F Snead ML Maxson R Regulation of the Msx2 homeobox gene during mouse embryogenesis: a transgene with 439 bp of 5′ flanking sequence is expressed exclusively in the apical ectodermal ridge of the developing limb.Mech Dev. 1994; 48: 187-197Crossref PubMed Scopus (61) Google Scholar The founder K14-Noggin mice were then backcrossed onto C57BL/6J background for six generations. All phenotypical features of K14-Noggin mice showed high penetrance. Mice were screened for transgene presence by polymerase chain reaction (PCR) using chicken Noggin construct-specific primers: 5′-CCAGATCTATGGATCATTCCCAGTGC-3′ and 5′-GGAGATCTCTAGCAGGAGCACTTGCA-3′. Tail genomic DNA was extracted as described in manufacturer's protocol (Qiagen, Valencia, CA). PCR products were amplified in separate reactions using the three-stage PCR program: 94°C for 2 minutes; 94°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute (30 cycles); 72°C for 10 minutes. The identities of the founder mice were confirmed by Southern blot analyses. Quantitative genotyping of K14-Noggin mice was done by real-time quantitative PCR using SYBR Green technology.37Dhar AK Roux MM Klimpel KR Detection and quantification of infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot virus (WSV) of shrimp by real-time quantitative PCR and SYBR chemistry.J Clin Microbiol. 2001; 39: 2835-2845Crossref PubMed Scopus (133) Google Scholar The reaction and detection were performed in GeneAmp 5700 (see the User Manual; PE Applied Biosystems, Foster City, CA). A separate set of primers was designed for chicken Noggin to be used for real-time quantitative PCR: 5′-TCTGTCCCAGAAGGCATGGT-3′ and 5′-CGCCACCTCAGGATCGTTAA-3′. To control differences in the quantity of DNA template, the mouse L-32 gene was amplified in parallel for each sample and was used as a normalization factor to calculate the relative amount of chicken Noggin in mouse genomic DNA. The following L-32-specific primers were used: 5′-TGGTTTTCTTGTTGCTCCCATA-3′ and 5′-GGGTGCGGAGAAGGTTCAA-3′. A detailed protocol for real-time quantitative PCR is described elsewhere.38Hizer SE Dhar AK Klimpel KR Garcia DK RAPD markers as predictors of infectious hypodermal and hematopoietic necrosis virus (IHHNV) resistance in shrimp (Litopenaeus stylirostris).Genome. 2002; 45: 1-7Crossref PubMed Scopus (25) Google Scholar Among all K14-Noggin mice tested, we selected one mouse that showed the highest dCT value (5.5) on real-time quantitative PCR (CT, cycle threshold value; dCT = CT of Noggin − CT of L-32 for the same sample). This mouse contained the lowest number of K14-Noggin in the genome. Relative amount of K14-Noggin in all other mice was calculated as following: fold difference = 2 × (5.5 − dCT). The amount of chicken Noggin mRNA in K14-Noggin mice tissue was measured using the real-time quantitative RT-PCR method, based on SYBR Green technology, mentioned above. RNA was extracted from the ear pinna using the RNeasy mini kit, following the manufacturer's protocol (Qiagen). Ear pinna was selected for this experiment because it contains two layers of K14-expressing epidermis within relatively small amount of tissue. Real-time PCR was performed using identical set of primers and following the same protocol as for the quantitative genotyping described above. Relative amount of Noggin mRNA in mice tissue was calculated as following: fold difference = 2 × (9.45 − dCT), where 9.45 is the dCT value for the mouse with the lowest level of chicken Noggin mRNA in the tested tissue. Tissues were collected and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated, embedded in paraffin, and sectioned at 5 to 6 μm. When necessary, specimens were additionally decalcified in Immunocal solution (American Mastertech Scientific, Lodi, CA) for 48 hours at 4°C after fixation. Standard hematoxylin and eosin (H&E) staining was performed for basic histological analysis. Frozen tissue sections were used for the tyramide-based tyrosinase assay.39Han R Baden HP Brissette JL Weiner L Redefining the skin's pigmentary system with a novel tyrosinase assay.Pigment Cell Res. 2002; 15: 290-297Crossref PubMed Scopus (43) Google Scholar Briefly, after 3% peroxide treatment, 5% bovine serum albumin (fraction V) and avidin/biotin were used to block nonspecific binding (Vector Laboratories, Burlingame, CA). Next, biotin-tyramide in 1× application diluent (Perkin-Elmer Life Sciences, Emeryville, CA) was applied. After a washing step, streptavidin-CY3 (1:600; Sigma Chemical Co., St. Louis, MO) was applied. Immunostaining was performed using the Ventana Discovery automated immunostaining module (Ventana Medical Systems, Tucson, AZ). The primary antibodies used were mouse monoclonal anti-proliferating cell nuclear antigen (PCNA) (1:500; Chemicon, Temecula, CA), rabbit anti-K14 (1:400; Berkeley Antibody Company, Richmond, CA), and anti-K10 (1:200, Sigma). The DAB detection kit (Ventana Medical Systems) was used for color development. Mouse tissues from various ages were used for section in in situ hybridization. Section in situ samples were fixed and dehydrated according to the standard protocol. All solutions used for the procedure were diethyl pyrocarbonate-treated to inactivate RNase. To detect the RNA expression, the tissue was hybridized with digoxigenin-labeled probes. The signals were detected by using an anti-digoxigenin antibody coupled to alkaline phosphatase. Some samples were processed using the Discovery automated in situ hybridization instrument (Ventana Medical Systems). Whole mount in situ procedure was performed on E15 mouse embryos. Specimens were fixed in 4% paraformaldehyde in diethyl pyrocarbonate-treated PBS. Tissue samples were then dehydrated in a series of methanol in PBS and 0.1% Tween 20 (PBT buffer) and stored in absolute methanol at −20°C before the actual staining procedure. Whole mount in situ hybridization procedures were performed using the InsituPro automated in situ detection module (Intavis AG, Koeln, Germany). Analysis was performed according to the standard whole mount in situ protocol.40Jiang TX Jung HS Widelitz RB Chuong CM Self-organization of periodic patterns by dissociated feather mesenchymal cells and the regulation of size, number and spacing of primordia.Development. 