Timothy A. Evans

Axon guidance at the midline: of mice and flies

In bilaterally symmetric organisms, the midline is a critical organizing center for the developing central nervous system. There is a striking conservation of the molecules and mechanisms that control axon path finding at the midline in vertebrate and invertebrate nervous systems. The majority of axons in the CNS cross the midline before projecting to their contralateral synaptic targets and this crossing decision is under exquisite spatial and temporal regulation. Growing commissural axons initially respond to attractive signals, while inhibiting responses to repulsive signals. Once across, repulsion dominates, allowing axons to leave and preventing them from re-entering the midline. Here we review recent advances in flies and mice that illuminate the molecular mechanisms underlying the establishment of precise connectivity at the midline.

Functional Diversity of Robo Receptor Immunoglobulin Domains Promotes Distinct Axon Guidance Decisions

Recognition molecules of the immunoglobulin (Ig) superfamily control axon guidance in the developing nervous system. Ig-like domains are among the most widely represented protein domains in the human genome, and the number of Ig superfamily proteins is strongly correlated with cellular complexity. In Drosophila, three Roundabout (Robo) Ig superfamily receptors respond to their common Slit ligand to regulate axon guidance at the midline: Robo and Robo2 mediate midline repulsion, Robo2 and Robo3 control longitudinal pathway selection, and Robo2 can promote midline crossing. How these closely related receptors mediate distinct guidance functions is not understood. We report that the differential functions of Robo2 and Robo3 are specified by their ectodomains and do not reflect differences in cytoplasmic signaling. Functional modularity of Robo2s ectodomain facilitates multiple guidance decisions: Ig1 and Ig3 of Robo2 confer lateral positioning activity, whereas Ig2 confers promidline crossing activity. Robo2s distinct functions are not dependent on greater Slit affinity but are instead due in part to differences in multimerization and receptor-ligand stoichiometry conferred by Robo2s Ig domains. Together, our findings suggest that diverse responses to the Slit guidance cue are imparted by intrinsic structural differences encoded in the extracellular Ig domains of the Robo receptors.

Robo2 acts in trans to inhibit Slit-Robo1 repulsion in pre-crossing commissural axons

During nervous system development, commissural axons cross the midline despite the presence of repellant ligands. In Drosophila, commissural axons avoid premature responsiveness to the midline repellant Slit by expressing the endosomal sorting receptor Commissureless, which reduces surface expression of the Slit receptor Roundabout1 (Robo1). In this study, we describe a distinct mechanism to inhibit Robo1 repulsion and promote midline crossing, in which Roundabout2 (Robo2) binds to and prevents Robo1 signaling. Unexpectedly, we find that Robo2 is expressed in midline cells during the early stages of commissural axon guidance, and that over-expression of Robo2 can rescue robo2-dependent midline crossing defects non-cell autonomously. We show that the extracellular domains required for binding to Robo1 are also required for Robo2s ability to promote midline crossing, in both gain-of-function and rescue assays. These findings indicate that at least two independent mechanisms to overcome Slit-Robo1 repulsion in pre-crossing commissural axons have evolved in Drosophila. DOI: http://dx.doi.org/10.7554/eLife.08407.001

Slit/Robo-mediated axon guidance in Tribolium and Drosophila: divergent genetic programs build insect nervous systems.

As the complexity of animal nervous systems has increased during evolution, developmental control of neuronal connectivity has become increasingly refined. How has functional diversification within related axon guidance molecules contributed to the evolution of nervous systems? To address this question, we explore the evolution of functional diversity within the Roundabout (Robo) family of axon guidance receptors. In Drosophila, Robo and Robo2 promote midline repulsion, while Robo2 and Robo3 specify the position of longitudinal axon pathways. The Robo family has expanded by gene duplication in insects; robo2 and robo3 exist as distinct genes only within dipterans, while other insects, like the flour beetle Tribolium castaneum, retain an ancestral robo2/3 gene. Both Robos from Tribolium can mediate midline repulsion in Drosophila, but unlike the fly Robos cannot be down-regulated by Commissureless. The overall architecture and arrangement of longitudinal pathways are remarkably conserved in Tribolium, despite it having only two Robos. Loss of TcSlit causes midline collapse of axons in the beetle, a phenotype recapitulated by simultaneous knockdown of both Robos. Single gene knockdowns reveal that beetle Robos have specialized axon guidance functions: TcRobo is dedicated to midline repulsion, while TcRobo2/3 also regulates longitudinal pathway formation. TcRobo2/3 knockdown reproduces aspects of both Drosophila robo2 and robo3 mutants, suggesting that TcRobo2/3 has two functions that in Drosophila are divided between Robo2 and Robo3. The ability of Tribolium to organize longitudinal axons into three discrete medial-lateral zones with only two Robo receptors demonstrates that beetle and fly achieve equivalent developmental outcomes using divergent genetic programs.

