Franco Cotelli | Università degli Studi di Milano - State University of Milan (Italy) (original) (raw)

Papers by Franco Cotelli

The zebrafish (Danio rerio)/tumor xenograft model represents a powerful new model system in cance... more The zebrafish (Danio rerio)/tumor xenograft model represents a powerful new model system in cancer. Here, we describe a novel exploitation of the zebrafish model to investigate tumor angiogenesis, a pivotal step in cancer progression and target for antitumor therapies. Human and murine tumor cell lines that express the angiogenic fibroblast growth factor (FGF) 2 and/or vascular endothelial growth factor (VEGF) induce the rapid formation of a new microvasculature when grafted close to the developing subintestinal vessels of zebrafish embryos at 48 h postfertilization. Instead, no angiogenic response was exerted by related cell clones defective in the production of these angiogenic growth factors. The newly formed blood vessels sprout from the subintestinal plexus of the zebrafish embryo, penetrate the tumor graft, and express the transcripts for the zebrafish orthologues of the early endothelial markers Fli-1, VEGF receptor-2 (VEGFR2/KDR), and VE-cadherin. Accordingly, green fluorescent protein-positive neovessels infiltrate the graft when tumor cells are injected in transgenic VEGFR2:G-RCFP zebrafish embryos that express green fluorescent protein under the control of the VEGFR2/KDR promoter. Systemic exposure of zebrafish embryos immediately after tumor cell injection to prototypic antiangiogenic inhibitors, including the FGF receptor tyrosine kinase inhibitor SU5402 and the VEGFR2/KDR tyrosine kinase inhibitor SU5416, suppresses tumor-induced angiogenesis without affecting normal blood vessel development. Accordingly, VE-cadherin gene inactivation by antisense morpholino oligonucleotide injection inhibits tumor neovascularization without affecting the development of intersegmental and subintestinal vessels. These data show that the zebrafish/ tumor xenograft model represents a novel tool for investigating the neovascularization process exploitable for drug discovery and gene targeting in tumor angiogenesis.

Cancer Research, 2007

Mechanisms of Development, 1999

We have cloned three sox genes in zebrafish (Danio rerio), one related to human and chicken SOX21... more We have cloned three sox genes in zebrafish (Danio rerio), one related to human and chicken SOX21, and two related to mammalian and chicken Sox-11. Zebrafish sox21, sox11A and sox11B transcripts are accumulated in the egg, are present in all cells until gastrulation and become restricted later to the developing central nervous system (CNS); expression in adults is undetectable. sox21 is expressed in the forebrain, midbrain and hindbrain, but maximally at the midbrain-hindbrain junction; sox11A,B have a widespread and dynamic expression in the CNS, but in contrast to sox21 are absent at the midbrain-hindbrain boundary.

Mechanisms of Development, 2004

Sox proteins are DNA-binding proteins belonging to the HMG box superfamily and they play key role... more Sox proteins are DNA-binding proteins belonging to the HMG box superfamily and they play key roles in animal embryonic development. Zebrafish Sox21a is part of group B Sox proteins and its chicken and mouse orthologs have been described as transcriptional repressor and activator, respectively, in two different target gene contexts. Zebrafish sox21a is present as a maternal transcript in the oocyte and is mainly expressed at the developing midbrain -hindbrain boundary from the onset of neurulation. In order to understand its role in vivo, we ectopically expressed sox21a by microinjection. Ectopic expression of full length sox21a leads to dorsalization of the embryos. A subset of the dorsalized embryos shows a partial axis splitting, and hence an ectopic neural tube, as an additional phenotype. At gastrulation, injected embryos show expansion of the expression domains of organizer-specific genes, such as chordin and goosecoid. Molecular markers used in somitogenesis highlight that sox21a-injected embryos have shortened AP axis, undulating axial structures, enlarged or even radialized paraxial territory. The developmental abnormalities caused by ectopic expression of sox21a are suggestive of defects in convergenceextension morphogenetic movements. Antisense morpholino oligonucleotides, designed to functionally knockdown sox21a, cause ventralization of the embryos. Moreover, gain-of-function experiments with chimeric constructs, where Sox21a DNA-binding domain is fused to a transcriptional activator (VP16) or repressor (EnR) domain, suggests that zebrafish Sox21a acts as a repressor in dorso-ventral patterning. q

