Harald Hutter (2002) European Worm Meeting "Axon guidance in the ventral cord of C. elegans"

Harald Hutter

[European Worm Meeting, 2002]

The ventral cord is the major longitudinal axon tract in C. elegans containing essential components of the motor circuit. It consists of two bundles running on either side of the ventral midline. Most axons enter the ventral cord after exiting the nerve ring in the head of the animal. Essentially all axons cross over to the right side near the retrovesicular ganglion leading to a highly asymmetrical ventral cord. Motorneuron axons originating from cell bodies at the ventral midline also exclusively grow in the right axon tract. The arrangement of processes within the cord is highly invariant implying that there is a precise navigational system guiding axons not just to the cord but also within the cord. The order of outgrowth of axons in the ventral cord during embryonic development has been studied by Richard Durbin1, who also initiated a first analysis on the pioneering function of certain early outgrowing axons. A number of genes are now known to affect guidance of axons in the ventral cord. The importance of particular guidance cues and pioneers for the navigation of individual classes of axons in the ventral cord can now be studied more systematically, since it is possible to visualize all major groups of ventral cord axons at the light microscopic level with GFP markers. To analyse pioneer-follower relationships several strains were made which express color variants of GFP in different groups of axons. The importance of pioneers like AVG was tested in laser ablation experiments. It was found that interneurons (glr-1 expressing) and D-type motorneurons are highly dependant on the pioneer AVG, whereas DA/DB motorneurons are almost not affected in the absence of AVG. Navigational mistakes are made individually. Errors of early outgrowing axons (DD) typically do not lead to mistakes of later outgrowing axons. Order of outgrowth apparently does not always reflect a pioneer-follower relationship. Similar observations were made in the absence of guidance cues like UNC-6. AVG and UNC-6 appear to be the main sources of navigational information for D-type motorneuron axons, but not so much for DA/DB motorneuron or interneuron axons indicating that different classes of neurons with axons in the ventral cord use different navigational cues. This analysis is currently extended to include other guidance cues and to test for redundancy among early outgrowing axons for the guidance of later outgrowing ones.

H Hutter et al. (1999) International C. elegans Meeting "Genes required for axonal fasciculation/target recognition"

EM Hedgecock, H Hutter

[International C. elegans Meeting, 1999]

One of the central problems in developmental neurobiology is the question of how the growing axons of neurons find their way through the complex environment of the developing embryo to their target areas, where synaptic partners are choosen. Since synapses in C. elegans are made en passant between neighbouring axons in an axon bundle the correct fasciculation of axons even within a bundle is crucial for the correct wiring of the nervous system. To study the molecular basis of this axon-axon recognition mechanism, we have begun screens for mutants with fasciculation defects in a defined subset of axons, the interneuron axons in the ventral cord (visualized in a strain expressing a glr-1::GFP marker). Thus far, we have recovered mutants with defects ranging from rather minor defasciculations over short distances to a severe disorganization of the ventral cord that extends to other classes of neurons as well. The interneuron axons affected in the mutants are part of the motor circuit, yet not all of the mutants show obvious movement defects as might be expected from a disruption of the motor circuit. This indicates a certain amount of redundancy in the motor circuit. Four of the mutants defining four different genes are being studied in more detail. rh299 and rh300 both show strong fasciculation defects of interneuron axons and no obvious defects in the outgrowth of other axons as judged by various GFP markers. These mutants might define genes specifically involved in the target recognition of the interneurons of the motorcircuit. In rh309 and rh315 mutants interneuron axons not only are defasciculated in the ventral cord, they sometimes don't reach the cord at all and extend at abnormal lateral positions. Many other axons are also affected and show variable outgrowth defects. Mutant animals are Unc. These mutants define genes that are more generally required for axonal outgrowth. Based on their map position and phenotype these mutants seem to define previously unidentified genes: rh299 is close to sem-4 on chromosome I, rh300 is between dpy-10 and unc-104 on II, rh309 is not far to the right of unc-68 on IV and rh315 is near daf-4 on III.

