Myocardial Afterload Is a Key Biomechanical Regulator of Atrioventricular Myocyte Differentiation in Zebrafish

Heart valve development is governed by both genetic and biomechanical inputs. Prior work has demonstrated that oscillating shear stress associated with blood flow is required for normal atrioventricular (AV) valve development. Cardiac afterload is defined as the pressure the ventricle must overcome in order to pump blood throughout the circulatory system. In human patients, conditions of high afterload can cause valve pathology. Whether high afterload adversely affects embryonic valve development remains poorly understood. Here we describe a zebrafish model exhibiting increased myocardial afterload, caused by vasopressin, a vasoconstrictive drug. We show that the application of vasopressin reliably produces an increase in afterload without directly acting on cardiac tissue in zebrafish embryos. We have found that increased afterload alters the rate of growth of the cardiac chambers and causes remodeling of cardiomyocytes. Consistent with pathology seen in patients with clinically high afterload, we see defects in both the form and the function of the valve leaflets. Our results suggest that valve defects are due to changes in atrioventricular myocyte signaling, rather than pressure directly acting on the endothelial valve leaflet cells. Cardiac afterload should therefore be considered a biomechanical factor that particularly impacts embryonic valve development.

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Lignans from Bursera fagaroides Affect In Vivo Cell Behavior by Disturbing the Tubulin Cytoskeleton in Zebrafish Embryos

Molecules ◽

10.3390/molecules24010008 ◽

2018 ◽

Vol 24 (1) ◽

pp. 8 ◽

Cited By ~ 2

Author(s):

Mayra Antúnez-Mojica ◽

Andrés Rojas-Sepúlveda ◽

Mario Mendieta-Serrano ◽

Leticia Gonzalez-Maya ◽

Silvia Marquina ◽

...

Keyword(s):

Cell Migration ◽

Biological Effects ◽

In Vitro Models ◽

In Vivo Model ◽

Zebrafish Embryos ◽

Microtubule Cytoskeleton ◽

Zebrafish Model ◽

Bioassay Guided Fractionation

By using a zebrafish embryo model to guide the chromatographic fractionation of antimitotic secondary metabolites, seven podophyllotoxin-type lignans were isolated from a hydroalcoholic extract obtained from the steam bark of Bursera fagaroides. The compounds were identified as podophyllotoxin (1), β-peltatin-A-methylether (2), 5′-desmethoxy-β-peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7). The biological effects on mitosis, cell migration, and microtubule cytoskeleton remodeling of lignans 1–7 were further evaluated in zebrafish embryos by whole-mount immunolocalization of the mitotic marker phospho-histone H3 and by a tubulin antibody. We found that lignans 1, 2, 4, and 7 induced mitotic arrest, delayed cell migration, and disrupted the microtubule cytoskeleton in zebrafish embryos. Furthermore, microtubule cytoskeleton destabilization was observed also in PC3 cells, except for 7. Therefore, these results demonstrate that the cytotoxic activity of 1, 2, and 4 is mediated by their microtubule-destabilizing activity. In general, the in vivo and in vitro models here used displayed equivalent mitotic effects, which allows us to conclude that the zebrafish model can be a fast and cheap in vivo model that can be used to identify antimitotic natural products through bioassay-guided fractionation.

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High-resolution cardiovascular function confirms functional orthology of myocardial contractility pathways in zebrafish

Physiological Genomics ◽

10.1152/physiolgenomics.00206.2009 ◽

2010 ◽

Vol 42 (2) ◽

pp. 300-309 ◽

Cited By ~ 44

Author(s):

Jordan T. Shin ◽

Eugene V. Pomerantsev ◽

John D. Mably ◽

Calum A. MacRae

Keyword(s):

