Flamingo,a seven‐pass transmembrane cadherin,cooperates with Netrin/Frazzled in Drosophila midline guidance

During central nervous system development, several guidance cues and receptors, as well as cell adhesion molecules, are required for guiding axons across the midline and along the anterior–posterior axis. In Drosophila, commissural axons sense the midline attractants Netrin A and B (Net) through Frazzled (Fra) receptors. Despite their importance, lack of Net or fra affects only some commissures, suggesting that additional molecules can fulfill this function. Recently, planar cell polarity (PCP) proteins have been implicated in midline axon guidance in both vertebrate and invertebrate systems. Here, we report that the atypical cadherin and PCP molecule Flamingo/Starry night (Fmi/Stan) acts jointly with Net/Fra signaling during midline development. Additional removal of fmi strongly increases the guidance defects in Net/fra mutants. Rescue and domain deletion experiments suggest that Fmi signaling facilitates commissural pathfinding potentially by mediating axonal fasciculation in a partly homophilic manner. Altogether, our results indicate that contact‐mediated cell adhesion via Fmi acts in addition to the Net/Fra guidance system during axon pathfinding across the midline, underlining the importance of PCP molecules during vertebrates and invertebrates midline development.     

Midline radial glia translocation and corpus callosum formation require FGF signaling

Midline astroglia in the cerebral cortex develop earlier than other astrocytes through mechanisms that are still unknown. We show that radial glia in dorsomedial cortex retract their apical endfeet at midneurogenesis and translocate to the overlaying pia, forming the indusium griseum. These cells require the fibroblast growth factor receptor 1 (Fgfr1) gene for their precocious somal translocation to the dorsal midline, as demonstrated by inactivating the Fgfr1 gene in radial glial cells and by RNAi knockdown of Fgfr1 in vivo. Dysfunctional astroglial migration underlies the callosal dysgenesis in conditional Fgfr1 knockout mice, suggesting that precise targeting of astroglia to the cortex has unexpected roles in axon guidance. FGF signaling is sufficient to induce somal translocation of radial glial cells throughout the cortex; furthermore, the targeting of astroglia to dorsolateral cortex requires FGFr2 signaling after neurogenesis. Hence, FGFs have an important role in the transition from radial glia to astrocytes by stimulating somal translocation of radial glial cells.     

her9 promotes floor plate development in zebrafish.

Notochord, floor plate, and in anamniotes hypochord, are vertebrate embryonic midline structures that are the sources of molecules that pattern the nervous system, somites, and dorsal aorta. Midline precursor cells arise from the dorsal organizer during gastrulation, and Notch signaling is an important regulator of midline cell fate specification. To understand fully how Notch signaling regulates midline development, we investigated the role of potential Notch target genes. We show here that midline precursors express her9, a member of the hairy/Enhancer of split gene family. Although her9 inhibits notochord development and promotes floor plate specification, her9 expression in floor plate cells appears not to require Notch signaling. We show that, instead, her9 is a downstream effector of Nodal signaling for floor plate specification.     

Netrins guide Drosophila commissural axons at short range

Netrins are secreted axon guidance molecules required for commissure formation in a wide range of animal species, including Caenorhabditis elegans, Drosophila melanogaster and mice. They are generally thought to function as chemoattractants, acting at a distance to direct commissural axon growth toward the midline of the central nervous system. We show here, however, that D. melanogaster commissural axons still orient normally and reach the midline even in the complete absence of netrins, though some of them fail to cross the midline. Tethering endogenous netrin to the membrane selectively disrupts its long-range but not short-range activity, yet still allows normal commissure formation. We therefore propose that netrins act in commissural axon guidance as short-range cues that promote midline crossing, not as long-range chemoattractants.     

Axon guidance at the midline choice point.

