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An in vivo functional analysis system for renal gene discovery in Drosophila pericardial nephrocytes.

ABSTRACT: The difficulty in accessing mammalian nephrons in vivo hinders the study of podocyte biology. The Drosophila nephrocyte shares remarkable similarities to the glomerular podocyte, but the lack of a functional readout for nephrocytes makes it challenging to study this model of the podocyte, which could potentially harness the power of Drosophila genetics. Here, we present a functional analysis of nephrocytes and establish an in vivo system to screen for renal genes. We found that nephrocytes efficiently take up secreted fluorescent protein, and therefore, we generated a transgenic line carrying secreted fluorescent protein and combined it with a nephrocyte-specific driver for targeted gene knockdown, allowing the identification of genes required for nephrocyte function. To validate this system, we examined the effects of knocking down sns and duf, the Drosophila homologs of nephrin and Neph1, respectively, in pericardial nephrocytes. Knockdown of sns or duf completely abolished the accumulation of the fluorescent protein in pericardial nephrocytes. Examining the ultrastructure revealed that the formation of the nephrocyte diaphragm and lacunar structure, which is essential for protein uptake, requires sns. Our preliminary genetic screen also identified Mec2, which encodes the homolog of mammalian Podocin. Taken together, these data suggest that the Drosophila pericardial nephrocyte is a useful in vivo model to help identify genes involved in podocyte biology and facilitate the discovery of renal disease genes.

SUBMITTER: Zhang F

PROVIDER: S-EPMC3559487 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIn animals with bilateral symmetry, midline crossing of axons in the developing central nervous system is regulated by Slit ligands and their neuronal Roundabout (Robo) receptors. Multiple structural domains are present in an evolutionarily conserved arrangement in Robo family proteins, but our understanding of the functional importance of individual domains for midline repulsive signaling is limited.MethodsWe have examined the functional importance of each of the five conserved immunoglobulin-like (Ig) domains within the Drosophila Robo1 receptor. We generated a series of Robo1 variants, each lacking one of the five Ig domains (Ig1-5), and tested each for their ability to bind Slit when expressed in cultured Drosophila cells. We used a transgenic approach to express each variant in robo1's normal expression pattern in wild-type and robo1 mutant embryos, and examined the effects of deleting each domain on receptor expression, axonal localization, regulation, and midline repulsive signaling in vivo.ResultsWe show that individual deletion of Ig domains 2-5 does not interfere with Robo1's ability to bind Slit, while deletion of Ig1 strongly disrupts Slit binding. None of the five Ig domains (Ig1-5) are individually required for proper expression of Robo1 in embryonic neurons, for exclusion from commissural axon segments in wild-type embryos, or for downregulation by Commissureless (Comm), a negative regulator of Slit-Robo repulsion in Drosophila. Each of the Robo1 Ig deletion variants (with the exception of Robo1∆Ig1) were able to restore midline crossing in robo1 mutant embryos to nearly the same extent as full-length Robo1, indicating that Ig domains 2-5 are individually dispensable for midline repulsive signaling in vivo.ConclusionsOur findings indicate that four of the five Ig domains within Drosophila Robo1 are dispensable for its role in midline repulsion, despite their strong evolutionary conservation, and highlight a unique requirement for the Slit-binding Ig1 domain in the regulation of midline crossing.

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An in vivo functional analysis system for renal gene discovery in Drosophila pericardial nephrocytes.

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