labial acts to initiate neuronal fate specification, but not axon pathfinding, in the embryonic brain of Drosophila

Drosophila embryos lacking the homeotic gene labial (lab) show two types of defects in brain development: (1) cells in the brain lab domain do not express neuronal markers or extend axons, and (2) axons originating from outside the lab domain stop at this region or project ectopically. A severe disruption of neuronal patterning and axon scaffolding is the net result. It is not clear how the absence of Lab can result in both neuronal fate defects and axon pathfinding defects. I have expressed Lab in short pulses in lab loss-of- function embryos, and this gave almost complete rescue; for example, the tritocerebral commissure was restored. Rescue only occurred when Lab was provided at the time when cells in the brain are adopting a neuronal fate. Lab expression later, when the first axons are seen in the lab domain, did not give rescue. I conclude that Lab expression helps to establish neuronal identity in the lab domain, and these neurons act as a permissive substrate for axon extension. However, Lab itself is not required at the time of axon pathfinding through this region. Received: 31 May 2000 / Accepted: 5 July 2000     

The morphogenic properties of oligomeric endostatin are dependent on cell surface heparan sulfate

Endostatin has attracted considerable attention because of its ability to inhibit angiogenesis. This property of monomeric endostatin contrasts with that of the trimeric endostatin moiety generated from the intact C-terminal domain of collagen XVIII that induces a promigratory phenotype in endothelial cells. This activity is inhibited by monomeric endostatin. In this study we demonstrate that the effect of oligomeric endostatin can also be inhibited by exogenous glycosaminoglycans in a size-dependent manner, with heparin oligosaccharides containing more than 20 monosaccharide residues having optimal inhibitory activity. Oligomeric endostatin was also found to induce morphological changes in Chinese hamster ovary cells, an epithelial cell line. This novel observation allowed the utilization of a panel of Chinese hamster ovary cell mutants with defined glycosaminoglycan biosynthetic defects. The action of oligomeric endostatin on these cells was shown to be dependent on cell surface glycosaminoglycans, principally heparan sulfate with N- and 6-O-sulfation of glucosamine residues rather than iduronate 2-O-sulfation being important for bioactivity. The responsiveness of a cell line (pgsE-606) with globally reduced heparan sulfate sulfation and shortened S domains, however, indicates that overall heparan sulfate domain patterning is the key determinant of the bioactivity of oligomeric endostatin. Purified heparin-monomeric endostatin constructs generated by zero-length cross-linking techniques were found to be unable to inhibit the action of oligomeric endostatin. This indicates a mechanism for the perturbation of oligomeric endostatin action by its monomeric counterpart via competition for glycosaminoglycan attachment sites at the cell surface.     

The role of Drosophila hyperplastic discs gene in spermatogenesis

In Drosophila, the ubiquitin ligase Hyd (hyperplastic disc) is required for regulation of cell proliferation during development [ Martin et al. (1977) Dev Biol 55 , 213–232; Mansfield et al. (1994) Dev Biol 165 , 507–526]. Earlier, we demonstrated that the Drosophila tumour suppressor Merlin participates not only in imaginal discs proliferation control, but also performs a separate Nebenkern structural function in Drosophila spermatogenesis [ Dorogova et al. (2008) BMC Cell Biol 9 , 1. Here, we show that the hyd mutants also have spermatogenesis defects: chromosome condensation and attachment to the spindle, centrosome behaviour and cytokinesis in meiosis. The process of spermatid elongation was also greatly affected: nuclei were scattered along the cyst and had an abnormal shape, Nebenkern–axoneme angular relation and attachment was distorted, axonemes themselves lost correct structure. Since Hyd and pAbp protein families share a common PABC [poly(A)‐binding protein C‐terminal] protein domain, we also studied spermatogenesis in pAbp homozygotes and found defects in cytokinesis and spermatid elongation. However, our study of hyd and pAbp genetic interaction revealed only the phenotype of defective nuclei shape at the final stage of spermatid differentiation. So, the PABC domain is unlikely to be responsible for meiotic defects. Thus, our data document that, in addition to the tumour suppressor Merlin, another tumour suppressor, Hyd, also has a function in spermatogenesis.     

