Nicola Facchinello

The effects of Ankrd2 alteration indicate its involvement in cell cycle regulation during muscle differentiation

Ankrd2 is a member of the Muscle Ankyrin Repeat Protein family (MARPs), consisting of sarcomere-associated proteins that can also localize in the nucleus. There are indications that MARPs might function as shuttle proteins between the cytoplasm and nucleus, likely sending information to the nucleus concerning the changes in the structure or function of the contractile machinery. Even though recent findings suggest that the MARP gene family is not essential for the basal functioning of skeletal muscle, its influence on the gene expression program of skeletal muscle cells was highlighted. To investigate this regulatory role we produced and examined both morphological and functional features of myocytes stable overexpressing or silencing the Ankrd2 protein. The transcriptional profiles of the myocytes revealed that the molecular pathways perturbed by changes in Ankrd2 protein level are congruent with the morpho-physiological and biochemical data obtained in Ankrd2-modified myoblasts induced to differentiate. Our results suggest that Ankrd2 gives an important contribution to the coordination of proliferation and apoptosis during myogenic differentiation in vitro, mainly through the p53 network.

Vascular Smooth Muscle Emilin-1 Is a Regulator of Arteriolar Myogenic Response and Blood Pressure

Objective—Emilin-1 is a protein of elastic extracellular matrix involved in blood pressure (BP) control by negatively affecting transforming growth factor (TGF)-&bgr; processing. Emilin1 null mice are hypertensive. This study investigates how Emilin-1 deals with vascular mechanisms regulating BP. Methods and Results—This study uses a phenotype rescue approach in which Emilin-1 is expressed in either endothelial cells or vascular smooth muscle cells of transgenic animals with the Emilin1−/− background. We found that normalization of BP required Emilin-1 expression in smooth muscle cells, whereas expression of the protein in endothelial cells did not modify the hypertensive phenotype of Emilin1−/− mice. We also explored the effect of treatment with anti-TGF-&bgr; antibodies on the hypertensive phenotype of Emilin1−/− mice, finding that neutralization of TGF-&bgr; in Emilin1 null mice normalized BP quite rapidly (2 weeks). Finally, we evaluated the vasoconstriction response of resistance arteries to perfusion pressure and neurohumoral agents in different transgenic mouse lines. Interestingly, we found that the hypertensive phenotype was coupled with an increased arteriolar myogenic response to perfusion pressure, while the vasoconstriction induced by neurohumoral agents remained unaffected. We further elucidate that, as for the hypertensive phenotype, the increased myogenic response was attributable to increased TGF-&bgr; activity. Conclusion—Our findings clarify that Emilin-1 produced by vascular smooth muscle cells acts as a main regulator of resting BP levels by controlling the myogenic response in resistance arteries through TGF-&bgr;.

A Smad3 transgenic reporter reveals TGF-beta control of zebrafish spinal cord development.

TGF-beta (TGFβ) family mediated Smad signaling is involved in mesoderm and endoderm specifications, left-right asymmetry formation and neural tube development. The TGFβ1/2/3 and Activin/Nodal signal transduction cascades culminate with activation of SMAD2 and/or SMAD3 transcription factors and their overactivation are involved in different pathologies with an inflammatory and/or uncontrolled cell proliferation basis, such as cancer and fibrosis. We have developed a transgenic zebrafish reporter line responsive to Smad3 activity. Through chemical, genetic and molecular approaches we have seen that this transgenic line consistently reproduces in vivo Smad3-mediated TGFβ signaling. Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. In the CNS, Smad3 reporter activity is particularly high in the subpallium, tegumentum, cerebellar plate, medulla oblongata and the retina proliferative zone. In the spinal cord, the reporter is activated at the ventricular zone, where neuronal progenitor cells are located. Colocalization methods show in vivo that TGFβ signaling is particularly active in neuroD+ precursors. Using neuronal transgenic lines, we observed that TGFβ chemical inhibition leads to a decrease of differentiating cells and an increase of proliferation. Similarly, smad3a and 3b knock-down alter neural differentiation showing that both paralogues play a positive role in neural differentiation. EdU proliferation assay and pH3 staining confirmed that Smad3 is mainly active in post-mitotic, non-proliferating cells. In summary, we demonstrate that the Smad3 reporter line allows us to follow in vivo Smad3 transcriptional activity and that Smad3, by controlling neural differentiation, promotes the progenitor to precursor switch allowing neural progenitors to exit cell cycle and differentiate.

