Eph:ephrin-B1 forward signaling controls fasciculation of sensory and motor axons

Axon fasciculation is one of the processes controlling topographic innervation during embryonic development. While axon guidance steers extending axons in the accurate direction, axon fasciculation allows sets of co-extending axons to grow in tight bundles. The Eph:ephrin family has been involved both in axon guidance and fasciculation, yet it remains unclear how these two distinct types of responses are elicited. Herein we have characterized the role of ephrin-B1, a member of the ephrinB family in sensory and motor innervation of the limb. We show that ephrin-B1 is expressed in sensory axons and in the limb bud mesenchyme while EphB2 is expressed in motor and sensory axons. Loss of ephrin-B1 had no impact on the accurate dorso-ventral innervation of the limb by motor axons, yet EfnB1 mutants exhibited decreased fasciculation of peripheral motor and sensory nerves. Using tissue-specific excision of EfnB1 and in vitro experiments, we demonstrate that ephrin-B1 controls fasciculation of axons via a surround repulsion mechanism involving growth cone collapse of EphB2-expressing axons. Altogether, our results highlight the complex role of Eph:ephrin signaling in the development of the sensory-motor circuit innervating the limb.     

Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons

The identification of surface proteins restricted to subsets of embryonic axons and growth cones may provide information on the mechanisms underlying axon fasciculation and pathway selection in the vertebrate nervous system. We describe here the characterization of a 135 kd cell surface glycoprotein, TAG-1, that is expressed transiently on subsets of embryonic spinal cord axons and growth cones. TAG-1 is immunochemically distinct from the cell adhesion molecules N-CAM and L1 (NILE) and is expressed on commissural and motor neurons over the period of initial axon extension. Moreover, TAG-1 and L1 appear to be segregated on different segments of the same embryonic spinal axons. These observations provide evidence that axonal guidance and pathway selection in vertebrates may be regulated in part by the transient and selective expression of distinct surface glycoproteins on subsets of developing neurons.     

DCC plays a role in navigation of forebrain axons across the ventral midbrain commissure in embryonic xenopus

In the developing vertebrate brain, growing axons establish a scaffold of axon tracts connected across the midline via commissures. We have previously identified a population of telencephalic neurons that express NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM that is involved in axon guidance in the forebrain. These axons arise from the presumptive telencephalic nucleus, course caudally along the principal longitudinal tract of the forebrain, cross the ventral midline in the midbrain, and then project to the contralateral side of the brain. In the present study we have investigated mechanisms controlling the growth of these axons across the ventral midline of the midbrain. The axon guidance receptor DCC is expressed by the NOC-2 population of axons both within the longitudinal tract and within the ventral midbrain commissure. Disruption of DCC-dependent interactions, both in vitro and in vivo, inhibited the NOC-2 axons from crossing the ventral midbrain. Instead, these axons grew along aberrant trajectories away from the midline, suggesting that DCC-dependent interactions are important for overcoming inhibitory mechanisms within the midbrain of the embryonic vertebrate brain. Thus, coordinated responsiveness of forebrain axons to both chemostimulatory and chemorepulsive cues appears to determine whether they cross the ventral midline in the midbrain.     

BMP signaling regulates murine enteric nervous system precursor migration, neurite fasciculation, and patterning via altered Ncam1 polysialic acid addition

The enteric nervous system (ENS) forms from migrating neural crest-derived precursors that differentiate into neurons and glia, aggregate into ganglion cell clusters, and extend neuronal processes to form a complex interacting network that controls many aspects of intestinal function. Bone morphogenetic proteins (BMPs) have diverse roles in development and influence the differentiation, proliferation, and survival of ENS precursors. We hypothesized that BMP signaling might also be important for the ENS precursor migration, ganglion cell aggregation, and neurite fasciculation necessary to form the enteric nervous system. We now demonstrate that BMP signaling restricts murine ENS precursors to the outer bowel wall during migration. In addition, blocking BMP signaling causes faster colonization of the murine colon, reduces ganglion cell aggregation, and reduces neurite fasciculation. BMP signaling also influences patterns of neurite extension within the developing bowel wall. These effects on ENS precursor migration and neurite fasciculation appear to be mediated at least in part by increased polysialic acid addition to neural cell adhesion molecule (Ncam1) in response to BMP. Removing PSA enzymatically reverses the BMP effects on ENS precursor migration and neurite fasciculation. These studies demonstrate several novel roles for BMP signaling and highlight new functions for sialyltransferases in the developing ENS.     

