ABSTRACT
Introduction: Lung carcinoids (LCs) are a type of neuroendocrine tumor (NET) that originate in the bronchopulmonary tract. LCs account for 20-25% of all NETs and approximately 1-2% of lung cancers. Given the highly vascularized nature of NETs and their tendency to overexpress vascular growth factor receptors (VEGFR), inhibiting angiogenesis appears as a potential therapeutic target in slowing down tumor growth and spread. This study evaluated the long-term antitumor activity and related mechanisms of axitinib (AXI), a VEGFR-targeting drug, in LC cell lines. Methods: Three LC cell lines (NCI-H727, UMC-11 and NCI-H835) were incubated with their respective EC50 AXI concentrations for 6 days. At the end of the incubation, FACS experiments and Western blot analyses were performed to examine changes in the cell cycle and the activation of apoptosis. Microscopy analyses were added to describe the mechanisms of senescence and mitotic catastrophe when present. Results: The primary effect of AXI on LC cell lines is to arrest tumor growth through an indirect DNA damage. Notably, AXI triggers this response in diverse manners among the cell lines, such as inducing senescence or mitotic catastrophe. The drug seems to lose its efficacy when the DNA damage is mitigated, as observed in NCI-H835 cells. Conclusion: The ability of AXI to affect cell viability and proliferation in LC tumor cells highlights its potential as a therapeutic agent. The role of DNA damage and the consequent activation of senescence seem to be a prerequisite for AXI to exert its function.
Subject(s)
Apoptosis , Axitinib , Carcinoid Tumor , Cell Proliferation , Lung Neoplasms , Humans , Axitinib/pharmacology , Axitinib/therapeutic use , Carcinoid Tumor/drug therapy , Carcinoid Tumor/pathology , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Cycle/drug effectsABSTRACT
Lung carcinoids (LCs) comprise well-differentiated neuroendocrine tumors classified as typical (TCs) and atypical (ACs) carcinoids. Unfortunately, curative therapies remain elusive for metastatic LCs, which account for 25-30% of cases. This study evaluated the antitumor activity of axitinib (AXI), a second-generation tyrosine kinase inhibitor selectively targeting vascular endothelial growth factor receptors (VEGFR-1, VEGFR-2, VEGFR-3) in human lung TC (NCI-H727, UMC-11, NCI-H835) and AC (NCI-H720) cell lines. In vitro and in vivo (zebrafish) assays were performed following AXI treatment to gather several read-outs about cell viability, cell cycle, the secretion of proangiogenic factors, apoptosis, tumor-induced angiogenesis and migration. AXI demonstrated relevant antitumor activity in human LC cells, with pronounced effects observed in UMC-11 and NCI-H720, characterized by cell cycle perturbation and apoptosis induction. AXI significantly hindered tumor induced-angiogenesis in Tg(fli1a:EGFP)y1 zebrafish embryos implanted with all LC cell lines and also reduced the invasiveness of NCI-H720 cells, as well as the secretion of several proangiogenic factors. In conclusion, our study provides initial evidence supporting the potential anti-tumor activity of AXI in LC, offering a promising basis for future investigations in mammalian animal models and, eventually, progressing to clinical trials.
ABSTRACT
Neuroendocrine tumors (NETs) are highly vascularized malignancies in which angiogenesis may entail cell proliferation and survival. Among the emerging compounds with antivascular properties, cabozantinib (CAB) appeared promising. We analyzed the antitumor activity of CAB against NETs utilizing in vitro and in vivo models. For cell cultures, we used BON-1, NCI-H727 and NCI-H720 cell lines. Cell viability was assessed by manual count coupled with quantification of cell death, performed through fluorescence-activated cell sorting analysis as propidium iodide exclusion assay. In addition, we investigated the modulation of the antiapoptotic myeloid cell leukemia 1 protein under CAB exposure, as a putative adaptive pro-survival mechanism, and compared the responses with sunitinib. The activity of CAB was also tested in mouse and zebrafish xenograft tumor models. Cabozantinib showed a dose-dependent and time-dependent effect on cell viability and proliferation in human NET cultures, besides a halting of cell cycle progression for endoduplication, never reported for other tyrosine kinase inhibitors. In a transplantable zebrafish model, CAB drastically inhibited NET-induced angiogenesis and migration of implanted cells through the embryo body. CAB showed encouraging activity in NETs, both in vitro and in vivo models. On this basis, we envisage future research to further investigate along these promising lines.
