Murine Model of the Ehlers-Danlos Syndrome

The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of proα1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS. The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of proα1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS. Abnormal collagen fibril formation is a hallmark of several forms of the Ehlers-Danlos syndrome. The classic form of the Ehlers-Danlos syndrome (EDS 2The abbreviations used are: EDS, Ehlers-Danlos syndrome; RPA, ribonuclease protection assay; PBS, phosphate-buffered saline; CI, confidence interval. 2The abbreviations used are: EDS, Ehlers-Danlos syndrome; RPA, ribonuclease protection assay; PBS, phosphate-buffered saline; CI, confidence interval. types I, II) is characterized by fragile, hyperextensible skin, widened, atrophic scars, bruisability, joint laxity, a high prevalence of aortic root dilation, and other manifestations of connective tissue weakness, including inguinal hernia and prolapse of the uterine cervix or rectum (1Beighton P. Beighton P. 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Symoens S. Loeys B. Nuytinck L. De Paepe A. Hum. Mutat. 2005; 25: 28-37Crossref PubMed Scopus (96) Google Scholar). Type V collagen is a quantitatively minor fibril-forming collagen. Several isoforms of type V collagen exist, differing in the type and ratios of constituent α chains, including heterotrimeric molecules containing type XI collagen chains. The proα1(V) chain, encoded by COL5A1 at human chromosomal locus 9q34, is the rate-limiting component of type V collagen trimer assembly by virtue of the eight-cysteine motif in the NC1 domain (19Lees J.F. Bulleid N.J. J. Biol. Chem. 1994; 269: 24354-24360Abstract Full Text PDF PubMed Google Scholar). The most abundant and most widely distributed isoform of type V collagen is the [α1(V)]2 α2(V)] heterotrimer that co-assembles with type I collagen as heterotypic fibrils (20Chanut-Delalande H. Fichard A. Bernocco S. Garrone R. Hulmes D.J. Ruggiero F. J. Biol. Chem. 2001; 276: 24352-24359Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). This isoform of type V collagen retains a non-collagenous, N-terminal domain that is present on the fibril surface, and this domain has been demonstrated to have regulatory functions (21Birk D.E. Micron. 2001; 32: 223-237Crossref PubMed Scopus (315) Google Scholar, 22Birk D.E. Fitch M. Babiarz J.P. Linsenmayer T.F. J. Cell Biol. 1988; 106: 999-1008Crossref PubMed Scopus (291) Google Scholar, 23Niyibizi C. Eyre D.R. Biochim. Biophys. Acta. 1993; 1203: 304-309Crossref PubMed Scopus (31) Google Scholar). Disruption of [α1(V)]2 [α2(V)] heterotrimer synthesis using a dominant negative approach (24Marchant J.K. Hahn R.A. Linsenmayer T.F. Birk D.E. J. Cell Biol. 1996; 135: 1415-1426Crossref PubMed Scopus (97) Google Scholar) or utilizing fibroblasts from EDS patients with characterized mutations in COL5A1 have demonstrated that heterotypic collagen I/V interactions are involved in regulation of fibril diameter and fibril number in vitro (25Wenstrup R.J. Florer J.B. Cole W.G. Willing M.C. Birk D.E. J. Cell. Biochem. 2004; 92: 113-124Crossref PubMed Scopus (50) Google Scholar). In the total absence of theα1(V) chain, collagen fibrils were virtually absent though embryonal fibroblasts from col5a1-/-mice synthesize and secrete normal amounts of type I collagen; mice died at the onset of organogenesis (26Wenstrup R.J. Florer J.B. Brunskill E.W. Bell S.M. Chervoneva I. Birk D.E. J. Biol. Chem. 2004; 279: 53331-53337Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). In addition, severe reduction of type V heterotrimers in favor of homotrimers leads to deposition of an abnormal dermal matrix (27Chanut-Delalande H. Bonod-Bidaud C. Cogne S. Malbouyres M. Ramirez F. Fichard A. Ruggiero F. Mol. Cell. Biol. 2004; 24: 6049-6057Crossref PubMed Scopus (62) Google Scholar). Evidence indicates that the [α1(V)]3 homotrimer does not associate with type I collagen in fibrils (20Chanut-Delalande H. Fichard