The central nervous system (CNS) of higher organisms is bilaterally-symmetric. The transfer of information between the two sides of the nervous system occurs through commissures formed by neurons that project axons across the midline to the contralateral side of the CNS. Interestingly, these axons cross the midline only once. Other neurons extend axons that never cross the midline; they project exclusively on their own (ipsilateral) side of the CNS. Thus, the midline is an important choice point for several classes of pathfinding axons. Recent studies demonstrate that specialized midline cells play critical roles in regulating the guidance of both crossing and non-crossing axons at the ventral midline of the developing vertebrate spinal cord and the Drosophila ventral nerve cord. For example, these cells secrete attractive cues that guide commissural axons over long distances to the midline of the CNS. Furthermore, short-range interactions between guidance cues present on the surfaces of midline cells, and their receptors expressed on the surfaces of pathfinding axons, allow commissural axons to cross the midline only once and prevent ipsilaterally-projecting axons from entering the midline. Remarkably, the molecular composition of commissural axon surfaces is dynamically-altered as they cross the midline. Consequently, commissural axons become responsive to repulsive midline guidance cues that they had previously ignored on the ipsilateral side of the midline. Concomitantly, commissural axons lose responsiveness to attractive guidance cues that had initially attracted them to the midline. Thus, these exquisitely regulated guidance systems prevent commissural axons from lingering within the confines of the midline and allow them to pioneer an appropriate pathway on the contralateral side of the CNS. Many aspects of midline guidance are controlled by mechanistically and evolutionarily-conserved ligand-receptor systems. Strikingly, recent studies demonstrate that these receptors are modular; the ectodomains determine ligand recognition and the cytoplasmic domains specify the response of an axon to a given guidance cue. Despite rapid and dramatic progress in elucidating the molecular mechanisms that control midline guidance, many questions remain.     

Comm function in commissural axon guidance: cell-autonomous sorting of Robo in vivo

Commissureless (Comm) controls axon guidance across the Drosophila melanogaster midline by regulating surface levels of Robo, the receptor for the midline repellent Slit. Two different models have been proposed for how Comm regulates Robo: a 'sorting' model and a 'clearance' model, both based on studies using heterologous cells in vitro. Here, we test these two models in vivo. We establish a genetic rescue assay for Comm, and use this assay to show that midline crossing does not require the presence of Comm in midline cells, as proposed by the clearance model. Moreover, by monitoring the trafficking of a Robo-green fluorescent protein (GFP) fusion in living embryos, we demonstrate that Comm prevents the delivery of Robo-GFP to the growth cone, as predicted by the sorting model. It has also been suggested that Comm must be ubiquitinated by the Nedd4 ubiquitin ligase. We show here, however, that ubiquitination of Comm is not required for its function in vitro or in vivo, and that Nedd4 is unlikely to function in axon guidance at the midline.     

Expression and role of Roundabout-1 in embryonic Xenopus forebrain.

The receptor Roundabout-1 (Robo1) and its ligand Slit are known to influence axon guidance and central nervous system (CNS) patterning in both vertebrate and nonvertebrate systems. Although Robo-Slit interactions mediate axon guidance in the Drosophila CNS, their role in establishing the early axon scaffold in the embryonic vertebrate brain remains unclear. We report here the identification and expression of a Xenopus Robo1 orthologue that is highly homologous to mammalian Robo1. By using overexpression studies and immunohistochemical and in situ hybridization techniques, we have investigated the role of Robo1 in the development of a subset of neurons and axon tracts in the Xenopus forebrain. Robo1 is expressed in forebrain nuclei and in neuroepithelial cells underlying the main axon tracts. Misexpression of Robo1 led to aberrant development of axon tracts as well as the ectopic differentiation of forebrain neurons. These results implicate Robo1 in both neuronal differentiation and axon guidance in embryonic vertebrate forebrain.     

Soluble adenylyl cyclase is required for netrin-1 signaling in nerve growth cones

Growth cones at the tips of nascent and regenerating axons direct axon elongation. Netrin-1, a secreted molecule that promotes axon outgrowth and regulates axon pathfinding, elevates cyclic AMP (cAMP) levels in growth cones and regulates growth cone morphology and axonal outgrowth. These morphological effects depend on the intracellular levels of cAMP. However, the specific pathways that regulate cAMP levels in response to netrin-1 signaling are unclear. Here we show that 'soluble' adenylyl cyclase (sAC), an atypical calcium-regulated cAMP-generating enzyme previously implicated in sperm maturation, is expressed in developing rat axons and generates cAMP in response to netrin-1. Overexpression of sAC results in axonal outgrowth and growth cone elaboration, whereas inhibition of sAC blocks netrin-1-induced axon outgrowth and growth cone elaboration. Taken together, these results indicate that netrin-1 signals through sAC-generated cAMP, and identify a fundamental role for sAC in axonal development.     

