Supplementary Figure 2 from Mammalian Tumor Xenografts Induce Neovascularization in Zebrafish Embryos

Supplementary Figure 1 from Mammalian Tumor Xenografts Induce Neovascularization in Zebrafish Embryos

Classical dynamins (DNMs) are GTPase proteins engaged in endocytosis, a fundamental process for cargo internalization from the plasma membrane. In mammals, three DNM genes are present with different expression patterns. DNM1 is expressed at high levels in neurons, where it takes place in the recycling of synaptic vesicles; DNM2 is ubiquitously expressed, while DNM3 is found in the brain and in the testis. Due to the conservation of genes in comparison to mammals, we took advantage of a zebrafish model for functional characterization of dnm1a, ortholog of mammalian DNM1. Our data strongly demonstrated that dnm1a has a nervous tissue-specific expression pattern and plays a role in the formation of both axon and synapse. This is the first in vivo study that collects evidence about the effects of dnm1a loss of function in zebrafish, thus providing a new excellent model to be used in different scientific fields.

BACKGROUND: We tested the hypothesis that whole-body vibration (WBV) positively affects the fatigue process ensuing from repeated bouts of maximal efforts, as induced by repeated sprints' ability (RSA). Eleven male soccer players performed three sets of six repeated shuttle sprints (40 meters). METHODS: Eleven male soccer players (age 23.6 +/- 4.5 years) were cross-randomized to perform WBW before RSA and during the recovery between sets (WBV-with) or to warm-up and passive recovery between sets (WBV-without). The effects of WBV were quantified by sprint time (ST) and blood lactate concentration (LA), collected up to 15 min after completion of tests. RESULTS: ST during RSA showed a better maintenance of performance in the WBV-with compared to WBV-without condition in all three sets, reaching a statistical significance between-groups during the 2nd and 3rd set (P<0.05). No significant differences in ST over the sets were detected in WEIV-with, whereas a significant decrease was observed in the WBV-without condition (P<0.001). LA recovered significantly faster from the 9th to 15th minute of recovery in WBV-with as compared to WBV-without (P<0.05). CONCLUSIONS: These findings would indicate that WBV performed during recovery between RSA sets can delay the onset of muscle fatigue resulting in a better maintenance of sprint performance.

SMYD3 (SET and MYND domain containing protein 3) is a methylase over-expressed in cancer cells and involved in oncogenesis. While several studies uncovered key functions for SMYD3 in cancer models, the SMYD3 role in physiological conditions has not been fully elucidated yet. Here, we dissect the role of SMYD3 at early stages of development, employing mouse embryonic stem cells (ESCs) and zebrafish as model systems. We report that SMYD3 depletion promotes the induction of the mesodermal pattern during in vitro differentiation of ESCs and is linked to an upregulation of cardiovascular lineage markers at later stages. In vivo, smyd3 knockdown in zebrafish favors the upregulation of mesendodermal markers during zebrafish gastrulation. Overall, our study reveals that SMYD3 modulates levels of mesendodermal markers, both in development and in embryonic stem cell differentiation.

Objective: Squamous cell carcinoma（SCC） represents the most common histotype of all head and neck malignancies and includes oropharyngeal squamous cell carcinoma（OSCC）, a tumor associated with different clinical outcomes and linked to human papilloma virus（HPV） status. Translational research has few available in vitro models with which to study the different pathophysiological behavior of OSCCs. The present study proposes a 3-dimensional（3 D） biomimetic collagen-based scaffold to mimic the tumor microenvironment and the crosstalk between the extracellular matrix（ECM） and cancer cells.Methods: We compared the phenotypic and genetic features of HPV-positive and HPV-negative OSCC cell lines cultured on common monolayer supports and on scaffolds. We also explored cancer cell adaptation to the 3 D microenvironment and its impact on the efficacy of drugs tested on cell lines and primary cultures.Results: HPV-positive and HPV-negative cell lines were successfully grown in the 3 D model and displayed different collagen fiber organization. The 3 D cultures induced an increased expression of markers related to epithelial–mesenchymal transition（EMT） and to matrix interactions and showed different migration behavior, as confirmed by zebrafish embryo xenografts. The expression of hypoxia-inducible factor 1α（1α） and glycolysis markers were indicative of the development of a hypoxic microenvironment inside the scaffold area. Furthermore, the 3 D cultures activated drug-resistance signaling pathways in both cell lines and primary cultures.Conclusions: Our results suggest that collagen-based scaffolds could be a suitable model for the reproduction of the pathophysiological features of OSCCs. Moreover, 3 D architecture appears capable of inducing drug-resistance processes that can be studied to better our understanding of the different clinical outcomes of HPV-positive and HPV-negative patients with OSCCs.