A. Bernocco S. Garrone R. Hulmes D.J. Ruggiero F. J. Biol. Chem. 2001; 276: 24352-24359Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). These data support a critical role for the [α1(V)]2 [α2(V)] heterotrimer in the nucleation of fibril assembly, consistent with its early evolutionary appearance. Mice heterozygous for a col5a1 mutation show a 50% reduction in type V collagen and recapitulate many of the clinical, biomechanical, morphologic, and biochemical features of the Ehlers-Danlos syndrome, classic type and are thus excellent models for the classic form of EDS for use in further studies in the regulation of collagen biogenesis and for potential therapeutic interventions. Analysis of heterotypic, structurally abnormal fibrils from dermis of col5a1-haploinsufficient mice provides insights into the mechanisms by which collagen fibrillogenesis is regulated and the mechanism of abnormal connective biosynthesis underlying the Ehlers-Danlos syndrome. Generation of col5a1-deficient Mice—The construction of the targeting vector was previously described in detail (25Wenstrup R.J. Florer J.B. Cole W.G. Willing M.C. Birk D.E. J. Cell. Biochem. 2004; 92: 113-124Crossref PubMed Scopus (50) Google Scholar). The col5a1-targeting vector was generated using the col5a1 sequence obtained from the Celera mouse genomic data base. Gene-specific primers amplified a 5′-targeting arm from a region that included part of exon 3 and a 3′-targeting arm from a region that included part of exon 4 from KG-1 embryonic stem cell DNA (see Fig. 1D for location of the recombination site relative to functional domains within col5a1). Germ line transmission was obtained by breeding chimeric animals to C57BL/6. Quantitation of mRNA—Quantitation of col5a1, col5a2, and col1a1 mRNA in total RNA from mice tails was examined by ribonuclease protection assay (RPA III; Ambion). The probes were obtained using PCR to attach the T7 promoter sequence to the 5′-end of the antisense sequence for transcription of an antisense RNA probe. The primer pairs for the probes were as follows: col5a1,(5′-GAACAGATGAAGCGACCAC-3′) and (5′-GATCCTAATACGACTCACTATAGGGAGGGCCTTCAGCATCCAC-3′); col5a2, (5′-CCATCTTACTGCTGCTCTTG-3′) (5′-CGCCTAATACGACTCACTATAGGGAGGATT CACCGCTCTGCTTTG-3′); col1a1, (5′-GTCTCAAGATGGTGACCGCTAC-3′ (5′-GAGCCTAATACGACTCACTATAGGGAGGC TCTCCGCTCTTCCAGTC-3′). The col5a1 probe used for RPA was complementary to sequences in exon 64, downstream from the recombination-mediated deletions of exons 3-4 (Fig. 1D). RPA with this probe was designed to exclude the possibility that a transcript encoding a truncated peptide containing a reconstituted C-propeptide could have a dominant negative effect by co-assembling with wild-type proα1(V) or proα2(V) chains. Amplified DNA was used directly in the transcription reaction; 10 ng was transcribed by T7 RNA polymerase in the presence of [32P]UTP using reagents for in vitro transcription from Ambion (MaxiScript). The labeled probes were gel purified following the manufacturer's suggestions. Total cellular RNA (2.5-10 μg) and at least 2 fmol of each labeled probe were used for each assay following the manufacturer's instructions. Hybridization was carried out at 68 °C for 18 h followed by RNase digestion at 37 °C for 30 min using 2.5 units/ml of RNase A and 100 units/ml of RNase T1. Protected fragments were separated on a 5% acrylamide/8 m urea gel and detected by exposure to X-OMAT (Kodak) for 18 h at -80 °C with an intensifying screen. Films were scanned and evaluated using Pharmacia GSXL system. Measurement of Total Collagen Deposited in the Skin—Skin was harvested from animals sacrificed at postnatal days P0 and P10, and at 4, 6, 8, 12, and 20 weeks. Two punch biopsies were taken from each animal, to the right and left of midline on the upper back. Samples were hydrolyzed in 6 N HCl at 100°C for 18 h. Colorimetric analysis of the hydroxyproline content of each skin sample was adapted from the method of Berg (25Wenstrup R.J. Florer J.B. Cole W.G. Willing M.C. Birk D.E. J. Cell. Biochem. 