Kekkon5 is an extracellular regulator of BMP signaling.

Precise spatial and temporal control of Drosophila Bone Morphogenetic Protein (BMP) signaling is achieved by a host of extracellular factors that modulate ligand distribution and activity. Here we describe Kekkon5 (Kek5), a transmembrane protein containing leucine-rich repeats (LRRs), as a novel regulator of BMP signaling in Drosophila. We find that loss or gain of kek5 disrupts crossvein development and alters the early profile of phosphorylated Mad and dSRF in presumptive crossvein cells. kek5 phenotypic effects closely mimic those observed with Short gastrulation (Sog), but do not completely recapitulate the effects of dominant negative BMP receptors. We further demonstrate that Kek5 is able to antagonize the BMP ligand Glass bottom boat (Gbb) and that the Kek5 LRRs are required for BMP inhibitory activity, while the Ig domain is dispensable in this context. Our identification of Kek5 as a modulator of BMP signaling supports the emerging notion that LIG proteins function as diverse regulators of cellular communication.

Slit Binding via the Ig1 Domain Is Essential for Midline Repulsion by Drosophila Robo1 but Dispensable for Receptor Expression, Localization, and Regulation in Vivo

The midline repellant ligand Slit and its Roundabout (Robo) family receptors constitute the major midline repulsive pathway in bilaterians. Slit proteins produced at the midline of the central nervous system (CNS) signal through Robo receptors expressed on axons to prevent them from crossing the midline, and thus regulate connectivity between the two sides of the nervous system. Biochemical structure and interaction studies support a model in which Slit binding to the first immunoglobulin-like (Ig1) domain of Robo receptors activates a repulsive signaling pathway in axonal growth cones. Here, we examine the in vivo functional importance of the Ig1 domain of the Drosophila Robo1 receptor, which controls midline crossing of axons in response to Slit during development of the embryonic CNS. We show that deleting Ig1 from Robo1 disrupts Slit binding in cultured Drosophila cells, and that a Robo1 variant lacking Ig1 (Robo1∆Ig1) is unable to promote ectopic midline repulsion in gain-of-function studies in the Drosophila embryonic CNS. We show that the Ig1 domain is not required for proper expression, axonal localization, or Commissureless (Comm)-dependent regulation of Robo1 in vivo, and we use a genetic rescue assay to show that Robo1∆Ig1 is unable to substitute for full-length Robo1 to properly regulate midline crossing of axons. These results establish a direct link between in vitro biochemical studies of Slit–Robo interactions and in vivo genetic studies of Slit-Robo signaling during midline axon guidance, and distinguish Slit-dependent from Slit-independent aspects of Robo1 expression, regulation, and activity during embryonic development.

Embryonic axon guidance: insights from Drosophila and other insects

During embryonic development, growing axons are guided by cellular signaling pathways that control a series of individual axon guidance decisions. In Drosophila, two major pathways (Netrin-Frazzled/DCC and Slit-Robo) regulate axon guidance in the embryonic ventral nerve cord, including the critical decision of whether or not to cross the midline. Studies in the fruit fly have revealed a complex picture of precise regulation and cross-talk between these pathways. In addition, Robo receptors in Drosophila have diversified their activities to regulate additional axon guidance decisions in the developing embryo. Here, I discuss recent advances in understanding roles and regulation of the Net-Fra and Slit-Robo signaling pathways in Drosophila, and examine the evolutionary conservation of these signaling mechanisms across insects and other arthropods.

