One of the features common among olfactory systems for vertebrate and invertebrate species is the division of the primary processing area into distinct clumps of synaptic neuropil, called glomeruli. The olfactory glomeruli appear to serve as functional units of olfaction and are the location of the primary processing between chemosensory afferents and second-order neurons. Although glomeruli are found across all phyla, their numbers and size appear to be characteristic for each species, giving rise to the speculation that there is a relationship between glomerular number and function. It has been hypothesized, for example, that animals with more glomeruli may be able to resolve a wider range of odors. Crustacean species are distributed among freshwater, marine, and terrestrial habitats in arctic, temperate, and tropical climates. They also exhibit a variety of lifestyles and behaviors in which olfaction may play a dominant role. Feeding, for example, ranges from carnivorous, through subaquatic and terrestrial omnivorous scavenging, to filter feeding. Mating and territorial behaviors also are known to involve chemical signals. The current study examines glomerular numbers in the olfactory lobes of 17 crustacean species from six of the seven taxa now included in the reptantian decapods. Estimates of the glomerular numbers were obtained from the analysis of sectioned material treated immunocytochemically with an antibody against synapsin that labels proteins contained in neuronal terminals. The numbers of glomeruli found in the different species were then compared with the volume of the glomerular neuropil, numbers of olfactory sensilla, life styles, habitat, and phylogenetic affinities. The picture that emerges from these correlations is that the decapod crustaceans have exploited various strategies in the construction of their olfactory systems in which the problems of size, sensitivity, and selectivity have all interacted. We find a continuum across the groups ranging from those that favor a high convergence of receptor neurons onto a few glomeruli to those that share a small number of receptor neurons among many glomeruli. The potential functional consequences of these differences are discussed.

Neurofibromatosis type 1 (NF1), a genetic disease that affects 1 in 3,000, is caused by loss of a large evolutionary conserved protein that serves as a GTPase Activating Protein (GAP) for Ras. Among Drosophila melanogaster Nf1 (dNf1) null mutant phenotypes, learning/memory deficits and reduced overall growth resemble human NF1 symptoms. These and other dNf1 defects are relatively insensitive to manipulations that reduce Ras signaling strength but are suppressed by increasing signaling through the 3′-5′ cyclic adenosine monophosphate (cAMP) dependent Protein Kinase A (PKA) pathway, or phenocopied by inhibiting this pathway. However, whether dNf1 affects cAMP/PKA signaling directly or indirectly remains controversial. To shed light on this issue we screened 486 1st and 2nd chromosome deficiencies that uncover >80% of annotated genes for dominant modifiers of the dNf1 pupal size defect, identifying responsible genes in crosses with mutant alleles or by tissue-specific RNA interference (RNAi) knockdown. Validating the screen, identified suppressors include the previously implicated dAlk tyrosine kinase, its activating ligand jelly belly (jeb), two other genes involved in Ras/ERK signal transduction and several involved in cAMP/PKA signaling. Novel modifiers that implicate synaptic defects in the dNf1 growth deficiency include the intersectin-related synaptic scaffold protein Dap160 and the cholecystokinin receptor-related CCKLR-17D1 drosulfakinin receptor. Providing mechanistic clues, we show that dAlk, jeb and CCKLR-17D1 are among mutants that also suppress a recently identified dNf1 neuromuscular junction (NMJ) overgrowth phenotype and that manipulations that increase cAMP/PKA signaling in adipokinetic hormone (AKH)-producing cells at the base of the neuroendocrine ring gland restore the dNf1 growth deficiency. Finally, supporting our previous contention that ALK might be a therapeutic target in NF1, we report that human ALK is expressed in cells that give rise to NF1 tumors and that NF1 regulated ALK/RAS/ERK signaling appears conserved in man.