Down-regulation of coasy, the gene associated with NBIA-VI, reduces Bmp signaling, perturbs dorso-ventral patterning and alters neuronal development in zebrafish

Mutations in Pantothenate kinase 2 and Coenzyme A (CoA) synthase (COASY), genes involved in CoA biosynthesis, are associated with rare neurodegenerative disorders with brain iron accumulation. We showed that zebrafish pank2 gene plays an essential role in brain and vasculature development. Now we extended our study to coasy. The gene has high level of sequence identity with the human ortholog and is ubiquitously expressed from the earliest stages of development. The abrogation of its expression led to strong reduction of CoA content, high lethality and a phenotype resembling to that of dorsalized mutants. Lower doses of morpholino resulted in a milder phenotype, with evident perturbation in neurogenesis and formation of vascular arborization; the dorso-ventral patterning was severely affected, the expression of bone morphogenetic protein (Bmp) receptors and activity were decreased, while cell death increased. These features specifically correlated with the block in CoA biosynthesis and were rescued by the addition of CoA to fish water and the overexpression of the human wild-type, but not mutant gene. These results confirm the absolute requirement for adequate levels of CoA for proper neural and vascular development in zebrafish and point to the Bmp pathway as a possible molecular connection underlining the observed phenotype.

Lentiviral-mediated RNAi in vivo silencing of Col6a1, a gene with complex tissue specific expression pattern

RNA interference (RNAi) through the use of lentiviral vectors is a valuable technique to induce loss of function mutations in mammals. Although very promising, the method has found only limited application and its general applicability remains to be established. Here we analyze how different factors influence RNAi mediated silencing of Col6a1, a gene of the extracellular matrix with a complex pattern of tissue specific expression. Our results, obtained with vectors pLVTHM and pLVPT-rtTRKRAB, point out three parameters as major determinants of the efficiency of interference: the choice of interfering sequence, the number of proviral copies integrated into the mouse genome and the site of insertion of the provirus. Although low copy number may produce efficient interference with low frequency, the general trend is that the number of integrated proviral copies determines the level of silencing and the severity of phenotypic traits. The site of insertion not only determines the overall intensity of expression of the small interfering RNA (siRNA), but also introduces slight variability of silencing in different organs. A lentiviral vector (pLVPT-rtTRKRAB) with doxycycline-inducible production of siRNA was also tested. Control of expression by the drug was stringent in many tissues; however, in some tissues turning off of siRNA synthesis was not complete. The data support the application of lentiviral vectors used here in transgenesis.

Treponema pallidum (syphilis) antigen TpF1 induces angiogenesis through the activation of the IL-8 pathway

Over 10 million people every year become infected by Treponema pallidum and develop syphilis, a disease with broad symptomatology that, due to the difficulty to eradicate the pathogen from the highly vascularized secondary sites of infection, is still treated with injections of penicillin. Unlike most other bacterial pathogens, T. pallidum infection produces indeed a strong angiogenic response whose mechanism of activation, however, remains unknown. Here, we report that one of the major antigen of T. pallidum, the TpF1 protein, has growth factor-like activity on primary cultures of human endothelial cells and activates specific T cells able to promote tissue factor production. The growth factor-like activity is mediated by the secretion of IL-8 but not of VEGF, two known angiogenic factors. The pathogen’s factor signals IL-8 secretion through the activation of the CREB/NF-κB signalling pathway. These findings are recapitulated in an animal model, zebrafish, where we observed that TpF1 injection stimulates angiogenesis and IL-8, but not VEGF, secretion. This study suggests that the angiogenic response observed during secondary syphilis is triggered by TpF1 and that pharmacological therapies directed to inhibit IL-8 response in patients should be explored to treat this disease.

nr3c1 null mutant zebrafish are viable and reveal DNA-binding-independent activities of the glucocorticoid receptor

Glucocorticoids (GCs) play important roles in developmental and physiological processes through the transcriptional activity of their cognate receptor (Gr). Using CRISPR/Cas9 technology, we established a zebrafish null Gr mutant line and compared its phenotypes with wild type and a zebrafish line with partially silenced gr (grs357/s357). Homozygous gr−/− larvae are morphologically inconspicuous and, in contrast to GR−/− knockout mice, viable through adulthood, although with reduced fitness and early life survival. Mutants gr−/− are fertile, but their reproductive capabilities fall at around 10 months of age, when, together with cardiac and intestinal abnormalities already visible at earlier stages, increased fat deposits are also observed. Mutants show higher levels of whole-body cortisol associated with overstimulated basal levels of crh and pomca transcripts along the HPI axis, which is unresponsive to a mechanical stressor. Transcriptional activity linked to immune response is also hampered in the gr−/− line: after intestinal damage by dextran sodium sulphate exposure, there are neither inflammatory nor anti-inflammatory cytokine gene responses, substantiating the hypothesis of a dual-action of the GC-GR complex on the immune system. Hence, the zebrafish gr mutant line appears as a useful tool to investigate Gr functions in an integrated in vivo model.