2004; 92: 113-124Crossref PubMed Scopus (50) Google Scholar, 28Berg R.A. Methods Enzymol. 1982; 82: 372-398Crossref PubMed Scopus (179) Google Scholar). The mean collagen content from the two samples per animal was calculated and normalized to skin area in millimeters squared. The conversion ratio of 0.12:1.0 was used to convert micrograms of hydroxyproline to total collagen. Semiquantitative measurement of type V content was performed after dermis was directly extracted into SDS buffer and analyzed by Western blot, using an antisera to a peptide sequence encoded by exon 6 in the NC3 domain (25Wenstrup R.J. Florer J.B. Cole W.G. Willing M.C. Birk D.E. J. Cell. Biochem. 2004; 92: 113-124Crossref PubMed Scopus (50) Google Scholar), downstream from the exon 3-4 region targeted by homologous recombination DNA (see Fig. 1D for location of the peptide epitope relative to the recombination site within col5a1). Biomechanical Analysis—The thoracic aortas were dissected, separated into ascending and descending portions, threaded with stainless steel hooks, and circumferential load-extension curves were obtained using a TA-XT2 texture analyzer (Stable MicroSystems) as described previously (29Vouyouka A.G. Pfeiffer B.J. Liem T.K. Taylor T.A. Mudaliar J. Phillips C.L. J. Vasc. Surg. 2001; 33: 1263-1270Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Load-extension curves were analyzed for thoracic aortic maximal breaking strength (Fmax) and incremental elastic modulus as described previously (29Vouyouka A.G. Pfeiffer B.J. Liem T.K. Taylor T.A. Mudaliar J. Phillips C.L. J. Vasc. Surg. 2001; 33: 1263-1270Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Tensile testing of wounded and unwounded skin was performed as previously described (30Liaw L. Birk D.E. Ballas C.B. Whitsitt J.S. Davidson J.M. Hogan B.L. J. Clin. Investig. 1998; 101: 1468-1478Crossref PubMed Google Scholar). Transmission Electron Microscopy—Wild-type and col5a1+/-postnatal day 10 (P10) as well as 6-, 12-, and 20-week male mice were used in these experiments. The subscapular dermis was analyzed for wild-type and haploinsufficient postnatal animals. Tissues were prepared for transmission electron microscopy as previously described (17Wenstrup R.J. Florer J.B. Willing M.C. Giunta C. Steinmann B. Young F. Susic M. Cole W.G. Am. J. Hum. Genet. 2000; 66: 1766-1776Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 31Birk D.E. Trelstad R.L. J. Cell Biol. 1984; 99: 2024-2033Crossref PubMed Scopus (222) Google Scholar). Briefly, fixation was in 4% paraformaldehyde/2.5% glutaraldehyde/0.1 m sodium cacodylate, pH 7.4 with 8.0 mm CaCl2 for 2 h on ice, followed by post-fixation with 1% osmium tetroxide for 1 h. After dehydration in a graded ethanol series followed by propylene oxide, the tissues were infiltrated and embedded in a mixture of Embed 812, nadic methyl anhydride, dodecenylsuccinic anhydride, and DMP-30 (EM Sciences, Hatfield, PA). Thick sections (1 μm) were cut and stained with methylene blue-azure B for light microscopy and selection of specific regions for further analysis. Thin sections were prepared using a Reichert UCT ultramicrotome and a diamond knife. Staining was with 2% aqueous uranyl acetate followed by 1% phosphotungstic acid, pH 3.2. Sections were examined and photographed at 75 kV using a Hitachi 7000 transmission electron microscope. The microscope was calibrated using a line grating. For measurement of collagen fibril density, the dermis was divided into four equal regions. Analysis was done for both the superficial and deep dermis. The superficial dermis was defined as the region subjacent to the epidermis, and the deep dermis was the lower quarter. Both regions were photographed in the central portion. Micrographs were taken at ×31,680. Calibrated micrographs from each region were randomly chosen in a masked manner from the different regions. The micrographs were digitized, and all diameters were measured within a 1.6-μm mask. The