Sympathetic nerve activity regulates blood pressure by altering peripheral vascular resistance. Variations in vascular sympathetic innervation suggest that vascular-derived cues promote selective innervation of particular vessels during development. As axons extend towards peripheral targets, they migrate along arterial networks following gradients of guidance cues. Collective ratios of these gradients may determine whether axons grow towards and innervate vessels or continue past non-innervated vessels towards peripheral targets. Utilizing directed neurite outgrowth in a three-dimensional (3D) co-culture, we observed increased axon growth from superior cervical ganglion explants (SCG) towards innervated compared to non-innervated vessels, mediated in part by vascular endothelial growth factor (VEGF-A) and Semaphorin3A (Sema3A) which both signal via neuropilin-1 (Nrp1). Exogenous VEGF-A, delivered by high-expressing VEGF-A–LacZ vessels or by rhVEGF-A/alginate spheres, increased sympathetic neurite outgrowth while exogenous rhSema3A/Fc decreased neurite outgrowth. VEGF-A expression is similar between the innervated and non-innervated vessels examined. Sema3A expression is higher in non-innervated vessels. Spatial gradients of Sema3A and VEGF-A may promote differential Nrp1 binding. Vessels expressing high levels of Sema3A favor Nrp1-PlexinA1 signaling, producing chemorepulsive cues limiting sympathetic neurite outgrowth and vascular innervation; while low Sema3A expressing vessels favor Nrp1-VEGFR2 signaling providing chemoattractive cues for sympathetic neurite outgrowth and vascular innervation.

The Abelson (Abl) non-receptor tyrosine kinase regulates the cytoskeleton during multiple stages of neural development, from neurulation, to the articulation of axons and dendrites, to synapse formation and maintenance. We previously showed that Abl is genetically linked to the microtubule (MT) plus end tracking protein (+TIP) CLASP in Drosophila. Here we show in vertebrate cells that Abl binds to CLASP and phosphorylates it in response to serum or PDGF stimulation. In vitro, Abl phosphorylates CLASP with a Km of 1.89 µM, indicating that CLASP is a bona fide substrate. Abl-phosphorylated tyrosine residues that we detect in CLASP by mass spectrometry lie within previously mapped F-actin and MT plus end interaction domains. Using purified proteins, we find that Abl phosphorylation modulates direct binding between purified CLASP2 with both MTs and actin. Consistent with these observations, Abl-induced phosphorylation of CLASP2 modulates its localization as well as the distribution of F-actin structures in spinal cord growth cones. Our data suggest that the functional relationship between Abl and CLASP2 is conserved and provides a means to control the CLASP2 association with the cytoskeleton. © 2014 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.

Perivascular sympathetic innervation density (PSID) is a key determinant of vasomotor responses to sympathetic nerve activity. However, total axonal length (for en passant neurotransmission) per vessel surface area has not been well defined, particularly while preserving 3-dimensional vascular structure. We developed a novel method for quantifying PSID using 3-dimensional anatomical reconstruction and compare a variety of blood vessels in Young (3 months) and Old (20 months) male C57BL/6 mice. Individual vessels were dissected and immunolabeled for tyrosine hydroxylase. The total length of fluorescent axons in defined vessel surface areas was quantified by mapping Zstack images (magnification = 760×). For young mice, innervation densities (μm axon length/μm 2 vessel surface area) in mesenteric (0.075 ± 0.002) and femoral (0.080 ± 0.003) arteries were greater (P<0.05) than mesenteric veins (0.052 ± 0.002) and gracilis muscle feed arteries (0.040 ± 0.002). Carotid arteries and gracilis muscle veins were not immunoreactive nor were there significant differences in PSID between Young and Old animals. We demonstrate a novel approach to quantify sympathetic innervation of the vasculature while preserving its 3-dimensional structure and document regional variation in PSID that persists with aging in mice. This analytical approach may used for quantifying PSID in other tissues that have superficial vessels which can be studied in situ or from which embedded vessels can be excised. With appropriate visualization of neuronal projections, it may also be applied to tissues that have other sources of superficial innervation.

