A ligand-insensitive UNC5B splicing isoform regulates angiogenesis by promoting apoptosis.

The Netrin-1 receptor UNC5B is an axon guidance regulator that is also expressed in endothelial cells (ECs), where it finely controls developmental and tumor angiogenesis. In the absence of Netrin-1, UNC5B induces apoptosis that is blocked upon Netrin-1 binding. Here, we identify an UNC5B splicing isoform (called UNC5B-Δ8) expressed exclusively by ECs and generated through exon skipping by NOVA2, an alternative splicing factor regulating vascular development. We show that UNC5B-Δ8 is a constitutively pro-apoptotic splicing isoform insensitive to Netrin-1 and required for specific blood vessel development in an apoptosis-dependent manner. Like NOVA2, UNC5B-Δ8 is aberrantly expressed in colon cancer vasculature where its expression correlates with tumor angiogenesis and poor patient outcome. Collectively, our data identify a mechanism controlling UNC5B's necessary apoptotic function in ECs and suggest that the NOVA2/UNC5B circuit represents a post-transcriptional pathway regulating angiogenesis.

MITO-Luc/GFP zebrafish model to assess spatial and temporal evolution of cell proliferation in vivo.

We developed a novel reporter transgenic zebrafish model called MITO-Luc/GFP zebrafish in which GFP and luciferase expression are under the control of the master regulator of proliferation NF-Y. In MITO-Luc/GFP zebrafish it is possible to visualize cell proliferation in vivo by fluorescence and bioluminescence. In this animal model, GFP and luciferase expression occur in early living embryos, becoming tissue specific in juvenile and adult zebrafish. By in vitro and ex vivo experiments we demonstrate that luciferase activity in adult animals occurs in intestine, kidney and gonads, where detectable proliferating cells are located. Further, by time lapse experiments in live embryos, we observed a wave of GFP positive cells following fin clip. In adult zebrafish, in addition to a bright bioluminescence signal on the regenerating tail, an early unexpected signal coming from the kidney occurs indicating not only a fin cell proliferation, but also a systemic response to tissue damage. Finally, we observed that luciferase activity was inhibited by anti-proliferative interventions, i.e. 5FU, cell cycle inhibitors and X-Rays. In conclusion, MITO-Luc/GFP zebrafish is a novel animal model that may be crucial to assess the spatial and temporal evolution of cell proliferation in vivo.

HDAC8: A Promising Therapeutic Target for Acute Myeloid Leukemia.

Histone deacetylase 8 (HDAC8), a class I HDAC that modifies non-histone proteins such as p53, is highly expressed in different hematological neoplasms including a subtype of acute myeloid leukemia (AML) bearing inversion of chromosome 16 [inv(16)]. To investigate HDAC8 contribution to hematopoietic stem cell maintenance and myeloid leukemic transformation, we generated a zebrafish model with Hdac8 overexpression and observed an increase in hematopoietic stem/progenitor cells, a phenotype that could be reverted using a specific HDAC8 inhibitor, PCI-34051 (PCI). In addition, we demonstrated that AML cell lines respond differently to PCI treatment: HDAC8 inhibition elicits cytotoxic effect with cell cycle arrest followed by apoptosis in THP-1 cells, and cytostatic effect in HL60 cells that lack p53. A combination of cytarabine, a standard anti-AML chemotherapeutic, with PCI resulted in a synergistic effect in all the cell lines tested. We, then, searched for a mechanism behind cell cycle arrest caused by HDAC8 inhibition in the absence of functional p53 and demonstrated an involvement of the canonical WNT signaling in zebrafish and in cell lines. Together, we provide the evidence for the role of HDAC8 in hematopoietic stem cell differentiation in zebrafish and AML cell lines, suggesting HDAC8 inhibition as a therapeutic target in hematological malignancies. Accordingly, we demonstrated the utility of a highly specific HDAC8 inhibition as a therapeutic strategy in combination with standard chemotherapy.

IRSp53 controls plasma membrane shape and polarized transport at the nascent lumen in epithelial tubules.

It is unclear whether the establishment of apical-basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane.