The ubiquitin ligase Rnf6 regulates local LIM kinase 1 levels in axonal growth cones

LIM kinase 1 (LIMK1) controls important cellular functions such as morphogenesis, cell motility, tumor cell metastasis, development of neuronal projections, and growth cone actin dynamics. We have investigated the role of the RING finger protein Rnf6 during neuronal development and detected high Rnf6 protein levels in developing axonal projections of motor and DRG neurons during mouse embryogenesis as well as cultured hippocampal neurons. RNAi-mediated knock-down experiments in primary hippocampal neurons identified Rnf6 as a regulator of axon outgrowth. Consistent with a role in axonal growth, we found that Rnf6 binds to, polyubiquitinates, and targets LIMK1 for proteasomal degradation in growth cones of primary hippocampal neurons. Rnf6 is functionally linked to LIMK1 during the development of axons, as the changes in axon outgrowth induced by up- or down-regulation of Rnf6 levels can be restored by modulation of LIMK1 expression. Thus, these results assign a specific role for Rnf6 in the control of cellular LIMK1 concentrations and indicate a new function for the ubiquitin/proteasome system in regulating local growth cone actin dynamics.     

A midline switch of receptor processing regulates commissural axon guidance in vertebrates

Commissural axon guidance requires complex modulations of growth cone sensitivity to midline-derived cues, but underlying mechanisms in vertebrates remain largely unknown. By using combinations of ex vivo and in vivo approaches, we uncovered a molecular pathway controlling the gain of response to a midline repellent, Semaphorin3B (Sema3B). First, we provide evidence that Semaphorin3B/Plexin-A1 signaling participates in the guidance of commissural projections at the vertebrate ventral midline. Second, we show that, at the precrossing stage, commissural neurons synthesize the Neuropilin-2 and Plexin-A1 Semaphorin3B receptor subunits, but Plexin-A1 expression is prevented by a calpain1-mediated processing, resulting in silencing commissural responsiveness. Third, we report that, during floor plate (FP) in-growth, calpain1 activity is suppressed by local signals, allowing Plexin-A1 accumulation in the growth cone and sensitization to Sema3B. Finally, we show that the FP cue NrCAM mediates the switch of Plexin-A1 processing underlying growth cone sensitization to Sema3B. This reveals pathway-dependent modulation of guidance receptor processing as a novel mechanism for regulating guidance decisions at intermediate targets.     

Midline axon guidance and human genetic disorders

In bilaterally symmetric animals, many axons cross the midline to interconnect the left and right sides of the central nervous system (CNS). This process is critical for the establishment of neural circuits that control the proper integration of information perceived by the organism and the resulting response. While neurons at different levels of the CNS project axons across the midline, the molecules that regulate this process are common to many if not all midline-crossing regions. This article reviews the molecules that function as guidance cues at the midline in the developing vertebrate spinal cord, cortico-spinal tract and corpus callosum. As well, we describe the mutations that have been identified in humans that are linked to axon guidance and midline-crossing defects.     

Spinal nerve segmentation in higher vertebrates: axon guidance by repulsion and attraction

The development of spinal nerve segmentation in higher vertebrate embryos provides a convenient experimental system for the analysis of axon guidance mechanisms. We review evidence from chick embryo experiments that segmentation of motor and sensory axons results from a combination of contact repulsion of axon growth cones by posterior somite cells and chemoattraction of growth cones by anterior cells. We also review progress in identifying the underlying molecular mechanisms in this system, and suggest a prominent role for carbohydrate groups in mediating growth cone repulsion.     