Pompe disease (PD) is an autosomal recessive muscular disorder caused by deficiency of the glycogen hydrolytic enzyme acid α-glucosidase (GAA). The enzyme replacement therapy, currently the only available therapy for PD patients, is efficacious in improving cardiomyopathy in the infantile form, but not equally effective in the late onset cases with involvement of skeletal muscle. Correction of the skeletal muscle phenotype has indeed been challenging, probably due to concomitant dysfunctional autophagy. The increasing attention to the pathogenic mechanisms of PD and the search of new therapeutic strategies prompted us to generate and characterize a novel transient PD model, using zebrafish. Our model presented increased glycogen content, markedly altered motor behavior and increased lysosome content, in addition to altered expression of the autophagy-related transcripts and proteins Beclin1, p62 and Lc3b. Furthermore, the model was used to assess the beneficial effects of 3-bromopyruvic acid (3-BrPA). Treatment with 3-BrPA induced amelioration of the model phenotypes regarding glycogen storage, motility behavior and autophagy-related transcripts and proteins. Our zebrafish PD model recapitulates most of the defects observed in human patients, proving to be a powerful translational model. Moreover, 3-BrPA unveiled to be a promising compound for treatment of conditions with glycogen accumulation.

Cornelia de Lange syndrome (CdLS), which is reported to affect similar to 1 in 10 000 to 30 000 newborns, is a multisystem organ developmental disorder with relatively mild to severe effects. Among others, intellectual disability represents an important feature of this condition. CdLS can result from mutations in at least five genes: nipped-B-like protein, structural maintenance of chromosomes 1A, structural maintenance of chromosomes 3, RAD21 cohesin complex component and histone deacetylase 8 (HDAC8). It is believed that mutations in these genes cause CdLS by impairing the function of the cohesin complex (to which all the aforementioned genes contribute to the structure or function), disrupting gene regulation during critical stages of early development. Since intellectual disorder might result from alterations in neural development, in this work, we studied the role of Hdac8 gene in mouse neural stem cells (NSCs) and in vertebrate (Danio rerio) brain development by knockdown and chemical inhibition experiments. Underlying features of Hdac8 deficiency is an increased cell death in the developing neural tissues, either in mouse NSCs or in zebrafish embryos.

SMYD3 is a methylase previously linked to cancer cell invasion and migration. Here we show that SMYD3 favors TGF-induced epithelial-mesenchymal transition (EMT) in mammary epithelial cells, promoting mesenchymal and EMT transcription factors expression. SMYD3 directly interacts with SMAD3 but it is unnecessary for SMAD2/3 phosphorylation and nuclear translocation. Conversely, SMYD3 is indispensable for SMAD3 direct association to EMT genes regulatory regions. Accordingly, SMYD3 knockdown or its pharmacological blockade with the BCI121 inhibitor dramatically reduce TGF-induced SMAD3 association to the chromatin. Remarkably, BCI121 treatment attenuates mesenchymal genes transcription in the mesenchymal-like MDA-MB-231 cell line and reduces their invasive ability in vivo, in a zebrafish xenograft model. In addition, clinical datasets analysis revealed that higher SMYD3 levels are linked to a less favorable prognosis in claudin-low breast cancers and to a reduced metastasis free survival in breast cancer patients. Overall, our data point at SMYD3 as a pivotal SMAD3 cofactor that promotes TGF-dependent mesenchymal gene expression and cell migration in breast cancer, and support SMYD3 as a promising pharmacological target for anti-cancer therapy.

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.

SEL1L (suppressor/enhancer of Lin‐12‐like) is a highly conserved gene associated with the endoplasmic reticulum‐associated degradation (ERAD) pathway and involved in mediating the balance between stem cells self‐renewal and differentiation of neural progenitors. It has been recently shown that SEL1L KO mice are embryonic lethal and display altered organogenesis. To better characterize the function of SEL1L in the early stages of embryonic development, we turned to the zebrafish model ( Danio rerio ). After exploring sel1l expression by RT‐PCR and in situ hybridization, we employed a morpholino‐mediated down‐regulation approach. Results showed extensive impairments in the vasculature, which supports the mice knock‐out findings.

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.

Ramification of blood circulation is relevant in a number of physiological and pathological conditions. The oxygen exchange occurs largely in the capillary bed, and the cancer progression is closely linked to the angiogenesis around the tumor mass. Optical microscopy has made impressive improvements in in vivo imaging and dynamic studies based on correlation analysis of time stacks of images. Here, we develop and test advanced methods that allow mapping the flow fields in branched vessel networks at the resolution of 10 to 20 mu m. The methods, based on the application of spatiotemporal image correlation spectroscopy and its extension to cross-correlation analysis, are applied here to the case of early stage embryos of zebrafish. (C) 2017 Society of Photo-Optical Instrumentation Engineers (SPIE).