Harald Hutter et al. (2005) International Worm Meeting "The astacin family in C. elegans: evolution, expression and function"

Robert Johnsen, Marcus Schupp, Frank Mohrlen, Harald Hutter, Allan Mah, Donald Moerman

[International Worm Meeting, 2005]

Astacins are an evolutionary conserved family of secreted metalloproteases found in all animals. Members of this family are implicated in such different processes as digestion of food or pattern formation during embryonic development. C. elegans has the largest number of astacin genes among animals (39 genes). A comparison with C. briggsae indicates that most members of this family originated before these two species were separated in evolution, since only four astacins are unique to C. elegans and only one is unique to C. briggsae. Only three members of this gene family have been functionally characterized and these are involved in either hatching (hch-1), molting (nas-37) or cuticular collagen processing (dpy-31/toh-2). In order to get an idea of the functional diversity of this gene family in C. elegans we analysed the expression pattern of all astacins with promoter-GFP constructs and SAGE analysis (see http://elegans.bcgsc.bc.ca/ for link to this data). One third of the astacins are not represented in any of the developmental or tissue specific SAGE libraries posted to date, and for one quarter we did not observe GFP expression in the reporter strain. However, combining SAGE and GFP data we obtained tissue specfic expression information for all but three astacins, indicating that the two approaches can be complementary. With only a few exceptions, astacins tags in SAGE libraries were not abundant. GFP expression levels reflected this low message abundance for many family members. Several astacins not represented in SAGE libraries show clear GFP expression limited to a few cells or to certain stages in development indicating that there is a detection limit for SAGE when the abundance of a message is either very low or limited to a few cells. The digestive tract (pharynx, gut) and the hypodermis are tissues expressing a variety of different astacins, suggesting that a large fraction of the astacins might play a role in food digestion and molting/cuticular collagen processing. Only a few members are expressed in body wall muscle or the nervous system. To initiate a functional analysis we selected certain members for targeted knock-out. So far we have isolated mutants in nas-4, nas-5 and nas-39 (tolloid homolog), which are viable and show no obvious developmental defects.

Hutter H et al. (1998) European Worm Meeting "Genes required for axonal fasciculation/target recognition"

Hutter H, Hedgecock EM

[European Worm Meeting, 1998]

One of the central problems in developmental neurobiology is the question of how the growing axons of neurons find their way through the complex environment of the developing embryo to their target areas. The final stages of axonal guidance involve the selection of synaptic partners within the target area. Since synapses in C. elegans are made en passant between neighbouring axons in an axon bundle the correct fasciculation of axons even within a bundle is crucial for the correct wiring of the nervous system. To study the molecular basis of this axon-axon recognition mechanism, we have begun screens for mutants with fasciculation defects in a defined subset of axons, the interneuron axons in the ventral cord. These axons can be visualized in strains expressing a glr-1::GFP marker, and form a tightly fasciculated subbundle within the right ventral cord. For the screens, F2 progeny of mutagenized animals are scored under the microscope for fasciculation defects and mutant animals are recovered from the slides. Thus far, we have recovered mutants with defects ranging from rather minor defasciculations over short distances to a severe disorganization of the ventral cord that extends to other classes of neurons as well. The interneuron axons affected in the mutants are part of the motor circuit, yet not all of the mutants show obvious movement defects as might be expected from a disruption of the motor circuit. This indicates a certain amount of redundancy in the motor circuit and also shows that direct screens for axonal outgrowth defects are very sensitive allowing us to obtain mutants with minor defects that do not lead to gross behavioral defects. Four of the mutants defining four different genes are being studied in more detail. rh299 and rh300 both show strong fasciculation defects of interneuron axons and no obvious defects in the outgrowth of other axons as judged by various GFP markers. These mutants might define genes specifically involved in the target recognition of the interneurons of the motorcircuit. In rh309 and rh315 mutants interneuron axons not only are defasciculated in the ventral cord, they sometimes don't reach the cord at all and extend at abnormal lateral positions. Many other axons are also affected and show variable outgrowth defects. Mutant animals are Unc. These mutants define genes that are more generally required for axonal outgrowth. Based on their map position and phenotype these mutants seem to define previously unidentified genes: rh299 is close to sem-4 on chromosome I, rh300 is between dpy-10 and unc-104 on II, rh309 is not far to the right of unc-68 on IV and rh315 is near daf-4 on III. Further mapping and transformation rescue expriments to clone the genes are in progress...

