A molecular mechanism that regulates medially oriented axonal growth of upper layer neurons in the developing neocortex

Abstract

During development, cortical neurons extend axons to their targets based on their laminar locations and cell types. Here we studied the molecular mechanism that regulates medially oriented axonal growth of upper layer neurons in the developing mouse cortex. Upper layer neurons were labeled with enhanced yellow fluorescent protein (EYFP) by in utero electroporation at E15.5. Cortical slices containing EYFP-labeled cells were dissected at E16, when axonal outgrowth from upper layer neurons is not initiated, and were cultured in an organotypic manner. After 3 days in culture, most labeled cells were found to extend axons medially in the same fashion as those observed in vivo. This oriented growth was disrupted when the lateral side of the cortical slice was removed, indicating that a laterally located repellent is involved in the medially oriented growth. Strikingly, the medially directed growth within the slices was reduced in the medium containing Semaphorin3A (Sema3A) or soluble form of Neuropilin-1 (Npn1), a receptor for Sema3A. Importantly, we found that Sema3A was expressed in a gradient from lateral-high to medial-low within the cortex, and callosal axons originating from upper layer neurons uniquely expressed Npn1. Consistent with these findings, ectopically expressed Sema3A repelled medially oriented elongation of upper layer cell axons in vivo. These results therefore suggest the operation of a repulsive mechanism for medially oriented axon growth of upper layer neurons, and further point to a role for a gradient expression of Sema3A in this directional axon growth along the mediolateral axis within the neocortex.

Axon Guidance Mechanisms for Establishment of Callosal Connections

“…The long-range guidance molecules are secreted by the midline glial populations, forming a concentration gradient and helping callosal axons pass through the CC with attractive (Figure 2(a)) and repulsive signals (Figures 2(b) and 2(c)). Slits [15, 24, 25], Wnts [26], Netrins [27, 28], Draxin [29], and Semaphorins [30] are some of the reported long-range guidance molecules. Recent studies have also clarified new roles for some of these guidance molecules.…”

PlexinA1 is crucial for the midline crossing of callosal axons during corpus callosum development in BALB/cAJ mice

“…Sema3A mRNA is expressed in the developing neocortex following the gradient from lateral-high to medial-low, and Npn1, the receptor of Sema3A is expressed on callosal axons extending from upper layer neurons [55]. Because Sema3A expressed ectopically suppresses the extension of callosal axons orienting medially, the gradient of Sema3A expression in the developing neocortex may be a mechanism in which callosal axons initially extend toward the midline from the developing neocortex [55]. To ask if the gradient of Sema3A exists in the developing neocortex of WT and KO mice under BALB/cAJ background, we analyzed the expression of Seam3A and Npn1 along the mediolateral axis of developing neocortex on E15.5–17.5.…”

“…The gradient of Sema3A expression from lateral-high to medial-low in the developing neocortex was observed in the region from developing neocortex to the midline of both WT and PlexinA1 KO mice, and Npn1+ axons extended medially from the lateral side in the brains of both genotypes (Fig 5). Previous studies have revealed the crucial roles of the Sema3A/Npn1 signal in the extension of callosal axons from the neocortex and axonal positioning in the CC during CC development [55, 59]. When axons of callosal projection neurons in the developing cortex initially begin to extend toward the midline, the gradient from high to low concentration of Sema3A may orient axons from the lateral to medial direction [55].…”

“…Previous studies have revealed the crucial roles of the Sema3A/Npn1 signal in the extension of callosal axons from the neocortex and axonal positioning in the CC during CC development [55, 59]. When axons of callosal projection neurons in the developing cortex initially begin to extend toward the midline, the gradient from high to low concentration of Sema3A may orient axons from the lateral to medial direction [55]. As discussed in their work [55], callosal axons may come close to the midline following another mechanism including local guidance from several guideposts in addition to the Sema3A gradient.…”

“…When axons of callosal projection neurons in the developing cortex initially begin to extend toward the midline, the gradient from high to low concentration of Sema3A may orient axons from the lateral to medial direction [55]. As discussed in their work [55], callosal axons may come close to the midline following another mechanism including local guidance from several guideposts in addition to the Sema3A gradient. The Sema3A/Npn1 signal in the cortical midline operates for the axonal positioning within CC, in which axons derived from the cortex near the midline are located dorsally and those from the neocortex located far from the midline are positioned ventrally in the CC [59].…”