Subject(s)
Neuroendocrine Tumors , Zebrafish , Humans , Animals , Mice , Signal Transduction , Neuroendocrine Tumors/pathology , Cell Line, TumorABSTRACT
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.
Subject(s)
Nerve Tissue , Zebrafish , Animals , Male , Axons , Neurons/metabolism , Synapses/metabolism , MammalsABSTRACT
Carcinoid syndrome represents a debilitating paraneoplastic disease, caused by the secretion of several substances, occurring in about 10-40% of patients with well-differentiated neuroendocrine tumors (NETs). The main signs and symptoms associated with carcinoid syndrome are flushing, diarrhea, hypotension, tachycardia, bronchoconstriction, venous telangiectasia, dyspnea and fibrotic complications (mesenteric and retroperitoneal fibrosis, and carcinoid heart disease). Although there are several drugs available for the treatment of carcinoid syndrome, the lack of therapeutic response, poor tolerance or resistance to drugs are often reported. Preclinical models are indispensable tools for investigating the pathogenesis, mechanisms for tumor progression and new therapeutic approaches for cancer. This paper provides a state-of-the-art overview of in vitro and in vivo models in NETs with carcinoid syndrome, highlighting the future developments and therapeutic approaches in this field.
Subject(s)
Malignant Carcinoid Syndrome , Neuroendocrine Tumors , Humans , Malignant Carcinoid Syndrome/complications , Malignant Carcinoid Syndrome/drug therapy , Neuroendocrine Tumors/therapy , Diarrhea/drug therapyABSTRACT
Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor arising from parafollicular C cells of the thyroid gland. In this preclinical study, we tested three tyrosine-kinase inhibitors (TKIs): SU5402, a selective inhibitor of fibroblast growth factor receptor (FGFR)-1 and vascular endothelial growth factor receptor (VEGFR)-2; sulfatinib, an inhibitor of FGFR-1 and VEGFR-1, -2, -3; and SPP86, a RET-specific inhibitor. The effects of these compounds were evaluated in vitro in two human MTC cell lines (TT and MZ-CRC-1), and in vivo using xenografts of MTC cells in zebrafish embryos. SU5402, sulfatinib and SPP86 decreased cell viability. Sulfatinib and SPP86 significantly induced apoptosis in both cell lines. Sulfatinib and SPP86 inhibited the migration of TT and MZCRC-1 cells, while SU5402 was able to inhibit migration only in TT cells. In vivo we observed a significant reduction in TT cell-induced angiogenesis in zebrafish embryos after incubation with sulfatinib and SPP86. In conclusion, sulfatinib and SPP86 displayed a relevant antitumor activity both in vitro and in vivo. Moreover, this work suggests the potential utility of targeting FGFR and VEGFR signaling pathways as an alternative therapy for MTC.
