scholarly journals Coagulation Factor IIIa (f3a) Knockdown in Zebrafish Leads to Defective Angiogenesis and Mild Bleeding Phenotype

2021 ◽  
Author(s):  
Saravanan Subramaniam ◽  
Jiandong LU ◽  
Craig FLETCHER ◽  
Ramani Ramchandran ◽  
HARTMUT WEILER
Keyword(s):  
Embryonic Development ◽  
Developmental Stages ◽  
Vascular Development ◽  
Coagulation Factor ◽  
Zebrafish Model ◽  
Vascular Patterning ◽  
Spatiotemporal Expression ◽  
Molecular Phylogenetic ◽  
Transgenic Lines

Aim: Tissue factor (TF), an initiator of the extrinsic coagulation pathway, is crucial for embryogenesis, as mice lacking TF are embryonically lethal (E10.5). This lethality may be attributed to defects in vascular development and circulatory failure, suggesting additional roles for TF in embryonic development beyond coagulation. In this study, we characterized the role of one of the TF paralogs (f3a) using a zebrafish model. Methods: To understand the TF evolution across different species, we performed molecular phylogenetic and sequence homology analysis. The expression of f3a during embryonic developmental stages was determined by RT-PCR. Endothelial-specific transgenic lines of zebrafish (flk1:egfp-NLS/kdrl:mCherry-CAAX) was used to image the vascular development. The role of f3a during embryonic development was investigated by mRNA knockdown using Morpholinos (MO), an antisense-based oligonucleotide strategy. The f3a morphants were examined at 52 hpf for defects in morphological appearance, bleeding, and vascular patterning. Results: Spatiotemporal expression of f3a by qPCR revealed expression in all developmental stages, suggesting that f3a transcripts are both maternally and zygotically expressed. High expression of f3a from 28 hpf to 36 hpf confirmed the role of in the development of the yolk sac, circulation, and fins. f3a MO-injected embryos showed morphological abnormalities, including shorter body lengths and crooked tails. O-dianisidine staining showed f3a MO-injected embryos exhibited bleeding in the trunk (5.44%) and head (9.52%) regions. Using endothelial-specific transgenic lines of zebrafish (flk1:egfp-NLS/kdrl:mCherry-CAAX), imaging of caudal vein plexus, which forms immediately following the onset of circulation and sprouting, showed a 3-fold decrease in f3a morphants versus controls at 48 hpf, suggesting a potential role for f3a in flow-induced angiogenesis.

scholarly journals Deficiency of the ywhaz gene, involved in neurodevelopmental disorders, alters brain activity and behaviour in zebrafish

2021 ◽  
Author(s):  
Ester Anton-Galindo ◽  
Elisa Dalla Vecchia ◽  
Javier G Orlandi ◽  
Gustavo Castro ◽  
Emilio Gualda ◽  
...  
Keyword(s):  
Neural Development ◽  
Neurodevelopmental Disorders ◽  
Genome Engineering ◽  
Developmental Stages ◽  
Brain Activity ◽  
Autism Spectrum ◽  
Wild Type ◽  
Zebrafish Model ◽  
Risk Variants

Genetic risk variants in YWHAZ, encoding 14-3-ζ, have been found to contribute to psychiatric disorders such as autism spectrum disorder and schizophrenia, and have been related to an impaired neurodevelopment in humans and mice. Here, we have used a zebrafish model to further understand the mechanisms by which YWHAZ contribute to neurodevelopmental disorders. We first observed pan-neuronal expression of ywhaz during developmental stages, suggesting an important role of this gene in neural development. During adulthood ywhaz expression was restricted to Purkinje cells in the cerebellum, a region that shows alterations in autistic patients. We then established a novel stable ywhaz knockout (KO) zebrafish line using CRISPR/Cas9 genome engineering. We performed whole-brain calcium imaging in wild-type (WT) and ywhaz KO larvae and found altered neural activity and functional connectivity in the hindbrain. Interestingly, adult ywhaz KO fish also display decreased levels of dopamine and serotonin in the hindbrain and freeze when exposed to novel stimuli, a phenotype that can be reversed with fluoxetine and quinpirole, drugs that target serotonin and dopamine neurotransmission. Together, these findings suggest an important role for ywhaz in establishing neuronal connectivity during developmental stages. ywhaz deficiency leads to impaired dopamine and serotonin neurotransmission that may underlie the altered behaviour observed during adulthood.

