Vav GEFs regulate macrophage morphology and adhesion-induced Rac and Rho activation

The Vav family of proteins have the potential to act as both signalling adapters and GEFs for Rho GTPases. They have therefore been proposed as regulators of the cytoskeleton in various cell types. We have used macrophages from mice deficient in all three Vav isoforms to determine how their function affects cell morphology and migration. Macrophages lacking Vav proteins adopt an elongated morphology and have enhanced migratory persistence in culture. To investigate the pathways through which Vav proteins exert their effects we analysed the responses of macrophages to the chemoattractant CSF-1 and to adhesion. We found that morphological and signalling responses of macrophages to CSF-1 did not require Vav proteins. In contrast, adhesion-induced cell spreading, RhoA and Rac1 activation and cell signalling were all dependent on Vav proteins. We propose that Vav proteins affect macrophage morphology and motile behaviour by coupling adhesion receptors to Rac1 and RhoA activity and regulating adhesion signalling events such as paxillin and ERK1/2 phosphorylation by acting as adapters.     

Metazoan Scc4 homologs link sister chromatid cohesion to cell and axon migration guidance

Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such as Drosophila Nipped-B, a regulator of cut and Ultrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs of Caenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein–protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation in C. elegans embryos. Using antisense morpholino oligonucleotides to knock down Xenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development.     

Evolution and diversity of axon guidance Robo receptor family genes

The diversity of axon guidance (AG) receptors reflects gains in complexity of the animal nervous system during evolution. Members of the Roundabout (Robo) family of receptors interact with Slit proteins and play important roles in many developmental processes, including AG and neural crest cell migration. There are four members of the Robo gene family. However, the evolutionary history of Robo family genes remain obscure. We analyzed the distribution of Robo family members in metazoan species ranging in complexity from hydras to humans. We undertook a phylogenetic analysis in metazoans, synteny analysis, and ancestral chromosome mapping in vertebrates, and detected selection pressure and functional divergence among four mammalian Robo paralogs. Based on our analysis, we proposed that the ancestral Robo gene could have undergone a tandem duplication in the vertebrate ancestor; then one round of whole genome duplication events occurred before the divergence of ancestral lamprey and gnathostome, generating four paralogs in early vertebrates. Robo4 paralog underwent segmental loss in the following evolutionary process. Our results showed that Robo3 paralog is under more powerful purifying selection pressure compared with other three paralogs, which could correlate with its unique expression pattern and function. Furthermore, we found four sites under positive selection pressure on the Ig1‐2 domains of Robo4 that might interfere with its binding to Slits ligand. Diverge analysis at the amino acid level showed that Robo4 paralog have relatively greater functional diversifications than other Robo paralogs. This coincides with the fact that Robo4 predominantly functions in vascular endothelial cells but not the nervous system.     

A family of proteins implicated in axon guidance and outgrowth.

Rapid progress in the identification and characterization of axon guidance molecules and their receptors has left the field poised to explore the intracellular mechanisms by which signals are transduced into growth cone responses. The TUC (TOAD/Ulip/CRMP) family of proteins has emerged as a strong candidate for a role in growth cone signaling. The TUC family members reach their highest expression levels in all neurons during their peak periods of axonal growth and are strongly down-regulated afterward. When axonal regrowth in the adult is triggered by axotomy, TUC-4 is reexpressed during the period of regrowth. Mutations in unc-33, a homologous nematode gene, lead to severe axon guidance errors in all neurons. Furthermore, the TUC family is required for the growth cone-collapsing activity of collapsin-1. An important role for the TUC family is also suggested by its high degree of interspecies amino acid sequence identity, with the rat TUC-2 protein showing 98% identity with its chick ortholog and 89% identity with its Xenopus ortholog. Information gained from the study of the TUC family will be of key importance in understanding how growth cones find their targets.     

