Giorgio M. Bressan

Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors.

Wnt/β-catenin signaling plays key roles in several developmental and pathological processes. Domains of Wnt expression have been extensively investigated in the mouse, but the tissues receiving the signal remain largely unidentified. To define which cells respond to activated β-catenin during mammalian development, we generated the β-catenin-activated transgene driving expression of nuclear β-galactosidase reporter (BAT-gal) transgenic mice, expressing the lacZ gene under the control of β-catenin/T cell factor responsive elements. Reporter gene activity is found in known organizing centers, such as the midhindbrain border and the limb apical ectodermal ridge. Moreover, BAT-gal expression identifies novel sites of Wnt signaling, like notochord, endothelia, and areas of the adult brain, revealing an unsuspected dynamic pattern of β-catenin transcriptional activity. Expression of the transgene was analyzed in mutant backgrounds. In lipoprotein receptor-related protein 6-null homozygous mice, which lack a Wnt coreceptor, BAT-gal staining is absent in mutant tissues, indicating that BAT-gal mice are bona fide in vivo indicators of Wnt/β-catenin signaling. Analyses of BAT-gal expression in the adenomatous polyposis coli (multiple intestinal neoplasia/+) background revealed βcatenin transcriptional activity in intestinal adenomas but surprisingly not in normal crypt cells. In summary, BAT-gal mice unveil the entire complexity of Wnt/β-catenin signaling in mammals and have broad application potentials for the identification of Wnt-responsive cell populations in development and disease.

Mitochondrial dysfunction and apoptosis in myopathic mice with collagen VI deficiency

Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. Inherited mutations in genes encoding collagen VI in humans cause two muscle diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy. We previously generated collagen VI–deficient (Col6a1−/−) mice and showed that they have a muscle phenotype that strongly resembles Bethlem myopathy. The pathophysiological defects and mechanisms leading to the myopathic disorder were not known. Here we show that Col6a1−/− muscles have a loss of contractile strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis. We found a latent mitochondrial dysfunction in myofibers of Col6a1−/− mice on incubation with the selective F1FO-ATPase inhibitor oligomycin, which caused mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they could be normalized by plating Col6a1−/− myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Treatment of Col6a1−/− mice with CsA rescued the muscle ultrastructural defects and markedly decreased the number of apoptotic nuclei in vivo. These findings indicate that collagen VI myopathies have an unexpected mitochondrial pathogenesis that could be exploited for therapeutic intervention.

Emilin1 links TGF-β maturation to blood pressure homeostasis

TGF-beta proteins are main regulators of blood vessel development and maintenance. Here, we report an unprecedented link between TGF-beta signaling and arterial hypertension based on the analysis of mice mutant for Emilin1, a cysteine-rich secreted glycoprotein expressed in the vascular tree. Emilin1 knockout animals display increased blood pressure, increased peripheral vascular resistance, and reduced vessel size. Mechanistically, we found that Emilin1 inhibits TGF-beta signaling by binding specifically to the proTGF-beta precursor and preventing its maturation by furin convertases in the extracellular space. In support of these findings, genetic inactivation of Emilin1 causes increased TGF-beta signaling in the vascular wall. Strikingly, high blood pressure observed in Emilin1 mutants is rescued to normal levels upon inactivation of a single TGF-beta1 allele. This study highlights the importance of modulation of TGF-beta availability in the pathogenesis of hypertension.

EMILIN-1 deficiency induces elastogenesis and vascular cell defects

ABSTRACT EMILINs constitute a family of genes of the extracellular matrix with high structural similarity. Four genes have been identified so far in human and mouse. To gain insight into the function of this gene family, EMILIN-1 has been inactivated in the mouse by gene targeting. The homozygous animals were fertile and did not show obvious abnormalities. However, histological and ultrastructural examination revealed alterations of elastic fibers in aorta and skin. Formation of elastic fibers by mutant embryonic fibroblasts in culture was also abnormal. Additional alterations were observed in cell morphology and anchorage of endothelial and smooth muscle cells to elastic lamellae. Considering that EMILIN-1 is adhesive for cells and that the protein binds to elastin and fibulin-5, EMILIN-1 may regulate elastogenesis and vascular cell maintenance by stabilizing molecular interactions between elastic fiber components and by endowing elastic fibers with specific cell adhesion properties.

