22 Papers found (0.01 seconds)

Hannes E Buelow et al. (2005) International Worm Meeting "Complex heparan sulfate modifications instructively affect axon guidance choices"

Hannes E Buelow, Dominic Didiano, Oliver Hobert

[

International Worm Meeting,

2005]

Heparan sulfate proteoglycans (HSPG) are components of the extracellular matrix through which axons navigate to reach their target cells. The heparan sulfate (HS) side chains of HSPGs show complex and differentially regulated patterns of secondary modifications, including sulfations of distinct hydroxyl groups and epimerization of an asymmetric carbon atom. These modifications endow the HSPG-containing extracellular matrix with the potential to code for an enormous molecular diversity (1). We sought to decode this potential diversity by analyzing mutations in three C.elegans enzymes that catalyze secondary modifications in HS, namely the glucuronyl C5 epimerase, the heparan 6O-sulfotransferase and 2O-sulfotransferase. Each of the mutants are viable yet exhibit distinct as well as overlapping axonal and cellular guidance defects in specific subsets of neuron classes, demonstrating that different modifications are crucial for distinct aspects of neuronal development. We have linked individual HS modifications to two specific guidance systems, the Slit/Robo and kal-1/Anosmin-1 system. We show that the activity of both guidance systems is dependent on different HS modifications in different cellular contexts (2). To distinguish whether combinations of HS modifications have a permissive or instructive role in axon guidance choices we misexpressed specific HS modifying enzymes. These experiments establish that specific misexpression of HS modifying enzymes (presumably leading to aberrant HS modification patterns) elicit defined axonal defects in some neurons but not in others indicating that specifically modified HS has the potential to play an instructive role during neuronal development. Using genetic and molecular approaches we have begun to identify the genes that define this instructive role of HS and that respond to it. Our results demonstrate that the molecular diversity in HS encodes information leading us to propose the existence of a heparan sulfate code that instructively controls different aspects of nervous system development. (1) Lee, J.S., and Chien, C.B. (2004). When sugars guide axons: insights from heparan sulphate proteoglycan mutants. Nat. Rev. Genet. 5, 923-935. (2) Blow, H.E., and Hobert, O. ( 2004). Differential sulfations and epimerization define heparan sulfate specificity in nervous system development. Neuron 41, 723-736.

Hannes E Buelow et al. (2001) International C. elegans Meeting "Genetic Analysis of the Kallmann Syndrome Protein Ortholog Cekal-1 in C. elegans"

Hannes E Buelow, Oliver Hobert, Katherine L Berry

[

International C. elegans Meeting,

2001]