A genetic model of CEDNIK syndrome in zebrafish highlights the role of the SNARE protein Snap29 in neuromotor and epidermal development.

Homozygous mutations in SNAP29, encoding a SNARE protein mainly involved in membrane fusion, cause CEDNIK (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma), a rare congenital neurocutaneous syndrome associated with short life expectancy, whose pathogenesis is unclear. Here, we report the analysis of the first genetic model of CEDNIK in zebrafish. Strikingly, homozygous snap29 mutant larvae display CEDNIK-like features, such as microcephaly and skin defects. Consistent with Snap29 role in membrane fusion during autophagy, we observe accumulation of the autophagy markers p62 and LC3, and formation of aberrant multilamellar organelles and mitochondria. Importantly, we find high levels of apoptotic cell death during early development that might play a yet uncharacterized role in CEDNIK pathogenesis. Mutant larvae also display mouth opening problems, feeding impairment and swimming difficulties. These alterations correlate with defective trigeminal nerve formation and excess axonal branching. Since the paralog Snap25 is known to promote axonal branching, Snap29 might act in opposition with, or modulate Snap25 activity during neurodevelopment. Our vertebrate genetic model of CEDNIK extends the description in vivo of the multisystem defects due to loss of Snap29 and could provide the base to test compounds that might ameliorate traits of the disease.

Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model.

Cystic fibrosis (CF) is a hereditary disease due to mutations in the CFTR gene and causes mortality in humans mainly due to respiratory infections caused by Pseudomonas aeruginosa. In a previous work we used phage therapy, which is a treatment with a mix of phages, to actively counteract acute P. aeruginosa infections in mice and Galleria mellonella larvae. In this work we apply phage therapy to the treatment of P. aeruginosa PAO1 infections in a CF zebrafish model. The structure of the CFTR channel is evolutionary conserved between fish and mammals and cftr-loss-of-function zebrafish embryos show a phenotype that recapitulates the human disease, in particular with destruction of the pancreas. We show that phage therapy is able to decrease lethality, bacterial burden, and the pro-inflammatory response caused by PAO1 infection. In addition, phage administration relieves the constitutive inflammatory state of CF embryos. To our knowledge, this is the first time that phage therapy is used to cure P. aeruginosa infections in a CF animal model. We also find that the curative effect against PAO1 infections is improved by combining phages and antibiotic treatments, opening a useful therapeutic approach that could reduce antibiotic doses and time of administration.

NIPBL: a new player in myeloid cell differentiation.

The nucleophosmin 1 gene (NPM1) is the most frequently mutated gene in acute myeloid leukemia. Notably, NPM1 mutations are always accompanied by additional mutations such as those in cohesin genes RAD21, SMC1A, SMC3, and STAG2 but not in the cohesin regulator, nipped B-like (NIPBL). In this work, we analyzed a cohort of adult patients with acute myeloid leukemia and NPM1 mutation and observed a specific reduction in the expression of NIPBL but not in other cohesin genes. In our zebrafish model, overexpression of the mutated form of NPM1 also induced downregulation of nipblb, the zebrafish ortholog of human NIPBL To investigate the hematopoietic phenotype and the interaction between mutated NPM1 and nipblb, we generated a zebrafish model with nipblb downregulation which showed an increased number of myeloid progenitors. This phenotype was due to hyper-activation of the canonical Wnt pathway: myeloid cells blocked in an undifferentiated state could be rescued when the Wnt pathway was inhibited by dkk1b mRNA injection or indomethacin administration. Our results reveal, for the first time, a role for NIPBL during zebrafish hematopoiesis and suggest that an interplay between NIPBL/NPM1 may regulate myeloid differentiation in zebrafish and humans through the canonical Wnt pathway and that dysregulation of these interactions may drive leukemic transformation.

Zebrafish Tmem230a cooperates with the Delta/Notch signaling pathway to modulate endothelial cell number in angiogenic vessels.