ADAMTS13-/-zebrafish Model Created By Crispr/Cas Reveals a Novel Role of ADAMTS13 in Vascular Development and Prothrombotic Phenotype

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1446-1446
Author(s):  
Liang Zheng ◽  
X. Long Zheng
Keyword(s):  
Vascular Development ◽  
Fluorescent Microscopy ◽  
Premature Stop Codon ◽  
The Body ◽  
Fluorescence Signal ◽  
Wild Type ◽  
Zebrafish Model ◽  
Growth Environment ◽  
Caudal Vein

Abstract Background: Deficiency of plasma ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), a von Willebrand factor (VWF)-cleaving enzyme, in humans results in thrombotic thrombocytopenic purpura (TTP), a potentially fatal syndrome. Previous studies have demonstrated that ADAMTS13, particularly the C-terminal domains of ADAMTS13 may stimulate or inhibit angiogenesis, depending on the growth environment. However, the role of ADAMTS13 in vascular development in vivo is not known. Objective: To identify the novel function of ADAMTS13 beyond the proteolysis of VWF, we generated ADAMTS13 knockdown and knockout zebrafish to determine the vascular development and propensity for development of thrombosis. Methods: Morpholino anti-sense RNA was microinjected to the embryos of a double transgenic Tg (gata1:dsRed; fli1:eGFP)zebrafish to transiently block ADAMTS13 translation. Also, CRSIPR/Cas9 system was employed to generate ADAMTS13 knockout zebrafish. The vascular development and thrombus formation were determined by fluorescent microscopy. Results: Approximately 30% of ADAMTS13 knockdown zebrafish exhibited vascular abnormalities in the dwarf intersegmental vessels and narrow caudal vein plexus (Fig. 1A and B). To confirm the phenotype, ADAMTS13 knockout using CRSIPR/Cas system was performed. Approximately 85% of the F0 fish carried insertion or deletion mutations in the targeted region. The founder fish were outcrossed to wild type fish to generate heterozygous F1 fish. Fourteen different mutation sequences with germ line transmission were identified in 33 F1 mutants. The siblings carrying an 8-bp deletion mutation, which created a premature stop codon in the signal peptide region of ADAMTS13, were then crossed to produce F2 progeny. The genotypes and protein expression were further confirmed by sequencing and by Western blot, respectively. As shown, a significant higher rate of vascular development defects, particularly in intersegmental vessels and caudal vein, was observed in homozygous ADAMTS13 knockout fish compared to the heterozygous and wild type fish (Fig. 1C). Thrombosis was triggered by incubation of larvae with various low concentrations (0.06%-0.25%) of FeCl3 and determined by the real-time change of fluorescence intensity within 10 min in the head region of larvae under a fluorescent microscope. When compared with wild type fish, ADAMTS13-/- fish demonstrated dramatically decreased fluorescence signal (Fig.1D), indicative of the cessation of blood flow elsewhere in the body after FeCl3injury. Conclusion: Our results demonstrate that severe deficiency of ADAMTS13 in zebrafish results in abnormalities of vascular development and increases the propensity of thrombosis after oxidative injury in fish larvae. These results suggest that ADAMTS13 may have other substrates than VWF. The zebrafish model may turn out to be a powerful tool for identifying a novel pro-angiogenic or anti-thrombotic agent. Disclosures Zheng: Alexion: Research Funding; Ablynx: Consultancy.

