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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.04.017 Byline: Lwaki Ebarasi (a), Liqun He (b), Kjell Hultenby (c), Minoru Takemoto (d), Christer Betsholtz (b), Karl Tryggvason (a), Arindam Majumdar (a) Keywords: Podocyte; Pronephros; Glomerulus; Glomerular filtration barrier; Nephrin; Zebrafish; Crumbs; Slit diaphragm; Differentiation; Cell polarity Abbreviations: GBM, glomerular basement membrane; dpf, days post fertilization; crb, crumbs Abstract: The glomerular filtration barrier is necessary for the selective passage of low molecular weight waste products and the retention of blood plasma proteins. Damage to the filter results in proteinuria. The filtration barrier is the major pathogenic site in almost all glomerular diseases and its study is therefore of clinical significance. We have taken advantage of the zebrafish pronephros as a system for studying glomerular filtration. In order to identify new regulators of filtration barrier assembly, we have performed a reverse genetic screen in the zebrafish testing a group of genes which are enriched in their expression within the mammalian glomerulus. In this novel 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. Screening 20 genes resulted in the identification of ralgps1, rapgef2, rabgef1, and crb2b. The crumbs (crb) genes encode a family of evolutionarily conserved proteins important for apical-basal polarity within epithelia. The crb2b gene is expressed in zebrafish podocytes. Electron microscopic analysis of crb2b morphants reveals a gross disorganization of podocyte foot process architecture and loss of slit diaphragms while overall polarity is maintained. Nephrin, a major component of the slit diaphragm, is apically mis-localized in podocytes from crb2b morphants suggesting that crb2b is required for the proper protein trafficking of Nephrin. This report is the first to show a role for crb function in podocyte differentiation. Furthermore, these results suggest a novel link between epithelial polarization and the maintenance of a functional filtration barrier. Author Affiliation: (a) Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheeles vag 2, Plan 4 B1, SE-171 77 Stockholm, Sweden (b) Laboratory of Vascular Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 17177 Stockholm, Sweden (c) Department of Pathology, Karolinska University Hospital/Huddinge, Karolinska Institutet, Stockholm, Sweden (d) Department of Clinical Cell Biology and Medicine, Chiba University Graduate School of Medicine, Chiba, Japan Article History: Received 27 October 2008; Revised 26 March 2009; Accepted 16 April 2009

The importance of angiomotin (Amot) during embryonic angiogenesis is studied. Results demonstrate an important role for Amot during tube formation, with defects in endothelial cell (EC) polarization and growth factor-induced migration.

Podocytes are highly specialized epithelial cells located at the outer aspects of the glomerular capillary tuft and critical components of the kidney filtration barrier. To maintain their unique features, podocytes express a number of proteins that are only sparsely found elsewhere in the body. In this study, we have identified four (Tmem234, Znf185, Lrrc49, and Slfn5) new highly podocyte- enriched proteins. The proteins are strongly expressed by podocytes, while other parts of the kidney show only weak or no expression. Tmem234, Slfn5, and Lrrc49 are located in foot processes, whereas Znf185 is found in both foot and major processes. Expressional studies in developing kidneys show that these proteins are first expressed at the capillary stage glomerulus, the same stage when the formation of major and foot processes begins. We identified zebrafish orthologs for Tmem234 and Znf185 genes and knocked down their expression using morpholino technology. Studies in zebrafish larvae indicate that Tmem234 is essential for the organization and functional integrity of the pronephric glomerulus filtration barrier, as inactivation of Tmem234 expression results in foot process effacement and proteinuria. In summary, we have identified four novel highly podocyte-enriched proteins and show that one of them, Tmem234, is essential for the normal filtration barrier in the zebrafish pronephric glomerulus. Identification of new molecular components of the kidney filtration barrier opens up possibilities to study their role in glomerulus biology and diseases. [ABSTRACT FROM AUTHOR]