A defining characteristic of neuronal cell type is the growth of axons and dendrites into specific layers and columns of the brain. Although differences in cell surface receptors and adhesion molecules are known to cause differences in synaptic specificity, differences in downstream signaling mechanisms that determine cell type-appropriate targeting patterns are unknown. Using a forward genetic screen in Drosophila, the GTPase effector Genghis khan (Gek) was identified as playing a crucial role in the ability of a subset of photoreceptor (R cell) axons to innervate appropriate target columns. In particular, single-cell mosaic analyses demonstrate that R cell growth cones lacking Gek function grow to the appropriate ganglion, but frequently fail to innervate the correct target column. Further studies reveal that R cell axons lacking the activity of the small GTPase Cdc42 display similar defects, providing evidence that these proteins regulate a common set of processes. Gek is expressed in all R cells, and a detailed structure-function analysis reveals a set of regulatory domains with activities that restrict Gek function to the growth cone. Although Gek does not normally regulate layer-specific targeting, ectopic expression of Gek is sufficient to alter the targeting choices made by another R cell type, the targeting of which is normally Gek independent. Thus, specific regulation of cytoskeletal responses to targeting cues is necessary for cell type-appropriate synaptic specificity (Gontang, 2011).

Sequences of genetically programmed developmental decisions play an important role in determining the complex architecture of the adult brain. At the level of single neurons, these decisions produce cellular morphologies that are characteristic of each cell type and reflect specific guidance and targeting choices made by both axons and dendrites. Although many molecules have been identified that affect synaptic specificity, the molecular mechanisms that allow closely related cell types to make distinct targeting decisions in vivo are almost completely unknown. This study examined these mechanisms in the Drosophila visual system, and demonstrates that cell type-specific utilization of a cytoskeletal regulatory pathway plays a crucial role in the targeting choices made by a subset of R cells (Gontang, 2011).

The visual systems of many animals are characterized by a broad organization of axons and dendrites into columns and layers (Sanes and Zipursky, 2010). Columns are organized into retinotopic arrays to allow the brain to process, in parallel, information from different points of visual space. Within each column, layers process different qualities of light and reflect different combinations of pre- and post-synaptic inputs. Closely related cell types make different choices with respect to these architectural features. How axons can be genetically programmed to innervate both the correct column and the appropriate layer, making cell type-specific targeting decisions, remains an open question (Gontang, 2011).

Photoreceptor axons in Drosophila display both columnar and layer-specific targeting. The Drosophila visual system comprises the compound eye and four optic ganglia: the lamina, the medulla, the lobula and the lobula plate. Each facet of the eye, or ommatidium, contains eight R cells, which can be divided into three distinct types. R1-R6 cells, the outer photoreceptors in each ommatidium, project their axons into the lamina. Each of these axons innervates a specific column of post-synaptic target neurons, reconstructing a topographic map of the visual world in the brain. By contrast, the inner R7 and R8 cells project axons that terminate in two distinct layers in the medulla, making synaptic connections across multiple layers. Cell ablation studies and genetic manipulations demonstrate that the targeting of R1-R6 axons to specific targets is both genetically hard-wired and dependent on interactions amongst afferent axons, rather than on specific interactions between afferents and their particular targets. The layer-specific targeting of R7 and R8 cells, however, develops in a precise temporal sequence and is dependent upon specific interactions with presumed post-synaptic cells and intermediate targets. Thus, the cellular mechanisms that underlie columnar versus layer-specific targeting in this system are at least partially distinct (Gontang, 2011).

Both histological and behavioral screens have identified many genes involved in the targeting of R1-R6 axons to appropriate columns within the lamina and in the targeting of R7 and R8 axons to appropriate medulla layers. For example, the classical cadherin N-cadherin mediates interactions between pre- and post-synaptic cells that are required for the targeting choices made by R1-R6 cells, and by R7 cells. The receptor tyrosine phosphatases Lar and Ptp69D, as well as the Lar-interacting protein Liprin-α, stabilize these connections. Dynamic regulation of the expression of N-cadherin and Lar is also crucial to the layer-specific targeting decisions of R7 and R8. The non-classical cadherin Flamingo (Starry night) is required for appropriate topographic mapping, the layer-specific targeting of R8, and the columnar targeting choices made by R1-R6. Intriguingly, for all genes where mutant phenotypes have been assessed in R1-R6, R7 and R8 photoreceptors, both columnar targeting by R1-R6, as well as layer-specific targeting by R7 and/or R8, were affected, even though the selection criteria under which they were initially identified were specific to only one of these phenotypes. Thus, these two different types of targeting decisions clearly use many of the same molecular components. However, it remains unclear whether additional components are required specifically for either the columnar targeting decisions of R1-R6 or the layer-specific targeting of R7 and R8 (Gontang, 2011 and references therein).