Spatiotemporal Expression Profile of the Pumilio Gene in the Embryonic Development of Silkworm

2014 ◽  
Vol 69 (7-8) ◽  
pp. 317-324
Author(s):  
Liang Chen ◽  
Zaizhi You ◽  
Hengchuan Xia ◽  
Qi Tang ◽  
Yang Zhou ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Bombyx Mori ◽  
Pcr Analysis ◽  
Germ Band ◽  
Band Formation ◽  
Transcript Levels ◽  
Spatiotemporal Expression ◽  
Formation Stage ◽  
Stage 1

We previously identified a pumilio gene in silkworm (Bombyx mori L.), designated BmPUM, which was specifically expressed in the ovary and testis. To further characterize this gene’s involvement in silkworm development, we have determined the spatiotemporal expression pattern of BmPUM during all embryonic stages. Real-time polymerase chain reaction (RT-PCR) analysis revealed that BmPUM was expressed in all stages of silkworm embryos and that its transcript levels displayed two distinct peaks. The first was observed at the germ-band formation stage (1 d after oviposition) and dropped to a low level at the gonad formation stage (5 d after oviposition). The second was detected at the stage of bristle follicle occurrence (6 d after oviposition), which was confirmed by Western blot analysis and immunohistochemistry. Nanos (Nos), functioning together with Pum in abdomen formation of Drosophila embryos, was also highly expressed at the beginning (0 h to 1 d after oviposition) of embryogenesis, but its transcript levels were very low after the stage of germ-band formation. These results suggest that BmPUM functions with Bombyx mori nanos (Bm-nanos) at the early stages of silkworm embryonic development, and may then play a role in gonad formation and the occurrence of bristle follicles. Our data thus provide a foundation to uncover the role of BmPUM during silkworm development.

Hedgehog regulates distinct vascular patterning events through VEGF-dependent and -independent mechanisms

Blood ◽  
2010 ◽  
Vol 116 (4) ◽  
pp. 653-660 ◽  
Author(s):  
Leigh Coultas ◽  
Erica Nieuwenhuis ◽  
Gregory A. Anderson ◽  
Jorge Cabezas ◽  
Andras Nagy ◽  
...  
Keyword(s):  
Vascular Development ◽  
Tube Formation ◽  
Negative Regulator ◽  
Vascular Density ◽  
Genetic Rescue ◽  
Vascular Patterning ◽  
Endothelial Tube Formation ◽  
Hh Signaling ◽  
Endothelial Growth Factor

Abstract Despite the clear importance of Hedgehog (Hh) signaling in blood vascular development as shown by genetic analysis, its mechanism of action is still uncertain. To better understand the role of Hh in vascular development, we further characterized its roles in vascular development in mouse embryos and examined its interaction with vascular endothelial growth factor (VEGF), a well-known signaling pathway essential to blood vascular development. We found that VEGF expression in the mouse embryo depended on Hh signaling, and by using genetic rescue approaches, we demonstrated that the role of Hh both in endothelial tube formation and Notch-dependent arterial identity was solely dependent on its regulation of VEGF. In contrast, overactivation of the Hh pathway through deletion of Patched1 (Ptch1), a negative regulator of Hh signaling, resulted in reduced vascular density and increased Delta-like ligand 4 expression. The Ptch1 phenotype was independent of VEGF pathway dysregulation and was not rescued when Delta-like ligand 4 levels were restored to normal. These findings establish that Hh uses both VEGF- and Notch-dependent and -independent mechanisms to pattern specific events in early blood vascular development.