Emilin1 Deficiency Causes Structural and Functional Defects of Lymphatic Vasculature

ABSTRACT Lymphatic-vasculature function critically depends on extracellular matrix (ECM) and on its connections with lymphatic endothelial cells (LECs). However, the composition and the architecture of ECM have not been fully taken into consideration in studying the biology and the pathology of the lymphatic system. EMILIN1, an elastic microfibril-associated protein, is highly expressed by LECs in vitro and colocalizes with lymphatic vessels in several mouse tissues. A comparative study between WT and Emilin1−/− mice highlighted the fact that Emilin1 deficiency in both CD1 and C57BL/6 backgrounds results in hyperplasia, enlargement, and frequently an irregular pattern of superficial and visceral lymphatic vessels and in a significant reduction of anchoring filaments. Emilin1-deficient mice also develop larger lymphangiomas than WT mice. Lymphatic vascular morphological alterations are accompanied by functional defects, such as mild lymphedema, a highly significant drop in lymph drainage, and enhanced lymph leakage. Our findings demonstrate that EMILIN1 is involved in the regulation of the growth and in the maintenance of the integrity of lymphatic vessels, a fundamental requirement for efficient function. The phenotype displayed by Emilin1−/− mice is the first abnormal lymphatic phenotype associated with the deficiency of an ECM protein and identifies EMILIN1 as a novel local regulator of lymphangiogenesis.

Relevance of aggregation properties of tropoelastin to the assembly and structure of elastic fibers

Solutions of tropoelastin incubated under different experimental conditions were examined by electron microscopy after negative staining and after fixation and embedding. Below 37 degrees C only polymorphous structureless elements of variable size could be found. In samples kept for a few minutes at 40 degrees C, flexible, isolated filaments of 5 nm diameter and variable length, together with a few small aggregates of filaments, were seen. No single filaments, but only bundles of filaments were detectable after incubation at 40 degrees C for longer than 5-10 min. Tropoelastin kept at 40 degrees C for longer than 10 hr formed a white precipitate, which, when fixed and embedded as in conventional electron microscopy, consisted of 0.5-2 microns thick, amorphous and branching fibers, identical to those seen in identically processed normal tissues. From these observations a model for the assembly and structure of elastic fibers is proposed.

Negative control of Smad activity by ectodermin/Tif1γ patterns the mammalian embryo

The definition of embryonic potency and induction of specific cell fates are intimately linked to the tight control over TGFβ signaling. Although extracellular regulation of ligand availability has received considerable attention in recent years, surprisingly little is known about the intracellular factors that negatively control Smad activity in mammalian tissues. By means of genetic ablation, we show that the Smad4 inhibitor ectodermin (Ecto, also known as Trim33 or Tif1γ) is required to limit Nodal responsiveness in vivo. New phenotypes, which are linked to excessive Nodal activity, emerge from such a modified landscape of Smad responsiveness in both embryonic and extra-embryonic territories. In extra-embryonic endoderm, Ecto is required to confine expression of Nodal antagonists to the anterior visceral endoderm. In trophoblast cells, Ecto precisely doses Nodal activity, balancing stem cell self-renewal and differentiation. Epiblast-specific Ecto deficiency shifts mesoderm fates towards node/organizer fates, revealing the requirement of Smad inhibition for the precise allocation of cells along the primitive streak. This study unveils that intracellular negative control of Smad function by ectodermin/Tif1γ is a crucial element in the cellular response to TGFβ signals in mammalian tissues.

Banded fibers in tropoelastin coacervates at physiological temperatures.