Hutter H et al. (1996) West Coast Worm Meeting "SCREENING FOR MUTANTS WITH FASCICULATION DEFECTS IN C. ELEGANS"

Hutter H, Hedgecock EM

[West Coast Worm Meeting, 1996]

The selective fasciculation of axons within an axon bundle is important for the correct wiring of the nervous system. Markers that allow the visualization of neighboring axons are essential for studying selective fasciculation. Especially useful are GFP-derived markers because they would allow visualization of the dynamics of axonal outgrowth and fasciculation in the living animal. Fortunately many promoters giving expression in the nervous system are now available. Currently we are determining the usefulness of various GFP-constructs for fasciculation studies. As criteria, the level of expression should be high enough so that axons are visible over their entire length and the pattern of expression should contain few neighboring axons. Ideally expression should start at early stages and continue through the life cycle. Once a suitable marker (or a combination of markers) has been found, we are going to screen for mutants with fasciculation defects. Since such defects do not necessarily lead to a behavioral defect and since some mutants with fasciculation defects are lethal (e.g. FasII in Drosophila) we intend to screen without any bias towards a certain phenotype beyond the visible defect in fasciculation as revealed by the marker we are using. This unbiased screen should identify new genes involved in the control of the selective fasciculation of axons.

H Hutter et al. (2004) European Worm Meeting "PIONEER-FOLLOWER RELATIONSHIPS IN THE VENTRAL CORD OF C. ELEGANS"

H Hutter, I Wacker

[European Worm Meeting, 2004]

The ventral cord is the major longitudinal axon tract in C. elegans containing essential components of the motor circuit. The AVG neuron has been identified as the pioneer sending a first axon into the right axon tract, which eventually contains all but four of the axons running in the ventral cord (R. Durbin, Thesis, Cambridge 1987). Laser ablation experiments revealed that the AVG axon is not responsible for setting up the asymmetry of the ventral cord, which still develops in the absence of AVG. The pioneer is not even essential for the correct outgrowth of follower interneurons and motoneurons, since axonal defects are found in less than half of the animals where AVG was ablated. Detailed analysis of dependencies between early and late outgrowing axons revealed that strict pioneer-followers relationships are rare - the only example found in the ventral cord was between PVP and PVQ - the PVQ axons always following the PVP axons. In genetic screens we identified a number of mutants affecting pioneer-follower relationship or ventral cord asymmetry. Ventral cord defects in lin-11 mutants resemble defects seen after AVG laser ablation. lin-11, encoding a LIM-homeobox transcription factor apparently affects differentiation of the AVG neuron, since AVG specific markers are no longer expressed in lin-11 mutants. In rh308 mutants the PVQ axons no longer follow the PVP axons, indicating that this pioneer-follower relationship is disturbed. In rh311 and hd11 mutants we observe a partial breakdown of the ventral cord asymmetry, a phenotype also observed in nid-1 mutants. Further characterisation of these genes will tell us more about the molecular nature of the cues controlling pioneer-follower relationship and ventral cord asymmetry in C. elegans.

Hutter H et al. (1998) East Coast Worm Meeting "Genes required for axonal fasciculation/target recognition"

Hutter H, Hedgecock EM

[East Coast Worm Meeting, 1998]

One of the central problems in developmental neurobiology is the question of how the growing axons of neurons find their way through the complex environment of the developing embryo to their target areas. The final stages of axonal guidance involve the selection of synaptic partners within the target area. Since synapses in C. elegans are made en passant between neighbouring axons in an axon bundle the correct fasciculation of axons even within a bundle is crucial for the correct wiring of the nervous system and it has been proposed that the sorting of axons within a bundle is a major determinant of the synaptic connectivity. To study the molecular basis of this axon-axon recognition mechanism, we have begun screens for mutants with fasciculation defects in a defined subset of axons, the interneuron axons in the ventral cord, which can be visualized in strains expressing a glr-1::GFP marker. These axons form a tightly fasciculated subbundle within the right ventral cord. For the screens, F2 progeny of mutagenized animals are scored under the microscope for fasciculation defects and mutant animals are recovered from the slides. Thus far, we have recovered mutants with defects ranging from rather minor defasciculations over short distances to a severe disorganisation of the ventral cord that extends to other classes of neurons as well. The interneuron axons affected in the mutants are part of the motor circuit, yet not all of the mutants show obvious movement defects as might be expected from a disruption of the motor circuit. This indicates a certain amount of redundancy in the motor circuit and also shows that direct screens for axonal outgrowth defects are very sensitive allowing us to obtain mutants with minor defects that do not lead to gross behavioral defects. Four of the mutants defining four different genes are currently studied in more detail. rh299 and rh300 both show strong fasciculation defects of interneuron axons and no obvious defects in the outgrowth of other axons as judged by various GFP markers. These mutants might define genes specifically involved in the target recognition of the interneurons of the motorcircuit. In rh309 and rh315 mutant animals interneurons axons not only are defasciculated in the ventral cord, they sometimes don't reach the cord in the first place and extend at abnormal lateral positions. Many other axons are also affected and show variable outgrowth defects and mutant animals are penetrant Unc. These mutants define genes that are more generally required in axonal outgrowth. Based on their map position and phenotype these mutants seem to define previously unidentified genes: rh299 is close to sem-4 on chromosome I, rh300 is between dpy-10 and unc-104 on II, rh309 is not far to the right of unc-68 on IV and rh315 is near daf-4 on III. Further mapping and transformation rescue expriments to clone the genes are in progress...