mask was placed based on fibril orientation, i.e. cross-section and absence of cells. Diameters were measured along the minor axis of cross-sections using a RM Biometrics-Bioquant Image Analysis System (Memphis, TN). Quantitation of fibrils/μm2 was measured from transmission electron micrographs of cross-sections from the deep dermis of the subscapular region of 10-day, 6-week, and 20-week wild-type and heterozygous mice. Immunoelectron Microscopy—The subscapular dermis of col5a1+/- and wild-type mice (15 weeks) was fixed in 4% paraformaldehyde in PBS for 30 min at 4 °C. The tissues were washed in PBS, free aldehydes were reduced by incubation in 0.05% sodium borohydride in PBS, and then tissues were rinsed and blocked with 50 mm glycine in PBS. The samples were dehydrated to 70% ethanol and then infiltrated and embedded in LR White resin (EMS, Hatfield, PA). Thin sections were cut and picked up onto formvar-coated nickel grids. For antibody labeling, sections were blocked with 5% normal goat serum in PBS for 60 min at room temperature and then either anti-collagen V antibody (25 μg/ml) diluted in PBS containing 0.1% bovine serum albumin and 0.05% Tween 20 or buffer alone overnight at 4 °C. After four washes, the sections were incubated with goat anti-rabbit secondary antibody conjugated to 10 nm colloidal gold (Ted Pella, Redding, CA) for 1 h at room temperature, washed four times, rinsed with deionized water, and post-stained with 2% aqueous uranyl acetate. Sections were examined using a Tecnai 12 transmission electron microscope with a Gatan Multiscan 1000 camera. Statistical Methods—For each micrograph, the number of fibrils within a 1.6-μm2 mask was divided by the area of the box to compute the fibril density. For the fibril diameters, it was not adequate to assume normal distribution within micrographs, even for the diameters trimmed of outliers (mostly abnormally large fibrils). In each micrograph, the median, which is robust (minimally affected by outliers), was used as a measure of the central location of the fibril diameter distribution. The micrograph-specific fibril densities and medians were analyzed separately by fitting a linear mixed effects model (33Vonesh E.F. Chinchilli V.M. Linear and Nonlinear Models for the Analysis of Repeated Measures. Marcel Dekker, New York1997Google Scholar) incorporating animal-to-animal and micrograph-to-micrograph variability. The fibril densities were log transformed to satisfy the normality assumptions of the model (33Vonesh E.F. Chinchilli V.M. Linear and Nonlinear Models for the Analysis of Repeated Measures. Marcel Dekker, New York1997Google Scholar). Based on examination of residuals from the fitted models, the model assumptions were adequate. Targeted Disruption of col5a1 Causes Haploinsufficiency—The most common mutation resulting in classic EDS is the functional loss of one COL5A1 allele. We created a mouse model to study the molecular mechanism generating the clinical phenotype by targeted disruption of col5a1. RPA of mRNA isolated from col5a1+/+ and col5a1+/-animals showed half-reduction of col5a1-derived mRNA compared with col5a2-dervived mRNA by densitometric analysis of the autoradiograms, 0.89 ± 0.14 (n = 8) versus 0.36 ± 0.03 (n = 10), respectively. There was no difference in the steady state levels of col1a1 mRNA expression levels between col5a1+/- and wild-type mice (Fig. 1, A and B). Densitometric scanning of Western blots of dermal proteins from P10 animals indicated that there was a 50% decrease in the type V collagen content in skin from the col5a1+/-animals compared with wild-type littermates, demonstrating a direct relationship between mRNA and protein content. Corneal tissue, which has substantially higher type V collagen content as a proportion of total collagen than other connective tissues, was also analyzed. The type V collagen content of col5a1+/-mouse corneas was only 55% that of wild-type littermates by densitometric scanning of Western blots (data not shown). Vascular