Cooperative slit and netrin signaling in contralateralization of the mouse trigeminothalamic pathway.

The trigeminothalamic projections (a.k.a. trigeminal lemnniscus) arising from the principal sensory nucleus of the trigeminal nerve (PrV) form the orofacial component of the major ascending somatosensory pathway, the medial lemniscus. This pathway is entirely contralateral and terminates in the ventroposteromedial (VPM) nucleus of the thalamus (Erzurumlu et al., 1980; Ding et al., 2003). The somatosensory map of the body is also conveyed contralateral to the ventroposterolateral (VPL) nucleus of the thalamus via the dorsal column nuclei (reviewed in Tracey and Waite, 1995). The somatotopic face and body maps formed in these two thalamic nuclei are then conveyed to the primary somatosensory cortex via the thalamocortical projections. In rodents, relatively large volume of neural substrate is devoted to the point-to-point representation of the whiskers on the snout in the VPM (“barreloids”) and in layer IV (“barrels”) of the primary somatosensory cortex; these whisker-specific patterns are first initiated in the PrV and then sequentially relayed to the thalamus and cortex (reviewed in Sehara and Kawasaki, 2011). Numerous studies have focused on the development of the thalamocortical pathway and formation of the barrels (Erzurumlu and Kind, 2001; Inan and Crair, 2007; Pinon et al., 2009; Wu et al., 2011). In particular studies in mice with targeted gene mutations have revealed many of the underlying molecular mechanisms in thalamocortical axon guidance (Garel and Rubenstein, 2004). However, very little is known about the development of the trigeminal lemniscal pathway and the molecular guidance these axons use as they form a ribbon-shaped (lemniscus) pathway heading towards the contralateral thalamus. In the preceding paper we describe the normal development of the mouse trigeminothalamic pathway and here we address the molecular mechanisms of axon guidance as this tract acquires a contralateral course. Specialized midline cells of the vertebrate central nervous system secrete a variety of molecular signals, which attract or repel growing axons (Kennedy et al., 1994; Kidd et al., 1999). Commissural pathways and long axonal tracts such as the corticospinal and medial lemniscal pathways all interact with the neuroaxial midline during their establishment. Developmental regulation of the midline cues and specific receptors for them on the growing tips of axons play an essential role in contralateralization of these pathways. Slit and netrin proteins are abundant along the developing hindbrain and spinal cord midline, and the ascending somatosensory axons are endowed with cognate receptors. Three types of netrins (netrin-1, -3 and -4) have been identified in mammals (Moore et al., 2007). Netrin-1 is essential for proper extension of spinal commissural axons, corpus callosum and hippocampal commissure (Serafini et al., 1996). In the spinal cord, floor plate glial cells secrete netrin-1 and attract commissural axons towards the floor plate (Kennedy et al., 1994; Kennedy et al., 2006). In vertebrates netrin receptors are “deleted in colorectal cancer”(dcc), neogenin and four “uncoordinated” (unc) 5 proteins (unc5A-D). Axon attraction occurs when netrin-1 binds dcc (Moore et al., 2007). In Drosophila, “frazzled” is the name given to the member of the dcc gene family (Kolodziej et al., 1996) and mediates axon attraction upon binding netrin. Genetic and functional blockade of dcc results in loss of attraction towards the midline by commissural axons (Keino-Masu et al., 1996; Fazeli et al., 1997). Similarly, failure in midline crossing of internal arcuate fibers arising from the dorsal column nuclei has been reported for netrin-1 knockout mice (Kubota et al., 2004). Slit is an axon repellent protein, also secreted by the midline glia (Kidd et al., 1999). Three slit proteins (slit1-3) have been identified in mammals. They bind with roundabout or robo (robo1-4) receptors. Slit2 and slit1/2 double knockout mice display axon pathfinding defects in the corpus callosum, hippocampal commissure and thalamocortical projections (Bagri et al., 2002). Axonal pathfinding defects were also reported in robo1 knockout mice (Andrews et al., 2006; Jaworski et al., 2010). Our in situ hybridization assays indicated that the message for the slit and netrin proteins and their receptors are found at the right time and place during the development of the mouse trigeminal lemniscal pathway. We used dcc and slit 1, slit2, slit3 single or double knockout mice and observed marked path finding errors in trigeminothalamic projection axons of the PrV. Collectively, our results indicate that a cooperative signaling of attractant (netrin1) and repellent (slits) midline cues plays a fundamental role in guiding developing trigeminal lemniscal axons across the midline and contralateralization of the facial somatosensory inputs.