Kallmann Syndrome is a hereditary disease that exists in both X-linked and autosomal forms and is characterized by anosmia (inability to smell) and infertility. Additionally, affected patients display kidney malformations or even lack one kidney. Detailed analyses of the anosmic phenotype showed a specific defect in olfactory axons that fail to make connections with their interneuronal targets while their pathfinding seems unaffected. For the X-linked form a gene called Kal-1 was identified (1,2) that codes for a secreted protein containing FNIII modules and a protease inhibitor domain. However, the genetic lesions responsible for the autosomal forms, that could reside e.g. in a putative receptor gene, remain elusive. We identified Cekal-1 , an ortholog of the human Kallmann Syndrome gene Kal-1 , in C. elegans . CeKAL-1 shows a high structural homology to human Kal-1 and is expressed in subsets of neurons in C. elegans as well as in non-neuronal tissues e.g. the excretory cell. To identify loci that genetically interact with Cekal-1 we employed a two step approach. We first generated a gain-of-function ( gf ) phenotype by over/misexpressing the Cekal-1 cDNA and then searched for mutations that modified this phenotype by both testing candidate mutations and performing a genetic screen. When we overexpressed Cekal-1 in AIY, a pair of interneurons, in a strain carrying a GFP reporter that labeled these neurons, 100% of the animals displayed ectopic neurites originating from the cell body and/or the axons of AIY. We tested whether these neurites were specific for Cekal-1 by expressing a truncated form of CeKAL-1 in AIY. This, as well as expression of related proteins had no effect in AIY interneurons suggesting the phenotype in AIY to be specific for expression of CeKAL-1. Mis/overexpression using a pan-neuronal promoter also leads to visible neuroanatomical defects in many but not all neurons (see poster by Berry et al. ). The AIY specific neurite outgrowth defect could not be modified by mutations in genes required for axonal pathfinding including unc-6 , unc-5 , unc-40 , unc-73 , sax-3 , vab-1 , and ina-1 indicating that these genes do not have a role in the generation of the observed neurites. However, some exc genes that had previously been identified in a screen for animals with defects in the excretory canal (3) efficiently suppressed our gf phenotype. This is insofar of significance as the excretory canal is believed to be the functional equivalent of the mammalian kidney. Thus, the biological function of the Kallmann Syndrome protein in neuronal guidance as well as development of kidney-like structures could be conserved throughout evolution To identify additional interacting loci we performed a suppressor/modifier screen of the gf phenotype. In a small screen we identified at least 6 mutants ( ot16-ot20, ot24 ) in 4 complementation groups that efficiently suppressed the sprouting phenotype to various degrees between 25-80%. Interestingly we also found one mutant ( ot21 ) that qualitatively enhanced the axonal sprouting phenotype. We assessed the specificity of the modifier mutations by constructing double mutants of these mutations with ttx-3(ks5 ) or sax-2(ot10) , both mutants that lead to neurite outgrowth phenotypes in AIY or sensory neurons. Our modifier mutations did not modify these phenotypes suggesting specific modification of the Cekal-1 dependent neurites. We have started to characterize the isolated modifier mutations molecularly. Using a combination of 3-factor mapping, deficiencies and single nucleotide polymorphisms we were able to map two of the strong suppressor mutations ot16III and ot17X to a small intervals. ot16III maps to a six cosmid interval between unc-79 and emb-5 on LGIII and ot17X to seven cosmids between lon-2 and unc-97 on LGX. We are currently attempting tranformation rescue by injecting cosmids of the respective regions. 1. Franco B, Guiloli S, Pragliola A, et al. (1991) Nature , 353:529-536 2. Legouis R, Hardelin JP, et al. (1991) Cell , 67:423-435 3. Buechner et al. (1999) Dev. Biol. 214:227-241

Hannes E Buelow et al. (2005) International Worm Meeting "HOX genes define position-specific projection patterns of ventral nerve cord motor neurons"

Oliver Hobert, Hannes E Buelow, Nartono Tjoe

[

International Worm Meeting,

2005]

The developing nervous system of all animals consists of a multitude of neuronal cell types with characteristic position-specific projection and innervation patterns. In the developing spinal cord of vertebrates recent work has shown that homeodomain-containing transcription factors of the Hox-c class are sequentially expressed in overlapping domains along the anterior-posterior (ap) axis and thereby determine distinct neuronal cell fates (1). However, little is known about how neuronal fate and cell position is translated into the specific neuronal trajectories that are characteristic for their respective position along the ap axis. The cluster of Hox genes in C. elegans consists of at least four genes ceh-13, lin-39, mab-5, and egl-5 which have been studied widely for their role in different cell fate decisions along the ap axis (2). Furthermore, Hox genes in C.elegans have been shown to define the migratory behavior of neuroblasts along the ap axis, yet few if any axonal phenotypes have been reported for these mutants (3). We show here that in C. elegans mutations in Hox genes lead to homeotic transformations in neuronal cell fates of motor neurons in the ventral nerve cord where specific neurons at specific positions along the ap axis adopt fates of adjacent neurons. These transformations are evidenced by changes in axonal projection patterns of motor neurons along the ap axis and will allow to dissect how cell fate/position is linked to axonal projection patterns. We are currently working on two basic questions relating to the observed axonal phenotypes. Specifically we are asking whether (a) Hox genes act cell-autonomously in neurons to determine position-specific axonal projection patterns and (b) which guidance factors may be under (direct or indirect control) of the Hox genes to determine the axonal trajectories. We are using a combination of expression analyses and cell-specific rescue experiments to address the first question. To answer the second question we are employing a candidate gene approach to identify guidance cues which may be responsible for specific axonal projection patterns. For these experiments we are using both gfp reporters for various guidance cues to determine whether these reporters are under control of Hox genes as well as double mutant analyses. (1) Dasen JS et al., Nature. 2003;425: 926-33. (2) Wang BB et al., Cell. 1993;74(1): 29-42.; Clark, S. et al. Cell. 1993;74(1)43-55. Costa M, et al. Cell. 1988; 55(5):747-56.; Ferreira HB et al., Dev. Biol. 1999; 207: 215-228; Brunschwig K et al., Development. 1999;126(7): 1537-46. (3) Desai C et al. Nature. 1988; 336:638-46.