ENU-3 is a novel motor axon outgrowth and guidance protein in C. elegans

During the development of the nervous system, the migration of many cells and axons is guided by extracellular molecules. These molecules bind to receptors at the tips of the growth cones of migrating axons and trigger intracellular signaling to steer the axons along the correct trajectories. We have identified a novel mutant, enu-3 (enhancer of Unc), that enhances the motor neuron axon outgrowth defects observed in strains of Caenorhabditis elegans that lack either the UNC-5 receptor or its ligand UNC-6/Netrin. Specifically, the double-mutant strains have enhanced axonal outgrowth defects mainly in DB4, DB5 and DB6 motor neurons. enu-3 single mutants have weak motor neuron axon migration defects. Both outgrowth defects of double mutants and axon migration defects of enu-3 mutants were rescued by expression of the H04D03.1 gene product. ENU-3/H04D03.1 encodes a novel predicted putative trans-membrane protein of 204 amino acids. It is a member of a family of highly homologous proteins of previously unknown function in the C. elegans genome. ENU-3 is expressed in the PVT interneuron and is weakly expressed in many cell bodies along the ventral cord, including those of the DA and DB motor neurons. We conclude that ENU-3 is a novel C. elegans protein that affects both motor axon outgrowth and guidance.     

Regulated vnd expression is required for both neural and glial specification in Drosophila

The Drosophila embryonic CNS arises from the neuroectoderm, which is divided along the dorsal‐ventral axis into two halves by specialized mesectodermal cells at the ventral midline. The neuroectoderm is in turn divided into three longitudinal stripes—ventral, intermediate, and lateral. The ventral nervous system defective, or vnd, homeobox gene is expressed from cellularization throughout early neural development in ventral neuroectodermal cells, neuroblasts, and ganglion mother cells, and later in an unrelated pattern in neurons. Here, in the context of the dorsal‐ventral location of precursor cells, we reassess the vnd loss‐ and gain‐of‐function CNS phenotypes using cell specific markers. We find that over expression of vnd causes significantly more profound effects on CNS cell specification than vnd loss. The CNS defects seen in vnd mutants are partly caused by loss of progeny of ventral neuroblasts—the commissures are fused and the longitudinal connectives are aberrantly positioned close to the ventral midline. The commissural vnd phenotype is associated with defects in cells that arise from the mesectoderm, where the VUM neurons have pathfinding defects, the MP1 neurons are mis‐specified, and the midline glia are reduced in number. vnd over expression results in the mis‐specification of progeny arising from all regions of the neuroectoderm, including the ventral neuroblasts that normally express the gene. The CNS of embryos that over express vnd is highly disrupted, with weak longitudinal connectives that are placed too far from the ventral midline and severely reduced commissural formation. The commissural defects seen in vnd gain‐of‐function mutants correlate with midline glial defects, whereas the mislocalization of interneurons coincides with longitudinal glial mis‐specification. Thus, Drosophila neural and glial specification requires that vnd expression by tightly regulated. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 118–136, 2002; DOI 10.1002/neu.10022     

Dmp53 is sequestered to nuclear bodies in spermatogonia of Drosophila melanogaster

p53 family members have been implicated in regulation of genomic integrity and apoptosis in a variety of tissues. The Drosophila family member, Dmp53, primarily functions to regulate apoptosis in developing and regenerating tissues but loss of function mutants are viable and fertile. Dmp53 exhibits a striking expression pattern in the male germline with high levels found in nuclear bodies in pre-meiotic germ cells. The localisation of Dmp53 to nuclear bodies is dependent upon Dmp53 complexes being able to bind DNA, and although dmp53 mutants do not affect germline stem cell (GSC) maintenance or differentiation, GSCs are sensitive to overexpression of Dmp53 but maturing spermatogonia are not. Dmp53 thus has differential effects depending upon the stage of male germline maturation.     