Hutter, Harald et al. (2009) International Worm Meeting "GExplore: Multi-gene data mining for worm geneticists."

Chen, Nansheng, Hutter, Harald, Ng, Man-Ping

[International Worm Meeting, 2009]

The C. elegans genome contains some 20,000 genes, the majority of which has not been functionally characterized. Numerous genome wide data sets providing hints for gene function exist, yet mining those data to retrieve potential candidate genes for a particular function remains a challenge. Data most relevant for gene/protein function are - phenotype and expression (if known) - homology to other proteins (which might have been characterized already) - protein domains, which are frequently diagnostic for a biochemical function GExplore provides a simple database interface for worm biologists/geneticists for data mining at the gene/protein function level to help in experimental planning. It combines data downloaded from Wormbase with selected data sets related to gene expression or function, like SAGE data sets containing stage and tissue specific expression profiles. Data are preprocessed and organized for simple searches. The interface allows individual or combinatorial searches for proteins with certain domains, expression in certain tissues and/or certain phenotypes. Genes in a given genetic or physical interval can be listed to provide a convenient way to screen for candidate genes in mapping/cloning experiments. Information about available alleles can be displayed to simplify experimental planning. Literature related to entire lists of genes can be quickly retrieved and surveyed. GExplore operates on a local database and has fast response times, which is essential for exploratory searches. The interface is simple yet flexible enough to accommodate the addition of more data sets in the future. Since it is mainly based on data extracted from Wormbase it can be easily updated as new stable releases become available. GExplore is hosted under: http://genome.sfu.ca/gexplore/.

Harald Hutter (2003) International Worm Meeting "Axon guidance in the ventral cord of C. elegans: The role of pioneers and extracellular guidance cues"

Harald Hutter

[International Worm Meeting, 2003]

The ventral cord is the major longitudinal axon tract in C. elegans consisting of two bundles running on either side of the ventral midline. The majority of the axons runs in the right axon tract, despite an overall left-right symmetry of the nervous system. The arrangement of neuronal processes within the ventral cord is highly invariant implying that there is a precise navigational system guiding axons not just to the cord but also within the cord. Ventral cord axons grow out sequentially, suggesting that there might be dependancies between early and late outgrowing axons (pioneer-follower relationship). To systematically analyse such dependancies, transgenic strains were made expressing two or three color variants of GFP in different groups of axons. The importance of pioneers like the AVG neuron, which is the first neuron to send an axon into the right ventral cord tract, was tested in laser ablation experiments. It was found that certain classes of interneurons and motorneurons are highly dependant on this pioneer, whereas other classes of motorneurons are almost not affected by its absence. Surprisingly, neither AVG nor pioneers supposedly leading nerve ring axons to the right side of the ventral cord (RIF, RIG, SABV) are essential for the generation of left-right asymmetry of the ventral cord. Navigational errors like crossing of the ventral midline by individual axons do not seem to influence other nearby axons, which continue on their normal path, but might commit a similar mistake independantly. Frequently misrouting of early outgrowing axons does not lead to errors of later outgrowing axons, suggesting that the order of outgrowth does not necessarily reflect a strict pioneer-follower relationship. On the other hand, a pairwise correlation of axon outgrowth defects of different classes of neurons revealed subtle dependancies for certain combinations, e.g. between interneurons and PVQ axons, indicating that early outgrowing axons have some influence on the behaviour of late outgrowing axons. A detailed analysis of defects in known axon guidance mutants revealed that the different classes of neurons use different combinations of guidance cues. Even early outgrowing axons like AVG and DD-type motorneuron axons, which should depend almost exclusively on extracellular cues, are affected rather differently by the absence of known extracellular guidance signals. Taken together this analysis shows: not all the early outgrowing axons act as pioneer; later outgrowing axons can interpret the same navigational cues as those used by the pioneers; different axons in the same axon bundle are fairly independant in their outgrowth using different combinations of guidance cues for their navigation.

Hutter H et al. (1993) International C. elegans Meeting "The left-right asymmetry of the C. elegans embryo is induced by the MS blastomere."

Schnabel R, Hutter H

[International C. elegans Meeting, 1993]