Maturation-Dependent Differences in the Re-innervation of the Denervated Dentate Gyrus by Sprouting Associational and Commissural Mossy Cell Axons in Organotypic Tissue Cultures of Entorhinal Cortex and Hippocampus

Sprouting of surviving axons is one of the major reorganization mechanisms of the injured brain contributing to a partial restoration of function. Of note, sprouting is maturation as well as age-dependent and strong in juvenile brains, moderate in adult and weak in aged brains. We have established a model system of complex organotypic tissue cultures to study sprouting in the dentate gyrus following entorhinal denervation. Entorhinal denervation performed after 2 weeks postnatally resulted in a robust, rapid, and very extensive sprouting response of commissural/associational fibers, which could be visualized using calretinin as an axonal marker. In the present study, we analyzed the effect of maturation on this form of sprouting and compared cultures denervated at 2 weeks postnatally with cultures denervated at 4 weeks postnatally. Calretinin immunofluorescence labeling as well as time-lapse imaging of virally-labeled (AAV2-hSyn1-GFP) commissural axons was employed to study the sprouting response in aged cultures. Compared to the young cultures commissural/associational sprouting was attenuated and showed a pattern similar to the one following entorhinal denervation in adult animals in vivo. We conclude that a maturation-dependent attenuation of sprouting occurs also in vitro, which now offers the chance to study, understand and influence maturation-dependent differences in brain repair in these culture preparations.

The Slit-binding Ig1 domain is required for multiple axon guidance activities of Drosophila Robo2

Drosophila Robo2 is a member of the evolutionarily conserved Roundabout (Robo) family of axon guidance receptors. The canonical role of Robo receptors is to signal midline repulsion in response to their cognate Slit ligands, which bind to the N-terminal Ig1 domain in most Robo family members. In the Drosophila embryonic ventral nerve cord, Robo1 and Robo2 cooperate to signal Slit-dependent midline repulsion, while Robo2 also regulates the medial-lateral position of longitudinal axon pathways and acts non-autonomously to promote midline crossing of commissural axons. Although it is clear that Robo2 signals midline repulsion in response to Slit, it is less clear whether Robo2's other activities are also Slit-dependent. To determine which of Robo2's axon guidance roles depend on its Slit-binding Ig1 domain, we have used a CRISPR/Cas9-based strategy replace the endogenous robo2 gene with a robo2 variant from which the Ig1 domain has been deleted (robo2ΔIg1). We compare the expression and localization of Robo2ΔIg1 protein with that of full-length Robo2 in embryonic neurons in vivo, and examine its ability to substitute for Robo2 to mediate midline repulsion and lateral axon pathway formation. We find that removal of the Ig1 domain from Robo2ΔIg1 disrupts both of these axon guidance activities. In addition, we find that the Ig1 domain of Robo2 is required for its proper subcellular localization in embryonic neurons, a role that is not shared by the Ig1 domain of Robo1. Finally, we report that although FasII-positive lateral axons are misguided in embryos expressing Robo2ΔIg1, the axons that normally express Robo2 are correctly guided to the lateral zone, suggesting that Robo2 may guide lateral longitudinal axons through a cell non-autonomous mechanism.

Robo recruitment of the Wave Regulatory Complex plays an essential and conserved role in midline repulsion

The Roundabout (Robo) guidance receptor family induces axon repulsion in response to its ligand Slit by inducing local cytoskeletal changes; however, the link to the cytoskeleton and the nature of these cytoskeletal changes are poorly understood. Here, we show that the heteropentameric Scar/Wave Regulatory Complex (WRC) which drives Arp2/3-induced branched actin polymerization, is a direct effector of Robo signaling. Biochemical evidence shows that Slit triggers WRC recruitment to the Robo receptor's WIRS motif. In Drosophila embryos, mutants of the WRC enhance Robo1-dependent midline crossing defects. Additionally, mutating Robo1's WIRS motif significantly reduces receptor activity in rescue assays in vivo, and CRISPR-Cas9 mutagenesis shows that the WIRS motif is essential for endogenous Robo1 function. Finally, axon guidance assays in mouse dorsal spinal commissural axons and gain-of-function experiments in chick embryos demonstrate that the WIRS motif is also required for Robo1 repulsion in mammals. Together, our data support an essential conserved role for the WIRS-WRC interaction in Robo1-mediated axon repulsion.