Von Willebrand disease and Weibel-Palade bodies

2010 ◽  
Vol 30 (03) ◽  
pp. 150-155 ◽  
Author(s):  
J. W. Wang ◽  
J. Eikenboom
Keyword(s):  
Platelet Adhesion ◽  
Coagulation Factor ◽  
Von Willebrand Disease ◽  
Inflammatory Factors ◽  
Limited Data ◽  
Storage Organelle ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryVon Willebrand factor (VWF) is a pivotal haemostatic protein mediating platelet adhesion to injured endothelium and carrying coagulation factor VIII (FVIII) in the circulation to protect it from premature clearance. Apart from the roles in haemostasis, VWF drives the formation of the endothelial cell specific Weibel-Palade bodies (WPBs), which serve as a regulated storage of VWF and other thrombotic and inflammatory factors. Defects in VWF could lead to the bleeding disorder von Willebrand disease (VWD).Extensive studies have shown that several mutations identified in VWD patients cause an intracellular retention of VWF. However, the effects of such mutations on the formation and function of its storage organelle are largely unknown. This review gives an overview on the role of VWF in WPB biogenesis and summarizes the limited data on the WPBs formed by VWD-causing mutant VWF.

Baboon Fibrinogen Adsorption and Platelet Adhesion to Polymeric Materials

1991 ◽  
Vol 65 (05) ◽  
pp. 608-617 ◽  
Author(s):  
Joseph A Chinn ◽  
Thomas A Horbett ◽  
Buddy D Ratner
Keyword(s):  
Platelet Adhesion ◽  
Coagulation Factor ◽  
Polymeric Materials ◽  
Platelet Suspension ◽  
Perfusion Chamber ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Fibrinogen Adsorption

SummaryThe role of fibrinogen in mediating platelet adhesion to polymers exposed to blood plasma was studied by comparison of the effect of plasma dilution on fibrinogen adsorption and platelet adhesion, and by the use of coagulation factor deficient plasmas. Polyetherurethane substrates were first preadsorbed with dilute plasma, then contacted with washed platelets suspended in a modified, apyrase containing Tyrode’s buffer. Platelet adhesion was studied under static conditions in Multiwell dishes, and also under shearing conditions using a parallel plate perfusion chamber. Fibrinogen adsorption and platelet adhesion were measured using 125I radiolabeled baboon fibrinogen and min radiolabeled baboon platelets, respectively. Surfaces were characterized by electron spectroscopy for chemical analysis (ESCA).When fibrinogen adsorption to Biomer was measured after 2 h contact with a series of dilute plasma solutions under static conditions, a peak in adsorption was observed from 0.26% plasma, i.e., adsorption was greater from 0.26% plasma than from either more or less dilute plasma. A peak in subsequent platelet adhesion to the plasma preadsorbed surfaces, measured after 2 h static incubation with washed platelets, was also observed but occurred on Biomer preadsorbed with 1.0% plasma.When fibrinogen adsorption was measured after 5 min contact under shearing conditions, the fibrinogen adsorption peak occurred on surfaces that had been exposed to 1.0% plasma. A peak in platelet adhesion to these preadsorbed surfaces, measured after 5 min contact with the platelet suspensions under shearing conditions, was observed on Biomer preadsorbed with 0.1% plasma. Shifts between the positions of the peaks in protein adsorption and platelet adhesion occurred on other polymers tested as well.Platelet adhesion was almost completely inhibited when baboon and human plasmas lacking fibrinogen (i. e., serum, heat defibrinogenated plasma, and congenitally afibrinogénémie plasma) were used. Platelet adhesion was restored to near normal when exogenous fibrinogen was added to fibrinogen deficient plasmas. Adhesion was also inhibited completely when a monoclonal antibody directed against the glycoprotein IIb/IIIa complex was added to the platelet suspension. Platelet adhesion to surfaces preadsorbed to von Willebrand factor deficient plasma was the same as to surfaces preadsorbed with normal plasma.While it appears that surface bound fibrinogen does mediate the initial attachment of platelets to Biomer, the observation that the fibrinogen adsorption and platelet adhesion maxima do not coincide exactly also suggests that the degree of subsequent platelet adhesion is dictated not only by the amount of surface bound fibrinogen but also by its conformation.

