Abstract
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.
Highlights
In 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
Apart from Ig1, which of the other domains in Drosophila Robo1 are required for midline repulsion, if any? Are any of the other Robo1 Ig or fibronectin type III (Fn) domains required for receptor expression, protein stability, axonal localization, or Slit binding? Here, we address these questions by individually deleting each of the five Robo1 Ig domains and examining the effects of these deletions on Slit binding as well as in vivo protein expression, localization, and Slit-dependent midline repulsive signaling
We have recently demonstrated that deletion of the Ig1 domain from Drosophila Robo1 prevents it from binding to Slit, and abolishes its ability to prevent midline crossing of axons in vivo [34]
Summary
In 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. Two regulatory mechanisms have been identified which prevent premature Slit-Robo repulsion in pre-crossing commissural axons in Drosophila. The endosomal sorting receptor Commissureless (Comm) prevents newly synthesized Robo proteins from reaching the growth cone surface as commissural axons are growing towards and across the midline [14, 19,20,21], and Robo acts non-autonomously to antagonize repulsive signaling by the remaining surface-localized Robo, facilitating midline crossing [15]. Orthologs of Comm and Robo have not been identified outside of insects, and vertebrates have acquired distinct regulatory mechanisms to prevent premature Slit-Robo repulsion in commissural axons [16, 23]
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