EMILIN1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis.

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.

Molecular alterations due to Col5a1 haploinsufficiency in a mouse model of classic Ehlers-Danlos syndrome.

Type V collagen is a regulatory fibrillar collagen essential for type I collagen fibril nucleation and organization and its deficiency leads to structurally abnormal extracellular matrix (ECM). Haploinsufficiency of the Col5a1 gene encoding α(1) chain of type V collagen is the primary cause of classic Ehlers-Danlos syndrome (EDS). The mechanisms by which this initial insult leads to the spectrum of clinical presentation are not fully understood. Using transcriptome analysis of skin and Achilles tendons from Col5a1 haploinsufficient (Col5a1+/-) mice, we recognized molecular alterations associated with the tissue phenotypes. We identified dysregulation of ECM components including thrombospondin-1, lysyl oxidase, and lumican in the skin of Col5a1+/- mice when compared with control. We also identified upregulation of transforming growth factor ß1 (Tgf-ß) in serum and increased expression of pSmad2 in skin from Col5a1+/- mice, suggesting Tgf-ß dysregulation is a contributor to abnormal wound healing and atrophic scarring seen in classic EDS. Together, these findings support altered matrix to cell signaling as a component of the pathogenesis of the tissue phenotype in classic EDS and point out potential downstream signaling pathways that may be targeted for the treatment of this disease.

Localized chondro-ossification underlies joint dysfunction and motor deficits in the Fkbp10 mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetic disorder that features wide-ranging defects in both skeletal and nonskeletal tissues. Previously, we and others reported that loss-of-function mutations in FK506 Binding Protein 10 (FKBP10) lead to skeletal deformities in conjunction with joint contractures. However, the pathogenic mechanisms underlying joint dysfunction in OI are poorly understood. In this study, we have generated a mouse model in which Fkbp10 is conditionally deleted in tendons and ligaments. Fkbp10 removal substantially reduced telopeptide lysyl hydroxylation of type I procollagen and collagen cross-linking in tendons. These biochemical alterations resulting from Fkbp10 ablation were associated with a site-specific induction of fibrosis, inflammation, and ectopic chondrogenesis followed by joint deformities in postnatal mice. We found that the ectopic chondrogenesis coincided with enhanced Gli1 expression, indicating dysregulated Hedgehog (Hh) signaling. Importantly, genetic inhibition of the Hh pathway attenuated ectopic chondrogenesis and joint deformities in Fkbp10 mutants. Furthermore, Hh inhibition restored alterations in gait parameters caused by Fkbp10 loss. Taken together, we identified a previously unappreciated role of Fkbp10 in tendons and ligaments and pathogenic mechanisms driving OI joint dysfunction.

Lysyl hydroxylase 3-mediated post-translational modifications are required for proper biosynthesis of collagen α1α1α2(IV).

Collagens are the most abundant proteins in the body and among the most biosynthetically complex. A molecular ensemble of over 20 endoplasmic reticulum resident proteins participates in collagen biosynthesis and contributes to heterogeneous post-translational modifications. Pathogenic variants in genes encoding collagens cause connective tissue disorders, including osteogenesis imperfecta, Ehlers-Danlos syndrome, and Gould syndrome (caused by mutations in COL4A1 and COL4A2), and pathogenic variants in genes encoding proteins required for collagen biosynthesis can cause similar but overlapping clinical phenotypes. Notably, pathogenic variants in lysyl hydroxylase 3 (LH3) cause a multisystem connective tissue disorder that exhibits pathophysiological features of collagen-related disorders. LH3 is a multifunctional collagen-modifying enzyme; however, its precise role(s) and substrate specificity during collagen biosynthesis has not been defined. To address this critical gap in knowledge, we generated LH3 KO cells and performed detailed quantitative and molecular analyses of collagen substrates. We found that LH3 deficiency severely impaired secretion of collagen α1α1α2(IV) but not collagens α1α1α2(I) or α1α1α1(III). Amino acid analysis revealed that LH3 is a selective LH for collagen α1α1α2(IV) but a general glucosyltransferase for collagens α1α1α2(IV), α1α1α2(I), and α1α1α1(III). Importantly, we identified rare variants that are predicted to be pathogenic in the gene encoding LH3 in two of 113 fetuses with intracranial hemorrhage-a cardinal feature of Gould syndrome. Collectively, our findings highlight a critical role of LH3 in α1α1α2(IV) biosynthesis and suggest that LH3 pathogenic variants might contribute to Gould syndrome.

