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In Vivo Functional Analysis of Drosophila Robo1 Fibronectin Type-III Repeats.

ABSTRACT: The repellant ligand Slit and its Roundabout (Robo) family receptors regulate midline crossing of axons during development of the embryonic central nervous system (CNS). Slit proteins are produced at the midline and signal through Robo receptors to repel axons from the midline. Disruption of Slit-Robo signaling causes ectopic midline-crossing phenotypes in the CNS of a broad range of animals, including insects and vertebrates. While previous studies have investigated the roles of Drosophila melanogaster Robo1's five Immunoglobulin-like (Ig) domains, little is known about the importance of the three evolutionarily conserved Fibronectin (Fn) type-III repeats. We have individually deleted each of Drosophila Robo1's three Fn repeats, and then tested these Robo1 variants in vitro to determine their ability to bind Slit in cultured Drosophila cells and in vivo to investigate the requirement for each domain in regulating Robo1's embryonic expression pattern, axonal localization, midline repulsive function, and sensitivity to Commissureless (Comm) downregulation. We demonstrate that the Fn repeats are not required for Robo1 to bind Slit or for proper expression of Robo1 in Drosophila embryonic neurons. When expressed in a robo1 mutant background, these variants are able to restore midline repulsion to an extent equivalent to full-length Robo1. We identify a novel requirement for Fn3 in the exclusion of Robo1 from commissures and downregulation of Robo1 by Comm. Our results indicate that each of the Drosophila Robo1 Fn repeats are individually dispensable for the protein's role in midline repulsion, despite the evolutionarily conserved "5 + 3" protein structure.

SUBMITTER: Brown HE

PROVIDER: S-EPMC5919748 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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In Vivo Functional Analysis of Drosophila Robo1 Fibronectin Type-III Repeats.

Brown Haley E HE Reichert Marie C MC Evans Timothy A TA

G3 (Bethesda, Md.) 20180202 2

The repellant ligand Slit and its Roundabout (Robo) family receptors regulate midline crossing of axons during development of the embryonic central nervous system (CNS). Slit proteins are produced at the midline and signal through Robo receptors to repel axons from the midline. Disruption of Slit-Robo signaling causes ectopic midline-crossing phenotypes in the CNS of a broad range of animals, including insects and vertebrates. While previous studies have investigated the roles of Drosophila m ...[more]

PMID: 29217730

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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.

Dataset Information

In Vivo Functional Analysis of Drosophila Robo1 Fibronectin Type-III Repeats.

Publications

<i>In Vivo</i> Functional Analysis of <i>Drosophila</i> Robo1 Fibronectin Type-III Repeats.

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