The NC1/endostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance

Type XVIII collagen is a homotrimeric basement membrane molecule of unknown function, whose COOH-terminal NC1 domain contains endostatin (ES), a potent antiangiogenic agent. The Caenorhabditis elegans collagen XVIII homologue, cle-1, encodes three developmentally regulated protein isoforms expressed predominantly in neurons. The CLE-1 protein is found in low amounts in all basement membranes but accumulates at high levels in the nervous system. Deletion of the cle-1 NC1 domain results in viable fertile animals that display multiple cell migration and axon guidance defects. Particular defects can be rescued by ectopic expression of the NC1 domain, which is shown to be capable of forming trimers. In contrast, expression of monomeric ES does not rescue but dominantly causes cell and axon migration defects that phenocopy the NC1 deletion, suggesting that ES inhibits the promigratory activity of the NC1 domain. These results indicate that the cle-1 NC1/ES domain regulates cell and axon migrations in C. elegans.     

Diverse mechanisms for assembly of branchiomeric nerves

The formation of branchiomeric nerves (cranial nerves V, VII, IX and X) from their sensory, motor and glial components is poorly understood. The current model for cranial nerve formation is based on the Vth nerve, in which sensory afferents are formed first and must enter the hindbrain in order for the motor efferents to exit. Using transgenic zebrafish lines to discriminate between motor neurons, sensory neurons and peripheral glia, we show that this model does not apply to the remaining three branchiomeric nerves. For these nerves, the motor efferents form prior to the sensory afferents, and their pathfinding show no dependence on sensory axons, as ablation of cranial sensory neurons by ngn1 knockdown had no effect. In contrast, the sensory limbs of the IXth and Xth nerves (but not the Vth or VIIth) were misrouted in gli1 mutants, which lack hindbrain bmn, suggesting that the motor efferents are crucial for appropriate sensory axon projection in some branchiomeric nerves. For all four nerves, peripheral glia were the intermediate component added and had a critical role in nerve integrity but not in axon guidance, as foxd3 null mutants lacking peripheral glia exhibited defasciculation of gVII, gIX, and gX axons. The bmn efferents were unaffected in these mutants. These data demonstrate that multiple mechanisms underlie formation of the four branchiomeric nerves. For the Vth, sensory axons initiate nerve formation, for the VIIth the sensory and motor limbs are independent, and for the IXth/Xth the motor axons initiate formation. In all cases the glia are patterned by the initiating set of axons and are needed to maintain axon fasciculation. These results reveal that coordinated interactions between the three neural cell types in branchiomeric nerves differ according to their axial position.     

C. elegans dystroglycan coordinates responsiveness of follower axons to dorsal/ventral and anterior/posterior guidance cues

Neural development in metazoans is characterized by the establishment of initial process tracts by pioneer axons and the subsequent extension of follower axons along these pioneer processes. Mechanisms governing the fidelity of follower extension along pioneered routes are largely unknown. In C. elegans, formation of the right angle‐shaped lumbar commissure connecting the lumbar and preanal ganglia is an example of pioneer/follower dynamics. We find that the dystroglycan ortholog DGN‐1 mediates the fidelity of follower lumbar commissure axon extension along the pioneer axon route. In dgn‐1 mutants, the axon of the pioneer PVQ neuron faithfully establishes the lumbar commissure, but axons of follower lumbar neurons, such as PVC, frequently bypass the lumbar commissure and extend along an oblique trajectory directly toward the preanal ganglion. In contrast, disruption of the UNC‐6/netrin guidance pathway principally perturbs PVQ ventral guidance to pioneer the lumbar commissure. Loss of DGN‐1 in unc‐6 mutants has a quantitatively similar effect on follower axon guidance regardless of PVQ axon route, indicating that DGN‐1 does not mediate follower/pioneer adhesion. Instead, DGN‐1 appears to block premature responsiveness of follower axons to a preanal ganglion‐directed guidance cue, which mediates ventral‐to‐anterior reorientation of lumbar commissure axons. Deletion analysis shows that only the most N‐terminal DGN‐1 domain is required for these activities. These studies suggest that dystroglycan modulation of growth cone responsiveness to conflicting guidance cues is important for restricting follower axon extension to the tracts laid down by pioneers. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Regulation of axon arborization pattern in the developing chick ciliary ganglion: Possible involvement of caspase 3