High Speed ◽

Dynamic Range ◽

Model Organism ◽

Cardiovascular Physiology ◽

Zebrafish Embryos ◽

Cardiovascular Development ◽

Zebrafish Model ◽

Ventricular Performance ◽

Pharmacological Agents

Phenotype-driven screens in larval zebrafish have transformed our understanding of the molecular basis of cardiovascular development. Screens to define the genetic determinants of physiological phenotypes have been slow to materialize as a result of the limited number of validated in vivo assays with relevant dynamic range. To enable rigorous assessment of cardiovascular physiology in living zebrafish embryos, we developed a suite of software tools for the analysis of high-speed video microscopic images and validated these, using established cardiomyopathy models in zebrafish as well as modulation of the nitric oxide (NO) pathway. Quantitative analysis in wild-type fish exposed to NO or in a zebrafish model of dilated cardiomyopathy demonstrated that these tools detect significant differences in ventricular chamber size, ventricular performance, and aortic flow velocity in zebrafish embryos across a large dynamic range. These methods also were able to establish the effects of the classic pharmacological agents isoproterenol, ouabain, and verapamil on cardiovascular physiology in zebrafish embryos. Sequence conservation between zebrafish and mammals of key amino acids in the pharmacological targets of these agents correlated with the functional orthology of the physiological response. These data provide evidence that the quantitative evaluation of subtle physiological differences in zebrafish can be accomplished at a resolution and with a dynamic range comparable to those achieved in mammals and provides a mechanism for genetic and small-molecule dissection of functional pathways in this model organism.

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From cushions to leaflets: morphogenesis of cardiac atrioventricular valves

10.1093/med/9780198757269.003.0017 ◽

2018 ◽

Author(s):

Donal MacGrogan ◽

José Maria Pérez-Pomares ◽

Bill Chaudhry ◽

José Luis de la Pompa ◽

Deborah J. Henderson

Keyword(s):

Heart Development ◽

Cellular Proliferation ◽

Valve Disease ◽

Signalling Pathways ◽

Valve Leaflet ◽

Endocardial Cushions ◽

Developmental Defects ◽

Mesenchymal Transition ◽

Valve Development ◽

Developmental Signalling

At the looping stage of heart development, tissue patterning of myocardium and endocardium at the atrioventricular (AV) junction defines a morphogenic field competent to form valves that initially appear as protrusions of proteoglycan-rich extracellular matrix (ECM) called endocardial cushions (ECs) which are cellularized by an endocardial-mesenchymal transition (EMT). Cellular proliferation results in fusion of the major AV mesenchymal cushions and AV septation, whereas smaller cushions receive a supply from epicardially derived cells. These various sources of mesenchyme precursors give rise to most of the valve structures, leaflets, annuli, and supporting tension apparatus. During valve leaflet maturation, the ECM matrix accumulates collagen and elastin and assembles into a thin flexible fibrous structure, which is remarkably tough. Valve development is regulated by the cross-talk between developmental signalling pathways. Pathogenic mutations in a subset of developmentally important genes have been linked to valve disease, suggesting that developmental defects may underlie valve disease in adulthood.

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The Genome-Wide Impact of Nipblb Loss-of-Function on Zebrafish Gene Expression

International Journal of Molecular Sciences ◽

10.3390/ijms21249719 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9719

Author(s):

Marco Spreafico ◽

Eleonora Mangano ◽

Mara Mazzola ◽

Clarissa Consolandi ◽

Roberta Bordoni ◽

...

Keyword(s):

