Dino Volpin

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.

The ultrastructural organization of elastin

The ultrastructural organization of elastin has been studied by electron microscopy of negatively stained specimens. Fine suspensions of elastin from ligamentum nuchae were obtained by mechanical stirring or by sonication and subsequently were purified under carefully controlled procedures. In the presence of various negative stains, the elastin fibers were seen to be composed of a network of branched bundles possessing a wide range of diameters. High resolution micrographs revealed that the fibers consisted of slender filaments of about 30–40 A diameter arranged roughly parallel to the fiber long axis. A regular periodicity of about 40 A along the filaments was also resolved. The images from negatively stained specimens are discussed in relation to the structures seen in thin-sectioned material together with current views concerning the interpretation of the submicroscopic and molecular architecture of elastin.

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.

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.

Studies by electron microscopy on the structure of coacervates of synthetic polypeptides of tropoelastin

Abstract Studies by electron microscopy of negatively stained coacervates of the synthetic polypentapeptide and polyhexapeptide of tropoelastin are reported. Both coacervates are seen to be filamentous. When the micrographs were analyzed by means of optical diffraction, the coacervates gave a 5.0–5.5nm equatoral reflection which corresponds to the inter filament distance. Additional reflections were also observed which indicate the presence of filament sub-structure. The results are discussed in terms of solution conformations previously proposed for these polypenta- and polyhexapeptides and are compared with ultrastructural studies on native elastin.

Electron microscopy and optical diffraction of elastin

Abstract The ultrastructural organization of purified elastin has been studied with the aid of electron microscopy and optical diffraction. Elastin fibril bundles were observed to have a considerable range of diameters. The basic morphological component resolved in negatively stained preparations of elastin bundles was seen as a slender filament of undefined length arranged roughly parallel to the long fibre axis. The diameter of the filaments was 3–4nm with an average centre-to-centre distance of 5nm. A regular periodicity of about 4–4.5nm was resolved along the individual filaments. Evidence of ‘cross-bridging’ spaced at about 3–3.5nm along the filaments was also resolved in many electron micrographs. Analysis of the electron micrographs with the aid of optical diffraction showed spectra with an equatorial reflection of 5nm and weak near meridional spectra at 4nm. The electron microscope images when considered together with independent X-ray diffraction data, suggested a model for the elastin filament as being a rope-like structure of about 3.5–4.Onm dia and composed of paired sub-filaments of about 1.5nm dia. The proposed model also offered a possible explanation for the small changes observed in the axial periodicity along the elastin filaments, on the basis of their being in a stretched or relaxed state.

Optical diffraction of tropelastin and α-elastin coacervates

Optical diffraction applied to micrographs of coacervated tropoelastin and alpha-elastin show an equatorial repeat around 50 A. This confirms a 50 A center-to-center distance of parallel aligned filaments to be a fundamental property of the tropoelastin and alpha-elastin coacervates. This periodicity is similar to that of mature cross-linked elastin. These results allow the conclusion that hydrophobic association is the predominant driving force for formation of filamentous elastin in vitro. It is suggested that the coacervate is a model for relaxed fibrous elastin.