Shh signaling guides spatial pathfinding of raphespinal tract axons by multidirectional repulsion

Relatively little is known about the molecular mechanisms underlying spatial pathfinding in the descending serotonergic raphespinal tract (RST) in the developing spinal cord, one of the most important nerve pathways for pain, sensory and motor functions. We provide evidence that ventral floor plate-secreted Sonic hedgehog (Shh) is responsible for the establishment of decreasing gradients in both the anterior-to-posterior (A-P) and the medial-to-lateral (M-L) directions in the ventral spinal cord during serotonergic RST axon projection. Downstream components of the Shh pathway, Patched 1 (Ptch1) and Smoothened (Smo), were expressed in the serotonergic caudal raphe nuclei and enriched in the descending serotonergic RST axons. Diffusible Shh repulsion of serotonergic RST axons was shown to be mediated by Shh-Ptch1 interactions and derepression of Smo. Using a co-culture assay, we showed that A-P graded repulsion mediated by Shh signaling pushed the serotonergic axons caudally through the ventral spinal cord and M-L graded repulsion mediated by Shh signaling simultaneously restricted the serotonergic axons to the ventral and ventral-lateral funiculus. Prominent pathfinding errors of serotonergic RST axons were observed in various Shh, Ptch1 and Smo mutants. We conclude that Shh signaling-mediated multidirectional repulsion is required to push descending serotonergic RST axons in the A-P direction, and to restrict these axons to the ventral and ventral-lateral funiculus in the M-L direction. This is the first demonstration that Shh signaling-mediated multidirectional repulsion of serotonergic RST axons maintains spatial axon pathfinding in the developing spinal cord.     

Dendrite self-avoidance is controlled by Dscam

Dendrites distinguish between sister branches and those of other cells. Self-recognition can often lead to repulsion, a process termed "self-avoidance." Here we demonstrate that dendrite self-avoidance in Drosophila da sensory neurons requires cell-recognition molecules encoded by the Dscam locus. By alternative splicing, Dscam encodes a vast number of cell-surface proteins of the immunoglobulin superfamily. We demonstrate that interactions between identical Dscam isoforms on the cell surface underlie self-recognition, while the cytoplasmic tail converts this recognition to dendrite repulsion. Sister dendrites expressing the same isoforms engage in homophilic repulsion. By contrast, Dscam diversity ensures that inappropriate repulsive interactions between dendrites sharing the same receptive field do not occur. The selectivity of Dscam-mediated cell interactions is likely to be widely important in the developing fly nervous system, where processes of cells must distinguish between self and nonself during the construction of neural circuits.     

Drosophila Dscam is required for divergent segregation of sister branches and suppresses ectopic bifurcation of axons

Axon bifurcation results in the formation of sister branches, and divergent segregation of the sister branches is essential for efficient innervation of multiple targets. From a genetic mosaic screen, we find that a lethal mutation in the Drosophila Down syndrome cell adhesion molecule (Dscam) specifically perturbs segregation of axonal branches in the mushroom bodies. Single axon analysis further reveals that Dscam mutant axons generate additional branches, which randomly segregate among the available targets. Moreover, when only one target remains, branching is suppressed in wild-type axons while Dscam mutant axons still form multiple branches at the original bifurcation point. Taken together, we conclude that Dscam controls axon branching and guidance such that a neuron can innervate multiple targets with minimal branching.     

Dscam and neuronal uniqueness

In the fruit fly Drosophila the gene encoding the cell adhesion molecule Dscam generates alternatively spliced mRNAs that can be translated into thousands of different protein isoforms. Three recent papers show that isoform-specific homophilic Dscam interactions cause dendritic branches of the same neuron to avoid each other (Hughes et al., 2007; Soba et al., 2007; Matthews et al., 2007). This process ensures the correct patterning of dendrites in the peripheral nervous system.     

EphB2 guides axons at the midline and is necessary for normal vestibular function

Mice lacking the EphB2 receptor tyrosine kinase display a cell-autonomous, strain-specific circling behavior that is associated with vestibular phenotypes. In mutant embryos, the contralateral inner ear efferent growth cones exhibit inappropriate pathway selection at the midline, while in mutant adults, the endolymph-filled lumen of the semicircular canals is severely reduced. EphB2 is expressed in the endolymph-producing dark cells in the inner ear epithelium, and these cells show ultrastructural defects in the mutants. A molecular link to fluid regulation is provided by demonstrating that PDZ domain-containing proteins that bind the C termini of EphB2 and B-ephrins can also recognize the cytoplasmic tails of anion exchangers and aquaporins. This suggests EphB2 may regulate ionic homeostasis and endolymph fluid production through macromolecular associations with membrane channels that transport chloride, bicarbonate, and water.     

Protein synthesis in axons and its possible functions

Brain Cell Biology - Proteins synthesized in neuronal cell bodies are transported along axons by fast and slow axonal transport. Cytoskeletal proteins and cytosolic proteins that travel by slow...     

