A new model for netrin1 in commissural axon guidance

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“Chemotropism may be only one of several guidance mechanisms that operate successively or coordinately to define the trajectory of commissural axons in vivo” (Tessier‐Lavigne, Placzek, Lumsden, Dodd, & Jessell, 1988).
The ingenious efforts of a handful of laboratories in the 1980's and 1990's answered a century‐old question, which went back to Santiago Ramón y Cajal's pioneering observations of neuronal axons (Ramón y Cajal, 1890): what mechanism allows developing nerve fibers to navigate through the embryonic tissues and find their targets?
The answer is, axonal tips called growth cones are equipped with membrane‐tethered molecular receptors, which bind guidance cue proteins scattered along their paths. These proteins trigger intracellular signaling cascades within the growth cone that bias its direction of extension and result in axons being attracted or repelled from the cue‐expressing tissues. Initial investigations made a key distinction between short‐range, contact‐mediated cues and long‐range, diffusible cues (Tessier‐Lavigne & Goodman, 1996). The latter was thought to create concentration gradients that axons could track.
One of the first molecules found to exemplify the chemotactic gradient model was netrin1 (Kennedy, Serafini, La Torre, & Tessier‐Lavigne, 1994). A cartoon illustration, currently present in virtually all neuroscience textbooks, shows a cross‐section of a spinal cord with a few dorsally positioned interneurons extending their axons towards the ventral‐most central tissue in the spinal cord. This is called the floor plate (FP), which is shown as the source of dorsally diffusing gradients of netrin1 protein that the axons are following (Figure 1d). These nerve fibers are known as commissural axons, and they reach the FP, cross the midline, and subsequently turn to extend toward rostral targets.
Much work has linked commissural axon guidance to netrin1 signaling, resulting in the ubiquitous model figure. Early in vitro outgrowth and orientation experiments showed that FP tissue positioned a distance away from dorsal spinal cord explants was sufficient for commissural axon turning (Figure 1a; Tessier‐Lavigne et al., 1988; Placzek, Tessier‐Lavigne, Jessell, & Dodd, 1990a; Placzek, Tessier‐Lavigne, Yamada, Dodd, & Jessell, 1990b). In vivo evidence was produced by grafting rat FP onto chicken spinal cords, which resulted in dramatic commissural axon attraction away from their normal trajectories (Figure 1b; Placzek et al., 1990b). Later replicas of the in vitro orientation experiments replaced FP with netrin1‐expressing COS cells and found the same result (Kennedy et al., 1994), confirming netrin1 as the FP attractant (or at least one of them). Indeed, some characterizations of netrin1 message and protein showed an enrichment in the FP and suggested the existence of a concentration gradient in chicken, mouse, and rat spinal cords (Kennedy et al., 1994; Serafini et al., 1996; Kennedy, Wang, Marshall, & Tessier‐Lavigne, 2006). It must be said, though, that early experiments by others failed to detect the netrin1 gradient (MacLennan et al., 1997), its existence remaining a controversial topic in the field due in part to the poor resolution of early in situ hybridization experiments and the fact that the initial antibodies were homemade and not commercially available. However, the netrin1 receptor, deleted in colorectal carcinoma (DCC), was found to be enriched in commissural axons (Keino‐Masu et al., 1996; Fazeli et al., 1997), and the turning of these axons was elicited by puffing recombinant netrin1 from a pipette (Figure 1c; Ming et al., 1997) or engineering a gradient in a culture chamber (Sloan, Qasaimeh, Juncker, Yam, & Charron, 2015). Moreover, netrin1 was confirmed to be exocytosed, even though it was always clear that its laminin‐like structure made for a “sticky” molecule that, although found in the conditioned medium of FP tissue culture, mostly remained in cell‐associated fractions the way matrix proteins do (Kennedy et al., 2006).
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