Rho family GTPases regulate many stages of neuronal growth, including growth cone motility, axonal migration and dendritic spine morphogenesis. In the Drosophila nervous system, these GTPases regulate axon growth and guidance. In the Drosophila visual system, Rac is an upstream activator of the Dock-Pak signaling pathway, affecting R cell axon targeting to the lamina and the medulla. However, understanding of the links between small GTPases and specific axonal targeting decisions remains incomplete, and only a limited set of GTPase effector proteins have been identified. One of these, Genghis khan (Gek), the Drosophila homolog of mammalian Cdc42-binding protein kinase alpha [CDC42BPα (or MRCKα); myotonic dystrophy-related Cdc42-binding kinase (MRCK-1) in C. elegans], was originally identified using biochemical approaches to identify effectors of the small GTPase Cdc42 (Leung, 1998; Luo, 1997). These studies demonstrated that Gek binds specifically to activated Cdc42, that this interaction increases the kinase activity of Gek in vitro, and that Gek activity alters actin cytoskeletal organization (Luo, 1997). Finally, genetic studies of asymmetric cell division and epithelial morphogenesis in C. elegans have demonstrated that MRCK-1 functions downstream of Cdc42 in vivo (Gally, 2009; Kumfer, 2010). However, the function of Gek in neurons is unknown. This study examined the role of Gek in controlling axon outgrowth and target selection in all subtypes of R cells in the Drosophila eye (Gontang, 2011).

The data demonstrate that Gek and Cdc42 function as regulators of the actin cytoskeleton within R1-R6 cell growth cones. R1-R6 growth cones lacking Gek or Cdc42 activity display two prevalent targeting phenotypes, as they can either fail to extend to their terminal targets or can extend to one or more inappropriate targets within the lamina. Taken together with the biochemical studies of MRCK, it is proposed that localized activation of Gek by Cdc42 within subdomains of R1-R6 growth cones is a necessary step in making a directed extension towards a specific target. In this view, Gek acts downstream of adhesive cues and/or targeting signals generated on the cell surface, integrating these cues and altering the organization of the actin cytoskeleton so as to promote lateral axon extension. gek mutant growth cones that fail to extend presumably fail to define an extension domain; those that mistarget or that extend to multiple targets must initiate extensions in multiple, spatially inappropriate domains. These latter cases might reflect perduring Gek or Cdc42 protein in the single-cell clones, or might result from the activities of other regulators of protrusion. These data also demonstrate that the known regulators of R cell axon targeting, including the cadherins Flamingo and N-cadherin as well as the tyrosine phosphatase Lar, are not individually required to regulate Gek or Cdc42. Thus, either these regulators act redundantly to control Gek, or an as yet unknown pathway is required for columnar targeting of R1-R6 growth cones (Gontang, 2011).

