Hubrecht Institute KNAW and UMC Utrecht

Utrecht, Netherlands

Hubrecht Institute KNAW and UMC Utrecht

Utrecht, Netherlands
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Coxam B.,University of Queensland | Sabine A.,University of Lausanne | Bower N.I.,University of Queensland | Smith K.A.,University of Queensland | And 12 more authors.
Cell Reports | Year: 2014

Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by known signaling and transcriptional mechanisms. The ongoing elaboration of vessels to form a network is less well understood. This involves cell polarization, coordinated migration, adhesion, mixing, regression, and shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. A mutation in polycystic kidney disease 1a was responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial lymphatic precursor sprouting is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice has no effect on precursor sprouting but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation, and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development. © 2014 The Authors.


Apschner A.,Hubrecht Institute KNAW and UMC Utrecht | Schulte-Merker S.,Hubrecht Institute KNAW and UMC Utrecht | Witten P.E.,Nutreco Aquaculture Research Center | Witten P.E.,Ghent University
Methods in Cell Biology | Year: 2011

Developmental osteogenesis and pathologies of mineralized tissues are areas of intense investigations in the mammalian field, but different from other areas of organ formation and developmental biology, zebrafish have been somewhat slow in joining the area of bone research. In recent years, however, genetic screens have provided a number of exciting mutants, and transgenic lines have been developed that permit visualization of osteoblasts and osteoclasts in vivo. We here review some of the recent literature and provide examples where insights from studies in zebrafish have complemented the information available from mammalian models or clinical studies. Furthermore, we provide a comparative overview about different forms of bone within the teleost lineage, and between teleosts and mammals. © 2011 Elsevier Inc.


van Impel A.,Hubrecht Institute KNAW and UMC Utrecht | Zhao Z.,A STAR Institute of Molecular and Cell Biology | Hermkens D.M.A.,Hubrecht Institute KNAW and UMC Utrecht | Roukens M.G.,Hubrecht Institute KNAW and UMC Utrecht | And 9 more authors.
Development (Cambridge) | Year: 2014

In mammals, the homeodomain transcription factor Prox1 acts as the central regulator of lymphatic cell fate. Its restricted expression in a subset of cardinal vein cells leads to a switch towards lymphatic specification and hence represents a prerequisite for the initiation of lymphangiogenesis. Murine Prox1-null embryos lack lymphatic structures, and sustained expression of Prox1 is indispensable for the maintenance of lymphatic cell fate even at adult stages, highlighting the unique importance of this gene for the lymphatic lineage. Whether this pre-eminent role of Prox1 within the lymphatic vasculature is conserved in other vertebrate classes has remained unresolved, mainly owing to the lack of availability of loss-of-function mutants. Here, we re-examine the role of Prox1a in zebrafish lymphangiogenesis. First, using a transgenic reporter line, we show that prox1a is initially expressed in different endothelial compartments, becoming restricted to lymphatic endothelial cells only at later stages. Second, using targeted mutagenesis, we show that Prox1a is dispensable for lymphatic specification and subsequent lymphangiogenesis in zebrafish. In line with this result, we found that the functionally related transcription factors Coup-TFII and Sox18 are also dispensable for lymphangiogenesis. Together, these findings suggest that lymphatic commitment in zebrafish and mice is controlled in fundamentally different ways. © 2014. Published by The Company of Biologists Ltd.


PubMed | University of Lausanne, University of Auckland, Center for Cancer Biology, University of Queensland and Hubrecht Institute KNAW and UMC Utrecht
Type: Journal Article | Journal: Cell reports | Year: 2014

Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by known signaling and transcriptional mechanisms. The ongoing elaboration of vessels to form a network is less well understood. This involves cell polarization, coordinated migration, adhesion, mixing, regression, and shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. A mutation in polycystic kidney disease 1a was responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial lymphatic precursor sprouting is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice has no effect on precursor sprouting but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation, and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development.


de Vrieze E.,Radboud University Nijmegen | Zethof J.,Radboud University Nijmegen | Schulte-Merker S.,Hubrecht Institute KNAW and UMC Utrecht | Schulte-Merker S.,Institute for Cardiovascular Organogenesis and Regeneration | And 3 more authors.
Bone | Year: 2015

Tight interactions among different cell types contributing to bone formation are of key importance in the maintenance of bone homeostasis. Based on the high similarity in responses to (anti)osteogenic signals between zebrafish scales and mammalian bone, we developed and validated a model to screen large numbers of compounds using ex-vivo cultured scales of a sp7:luciferase transgenic zebrafish. This model combines the high predictive value of explant cultures with quick, sensitive, and quantifiable readout converging the effects via various pathways including WNT-signaling, to SP7/osterix promoter activity. Sp7 is pivotal in osteoblast differentiation and activity and its promoter activity provides an excellent surrogate for sp7 expression. Bmp-2a was shown to dose-dependently increase sp7-driven luciferase activity ex vivo. Next, we identified novel effects on bone for 51.7% of the compounds from a small library of WNT-signaling modulators, including a strong osteogenic effect for niclosamide. From all previously characterized compounds, the effect on bone was correctly predicted for 70% of compounds, resulting in a 7% false positive- and 21% false negative rate. The proposed sp7:luciferase zebrafish scale model is unique, powerful and efficient new tool to assess compounds with osteogenic effects, prior to further testing in rodents. © 2015 Elsevier Inc.