Endoglycan plays a role in axon guidance by modulating cell adhesion

Axon navigation depends on the interactions between guidance molecules along the trajectory and specific receptors on the growth cone. However, our in vitro and in vivo studies on the role of Endoglycan demonstrate that in addition to specific guidance cue – receptor interactions, axon guidance depends on fine-tuning of cell-cell adhesion. Endoglycan, a sialomucin, plays a role in axon guidance in the central nervous system of chicken embryos, but it is neither an axon guidance cue nor a receptor. Rather, Endoglycan acts as a negative regulator of molecular interactions based on evidence from in vitro experiments demonstrating reduced adhesion of growth cones. In the absence of Endoglycan, commissural axons fail to properly navigate the midline of the spinal cord. Taken together, our in vivo and in vitro results support the hypothesis that Endoglycan acts as a negative regulator of cell-cell adhesion in commissural axon guidance.

SlitC-PlexinA1 mediates iterative inhibition for orderly passage of spinal commissural axons through the floor plate

Spinal commissural axon navigation across the midline in the floor plate requires repulsive forces from local Slit repellents. The long-held view is that Slits push growth cones forward and prevent them from turning back once they became sensitized to these cues after midline crossing. We analyzed with fluorescent reporters Slits distribution and FP glia morphology. We observed clusters of Slit-N and Slit-C fragments decorating a complex architecture of glial basal process ramifications. We found that PC2 proprotein convertase activity contributes to this pattern of ligands. Next, we studied Slit-C acting via PlexinA1 receptor shared with another FP repellent, the Semaphorin3B, through generation of a mouse model baring PlexinA1Y1815F mutation abrogating SlitC but not Sema3B responsiveness, manipulations in the chicken embryo, and ex vivo live imaging. This revealed a guidance mechanism by which SlitC constantly limits growth cone exploration, imposing ordered and forward-directed progression through aligned corridors formed by FP basal ramifications.

Robo recruitment of the Wave Regulatory Complex plays an essential and conserved role in midline repulsion

SUMMARYThe Roundabout (Robo) guidance receptor family induces axon repulsion in response to its ligand Slit by inducing local cytoskeletal changes; however, the link to the cytoskeleton and the nature of these cytoskeletal changes are unclear. Here we show that the heteropentameric Scar/Wave Regulatory Complex (WRC) which drives Arp2/3-induced branched actin polymerization, is a direct effector of Robo signaling. Biochemical evidence shows that Slit triggers WRC recruitment to the Robo receptor’s WIRS motif. In Drosophila embryos, mutants of the WRC enhance Robol-dependent midline crossing defects. Additionally, mutating Robo1’s WIRS motif significantly reduces receptor activity in rescue assays in vivo, and CRISPR-Cas9 mutagenesis shows that the WIRS motif is essential for endogenous Robo1 function. Finally, axon guidance assays in mouse dorsal spinal commissural axons demonstrate that the WIRS motif is also required for Robo1 repulsion in mammals. Together, our data support an essential conserved role for the WRC in commissural axon repulsion.

Axon guidance at the midline – a live imaging perspective

ABSTRACTDuring neural circuit formation, axons navigate several choice points to reach their final target. At each one of these intermediate targets, growth cones need to switch responsiveness from attraction to repulsion in order to move on. Molecular mechanisms that allow for the precise timing of surface expression of a new set of receptors that support the switch in responsiveness are difficult to study in vivo. Mostly, mechanisms are inferred from the observation of snapshots of many different growth cones analyzed in different preparations of tissue harvested at distinct time points. However, to really understand the behavior of growth cones at choice points, a single growth cone should be followed arriving at and leaving the intermediate target.Here, we describe a spinal cord preparation that allows for live imaging of individual axons during navigation in their intact environment. The possibility to observe single growth cones navigating their intermediate target allows for measuring growth speed, changes in morphology, or aberrant behavior. Moreover, observation of the intermediate target – the floor plate – revealed its active participation and interaction with commissural axons during midline crossing.Summary statementLive tracking of single growth cones is more informative about axonal behavior during navigation than inference of behavior from the analyses of snapshots of different growth cones.