1999; 126: 4997-5009PubMed Google Scholar All surgical procedures were performed on anesthetized mice (ketamine HCl:xylazine mixture was used). For the whole mount skin preparation, anagen skin was collected, inverted, and subcutaneous tissue was removed. These samples were fixed and dehydrated in a stretched condition. After dehydration, skin samples were cleared with xylene and photographed. Morphometric analyses were then performed using Adobe PhotoShop. Analysis of hair shaft structure was performed under the inverted microscope according to a previously described protocol.41Millar SE Willert K Salinas PC Roelink H Nusse R Sussman DJ Barsh GS WNT signaling in the control of hair growth and structure.Dev Biol. 1999; 207: 133-149Crossref PubMed Scopus (242) Google Scholar, 42Nakamura M Sundberg JP Paus R Mutant laboratory mice with abnormalities in hair follicle morphogenesis, cycling, and/or structure: annotated tables.Exp Dermatol. 2001; 10: 369-390Crossref PubMed Scopus (77) Google Scholar The relative number of guard, awl, auchene, and zigzag hairs was determined from the fur of the dorsal skin. Lengths of the hair growth cycle stages were based on the change of the skin color from pink during telogen to black during anagen. These changes occur because of the active melanogenesis in the HFs during anagen and were proven to be valid criteria for the hair cycle staging elsewhere.43Slominski A Paus R Plonka P Chakraborty A Maurer M Pruski D Lukiewicz S Melanogenesis during the anagen-catagen-telogen transformation of the murine hair cycle.J Invest Dermatol. 1994; 102: 862-869Abstract Full Text PDF PubMed Google Scholar All observations were performed on shaved mice (n = 6). Several consecutive hair growth cycles were analyzed on the same mice for 4 months. We started when they were 2 months old and ended when they were 6 month old. The lengths of the anagen and telogen stages of the hair growth cycle were established. External genital measurements were performed on anesthetized animals. Nonerect genitalia were measured in both control and K14-Noggin animals. Tissues were prepared according to the standard scanning electron microscopy protocol. Briefly, it includes fixation in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate, dehydration, and critical point drying from ethanol. Next, samples were coated with gold in a sputter coat chamber. They were examined by scanning electron microscopy in the Doheny Eye Institute Core Facility, University of Southern California. The chicken Noggin cDNA fragment was subcloned into the human K14 vector (Figure 1A).44Oro AE Higgins K Hair cycle regulation of Hedgehog signal reception.Dev Biol. 2003; 255: 238-248Crossref PubMed Scopus (192) Google Scholar K14-Noggin-PolyA inserts were released from plasmids and used for injection. Three independent transgenic lines were produced with similar phenotypes. Identities of K14-Noggin mice were confirmed by PCR-genotyping using chicken Noggin-specific primers (Figure 1B). We have isolated several lines and found a range of phenotypes. There were mice with severe phenotypic changes (Figure 1C) and mice with moderate alterations. The more severe phenotypes included absence of claws, interdigital webbing of the paws, hypertrichosis all over the body, shortening of the telogen period of the hair growth cycle, and increased size of the genitalia. In other mice pathological changes were milder. We performed quantitative genotyping of the K14-Noggin mice and quantitative RT-PCR for the Noggin mRNA to establish whether strength of phenotypic changes correlates with K14-Noggin transgene copy number in the mouse genome and Noggin expression level. We studied several key phenotypic features of the limbs of every mouse and correlated them with the fold difference values (see Materials and Methods). Based on this we divided all K14-Noggin mice into low-transgenic (TG) copy number and high-TG copy number with fold difference value for high-TG copy number animals equal to 3 and higher (Table 1).Table 1Phenotypic Changes in Low- and High-TG Copy Number in K14-Noggin MiceHigh-TG copy number mice (n = 8)*Number of animals studied.Low-TG copy number mice (n = 6)*Number of animals studied.Genotyping4.2 ± 0.6†Fold difference, calculated as described in Materials and Methods.1Noggin transcript level2.9 ± 0.7†Fold difference, calculated as described in Materials and Methods.1Percentage showing phenotypeFusion of forelimb digits100%12.5 – 25%Fusion of hindlimb digits80 –100%0 – 25%Absence of forelimb claws95 –100%29 – 75%Absence of hindlimb claws63 –100%33 – 58%Hyperpigmentation of forelimb claws100%0 – 60%Hyperpigmentation of hindlimb claws70 –100%40 – 66%* Number of animals studied.† Fold difference, calculated as described in Materials and Methods. Open table in a new tab Adult high-TG copy number K14-Noggin mice had smaller eye openings (Figure 2, A and B), whereas the diameters of the eyeballs were not significantly different (3.9 ± 0.5 mm in control mice and 3.75 ± 0.05 mm in high-TG copy number K14-Noggin mice, n = 3). Smaller eyelid openings were already obvious as early as postnatal day 14. However, no significant delay of eyelid opening was seen in K14-Noggin mice in comparison with the control mice. In addition, adult eyelids of high-TG copy number K14-Noggin mice exhibited abnormalities. Most significantly, there w
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