The CFDP1 proteins have been linked to craniofacial development and osteogenesis in vertebrates, though specific human syndromes have not yet been identified. Alterations of craniofacial development represent the main cause of infant disability and mortality in humans. For this reason, it is crucial to understand the cellular functions and mechanism of action of the CFDP1 protein in model vertebrate organisms. Using a combination of genomic, molecular and cell biology approaches, we have performed a functional analysis of the cfdp1 gene and its encoded protein, zCFDP1, in the zebrafish model system. We found that zCFDP1 is present in the zygote, is rapidly produced after MTZ transition and is highly abundant in the head structures. Depletion of zCFDP1, induced by an ATG-blocking morpholino, produces considerable defects in craniofacial structures and bone mineralization. Together, our results show that zCFDP1 is an essential protein required for proper development and provide the first experimental evidence showing that in vertebrates it actively participates to the morphogenesis of craniofacial territories.

In vivo studies of blood circulation pathologies have great medical relevance and need methods for the characterization of time varying flows at high spatial and time resolution in small animal models. We test here the efficacy of the combination of image correlation techniques and single plane illumination microscopy (SPIM) in characterizing time varying flows in vitro and in vivo. As indicated by numerical simulations and by in vitro experiments on straight capillaries, the complex analytical form of the cross-correlation function for SPIM detection can be simplified, in conditions of interest for hemodynamics, to a superposition of Gaussian components, easily amenable to the analysis of variable flows. The possibility to select a wide field of view with a good spatial resolution along the collection optical axis and to compute the cross-correlation between regions of interest at varying distances on a single time stack of images allows one to single out periodic flow components from spurious peaks on the cross-correlation functions and to infer the duration of each flow component. We apply this cross-correlation analysis to the blood flow in Zebrafish embryos at 4 days after fertilization, measuring the average speed and the duration of the systolic and diastolic phases.

Resistance to thyroid hormone can be due to heterozygous, dominant negative (DN) THRA (RTH alpha) or THRB (RTH beta) mutations, but the underlying mechanisms are incompletely understood. Here, we delineate the spatiotemporal expression of TH receptors (TRs) in zebrafish and generated morphants expressing equivalent amounts of wild-type and DN TR alpha s (thraa_MOs) and TR beta s (thrb_MOs) in vivo. Both morphants show severe developmental abnormalities. The phenotype of thraa_MOs includes brain and cardiac defects, but normal thyroid volume and tshba expression. A combined modification of dio2 and dio3 expression can explain the high T3/T4 ratio seen in thraa_MOs, as in RTH alpha. Thrb_MOs show abnormal eyes and otoliths, with a typical RTH beta pattern of thyroid axis. The coexpression of wild-type, but not mutant, human TRs can rescue the phenotype in both morphants. High T3 doses can partially revert the dominant negative action of mutant TRs in morphant fish.Therefore, our morphants recapitulate the RTH alpha and RTH beta key manifestations-representing new models in which the functional consequences of human TR mutations can be rapidly and faithfully evaluated. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Background: Heterozygous mutations in the thyroid hormone receptor alpha (THRA) gene cause resistance to thyroid hormone alpha (RTHα), a disease characterized by variable manifestations reminiscent of untreated congenital hypothyroidism but a raised triiodothyronine/thyroxine ratio and normal thyrotropin levels. It was recently described that zebrafish embryos expressing a dominant negative (DN) form of thraa recapitulate the key features of RTHα, and that zebrafish and human receptors are functionally interchangeable. Methods: This study expressed several human thyroid hormone receptor alpha (hTRα) variants in zebrafish embryos and analyzed the resulting phenotypes. Results: All hTRα-injected embryos showed variable defects, including cerebral and cardiac edema likely caused by an aberrant looping during heart development, anemia, and an incomplete formation of the vascular network. Moreover, the hTRα-injected embryos presented severe defects of motorneurons and craniofacial development, thus affecting their autonomous feeding and swimming behaviors. Surprisingly, expression of all hTRα mutants had no detectable effect on thyrotropin beta and thyrotropin-releasing hormone transcripts, indicating that their DN action is limited on the thyroid hormone reception beta 2 targets at the hypothalamic/pituitary level in vivo. As previously described in vitro, treatment with high triiodothyronine doses can efficiently revert the observed defects only in embryos injected with missense hTRα variants. Conclusion: Injection of human THRA variants in zebrafish embryos causes tissue-specific defects recapitulating most of the RTHα clinical and biochemical manifestations. The described manipulation of zebrafish embryos represents a novel in vivo model to screen the functional consequences of THRA variants and the rescue potential of new therapeutic compounds.