TrkB-dependent EphrinA reverse signaling regulates callosal axon fasciculate growth downstream of Neurod2/6

Abstract

Abnormal development of corpus callosum is relatively common and causes a broad spectrum of cognitive impairments in humans. We use acallosal Neurod2/6-deficient mice to study callosal axon guidance within the ipsilateral cerebral cortex. Initial callosal tracts form but fail to traverse the ipsilateral cingulum and are not attracted towards the midline in the absence of Neurod2/6. We show that the restoration of Ephrin-A4 (EfnA4) expression in the embryonic neocortex of Neurod2/6-deficient embryos is sufficient to partially rescue targeted callosal axon growth towards the midline. EfnA4 cannot directly mediate reverse signaling within outgrowing axons, but it forms co-receptor complexes with TrkB (Ntrk2). The ability of EfnA4 to rescue the guided growth of a subset of callosal axons in Neurod2/6-deficient mice is abolished by the co-expression of dominant negative TrkBK571N (kinase-dead) or TrkBY515F (SHC-binding deficient) variants, but not by TrkBY816F (PLCγ1-binding deficient). Additionally, EphA4 is repulsive to EfnA4-positive medially projecting axons in organotypic brain slice culture. Collectively, we suggest that EfnA4-mediated reverse signaling acts via TrkB-SHC and is required for ipsilateral callosal axon growth accuracy towards the midline downstream of Neurod family factors.

Lmo4 synergizes with Fezf2 to promote direct in vivo reprogramming of upper layer cortical neurons and cortical glia towards deep-layer neuron identities

“…Given the high efficiency of Fezf2 and Lmo4 in reprogramming postmitotic UL neurons at embryonic stages, we next tested their potential at postnatal stages ( Fig 4 ). To this purpose, we subcloned Fezf2 or Lmo4 into the inducible vector pCAG-fl-mutCherry-fl-IRES-EGFP [ 44 ] resulting in pCAG-Ind-GFP (iGFP) , pCAG-Ind-Fezf2 (iFezf2) , and pCAG-Ind-Lmo4 (iLmo4) ( Fig 4A ). All vectors were co-electroporated with the pCAG-CRE-ER T2 plasmid at E14.5, and 4-hydroxytamoxifen (TAM) was administrated at different postnatal stages to drive Cre -recombinase activation [ 45 ].…”

“…pCdk5r-Fezf2-IRES-GFP and pCdk5r-IRES-GFP were donated by the lab of Paola Arlotta [2], while pCdk5r-Lmo4-IRES-GFP was generated in the lab (Harb and colleagues). All plasmids were tested for correct expression (S1 Fig) . To induce the expression of Lmo4 and Fezf2 at postnatal stages upon IUE, Fezf2 and Lmo4 cDNAs were subcloned into the pCAG-fl-mutCherry-fl-IRES-EGFP vector [44] by substituting the mutCherry with Fezf2 or Lmo4, resulting in pCAG-Ind-GFP (iGFP), pCAG-Ind-Fezf2 (iFezf2), and pCAG-Ind-Lmo4 (iLmo4). The plasmids consist of a stop sequence flanked by loxP sites in the same directional orientation as the inserted cDNAs and followed by an IRE-S-EGFP sequence to allow EGFP reporter expression.…”

Molecular mechanisms of corpus callosum development: a four-step journey

“… Callosal axons migrate within a permissive corridor between Ventricular Zone (VZ) and Cortical Plate (CP). EphA receptors are expressed in both VZ and CP (purple arrows) and repel EfnA4-positive callosal axons that remain bundled (modified from Yan et al, 2023 ). …”

“…EfnA4 expressing axons are repelled by EphA expressing cells. This repulsive interaction keeps callosal axons as a bundle and prevents them from entering both VZ and CP In the developing neocortex, these interactions are required to keep the growing axons within a “permissive corridor” at the border between the SVZ and CP ( Yan et al, 2023 ; Figure 4 ). Eph signaling is also important for midline crossing (see below).…”