Gene Expression ◽

Developmental Stages ◽

System Development ◽

Expression Patterns ◽

Nervous System Development ◽

Zebrafish Embryos ◽

Loss Of Function ◽

Zebrafish Model ◽

Genome Wide ◽

Transcriptional Effect

Transcriptional changes normally occur during development but also underlie differences between healthy and pathological conditions. Transcription factors or chromatin modifiers are involved in orchestrating gene activity, such as the cohesin genes and their regulator NIPBL. In our previous studies, using a zebrafish model for nipblb knockdown, we described the effect of nipblb loss-of-function in specific contexts, such as central nervous system development and hematopoiesis. However, the genome-wide transcriptional impact of nipblb loss-of-function in zebrafish embryos at diverse developmental stages remains under investigation. By RNA-seq analyses in zebrafish embryos at 24 h post-fertilization, we examined genome-wide effects of nipblb knockdown on transcriptional programs. Differential gene expression analysis revealed that nipblb loss-of-function has an impact on gene expression at 24 h post fertilization, mainly resulting in gene inactivation. A similar transcriptional effect has also been reported in other organisms, supporting the use of zebrafish as a model to understand the role of Nipbl in gene regulation during early vertebrate development. Moreover, we unraveled a connection between nipblb-dependent differential expression and gene expression patterns of hematological cell populations and AML subtypes, enforcing our previous evidence on the involvement of NIPBL-related transcriptional dysregulation in hematological malignancies.

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Infection-induced miR-126 suppresses tsc1- and cxcl12a-dependent permissive macrophages during mycobacterial infection

10.1101/2021.10.07.463594 ◽

2021 ◽

Author(s):

Kathryn Wright ◽

Kumudika de Silva ◽

Karren M. Plain ◽

Auriol C. Purdie ◽

Warwick J. Britton ◽

...

Keyword(s):

Immune Response ◽

Detrimental Effect ◽

Mycobacterial Infection ◽

Protective Role ◽

Zebrafish Embryos ◽

Zebrafish Model ◽

Effector Pathways ◽

The Impact ◽

Mtor Signalling

AbstractRegulation of host microRNA (miRNA) expression is a contested node that controls the host immune response to mycobacterial infection. The host must overcome concerted subversive efforts of pathogenic mycobacteria to launch and maintain a protective immune response. Here we examine the role of miR-126 in the zebrafish model of Mycobacterium marinum infection and identify a protective role for this infection-induced miRNA through multiple effector pathways. Specifically, we analyse the impact of the miR-126 knockdown-induced tsc1a and cxcl12a/ccl2/ccr2 signalling axes during early host-M. marinum interactions. We find a strong detrimental effect of tsc1a upregulation that renders zebrafish embryos susceptible to higher bacterial burden and increased cell death despite dramatically higher recruitment of macrophages to the site of infection. We demonstrate that infection-induced miR-126 suppresses tsc1 and cxcl12a expression thus improving macrophage function early in infection, partially through activation of mTOR signalling and strongly through preventing the recruitment of Ccr2+ permissive macrophages, resulting in the recruitment of protective tnfa-expressing macrophages. Together our results demonstrate an important role for infection-induced miR-126 in shaping an effective immune response to M. marinum infection in zebrafish embryos.

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Teratogenic potential of traditional Malaysian vegetables (ulam) in the zebrafish model

British Food Journal ◽

10.1108/bfj-02-2020-0118 ◽

2020 ◽

Vol 122 (10) ◽

pp. 3089-3098

Author(s):

Alya Hanisah Omar ◽

Chin Siang Kue ◽

Roza Dianita ◽

Ke-Xin Yu

Keyword(s):

