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

Abstract

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.

Contribution of the neuron-specific ATP1A3 to embryonic spinal circuit emergence

“…To make sure that spinal cord patterning was maintained and that floor plate functions were preserved, we performed HCR RNA-FISH on E4 operated embryos. Consistently with the fact that ex vivo spinal cord open-books can be used to study DIN axon navigation across the floor plate 61,62 , we showed that the surgical manipulation did not interfere with expression of key guidance molecules such as Slit2 or neuron specification such as Isl1 expression (Figure 4d-e). As expected, Isl1 expression was still detected in a dorsal population of neurons (Figure 4d-e).…”

3D exploration of gene expression in chicken embryos through combined RNA fluorescence in situ hybridization, immunofluorescence, and clearing

“…As expected, in the spinal cord, GFP positive axons of the dorsal interneurons were observed to cross the midline below the soma layer of the floor plate that strongly expresses SLIT2 (Fig. 4 c) [ 64 ]. The resolution even allowed us to observe the individual axons navigating across the floor plate (Fig.…”

Regulation of axon pathfinding by astroglia across genetic model organisms

“…Yet, in contrast to regulators of fate specification and migration of glial cells, mechanisms driving their morphogenesis remain even more understudied. And yet this architecture may be key for axon pathfinding, either through physical interactions of adhesion or by presenting specific subcellular localization of membrane-bound guidance factors, such as the aforementioned Slit and Semaphorin3B clusters on endfeet of radial glial cells in the developing mouse spinal cord ( Ducuing et al, 2020 ). Thus, studying how astroglial cells establish their morphological cell architecture should be another important forefront in the investigations of neuropil development and axon navigation.…”

“…Growth cones establish contact with these glial cell surfaces during midline crossing. The clusters of Slit and Semaphorin3B fragments on these endfeet produce constraints protecting the growth cones from the risk of aberrant deviations due to active exploration of other cues in their environment ( Ducuing et al, 2020 ).…”