Monitoring Wnt Signaling in Zebrafish Using Fluorescent Biosensors

In this chapter, we are presenting methods to monitor and quantify in vivo canonical Wnt signaling activities at single-cell resolution in zebrafish. Our technology is based on artificial enhancers, obtained by polymerization of TCF binding elements, cloned upstream to ubiquitous or tissue-specific promoters. The different promoter/enhancer combinations are used to drive fluorescent protein reporter constructs integrated in the zebrafish germline by microinjection of fertilized zebrafish eggs. Fish with a single integration site are selected by Mendelian analysis of fluorescent carriers, and heterozygous offspring are used to monitor and quantify canonical Wnt activities. Open source public domain software such as ImageJ/Fiji is used to calculate the integrated densities in the region of interest and compare the effect of experimental conditions on control and treated animals.

Tcf7l2 plays pleiotropic roles in the control of glucose homeostasis, pancreas morphology, vascularization and regeneration

Type 2 diabetes (T2D) is a disease characterized by impaired insulin secretion. The Wnt signaling transcription factor Tcf7l2 is to date the T2D-associated gene with the largest effect on disease susceptibility. However, the mechanisms by which TCF7L2 variants affect insulin release from β-cells are not yet fully understood. By taking advantage of a tcf7l2 zebrafish mutant line, we first show that these animals are characterized by hyperglycemia and impaired islet development. Moreover, we demonstrate that the zebrafish tcf7l2 gene is highly expressed in the exocrine pancreas, suggesting potential bystander effects on β-cell growth, differentiation and regeneration. Finally, we describe a peculiar vascular phenotype in tcf7l2 mutant larvae, characterized by significant reduction in the average number and diameter of pancreatic islet capillaries. Overall, the zebrafish Tcf7l2 mutant, characterized by hyperglycemia, pancreatic and vascular defects, and reduced regeneration proves to be a suitable model to study the mechanism of action and the pleiotropic effects of Tcf7l2, the most relevant T2D GWAS hit in human populations.

Glucocorticoids promote Von Hippel Lindau degradation and Hif-1α stabilization

Significance An in vivo chemical screen in zebrafish identified glucocorticoids (GCs) as activators of hypoxia-inducible factor transcriptional responses in the liver. This cross-talk is conserved in human liver and requires glucocorticoid receptor signaling but not DNA binding. In human liver cells, GCs down-regulate Von Hippel Lindau expression at a posttranscriptional level most likely through c-src–mediated proteasomal degradation. Since the liver is an important regulator of blood glucose and hypoxia-inducible factors regulate gluconeogenesis/glycogen synthesis, cross-talk between these transcriptional regulators may be essential to control glucose metabolism in the liver. This identified, conserved, noncanonical pathway may have wider physiological significance in health and disease. Glucocorticoid (GC) and hypoxic transcriptional responses play a central role in tissue homeostasis and regulate the cellular response to stress and inflammation, highlighting the potential for cross-talk between these two signaling pathways. We present results from an unbiased in vivo chemical screen in zebrafish that identifies GCs as activators of hypoxia-inducible factors (HIFs) in the liver. GCs activated consensus hypoxia response element (HRE) reporters in a glucocorticoid receptor (GR)-dependent manner. Importantly, GCs activated HIF transcriptional responses in a zebrafish mutant line harboring a point mutation in the GR DNA-binding domain, suggesting a nontranscriptional route for GR to activate HIF signaling. We noted that GCs increase the transcription of several key regulators of glucose metabolism that contain HREs, suggesting a role for GC/HIF cross-talk in regulating glucose homeostasis. Importantly, we show that GCs stabilize HIF protein in intact human liver tissue and isolated hepatocytes. We find that GCs limit the expression of Von Hippel Lindau protein (pVHL), a negative regulator of HIF, and that treatment with the c-src inhibitor PP2 rescued this effect, suggesting a role for GCs in promoting c-src–mediated proteosomal degradation of pVHL. Our data support a model for GCs to stabilize HIF through activation of c-src and subsequent destabilization of pVHL.