Tecle, Eillen et al. (2009) International Worm Meeting "The Role of 3-O Sulfation of Heparan Sulfate in Neuronal Development in C. elegans. ."

Buelow, Hannes, Tecle, Eillen

[

International Worm Meeting,

2009]

Our lab is interested in how Heparan Sulfate (HS) modifications regulate neuronal connectivity and patterning in C. elegans. HS is a highly modified un-branched glycosaminoglycan exhibiting substantial molecular diversity due to multiple modifications such as sulfations, epimerization and acetylation. HS modifications have been documented to have specific and instructive roles (Bulow and Hobert, 2004; Bulow et al., 2008) in neuronal development leading to the hypothesis of a HS code that regulates nervous system patterning. However, the role of the 3-O sulfation modification of HS, introduced by heparan sulfotransferase 3 enzymes (HST-3s), has not been established. Vertebrate genomes code for at least seven members of the HST-3 gene family that are grouped into two distinct classes. A subset of the vertebrate HST-3s display temporarily and spatially restricted expression patterns in the developing and post-natal brain, however, very little is known about the in vivo function of these enzymes in neuronal development due to functional redundancy. We have identified one gene coding for a predicted HST-3 of each class in the C. elegans genome: hst-3.1 and hst-3.2. Analysis of neuronal patterning in null mutants of hst-3.1 and hst-3.2 indicates that both genes are required for synaptic branch formation in a subset of C. elegans neurons. In addition, hst-3.2 may play a role in axon termination. These phenotypes are reminiscent of mutations in genes regulating synaptic maturation and/or function. Therefore, on going experiments are focused on determining if loss of hst-3.2 and/or hst-3.1 results in synaptic disorganization and if either gene acts in known synaptic pathways. We have recently elucidated that several other null mutants of HS modification enzymes and HS core proteins share the phenotypes that we identified in null mutants of hst-3.1 and hst-3.2. We are investigating the interaction of HS modifications and core proteins, via double and triple mutant analysis, in various cellular contexts. Furthermore, we are determining the cellular focus of action of the HS 3O-sulfotransferases. This analysis will provide invaluable insight into possible mechanisms by which the HS code regulates neuronal development and patterning.

Attreed, Matthew et al. (2011) International Worm Meeting "Direct visualization and functional modulation suggest a role for specific heparan sulfate motifs in cholinergic synaptic transmission in C. elegans."

Attreed, Matthew, Buelow, Hannes

[

International Worm Meeting,

2011]

Heparan sulfates (HS) are unbranched, variably modified polysaccharide chains in the extracellular space that are invariably attached to proteins. The modification process of the sugars results in HS chains harboring different motifs of specifically arranged modifications. We and others have shown that individual HS modifications are crucial for various aspects of neural development in metazoans. Yet, little is known about the dynamics and function of defined HS motifs in the nervous system. In order to address this question experimentally, we have developed a tool that allows for live imaging of HS sugar motifs. By transgenically expressing secreted single chain variable fragment (scFv) antibody::GFP fusions, which recognize different HS motifs, we can visualize for the first time HS sugars with distinct modification patterns in living animals. We detect a highly refined distribution of HS motifs in both neuronal and non-neuronal tissues that often exhibit dynamic changes during development. Specifically, we find different HS motifs associated with the nervous system from late embryonic stages into adulthood. Intriguingly, our data suggest the existence of neuron-specific HS motifs. Genetic experiments and colocalization studies show that some HS motifs are associated with synaptic markers in the nerve cords. The possibility of functional neutralization of the HS motifs recognized by different scFv antibodies prompted us to study the nervous system of the transgenic animals in more detail. We find that the gross neuroanatomy in transgenic animals expressing different scFv antibody constructs is indistinguishable from wild type animals. However, based on aldicarb and levamisole assays, we detect defects in cholinergic synaptic transmission in animals expressing certain antibodies, possibly as a result of functional neutralization by the antibody fusions. These findings suggest that specific HS motifs serve a role in cholinergic transmission. In conclusion, we have developed a novel tool for the visualization and modulation of HS sugar motifs in vivo. Our experiments indicate that HS motifs are expressed dynamically and, possibly, in a neuron-specific manner. Moreover, distinct HS motifs may serve a role in modulating synaptic transmission.

Desbois, Muriel et al. (2013) International Worm Meeting "A non-trapping method for live imaging of specific neuronal connections."