Netrin-3 protein is localized to the axons of motor, sensory, and sympathetic neurons

The netrin family of axon guidance cues has been shown to play a pivotal role in the guidance of a variety of axon projections during embryonic development, both in the vertebrate and invertebrate. While the guidance potential of netrin-1 has been examined in depth in many regions of the developing mouse brain very little information is available on the expression and activity of netrin-3. Here we show that the netrin-3 protein is present on motor neurons and subpopulations of neurons within sensory and sympathetic ganglia. Moreover, significant levels of netrin-3 protein were found to be associated with the axons projecting from these neurons suggesting a role for netrin-3 in axon pathfinding and fasciculation within the peripheral nervous system.     

Deficits of olfactory interneurons in polysialyltransferase‐ and NCAM‐deficient mice

The neurogenic niche of the anterior subventricular zone (SVZ) persistently generates neuroblasts, which migrate along the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into granule and periglomerular cells. Loss of the neural cell adhesion molecule NCAM or its post‐translational modification polysialic acid (polySia) impairs migration causing accumulations of cells in the proximal RMS and decreased OB volume. Polysialylation of NCAM is implemented by two polysialyltransferases, ST8SIA2 and ST8SIA4, with overlapping functions. Here, we used mice with Ncam1 and polysialyltransferase deletions to analyze how partial or complete loss of polySia synthesis or a combined loss of polySia and NCAM affects the RMS and the interneuron composition in the OB. Numerous calretinin (CR)‐positive cells were detected dispersed around the RMS in Ncam1 knockout, St8sia2, St8sia4 double‐knockout, and St8sia2, St8sia4, Ncam1 triple‐knockout mice, as well as in St8sia2 ^?/? but not in St8sia4 ^?/? mice. These changes were not reflected by reductions of CR‐positive cells in the granule or glomerular layer of the OB. Instead, calbindin‐positive periglomerular interneurons were strongly reduced in all polySia‐NCAM negative mice and slightly attenuated in St8sia2 ^?/? as well as in the St8sia4 ^?/? mice, which were devoid of ectopic CR‐positive cells along the RMS. Consistent with the early developmental generation of calbindin‐ as compared with CR‐positive OB interneurons, this phenotype was fully developed at postnatal day 5. Together, these results demonstrate that the early development of calbindin‐positive periglomerular interneurons depends on the presentation of polySia on NCAM and requires the activity of both polysialyltransferases. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 421–433, 2016     

Distinct roles for secreted semaphorin signaling in spinal motor axon guidance

Neuropilins, secreted semaphorin coreceptors, are expressed in discrete populations of spinal motor neurons, suggesting they provide critical guidance information for the establishment of functional motor circuitry. We show here that motor axon growth and guidance are impaired in the absence of Sema3A-Npn-1 signaling. Motor axons enter the limb precociously, showing that Sema3A controls the timing of motor axon in-growth to the limb. Lateral motor column (LMC) motor axons within spinal nerves are defasciculated as they grow toward the limb and converge in the plexus region. Medial and lateral LMC motor axons show dorso-ventral guidance defects in the forelimb. In contrast, Sema3F-Npn-2 signaling guides the axons of a medial subset of LMC neurons to the ventral limb, but plays no major role in regulating their fasciculation. Thus, Sema3A-Npn-1 and Sema3F-Npn-2 signaling control distinct steps of motor axon growth and guidance during the formation of spinal motor connections.     