Local Community ◽

Lethal Effect ◽

Negative Control ◽

Teratogenic Effect ◽

Zebrafish Embryos ◽

Zebrafish Model ◽

Fish Embryo ◽

Content Type ◽

Cancer Pathways ◽

Teratogenic Potential

PurposeTraditional Malaysian vegetables, also known as ulam, are believed to have healing properties among the local community. Ulam is commonly reported to have high antioxidant content which makes it a popular food. The purpose of this paper is to determine the teratogenic potential of eight ulam, using zebrafish model. The tested ulam were Cosmos caudatus, Gynura procumbens, Labisia pumila var. alata, Phaleria macrocarpa, Polygonum minus, Piper sarmentosum, Premna foetida and Sauropus androgynous.Design/methodology/approachMethanol extract of ulam was prepared using the maceration method. Various concentrations of extracts were tested against fish embryo short-term toxicity test. The lethal concentration (LC50) and teratogenic effect of the ulam were determined.FindingsAmong all tested species, L. pumila, P. foetida and S. androgynous showed 100% lethal effect towards zebrafish embryos at concentrations of 10 µg/mL, 1,000 µg/mL and 100 µg/mL, respectively. The three ulam have exhibited teratogenic effect on zebrafish embryos after 72 h post-fertilization. L. pumila had induced yolk sac edema at 1.0 µg/mL for normalized measurement of 108.3 ± 2.0% (which is higher than negative control, p < 0.05, median = 110.7%), while P. foetida had induced pericardial edema at 100 µg/mL for normalized measurement of 124.0 ± 4.6% (which is higher than negative control, p < 0.05, median = 124.3%). On the other hand, S. androgynus induced curve trunk at 30 µg/mL for the presence of 70.9 ± 4.2%.Originality/valueThe teratogenic effect of L. pumila, P. foetida and S. androgynous suggests the possible disruption in the embryogenesis in zebrafish, namely Notch, vascular endothelial growth factor (VEGF) and retinoic acid pathways. The results of ulam gave possible implications and insights on the cancer pathways involved, which could be a useful target for cancer research. This is the first report on teratogenicity evaluation of Malaysian ulam showing relationship to cancer pathways by using zebrafish embryo model.

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A New Model for Studying the Dissemination of Myeloma Cells throughout the Bone Marrow Using Embryonic Zebrafish

Blood ◽

10.1182/blood.v126.23.915.915 ◽

2015 ◽

Vol 126 (23) ◽

pp. 915-915

Author(s):

Aldo M Roccaro ◽

Antonio Sacco ◽

Dongdong Ma ◽

Jiantao Shi ◽

Yuji Mishima ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Zebrafish Embryos ◽

Zebrafish Model ◽

Hematopoietic Stem ◽

Myeloma Cells ◽

Cell Homing ◽

Qrt Pcr ◽

Hematopoietic Niche

Abstract Multiple myeloma develops from a pre-malignant clonal proliferation of plasma cells. The dissemination of myeloma cells throughout the bone marrow (BM) is an important early step in myeloma pathogenesis. Studies of myeloma cell homing in mouse models are not quantitative and cannot be used for functional genomics or drug screening. To overcome these limitations, we have developed a novel in vivo model to assess multiple myeloma (MM) cell homing, that takes advantage of the optical clarity of zebrafish (Danio rerio) embryos. We performed intra-cardiac (i.c.) injection of GFP+MM.1S cells into zebrafish embryos 48 hpf, and assessed the ability of the injected cells to enter the peripheral circulation and then traced their homing to the trunk region of zebrafish embryos, an area referred as the caudal hematopoietic tissue (CHT), by using intra-vital confocal microscopy. We next injected primary CD138+ cells derived from MM patient BM that had been stained with either DiO or DiD and demonstrated that they also homed to CHT. As a control, we injected DiO-labeled CD41-GFPlow zebrafish hematopoietic stem cells (HSCs) that are known to home to the CHT hematopoietic niche; and observed that zebrafish-derived CD41-HSCs homed to the same area as MM cells. We next analyzed changes in the transcriptome of those MM cells that homed to the CHT-niche. We dissected the zebrafish embryos to separate the CHT from other tissue containing non-adherent MM cells and performed whole human exome enrichment prior to sequencing of total RNA. We had an alignment rate of 10-15%, with a high intragenic rate an exonic rate (> 95%) and a low mismatch rate (~0.5%). RNAseq revealed that the MM cells that homed to the CHT were enriched in transcripts important for cytokine/chemokine mediated signaling, the IL-6 signalling pathway, cell-cell adhesion and angiogenesis (FDR<0.25; P<0.05). Overall, these findings indicate that the changes observed in MM cells that have homed to the CHT mirror those that are seen in MM cells that are resident in the human BM. In order to investigate the functional relevance of the zebrafish model, we established CXCR4-, VLA-4- and FAK-silenced MM cells and compared their ability to home to CHT to that of control scrambled shRNA-transfected cells. DiO-labeled-CXCR4-silenced and DiO-labeled-scrambled-probe control MM cells were mixed in equal numbers and subsequently injected into recipient zebrafish. We found a significant reduction in the number of CXCR4-silenced MM cells homing to the CHT, compared to the control cells (P<0.00). We then examined VLA-4- and FAK-knock-down MM cells and observed that the homing of MM cells to CHT was impaired when either VLA-4 or FAK were silenced (P<0.001; Fig. 3C-D). Having demonstrated the role of CXCF4, FAK and VLA4 in MM cell homing to the CHT niche, we next performed qRT-PCR for those transcripts and confirmed that MM cells harvested from the CHT areas expressed higher levels of CXCR4, FAK and VLA4, compared to MM cells harvested from non-CHT areas. (P<0.05). To ascertain whether homing to zebrafish embryo CHT is occurs in other hematologic malignancies that are known to home to the human and murine BM, we used a cultured cell lined derived from a patient with Waldenstrom's Macroglobulinemia (WM). We injected either CXCR4-overexpressing or CXCR4-silenced WM cells and found that increased CXCR4 expression in WM cells led to enhanced CHT-homing of WM cells (P<0.001), while the homing of CXCR4-silenced WM cells to the CHT was reduced compared to scrambled control (P<0.001). These findings demonstrate that zebrafish can be used to study the homing of human myeloma cells to a hematopoietic niche. The rapidity of homing to CHT, which occurs within seconds of cell injection, suggests that a fraction of the CD138+ harvested from patient bone marrow already express those RNA transcripts and proteins needed for stable adhesion and residence in CHT. This hypothesis is confirmed by the RNA seq and qRT-PCR studies which directly demonstrate increased expression of relevant transcripts in adherent cells. This zebrafish model may provide new insights into the pathogenesis of MM and may be useful as a means to screen for agents which can disrupt homing and dissemination of MM cells. Disclosures No relevant conflicts of interest to declare.

