Franco Cotelli

Mammalian Tumor Xenografts Induce Neovascularization in Zebrafish Embryos

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.

Retinoic acid induces stage-specific antero-posterior transformation of rostral central nervous system

We report a time-course analysis of the effect of retinoic acid (RA) on the development of the mouse central nervous system (CNS) from the beginning of gastrulation throughout induction and patterning of the neural tube. RA administration induces three different, stage-specific alterations of brain development, indicating perturbation of different morphogenetic steps during the establishment of a neural pattern. In particular, treatment at mid-late streak stage (7.2-7.4 days post coitum (d.p.c.)) results in early repression of Otx2 expression in the posterior neuroectoderm of the head fold and in the ventral mid line, including the prechordal plate and the rostralmost endoderm, followed by loss of forebrain morphological and molecular identities, as revealed by analysis of the expression of regionally-restricted brain genes (Otx2, Otx1, Emx2, Emx1 and Dlx1). In these embryos, reduction of the Otx2 expression domain correlates with hindbrain expansion marked by rostral extension of the Hoxb-1 expression domain. Our analysis indicates that RA interferes with the correct definition of both planar and vertical morphogenetic signals at specific developmental stages by affecting gene expression in the regions which are likely either to produce or to respond to these signals. We suggest that retinoids may contribute to early definition of head from trunk structures by selecting different sets of regulatory genes.

Role of delta-like-4/notch in the formation and wiring of the lymphatic network in zebrafish

Objective—To study whether Notch signaling, which regulates cell fate decisions and vessel morphogenesis, controls lymphatic development. Methods and Results—In zebrafish embryos, sprouts from the axial vein have lymphangiogenic potential because they give rise to the first lymphatics. Knockdown of delta-like-4 (Dll4) or its receptors Notch-1b or Notch-6 in zebrafish impaired lymphangiogenesis. Dll4/Notch silencing reduced the number of sprouts producing the string of parchordal lymphangioblasts; instead, sprouts connecting to the intersomitic vessels were formed. At a later phase, Notch silencing impaired navigation of lymphatic intersomitic vessels along their arterial templates. Conclusion—These studies imply critical roles for Notch signaling in the formation and wiring of the lymphatic network.

Stable Vascular Connections and Remodeling Require Full Expression of VE-Cadherin in Zebrafish Embryos

Background VE-cadherin is an endothelial specific, transmembrane protein, that clusters at adherens junctions where it promotes homotypic cell-cell adhesion. VE-cadherin null mutation in the mouse results in early fetal lethality due to altered vascular development. However, the mechanism of action of VE-cadherin is complex and, in the mouse embryo, it is difficult to define the specific steps of vascular development in which this protein is involved. Methodology and Principal Findings In order to study the role VE-cadherin in the development of the vascular system in a more suitable model, we knocked down the expression of the coding gene in zebrafish. The novel findings reported here are: 1) partial reduction of VE-cadherin expression using low doses of morpholinos causes vascular fragility, head hemorrhages and increase in permeability; this has not been described before and suggests that the total amount of the protein expressed is an important determinant of vascular stability; 2) concentrations of morpholinos which abrogate VE-cadherin expression prevent vessels to establish successful reciprocal contacts and, as a consequence, vascular sprouting activity is not inhibited. This likely explains the observed vascular hyper-sprouting and the presence of several small, collapsing vessels; 3) the common cardinal vein lacks a correct connection with the endocardium leaving the heart separated from the rest of the circulatory system. The lack of closure of the circulatory loop has never been described before and may explain some downstream defects of the phenotype such as the lack of a correct vascular remodeling. Conclusions and Significance Our observations identify several steps of vascular development in which VE-cadherin plays an essential role. While it does not appear to regulate vascular patterning it is implicated in vascular connection and inhibition of sprouting activity. These processes require stable cell-cell junctions which are defective in absence of VE-cadherin. Notably, also partial modifications in VE-cadherin expression prevent the formation of a stable vasculature. This suggests that partial internalization or change of function of this protein may strongly affect vascular stability and organization.

Inactivation of the zebrafish homologue of Chx10 by antisense oligonucleotides causes eye malformations similar to the ocular retardation phenotype

We report the cloning of a zebrafish paired-type homeobox gene, Alx, closely related to the murine Chx10 and the gold fish Vsx-I homeodomain proteins. Alx is first expressed at about 12 h post-fertilization (hpf) when optic vesicles appear. Its expression is restricted to the early retinal neuroepithelium, whereas no signal can be detected in the optic placode. Later, Alx expression follows the differentiation of the neural retina. Inhibition experiments with antisense oligonucleotides resulted in specific eye malformations which are reminiscent of the phenotype of ocular retardation (or) mice, caused by a spontaneous Chx10 mutation. The expression of other developmentally relevant genes such as pax(zf-a), pax(zf-b) and krx-20 was not affected in the antisense treated embryos.