Tropoelastin was purified from aortas of chicks grown on a beta-aminopropionitrile-containing diet. The preparation could be considered pure following the criteria of amino acid composition and gel electrophoresis. When aqueous solutions of tropoelastin (5 mg/ml) were warmed to 40 degrees C (physiological temperature for chicken) for 10 min, and observed by negative-staining electron microscopy, it revealed the presence of two kinds of ordered structures. One consisted of densely packed parallel filaments with a center-to-center distance of about 5 nm, and the other of banded fibers, 100-150 nm in diameter, with a cross periodicity of about 55 nm. In some areas the fibers appeared to be formed by lateral aggregation of 1.5-2-nm-thick microfilaments. The fibers were similar to those previously obtained with the synthetic polypentapeptide of elastin (Val-Pro-Gly-Val-Gly)n and degradation products of elastin at temperatures much higher than the physiological one. The results indicate that the property of tropoelastin to form ordered structures is intrinsic to some of the polypeptide sequences of the molecule and that hydrophobic forces are involved in the formation of the aggregates.

The EMILIN/Multimerin Family

Elastin microfibrillar interface proteins (EMILINs) and Multimerins (EMILIN1, EMILIN2, Multimerin1, and Multimerin2) constitute a four member family that in addition to the shared C-terminus gC1q domain typical of the gC1q/TNF superfamily members contain a N-terminus unique cysteine-rich EMI domain. These glycoproteins are homotrimeric and assemble into high molecular weight multimers. They are predominantly expressed in the extracellular matrix and contribute to several cellular functions in part associated with the gC1q domain and in part not yet assigned nor linked to other specific regions of the sequence. Among the latter is the control of arterial blood pressure, the inhibition of Bacillus anthracis cell cytotoxicity, the promotion of cell death, the proangiogenic function, and a role in platelet hemostasis. The focus of this review is to highlight the multiplicity of functions and domains of the EMILIN/Multimerin family with a particular emphasis on the regulatory role played by the ligand–receptor interactions of the gC1q domain. EMILIN1 is the most extensively studied member both from the structural and functional point of view. The structure of the gC1q of EMILIN1 solved by NMR highlights unique characteristics compared to other gC1q domains: it shows a marked decrease of the contact surface of the trimeric assembly and while conserving the jelly-roll topology with two β-sheets of antiparallel strands it presents a nine-stranded β-sandwich fold instead of the usual 10-stranded fold. This is likely due to the insertion of nine residues that disrupt the ordered strand organization and forma a highly dynamic protruding loop. In this loop the residue E933 is the site of interaction between gC1q and the α4β1 and α9β1 integrins, and contrary to integrin occupancy that usually upregulates cell growth, when gC1q is ligated by the integrin the cells reduce their proliferative activity.

Spatial and temporal changes of typeVI collagen expression during mouse development

The expression of type VI collagen has been studied in mouse tissues. By Northern blotting, the mRNA for the α1(VI) chain was detectable in whole embryos at 10.5 days postcoitum and steeply increased afterward. The messenger levels were high at birth, but decreased rapidly in the following days, reaching low levels in adult animals. In 2‐month‐old mice, lung, skin, adrenal gland, heart, skeletal muscle and tail and fat were among the most active producers of α1(VI) mRNA. In situ hybridization first identified mRNA for α1(VI) collagen in mesenchymal cells of 10.5‐day embryos in various locations, including serosae, branchial arches, large blood vessels and the cephalic mesenchyme. Staining increased at later stages of development and most connective tissues were positive at 16.5 days and later. Strongly staining tissues were joints, intervertebral disks, perichondrium, periostium, dermis, skeletal muscle and heart valves, whereas cartilage and bone were very poorly labelled. Epithelia and the central nervous system were completely negative. In several organs, notably lung, salivary glands and the digestive tract, staining was concentrated underneath epithelia. This staining pattern was different from that for collagen type I, which was evenly distributed in the subepithelial mesenchyme. The pattern of distribution of the protein, revealed by immunocytochemistry, was coincident with that of the α1(VI) mRNA. In addition, the results confirmed that type VI collagen is preferentially deposited in the pericellular environment. This was particularly evident in skeletal muscle. The data show that type VI collagen is mainly produced by mesenchymal cells and suggest a role for the protein in delineating the boundary of distinct domains in connective tissue.