The Fraser Complex Proteins (Frem1, Frem2, and Fras1) Can Form Anchoring Cords in the Absence of AMACO at the Dermal-Epidermal Junction of Mouse Skin.

AMACO (VWA2 protein), secreted by epithelial cells, is strongly expressed at basement membranes when budding or invagination occurs in embryos. In skin, AMACO associates with proteins of the Fraser complex, which form anchoring cords. These, during development, temporally stabilize the dermal-epidermal junction, pending the formation of collagen VII-containing anchoring fibrils. Fraser syndrome in humans results if any of the core members of the Fraser complex (Fras1, Frem1, Frem2) are mutated. Fraser syndrome is characterized by subepidermal blistering, cryptophthalmos, and syndactyly. In an attempt to determine AMACO function, we generated and characterized AMACO-deficient mice. In contrast to Fraser complex mutant mice, AMACO-deficient animals lack an obvious phenotype. The mutually interdependent basement membrane deposition of the Fraser complex proteins, and the formation of anchoring cords, are not affected. Furthermore, hair follicle development in newborn AMACO-deficient mice showed no gross aberration. Surprisingly, it appears that, while AMACO is a component of the anchoring cords, it is not essential for their formation or function.

Loss of Smad4 in the scleraxis cell lineage results in postnatal joint contracture.

Growth of the musculoskeletal system requires precise coordination between bone, muscle, and tendon during development. Insufficient elongation of the muscle-tendon unit relative to bone growth results in joint contracture, a condition characterized by reduction or complete loss of joint range of motion. Here we establish a novel murine model of joint contracture by targeting Smad4 for deletion in the tendon cell lineage using Scleraxis-Cre (ScxCre). Smad4ScxCre mutants develop a joint contracture shortly after birth. The contracture is stochastic in direction and increases in severity with age. Smad4ScxCre mutant tendons exhibited a stable reduction in cellularity and a progressive reduction in extracellular matrix volume. Collagen fibril diameters were reduced in the Smad4ScxCre mutants, suggesting a role for Smad4 signaling in the regulation of matrix accumulation. Although ScxCre also has sporadic activity in both cartilage and muscle, we demonstrate an essential role for Smad4 loss in tendons for the development of joint contractures. Disrupting the canonical TGFß-pathway in Smad2;3ScxCre mutants did not result in joint contractures. Conversely, disrupting the BMP pathway by targeting BMP receptors (Alk3ScxCre/Alk6null) recapitulated many features of the Smad4ScxCre contracture phenotype, suggesting that joint contracture in Smad4ScxCre mutants is caused by disruption of BMP signaling. Overall, these results establish a model of murine postnatal joint contracture and a role for BMP signaling in tendon elongation and extracellular matrix accumulation.

Type I and type V procollagen triple helix uses different subsets of the molecular ensemble for lysine posttranslational modifications in the rER.

Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.

Reversion-inducing cysteine-rich protein with Kazal motifs and MT1-MMP promote the formation of robust fibrillin fibers.

Fibrillins (FBNs) form mesh-like structures of microfibrils in various elastic tissues. RECK and FBN1 are co-expressed in many human tissues, suggesting a functional relationship. We found that dermal FBN1 fibers show atypical morphology in mice with reduced RECK expression (RECK-Hypo mice). Dermal FBN1 fibers in mice-lacking membrane-type 1-matrix metalloproteinase (MT1-MMP) show a similar atypical morphology, despite the current notion that MT1-MMP (a membrane-bound protease) and RECK (a membrane-bound protease inhibitor) have opposing functions. Our experiments using dermal fibroblasts indicated that RECK promotes pro-MT1-MMP activation, increases cell-associated gelatinase/collagenase activity, and decreases diffusible gelatinase/collagenase activity, while MT1-MMP stabilizes RECK in these cells. Experiments using purified proteins indicate that RECK and its binding partner ADAMTS10 keep the proteolytic activity of MT1-MMP within a certain range. These findings suggest that RECK, ADAMTS10, and MT1-MMP cooperate to support the formation of robust FBN1 fibers.

Surface-exposed loops and an acidic patch in the Scl1 protein of group A Streptococcus enable Scl1 binding to wound-associated fibronectin.