An evolutionary recent neuroepithelial cell adhesion function of huntingtin implicates ADAM10-Ncadherin

The Huntingtons disease gene product, huntingtin, is indispensable for neural tube formation, but its role is obscure. We studied neurulation in htt-null embryonic stem cells and htt-morpholino zebrafish embryos and found a previously unknown, evolutionarily recent function for this ancient protein. We found that htt was essential for homotypic interactions between neuroepithelial cells; it permitted neurulation and rosette formation by regulating metalloprotease ADAM10 activity and Ncadherin cleavage. This function was embedded in the N terminus of htt and was phenocopied by treatment of htt knockdown zebrafish with an ADAM10 inhibitor. Notably, in htt-null cells, reversion of the rosetteless phenotype occurred only with expression of evolutionarily recent htt heterologues from deuterostome organisms. Conversely, all of the heterologues that we tested, including htt from Drosophila melanogaster and Dictyostelium discoideum, exhibited anti-apoptotic activity. Thus, anti-apoptosis may have been one of htts ancestral function(s), but, in deuterostomes, htt evolved to acquire a unique regulatory activity for controlling neural adhesion via ADAM10-Ncadherin, with implications for brain evolution and development.

Differential regulation of the zebrafish orthopedia1 gene during fate determination of diencephalic neurons

BackgroundThe homeodomain transcription factor Orthopedia (Otp) is essential in restricting the fate of multiple classes of secreting neurons in the neuroendocrine hypothalamus of vertebrates. However, there is little information on the intercellular factors that regulate Otp expression during development.ResultsHere, we identified two otp orthologues in zebrafish (otp1 and otp2) and explored otp1 in the context of the morphogenetic pathways that specify neuroectodermal regions. During forebrain development, otp1 is expressed in anterior groups of diencephalic cells, positioned in the preoptic area (PO) (anterior alar plate) and the posterior tuberculum (PT) (posterior basal plate). The latter structure is characterized by Tyrosine Hydroxylase (TH)-positive cells, suggesting a role for otp1 in the lineage restriction of catecholaminergic (CA) neurons. Disruptions of Hedgehog (HH) and Fibroblast Growth Factor (FGF) pathways point to the ability of SHH protein to trigger otp1 expression in PO presumptive neuroblasts, with the attenuating effect of Dzip1 and FGF8. In addition, our data disclose otp1 as a determinant of CA neurons in the PT, where otp1 activity is strictly dependent on Nodal signaling and it is not responsive to SHH and FGF.ConclusionIn this study, we pinpoint the evolutionary importance of otp1 transcription factor in cell states of the diencephalon anlage and early neuronal progenitors. Furthermore, our data indicate that morphogenetic mechanisms differentially regulate otp1 expression in alar and basal plates.

Structure and macromolecular composition of the zebrafish egg chorion

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.

Aβ peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis

Cerebral amyloid angiopathy (CAA) caused by amyloid β (Aß) deposition around brain microvessels results in vascular degenerative changes. Antiangiogenic Aß properties are known to contribute to the compromised cerebrovascular architecture. Here we hypothesize that Aß peptides impair angiogenesis by causing endothelial cells to enter senescence at an early stage of vascular development. Wild‐type (WT) Aß and its mutated variant E22Q peptide, endowed with marked vascular tropism, were used in this study. In vivo, in zebrafish embryos, the WT or E22Q peptides reduced embryo survival with an IC50 of 6.1 and 4.7 μM, respectively. The 2.5 μM concentration, showing minimal toxicity, was chosen. Alkaline phosphatase staining revealed disorganized vessel patterning, narrowing, and reduced branching of vessels. ß‐Galactosidase staining and the cyclindependent kinase inhibitor p21 expression, indicative of senescence, were increased. In vitro, WT and E22Q reduced endothelial cell survival with an IC50 of 12.3 and 8.8 μM, respectively. The 5 μM concentration, devoid of acute effects on the endothelium, was applied chronically to long‐term cultured human umbilical vein endothelial cells (HUVECs). We observed reduced cumulative population doubling, which coincided with ß‐galactosidase accumulation, down‐regulation of telomerase reversetranscriptase mRNA expression, decreased telomerase activity, and p21 activation. Senescent HUVECs showed marked angiogenesis impairment, as Aß treatment reduced tube sprouting. The endothelial injuries caused by the E22Q peptide were much more aggressive than those induced by the WT peptide. Premature Aß‐induced senescence of the endothelium, producing progressive alterations of microvessel morphology and functions, may represent one of the underlying mechanisms for sporadic or heritable CAA.—Donnini, S., Solito, R., Cetti, E., Corti, F., Giachetti, A., Carra, S., Beltrame, M., Cotelli, F., Ziche, M. Aß peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis. FASEBJ. 24, 2385–2395 (2010). www.fasebj.org

Expression patterns of zebrafish sox11A, sox11B and sox21.

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.