Robo1 and Robo2 have distinct roles in pioneer longitudinal axon guidance

Abstract

Pioneer longitudinal axons grow long distances parallel to the floor plate and precisely maintain their positions using guidance molecules released from the floor plate. Two receptors, Robo1 and Robo2, are critical for longitudinal axon guidance by the Slit family of chemorepellents. Previous studies showed that Robo1−/−;2−/− double mutant mouse embryos have disruptions in both ventral and dorsal longitudinal tracts. However, the role of each Robo isoform remained unclear, because Robo 1 or 2 single mutants have mild or no errors. Here we utilized a more sensitive genetic strategy to reduce Robo levels for determining any separate functions of the Robo1 and 2 isoforms. We found that Robo1 is the predominant receptor for guiding axons in ventral tracts and prevents midline crossing. In contrast, Robo2 is the main receptor for directing axons within dorsal tracts. Robo2 also has a distinct function in repelling neuron cell bodies from the floor plate. Therefore, while Robo1 and 2 have some genetic overlap to cooperate in guiding longitudinal axons, each isoform has distinct functions in specific longitudinal axon populations.

Motor neuron cell bodies are actively positioned by Slit/Robo repulsion and Netrin/DCC attraction

“…Unexpectedly, we previously observed that βIII-tubulin + cell bodies are located within the ventral midline of the hindbrain in E10.5 Robo1 −/− ; 2 −/− mutants (Kim et al, 2011). …”

“…Primary antibodies were applied in PBST for 2–3 days. Our previous characterization of the Robo1 and Robo2 antisera showed specific labeling by immunofluorescence that was lost in homozygous mutants (Kim et al, 2011). After washing in PBST overnight, secondary antibodies (Jackson Immuno Laboratories) were applied in PBST for 2–3 days, followed by overnight washes.…”

“…The present study is based on our unexpected observation that neuronal cell bodies were present in the hindbrain floor plate of early Slit and Robo mutant mouse embryos (Kim et al, 2011). This observation led us to focus on the earliest stages of motor neuron development in the brain stem and spinal cord to reevaluate the in vivo roles of Slit/Robo and Netrin1/DCC signals in positioning motor neuron cell bodies.…”

Genetics and the Language Sciences

“…As noted in Section 3.1, this gene encodes a guidance molecule that helps neurons migrate to their correct location (Park et al 2002, Gonda et al 2013). However, the same molecule also becomes important in a different context, guiding the developing growth cones of axons as they extend to find the correct connectivity partners (Kim et al 2011, Kolodkin & Tessier-Lavigne 2011. The successful wiring up of a human brain is an amazing feat, considering that it is estimated to comprise ∼80 billion neurons (FonsecaAzevedo & Herculano-Houzel 2012) and that many of the connections are made between neurons located in distant regions.…”

Role of Slit and Robo Proteins in the Development of Dopaminergic Neurons

“…Slit proteins affect the trajectories of tracts that course longitudinally through the forebrain or rhombencephalic/spinal cord regions and whose origin is outside these regions [19]. This is the case of the ascending dopaminergic projection from the substantia nigra/ventral tegmental area complex that innervates the forebrain.…”