Structure-function analyses of Gek, combined with biochemical studies examining MRCK regulation in vitro, provide insight into the mechanism of Gek regulation. Work in both contexts demonstrates that the kinase domain is the crucial effector of the protein, as deletion or inactivation of this domain removes all detectable activity both in vivo and in vitro (Gontang, 2011; Luo, 1997; Leung, 1998). Previous studies of MRCK uncovered multiple negative regulatory mechanisms (Tan, 2001a; Tan, 2001b; Dong, 2002). In particular, the coiled-coil domain drives oligomerization and contains a kinase-inhibitory motif (KIM) that sequesters the kinase domain when the protein is inactive (Tan, 2001a; Dong, 2002; Garcia, 2006). Inhibitory activity has also been associated with the pleckstrin homology domain and the citron homology domain (Chen, 1999). Consistent with these observations, when any one of these three domains were deleted from Gek and these constructs were expressed in gek mutant R cells, premature stopping of R cell axons above the lamina were observed or defects in cell morphogenesis, depending on the level and timing of transgene expression. However, these mutant forms of Gek do not correspond to simple 'hyperactivated' proteins, as adding additional wild-type Gek protein eliminates, rather than enhances, the premature axon termination phenotype. Thus, the presence of wild-type protein restores normal inhibition to these mutant forms of Gek. These results are consistent with biochemical studies of MRCK showing that a conserved N-terminal domain can mediate dimerization, and that kinase activation is critically dependent on transautophosphorylation (Tan, 2001a). In this model, activation of a Gek dimer requires that both monomers are independently disinhibited, making them competent to be transphosphorylated by their partner. Thus, in a heterodimer comprising one wild-type subunit and one subunit lacking an autoinhibitory domain (Gek-δCC, Gek-δPH or Gek-δCH), the truncated monomer cannot phosphorylate its inactive wild-type partner (which remains autoinhibited) and does not get phosphorylated by it in turn. As a result, the presence of wild-type protein will suppress an otherwise gain-of-function mutant protein. Conversely, in the absence of wild-type protein, homodimers comprising only truncated subunits will transactivate throughout the cell, making growth cones competent to extend laterally, but also disrupting morphogenesis. Of course, because it is not possible to directly measure Gek activity within R cell growth cones, the possibility cannot be excluded that the gain-of-function gek mutations that were generated act differently from the inhibitory activities that have been associated with these domains in MRCK in vitro (Gontang, 2011).

Of the many molecules known to be involved in R cell axon target selection, Gek is the first to be specifically required for R1-R6 axons, but not R7 or R8 axons, to reach their targets. A simple mechanism to achieve such functional specificity would be cell type-specific expression of Gek. However, Gek is strongly expressed in all photoreceptor axons at developmentally appropriate stages. Thus, cell type-specific function must emerge through post-translational regulation of Gek activity. It is proposed that the cell type-specific requirement for Gek emerges from differences in how upstream signaling pathways control common regulators of the actin cytoskeleton to achieve columnar versus laminar targeting (Gontang, 2011).

MRCK function has been linked to two important regulators of the actin cytoskeleton, LIMK and MRLC, the latter through both direct and indirect pathways (Gally, 2009; Kumfer, 2010; Wilkinson, 2005; Dong, 2002; Sumi, 2001; Tan, 2001b). These data are consistent with the notion that at least one of these, MRLC, is a regulatory target of both Gek and Cdc42 in R cell growth cones, albeit one of as yet untested functional significance. It is proposed that the cell type-specific requirement for Gek in R1-R6 axons reflects the fact that an as yet unknown upstream signaling pathway requires a specific pattern of modulation of MRLC (achieved via Gek and Cdc42) for R1-R6 axons to be competent to achieve columnar target selection. In this view, the various cell surface interactions that are required for layer-specific targeting of R7 do not signal through Gek, but do use downstream effectors that can be modulated by Gek. When Gek is overexpressed in R7, mistargeting is caused by high levels of Gek bypassing this upstream regulation, causing abnormal activation of relatively ‘generic’ effector proteins in R7 growth cones. Thus, the columnar targeting of R1-R6 axons and the layer-specific targeting of R7 axons reflect the modulation of at least some of the same effector genes, but the upstream signals that direct columnar targeting are uniquely dependent on Gek function. More broadly, given that axon targeting in most systems is generally dependent on multiple, partially redundant cell surface interactions, whereas many of the direct cytoskeletal regulators (such as MRLC) are essential and pleiotropic, these results demonstrate that one way by which cell type-appropriate differences in targeting can be achieved is through the use of specific cytoskeletal modulators like Gek. In this way, subtle differences in the control of the cytoskeleton can cause significant differences in the overall pattern of target selection, making one group of cells competent to target in a columnar fashion. Thus, cell type-specific morphologies can emerge both through differences in cell surface interactions and through differences in cytoplasmic signaling mechanisms (Gontang, 2011).