Desbois, Muriel, Buelow, Hannes

[

International Worm Meeting,

2013]

The nervous system is a complex network that senses and processes information and is essential for the survival of both vertebrates and invertebrates. Information within the network is transmitted through specialized cell-cell contacts, including synaptic connections and GAP junctions. Importantly, the network is not static and is believed to change during development, learning but also during pathological or normal age-related decline. Previous strategies to label specific synapses in living animals 'trap' the synapses by fixing the connections, thus precluding dynamic studies. To circumvent this problem, we are adapting a technique called BLINC (Biotin Labeling of INtercellular Contacts) for live imaging of specific synapses in C. elegans. BLINC is based on the biotinylation of an acceptor peptide by the E. Coli biotin ligase BirA, both are fused to two interacting proteins. In analogy to work in cell culture, we fused the BirA ligase to neurexin and expressed the fusion in a set of neurons. We then fused the acceptor peptide to neuroligin and expressed this construct in another group of neurons, known to form connections with the former set of neurons. During the formation of synapses/connections between the two groups of neurons, the neuroligin and the neurexin form a complex, leading the ligase to transfer a biotin to the acceptor peptide. This "biotin mark" is specifically detected by a monovalent streptavidin fused to a fluorescent protein which is transgenically secreted from the coelomocytes. In preliminary findings we observe specific staining in living animals consistent with known connections between both sets of neurons. We will report on our progress, but expect that this new technique will allow to visualize formation and dynamics of specific neuronal connections in vivo under different experimental conditions.

Tecle, Eillen et al. (2011) International Worm Meeting "The Role of 3-O Sulfation of Heparan Sulfate in Neuronal Development in C. elegans."

Buelow, Hannes, Tecle, Eillen

[

International Worm Meeting,

2011]

Our lab is interested in how Heparan Sulfate (HS) modifications regulate neuronal connectivity and patterning in C. elegans. HS is a highly modified un-branched glycosaminoglycan exhibiting substantial molecular diversity of modifications, including sulfation, epimerization and acetylation. HS modifications have been documented to have specific and instructive roles (Bulow and Hobert, 2004; Bulow et al., 2008) in neuronal development leading to the hypothesis of a HS code that regulates the patterning of the nervous system. However, the role of HS 3-O sulfation, the most rare HS modification has not been established. Vertebrate genomes code for at least seven members of the HS 3O-sulfotranferase gene family and are grouped into two distinct classes. We have identified one gene coding for a predicted HS 3O-sulfotranferase of each class in the C. elegans genome which we have named hst-3.1 and hst-3.2, respectively. Reporter analyses of hst-3.1 and hst-3.2 reveal discrete, largely complementary expression patterns. The hst-3.2 reporter is primarily expressed in ectodermal tissues (hypodermis and neurons) whereas the hst-3.1 reporter shows expression in body wall muscle and a few select neurons. Defects in synaptic branch formation and axon termination in select neurons are observed in hst-3.2 mutants; indicating that hst-3.2 is required for the proper development of subset of neurons. In addition, perturbations in behaviors regulated by the affected neurons are observed in the hst-3.2 mutants. Some of the phenotypes identified in the hst-3.2 mutants are shared by mutations in rpm-1, a conserved regulator of pre-synaptic maturation (Schaefer et al., 2000; Zhen et al., 2000). Double and triple mutant analyses indicate that rpm-1 and hst-3.2 act in parallel genetic pathways. Our data suggests that HS 3-O sulfation, introduced by hst-3.2, may be a crucial determinant of and/or be part of a novel pathway in the establishment of neuronal connectivity.

Townley, Robert et al. (2009) International Worm Meeting "Genetic analysis of the complete heparan sulfate modification network."

Townley, Robert, Buelow, Hannes

[

International Worm Meeting,

2009]

