Morpholino knockdown of the ubiquitously expressed transmembrane serine protease TMPRSS4a in zebrafish embryos exhibits severe defects in organogenesis and cell adhesion

2011 ◽  
Vol 392 (7) ◽  
Author(s):  
Anke Ohler ◽  
Christoph Becker-Pauly
Keyword(s):  
Cell Differentiation ◽  
Serine Protease ◽  
Vascular System ◽  
Human Cancer ◽  
Zebrafish Embryos ◽  
Tissue Development ◽  
Rt Pcr ◽  
Morpholino Knockdown ◽  
Transmembrane Serine Protease

Abstract Over the past years the members of the type II transmembrane serine protease (TTSP) family have emerged as new players in mammalian biology. TMPRSS4 (transmembrane protease/serine) is overexpressed in several human cancer tissues, promoting invasion, migration, and metastasis. However, the physiological function has not yet been elucidated. Here, we present morpholino knockdown studies targeting TMPRSS4a, a homolog of human TMPRSS4 in zebrafish embryos. By RT-PCR, we could demonstrate an expression of this protease already 5 h post-fertilization, suggesting important functions in the early stages of embryonic development. Indeed, in vivo gene silencing caused severe defects in tissue development and cell differentiation including a disturbed skeletal muscle formation, a decelerated heartbeat, and a degenerated vascular system. Scanning electron microscopy revealed strong defects in epidermal skin organization, with clearly altered cell-cell contacts, resulting in the detachment of keratinocytes from the underneath tissue. The disturbed organogenesis in general is consistent with RT-PCR results which exhibited a ubiquitous expression of TMPRSS4a, predominantly in kidney, skin, heart, and gills. Our results demonstrate the importance of TMPRSS4a in tissue development and cell differentiation. Whether its proteolytic activity is directed towards adhesion molecules or leads to the activation of other proteases needs to be investigated further.

scholarly journals Phenotypic Analysis of Mice Lacking the Tmprss2-Encoded Protease

2006 ◽  
Vol 26 (3) ◽  
pp. 965-975 ◽  
Author(s):  
Tom S. Kim ◽  
Cynthia Heinlein ◽  
Robert C. Hackman ◽  
Peter S. Nelson
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Biological Activities ◽  
Type Ii ◽  
Phenotypic Analysis ◽  
Normal Prostate ◽  
Prostate Hyperplasia ◽  
Transmembrane Serine Protease

ABSTRACT Tmprss2 encodes an androgen-regulated type II transmembrane serine protease (TTSP) expressed highly in normal prostate epithelium and has been implicated in prostate carcinogenesis. Although in vitro studies suggest protease-activated receptor 2 may be a substrate for TMPRSS2, the in vivo biological activities of TMPRSS2 remain unknown. We generated Tmprss2 −/− mice by disrupting the serine protease domain through homologous recombination. Compared to wild-type littermates, Tmprss2 −/− mice developed normally, survived to adulthood with no differences in protein levels of prostatic secretions, and exhibited no discernible abnormalities in organ histology or function. Loss of TMPRSS2 serine protease activity did not influence fertility, reduce survival, result in prostate hyperplasia or carcinoma, or alter prostatic luminal epithelial cell regrowth following castration and androgen replacement. Lack of an observable phenotype in Tmprss2 −/− mice was not due to transcriptional compensation by closely related Tmprss2 homologs. We conclude that the lack of a discernible phenotype in Tmprss2 −/− mice suggests functional redundancy involving one or more of the type II transmembrane serine protease family members or other serine proteases. Alternatively, TMPRSS2 may contribute a specialized but nonvital function that is apparent only in the context of stress, disease, or other systemic perturbation.