During embryonic development, new arteries, and veins form from preexisting vessels in response to specific angiogenic signals. Angiogenic signaling is complex since not all endothelial cells exposed to angiogenic signals respond equally. Some cells will be selected to become tip cells and acquire migration and proliferation capacity necessary for vessel growth while others, the stalk cells become trailer cells that stay connected with pre-existing vessels and act as a linkage to new forming vessels. Additionally, stalk and tip cells have the capacity to interchange their roles. Stalk and tip cellular responses are mediated in part by the interactions of components of the Delta/Notch and Vegf signaling pathways. We have identified in zebrafish, that the transmembrane protein Tmem230a is a novel regulator of angiogenesis by its capacity to regulate the number of the endothelial cells in intersegmental vessels by co-operating with the Delta/Notch signaling pathway. Modulation of Tmem230a expression by itself is sufficient to rescue improper number of endothelial cells induced by aberrant expression or inhibition of the activity of genes associated with the Dll4/Notch pathway in zebrafish. Therefore, Tmem230a may have a modulatory role in vessel-network formation and growth. As the Tmem230 sequence is conserved in human, Tmem230 may represent a promising novel target for drug discovery and for disease therapy and regenerative medicine in promoting or restricting angiogenesis.

Endocytic reawakening of motility in jammed epithelia.

Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination.

Nucleophosmin leukemogenic mutant activates Wnt signaling during zebrafish development.

Nucleophosmin (NPM1) is a ubiquitous multifunctional phosphoprotein with both oncogenic and tumor suppressor functions. Mutations of the NPM1 gene are the most frequent genetic alterations in acute myeloid leukemia (AML) and result in the expression of a mutant protein with aberrant cytoplasmic localization, NPMc+. Although NPMc+ causes myeloproliferation and AML in animal models, its mechanism of action remains largely unknown. Here we report that NPMc+ activates canonical Wnt signaling during the early phases of zebrafish development and determines a Wnt-dependent increase in the number of progenitor cells during primitive hematopoiesis. Coherently, the canonical Wnt pathway is active in AML blasts bearing NPMc+ and depletion of the mutant protein in the patient derived OCI-AML3 cell line leads to a decrease in the levels of active ß-catenin and of Wnt target genes. Our results reveal a novel function of NPMc+ and provide insight into the molecular pathogenesis of AML bearing NPM1 mutations.

Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development.

Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology.

The alternative splicing factor Nova2 regulates vascular development and lumen formation.

Vascular lumen formation is a fundamental step during angiogenesis; yet, the molecular mechanisms underlying this process are poorly understood. Recent studies have shown that neural and vascular systems share common anatomical, functional and molecular similarities. Here we show that the organization of endothelial lumen is controlled at the post-transcriptional level by the alternative splicing (AS) regulator Nova2, which was previously considered to be neural cell-specific. Nova2 is expressed during angiogenesis and its depletion disrupts vascular lumen formation in vivo. Similarly, Nova2 depletion in cultured endothelial cells (ECs) impairs the apical distribution and the downstream signalling of the Par polarity complex, resulting in altered EC polarity, a process required for vascular lumen formation. These defects are linked to AS changes of Nova2 target exons affecting the Par complex and its regulators. Collectively, our results reveal that Nova2 functions as an AS regulator in angiogenesis and is a novel member of the 'angioneurins' family.

DEPDC1B coordinates de-adhesion events and cell-cycle progression at mitosis.

Cells entering mitosis become rounded, lose attachment to the substrate, and increase their cortical rigidity. Pivotal to these events is the dismantling of focal adhesions (FAs). How mitotic reshaping is linked to commitment to divide is unclear. Here, we show that DEPDC1B, a protein that accumulates in G2, coordinates de-adhesion events and cell-cycle progression at mitosis. DEPDC1B functions as an inhibitor of a RhoA-based signaling complex, which assembles on the FA-associated protein tyrosine phosphatase, receptor type, F (PTPRF) and mediates the integrity of FAs. By competing with RhoA for the interaction with PTPRF, DEPDC1B promotes the dismantling of FAs, which is necessary for the morphological changes preceding mitosis. The circuitry is relevant in whole organisms, as shown by the control exerted by the DEPDC1B/RhoA/PTPRF axis on mitotic dynamics during zebrafish development. Our results uncover an adhesion-dependent signaling mechanism that coordinates adhesion events with the control of cell-cycle progression.