Heparan sulfates (HS) are unbranched polysaccharides and a defining component of the extracellular matrix in metazoans. HS functions to regulate cellular communication through HS motifs that are composed of blocks of modified saccharide subunits. These motifs are generated by a set of conserved enzymes that catalyze specific sulfations and epimerizations. The molecular and cellular mechanisms that govern the synthesis of HS motifs in vivo are only poorly understood. In order to gain insight into this important process, we have undertaken an integrated genetic and biochemical analysis of the complete set of known modifying enzymes. We have measured the disaccharide composition of heparan sulfate purified from worms with defined deletions or point mutations in one or more Hs modifying enzymes. From these measurements we have developed a comprehensive model of heparan sulfate synthesis that contains novel quantitative and qualitative features. We find that the overall sulfate content of the heparan polymer is initially determined by the amount of sulfate-donor (PAPS) that is available in the lumen of the Golgi. PAPS is transported into the Golgi by a selective transporter (pst-1). The phenotype of pst-1 mutants can account for the puzzling observation made previously that removal of 2-O or 6-O sulfation alone has little effect on overall sulfation. Additionally, our data define the order in which the modifications occur. Most published models and discussions place 2-O sulfation before 6-O, however, our data indicate 6-O and 2-O sulfation can occur concomitantly and/or 6-O can precede 2-O sulfation. Finally, measurements of disaccharide composition in the 6-O endosulfatase (sul-1) mutant that selectively removes individual sulfations, quantify the extent to which the sulfation motifs initially established are dynamic and reversible. Taken together our results establish for the first time for any organism the genetic ground state of the complete heparan modification network.

Celestrin, Kevin et al. (2013) International Worm Meeting "A role for muscle-skin interactions in shaping PVD sensory dendrites."

Buelow, Hannes, Kaprielian, Zaven, Celestrin, Kevin

[

International Worm Meeting,

2013]

Complex dendritic arbors facilitate the reception and transduction/integration of a wide variety of environmental stimuli, such as temperature, touch, and pain. Similarly, the complex dendritic arbors of cortical neurons in the central nervous system allow the cortex to function as the center for reasoning, learning and memory. The loss of these complex dendritic arbors represents a hallmark of psychiatric disorders (e.g. schizophrenia) and neurodegenerative diseases (e.g. Alzheimer's). A wide variety of genes have been implicated in the regulation of dendrite growth including transcription factors, regulators of intracellular transport, and modulators of Golgi-ER and endosomal dynamics. However, the detailed molecular mechanisms and genetic pathways that regulate dendrite branching and dendritic arbor formation remain poorly understood. Specifically, the mechanism of how extracellular factors regulate dendrite development. It has recently been shown that integrins, receptors for ECM proteins, are required for proper dendrite branching in the Drosophila nervous system supporting a significant role for coordinated tissue interaction in dendritic development. In preliminary studies utilizing the highly branched mechanosensory neuron PVD in Caenorhabditis elegans, We have identified a role for several genes, such as integrin, PINCH (an adaptor protein that interacts with integrin) and perlecan in the organization of PVD. These genes are part of a complex that coordinates tissue interactions between the muscle and skin suggesting that these may play a role in shaping dendrite arbors. We will report on our progress to elucidate how muscle-skin interactions coordinate PVD dendritic arbor formation.

Aguirre-Chen, Cristina et al. (2009) International Worm Meeting "Molecular Mechanisms that Control Dendritic Branch Formation in C.elegans."

Aguirre-Chen, Cristina, Kaprielian, Zaven, Buelow, Hannes E.

[

International Worm Meeting,

2009]

The establishment of proper dendritic arborization patterns is a key phase of neuronal development that is required for the proper functioning of the nervous system. However, the molecular mechanisms that control dendritic branch formation are poorly understood across species. The C.elegans PVD neurons are a pair of putative mechanosensory neurons that respond to harsh mechanical stimuli. In both midlarval- and adult-staged animals, the PVD neurons exhibit an elaborate branching pattern. Aside from the finding that mec-3, a LIM homeodomain-containing gene, is required for the elaboration of PVD-associated arbors, the mechanisms that regulate branch formation in these neurons remain unclear. To identify novel regulators of PVD branching, both a small-scale, candidate-based (140 genes) and a large-scale RNAi screen of the approximately 3000 genes on Chromosome IV were carried out in a PVD reporter background. In total, twelve genes were retrieved that either promote or suppress the formation of PVD branches. The focus of our current studies is the bicd-1 gene, whose RNAi phenotype indicates a role for it in suppressing PVD branch formation and whose homologs have been shown to play a crucial role in the localization of mRNAs and organelles via its interactions with dynein. Preliminary characterization of the bicd-1 deletion allele, ok2731, confirms that this mutant phenocopies the enhanced branching phenotype exhibited by bicd-1 RNAi-treated animals. Additionally, a bicd-1 transcriptional reporter line that we recently generated displays robust and rather specific labeling of both the PVD and FLP neurons, the two most highly branched neurons in the C. elegans nervous system, as well as the H-shaped excretory cell. Currently, we are carrying out cell type-specific rescue experiments to determine the site of action of bicd-1 and double mutant analyses to identify genes that genetically interact with bicd-1.

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