ABSTRACT
Lung carcinoids are neuroendocrine tumors that comprise well-differentiated typical (TCs) and atypical carcinoids (ACs). Preclinical models are indispensable for cancer drug screening since current therapies for advanced carcinoids are not curative. We aimed to develop a novel in vivo model of lung carcinoids based on the xenograft of lung TC (NCI-H835, UMC-11, and NCI-H727) and AC (NCI-H720) cell lines and patient-derived cell cultures in Tg(fli1a:EGFP)y1 zebrafish embryos. We exploited this platform to test the anti-tumor activity of sulfatinib. The tumorigenic potential of TC and AC implanted cells was evaluated by the quantification of tumor-induced angiogenesis and tumor cell migration as early as 24 h post-injection (hpi). The characterization of tumor-induced angiogenesis was performed in vivo and in real time, coupling the tumor xenograft with selective plane illumination microscopy on implanted zebrafish embryos. TC-implanted cells displayed a higher pro-angiogenic potential compared to AC cells, which inversely showed a relevant migratory behavior within 48 hpi. Sulfatinib inhibited tumor-induced angiogenesis, without affecting tumor cell spread in both TC and AC implanted embryos. In conclusion, zebrafish embryos implanted with TC and AC cells faithfully recapitulate the tumor behavior of human lung carcinoids and appear to be a promising platform for drug screening.
Subject(s)
Carcinoid Tumor , Carcinoma, Neuroendocrine , Lung Neoplasms , Animals , Carcinoid Tumor/drug therapy , Carcinoma, Neuroendocrine/pathology , Heterografts , Humans , Lung/pathology , Lung Neoplasms/metabolism , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , ZebrafishSubject(s)
Intestinal Neoplasms , Neuroendocrine Tumors , Pancreatic Neoplasms , Stomach Neoplasms , Humans , Obesity/epidemiologyABSTRACT
Medullary thyroid carcinoma (MTC) is a tumor deriving from the thyroid C cells. Vandetanib (VAN) and cabozantinib (CAB) are two tyrosine kinase inhibitors targeting REarranged during Transfection (RET) and other kinase receptors and are approved for the treatment of advanced MTC. We aim to compare the in vitro and in vivo anti-tumor activity of VAN and CAB in MTC. The effects of VAN and CAB on viability, cell cycle, and apoptosis of TT and MZ-CRC-1 cells are evaluated in vitro using an MTT assay, DNA flow cytometry with propidium iodide, and Annexin V-FITC/propidium iodide staining, respectively. In vivo, the anti-angiogenic potential of VAN and CAB is evaluated in Tg(fli1a:EGFP)y1 transgenic fluorescent zebrafish embryos by analyzing the effects on the physiological development of the sub-intestinal vein plexus and the tumor-induced angiogenesis after TT and MZ-CRC-1 xenotransplantation. VAN and CAB exert comparable effects on TT and MZ-CRC-1 viability inhibition and cell cycle perturbation, and stimulated apoptosis with a prominent effect by VAN in MZ-CRC-1 and CAB in TT cells. Regarding zebrafish, both drugs inhibit angiogenesis in a dose-dependent manner, in particular CAB shows a more potent anti-angiogenic activity than VAN. To conclude, although VAN and CAB show comparable antiproliferative effects in MTC, the anti-angiogenic activity of CAB appears to be more relevant.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Anilides/therapeutic use , Carcinoma, Neuroendocrine/drug therapy , Piperidines/therapeutic use , Pyridines/therapeutic use , Quinazolines/therapeutic use , Thyroid Neoplasms/drug therapy , Zebrafish/physiology , Anilides/pharmacology , Animals , Apoptosis/drug effects , Carcinoma, Neuroendocrine/blood supply , Carcinoma, Neuroendocrine/pathology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Disease Models, Animal , Embryo, Nonmammalian/blood supply , Embryo, Nonmammalian/drug effects , Humans , Neovascularization, Pathologic/drug therapy , Neovascularization, Physiologic/drug effects , Pyridines/pharmacology , Thyroid Neoplasms/blood supply , Thyroid Neoplasms/pathology , Zebrafish/embryologyABSTRACT