During a certain critical period in the development of the central and peripheral nervous systems, axonal branches and synapses are massively reorganized to form mature connections. In this process, neurons search their appropriate targets, expanding and/or retracting their axons. Recent work suggested that the caspase superfamily regulates the axon morphology. Here, we tested the hypothesis that caspase 3, which is one of the major executioners in apoptotic cell death, is involved in regulating the axon arborization. The embryonic chicken ciliary ganglion was used as a model system of synapse reorganization. A dominant negative mutant of caspase‐3 precursor (C3DN) was made and overexpressed in presynaptic neurons in the midbrain to interfere with the intrinsic caspase‐3 activity using an in ovo electroporation method. The axon arborization pattern was 3‐dimensionally and quantitatively analyzed in the ciliary ganglion. The overexpression of C3DN significantly reduced the number of branching points, the branch order and the complexity index, whereas it significantly elongated the terminal branches at E6. It also increased the internodal distance significantly at E8. But, these effects were negligible at E10 or later. During E6–8, there appeared to be a dynamic balance in the axon arborization pattern between the “targeting” mode, which is accompanied by elongation of terminal branches and the pruning of collateral branches, and the “pathfinding” mode, which is accompanied by the retraction of terminal branches and the sprouting of new collateral branches. The local and transient activation of caspase 3 could direct the balance towards the pathfinding mode.     

Jelly belly trans‐synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture

In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans‐synaptic signaling. Here, we show that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb–Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild‐type postsynaptic Alk expression in Alk partial loss‐of‐function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb–Alk signaling triggers the Ras‐MAP kinase cascade in both pre‐ and postsynaptic compartments. These novel roles for Jeb–Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Crustacean frequenins: Molecular cloning and differential localization at neuromuscular junctions

Crustacean muscles are innervated by phasic and tonic motor neurons that display differential physiology and have morphologically distinct synaptic terminals. Phasic motor neurons release much more transmitter per impulse and have filiform terminals, whereas tonic motor neurons release less transmitter and have larger terminals with prominent varicosities. Using an antibody raised against Drosophila frequenin (frq), a calcium‐binding protein that enhances transmitter release in Drosophila synaptic terminals, we found that frq‐like immunoreactivity is prominent in many of the phasic, but not tonic nerve endings of crayfish motor neurons. In contrast, synapsin‐ and dynamin‐like immunoreactivities are strongly expressed in both types of terminal. The immunocytochemical findings strongly suggested the presence of an frq‐like molecule in crayfish, and its differential expression indicated a possible modulatory role in transmitter release. Therefore, we cloned the cDNA sequences for the crayfish and lobster homologues of Drosophila frq. Crustacean frequenins are very similar in sequence to their Drosophila counterpart, and calcium‐binding regions (EF hands) are conserved. The widespread occurrence of frq‐like molecules and their differential localization in crayfish motor neurons indicate a significant role in physiology or development of these neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 165–175, 1999     

Molecular Mechanisms Underlying Motor Axon Guidance in Drosophila

Decoding the molecular mechanisms underlying axon guidance is key to precise understanding of how complex neural circuits form during neural development. Although substantial progress has been made over the last three decades in identifying numerous axon guidance molecules and their functional roles, little is known about how these guidance molecules collaborate to steer growth cones to their correct targets. Recent studies in Drosophila point to the importance of the combinatorial action of guidance molecules, and further show that selective fasciculation and defasciculation at specific choice points serve as a fundamental strategy for motor axon guidance. Here, I discuss how attractive and repulsive guidance cues cooperate to ensure the recognition of specific choice points that are inextricably linked to selective fasciculation and defasciculation, and correct pathfinding decision-making.     