Matrix metalloproteinases promote motor axon fasciculation in the Drosophila embryo

Matrix metalloproteinases (MMPs) are a large conserved family of extracellular proteases, a number of which are expressed during neuronal development and upregulated in nervous system diseases. Primarily on the basis of studies using pharmaceutical inhibitors, MMPs have been proposed to degrade the extracellular matrix to allow growth cone advance during development and hence play largely permissive roles in axon extension. Here we show that MMPs are not required for axon extension in the Drosophila embryo, but rather are specifically required for the execution of several stereotyped motor axon pathfinding decisions. The Drosophila genome contains only two MMP homologs, Mmp1 and Mmp2. We isolated Mmp1 in a misexpression screen to identify molecules required for motoneuron development. Misexpression of either MMP inhibits the regulated separation/defasciculation of motor axons at defined choice points. Conversely, motor nerves in Mmp1 and Mmp2 single mutants and Mmp1 Mmp2 double mutant embryos are loosely bundled/fasciculated, with ectopic axonal projections. Quantification of these phenotypes reveals that the genetic requirement for Mmp1 and Mmp2 is distinct in different nerve branches, although generally Mmp2 plays the predominant role in pathfinding. Using both an endogenous MMP inhibitor and MMP dominant-negative constructs, we demonstrate that MMP catalytic activity is required for motor axon fasciculation. In support of the model that MMPs promote fasciculation, we find that the defasciculation observed when MMP activity is compromised is suppressed by otherwise elevating interaxonal adhesion -- either by overexpressing Fas2 or by reducing Sema-1a dosage. These data demonstrate that MMP activity is essential for embryonic motor axon fasciculation.     

Neurotractin, a novel neurite outgrowth-promoting Ig-like protein that interacts with CEPU-1 and LAMP.

The formation of axon tracts in nervous system histogenesis is the result of selective axon fasciculation and specific growth cone guidance in embryonic development. One group of proteins implicated in neurite outgrowth, fasciculation, and guidance is the neural members of the Ig superfamily (IgSF). In an attempt to identify and characterize new proteins of this superfamily in the developing nervous system, we used a PCR-based strategy with degenerated primers that represent conserved sequences around the characteristic cysteine residues of Ig-like domains. Using this approach, we identified a novel neural IgSF member, termed neurotractin. This GPI-linked cell surface glycoprotein is composed of three Ig-like domains and belongs to the IgLON subgroup of neural IgSF members. It is expressed in two isoforms with apparent molecular masses of 50 and 37 kD, termed L-form and S-form, respectively. Monoclonal antibodies were used to analyze its biochemical features and histological distribution. Neurotractin is restricted to subsets of developing commissural and longitudinal axon tracts in the chick central nervous system. Recombinant neurotractin promotes neurite outgrowth of telencephalic neurons and interacts with the IgSF members CEPU-1 (KD = 3 x 10(-8) M) and LAMP. Our data suggest that neurotractin participates in the regulation of neurite outgrowth in the developing brain.     

The directed growth of retinal axons towards surgically transposed tecta in Xenopus; an examination of homing behaviour by retinal ganglion cell axons

The growth of optic axons towards experimentally rotated tecta has been studied. In stage 24/25 embryos, a piece of the dorsal neural tube, containing the dorsal midbrain rudiment, was rotated through 180 degrees. At later stages of development, the pathways of growing optic axons were investigated by labelling with either horseradish peroxidase or fluorescent dye. It is shown that retinal ganglion cell axons followed well-defined pathways, in spite of the abnormal structure of the brain, and were able to locate displaced tecta. This directed outgrowth of retinal axons in the optic tracts appears to be related either to the tectum or to some other component included in the graft operations. In tadpoles in which the midbrain rudiment was removed, optic axons still followed the normal course of the optic tract. This observation argues against long-range target attraction as being essential in guiding growing retinal axons towards the tectum. An alternative axon guidance mechanism, selective fasciculation, is discussed as a possible alternative to explain the directed axon outgrowth which occurs in both the normal and in these experimentally manipulated tadpoles.     