Phenotype in col5a1+/-Animals—Ehlers-Danlos syndrome classic type is associated with high prevalence of aortic root dilation (34Wenstrup R.J. Meyer R.A. Lyle J.S. Hoechstetter L. Rose P.S. Levy H.P. Francomano C.A. Genet. Med. 2002; 4: 112-117Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and more rarely with rupture or dissection. Easy bruisability is a hallmark of the condition and is evidence of capillary fragility. The col5a1-/-mouse embryos and yolk sacs demonstrated blood pooling and reduced circulation at E10 before cessation of the heart's rhythmic contractions, indicating that cardiovascular insufficiency was a factor in embryonic demise (25Wenstrup R.J. Florer J.B. Cole W.G. Willing M.C. Birk D.E. J. Cell. Biochem. 2004; 92: 113-124Crossref PubMed Scopus (50) Google Scholar). To determine whether cardiovascular tissue was measurably compromised in col5a1-haploinsufficient animals, biomechanical analyses were performed on ascending and descending aortas from 12-week-old animals. The results indicated that col5a1 haploinsufficiency results in decreased aortic stiffness and breaking strength (Fig. 2). There was a 35.9% (97.4 ± 5.7 (5Hausser I. Anton-Lamprecht I. Hum. Genet. 1994; 93: 394-407Crossref PubMed Scopus (130) Google Scholar) g versus 62.4 ± 4.9 (8Toriello H.V. Glover T.W. Takahara K. Byers P.H. Miller D.E. Higgins J.V. Greenspan D.S. Nat. Genet. 1996; 13: 361-365Crossref PubMed Scopus (109) Google Scholar) g; p = 0.0008) decrease in breaking strength and a 36.8% (120.3 ± 7.1 (5Hausser I. Anton-Lamprecht I. Hum. Genet. 1994; 93: 394-407Crossref PubMed Scopus (130) Google Scholar) versus 76.0 ± 6.5 (8Toriello H.V. Glover T.W. Takahara K. Byers P.H. Miller D.E. Higgins J.V. Greenspan D.S. Nat. Genet. 1996; 13: 361-365Crossref PubMed Scopus (109) Google Scholar); p = 0.001) decrease in incremental elastic modulus for the ascending aorta. There was a 59.6% (104.2 ± 14.1 (5Hausser I. Anton-Lamprecht I. Hum. Genet. 1994; 93: 394-407Crossref PubMed Scopus (130) Google Scholar) g/mm versus 42.1 ± 4.2 (8Toriello H.V. Glover T.W. Takahara K. Byers P.H. Miller D.E. Higgins J.V. Greenspan D.S. Nat. Genet. 1996; 13: 361-365Crossref PubMed Scopus (109) Google Scholar) g/mm; p = 0.009) decrease in breaking strength and a 68.4% (194.1 ± 18.0 (5Hausser I. Anton-Lamprecht I. Hum. Genet. 1994; 93: 394-407Crossref PubMed Scopus (130) Google Scholar) versus 61.4 ± 10.2 (8Toriello H.V. Glover T.W. Takahara K. Byers P.H. Miller D.E. Higgins J.V. Greenspan D.S. Nat. Genet. 1996; 13: 361-365Crossref PubMed Scopus (109) Google Scholar); p <0.0001) decrease in incremental elastic modulus for the descending aorta. The difference between col5a1+/- and +/+ animals was greater in the descending aorta than in the ascending aorta, which is consistent with the current understanding that collagen and elastin bear the majority of the wall stress and determine the stiffness (compliance) of the aorta (35Dobrin P.B. Sidawy A. Sumpio B. DePalma R. The Basic Science of Vascular Disease. Futura Publishing, New York1997: 69-105Google Scholar). As the distance from the heart increases, the elastin content is known to decrease and the collagen content to increase, with a net result of higher collagen to elastin ratios in the descending aorta (36Clark J.M. Glagov S. Arteriosclerosis. 1985; 5: 19-34Crossref PubMed Google Scholar, 37Dobrin P.B. Baker W.H. Gley W.C. Arch. Surg. 1984; 119: 405-409Crossref PubMed Scopus (333) Google Scholar). Dermal Phenotype in col5a1+/-Animals—Dermal features of hyperelasticity, friability, and poor wound healing are unique and characteristic features of the Ehlers-Danlos syndrome, classic type. Dermal abnormalities were immediately apparent in col5a1+/-animals; the skin was hyperextensible in the col5a1+/-mice relative to wild-type controls, but there was no skin redundancy (Fig. 3A). The tensile strength of normal and wounded skin was reduced in the col5a1+/-mice relative to wild-type controls. Biomechanical testing of tensile (breaking) strength was performed on animals at ∼12 weeks of age. Unwounded skin of col5a1+/-animals failed at 27.05 ± 3.53 kilogram-feet tensile stress/centimeter squared (Kgf/cm2) compared with 51.99 ± 7.20 (t-test = 0.029625) for wild-type littermates (Fig. 3B). In addition, the tensile strength of incisional wounds on dorsal skin 8 days after wounding was analyzed. There was a significant reduction in wound strength in the col5a1+/- animals relative to wild-type controls, 5.57 ± 0.48 v. 9.50 ± 2.57 Kgf/cm2 (t-test = 0.006071), respectively. All parameters were compatible with those seen in patients with the classic form of EDS. Dermal thickness increases significantly in young adult C57BL/6 wild-type mice and is correlated with an increase in collagen content as determined by measurement of hydroxyproline. The col5a1+/-mice demonstrated a significant delay in dermal collagen accumulation between 4 and 8 weeks of age, but by 12 weeks the total quantity of dermal collagen of col5a1+/-mice approximates that of wild-type littermates (Fig. 4). The finding of quantitatively normal dermal collagen content in 12-week+/-animals was surprising in light of the observed reduction in biomechanical properties of the skin and suggested the possibility of qualitative defects in dermal architecture. Light microscopy of dermis from +/- animals at 12 weeks demonstrated that there was collagen fiber disarray and a general appearance of reduced density of dermal connective tissue compared with wild-type littermates (Fig. 5).FIGURE 5The dermis of col5a1+/-contains poorly organized and less densely packed fibers. Light microscopy of dermis from col5a1+/+ (left) and +/-(right) mice at 12 weeks of age. Dermal sections were stained with Massons trichrome and magnified at ×100.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Decreased Fibril Density in the col5a1+/-Dermis—Analysis of dermal collagen fibrils by transmission electron microscopy demonstrated that the fibril density (number of fibrils/μm2) was reduced by 38% to 46% relative to the wild-type controls at all developmental stages analyzed between postnatal day 10 and week 20 in the deep, subscapular dermis (Fig. 6). During this period of development and maturation of the dermis, the fibril density decreases as fibril diameter increases in the wild-type dermis (see also Fig. 7). The density of fibrils was on average 42% lower (95% CI: 34%, 50%; p <0.001) in mutant mice across all ages. Similar significant differences were observed at P10, 6 weeks, and 20 weeks. At P10 there were 185 fibrils/μm2 (95% CI: 154, 223) in the wild-type dermis and 114 fibrils/μm2 (95% CI: 95, 137) in the col5a1+/-mice. At 6 weeks the values were 89 fibrils/μm2 (95% CI: 76, 104) and 48 fibrils/μm2 (95% CI: 41, 57), and at 20 weeks there were 52 fibrils/μm2 (95% CI: 44, 60) and 29 fibrils/μm2 (95% CI: 25, 35) in wild-type and col5a1+/-mice, respectively (Fig. 6). This decrease was observed in papillary dermis as well as in dermis from the axillary region (data not shown). These data indicate a numerical reduction in fibril formation events in the col5a1-haploinsufficient dermis independent of developmental stage or tissue site.FIGURE 7The col5a1-haploinsufficient dermis assembles larger fibrils and a second very large population of structurally abnormal fibrils compared with the wild-type controls. The fibril diameter distributions from the dermis of postnatal day-10 (A), 6-week (B), and 20-week (C) wild-type (blue) and col5a1+/-(red) mice. Diameters were measured from transmission electron micrographs of cross-sections comparable with Fig. 8.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analyses of the fibril diameter distributions indicated two fibril subpopulations in the col5a1-haploinsufficient dermis at all stages analyzed between P10 and 20 weeks (Fig. 7). One was a relatively symmetrical subpopulation comparable with the wild-type distribution, only broader and shifted to larger diameters. The second was a subpopulation