The POU-domain protein Pdm3 regulates axonal targeting of R neurons in the Drosophila ellipsoid body

The ability of axons to project correctly to the target is essential for the formation of complex neural networks. The intrinsic regulation of this process is still unclear. Here, we show that POU domain motif 3 (Pdm3) is required in ring (R) neurons to control precise axon targeting to the Drosophila ellipsoid body (EB). Pdm3 is expressed in neurons of the central nervous system in larvae and adults and required for the normal development of the EB of the central complex in the adult brain. The normal EB structure is abolished in pdm3 mutants, and this phenotype is rescued by pdm3 expression in R neurons, suggesting that the defect in axonal targeting of R neurons is the major cause in EB malformation in pdm3 mutants. We show that cell fate determination, dendritic arborization, and initial axon projection of R neurons are normal while the axonal targeting to the EB is defective in pdm3 mutants. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Dbest1, a drosophila homolog of human bestrophin,is not required for viability or photoreceptor integrity

Summary: Best macular dystrophy (BMD) is an autosomal dominant human disease characterized by macular degeneration with juvenile onset (OMIM 153700). The disease is most often associated with mutations in Bestrophin, which encodes a novel protein with four putative transmembrane domains. However, complete loss‐of‐function mutations in Bestrophin have not been reported in humans or mice. We have identified three homologs of human Bestrophin in the Drosophila genome (dbest1‐3). The protein products of these three genes share significant homology to a 364 amino acid N‐terminal domain of human Bestrophin. We used P‐element mutagenesis to delete dbest1, which encodes a protein with the highest amino acid similarity to Bestrophin. Three independent dbest1 mutants were recovered from the mutagenesis screen. Homozygous null mutations in dbest1 do not significantly alter the viability or fertility of mutant flies. Moreover, dbest1 mutants have normal photoreceptor morphology and function. genesis 31:130–136, 2001. © 2001 Wiley‐Liss, Inc.     

Dissociation of rugose‐dependent short‐term memory component from memory consolidation in Drosophila

Extensive investigations show several molecular and neuroanatomical mechanisms underlying short‐lived and long‐lasting memory in Drosophila. At the molecular level, the genetic pathway of memory formation, which was obtained through mutant research, seems to occur sequentially. So far, studies of Drosophila mutants appear to support the idea that mutants defective in short‐term memory (STM) are always associated with long‐term memory (LTM) impairment. At the neuroanatomical level, distinct memory traces are partially independently distributed. However, whether memory phase dissociation also exists at the molecular level remains unclear. Here, we report on molecular separation of STM and consolidated memory through genetic dissection of rugose mutants. Mutants in the rugose gene, which encodes an evolutionarily conserved A‐kinase anchor protein, show immediate memory defects as assayed through aversive olfactory conditioning. Intriguingly, two well‐defined consolidated memory components, anesthesia‐resistant memory and protein synthesis‐dependent LTM, are both normal in spite of the defective immediate memory after 10‐session massed and spaced training. Moreover, rugose genetically interacts with cyclic AMP‐protein kinase A signaling during STM formation. Considering our previous study that AKAP Yu specifically participates in LTM formation, these results suggest that there exists a molecular level of memory phase dissociation with distinct AKAPs in Drosophila.     

Wild type and mutants of the HET‐s(218–289) prion show different flexibility at fibrillar ends: A simulation study

The C‐terminal segment (residues 218–289) of the HET‐s protein of the filamentous fungus Podosporina anserina is a prion‐forming domain. The structural model of the HET‐s(218–289) amyloid fibril based on solid‐state nuclear magnetic resonance (NMR) restraints shows a β solenoid topology which is comprised of a β‐sheet core and interconnecting loops. For the single‐point mutants Phe286Ala and Trp287Ala, slower aggregation rates in vitro and loss of prionic infectivity have been reported recently. Here we have used molecular dynamics to compare the flexibility of the mutants and wild type. The simulations, initiated from a trimeric aggregate extracted from the NMR structural model, show structural stability on a 100‐ns time scale for wild type and mutants. Analysis of the fluctuations along the simulations reveals that the mutants are less flexible than the wild type in the C‐terminal segment at only one of the two external monomers. Analysis of interaction energy and buried accessible surface indicates that residue Phe286 in particular is stabilized in the Trp287Ala mutant. The simulation results provide an atomistic explanation of the suggestion (based on indirect experimental evidence) that flexibility at the protofibril end(s) is required for fibril elongation. Moreover, they provide further evidence that the growth of the HET‐s amyloid fibril is directional. Proteins 2014; 82:399–404. © 2013 Wiley Periodicals, Inc.