Nck2 is essential for limb trajectory selection by spinal motor axons

Background: The development of a functioning nervous system requires precise assembly of neuronal connections, which can be achieved by the guidance of axonal growth cones to their proper targets. How axons are guided by signals transmitted to the cytoskeleton through cell surface‐expressed guidance receptors remains unclear. We investigated the function of Nck2 adaptor protein as an essential guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory into the limb. Results: Nck2 mRNA and protein are preferentially expressed in the medial subgroups of chick LMC neurons during axon trajectory into the limb. Nck2 loss‐ and gain‐of‐function in LMC neurons using in ovo electroporation perturb LMC axon trajectory selection demonstrating an essential role of Nck2 in motor axon guidance. We also showed that Nck2 knockdown and overexpression perturb the growth preference of LMC neurites against ephrins in vitro and Eph‐mediated redirection of LMC axons in vivo. Finally, the significant changes of LMC neurite growth preference against ephrins in the context of Nck2 and α2‐chimaerin loss‐ and gain‐of‐function implicated Nck2 function to modulate α2‐chimaerin activity. Conclusions: Here, we showed that Nck2 is required for Eph‐mediated axon trajectory selection from spinal motor neurons through possible interaction with α2‐chimaerin. Developmental Dynamics 247:1043‐1056, 2018. © 2018 Wiley Periodicals, Inc.     

Sonic Hedgehog及其受体Patched在小鼠视交叉发育过程中的表达

在小鼠胚胎发育过程中,胚胎第13d(E13)至15d(E15)是视交叉发育的主要阶段。在本研究中,我们观察了在E13～E15,Sonic Hedgehog(Shh)及其受体Patched(Ptc)在视觉传导通路的表达。结果发现:在视交叉和视束中,Shh在视神经纤维接近中线时表达上调,越过中线后表达下调,并且主要表达在较深的区域。Ptc在E13～E14的视网膜和E14～E15的视束中有表达,但在视交叉中无表达。Ptc,而不是Shh,表达在体外培养的生长锥中。Shh和Ptc在视觉传导通路发育中的表达提示Shh可能在引导视神经生长方面发挥一定作用。     

CNS-derived glia ensheath peripheral nerves and mediate motor root development

Motor function requires that motor axons extend from the spinal cord at regular intervals and that they are myelinated by Schwann cells. Little attention has been given to another cellular structure, the perineurium, which ensheaths the motor nerve, forming a flexible, protective barrier. Consequently, the origin of perineurial cells and their roles in motor nerve formation are poorly understood. Using time-lapse imaging in zebrafish, we show that perineurial cells are born in the CNS, arising as ventral spinal-cord glia before migrating into the periphery. In embryos lacking perineurial glia, motor neurons inappropriately migrated outside of the spinal cord and had aberrant axonal projections, indicating that perineurial glia carry out barrier and guidance functions at motor axon exit points. Additionally, reciprocal signaling between perineurial glia and Schwann cells was necessary for motor nerve ensheathment by both cell types. These insights reveal a new class of CNS-born glia that critically contributes to motor nerve development.     

Shared receptors in axon guidance: SAX-3/Robo signals via UNC-34/Enabled and a Netrin-independent UNC-40/DCC function

The C. elegans SAX-3/Robo receptor acts in anterior-posterior, dorsal-ventral and midline guidance decisions. Here we show that SAX-3 signaling involves the C. elegans Enabled protein UNC-34 and an unexpected Netrin-independent function of the Netrin receptor UNC-40/DCC. Genetic interactions with gain- and loss-of-function mutations suggest that unc-34 and unc-40 act together with sax-3 in several guidance decisions, but the C. elegans Netrin gene unc-6 does not act in the same genetic pathways. Within the migrating axon, sax-3, unc-34 and unc-40 all act cell-autonomously. Our results support a role for UNC-34/Enabled proteins in SAX-3-mediated repulsion, and show that UNC-40/DCC can potentiate SAX-3/Robo signaling via a mechanism that may involve direct binding of the two guidance receptors. A combinatorial logic dictates alternative functions for UNC-40/DCC, which can act in attraction to UNC-6/Netrin, repulsion from Netrin (with UNC-5), or repulsion from Slit (with SAX-3).     

Ephrin signaling in vivo: look both ways.

Eph receptors and ephrins have captured the interest of the developmental biology community in recent years for their pleiotropic functions during embryogenesis. Loss-of-function studies using various animal models have demonstrated the involvement of Ephs and ephrins in many aspects of embryogenesis including segmentation, neural crest cells migration, angiogenesis, and axon guidance. An essential property of this signaling pathway is the ability of both Ephs and ephrins to behave as receptors or ligands and their consequent cell autonomous and nonautonomous mode of action. While many reports did not discriminate between Eph autonomous signaling (forward) and ephrin autonomous signaling (reverse), recent genetic and in vivo studies have shown that both forward and reverse signaling play important roles during embryogenesis.