Pharmacologic inhibition of vacuolar H+ ATPase reduces physiologic and oncogenic Notch signaling.

Notch signaling in prominently involved in growth regulation in metazoan tissues. Because of this, Notch is often upregulated in cancer and current efforts point to developing drugs that block its activation. Notch receptor endocytosis towards acidic compartments is a recently appreciated determinant of signaling activation. Vacuolar H(+) ATPase (V-ATPase) is responsible for acidification of endocytic organelles and mutants in V-ATPase subunit encoding genes in model organisms have been recently shown to display loss of Notch signaling. Here, we show that administration of BafilomycinA1 (BafA1), a highly specific V-ATPase inhibitor decreases Notch signaling during Drosophila and Zebrafish development, and in human cells in culture. In normal breast cells, we find that BafA1 treatment leads to accumulation of Notch in the endo-lysosomal system, and reduces its processing and signaling activity. In Notch-addicted breast cancer cells, BafA1 treatment reduces growth in cells expressing membrane tethered forms of Notch, while sparing cells expressing cytoplasmic forms. In contrast, we find that V-ATPase inhibition reduces growth of leukemia cells, without affecting Notch activatory cleavage. However, consistent with the emerging roles of V-ATPase in controlling multiple signaling pathways, in these cells Akt activation is reduced, as it is also the case in BafA1-treated breast cancer cells. Our data support V-ATPase inhibition as a novel therapeutic approach to counteract tumor growth via signaling pathways regulated at the endo-lysosomal level.

Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase.

Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD(+)-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.

prep1.2 and aldh1a2 participate to a positive loop required for branchial arches development in zebrafish.

Segmentation is a key step in embryonic development. Acting in all germ layers, it is responsible for the generation of antero-posterior asymmetries. Hox genes, with their diverse expression in individual segments, are fundamental players in the determination of different segmental fates. In vertebrates, Hox gene products gain specificity for DNA sequences by interacting with Pbx, Prep and Meis homeodomain transcription factors. In this work we cloned and analysed prep1.2 in zebrafish. In-situ hybridization experiments show that prep1.2 is maternally and ubiquitously expressed up to early somitogenesis when its expression pattern becomes more restricted to the head and trunk mesenchyme. Experiments of loss of function with prep1.2 morpholinos change the shape of the hyoid and third pharyngeal cartilages while arches 4-7 and pectoral fins are absent, a phenotype strikingly similar to that caused by loss of retinoic acid (RA). In fact, we show that prep1.2 is positively regulated by RA and required for the normal expression of aldh1a2 at later stages, particularly in tissues involved in the development of the branchial arches and pectoral fins. Thus, prep1.2 and aldh1a2 are members of an indirect positive feedback loop required for pharyngeal endoderm and posterior branchial arches development. As the paralogue gene prep1.1 is more important in hindbrain patterning and neural crest chondrogenesis, we provide evidence of a functional specialization of prep genes in zebrafish head segmentation and morphogenesis.

Characterization of the regulatory region of the zebrafish Prep1.1 gene: analogies to the promoter of the human PREP1.

Prep1 is a developmentally essential TALE class homeodomain transcription factor. In zebrafish and mouse, Prep1 is already ubiquitously expressed at the earliest stages of development, with important tissue-specific peculiarities. The Prep1 gene in mouse is developmentally essential and has haploinsufficient tumor suppressor activity [1]. We have determined the human Prep1 transcription start site (TSS) by primer extension analysis and identified, within 20 bp, the transcription start region (TSR) of the zebrafish Prep1.1 promoter. The functions of the zebrafish 5' upstream sequences were analyzed both by transient transfections in Hela Cells and by injection in zebrafish embryos. This analysis revealed a complex promoter with regulatory sequences extending up to -1.8, possibly -5.0 Kb, responsible for tissue specific expression. Moreover, the first intron contains a conserved tissue-specific enhancer both in zebrafish and in human cells. Finally, a two nucleotides mutation of an EGR-1 site, conserved in all species including human and zebrafish and located at a short distance from the TSS, destroyed the promoter activity of the -5.0 Kb promoter. A transgenic fish expressing GFP under the -1.8 Kb zebrafish promoter/enhancer co-expressed GFP and endogenous Prep1.1 during embryonic development. In the adult fish, GFP was expressed in hematopoietic regions like the kidney, in agreement with the essential function of Prep1 in mouse hematopoiesis. Sequence comparison showed conservation from man to fish of the sequences around the TSS, within the first intron enhancer. Moreover, about 40% of the sequences spread throughout the 5 Kbof the zebrafish promoter are concentrated in the -3 to -5 Kb of the human upstream region.

