Disruption of the anterior commissure in Olig2 deficient mice.

In the present study, we examined neural circuit formation in the forebrain of the Olig2 knockout (Olig2-KO) mouse model and found disruption of the anterior commissure at the late foetal stage. Axon bundles of the anterior commissure encountered the wall of the third ventricle and ceased axonal extension. L1-CAM immunohistochemistry showed that Olig2-KO mice lose decussation formation in the basal forebrain. DiI tracing revealed that the thin bundles of the anterior commissure axons crossed the midline but ceased further extension into the deep part of the contralateral side. Furthermore, some fractions of DiI-labelled axons were oriented dorsolaterally, which was not observed in the control mouse forebrain. The rostral part of the third ventricle was much wider in the Olig2-KO mice than in wild-type mice, which likely resulted in the delay of midline fusion and subsequent delay and malformation of the anterior commissure. We analysed gene expression alterations in the Olig2-KO mice using a public database and found multiple genes, which are related to axon guidance and epithelial-mesenchymal transition, showing subtle expression changes. These results suggest that Olig2 is essential for anterior commissure formation, likely by regulating multiple biological processes.

Post-crossing segment of dI1 commissural axons forms collateral branches to motor neurons in the developing spinal cord.

The dI1 commissural axons in the developing spinal cord, upon crossing the midline through the floor plate, make a sharp turn to grow rostrally. These post-crossing axons initially just extend adjacent to the floor plate without entering nearby motor columns. However, it remains poorly characterized how these post-crossing dI1 axons behave subsequently to this process. In the present study, to address this issue, we examined in detail the behavior of post-crossing dI1 axons in mice, using the Atoh1 enhancer-based conditional expression system that enables selective and sparse labeling of individual dI1 axons, together with Hb9 and ChAT immunohistochemistry for precise identification of spinal motor neurons (MNs). We found unexpectedly that the post-crossing segment of dI1 axons later gave off collateral branches that extended laterally to invade motor columns. Interestingly, these collateral branches emerged at around the time when their primary growth cones initiated invasion into motor columns. In addition, although the length of the laterally growing collateral branches increased with age, the majority of them remained within motor columns. Strikingly, these collateral branches further gave rise to multiple secondary branches in the region of MNs that innervate muscles close to the body axis. Moreover, these axonal branches formed presynaptic terminals on MNs. These observations demonstrate that dI1 commissural neurons develop axonal projection to spinal MNs via collateral branches arising later from the post-crossing segment of these axons. Our findings thus reveal a previously unrecognized projection of dI1 commissural axons that may contribute directly to generating proper motor output.

Interstitial branch formation within the red nucleus by deep cerebellar nuclei-derived commissural axons during target recognition.

Target recognition by developing axons is one of the fundamental steps for establishing the proper pattern of neuronal connectivity during development. However, knowledge of the mechanisms that underlie this critical event is still limited. In this study, to examine how commissural axons in vertebrates recognize their targets after crossing the midline, we analyzed in detail the behavior of postcrossing commissural axons derived from the deep cerebellar nuclei (DCN) in the developing mouse cerebellum. For this, we employed a cell-type-specific genetic labeling approach to selectively visualize DCN axons during the time when these axons project to the red nucleus (RN), one of the well-characterized targets of DCN axons. We found that, when DCN axons initially entered the RN at its caudal end, these axons continued to grow rostrally through the RN without showing noticeable morphological signs of axon branching. Interestingly, after a delay, DCN axons started forming interstitial branches from the portion of the axon shaft selectively within the RN. Because commissural axons acquire responsiveness to several guidance cues when they cross the midline, we further addressed whether midline crossing is a prerequisite for subsequent targeting by using a Robo3 knockdown strategy. We found that DCN axons were still capable of forming interstitial branches within the RN even in the absence of midline crossing. These results therefore suggest that the mechanism of RN recognition by DCN axons involves a delayed interstitial branching, and that these axons possess an intrinsic ability to respond to the target-derived cues irrespective of midline crossing.

Dbx1 triggers crucial molecular programs required for midline crossing by midbrain commissural axons.

Axon guidance by commissural neurons has been well documented, providing us with a molecular logic of how midline crossing is achieved during development. Despite these advances, knowledge of the intrinsic genetic programs is still limited and it remains obscure whether the expression of a single transcription factor is sufficient to activate transcriptional programs that ultimately enable midline crossing. Here, we show in the mouse that the homeodomain transcription factor Dbx1 is expressed by a subset of progenitor cells that give rise to commissural neurons in the dorsal midbrain. Gain- and loss-of-function analyses indicate that the expression of Dbx1 alone is sufficient and necessary to trigger midline crossing in vivo. We also show that Robo3 controls midline crossing as a crucial downstream effector of the Dbx1-activated molecular programs. Furthermore, Dbx1 suppresses the expression of the transcriptional program for ipsilateral neuron differentiation in parallel. These results suggest that a single transcription factor, Dbx1, has an essential function in assigning midline-crossing identity, thereby contributing crucially to the establishment of the wiring laterality in the developing nervous system.

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

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.

T-Box transcription factor Tbx20 regulates a genetic program for cranial motor neuron cell body migration.