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Anticarcinogenic and Antioxidant Action of an Edible Aquatic Flora Jussiaea repens L. Using In Vitro Bioassays and In Vivo Zebrafish Model

Molecules ◽

10.3390/molecules26082291 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2291

Author(s):

Chongtham Rajiv ◽

Subhra Saikat Roy ◽

K. Tamreihao ◽

Pintubala Kshetri ◽

Thangjam Surchandra Singh ◽

...

Keyword(s):

Oxidative Stress ◽

Antiproliferative Activity ◽

Developmental Process ◽

In Vivo Studies ◽

Zebrafish Embryos ◽

Zebrafish Model ◽

Antioxidant Power ◽

Skov3 Cells

Oxidative stress is the major cause of many health conditions, and regular consumption of antioxidants helped to encounter and prevent such oxidative stress-related diseases. Due to safety concerns over long-term uses of synthetic antioxidants, natural antioxidants are more preferred. The purpose of this study is to investigate the antioxidant and anticancer activities of Jussiaea repens L., a wild edible flora found in Manipur, India. The antioxidant activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Ferric reducing antioxidant power (FRAP) assay and DNA-nicking assay. The anticancer activity was tested using five cancer lines viz., SKOV3 cells (ovarian), HeLa (cervical), MDA-MB-231 (breast), PANC-1 (pancreatic), and PC3 (prostate). The toxicity, developmental effect, antiproliferative activity was further tested using zebrafish embryos. The methanolic plant extract had higher polyphenol content than flavonoids. The in vitro study demonstrated a promising antioxidant capacity and DNA protection ability of this plant. The extract also showed cytotoxic activity against SKOV3, HeLa, MDA-MB-23, and PANC-1 cancer cell lines. The in vivo studies on zebrafish embryos demonstrated the extract’s ability to suppress the developmental process and elicited more cytotoxicity to cancer cells than developing zebrafish embryos. Moreover, the in vivo studies on zebrafish embryos also indicated the antiproliferative activity of J. repens L. extract.