Wnt5a regulates midbrain dopaminergic axon growth and guidance

During development, precise temporal and spatial gradients are responsible for guiding axons to their appropriate targets. Within the developing ventral midbrain (VM) the cues that guide dopaminergic (DA) axons to their forebrain targets remain to be fully elucidated. Wnts are morphogens that have been identified as axon guidance molecules. Several Wnts are expressed in the VM where they regulate the birth of DA neurons. Here, we describe that a precise temporo-spatial expression of Wnt5a accompanies the development of nigrostriatal projections by VM DA neurons. In mice at E11.5, Wnt5a is expressed in the VM where it was found to promote DA neurite and axonal growth in VM primary cultures. By E14.5, when DA axons are approaching their striatal target, Wnt5a causes DA neurite retraction in primary cultures. Co-culture of VM explants with Wnt5a-overexpressing cell aggregates revealed that Wnt5a is capable of repelling DA neurites. Antagonism experiments revealed that the effects of Wnt5a are mediated by the Frizzled receptors and by the small GTPase, Rac1 (a component of the non-canonical Wnt planar cell polarity pathway). Moreover, the effects were specific as they could be blocked by Wnt5a antibody, sFRPs and RYK-Fc. The importance of Wnt5a in DA axon morphogenesis was further verified in Wnt5a-/- mice, where fasciculation of the medial forebrain bundle (MFB) as well as the density of DA neurites in the MFB and striatal terminals were disrupted. Thus, our results identify a novel role of Wnt5a in DA axon growth and guidance.     

Genes necessary for directed axonal elongation or fasciculation in C. elegans.

The outgrowth of single axons through different cellular environments requires distinct sets of genes in the nematode C. elegans. Three genes are required for the pioneering circumferential outgrowth of identified motor neuron axons between the lateral hypodermal cell membrane and the basal lamina. Three other genes are required for the longitudinal outgrowth of these axons along preexisting axon bundles as well as for the fasciculation of axons within these neuron bundles. Five additional genes are required for circumferential outgrowth, longitudinal outgrowth, and fasciculation; mutations in three of these genes disrupt axon ultrastructure, suggesting that they function in axon formation rather than in axon guidance.     

Developmental profile of neural cell adhesion molecule glycoforms with a varying degree of polymerization of polysialic acid chains

More precise information on the degree of polymerization (DP) of polysialic acid (polySia) chains expressed on neural cell adhesion molecule (NCAM) and its developmental stage-dependent variation are considered important in understanding the mechanism of regulated polysialylation and fine-tuning of NCAM-mediated cell adhesion by polySia. In this paper, first we performed a kinetic study of acid-catalyzed hydrolysis of polySia and report our findings that (a) in (-->8Neu5Ac alpha 2-->)(n)-->8Neu5Ac alpha 2-->3Gal beta 1-->R, the proximal Neu5Ac residue alpha 2-->3 linked to Gal is cleaved about 2.5-4 times faster than the alpha 2-->8 linkages and (b) in contrary to general belief that alpha 2-->8 linkages in polySia are extremely labile, the kinetic consideration showed that they are not so unstable, and every ketosidic bond is hydrolyzed at the same rate. These findings are the basis of our strategy for DP analysis of polySia on NCAM. Second, using the recently developed method that provides base-line resolution of oligo/polySia from DP 2 to >80 with detection thresholds of 1.4 fmol per resolved peak, we have determined the DP of polySia chains expressed in embryonic chicken brains at different developmental stages. Our results support the presence of numerous NCAM glycoforms differing in DPs of oligo/polySia chains and a delicate change in their distribution during development.     

Origins and functional diversification of salinity‐responsive Na+, K+ ATPase α1 paralogs in salmonids

The Salmoniform whole‐genome duplication is hypothesized to have facilitated the evolution of anadromy, but little is known about the contribution of paralogs from this event to the physiological performance traits required for anadromy, such as salinity tolerance. Here, we determined when two candidate, salinity‐responsive paralogs of the Na⁺, K⁺ ATPase α subunit (α1a and α1b) evolved and studied their evolutionary trajectories and tissue‐specific expression patterns. We found that these paralogs arose during a small‐scale duplication event prior to the Salmoniform, but after the teleost, whole‐genome duplication. The ‘freshwater paralog’ (α1a) is primarily expressed in the gills of Salmoniformes and an unduplicated freshwater sister species (Esox lucius) and experienced positive selection in the freshwater ancestor of Salmoniformes and Esociformes. Contrary to our predictions, the ‘saltwater paralog’ (α1b), which is more widely expressed than α1a, did not experience positive selection during the evolution of anadromy in the Coregoninae and Salmonine. To determine whether parallel mutations in Na⁺, K⁺ ATPase α1 may contribute to salinity tolerance in other fishes, we studied independently evolved salinity‐responsive Na⁺, K⁺ ATPase α1 paralogs in Anabas testudineus and Oreochromis mossambicus. We found that a quarter of the mutations occurring between salmonid α1a and α1b in functionally important sites also evolved in parallel in at least one of these species. Together, these data argue that paralogs contributing to salinity tolerance evolved prior to the Salmoniform whole‐genome duplication and that strong selection and/or functional constraints have led to parallel evolution in salinity‐responsive Na⁺, K⁺ ATPase α1 paralogs in fishes.     