Members of the T-box transcription factor family (Tbx) are associated with several human syndromes during embryogenesis. Nevertheless, their functions within the developing CNS remain poorly characterized. Tbx20 is expressed by migrating branchiomotor/visceromotor (BM/VM) neurons within the hindbrain during neuronal circuit formation. We examined Tbx20 function in BM/VM cells using conditional Tbx20-null mutant mice to delete the gene in neurons. Hindbrain rhombomere patterning and the initial generation of post-mitotic BM/VM neurons were normal in Tbx20 mutants. However, Tbx20 was required for the tangential (caudal) migration of facial neurons, the lateral migration of trigeminal cells and the trans-median movement of vestibuloacoustic neurons. Facial cell soma migration defects were associated with the coordinate downregulation of multiple components of the planar cell polarity pathway including Fzd7, Wnt11, Prickle1, Vang1 and Vang2. Our study suggests that Tbx20 programs a variety of hindbrain motor neurons for migration, independent of directionality, and in facial neurons is a positive regulator of the non-canonical Wnt signaling pathway.

FGF as a target-derived chemoattractant for developing motor axons genetically programmed by the LIM code.

LIM transcription factors confer developing axons with specific navigational properties, but the downstream guidance receptors and ligands are not well defined. The dermomyotome, a transient structure from which axial muscles arise, is the source of a secreted long-range chemoattractant specific for medial-class spinal motor neuron axons (MMCm axons). We show that fibroblast growth factors (FGFs) produced by the dermomyotome selectively attract MMCm axons in vitro. FGF receptor 1 (FGFR1) expression is restricted to MMCm neurons, and conditional deletion of FGFR1 causes motor axon guidance defects. Furthermore, reprogramming the identity of limb-innervating motor neurons to that of dermomyotome-innervating MMCm cells using the LIM factor Lhx3 induces FGFR1 expression and shifts an increased number of motor axons to an FGF-responsive state. These results point to a role for FGF signaling in axon guidance and further unravel how downstream effectors of LIM codes direct wiring of the developing nervous system.

Coexpressed EphA receptors and ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains.

Contact-dependent signaling between membrane-linked ligands and receptors such as the ephrins and Eph receptor tyrosine kinases controls a wide range of developmental and pathological processes. Paradoxically, many cell types coexpress both ligands and receptors, raising the question of how specific signaling readouts are achieved under these conditions. Here, we studied the signaling activities exerted by coexpressed EphA receptors and GPI-linked ephrin-A ligands in spinal motor neuron growth cones. We demonstrate that coexpressed Eph and ephrin proteins segregate laterally into distinct membrane domains from which they signal opposing effects on the growth cone: EphAs direct growth cone collapse/repulsion and ephrin-As signal motor axon growth/attraction. This subcellular arrangement of Eph-ephrin proteins enables axons to discriminate between cis- versus trans-configurations of ligand/receptor proteins, thereby allowing the utilization of both Ephs and ephrins as functional guidance receptors within the same neuronal growth cone.

Beta1 integrins in muscle, but not in motor neurons, are required for skeletal muscle innervation.

In vitro studies have provided evidence that beta1 integrins in motor neurons promote neurite outgrowth, whereas beta1 integrins in myotubes regulate acetylcholine receptor (AChR) clustering. Surprisingly, using genetic studies in mice, we show here that motor axon outgrowth and neuromuscular junction (NMJ) formation in large part are unaffected when the integrin beta1 gene (Itgb1) is inactivated in motor neurons. In the absence of Itgb1 expression in skeletal muscle, interactions between motor neurons and muscle are defective, preventing normal presynaptic differentiation. Motor neurons fail to terminate their growth at the muscle midline, branch excessively, and develop abnormal nerve terminals. These defects resemble the phenotype of agrin-null mice, suggesting that signaling molecules such as agrin, which coordinate presynaptic and postsynaptic differentiation, are not presented properly to nerve terminals. We conclude that Itgb1 expression in muscle, but not in motor neurons, is critical for NMJ development.

Pathfinding and growth termination of primary trigeminal sensory afferents in the embryonic rat hindbrain.

Axons of the trigeminal ganglion convey sensory information from mechanoreceptors, thermoreceptors, and nociceptors in the face and nasal mucosa, then terminate on several groups of neurons including the principal sensory nucleus and the nuclei of the spinal trigeminal tract. To understand guidance mechanisms during the development of trigeminal sensory axons (TA) in the embryonic brain, we first investigated the growth pattern of TA in relation to organization in the hindbrain using flat whole-mount preparation from rat. We found that the primary TA from the trigeminal ganglion entered the brainstem and grew longitudinally within the hindbrain. Whereas descending axons ran just medial to the primary vestibular axons to innervate the spinal nucleus, ascending axons stayed near the entry point. In flat whole-mount culture, the TA extended both ascending and descending branches as they do in vivo. Rostral hindbrain was found to be a less permissive substrate for the TA compared to caudal hindbrain. In addition, the nonpermissive property of the ventral hindbrain substrate restricted the invasion of TA along the entire length of the hindbrain. Thus, cooperation of absolute and relative permissiveness of the substrate plays important roles in the guidance of TA to their targets.

Transcriptional codes and the control of neuronal identity.

The topographic assembly of neural circuits is dependent upon the generation of specific neuronal subtypes, each subtype displaying unique properties that direct the formation of selective connections with appropriate target cells. Studies of motor neuron development in the spinal cord have begun to elucidate the molecular mechanisms involved in controlling motor projections. In this review, we first describe the actions of transcription factors within motor neuron progenitors, which initiate a cascade of transcriptional interactions that lead to motor neuron specification. We next highlight the contribution of the LIM homeodomain (LIM-HD) transcription factors in establishing motor neuron subtype identity. Importantly, it has recently been shown that the combinatorial expression of LIM-HD transcription factors, the LIM code, confers motor neuron subtypes with the ability to select specific axon pathways to reach their distinct muscle targets. Finally, the downstream targets of the LIM code are discussed, especially in the context of subtype-specific motor axon pathfinding.