INTRODUCTION: Somatostatin and dopamine (DA) receptors have a pivotal role in controlling hormone secretion and cell proliferation in different neuroendocrine neoplasms, including medullary thyroid cancer (MTC). In the present preclinical study, we evaluated the anti-tumor activity of TBR-065 (formerly BIM-23B065), a second-generation somatostatin-DA chimera, in 2 human MTC cell lines. METHODS: The effects of lanreotide (LAN) and TBR-065 on cell growth and proliferation, calcitonin (CT) secretion, cell cycle, apoptosis, cell migration, and tumor-induced angiogenesis have been evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA flow cytometry with propidium iodide (PI), Annexin V-FITC/PI staining, electrochemiluminescence immuno assay, wound-healing assay, and zebrafish platform, respectively. RESULTS: TBR-065 exerted a more prominent anti-tumor activity than LAN in both MTC cell lines, as shown by inhibition of cell proliferation (maximal inhibition in TT: -50.3 and -37.6%, respectively; in MZ-CRC-1: -58.8 and -27%, respectively) and migration (in TT: -42.7 and -22.9%, respectively; in MZ-CRC-1: -75.5 and -58.2%, respectively). Only the new chimera decreased significantly the fraction of cells in S phase (TT: -33.8%; MZ-CRC-1: -18.8%) and increased cells in G2/M phase (TT: +13%; MZ-CRC-1: +30.5%). In addition, TBR-065 exerted a more prominent pro-apoptotic effect than LAN in TT cells. A concomitant decrease in CT secretion was observed after 2 days of incubation with both drugs, with a more relevant effect of TBR-065. However, neither LAN nor TBR-065 showed any effect on tumor-induced angiogenesis, as evaluated using a zebrafish/tumor xenograft model. DISCUSSION/CONCLUSION: In MTC cell lines, a second-generation somatostatin-DA analog, TBR-065, exerts a more relevant anti-tumor activity than LAN, through modulation of cell cycle, induction of apoptosis, and reduction in migration. Further studies are required to establish whether TBR-065 has comparable potent inhibitory effects on tumor growth in vivo.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Neuroendocrine/drug therapy , Dopamine/analysis , Somatostatin/analysis , Thyroid Neoplasms/drug therapy , Cell Line, Tumor , HumansABSTRACT
Neuroendocrine tumors (NETs) are a class of rare and heterogeneous neoplasms that originate from the neuroendocrine system. In several cases, these neoplasms can release bioactive hormones leading to characteristic clinical syndromes and hormonal dysregulations with detrimental impact on the quality of life and survival of these patients. Only few animal models are currently available to investigate pathogenesis, progression and functional syndromes in NETs and to identify new therapeutic strategies. The tropical teleost zebrafish (Danio rerio) is a popular vertebrate model system that offers unique advantages for the study of several biological processes, ranging from embryonic development to human diseases such as cancer. In this review, we summarize recent advances on zebrafish models for NET preclinical research that take advantage of modern genetic and transplantable technologies. In the future, these tools may have a role in the treatment decision-making and tertiary prevention of NETs.
Subject(s)
Neuroendocrine Tumors/pathology , Animals , Humans , ZebrafishABSTRACT
Neuroendocrine neoplasms (NENs) are a group of tumors originating from the neuroendocrine system. They mainly occur in the digestive system and the respiratory tract. It is well-know a strict interaction between neuroendocrine system and inflammation, which can play an important role in NEN carcinogenesis. Inflammatory mediators, which are produced by the tumor microenvironment, can favor cancer induction and progression, and can promote immune editing. On the other hand, a balanced immune system represents a relevant step in cancer prevention through the elimination of dysplastic and cancer cells. Therefore, an inflammatory response may be both pro- and anti-tumorigenic. In this review, we provide an overview concerning the complex interplay between inflammation and gastroenteropancreatic NENs, focusing on the tumorigenesis and clinical implications in these tumors.