Zebrafish Xenotransplantation and Focused Chemical Genomics: A Preclinical Therapeutic Model For T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2670-2670
Author(s):  
Victoria L Bentley ◽  
Chansey J Veinotte ◽  
Dale Corkery ◽  
Marissa A Leblanc ◽  
Karen Bedard ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Human Cancer ◽  
Lymphoblastic Leukemia ◽  
Zebrafish Embryos ◽  
Chemical Genomics ◽  
Cell Acute Lymphoblastic Leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subset of ALL, for which there is a need for new therapeutic strategies and efficient preclinical screening methods. We have pioneered an innovative zebrafish human cancer xenotransplantation (XT) model to examine drug-tumor interactions in vivo. T-ALL cell lines and primary patient T-ALL samples were microinjected into 48-hour zebrafish embryos, a stage at which the adaptive immune system has not yet developed. Fluorescent labelling of tumor cells prior to injection and use of casper pigment mutant fish facilitates evaluation of drug response both by direct observation in transparent fish and enumeration of human cells following embryo dissociation. Proliferation rates are rapidly determined by directly counting fluorescent cells using in silico-based programs and/or utilizing immunohistochemical approaches to distinguish human cancer cells from host cell populations. T-ALL cell lines harboring defined mutations in the NOTCH1, phosphoinositide 3-kinase (PI3K)/AKT and mTOR pathways differentially responded to targeted inhibition using the γ-secretase inhibitor Compound E, triciribine, and rapamycin, when xenografted into embryos, consistent with responses in vitro. Primary patient-derived T-ALL bone marrow samples similarly engrafted and proliferated in zebrafish embryos. Using this in vivo chemical genomic approach, a targetable mutation sensitive to γ-secretase inhibition was identified from the diagnostic bone marrow sample of a child with T-ALL, which was confirmed by exome Sanger sequencing, and validated as a gain-of-function mutation in the NOTCH1 gene by luciferase assay and Western blot. Focused chemical genomics using the zebrafish T-ALL XT model provides a means of tailoring therapy using a real time in vivo assay that more accurately recapitulates the tumor microenvironment than in vitro methods and more rapidly than mouse xenografts. Moreover, the efficiency and cost-effectiveness of this innovative platform provides a novel intermediary for the prioritization of much-needed drug candidates in the preclinical pipeline. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Extra-Intestinal Effects of C. difficile Toxin A and B: An In Vivo Study Using the Zebrafish Embryo Model

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2575
Author(s):  
Federica Tonon ◽  
Stefano Di Bella ◽  
Gabriele Grassi ◽  
Roberto Luzzati ◽  
Paolo Ascenzi ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Zebrafish Embryo ◽  
Vascular System ◽  
Morphological Changes ◽  
Vital Signs ◽  
Zebrafish Embryos ◽  
Toxin A ◽  
Cardiovascular Damage ◽  
Starting Point

C.difficile infection (CDI) is not a merely “gut-confined” disease as toxemia could drive the development of CDI-related extra-intestinal effects. These effects could explain the high CDI-associated mortality, not just justified by diarrhea and dehydration. Here, the extra-intestinal effects of toxin A (TcdA) and B (TcdB) produced by C. difficile have been studied in vivo using the zebrafish embryo model. Noteworthy, protective properties of human serum albumin (HSA) towards toxins-induced extra-intestinal effects were also addressed. Zebrafish embryos were treated with TcdA, TcdB and/or HSA at 24 h post-fertilization. Embryos were analyzed for 48 h after treatment to check vital signs and morphological changes. Markers related to cardio-vascular damage and inflammation were evaluated by Real-Time quantitative PCR and/or western blotting. Both toxins induced cardiovascular damage in zebrafish embryos by different mechanisms: (i) direct toxicity (i.e., pericardial edema, cardiac chambers enlargement, endothelial alteration); (ii) increased hormonal production and release (i.e., atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)), (iii) alteration of the vascular system through the increase of the vascular endothelial growth factor (VEGF-A) levels, as well as of its receptors, (iv) pro-inflammatory response through high cytokines production (i.e., CXCL8, IL1B, IL6 and TNFα) and (v) cell-mediated damage due to the increase in neutrophils number. In addition to cardiovascular damage, we observe skin alteration and inflammation. Finally, our data indicate a protective effect of HSA toward the toxins induced extra-intestinal effects. Together, our findings can serve as a starting point for humans’ studies to substantiate and understand the extra-intestinal effects observed in CDI patients.

Human Leukemia Xenotransplantation Into Zebrafish Embryos as An In Vivo Assay for Chemotherapy Drug Response

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1704-1704
Author(s):  
Dale Corkery ◽  
Graham Dellaire ◽  
Jason N Berman
Keyword(s):  
Human Cancer ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Therapeutic Window ◽  
Zebrafish Embryos ◽  
Human Leukemia ◽  
Proliferation Assay ◽  
Human Leukemia Cells ◽  
Anti Cancer

