Nicol X et al. (2011), Spatial and temporal second messenger codes for...

XB-ART-44615

Proc Natl Acad Sci U S A 2011 Aug 16;10833:13776-81. doi: 10.1073/pnas.1100247108.

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Spatial and temporal second messenger codes for growth cone turning.

Nicol X , Hong KP , Spitzer NC .

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Cyclic AMP (cAMP) and calcium are ubiquitous, interdependent second messengers that regulate a wide range of cellular processes. During development of neuronal networks they are critical for the first step of circuit formation, transducing signals required for axon pathfinding. Surprisingly, the spatial and temporal cAMP and calcium codes used by axon guidance molecules are unknown. Here, we identify characteristics of cAMP and calcium transients generated in growth cones during Netrin-1-dependent axon guidance. In filopodia, Netrin-1-dependent Deleted in Colorectal Cancer (DCC) receptor activation induces a transient increase in cAMP that causes a brief increase in calcium transient frequency. In contrast, activation of DCC in growth cone centers leads to a transient calcium-dependent cAMP increase and a sustained increase in frequency of calcium transients. We show that filopodial cAMP transients regulate spinal axon guidance in vitro and commissural axon pathfinding in vivo. These growth cone codes provide a basis for selective activation of specific downstream effectors.

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Species referenced: Xenopus
Genes referenced: camp dcc ntn1

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Fig. 7. Disruption of cAMP signaling prevents midline crossing of commissural axons in vivo. (A) Alexa 488 dextran-filled commissural axons imaged in the intact spinal cord of control embryos grow to the ventral midline (dashed line at the Alexa 568 border), cross it, and turn rostrally or caudally along the contralateral ventral fascicle (Left). Trajectories of control growth cones (Right). Orange horizontal bar represents the floor plate. (B) Commissural axons in a SQ 22536-treated spinal cord grow to the ventral midline but fail to cross it. (C) Treating the spinal cord with forskolin also prevents axons from crossing the midline. (Aï¿½C) n > 25 growth cones. All trajectories are shown in Fig. S7B. (D) Trajectories of individual axons were aligned to obtain an average trajectory for each experimental condition, and rostrally and caudally turning trajectories were superimposed. Averaged trajectories demonstrate a failure of midline crossing when spinal cords are treated with SQ22536 or forskolin. More than 25 trajectories were scored for each condition. (E) To distinguish effects on axon directionality and growth, we quantified the transverse velocity (toward the midline) and the longitudinal velocity (parallel to the midline) of axons from spinal cords exposed to control, SQ22536-, or forskolin-containing medium. The transverse velocity was reduced by high or low cAMP concentration before the midline and (F) further suppressed within a 40-μmï¿½wide area around the ventral midline. In contrast, the longitudinal velocity was only slightly affected by modifying the cAMP concentration. (E and F) Error bars, SEM; ***P < 0.001, *P < 0.05, ANOVA

Fig. 8. Local and pulsatile optogenetic elevation of cAMP concentration rescues defects of commissural axon pathfinding in vivo. (A) Before crossing the ventral midline, Alexa 488 dextran-filled commissural axons grow perpendicular to the midline (dashed line at the Alexa 568 border). Arrowheads identify growth cones. (B) Application of a blocking antibody against DCC prevents axons from growing perpendicular to the midline. Blue light illumination of growth cones that do not express PACα (blue areas; uninjected side of the animal, not labeled with Alexa 568) does not rescue this defect. (Inset) Outlined arrowhead indicates enlarged growth cone. (C) Local and pulsatile illumination (blue areas; 3 min three times per hour) of PACα-expressing growth cones (PACα and Alexa 568-coinjected side of the animal) maintain the orientation of axon growth toward the midline. (D) Mean angle of axon trajectory deviation from the perpendicular to the midline. Twelve or more trajectories were scored for each condition. Error bars, SEM; **P < 0.01, *P < 0.05, ANOVA.

References [+] :

Bhalla, Emergent properties of networks of biological signaling pathways. 1999, Pubmed