Subject(s)
Gastrointestinal Neoplasms/immunology , Inflammation Mediators/immunology , Inflammation/immunology , Intestinal Neoplasms/immunology , Neuroendocrine Tumors/immunology , Pancreatic Neoplasms/immunology , Stomach Neoplasms/immunology , Cytokines/metabolism , Gastrointestinal Neoplasms/pathology , Humans , Inflammation/metabolism , Inflammation Mediators/metabolism , Intestinal Neoplasms/pathology , Neuroendocrine Tumors/pathology , Pancreatic Neoplasms/pathology , Stomach Neoplasms/pathology , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Glycogen Storage Disease Type II/drug therapy , Glycogen/metabolism , Hexokinase/antagonists & inhibitors , Pyruvates/pharmacology , Animals , Animals, Genetically Modified , Autophagy/drug effects , Drug Evaluation, Preclinical , Gene Knockdown Techniques , Glycogen Storage Disease Type II/genetics , Glycogen Storage Disease Type II/pathology , Glycolysis/drug effects , Hexokinase/metabolism , Humans , Lysosomes , Microscopy, Electron , Morpholinos/administration & dosage , Morpholinos/genetics , Motor Activity/drug effects , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Muscle, Skeletal/ultrastructure , Pyruvates/therapeutic use , Zebrafish , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , alpha-Glucosidases/genetics , alpha-Glucosidases/metabolismABSTRACT
Neuroendocrine neoplasms (NENs) are traditionally considered as a single group of rare malignancies that originate from the highly spread neuroendocrine system. The clinical management is complex due to the high heterogeneity of these neoplasms in terms of clinical aggressiveness and response to the therapy. Indeed, a multidisciplinary approach is required to reach a personalization of the therapy, including cancer rehabilitation. In this review, we discuss the possibility to adopt a precision medicine (PM) approach in the management of NENs. To this purpose, we summarize current knowledge and future perspectives about biomarkers and preclinical in vitro and in vivo platforms, potentially useful to inform clinicians about the prognosis and for tailoring therapy in patients with NENs. This approach may represent a breakthrough in the therapy and tertiary prevention of these tumors.
Subject(s)
Endocrinology/trends , Neuroendocrine Tumors/therapy , Precision Medicine/trends , Forecasting , Humans , Patient Care Team/trendsABSTRACT
SMYD3 is a methylase previously linked to cancer cell invasion and migration. Here we show that SMYD3 favors TGFß-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 TGFß-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 TGFß-dependent mesenchymal gene expression and cell migration in breast cancer, and support SMYD3 as a promising pharmacological target for anti-cancer therapy.
Subject(s)
Breast Neoplasms/genetics , Histone-Lysine N-Methyltransferase/genetics , Smad3 Protein/genetics , Transforming Growth Factor beta/genetics , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement/genetics , Chromatin/drug effects , Epithelial-Mesenchymal Transition/drug effects , Female , Gene Knockdown Techniques , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Humans , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Phosphorylation , Signal Transduction/drug effects , Xenograft Model Antitumor Assays , ZebrafishABSTRACT
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.
Subject(s)
Embryonic Development/genetics , Endoplasmic Reticulum-Associated Degradation/genetics , Endoplasmic Reticulum/genetics , Ubiquitin-Protein Ligases/genetics , Animals , Blood Vessels/growth & development , Blood Vessels/metabolism , Endoplasmic Reticulum/metabolism , Endothelium/cytology , Gene Expression Regulation, Developmental/genetics , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish Proteins/geneticsABSTRACT
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.
Subject(s)
Cell Proliferation , Endothelial Cells/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Neovascularization, Physiologic , Receptors, Notch/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Base Sequence , Conserved Sequence , Gene Expression Regulation, Developmental , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Receptors, Notch/genetics , Signal Transduction , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
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.
Subject(s)
Gene Expression Regulation, Developmental , Osteogenesis/genetics , Phosphoproteins/genetics , Skull/metabolism , Zebrafish Proteins/genetics , Zebrafish/genetics , Amino Acid Sequence , Animals , Calcification, Physiologic/genetics , Cloning, Molecular , Embryo, Mammalian , Morpholinos/genetics , Morpholinos/metabolism , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Skull/growth & development , Zebrafish/embryology , Zebrafish/metabolism , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/metabolismABSTRACT
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 µm. 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.