Rho GTPases regulate PTPμ-mediated nasal neurite outgrowth and temporal repulsion of retinal ganglion cell neurons
Introduction
Spatiotemporal patterning of the visual system is a structured event in which retinal ganglion cell (RGC) axons from a particular region of the retina migrate and innervate a specific region of the optic tectum (McLaughlin et al., 2003, O'Leary and McLaughlin, 2005, Thanos and Mey, 2001, van Horck et al., 2004). For example, nasal axons innervate the posterior tectum, while temporal neurons innervate the anterior tectum. Studies have suggested that RGC projection to the tectum is partially due to the graded expression of Eph receptor tyrosine kinases and their ephrin ligands (McLaughlin et al., 2003, O'Leary and McLaughlin, 2005). Therefore, tyrosine phosphorylation is key to the development of the visual system but little is known about the function of tyrosine phosphatases in this system. Receptor tyrosine phosphatases (RPTPs), which catalyze the dephosphorylation of tyrosine residues, are expressed in the nervous system and recent evidence suggests that they may be involved in guiding retinal axons to their targets during development (Brady-Kalnay, 2001, Ensslen-Craig and Brady-Kalnay, 2004). A subfamily of RPTPs, including PTPμ, has cell adhesion molecule-like extracellular segments and intracellular domains with tyrosine phosphatase activity (Brady-Kalnay, 2001, Ensslen-Craig and Brady-Kalnay, 2004, Johnson and Van Vactor, 2003).
The PTPμ-like subfamily of RPTPs includes four members: PTPμ, PTPκ, PTPρ and PCP-2 (Brady-Kalnay, 2001). The PTPμ-like subfamily members contain motifs found in CAMs including a MAM domain, an immunoglobulin (Ig) domain and four FNIII repeats in their extracellular segment. The motifs present in PTPμ suggested that it might function in cell–cell adhesion. We demonstrated that expression of PTPμ induced the aggregation of nonadhesive Sf9 insect cells (Brady-Kalnay et al., 1993, Gebbink et al., 1993). PTPκ and PCP-2 have also been shown to mediate aggregation (Cheng et al., 1997, Sap et al., 1994). These studies demonstrated that the binding is homophilic (i.e. the “ligand” for a transmembrane PTP is an identical PTP molecule on an adjacent cell). Both PTPμ-dependent adhesion and neurite outgrowth are mediated by homophilic binding (Brady-Kalnay and Tonks, 1994, Ensslen-Craig and Brady-Kalnay, 2005). The homophilic binding site resides in the immunoglobulin domain (Brady-Kalnay and Tonks, 1994). The MAM domain of PTPμ also plays a role in cis dimerization (Aricescu et al., 2006, Cismasiu et al., 2004) and cell aggregation by sorting cells expressing PTPμ into distinct aggregates from cells expressing related molecules such as PTPκ (Zondag et al., 1995). Together, these studies demonstrate that PTPμ mediates cell–cell adhesion and suggest that it may transduce signals in response to adhesion that are required for neurite outgrowth.
The juxtamembrane domain of the PTPμ-like enzymes contains a region of homology to the intracellular domain of the cadherins (Brady-Kalnay, 2001). We demonstrated that PTPμ interacts specifically with N-cadherin, E-cadherin and R-cadherin (Brady-Kalnay et al., 1998). Both N- and R-cadherin are expressed in the retina and control retinal histogenesis (Redies, 1997, Redies and Takeichi, 1996). Importantly, we have demonstrated a functional role for the PTPμ/N-cadherin interaction because we found that PTPμ regulates N-cadherin-dependent neurite outgrowth of retinal ganglion cells (Burden-Gulley and Brady-Kalnay, 1999).
PTPμ is expressed in a gradient in both the retina and the tectum (Burden-Gulley et al., 2002). Since the axons of RGCs form the optic nerve and are the sole output from the retina to the brain, the expression of PTPμ on these cells was consistent with a putative role for PTPμ in axonal migration. The ability of retinal ganglion cell axons to grow out of a retinal explant onto a purified protein substrate has been used to study axonal (neurite) growth (Lemmon et al., 1992). To test whether PTPμ promoted neurite outgrowth, we used purified PTPμ as a substrate and demonstrated that it promoted neurite outgrowth from E8 retinal explants (Burden-Gulley and Brady-Kalnay, 1999). More importantly, PTPμ is a permissive substrate for neurite outgrowth from nasal RGC neurons, while it is inhibitory to temporal RGC neurons (Burden-Gulley et al., 2002). The molecular details of the intracellular signals that regulate axonal growth and guidance are not well defined.