The role of placental growth factor in regulating fetal brain vascular development

2014 ◽  
Author(s):  
Matthew T Ratsep ◽  
Bruno Zavan ◽  
Nicki Peterson ◽  
Leandra Tolusso ◽  
Vanessa Kay ◽  
...  
Keyword(s):  
Growth Factor ◽  
Vascular Development ◽  
Fetal Brain ◽  
Placental Growth Factor

scholarly journals Inhibition of Aurora Kinase B activity disrupts development and differentiation of salivary glands

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Abeer K. Shaalan ◽  
Tathyane H. N. Teshima ◽  
Abigail S. Tucker ◽  
Gordon B. Proctor
Keyword(s):  
Cell Cycle ◽  
Embryonic Development ◽  
Salivary Glands ◽  
Aurora Kinase ◽  
Serine Threonine Kinase ◽  
Aurora Kinase B ◽  
Development And Differentiation ◽  
Differentiation Marker ◽  
Key Events

AbstractLittle is known about the key molecules that regulate cell division during organogenesis. Here we determine the role of the cell cycle promoter aurora kinase B (AURKB) during development, using embryonic salivary glands (E-SGs) as a model. AURKB is a serine/threonine kinase that regulates key events in mitosis, which makes it an attractive target for tailored anticancer therapy. Many reports have elaborated on the role of AURKB in neoplasia and cancer; however, no previous study has shown its role during organ development. Our previous experiments have highlighted the essential requirement for AURKB during adult exocrine regeneration. To investigate if AURKB is similarly required for progression during embryonic development, we pharmacologically inhibited AURKB in developing submandibular glands (SMGs) at embryonic day (E)13.5 and E16.5, using the highly potent and selective drug Barasertib. Inhibition of AURKB interfered with the expansion of the embryonic buds. Interestingly, this effect on SMG development was also seen when the mature explants (E16.5) were incubated for 24 h with another cell cycle inhibitor Aphidicolin. Barasertib prompted apoptosis, DNA damage and senescence, the markers of which (cleaved caspase 3, γH2AX, SA-βgal and p21, respectively), were predominantly seen in the developing buds. In addition to a reduction in cell cycling and proliferation of the epithelial cells in response to AURKB inhibition, Barasertib treatment led to an excessive generation of reactive oxygen species (ROS) that resulted in downregulation of the acinar differentiation marker Mist1. Importantly, inhibition of ROS was able to rescue this loss of identity, with Mist1 expression maintained despite loss of AURKB. Together, these data identify AURKB as a key molecule in supporting embryonic development and differentiation, while inhibiting senescence-inducing signals during organogenesis.

scholarly journals Crayfish hemocytes develop along the granular cell lineage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Fang Li ◽  
Zaichao Zheng ◽  
Hongyu Li ◽  
Rongrong Fu ◽  
Limei Xu ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Cell Lineage ◽  
Granular Cell ◽  
Marker Genes ◽  
Hematopoietic Tissue ◽  
Differentiated Cells ◽  
Stage Of Development ◽  
Almost All ◽  
Relationship Of

AbstractDespite the central role of hemocytes in crustacean immunity, the process of hemocyte differentiation and maturation remains unclear. In some decapods, it has been proposed that the two main types of hemocytes, granular cells (GCs) and semigranular cells (SGCs), differentiate along separate lineages. However, our current findings challenge this model. By tracking newly produced hemocytes and transplanted cells, we demonstrate that almost all the circulating hemocytes of crayfish belong to the GC lineage. SGCs and GCs may represent hemocytes of different developmental stages rather than two types of fully differentiated cells. Hemocyte precursors produced by progenitor cells differentiate in the hematopoietic tissue (HPT) for 3 ~ 4 days. Immature hemocytes are released from HPT in the form of SGCs and take 1 ~ 3 months to mature in the circulation. GCs represent the terminal stage of development. They can survive for as long as 2 months. The changes in the expression pattern of marker genes during GC differentiation support our conclusions. Further analysis of hemocyte phagocytosis indicates the existence of functionally different subpopulations. These findings may reshape our understanding of crustacean hematopoiesis and may lead to reconsideration of the roles and relationship of circulating hemocytes.

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