Molecular Reproduction and Development, 1998

Using degenerate reverse transcription polymerase chain reaction (RT-PCR) techniques we have isol... more Using degenerate reverse transcription polymerase chain reaction (RT-PCR) techniques we have isolated a cDNA encoding a putative component of the zebrafish Danio rerio egg chorion, homologous to the mammalian ZP3 (ZPC). The predicted protein (zfZPC) has a calculated molecular mass of 58.4 kDa and contains a signal peptide (located in the N-terminal region) composed of 11 hydrophobic amino acid residues followed by a signal peptide cleavage site. The zfZPC contains the ZP domain, a characteristic amino acid sequence shared by all ZP proteins of the mammalian zona pellucida and of both amphibian and bird egg envelope components. The zfZPC also exhibits certain unique features including five N-terminal Q-rich tandem repeats presumably involved in the hardening of the chorion after the fertilization of the egg and a long C-terminal tail containing two potential sites of N-linked type glycosylation. RT-PCR and in situ hybridization revealed a restricted pattern of tissue distribution: the gene encoding zfZPC is transcribed only in the growing oocyte of sexually mature female fish.

Development Genes and Evolution, 2000

BMC Developmental Biology, 2008

Background: Prox1, the vertebrate homolog of prospero in Drosophila melanogaster, is a divergent ... more Background: Prox1, the vertebrate homolog of prospero in Drosophila melanogaster, is a divergent homeogene that regulates cell proliferation, fate determination and differentiation during vertebrate embryonic development.

Journal of Molecular Evolution, 1991

The study of the structural organization of the eukaryotic genome is one of the most important to... more The study of the structural organization of the eukaryotic genome is one of the most important tools for disclosing the evolutionary relationships between species.Artemia (Crustacea, Phyllopoda) offers a very interesting model for speciation studies. The genus, distributed all over the world, comprises both bisexual sibling species and parthenogenetic populations, exhibiting different chromosome numbers (diploidy, polyploidy, and heteroploidy). Digestion of genomic DNA of the parthenogeneticArtemia sp. from Tsing-Tao (China) with the restriction enzymes Eco RI and Alu I reveals that a highly repetitive sequence of 133 bp is present. The Eco RI fragment has been cloned and characterized by genomic organization. The distribution of the Eco RI family of repeats was also studied in several bisexual and parthenogeneticArtemia populations and compared with an Alu I repetitive fragment previously identified inArtemia franciscana.

Zygote, 1996

The chorion is the acellular envelope surrounding mature eggs of teleostean fish. The macromolecu... more The chorion is the acellular envelope surrounding mature eggs of teleostean fish. The macromolecular composition of the zebrafish (Danio rerio) egg chorion, organised as a three-layered structure, has been analysed. SDS-PAGE analysis, under reducing conditions, of isolated and purified chorions revealed a reproducible pattern of four major polypeptides (116, 97, 50 and 43 kDa) and several minor bands. Lectin binding assays showed that both the 116 kDa and 50 kDa proteins were recognised by concanavalin agglutinin (Con A), Galanthus nivalis agglutinin (GNA), Sambucus nigra bark agglutinin (SNA) and Ricinus communis agglutinin (RCA 120), suggesting that these polypeptides are N-linked glycoproteins. By contrast, neither the 97 kDa nor the 43 kDa polypeptides were stained by these lectins, indicating that these polypeptides are not glycosylated. Amino acid analysis also showed significant differences in the average content of some amino acids, for example serine and proline, when compared with previous reports.

Molecular Reproduction and Development, 1991

The extracellular coat surrounding the fish egg, commonly called the chorion, is a primary envelo... more The extracellular coat surrounding the fish egg, commonly called the chorion, is a primary envelope that confers biochemical and morphological identity typical of the species. Purified chorions can be easily isolated from either oocytes or ovulated eggs. The aim of this work was to analyze the macromolecular composition of the various chorion components in Oncorhynchus mykiss (Salmonids). SDS-PAGE analysis of purified chorion showed a reproducible pattern of four major components (129, 62, 54, and 47 kD), representing about 80% of total chorion proteins. The 129 and 47 kD polypeptides were periodic-acid Schiff (PAS) and concanavalin A positive. After chemical and enzymatic deglycosylation treatments only the 129 and 47 kD components proved to be glycosylated and to belong to the “asparagine-linked” glycoprotein family. Furthermore, peptide mapping performed on isolated polypeptides showed comigrating fragments on SDS-PAGE. These results suggest that the four main chorion polypeptides might share common structural features.