It is intriguing that PTPμ is able to mediate both permissive and repulsive responses in RGC neurons. PTPμ is expressed at low levels in nasal neurons and is permissive for nasal RGC neurite outgrowth (Burden-Gulley et al., 2002). PTPμ is expressed at high levels in temporal RGCs and is repulsive to those neurons (Burden-Gulley et al., 2002). Our recent data suggest that the gradient of PTPμ expression in the retina regulates whether PTPμ is a repulsive or permissive guidance cue (Ensslen-Craig and Brady-Kalnay, 2005). We found that expression of exogenous PTPμ in nasal neurites, which increases PTPμ levels to those found in temporal neurons, resulted in a phenotypic switch from a permissive to a repulsive response to a PTPμ substrate (Ensslen-Craig and Brady-Kalnay, 2005). Catalytic activity of PTPμ is necessary for both permissive and repulsive guidance events downstream of PTPμ binding. Since both expression and catalytic activity of PTPμ are required, this implies that PTPμ homophilic binding results in a tyrosine phosphatase-dependent signal that is necessary for PTPμ-mediated nasal outgrowth and temporal repulsion, adding support to the growing body of evidence that RPTP catalytic activity is important for axon guidance (Garrity et al., 1999, Johnson et al., 2001). Most importantly, these results demonstrate a functional significance for the observed gradients in retinotectal expression of PTPμ. In this manuscript, we investigate the molecular mechanisms by which PTPμ regulates both nasal outgrowth and temporal repulsion.
The Rho subfamily of small G-proteins plays a central role in regulating the actin cytoskeleton (Raftopoulou and Hall, 2004, Ridley, 2004). Activation of Cdc42 results in the formation of filopodia while Rac activation induces lamellipodia formation, and Rho activation results in stress fibers in fibroblasts (Nobes and Hall, 1995). Rho GTPases are molecular switches, cycling between GTP-bound “on” and GDP-bound “off” states. This cycle is controlled by GTPase activating proteins (GAPs), guanine nucleotide exchange factors (GEFs) and GDP-dissociation inhibitors (Bishop and Hall, 2000, Kaibuchi et al., 1999).
The role of Rho GTPase activation in the regulation of nasal outgrowth and temporal repulsion was tested by utilizing dominant negative and constitutively active TAT-tagged forms of Rac1, RhoA and Cdc42, which were directly taken up by cells. We prepared alternating stripes of PTPμ and laminin as substrates for single stripe assays and a combination of PTPμ mixed with laminin versus laminin only lanes for the mixed stripe assays. Single stripes measure permissive responses, i.e. the ability to grow or not on one adhesion molecule. Mixed stripes measure repulsive responses because a repulsive substrate-like PTPμ is mixed with a normally permissive cue, such as laminin. The TAT-Rho GTPase fusion proteins were added to the stripe assays and changes in nasal outgrowth or temporal crossing were observed. Nasal neurite outgrowth and temporal repulsion to a PTPμ substrate were substantially reduced by the addition of Cdc42-DN, while inhibition of Rac1 is required for temporal repulsion to PTPμ response. These data suggest that the key to PTPμ-dependent repulsion is inhibition of Rac1 activity in temporal RGC neurons.
Section snippets
Results
We cultured retinal explants from embryonic day 8 chicks in Bonhoeffer stripe assays (Vielmetter et al., 1990, Walter et al., 1987a, Walter et al., 1987b). The Bonhoeffer stripe assay examines the ability of neurons to grow on two protein substrates presented in alternating lanes. A permissive substrate must be present in a lane to observe any neurite outgrowth on that lane (Lemmon et al., 1992). Very few adhesion molecules promote neurite outgrowth of chick retinal ganglion cell (RGC) neurons (
Discussion
In this manuscript, we tested whether the Rho GTPases regulate PTPμ-dependent nasal outgrowth and temporal repulsion. The data presented here indicate that Cdc42 activity is required for both nasal outgrowth and temporal repulsion (Fig. 10), which is consistent with our previous study that demonstrated a requirement of Cdc42 activity for PTPμ-dependent growth cone rearrangement (Rosdahl et al., 2003). In the previous study, the stimulation of E6 retinal cultures with purified PTPμ resulted in a
Culturing of chick retinal explants
Chick embryonic day 8 embryos were staged according to Hamburger and Hamilton (Hamburger and Hamilton, 1951). Retinal explants were prepared as described (Drazba and Lemmon, 1990, Halfter et al., 1983). E8 (stage 32) neural retinas were dissected, flattened on concanavalin-coated nitrocellulose filters and cut into 350 μm wide explants. Explants were placed retinal ganglion side down on substrate-coated dishes and grown in 10% fetal bovine serum (Hyclone, Logan, UT), 2% chick serum 4(Sigma, St.
Acknowledgments
This research was supported by a grant from the National Institutes of Health Grant R01-EY12251 (S.B.K). Additional support was obtained from the Visual Sciences Research Center Core Grant PO-EY11373 from the National Eye Institute. We thank Dr. Steven Dowdy for providing the TAT-Rho GTPase fusion proteins. We thank Carol Luckey for technical support including preparation and purification of the TAT-fusion Rho GTPase proteins and members of the Brady-Kalnay lab for insightful discussions.
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