Multiple Guidance Mechanisms Regulate T-shaped Bifurcation of Dorsal Root Ganglion Axons in the Spinal Cord

Core Concepts

The formation of the stereotypic T-shaped bifurcation of dorsal root ganglion (DRG) sensory axons in the dorsal funiculus of the spinal cord requires multiple guidance mechanisms involving the extracellular molecules Netrin-1 and Slits.

Abstract

This study examines the molecular mechanisms that guide the formation of the T-shaped bifurcation of dorsal root ganglion (DRG) sensory axons in the dorsal funiculus of the spinal cord. The authors used mouse genetics and single axon labeling techniques to investigate the roles of the extracellular guidance cues Netrin-1 (Ntn1) and Slits.
Key findings:

Loss of Ntn1 leads to misprojections of DRG axons that escape from the dorsal funiculus at the time of bifurcation, suggesting Ntn1 is required for proper guidance of bifurcating axons.
Single axon analysis shows that Ntn1 affects the turning angles of bifurcated axons, but does not impact the bifurcation process itself.
Mice lacking both Ntn1 and the Slit signaling pathway (Slit1/2 and their receptors Robo1/2) exhibit a severe disorganization of the dorsal funiculus, with DRG axons misprojecting in both horizontal and dorsal directions.
The misprojections in Ntn1 and Slit mutants have distinct trajectories, suggesting Ntn1 and Slits regulate different aspects of DRG axon guidance during bifurcation.
Receptor mutants (DCC for Ntn1, Robo1/2 for Slits) show similar guidance defects, confirming the independent roles of these two guidance pathways.

These results demonstrate that the formation of the stereotypic T-shaped bifurcation in the dorsal funiculus requires the coordinated action of multiple guidance mechanisms involving Ntn1 and Slit signaling to ensure the proper guidance of bifurcating DRG axons.

Stats

The number of misprojecting axonal fibers at proximal, medial, and distal locations from the dorsal funiculus is significantly higher in Ntn1β/β mutants compared to wild-type embryos.
The distance from the midline to the dorsal root entry zone (DREZ) is reduced in Slit1-/-;Slit2-/-;Ntn1β/β triple mutants compared to controls.
The total length of horizontal misprojections inside the spinal cord is increased in Robo1Robo2-/-;DCC-/- triple mutants compared to single mutants.

Quotes

"Loss of Ntn1 caused NF-labeled axons to escape from the DREZ."
"Ntn1 and Slits have different guidance functions that are both required for the formation of the dorsal funiculus."
"The guidance function is further supported by the similar defect found in mice lacking the Ntn1 receptor DCC."

Key Insights Distilled From

Multiple Guidance Mechanisms Control Axon Growth to Generate Precise T-shaped Bifurcation during Dorsal Funiculus Development in the Spinal Cord

by Curran,B. M.... at www.biorxiv.org 11-18-2023

https://www.biorxiv.org/content/10.1101/2023.11.17.567638v3

Deeper Inquiries

How do the guidance functions of Ntn1 and Slits interact to regulate the precise formation of the T-shaped bifurcation during development?

The guidance functions of Ntn1 and Slits interact to ensure the precise formation of the T-shaped bifurcation during development by regulating different aspects of DRG axon guidance at the DREZ. Ntn1 is involved in guiding bifurcating DRG axons at the time of bifurcation, ensuring that the axons grow along the DREZ and do not stray from the dorsal funiculus. On the other hand, Slits play a role in guiding the axons to turn into the DREZ initially and continue to grow along the correct trajectory. The loss of either Ntn1 or Slits results in distinct guidance errors, with Ntn1 mutants showing wavy and randomly oriented misprojections that stay along the pial surface, while Slit mutants exhibit relatively straight and horizontally oriented misprojections that enter the spinal cord. When both pathways are impaired, the guidance errors are more severe, leading to a complete disorganization and loss of axonal bundles in the dorsal funiculus. This demonstrates the additive roles of Ntn1 and Slits in ensuring the proper formation of the T-shaped bifurcation during development.

How do the guidance functions of Ntn1 and Slits interact to regulate the precise formation of the T-shaped bifurcation during development?

The potential mechanisms by which Ntn1 and Slit signaling pathways differentially guide the two branches of the bifurcating DRG axons involve distinct actions and interactions at the DREZ. Ntn1 is likely to provide a permissive cue that encourages the growth of newly bifurcated branches but restricts their trajectory within the DREZ. This permissive function may involve the repulsive action of Ntn1 mediated by its receptor DCC, which could guide the axons to grow along the DREZ and prevent them from straying. On the other hand, Slits may guide the axons to turn into the DREZ initially and continue to grow along the correct trajectory. The differential actions of Ntn1 and Slits result in different types of misprojections when either pathway is impaired, with Ntn1 mutants showing dorsally oriented misprojections along the pial surface and Slit mutants exhibiting horizontally oriented misprojections that enter the spinal cord. The additive defects observed in triple mutants lacking both pathways suggest non-redundant functions of Ntn1 and Slits in guiding the bifurcating DRG axons.

Could the insights gained from this study on axon guidance during dorsal funiculus formation be applied to understand the development and regeneration of other branched neural circuits in the central nervous system?

The insights gained from this study on axon guidance during dorsal funiculus formation could be applied to understand the development and regeneration of other branched neural circuits in the central nervous system. Understanding the molecular mechanisms that regulate the precise formation of branched axonal structures, such as the T-shaped bifurcation in the dorsal funiculus, can provide valuable insights into how similar circuits are formed and maintained in other regions of the central nervous system. By studying the interactions between guidance molecules like Ntn1 and Slits, researchers can uncover common principles that govern the development of branched neural circuits and potentially apply this knowledge to promote regeneration and repair in cases of injury or neurodegenerative diseases affecting branched neural pathways. This study highlights the importance of multiple guidance mechanisms in ensuring the fidelity of branched circuits, which could have implications for understanding and manipulating neural circuit formation and regeneration in various regions of the central nervous system.