Keratinized epidermis constitutes a powerful barrier of the mucosa and skin, effectively preventing bacterial invasion, unless it is wounded and no longer protective. Wound healing involves deposition of distinct extracellular matrix (ECM) proteins enriched in cellular fibronectin (cFn) isoforms containing extra domain A (EDA). The streptococcal collagen-like protein 1 (Scl1) is a surface adhesin of group A Streptococcus (GAS), which contains an N-terminal variable (V) domain and a C-terminally located collagen-like domain. During wound infection, Scl1 selectively binds EDA/cFn isoforms and laminin, as well as low-density lipoprotein (LDL), through its V domain. The trimeric V domain has a six-helical bundle fold composed of three pairs of anti-parallel α-helices interconnected by hypervariable loops, but the roles of these structures in EDA/cFn binding are unclear. Here, using recombinant Scl (rScl) constructs to investigate structure-function determinants of the Scl1-EDA/cFn interaction, we found that full-length rScl1, containing both the globular V and the collagen domains, is necessary for EDA/cFn binding. We established that the surface-exposed loops, interconnecting conserved α-helices, guide recognition and binding of Scl1-V to EDA and binding to laminin and LDL. Moreover, electrostatic surface potential models of the Scl1-V domains pointed to a conserved, negatively charged pocket, surrounded by positively charged and neutral regions, as a determining factor for the binding. In light of these findings, we propose an updated model of EDA/cFn recognition by the Scl1 adhesin from GAS, representing a significant step in understanding the Scl1-ECM interactions within the wound microenvironment that underlie GAS pathogenesis.

Musculoskeletal integration at the wrist underlies the modular development of limb tendons.

The long tendons of the limb extend from muscles that reside in the zeugopod (arm/leg) to their skeletal insertions in the autopod (paw). How these connections are established along the length of the limb remains unknown. Here, we show that mouse limb tendons are formed in modular units that combine to form a functional contiguous structure; in muscle-less limbs, tendons develop in the autopod but do not extend into the zeugopod, and in the absence of limb cartilage the zeugopod segments of tendons develop despite the absence of tendons in the autopod. Analyses of cell lineage and proliferation indicate that distinct mechanisms govern the growth of autopod and zeugopod tendon segments. To elucidate the integration of these autopod and zeugopod developmental programs, we re-examined early tendon development. At E12.5, muscles extend across the full length of a very short zeugopod and connect through short anlagen of tendon progenitors at the presumptive wrist to their respective autopod tendon segment, thereby initiating musculoskeletal integration. Zeugopod tendon segments are subsequently generated by proximal elongation of the wrist tendon anlagen, in parallel with skeletal growth, underscoring the dependence of zeugopod tendon development on muscles for tendon anchoring. Moreover, a subset of extensor tendons initially form as fused structures due to initial attachment of their respective wrist tendon anlage to multiple muscles. Subsequent individuation of these tendons depends on muscle activity. These results establish an integrated model for limb tendon development that provides a framework for future analyses of tendon and musculoskeletal phenotypes.

Optimizing a 3D model system for molecular manipulation of tenogenesis.

PURPOSE: Tendon injuries are clinically challenging due to poor healing. A better understanding of the molecular events that regulate tendon differentiation would improve current strategies for repair. The mouse model system has been instrumental to tendon studies and several key molecules were initially established in mouse. However, the study of gene function has been limited by the absence of a standard in vitro tendon system for efficiently testing multiple mutations, physical manipulations, and mis-expression. The purpose of this study is therefore to establish such a system. METHODS: We adapted an existing design for generating three-dimensional (3D) tendon constructs for use with mouse progenitor cells harboring the ScxGFP tendon reporter and the Rosa26-TdTomato Cre reporter. Using these cells, we optimized the parameters for construct formation, inducing tenogenesis via transforming growth factor-ß2 (TGFß2), and genetic recombination via an adenovirus encoding Cre recombinase. Finally, for proof of concept, we used Smad4 floxed cells and tested the robustness of the system for gene knockdown. RESULTS: We found that TGFß2 treatment induced a tenogenic phenotype depending on the timing of initiation. Addition of TGFß2 after 3D "tensioning" enhanced tendon differentiation. Interestingly, while TGFß2-induced proliferation depended on Smad4, tenogenic parameters such as ScxGFP expression and fibril diameter were independent of Smad4. CONCLUSIONS: Our results demonstrate the feasibility of this optimized system for harnessing the power of mouse genetics for in vitro applications.

Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice.

Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

Molecular Mechanism Responsible for Fibronectin-controlled Alterations in Matrix Stiffness in Advanced Chronic Liver Fibrogenesis.