Semaphorin 5A is a bifunctional axon guidance cue for axial motoneurons in vivo

Semaphorins are a large class of proteins that function throughout the nervous system to guide axons. It had previously been shown that Semaphorin 5A (Sema5A) was a bifunctional axon guidance cue for mammalian midbrain neurons. We found that zebrafish sema5A was expressed in myotomes during the period of motor axon outgrowth. To determine whether Sema5A functioned in motor axon guidance, we knocked down Sema5A, which resulted in two phenotypes: a delay in motor axon extension into the ventral myotome and aberrant branching of these motor axons. Both phenotypes were rescued by injection of full-length rat Sema5A mRNA. However, adding back RNA encoding the sema domain alone significantly rescued the branching phenotype in sema5A morphants. Conversely, adding back RNA encoding the thrombospondin repeat (TSR) domain alone into sema5A morphants exclusively rescued delay in ventral motor axon extension. Together, these data show that Sema5A is a bifunctional axon guidance cue for vertebrate motor axons in vivo. The TSR domain promotes growth of developing motor axons into the ventral myotome whereas the sema domain mediates repulsion and keeps these motor axons from branching into surrounding myotome regions.     

A multifunctional cell surface developmental stage-specific antigen in the cockroach embryo: involvement in pathfinding by CNS pioneer axons

mAb DSS-8 binds to a 164-kD developmental stage-specific cell surface antigen in the nervous system of the cockroach, Periplaneta americana. The antigen is localized to different subsets of cells at various stages of development. The spatial and temporal distributions of DSS-8 binding were determined and are consistent with this antigen playing multiple roles in the development of the nervous system. Direct identification of some of these functions was made by perturbation experiments in which pioneer axon growth occurs in embryos that are cultured in vitro in the presence of mAb DSS-8 or its Fab fragment. Under these conditions the pioneer axons of the median fiber tract grow but follow altered pathways. In a smaller percentage of the ganglia, the immunoreagents additionally produce defasciculation of a subset of DSS-8 labeled axons. Therefore, direct roles for the DSS-8 antigen in both the guidance of pioneer axons and selective fasciculation have been demonstrated.     

UNC-6 expression by the vulval precursor cells of Caenorhabditis elegans is required for the complex axon guidance of the HSN neurons

Netrin is an evolutionarily conserved axon guidance molecule that has both axonal attraction and repulsion activities. In Caenorhabditis elegans, Netrin/UNC-6 is secreted by ventral cells, attracting some axons ventrally and repelling some axons, which extend dorsally. One axon guided by UNC-6 is that of the HSN neuron. The axon guidance process for HSN neurons is complex, consisting of ventral growth, dorsal growth, branching, second ventral growth, fasciculation with ventral nerve cords, and then anterior growth. The vulval precursor cells (VPC) and the PVP and PVQ neurons are required for the HSN axon guidance; however, the molecular mechanisms involved are completely unknown. In this study, we found that the VPC strongly expressed UNC-6 during HSN axon growth. Silencing of UNC-6 expression in only the VPC, using a novel tissue-specific RNAi technique, resulted in abnormal HSN axon guidance. The expression of Netrin/UNC-6 by only the VPC in unc-6 null mutants partially rescued the HSN ventral axon guidance. Furthermore, the expression of Netrin/UNC-6 by the VPC and the ventral nerve cord (VNC) in unc-6 null mutants restored the complex HSN axon guidance. These results suggest that UNC-6 expressed by the VPC and the VNC cooperatively regulates the complex HSN axon guidance.