Molecular Reproduction and Development, 1993

Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically ... more Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area.Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei.Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.

Development Growth & Differentiation, 1980

The process of acrosome formation in the course of spermatogenesis of Ciom intestinalis has been ... more The process of acrosome formation in the course of spermatogenesis of Ciom intestinalis has been investigated.

Journal of Ultrastructure Research, 1980

Proceedings of The National Academy of Sciences, 1982

The sperm receptors of the ascidian oocyte are located at the outer surface of the vitelline coat... more The sperm receptors of the ascidian oocyte are located at the outer surface of the vitelline coat (formerly called the chorion). The fucose residues are the receptor's most important components for sperm recognition and binding. We asked whether the fucosyl-containing glycoproteins of the vitelline coat are a product ofthe oocyte, the follicle cells, or the test cells. Ovaries of Ciona intestinalis were injected with L-[3H]fucose and the progress of its incorporation was followed by using autoradiography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the injected gonads and of the isolated vitelline coats. We found that incorporation of fucose begins within the vitellogenic oocytes, and fucose slowly accumulates in the differentiating vitelline coat. At no time could fucose incorporation be detected in the follicle cells or in the test cells. Sodium dodecyl sulfate/

Journal of Ultrastructure and Molecular Structure Research, 1986

Actin was identified in boar and mole spermatozoa by utilizing indirect immunofluorescence, immun... more Actin was identified in boar and mole spermatozoa by utilizing indirect immunofluorescence, immunoelectron microscopy, and SDS-PAGE, followed by blot and screening with an anti-actin monoclonal antibody. Actin was detected in two places in the sperm head: the equatorial segment of the acrosome and the postacrosomal region. The protein was present in a nonfilamentous form and was localized under the plasma membrane. A small amount of actin was also detected in the sperm tail. The function of actin in the sperm head is discussed. o 1986 Academic PESS, IX.

Journal of Ultrastructure and Molecular Structure Research, 1988

The mature egg of fishes is surrounded by an acellular coat conventionally called the chorion. In... more The mature egg of fishes is surrounded by an acellular coat conventionally called the chorion. In Carassius auratus three main layers can be recognized at the electron microscopic level, and we have termed them chl, ch2, and ch3 starting from the outer side to the inner side close to the egg plasma membrane. Isolated chorion from mature ovarian and ovulated eggs can be obtained by homogenization and further purification techniques. We analyzed these preparations on sodium dodecyl sulfate-palyacrylamide gel electrophoresis, and at least 20 polypeptide chains were reproducibly observed. The solubilization conditions were tested by means of various agents under reducing and nonreducing conditions. The present report focuses on the temporal and spatial pathway of chorion formation during oocyte growth, and we report the solubilization and partial characterization of protein and glycoprotein components of purified chorions.

Development Genes and Evolution, 1981

We have studied the differentiation of the vitelline coat (VC) of the ascidianCiona intestinalis.... more We have studied the differentiation of the vitelline coat (VC) of the ascidianCiona intestinalis. In the young previtellogenic oocyte the vitelline coat precursor material (VCPM) makes its first appearance as patches of fibrous material in close apposition to the outer surface of the oocyte. The presence of subcortical vescicles containing a fuzzy electron-dense material and their opening into the oocyte surface parallels the formation of VCPM. Numerous microvillar-like structures emerge from the oocyte surface. When the VCPM completely surrounds the oocyte the microvilli are withdrawn. An overall increase of VCPM parallels the growth of the oocyte. The next step in the differentiation of the vitelline coat consists in the packing of the constituent fibrils in a dense layer at its outer surface, i.e. the one in contact with the follicle cells. At this time the VC is penetrated by microvilli protruding both from the oocyte and follicle cells. The VC reaches its final structure and thickness at the time the test cells are extruded into the perivitelline space. The participation of the follicle cells in VC organization is also discussed.