Retinal axon growth cones respond to EphB extracellular domains as inhibitory axon guidance cues

Axon pathfinding relies on cellular signaling mediated by growth cone receptor proteins responding to ligands, or guidance cues, in the environment. Eph proteins are a family of receptor tyrosine kinases that govern axon pathway development, including retinal axon projections to CNS targets. Recent examination of EphB mutant mice, however, has shown that axon pathfinding within the retina to the optic disc is dependent on EphB receptors, but independent of their kinase activity. Here we show a function for EphB1, B2 and B3 receptor extracellular domains (ECDs) in inhibiting mouse retinal axons when presented either as substratum-bound proteins or as soluble proteins directly applied to growth cones via micropipettes. In substratum choice assays, retinal axons tended to avoid EphB-ECDs, while time-lapse microscopy showed that exposure to soluble EphB-ECD led to growth cone collapse or other inhibitory responses. These results demonstrate that, in addition to the conventional role of Eph proteins signaling as receptors, EphB receptor ECDs can also function in the opposite role as guidance cues to alter axon behavior. Furthermore, the data support a model in which dorsal retinal ganglion cell axons heading to the optic disc encounter a gradient of inhibitory EphB proteins which helps maintain tight axon fasciculation and prevents aberrant axon growth into ventral retina. In conclusion, development of neuronal connectivity may involve the combined activity of Eph proteins serving as guidance receptors and as axon guidance cues.     

Ephrin reverse signaling in axon guidance and synaptogenesis

Axon-cell and axon-dendrite contact is a highly regulated process necessary for the formation of precise neural circuits and a functional neural network. Eph-ephrin interacting molecules on the membranes of axon nerve terminals and target dendrites act as bidirectional ligands/receptors to transduce signals into both the Eph-expressing and ephrin-expressing cells to regulate cytoskeletal dynamics. In particular, recent evidence indicates that ephrin reverse signal transduction events are important in controlling both axonal and dendritic elaborations of neurons in the developing nervous system. Here we review how ephrin reverse signals are transduced into neurons to control maturation of axonal pre-synaptic and dendritic post-synaptic structures.     

Receptor tyrosine phosphatases in axon growth and guidance

Receptor-like protein tyrosine phosphatases (RPTPs) continue to emerge as important signalling molecules in axons and their growth cones. Recent findings show that Drosophila RPTPs play key roles in guiding retinal axons and in preventing midline crossing of longitudinal axons. Vertebrate RPTPs are now implicated in controlling axon outgrowth, and preliminary evidence suggests that they too may influence axon guidance.     

Conservation and divergence of axon guidance mechanisms.

Analysis of axon guidance mechanisms in vertebrates, Caenorhabditis elegans, and Drosophila melanogaster has led to the identification of several signaling pathways, many of which are strikingly conserved in function. Recent studies indicate that several axon guidance mechanisms are highly conserved in all animals, whereas others, though still conserved in a general sense, show strong evolutionary divergence at a detailed mechanistic level.     

Models of axon guidance and bundling during development

Diffusible chemoattractants and chemorepellants, together with contact attraction and repulsion, have been implicated in the establishment of connections between neurons and their targets. Here we study how such diffusible and contact signals can be involved in the whole sequence of events from bundling of axons, guidance of axon bundles towards their targets, to debundling and the final innervation of individual targets. By means of computer simulations, we investigate the strengths and weaknesses of a number of particular mechanisms that have been proposed for these processes.     

Nervous vascular parallels: axon guidance and beyond

The vascular and nervous systems are organized with well defined and accurate networks, which represent the anatomical structure enabling their functions. In recent years, it has been clearly demonstrated that these two systems share in common several mechanisms and specificities. For instance, the networking properties of the nervous and vascular systems are governed by common cues that in the brain regulate axon connections and in the vasculature remodel the primitive plexus towards the vascular tree. Here, we summarize the role of semaphorins as a paradigmatic example of the role of axon guidance molecules in physiological and pathological angiogenesis. Finally, we discuss the presence in blood vessels of neurexin and neuroligin, two proteins that finely modulate synaptic activity in the brain. This observation is suggestive of an intriguing new class of molecular and functional parallels between neurons and vascular cells.     