The microtubule (MT) plus-end tracking protein (؉TIP) CLASP mediates dynamic cellular behaviors and interacts with numerous cytoplasmic proteins. While the influence of some CLASP interactors on MT behavior is known, a comprehensive survey of the proteins in the CLASP interactome as MT regulators is missing. Ultimately, we are interested in understanding how CLASP collaborates with functionally linked proteins to regulate MT dynamics. Here, we utilize multiparametric analysis of time-lapse MT ؉TIP imaging data acquired in Drosophila melanogaster S2R؉ cells to assess the effects on individual microtubule dynamics for RNA interference-mediated depletion of 48 gene products previously identified to be in vivo genetic CLASP interactors. While our analysis corroborates previously described functions of several known CLASP interactors, its multiparametric resolution reveals more detailed functional profiles (fingerprints) that allow us to precisely classify the roles that CLASP-interacting genes play in MT regulation. Using these data, we identify subnetworks of proteins with novel yet overlapping MT-regulatory roles and also uncover subtle distinctions between the functions of proteins previously thought to act via similar mechanisms. The orchestration of cytoskeletal dynamics is critical for a broad range of cellular behaviors, including mitosis, polarity, motility, morphogenesis, and cell-cell interaction (1-3). Microtubule (MT) polymer networks participate in numerous signaling pathways, often helping to assemble and/or deliver effector protein complexes and to define the spatial organization of cellular responses. Many classes of cytoskeletal binding proteins regulate the configuration of MT arrays and often interact with other protein networks. However, our understanding of how these extended effector networks function to control cytoskeletal dynamics is still limited. Large-scale screens for MT regulators have primarily relied on endpoint phenotypes that affect mitosis (4-6). The mitotic spindle is a unique apparatus whose gross architecture can be severely disturbed by accumulated effects of altered MT dynamics and thus offers a simple readout for such studies. However, these readouts report screening hits only on the basis of indirect MT phenotypes in a large complex system without pinpointing the actual role that they play in terms of bona fide MT regulation. Direct detection of altered MT dynamics has been much more challenging. For this reason, we adopted a quantitative live-imaging approach that allowed us to identify with single-MT resolution shifts in MT dynamics induced by RNA interference (RNAi)-mediated depletion of putative MT regulators.CLASP (cytoplasmic linker protein [CLIP]-associated protein) is a well-conserved MT plus-end interacting protein (ϩTIP), which modulates dynamic instability and facilitates the interaction of MTs with other cellular structures, including the cell cortex (7, 8) and kinetochores (9-11). CLASP functions as an MT-stabilizing factor, promoting MT rescue both in cultured ...

Background: The process of axon guidance is important in establishing functional neural circuits. The differential expression of cell-autonomous axon guidance factors is crucial for allowing axons of different neurons to take unique trajectories in response to spatially and temporally restricted cell non-autonomous axon guidance factors. A key motivation in the field is to provide adequate explanations for axon behavior with respect to the differential expression of these factors. Results: We report the characterization of a predicted secreted semaphorin family member, semaphorin2b (Sema-2b) in Drosophila embryonic axon guidance. Misexpression of Sema-2b in neurons causes highly penetrant axon guidance phenotypes in specific longitudinal and motoneuron pathways; however, expression of Sema-2b in muscles traversed by these motoneurons has no effect on axon guidance. In Sema-2b loss-of-function embryos, specific motoneuron and interneuron axon pathways display guidance defects. Specific visualization of the neurons that normally express Sema-2b reveals that this neuronal cohort is strongly affected by Sema-2b loss-of-function alleles. Conclusions: While secreted semaphorins have been implicated as cell non-autonomous chemorepellants in a variety of contexts, here we report previously undescribed Sema-2b loss-of-function and misexpression phenotypes that are consistent with a cell-autonomous role for Sema-2b. Developmental Dynamics 242:861–873, 2013. © 2013 Wiley Periodicals, Inc.Key FindingsMisexpression of the secreted semaphorin Sema-2b in neurons results in specific axon guidance phenotypes.Both Sema-2b loss-of-function and misexpression phenotypes are congruent with a cell-autonomous role for Sema-2b.Novel axon guidance phenotypes caused by Sema-2b loss-of-function mutations are characterized.

Regulation of the synaptic cytoskeleton is essential to proper neuronal development and wiring. Perturbations in neuronal microtubules (MTs) are associated with numerous pathologies, yet it remains unclear how changes in MTs may be coupled to synapse morphogenesis. Studies have identified many MT regulators that promote synapse growth. However, less is known about the factors that restrict growth, despite the potential links of synaptic overgrowth to severe neurological conditions. Here, we report that dTACC, which is implicated in MT assembly and stability, prevents synapse overgrowth at the Drosophila neuromuscular junction by restricting addition of new boutons throughout larval development. dTACC localizes to the axonal MT lattice and is required to maintain tubulin levels and the integrity of higher‐order MT structures in motor axon terminals. While previous reports have demonstrated the roles of MT‐stabilizing proteins in promoting synapse growth, our findings suggest that in certain contexts, MT stabilization may correlate with restricted growth.