Abstract Abstract 1704 A major challenge in the treatment of human cancer is the variable response to therapy between patients. Any host or tumor-derived factors that produce variations in drug concentration, duration of exposure, or sensitivity of tumour cell populations to drug, may contribute to heterogeneity in the tumour response. The identification and characterization of specific drug-tumor interactions would enable the design of more personalized and targeted treatment with improved outcomes and reduced toxicity. To date, human cancer cell lines and murine models have provided some advances, however these approaches have been hindered by technical limitations, considerable expense and a lack of timely information to directly impact on a given therapeutic window. The zebrafish has emerged as a robust animal model of human malignancy due to conserved genetics and cell biology. In addition, their transparency provides exceptional opportunities for in vivo imaging, such as the unique ability to directly visualize the response of cancer cells to drugs in real time. Using zebrafish as a host organism, we have been developing a quantitative cell proliferation assay to monitor the response of human leukemia cells to anti-cancer agents in an environment that more closely recapitulates the human tumor microenvironment. As proof of principle, we have injected boluses of 50 fluorescently-labeled cells from the human K562 chronic myeloid leukemia cell (CML) line into the yolk sac of 48 hour old casper embryos, a combinatorial zebrafish mutant lacking both melanocytes and iridophores, thus enabling facile cell tracking and imaging. Injected embryos tolerate the presence of human leukemia cells for up to 7 days, during which time the engrafted leukemia cells proliferate and circulate within the embryonic bloodstream. Proliferation of the leukemia cells can be monitored by live-cell microscopy of engrafted embryos and quantified by their dissociation to a single cell suspension at 24 and 72 hours post-injection. Specifically, 20 embryos are dissociated at each time point and the number of leukemia cells is determined from fluorescent micrographs using a semi-automated cell quantification macro executed in ImageJ (NIH). Using this proliferation assay, we observed a reproducible increase in leukemia cell numbers within the embryo of approximately 5-fold after 48 hours. Furthermore, the proliferation of K562 cells in xenotransplanted zebrafish embryos could be significantly inhibited (by 45 ± 3% relative to untreated; p<0.001) following a 48 hour treatment with 20 μM of imatinib mesylate (Gleevec), a drug that targets the characteristic CML BCR-ABL translocation gene product harboured by these cells. In contrast, treatment with a second agent, all trans-retinoic acid (ATRA) that targets the PML-RARA fusion protein found in acute promyelocytic leukemia (APL), had no impact on proliferation. These results validate the use of zebrafish xenotransplantation in studying the response of human leukemia cells to anti-cancer agents and position this model as a unique in vivo tool to determine the sensitivity of primary patient tumor samples to current and novel chemotherapeutics. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Modified Riceberry rice extract suppresses melanogenesis-associated cell differentiation through tyrosinase-mediated MITF downregulation on B16 cells and in vivo zebrafish embryos

2020 ◽  
Vol 15 (5) ◽  
pp. 491
Author(s):  
Teerapat Rodboon ◽  
Sasithorn Sirilun ◽  
Seiji Okada ◽  
Ryusho Kariya ◽  
Thapana Chontananarth ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Zebrafish Embryos

In-vitro- und In-vivo-Wirkung von Schilddrüsenhormon auf das Wachstum von Neuroblastomzellen

1990 ◽  
Vol 29 (03) ◽  
pp. 120-124
Author(s):  
R. P. Baum ◽  
E. Rohrbach ◽  
G. Hör ◽  
B. Kornhuber ◽  
E. Busse
Keyword(s):  
Cell Differentiation ◽  
Neuroblastoma Cells ◽  
Control Group ◽  
Initial Value ◽  
Initial Rise ◽  
Biphasic Effect ◽  
Contrast Microscopy ◽  
Morphological Sign

The effect of triiodothyronine (T3) on the differentiation of cultured neuroblastoma (NB) cells was studied after 9 days of treatment with a dose of 10-4 M/106 cells per day. Using phase contrast microscopy, 30-50% of NB cells showed formation of neurites as a morphological sign of cellular differentiation. The initial rise of the mitosis rate was followed by a plateau. Changes in cyclic nucleotide content, in the triphosphates and in the activity of the enzyme ornithine decarboxylase (ODC) were assessed in 2 human and 2 murine cell lines to serve as biochemical parameters of the cell differentiation induced by T3. Whereas the cAMP level increased significantly (3 to 7 fold compared with its initial value), the cGMP value dropped to 30 to 50% of that of the control group. ATP and GTP increased about 200%, the ODC showed a decrease of about 50%. The present studies show a biphasic effect of T3 on neuroblastoma cells: the initial rise of mitotic activity is followed by increased cell differentiation starting from day 4 of the treatment.

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