Le Guen L.,University of Queensland | Le Guen L.,Montpellier University | Karpanen T.,Hubrecht Institute KNAW and UMC Utrecht | Schulte D.,Hubrecht Institute KNAW and UMC Utrecht | And 12 more authors.
Development (Cambridge) | Year: 2014

The VEGFC/VEGFR3 signaling pathway is essential for lymphangiogenesis (the formation of lymphatic vessels from preexisting vasculature) during embryonic development, tissue regeneration and tumor progression. The recently identified secreted protein CCBE1 is indispensible for lymphangiogenesis during development. The role of CCBE1 orthologs is highly conserved in zebrafish, mice and humans with mutations in CCBE1 causing generalized lymphatic dysplasia and lymphedema (Hennekam syndrome). To date, the mechanism by which CCBE1 acts remains unknown. Here, we find that ccbe1 genetically interacts with both vegfc and vegfr3 in zebrafish. In the embryo, phenotypes driven by increased Vegfc are suppressed in the absence of Ccbe1, and Vegfcdriven sprouting is enhanced by local Ccbe1 overexpression. Moreover, Vegfc- and Vegfr3-dependent Erk signaling is impaired in the absence of Ccbe1. Finally, CCBE1 is capable of upregulating the levels of fully processed, mature VEGFC in vitro and the overexpression of mature VEGFC rescues ccbe1 loss-of-function phenotypes in zebrafish. Taken together, these data identify Ccbe1 as a crucial component of the Vegfc/Vegfr3 pathway in the embryo. © 2014. Published by The Company of Biologists Ltd.


Geurtzen K.,TU Dresden | Knopf F.,TU Dresden | Knopf F.,Kennedy Institute of Rheumatology | Wehner D.,University of Ulm | And 4 more authors.
Development (Cambridge) | Year: 2014

Zebrafish have an unlimited capacity to regenerate bone after fin amputation. In this process, mature osteoblasts dedifferentiate to osteogenic precursor cells and thus represent an important source of newly forming bone. By contrast, differentiated osteoblasts do not appear to contribute to repair of bone injuries in mammals; rather, osteoblasts form anew from mesenchymal stem cells. This raises the question whether osteoblast dedifferentiation is specific to appendage regeneration, a special feature of the lepidotrichia bone of the fish fin, or a process found more generally in fish bone. Here, we show that dedifferentiation of mature osteoblasts is not restricted to fin regeneration afteramputation, but also occurs during repair of zebrafish fin fractures and skull injuries. In both models, mature osteoblasts surrounding the injury downregulate the expression of differentiation markers, upregulate markers of the pre-osteoblast state and become proliferative. Making use of photoconvertible Kaede protein as well as Cre-driven genetic fate mapping, we show that osteoblasts migrate to the site of injury to replace damaged tissue. Our findings suggest a fundamental role for osteoblast dedifferentiation in reparative bone formation in fish and indicate that adult fish osteoblasts display elevated cellular plasticity compared with mammalian bone-forming cells. © 2014. Published by The Company of Biologists Ltd.


Weijts B.G.M.W.,University Utrecht | van Impel A.,Hubrecht Institute KNAW and UMC Utrecht | Schulte-Merker S.,Hubrecht Institute KNAW and UMC Utrecht | Schulte-Merker S.,Wageningen University | de Bruin A.,University Utrecht
PLoS ONE | Year: 2013

Lymphatic vessels are derived from venous endothelial cells and their formation is governed by the Vascular endothelial growth factor C (VegfC)/Vegf receptor 3 (Vegfr3; Flt4) signaling pathway. Recent studies show that Collagen and Calcium Binding EGF domains 1 protein (Ccbe1) enhances VegfC-dependent lymphangiogenesis. Both Ccbe1 and Flt4 have been shown to be indispensable for lymphangiogenesis. However, how these essential players are transcriptionally regulated remains poorly understood. In the case of angiogenesis, atypical E2fs (E2f7 and E2f8) however have been recently shown to function as transcriptional activators for VegfA. Using a genome-wide approach we here identified both CCBE1 and FLT4 as direct targets of atypical E2Fs. E2F7/8 directly bind and stimulate the CCBE1 promoter, while recruitment of E2F7/8 inhibits the FLT4 promoter. Importantly, inactivation of e2f7/8 in zebrafish impaired venous sprouting and lymphangiogenesis with reduced ccbe1 expression and increased flt4 expression. Remarkably, over-expression of e2f7/8 rescued Ccbe1- and Flt4-dependent lymphangiogenesis phenotypes. Together these results identified E2f7/8 as novel in vivo transcriptional regulators of Ccbe1 and Flt4, both essential genes for venous sprouting and lymphangiogenesis. © 2013 Weijts et al.


Coxam B.,University of Queensland | Neyt C.,University of Queensland | Grassini D.R.,University of Queensland | Le Guen L.,University of Queensland | And 3 more authors.
Developmental Dynamics | Year: 2015

Background: The interplay between Notch and Vegf signaling regulates angiogenesis in the embryo. Notch signaling limits the responsiveness of endothelial cells to Vegf to control sprouting. Despite the importance of this regulatory relationship, much remains to be understood about extrinsic factors that modulate the pathway. Results: During a forward genetic screen for novel regulators of lymphangiogenesis, we isolated a mutant with reduced lymphatic vessel development. This mutant also exhibited hyperbranching arteries, reminiscent of Notch pathway mutants. Positional cloning identified a missense mutation in the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (cad) gene. Cad is essential for UDP biosynthesis, which is necessary for protein glycosylation and de novo biosynthesis of pyrimidine-based nucleotides. Using a transgenic reporter of Notch activity, we demonstrate that Notch signaling is significantly reduced in cadhu10125 mutants. In this context, genetic epistasis showed that increased endothelial cell responsiveness to Vegfc/Vegfr3 signaling drives excessive artery branching. Conclusions: These findings suggest important posttranslational modifications requiring Cad as an unappreciated mechanism that regulates Notch/Vegf signaling during angiogenesis. Developmental Dynamics 244:1-9, 2015. © 2014 Wiley Periodicals, Inc.

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