Focal adhesion kinase modulates Cdc42 activity downstream of positive and negative axon guidance cues.

There is biochemical, imaging and functional evidence that Rho GTPase signaling is a crucial regulator of actin-based structures such as lamellipodia and filopodia. However, although Rho GTPases are believed to serve similar functions in growth cones, the spatiotemporal dynamics of Rho GTPase signaling has not been examined in living growth cones in response to known axon guidance cues. Here we provide the first measurements of Cdc42 activity in living growth cones acutely stimulated with both growth-promoting and growth-inhibiting axon-guidance cues. Interestingly, we find that both permissive and repulsive factors can work by modulating Cdc42 activity, but in opposite directions. We find that the growth-promoting factors laminin and BDNF activate Cdc42, whereas the inhibitor Slit2 reduces Cdc42 activity in growth cones. Remarkably, we find that regulation of focal adhesion kinase (FAK) activity is a common upstream modulator of Cdc42 by BDNF, laminin and Slit. These findings suggest that rapid modulation of Cdc42 signaling through FAK by receptor activation underlies changes in growth cone motility in response to permissive and repulsive guidance cues.

Imaging adhesion and signaling dynamics in Xenopus laevis growth cones.

Xenopus laevis provides a robust model system to study cellular signaling and downstream processes during development both in vitro and in vivo. Intracellular signals must function within highly restricted spatial and temporal domains to activate specific downstream targets and cellular processes. Combining the versatility of developing Xenopus neurons with advances in fluorescent protein biosensors and imaging technologies has allowed many dynamic cellular processes to be visualized. This review will focus on the techniques we use to visualize and measure cell signaling, motility and adhesion by quantitative fluorescence microscopy in vitro and in vivo.

Focal adhesion kinase promotes integrin adhesion dynamics necessary for chemotropic turning of nerve growth cones.

The ability of extending axons to navigate using combinations of extracellular cues is essential for proper neural network formation. One intracellular signaling molecule that integrates convergent signals from both extracellular matrix (ECM) proteins and growth factors is focal adhesion kinase (FAK). Analysis of FAK function shows that it influences a variety of cellular activities, including cell motility, proliferation, and differentiation. Recent work in developing neurons has shown that FAK and Src function downstream of both attractive and repulsive growth factors, but little is known about the effectors or cellular mechanisms that FAK controls in growth cones on ECM proteins. We report that FAK functions downstream of brain-derived neurotrophic factor (BDNF) and laminin in the modulation of point contact dynamics, phosphotyrosine signaling at filopodial tips, and lamellipodial protrusion. BDNF stimulation accelerates paxillin-containing point contact turnover and formation. Knockdown of FAK function either with a FAK antisense morpholino or by expression of FRNK, a dominant-negative FAK isoform, blocks all aspects of the response to BDNF, including the acceleration of point contact dynamics. On the other hand, expression of specific FAK point mutants can selectively disrupt distinct aspects of the response to BDNF. We also show that growth cone turning depends on both signaling cascades tested here. Finally, we provide the first evidence that growth cone point contacts are asymmetrically regulated during turning to an attractive guidance cue.

Regulation of axonal outgrowth and pathfinding by integrin-ECM interactions.

Developing neurons use a combination of guidance cues to assemble a functional neural network. A variety of proteins immobilized within the extracellular matrix (ECM) provide specific binding sites for integrin receptors on neurons. Integrin receptors on growth cones associate with a number of cytosolic adaptor and signaling proteins that regulate cytoskeletal dynamics and cell adhesion. Recent evidence suggests that soluble growth factors and classic axon guidance cues may direct axon pathfinding by controlling integrin-based adhesion. Moreover, because classic axon guidance cues themselves are immobilized within the ECM and integrins modulate cellular responses to many axon guidance cues, interactions between activated receptors modulate cell signals and adhesion. Ultimately, growth cones control axon outgrowth and pathfinding behaviors by integrating distinct biochemical signals to promote the proper assembly of the nervous system. In this review, we discuss our current understanding how ECM proteins and their associated integrin receptors control neural network formation.

Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development.

Retinoic acid (RA) is essential for normal vertebrate development, including the patterning of the central nervous system. During early embryogenesis, RA is produced in the trunk mesoderm through the metabolism of vitamin A derived from the maternal diet and behaves as a morphogen in the developing hindbrain where it specifies nested domains of Hox gene expression. The loss of endogenous sources of RA can be rescued by treatment with a uniform concentration of exogenous RA, indicating that domains of RA responsiveness can be shaped by mechanisms other than the simple diffusion of RA from a localized posterior source. Here, we show that the cytochrome p450 enzymes of the Cyp26 class, which metabolize RA into polar derivatives, function redundantly to shape RA-dependent gene-expression domains during hindbrain development. In zebrafish embryos depleted of the orthologs of the three mammalian CYP26 genes CYP26A1, CYP26B1 and CYP26C1, the entire hindbrain expresses RA-responsive genes that are normally restricted to nested domains in the posterior hindbrain. Furthermore, we show that Cyp26 enzymes are essential for exogenous RA to rescue hindbrain patterning in RA-depleted embryos. We present a ;gradient-free' model for hindbrain patterning in which differential RA responsiveness along the hindbrain anterior-posterior axis is shaped primarily by the dynamic expression of RA-degrading enzymes.