| 1. |
- Aase, Karin, et al.
(author)
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Angiomotin regulates endothelial cell migration during embryonic angiogenesis
- 2007
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In: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 21:16, s. 2055-2068
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Journal article (peer-reviewed)abstract
- The development of the embryonic vascular system into a highly ordered network requires precise control over the migration and branching of endothelial cells (ECs). We have previously identified angiomotin (Amot) as a receptor for the angiogenesis inhibitor angiostatin. Furthermore, DNA vaccination targeting Amot inhibits angiogenesis and tumor growth. However, little is known regarding the role of Amot in physiological angiogenesis. We therefore investigated the role of Amot in embryonic neovascularization during zebrafish and mouse embryogenesis. Here we report that knockdown of Amot in zebrafish reduced the number of filopodia of endothelial tip cells and severely impaired the migration of intersegmental vessels. We further show that 75% of Amot knockout mice die between embryonic day 11 (E11) and E11.5 and exhibit severe vascular insufficiency in the intersomitic region as well as dilated vessels in the brain. Furthermore, using ECs differentiated from embryonic stem (ES) cells, we demonstrate that Amot-deficient cells have intact response to vascular endothelial growth factor (VEGF) in regard to differentiation and proliferation. However, the chemotactic response to VEGF was abolished in Amot-deficient cells. We provide evidence that Amot is important for endothelial polarization during migration and that Amot controls Rac1 activity in endothelial and epithelial cells. Our data demonstrate a critical role for Amot during vascular patterning and endothelial polarization.
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| 2. |
- Ando, Koji, et al.
(author)
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Conserved and context-dependent roles for pdgfrb signaling during zebrafish vascular mural cell development
- 2021
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In: Developmental Biology. - : Elsevier. - 0012-1606 .- 1095-564X. ; 479, s. 11-22
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Journal article (peer-reviewed)abstract
- Platelet derived growth factor beta and its receptor, Pdgfrb, play essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle cells. To determine if this role was conserved in zebrafish, we analyzed pdgfb and pdgfrb mutant lines. Similar to mouse, pdgfb and pdgfrb mutant zebrafish lack brain pericytes and exhibit anatomically selective loss of vascular smooth muscle coverage. Despite these defects, pdgfrb mutant zebrafish did not otherwise exhibit circulatory defects at larval stages. However, beginning at juvenile stages, we observed severe cranial hemorrhage and vessel dilation associated with loss of pericytes and vascular smooth muscle cells in pdgfrb mutants. Similar to mouse, pdgfrb mutant zebrafish also displayed structural defects in the glomerulus, but normal development of hepatic stellate cells. We also noted defective mural cell investment on coronary vessels with concomitant defects in their development. Together, our studies support a conserved requirement for Pdgfrb signaling in mural cells. In addition, these zebrafish mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects.
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| 3. |
- Ebarasi, Lwaki, et al.
(author)
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Defects of CRB2 Cause Steroid-Resistant Nephrotic Syndrome
- 2015
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In: American Journal of Human Genetics. - : Elsevier BV. - 0002-9297 .- 1537-6605. ; 96:1, s. 153-161
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Journal article (peer-reviewed)abstract
- Nephrotic syndrome (NS), the association of gross proteinuria, hypoalbuminaemia, edema, and hyperlipidemia, can be clinically divided into steroid-sensitive (SSNS) and steroid-resistant (SRNS) forms. SRNS regularly progresses to end-stage renal failure. By homozygosity mapping and whole exome sequencing, we here identify recessive mutations in Crumbs homolog 2 (CRB2) in four different families affected by SRNS. Previously, we established a requirement for zebrafish crb2b, a conserved regulator of epithelial polarity, in podocyte morphogenesis. By characterization of a loss-of-function mutation in zebrafish crb2b, we now show that zebrafish crb2b is required for podocyte foot process arborization, slit diaphragm formation, and proper nephrin trafficking. Furthermore, by complementation experiments in zebrafish, we demonstrate that CRB2 mutations result in loss of function and therefore constitute causative mutations leading to NS in humans. These results implicate defects in podocyte apico-basal polarity in the pathogenesis of NS.