Celsr3 and Fzd3 in axon guidance

The assembly of functional neuronal circuits depends on the correct wiring of axons and dendrites. To reach their targets, axons are guided by a variety of extracellular guidance cues, including Netrins, Ephrins, Semaphorins and Slits. Corresponding receptors in the growth cone, the dynamic structure at the tip of the growing axon, sense and integrate these positional signals, and activate downstream effectors to regulate cytoskeletal organization. In addition to the four canonical families of axon guidance cues mentioned above, some proteins that regulate planar cell polarity were recently found to be critical for axon guidance. The seven-transmembrane domain receptors Celsr3 and Fzd3, in particular, control the development of most longitudinal tracts in the central nervous system, and axon navigation in the peripheral, sympathetic and enteric nervous systems. Despite their unequivocally important role, however, underlying molecular mechanisms remain elusive. We do not know which extracellular ligands they recognize, whether they have co-receptors in the growth cone, and what their downstream effectors are. Here, we review some recent advances and discuss future trends in this emerging field.     

Wiring the zebrafish: axon guidance and synaptogenesis

Many zebrafish mutants have specific defects in axon guidance or synaptogenesis, particularly in the retinotectal and motor systems. Several mutants have now been characterized in detail and/or cloned. A combination of genetic studies, in vivo imaging and new techniques for misexpressing genes or blocking their function promises to reveal the molecules and principles that govern wiring of the vertebrate nervous system.     

Vav3 is involved in GABAergic axon guidance events important for the proper function of brainstem neurons controlling cardiovascular, respiratory, and renal parameters

Vav3 is a phosphorylation-dependent activator of Rho/Rac GTPases that has been implicated in hematopoietic, bone, cerebellar, and cardiovascular roles. Consistent with the latter function, Vav3-deficient mice develop hypertension, tachycardia, and renocardiovascular dysfunctions. The cause of those defects remains unknown as yet. Here, we show that Vav3 is expressed in GABAegic neurons of the ventrolateral medulla (VLM), a brainstem area that modulates respiratory rates and, via sympathetic efferents, a large number of physiological circuits controlling blood pressure. On Vav3 loss, GABAergic cells of the caudal VLM cannot innervate properly their postsynaptic targets in the rostral VLM, leading to reduced GABAergic transmission between these two areas. This results in an abnormal regulation of catecholamine blood levels and in improper control of blood pressure and respiration rates to GABAergic signals. By contrast, the reaction of the rostral VLM to excitatory signals is not impaired. Consistent with those observations, we also demonstrate that Vav3 plays important roles in axon branching and growth cone morphology in primary GABAergic cells. Our study discloses an essential and nonredundant role for this Vav family member in axon guidance events in brainstem neurons that control blood pressure and respiratory rates.     

The Beat generation: a multigene family encoding IgSF proteins related to the Beat axon guidance molecule in Drosophila.

A previous genetic screen led to the identification of the beaten path (beat Ia) gene in Drosophila. Beat Ia contains two immunoglobulin (Ig) domains and appears to function as an anti-adhesive factor secreted by specific growth cones to promote axon defasciculation. We identify a family of 14 beat-like genes in Drosophila. In contrast to beat Ia, four novel Beat-family genes encode membrane-bound proteins. Moreover, mutations in each gene lead to much more subtle guidance phenotypes than observed in beat Ia. Genetic interactions between beat Ic and beat Ia reveal complementary functions. Our data suggest a model whereby Beat Ic (and perhaps other membrane-bound family members) functions in a pro-adhesive fashion to regulate fasciculation, while Beat Ia (the original secreted Beat) functions in an anti-adhesive fashion to regulate defasciculation.