Converting Attraction to Repulsion

Abstract

During nervous system development, axonal growth cones respond to attractive and repulsive cues that help them navigate to their targets. Wen et al . discovered that, whereas embryonic Xenopus spinal axons in an in vitro culture system were attracted to a gradient of bone morphogenetic protein 7 (BMP7) presented 4 to 8 hours after being placed in culture, BMP7 repelled the same axons after they had been cultured overnight. The type II BMP receptor (BMPRII) mediated both attractive and repulsive responses, and overexpression of a shortened form of BMPRII [which lacks a region that mediates interactions with LIM kinase I (LIMKI) but is dispensable for activation of Smad signaling] abolished BMP7-induced turning. A 32-amino-acid peptide that competitively inhibits LIMKI abolished attractive turning to BMP7, as did a dominant-negative LIMKI mutant, whereas neither affected repulsive turning. In neurons cultured overnight that overexpressed a dominant-negative mutant of the phosphatase Slingshot (SSH), however, the response to BMP7 was converted from repulsion to attraction. A combination of live imaging of the actin cytoskeleton, immunofluorescence analysis of the phosphorylation of XAC [ Xenopus actin-depolymerizing factor (ADF)/cofilin], and expression of wild-type and mutant forms of XAC revealed that LIMKI and SSH regulated growth cone turning through phosphorylation of XAC. BMP7 elicited an increase in growth cone calcium concentration in neurons cultured overnight but not in those cultured for 6 hours. Manipulations designed to block calcium signaling failed to affect attractive turning in 4- to 8-hour cultures, whereas in overnight cultures these treatments abolished the BMP-dependent decrease in XAC phosphorylation and converted repulsive to attractive turning, as did pharmacological inhibition of calcineurin. The transient receptor potential channel TRPC1 was more abundant in overnight cultures than in 4- to 8-hour cultures, and expression of a dominant-negative TRPC1 mutant abolished BMP7-dependent repulsion in overnight cultures, whereas overexpression of wild-type TRPC1 elicited BMP7-dependent repulsion in 4- to 8-hour cultures. Thus, opposing actions of LIMKI and SSH on XAC phosphorylation appear to regulate the actin cytoskeleton and thereby growth cone turning, with calcium influx through TRPC1 switching attraction to repulsion by shifting the balance toward SSH. Z. Wen, L. Han, J. R. Bamburg, S. Shim, G.-l. Ming, J. Q. Zheng, BMP gradients steer nerve growth cones by a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofilin. J. Cell Biol. 178 , 107-119 (2007). [Abstract] [Full Text]