Ellen van Rooijen1,2, Glenn van de Hoek3,4, Ive Logister1,2, Henry Ajzenberg1,3, Nine V A M Knoers4, Freek van Eeden2, Emile E Voest1,5, Stefan Schulte-Merker2,6,7, Rachel H Giles1,3. 1. Department of Medical Oncology, University Medical Center Utrecht, Utrecht, the Netherlands. 2. Hubrecht Institute, KNAW and UMC Utrecht, Utrecht, the Netherlands. 3. Department Nephrology and Hypertension, Regenerative Medicine Center Utrecht, University Medical Center Utrecht, Utrecht, the Netherlands. 4. Department of Genetics, University Medical Center Utrecht, Utrecht, the Netherlands. 5. Medical Oncology, Netherlands Cancer Institute, Amsterdam, the Netherlands. 6. Institute of Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU, Münster, Germany. 7. CiM Cluster of Excellence (EXC1003-CiM), Münster, Germany.
Abstract
BACKGROUND: von Hippel-Lindau (VHL) disease is characterized by the development of benign and malignant tumours in many organ systems, including renal cysts and clear cell renal cell carcinoma. It is not completely understood what underlies the development of renal pathology, and the use of murine Vhl models has been challenging due to limitations in disease conservation. We previously described a zebrafish model bearing inactivating mutations in the orthologue of the human VHL gene. METHODS: We used histopathological and functional assays to investigate the pronephric and glomerular developmental defects in vhl mutant zebrafish, supported by human cell culture assays. RESULTS: Here, we report that vhl is required to maintain pronephric tubule and glomerulus integrity in zebrafish embryos. vhl mutant glomeruli are enlarged, cxcr4a+ capillary loops are dilated and the Bowman space is widened. While we did not observe pronephric cysts, the cells of the proximal convoluted and anterior proximal straight tubule are enlarged, periodic acid schiff (PAS) and Oil Red O positive, and display a clear cytoplasm after hematoxylin and eosine staining. Ultrastructural analysis showed the vhl-/- tubule to accumulate large numbers of vesicles of variable size and electron density. Microinjection of the endocytic fluorescent marker AM1-43 in zebrafish embryos revealed an accumulation of endocytic vesicles in the vhl mutant pronephric tubule, which we can recapitulate in human cells lacking VHL. CONCLUSIONS: Our data indicates that vhl is required to maintain pronephric tubule and glomerulus integrity during zebrafish development, and suggests a role for VHL in endocytic vesicle trafficking.
BACKGROUND:von Hippel-Lindau (VHL) disease is characterized by the development of benign and malignant tumours in many organ systems, including renal cysts and clear cell renal cell carcinoma. It is not completely understood what underlies the development of renal pathology, and the use of murineVhl models has been challenging due to limitations in disease conservation. We previously described a zebrafish model bearing inactivating mutations in the orthologue of the humanVHL gene. METHODS: We used histopathological and functional assays to investigate the pronephric and glomerular developmental defects in vhl mutant zebrafish, supported by human cell culture assays. RESULTS: Here, we report that vhl is required to maintain pronephric tubule and glomerulus integrity in zebrafish embryos. vhl mutant glomeruli are enlarged, cxcr4a+ capillary loops are dilated and the Bowman space is widened. While we did not observe pronephric cysts, the cells of the proximal convoluted and anterior proximal straight tubule are enlarged, periodic acid schiff (PAS) and Oil Red O positive, and display a clear cytoplasm after hematoxylin and eosine staining. Ultrastructural analysis showed the vhl-/- tubule to accumulate large numbers of vesicles of variable size and electron density. Microinjection of the endocytic fluorescent marker AM1-43 in zebrafish embryos revealed an accumulation of endocytic vesicles in the vhl mutant pronephric tubule, which we can recapitulate in human cells lacking VHL. CONCLUSIONS: Our data indicates that vhl is required to maintain pronephric tubule and glomerulus integrity during zebrafish development, and suggests a role for VHL in endocytic vesicle trafficking.
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