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| 4. |
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| 5. |
- Ebarasi, Lwaki
(author)
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Functional analysis of genes in the developing zebrafish pronephros and vasculature
- 2009
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Doctoral thesis (other academic/artistic)abstract
- During glomerulogenesis, the recruitment and assembly of visceral epithelial cells (podocytes), endothelial capillary cells, and smooth muscle pericytes (mesangial cells) result in the formation of the glomerular tuft. The mature glomerulus functions by passage of plasma under hemodynamic pressure across a filter, the glomerular filtration barrier, and the integrity of the barrier is crucial for proper function. A common pathology shared by virtually all glomerular diseases is the loss of filtration barrier function leading to proteinuria, the leakage of protein into the urine, and further glomerular and tubular damage. Thus, the podocyte slit diaphragm and its associated proteins have been the focus of intense research in the filtration barrier field. Even so, the understanding of how the podocytes, endothelia, and mesangial cells function together and communicate with each other within the mature glomerulus is at an early stage. With this question in mind, GlomBase, a bioinformatics database that describes the mammalian glomerular transcriptome, was created as a foundation from which to explore new aspects of glomerular biology. We have applied the zebrafish pronephric glomerulus as a model system to study novel aspects of glomerular biology. Our approach takes advantage of the rapid development and genetic accessibility of the renal system in combination with GlomBase to conduct a highthroughput functional analysis. We reasoned that if a gene is important for glomerular function in the zebrafish it might also be important in mammalian glomerular function. In this novel genetic screen, we have coupled gene knockdown using morpholinos with a physiological glomerular dye filtration assay to test for selective glomerular permeability in living zebrafish larvae. We identified the crb2b gene as a regulator of podocyte foot process formation. We found that Nephrin, a major slit diaphragm component, is apically mis-localized in podocytes lacking crb2b function. These observations suggest that Crb proteins may regulate protein trafficking and provide a way of understanding foot process formation within the larger context of apical-basal cell differentiation. The Angiomotin (Amot) family of proteins plays roles in endothelial migration, cell shape, and tube formation and members of this family are present within GlomBase. As a first step towards functionally characterizing Angiomotin family members in the zebrafish, we inactivated the amot gene in zebrafish using morpholinos within the Tg (fli1:EGFP)y1 transgenic line which expresses GFP within the developing vasculature. Zebrafish lacking amot function showed a clear, specific, and quantifiable defect in the formation of intersegmental vessels (ISVs) and this arose from a defect in endothelial cell migration and filopodia formation. These studies identified an evolutionarily conserved function for amot in blood vessel formation and paved the way for future studies of Amot family members within the glomerular vasculature. We then applied the zebrafish pronephros to the study of the third cell type of the glomerulus, the mesangial cell. Mesangial cells are specialized vascular pericytes within the glomerulus and associate intimately with the glomerular capillaries. However, a pericyte population within the zebrafish has not been thus far described. We found cells that express the early pericyte marker pdgfrb and these cells were also closely associated with vasculature in the eye, brain, and glomerulus. Morpholino knockdown of pdgfrb resulted in dilation of the glomerular capillaries phenocopying mouse knock out data and arguing for a conserved role for Pdgfrb signaling in recruiting mesangial cells during maturation of the glomerular tuft. These studies establish the zebrafish as a system for studying pericyte development.
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| 6. |
- Falkevall, Annelie, et al.
(author)
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Reducing VEGF-B Signaling Ameliorates Renal Lipotoxicity and Protects against Diabetic Kidney Disease
- 2017
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In: Cell Metabolism. - : Elsevier BV. - 1550-4131 .- 1932-7420. ; 25:3, s. 713-726
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Journal article (peer-reviewed)abstract
- Diabetic kidney disease (DKD) is the most common cause of severe renal disease, and few treatment options are available today that prevent the progressive loss of renal function. DKD is characterized by altered glomerular filtration and proteinuria. A common observation in DKD is the presence of renal steatosis, but the mechanism(s) underlying this observation and to what extent they contribute to disease progression are unknown. Vascular endothelial growth factor B (VEGF-B) controls muscle lipid accumulation through regulation of endothelial fatty acid transport. Here, we demonstrate in experimental mouse models of DKD that renal VEGF-B expression correlates with the severity of disease. Inhibiting VEGF-B signaling in DKD mouse models reduces renal lipotoxicity, re-sensitizes podocytes to insulin signaling, inhibits the development of DKD-associated pathologies, and prevents renal dysfunction. Further, we show that elevated VEGF-B levels are found in patients with DKD, suggesting that VEGF-B antagonism represents a novel approach to treat DKD.
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| 7. |
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| 8. |
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| 9. |
- He, Bing, et al.
(author)
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Lmx1b and FoxC Combinatorially Regulate Podocin Expression in Podocytes
- 2014
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In: Journal of the American Society of Nephrology. - 1046-6673 .- 1533-3450. ; 25:12, s. 2764-2777
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Journal article (peer-reviewed)abstract
- Podocin is a key protein of the kidney podocyte slit diaphragm protein complex, an important part of the glomerular filtration barrier. Mutations in the human podocin gene NPHS2 cause familial or sporadic forms of renal disease owing to the disruption of filtration barrier integrity. The exclusive expression of NPHS2 in podocytes reflects its unique function and raises interesting questions about its transcriptional regulation. Here, we further define a 2.5-kb zebrafish nphs2 promoter fragment previously described and identify a 49-bp podocyte-specific transcriptional enhancer using Tol2-mediated G(0) transgenesis in zebrafish. Within this enhancer, we identified a cis-acting element composed of two adjacent DNA-binding sites (FLAT-E and forkhead) bound by transcription factors Lnnx1b and FoxC. In zebrafish, double knockdown of Lmx1b and FoxC orthologs using morpholino doses that caused no or minimal phenotypic changes upon individual knockdown completely disrupted podocyte development in 40% of injected embryos. Co-overexpression of the two genes potently induced endogenous nphs2 expression in zebrafish podocytes. We found that the NPHS2 promoter also contains a cis-acting Lmx1b-FoxC motif that binds LMX1B and FoxC2. Furthermore, a genome-wide search identified several genes that carry the Lmx1b-FoxC motif in their promoter regions. Among these candidates, motif-driven podocyte enhancer activity of CCNC and MEIS2 was functionally analyzed in vivo. Our results show that podocyte expression of some genes is combinatorially regulated by two transcription factors interacting synergistically with a common enhancer. This finding provides insights into transcriptional mechanisms required for normal and pathologic podocyte functions.
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| 10. |
- Kok, Fatma O., et al.
(author)
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Reverse Genetic Screening Reveals Poor Correlation between Morpholino-Induced and Mutant Phenotypes in Zebrafish
- 2015
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In: Developmental Cell. - : Elsevier BV. - 1534-5807 .- 1878-1551. ; 32:1, s. 97-108
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Journal article (peer-reviewed)abstract
- The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.
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