Argos Mutants Define an Affinity Threshold for Spitz Inhibition in Vivo

Argos, a secreted antagonist of Drosophila epidermal growth factor receptor (dEGFR) signaling, acts by sequestering the activating ligand Spitz. To understand how different domains in Argos contribute to efficient Spitz sequestration, we performed a genetic screen aimed at uncovering modifiers of an Argos misexpression phenotype in the developing eye. We identified a series of suppressors mapping to the Argos transgene that affect its activity in multiple developmental contexts. These point mutations map to both the N- and C-terminal cysteine-rich regions, implicating both domains in Argos function. We show by surface plasmon resonance that these Argos mutants are deficient in their ability to bind Spitz in vitro. Our data indicate that a mere ∼2-fold decrease in KD is sufficient to compromise Argos activity in vivo. This effect could be recapitulated in a cell-based assay, where a higher molar concentration of mutant Argos was needed to inhibit Spitz-dependent dEGFR phosphorylation. In contrast, a ∼37-fold decrease in the binding constant nearly abolishes Argos activity in vivo and in cellular assays. In agreement with previously reported computational studies, our results define an affinity threshold for optimal Argos inhibition of dEGFR signaling during development.

In vivo functional analysis of Drosophila Robo1 immunoglobulin-like domains

BackgroundIn animals with bilateral symmetry, midline crossing of axons in the developing central nervous system is regulated by Slit ligands and their neuronal Roundabout (Robo) receptors. Multiple structural domains are present in an evolutionarily conserved arrangement in Robo family proteins, but our understanding of the functional importance of individual domains for midline repulsive signaling is limited.MethodsWe have examined the functional importance of each of the five conserved immunoglobulin-like (Ig) domains within the Drosophila Robo1 receptor. We generated a series of Robo1 variants, each lacking one of the five Ig domains (Ig1-5), and tested each for their ability to bind Slit when expressed in cultured Drosophila cells. We used a transgenic approach to express each variant in robo1’s normal expression pattern in wild-type and robo1 mutant embryos, and examined the effects of deleting each domain on receptor expression, axonal localization, regulation, and midline repulsive signaling in vivo.ResultsWe show that individual deletion of Ig domains 2–5 does not interfere with Robo1’s ability to bind Slit, while deletion of Ig1 strongly disrupts Slit binding. None of the five Ig domains (Ig1-5) are individually required for proper expression of Robo1 in embryonic neurons, for exclusion from commissural axon segments in wild-type embryos, or for downregulation by Commissureless (Comm), a negative regulator of Slit-Robo repulsion in Drosophila. Each of the Robo1 Ig deletion variants (with the exception of Robo1∆Ig1) were able to restore midline crossing in robo1 mutant embryos to nearly the same extent as full-length Robo1, indicating that Ig domains 2–5 are individually dispensable for midline repulsive signaling in vivo.ConclusionsOur findings indicate that four of the five Ig domains within Drosophila Robo1 are dispensable for its role in midline repulsion, despite their strong evolutionary conservation, and highlight a unique requirement for the Slit-binding Ig1 domain in the regulation of midline crossing.

CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3

BackgroundAxon guidance receptors of the Roundabout (Robo) family regulate a number of axon guidance outcomes in bilaterian animals in addition to their canonical role in Slit-dependent midline repulsion. In the fruit fly Drosophila melanogaster, three Robo paralogs (Robo1, Robo2, and Robo3) each have specialized roles in regulating midline crossing and the formation of longitudinal axon pathways in the embryonic ventral nerve cord. The number of robo genes differs in other insects, and it is unknown whether the roles and/or signaling mechanisms of Drosophila Robos are shared in other insect species. To directly compare the axon guidance activities of Robo receptors in Drosophila and the flour beetle Tribolium castaneum, I have used a CRISPR/Cas9-based approach to replace Drosophila robo3 with Tribolium robo2/3.ResultsI show that when expressed from the robo3 locus in Drosophila embryos, Tribolium Robo2/3 (TcRobo2/3) protein is properly translated and localized to axons, where it reproduces the normal expression pattern of Drosophila Robo3. In embryos expressing TcRobo2/3 in place of robo3, two distinct subsets of longitudinal axons are guided properly to their normal positions in the intermediate neuropile, indicating that TcRobo2/3 can promote Robo3-dependent axon guidance decisions in developing Drosophila neurons.ConclusionsThese observations suggest that the mechanism by which Drosophila Robo3 promotes longitudinal pathway formation is evolutionarily conserved in Tribolium, where it is performed by TcRobo2/3. The CRISPR/Cas9-based gene replacement approach described here can be applied to comparative evolutionary developmental studies of other Drosophila genes and their orthologs in other species.