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Loss of function mutations in CCDC32 cause a congenital syndrome characterized by craniofacial, cardiac and neurodevelopmental anomalies

Human Molecular Genetics ◽

10.1093/hmg/ddaa073 ◽

2020 ◽

Vol 29 (9) ◽

pp. 1489-1497 ◽

Cited By ~ 2

Author(s):

Tamar Harel ◽

John N Griffin ◽

Thomas Arbogast ◽

Tanner O Monroe ◽

Flavia Palombo ◽

...

Keyword(s):

Molecular Basis ◽

Congenital Malformations ◽

Mammalian Cell Culture ◽

Zebrafish Embryos ◽

Loss Of Function ◽

Zebrafish Model ◽

Functional Studies ◽

Whole Exome ◽

Cilia Formation ◽

Congenital Syndrome

Abstract Despite the wide use of genomics to investigate the molecular basis of rare congenital malformations, a significant fraction of patients remains bereft of diagnosis. As part of our continuous effort to recruit and perform genomic and functional studies on such cohorts, we investigated the genetic and mechanistic cause of disease in two independent consanguineous families affected by overlapping craniofacial, cardiac, laterality and neurodevelopmental anomalies. Using whole exome sequencing, we identified homozygous frameshift CCDC32 variants in three affected individuals. Functional analysis in a zebrafish model revealed that ccdc32 depletion recapitulates the human phenotypes. Because some of the patient phenotypes overlap defects common to ciliopathies, we asked if loss of CCDC32 might contribute to the dysfunction of this organelle. Consistent with this hypothesis, we show that ccdc32 is required for normal cilia formation in zebrafish embryos and mammalian cell culture, arguing that ciliary defects are at least partially involved in the pathomechanism of this disorder.

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SOX19b regulates the premature neuronal differentiation of neural stem cells through EZH2-mediated histone methylation in neural tube development of zebrafish

Stem Cell Research & Therapy ◽

10.1186/s13287-019-1495-3 ◽

2019 ◽

Vol 10 (1) ◽

Author(s):

Xian Li ◽

Wenjuan Zhou ◽

Xinyue Li ◽

Ming Gao ◽

Shufang Ji ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Neural Tube ◽

Neural Tube Defects ◽

Histone Methylation ◽

Zebrafish Embryos ◽

Loss Of Function ◽

Zebrafish Model ◽

Proliferation And Differentiation ◽

Neural Tube Development

Abstract Objective Neural tube defects (NTDs) are the most serious and common birth defects in the clinic. The SRY-related HMG box B1 (SoxB1) gene family has been implicated in different processes of early embryogenesis. Sox19b is a maternally expressed gene in the SoxB1 family that is found in the region of the presumptive central nervous system (CNS), but its role and mechanism in embryonic neural stem cells (NSCs) during neural tube development have not yet been explored. Considering that Sox19b is specific to bony fish, we intended to investigate the role and mechanism of Sox19b in neural tube development in zebrafish embryos. Material and methods Morpholino (MO) antisense oligonucleotides were used to construct a Sox19b loss-of-function zebrafish model. The phenotype and the expression of related genes were analysed by in situ hybridization and immunolabelling. Epigenetic modifications were detected by western blot and chromatin immunoprecipitation. Results In this study, we found that zebrafish embryos exhibited a reduced or even deleted forebrain phenotype after the expression of the Sox19b gene was inhibited. Moreover, we found for the first time that knockdown of Sox19b reduced the proliferation of NSCs; increased the transcription levels of Ngn1, Ascl1, HuC, Islet1, and cyclin-dependent kinase (CDK) inhibitors; and led to premature differentiation of NSCs. Finally, we found that knockdown of Sox19b decreased the levels of EZH2/H3K27me3 and decreased the level of H3K27me3 at the promoters of Ngn1 and ascl1a. Conclusion Together, our data demonstrate that Sox19b plays an essential role in early NSC proliferation and differentiation through EZH2-mediated histone methylation in neural tube development. This study established the role of transcription factor Sox19b and epigenetic factor EZH2 regulatory network on NSC development, which provides new clues and theoretical guidance for the clinical treatment of neural tube defects.

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