Telomerase-dependent and -independent chromosome healing in mouse embryonic stem cells

Telomeres play an important role in protecting the ends of chromosomes and preventing chromosome fusion. We have previously demonstrated that double-strand breaks near telomeres in mammalian cells result in either the addition of a new telomere at the site of the break, termed chromosome healing, or sister chromatid fusion that initiates chromosome instability. In the present study, we have investigated the role of telomerase in chromosome healing and the importance of chromosome healing in preventing chromosome instability. In embryonic stem cell lines that are wild type for the catalytic subunit of telomerase (TERT), chromosome healing at I-SceI-induced double-strand breaks near telomeres accounted for 22 of 35 rearrangements, with the new telomeres added directly at the site of the break in all but one instance. In contrast, in two TERT-knockout embryonic stem cell lines, chromosome healing accounted for only 1 of 62 rearrangements, with a 23 bp insertion at the site of the sole chromosome-healing event. However, in a third TERT-knockout embryonic stem cell line, 10PTKO-A, chromosome healing was a common event that accounted for 20 of 34 rearrangements. Although this chromosome healing also occurred at the I-SceI site, differences in the microhomology at the site of telomere addition demonstrated that the mechanism was distinct from that in wild-type embryonic stem cell lines. In addition, the newly added telomeres in 10PTKO-A shortened with time in culture, eventually resulting in either telomere elongation through a telomerase-independent mechanism or loss of the subtelomeric plasmid sequences entirely. The combined results demonstrate that chromosome healing can occur through both telomerase-dependent and -independent mechanisms, and that although both mechanisms can prevent degradation and sister chromatid fusion, neither mechanism is efficient enough to prevent sister chromatid fusion from occurring in many cells experiencing double-strand breaks near telomeres.     

Analysis of potassium channel functions in mammalian axons by gene knockouts

Mammalian axons express a rich repertoire of various K channel subtypes whose distribution is profoundly affected by myelination. In the past two decades, functional analysis of axonal K channels has been approached primarily through pharmacology. Recently, gene knockout techniques have been used to specifically delete a particular K channel subtype from axons. This is significant since the bulk of K channels in a myelinated nerve are covered by the myelin, making functional analysis of specific K channel subtypes by traditional means difficult. This review summarizes the first mutational analysis of this sort performed on an axonal fast K channel termed Kv1.1. This K channel is concealed by the myelin loops in the paranodes of all major myelinated fiber tracts, and exhibits highly heterogeneous distribution even in certain non-myelinated CNS axons. Physiological analysis of Kv1.1 null mutants suggest novel functions for this axonal K channel subtype, including modulation of conduction failures at branch points and stabilization of transition zones in myelinated nerves.     

Phosphatidylinositol 3-kinase-dependent and -independent cytolytic effector functions.

Two distinct forms of short-term cytolysis have been described for CD8+ CTLs, the perforin/granzyme- and Fas ligand/Fas (CD95 ligand (CD95L)/CD95)-mediated pathways. However, the difference in signal transduction events leading to these cytolytic mechanisms remains unclear. We used wortmannin, an irreversible antagonist of phosphatidylinositol 3-kinase (PI3-K) activity, to investigate the role of PI3-K in influenza-specific CD8+ CTL cytolytic effector function. We found that the addition of wortmannin at concentrations as low as 1 nM significantly inhibited both the Ag/MHC-induced cytolysis of CD95- target cells and serine esterase release. In strong contrast, W did not inhibit the Ag/MHC-induced CD95L expression or the CD95L/CD95-mediated cytolysis of CD95+ targets. A combination of wortmannin and blocking mAb against CD95L inhibited the cytolysis of CD95+ targets, indicating that the wortmannin-independent cytolysis was due to CD95L/CD95 mediated cytolysis. These findings suggest a differential role for PI3-K in mediating cytolysis and, thus far, the earliest difference between perforin/granzyme- and CD95L/CD95-dependent cytolysis. Our data reinforce the idea of a TCR with modular signal transduction pathways that can be triggered or inhibited selectively, resulting in differential effector function.     

Sema3a1 guides spinal motor axons in a cell- and stage-specific manner in zebrafish

In order for axons to reach their proper targets, both spatiotemporal regulation of guidance molecules and stepwise control of growth cone sensitivity to guidance molecules is required. Here, we show that, in zebrafish, Sema3a1, a secreted class 3 semaphorin, plays an essential role in guiding the caudal primary (CaP) motor axon that pioneers the initial region of the motor pathway. The expression pattern of Sema3a1 suggests that it delimits the pioneer CaP axons to the initial, common pathway via a repulsive action, but then CaP axons become insensitive to Sema3a1 beyond the common pathway. Indeed, nrp1a, which probably encodes a component of the Sema3a1 receptor, is specifically expressed by CaP during the early part of its outgrowth but not during later stages when extending into sema3a1-expressing muscle cells. To examine this hypothesis directly, expression of sema3a1 and/or nrp1a was manipulated in several ways. First, antisense knockdown of Sema3a1 induced CaP axons to branch excessively, stall and/or follow aberrant pathways. Furthermore, dynamic analysis showed they extended more lateral filopodia and often failed to pause at the horizontal myoseptal choice point. Second, antisense knockdown of Nrp1a and double knockdown of Nrp1a/Sema3a1 induced similar outgrowth defects in CaP. Third, CaP axons were inhibited by focally misexpressed sema3a1 along the initial common pathway but not along their pathway beyond the common pathway. Thus, as predicted, Sema3a1 is repulsive to CaP axons in the common region of the pathway, but not beyond the common pathway. Fourth, induced ubiquitous overexpression of sema3a1 caused the CaP axons but not the other primary motor axons to follow aberrant pathways. These results suggest that the repulsive response to Sema3a1 of the primary motor axons along the common pathway is both cell-type specific and dynamically regulated, perhaps via regulation of nrp1a.     

Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis

Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5β1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.     

Identification of proteins secreted from axons of embryonic dorsal-root-ganglia neurons

Secretion of proteins from the growth cone has been implicated in axon growth and synapse formation and might be involved in the transmission of a variety of axon-derived regulatory signals during neurogenesis. In order to identify axonally secreted proteins, dorsal-root-ganglia neurons from chicken embryos were cultured in a compartmentalized cell culture system that allows separate access to neuronal cell somas and axons. The proteins synthesized by the neurons were metabolically labeled by addition of [35S]methionine to the compartment containing the cell somas; the proteins released from the axons were harvested from the culture medium of the axonal compartment. Two-dimensional gel electrophoresis revealed two axonally secreted proteins with apparent molecular mass of 132-140 kDa and 54-60 kDa; they were termed axonin-1 and axonin-2, respectively. Both axonins were found to be secreted from a variety of neuronal cell cultures, but not from any of the nonneuronal cultures investigated, and hence might be neuron-specific. Virtual absence of these proteins from the axonal protein pattern suggests constitutive secretion. The information acquired on coordinates and spot morphology of these proteins in two-dimensional gel electrophoresis provides a useful assay for their purification.     

Spinal motor axons and neural crest cells use different molecular guides for segmental migration through the rostral half-somite

The peripheral nervous system in vertebrates is composed of repeating metameric units of spinal nerves. During development, factors differentially expressed in a rostrocaudal pattern in the somites confine the movement of spinal motor axons and neural crest cells to the rostral half of the somitic sclerotome. The expression patterns of transmembrane ephrin-B ligands and interacting EphB receptors suggest that these proteins are likely candidates for coordinating the segmentation of spinal motor axons and neural crest cells. In vitro, ephrin-B1 has indeed been shown to repel axons extending from the rodent neural tube (Wang & Anderson, 1997). In avians, blocking interactions between EphB3 expressed by neural crest cells and ephrin-B1 localized to the caudal half of the somite in vivo resulted in loss of the rostrocaudal patterning of trunk neural crest migration (Krull et al., 1997). The role of ephrin-B1 in patterning spinal motor axon outgrowth in avian embryos was investigated. Ephrin-B1 protein was found to be expressed in the caudal half-sclerotome and in the dermomyotome at the appropriate time to interact with the EphB2 receptor expressed on spinal motor axons. Treatment of avian embryo explants with soluble ephrin-B1, however, did not perturb the segmental outgrowth of spinal motor axons through the rostral half-somite. In contrast, under the same treatment conditions with soluble ephrin-B1, neural crest cells migrated aberrantly through both rostral and caudal somite halves. These results indicate that the interaction between ephrin-B1 and EphB2 is not required for patterning spinal motor axon segmentation. Even though spinal motor axons traverse the same somitic pathway as neural crest cells, different molecular guidance mechanisms appear to influence their movement.     

Ubiquitin-dependent and -independent functions of OTULIN in cell fate control and beyond

Ubiquitination, and its control by deubiquitinating enzymes (DUBs), mediates protein stability, function, signaling and cell fate. The ovarian tumor (OTU) family DUB OTULIN (FAM105B) exclusively cleaves linear (Met1-linked) poly-ubiquitin chains and plays important roles in auto-immunity, inflammation and infection. OTULIN regulates Met1-linked ubiquitination downstream of tumor necrosis factor receptor 1 (TNFR1), toll-like receptor (TLR) and nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) receptor activation and interacts with the Met1 ubiquitin-specific linear ubiquitin chain assembly complex (LUBAC) E3 ligase. However, despite extensive research efforts, the receptor and cytosolic roles of OTULIN and the distributions of multiple Met1 ubiquitin-associated E3-DUB complexes in the regulation of cell fate still remain controversial and unclear. Apart from that, novel ubiquitin-independent OTULIN functions have emerged highlighting an even more complex role of OTULIN in cellular homeostasis. For example, OTULIN interferes with endosome-to-plasma membrane trafficking and the OTULIN-related pseudo-DUB OTULINL (FAM105A) resides at the endoplasmic reticulum (ER). Here, we discuss how OTULIN contributes to cell fate control and highlight novel ubiquitin-dependent and -independent functions.Subject terms: Biochemistry, Cell biology