Fibrosis is characterized by extracellular matrix (ECM) remodeling and stiffening. However, the functional contribution of tissue stiffening to noncancer pathogenesis remains largely unknown. Fibronectin (Fn) is an ECM glycoprotein substantially expressed during tissue repair. Here we show in advanced chronic liver fibrogenesis using a mouse model lacking Fn that, unexpectedly, Fn-null livers lead to more extensive liver cirrhosis, which is accompanied by increased liver matrix stiffness and deteriorated hepatic functions. Furthermore, Fn-null livers exhibit more myofibroblast phenotypes and accumulate highly disorganized/diffuse collagenous ECM networks composed of thinner and significantly increased number of collagen fibrils during advanced chronic liver damage. Mechanistically, mutant livers show elevated local TGF-ß activity and lysyl oxidase expressions. A significant amount of active lysyl oxidase is released in Fn-null hepatic stellate cells in response to TGF-ß1 through canonical and noncanonical Smad such as PI3 kinase-mediated pathways. TGF-ß1-induced collagen fibril stiffness in Fn-null hepatic stellate cells is significantly higher compared with wild-type cells. Inhibition of lysyl oxidase significantly reduces collagen fibril stiffness, and treatment of Fn recovers collagen fibril stiffness to wild-type levels. Thus, our findings indicate an indispensable role for Fn in chronic liver fibrosis/cirrhosis in negatively regulating TGF-ß bioavailability, which in turn modulates ECM remodeling and stiffening and consequently preserves adult organ functions. Furthermore, this regulatory mechanism by Fn could be translated for a potential therapeutic target in a broader variety of chronic fibrotic diseases.

EMILIN3, an extracellular matrix molecule with restricted distribution in skin.

EMILIN3 is an extracellular matrix glycoprotein that displays a dynamic and restricted expression pattern in connective tissues during post-natal life. In this study, we report the characterization of EMILIN3 deposition in the skin. In addition, to unravel the functions of this protein in skin homeostasis, we generated Emilin3 null mice and provide evidence that EMILIN3 is dispensable for hair follicle growth and maintenance throughout adult life.

A trans-acting protein effect causes severe eye malformation in the Mp mouse.

Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene (Isoc1(Mp)) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene (Fbn2 (Mp)) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1-2646, exons 1-62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2(Mp) forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM - known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp "worse-than-null" eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing.

Safety and Wound Outcomes Following Genetically Corrected Autologous Epidermal Grafts in Patients With Recessive Dystrophic Epidermolysis Bullosa.

Importance: Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating, often fatal, inherited blistering disorder caused by mutations in the COL7A1 gene encoding type VII collagen. Support and palliation are the only current therapies. Objective: To evaluate the safety and wound outcomes following genetically corrected autologous epidermal grafts in patients with RDEB. Design, Setting, and Participants: Single-center phase 1 clinical trial conducted in the United States of 4 patients with severe RDEB with a measured area of wounds suitable for grafting of at least 100 cm2. Patients with undetectable type VII collagen keratinocyte expression were excluded. Interventions: Autologous keratinocytes isolated from biopsy samples collected from 4 patients with RDEB were transduced with good manufacturing practice-grade retrovirus carrying full-length human COL7A1 and assembled into epidermal sheet grafts. Type VII collagen gene-corrected grafts (approximately 35 cm2) were transplanted onto 6 wounds in each of the patients (n = 24 grafts). Main Outcomes and Measures: The primary safety outcomes were recombination competent retrovirus, cancer, and autoimmune reaction. Molecular correction was assessed as type VII collagen expression measured by immunofluorescence and immunoelectron microscopy. Wound healing was assessed using serial photographs taken at 3, 6, and 12 months after grafting. Results: The 4 patients (mean age, 23 years [range, 18-32 years]) were all male with an estimated body surface area affected with RDEB of 4% to 30%. All 24 grafts were well tolerated without serious adverse events. Type VII collagen expression at the dermal-epidermal junction was demonstrated on the graft sites by immunofluorescence microscopy in 9 of 10 biopsy samples (90%) at 3 months, in 8 of 12 samples (66%) at 6 months, and in 5 of 12 samples (42%) at 12 months, including correct type VII collagen localization to anchoring fibrils. Wounds with recombinant type VII collagen graft sites displayed 75% or greater healing at 3 months (21 intact graft sites of 24 wound sites; 87%), 6 months (16/24; 67%), and 12 months (12/24; 50%) compared with baseline wound sites. Conclusions and Relevance: In this preliminary study of 4 patients with RDEB, there was wound healing in some type VII collagen gene-corrected grafts, but the response was variable among patients and among grafted sites and generally declined over 1 year. Long-term follow-up is necessary for these patients, and controlled trials are needed with a broader range of patients to better understand the potential long-term efficacy of genetically corrected autologous epidermal grafts. Trial Registration: clinicaltrials.gov Identifier: NCT01263379.