Monoclonal antibodies isolated by large-scale screening are suitable for labeling adult zebrafish (Danio rerio) tissues and cell structures.

The lack of a sufficient number of antibodies represents an obstacle in the research performed using the zebrafish (Danio rerio) as a model organism. On the other hand, high-throughput generation of antibodies, especially those suitable for immunohistochemistry, is not an established methodology. Here we present the results of an immunization experiment with a zebrafish tissue lysate that allowed for the isolation of hundreds of monoclonal antibodies suitable for labeling of a large variety of zebrafish tissue and cell structures. Some of them were further characterized in terms of detailed localization and age-dependent expression. In addition, the antigen recognized by one of them was first immunoprecipitated and then identified by mass spectrometry. Furthermore, immunofluorescence-competent recombinant antibodies were also isolated by panning large repertoire phage display libraries, in both single-chain (scFv) and single-domain (VHH) format. Such selection alternative is simpler to organize and could contribute to limit the costs of antibody screening and production.

Expression of H-RASV12 in a zebrafish model of Costello syndrome causes cellular senescence in adult proliferating cells.

Constitutively active, 'oncogenic' H-RAS can drive proliferation and transformation in human cancer, or be a potent inducer of cellular senescence. Moreover, aberrant activation of the Ras pathway owing to germline mutations can cause severe developmental disorders. In this study we have generated transgenic zebrafish that constitutively express low levels, or can be induced to express high levels, of oncogenic H-RAS. We observed that fish carrying the integrated transgene in their germline display several hallmarks of Costello syndrome, a rare genetic disease caused by activating mutations in the gene H-RAS, and can be used as a model for the disease. In Costello-like fish, low levels of oncogenic H-RAS expression are associated with both reduced proliferation and an increase in senescence markers in adult progenitor cell compartments in the brain and heart, together with activated DNA damage responses. Overexpression of H-RAS through a heat-shock-inducible promoter in larvae led to hyperproliferation, activation of the DNA damage response and tp53-dependent cell cycle arrest. Thus, oncogene-induced senescence of adult proliferating cells contributes to the development of Costello syndrome and provides an alternative pathway to transformation in the presence of widespread constitutively active H-RAS expression.

The tumor suppressor PRDM5 regulates Wnt signaling at early stages of zebrafish development.

PRDM genes are a family of transcriptional regulators that modulate cellular processes such as differentiation, cell growth and apoptosis. Some family members are involved in tissue or organ maturation, and are differentially expressed in specific phases of embryonic development. PRDM5 is a recently identified family member that functions as a transcriptional repressor and behaves as a putative tumor suppressor in different types of cancer. Using gene expression profiling, we found that transcriptional targets of PRDM5 in human U2OS cells include critical genes involved in developmental processes, and specifically in regulating wnt signaling. We therefore assessed PRDM5 function in vivo by performing loss-of-function and gain-of-function experiments in zebrafish embryos. Depletion of prdm5 resulted in impairment of morphogenetic movements during gastrulation and increased the occurrence of the masterblind phenotype in axin+/- embryos, characterized by the loss of eyes and telencephalon. Overexpression of PRDM5 mRNA had opposite effects on the development of anterior neural structures, and resulted in embryos with a shorter body axis due to posterior truncation, a bigger head and abnormal somites. In situ hybridization experiments aimed at analyzing the integrity of wnt pathways during gastrulation at the level of the prechordal plate revealed inhibition of non canonical PCP wnt signaling in embryos overexpressing PRDM5, and over-activation of wnt/beta-catenin signaling in embryos lacking Prdm5. Our data demonstrate that PRDM5 regulates the expression of components of both canonical and non canonical wnt pathways and negatively modulates wnt signaling in vivo.