Somatic correction of junctional epidermolysis bullosa by a highly recombinogenic AAV variant.

Definitive correction of disease causing mutations in somatic cells by homologous recombination (HR) is an attractive therapeutic approach for the treatment of genetic diseases. However, HR-based somatic gene therapy is limited by the low efficiency of gene targeting in mammalian cells and replicative senescence of primary cells ex vivo, forcing investigators to explore alternative strategies such as retro- and lentiviral gene transfer, or genome editing in induced pluripotent stem cells. Here, we report correction of mutations at the LAMA3 locus in primary keratinocytes derived from a patient affected by recessive inherited Herlitz junctional epidermolysis bullosa (H-JEB) disorder using recombinant adenoassociated virus (rAAV)-mediated HR. We identified a highly recombinogenic AAV serotype, AAV-DJ, that mediates efficient gene targeting in keratinocytes at clinically relevant frequencies with a low rate of random integration. Targeted H-JEB patient cells were selected based on restoration of adhesion phenotype, which eliminated the need for foreign sequences in repaired cells, enhancing the clinical use and safety profile of our approach. Corrected pools of primary cells assembled functional laminin-332 heterotrimer and fully reversed the blistering phenotype both in vitro and in skin grafts. The efficient targeting of the LAMA3 locus by AAV-DJ using phenotypic selection, together with the observed low frequency of off-target events, makes AAV-DJ based somatic cell targeting a promising strategy for ex vivo therapy for this severe and often lethal epithelial disorder.

Microenvironmental regulation by fibrillin-1.

Fibrillin-1 is a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes. A role for fibrillin-1 in specifying tissue microenvironments has not been elucidated, even though the concept that fibrillin-1 provides extracellular control of growth factor signaling is currently appreciated. Mutations in FBN1 are mainly responsible for the Marfan syndrome (MFS), recognized by its pleiotropic clinical features including tall stature and arachnodactyly, aortic dilatation and dissection, and ectopia lentis. Each of the many different mutations in FBN1 known to cause MFS must lead to similar clinical features through common mechanisms, proceeding principally through the activation of TGFß signaling. Here we show that a novel FBN1 mutation in a family with Weill-Marchesani syndrome (WMS) causes thick skin, short stature, and brachydactyly when replicated in mice. WMS mice confirm that this mutation does not cause MFS. The mutation deletes three domains in fibrillin-1, abolishing a binding site utilized by ADAMTSLIKE-2, -3, -6, and papilin. Our results place these ADAMTSLIKE proteins in a molecular pathway involving fibrillin-1 and ADAMTS-10. Investigations of microfibril ultrastructure in WMS humans and mice demonstrate that modulation of the fibrillin microfibril scaffold can influence local tissue microenvironments and link fibrillin-1 function to skin homeostasis and the regulation of dermal collagen production. Hence, pathogenetic mechanisms caused by dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFß signaling in multiple tissues. We conclude that local tissue-specific microenvironments, affected in WMS, are maintained by a fibrillin-1 microfibril scaffold, modulated by ADAMTSLIKE proteins in concert with ADAMTS enzymes.

A review of color blindness for microscopists: guidelines and tools for accommodating and coping with color vision deficiency.

"Color blindness" is a variable trait, including individuals with just slight color vision deficiency to those rare individuals with a complete lack of color perception. Approximately 75% of those with color impairment are green diminished; most of those remaining are red diminished. Red-Green color impairment is sex linked with the vast majority being male. The deficiency results in reds and greens being perceived as shades of yellow; therefore red-green images presented to the public will not illustrate regions of distinction to these individuals. Tools are available to authors wishing to accommodate those with color vision deficiency; most notable are components in FIJI (an extension of ImageJ) and Adobe Photoshop. Using these tools, hues of magenta may be substituted for red in red-green images resulting in striking definition for both the color sighted and color impaired. Web-based tools may be used (importantly) by color challenged individuals to convert red-green images archived in web-accessible journal articles into two-color images, which they may then discern.

Posttranslational modifications in type I collagen from different tissues extracted from wild type and prolyl 3-hydroxylase 1 null mice.

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.