Unsoeld, Thomas et al. (2013) International Worm Meeting "Transmembrane Collagen COL-99 is Involved in Axon Guidance Along Major Longitudinal Axon Tracts and Ventral Nerve Cord Asymmetry in C. elegans."

Unsoeld, Thomas, Hutter, Harald, Taylor, Jesse

[International Worm Meeting, 2013]

The ventral nerve cord (VNC), the major longitudinal axon tract of C. elegans, consists of two fascicles separated by motor neuron cell bodies aligning at the ventral midline. The majority of neurons extend axons into the right tract creating the highly asymmetric structure of the VNC. Specifically, axons leaving the brain decussate into the right VNC tract shortly after descending from the nerve ring in the head. Mutants of known guidance cues and receptors have shown either no or very minor asymmetry defects in the VNC indicating existence of additional molecules involved in creating VNC asymmetry. Forward genetic screens in our lab revealed several candidate mutants displaying partially penetrant symmetrical VNC and midline crossing defects. One mutation, hd130, has been identified as an allele of the uncharacterized transmembrane collagen, col-99. col-99 encodes a type II transmembrane collagen consisting of a small intracellular domain and a large extracellular domain containing several collagen domains. Sequencing revealed an early nonsense mutation in col-99(hd130) likely resulting in complete loss-of-function. Further characterization of axonal trajectories in col-99 mutant animals identified significant axonal defects in most VNC neurons. Significant axonal navigation defects were observed in additional longitudinal axon tracts including the dorsal nerve cord and the two dorsal sublateral tracts. During axonal outgrowth in the embryo, col-99-GFP reporter expression is prominent in the hypodermis but not the nervous system suggesting a non cell-autonomous role for COL-99. col-99 interacts genetically with the collagen processing enzyme dpy-18, the basement membrane component nid-1 and the collagen-receptors ddr-1 and ddr-2. We currently explore the idea that the ectodomain of COL-99 is shed by proprotein convertases and localizes to the basement membrane. Our results demonstrate the importance of col-99 during axon navigation along major longitudinal tracts in C. elegans, and suggest a role in the decussation event establishing VNC asymmetry.

Thomas Unsoeld et al. (2005) International Worm Meeting "Identification of genes mediating pathfinding of the AVG pioneer axon and its followers"

Irene Wacker, Harald Hutter, Thomas Unsoeld

[International Worm Meeting, 2005]

In order to establish a correct neuronal network outgrowing axons face a challenging navigational problem due to the large numbers of neurons involved. The complexity of this problem is reduced by sequential axon outgrowth. The first axonal pathways are established by a single or few early outgrowing axons that path the way, the so called pioneers. Later outgrowing axons then frequently follow these pre-made pathways. In C. elegans the right ventral cord axon tract is pioneered by AVG. In a screen for axonal guidance defects, using EMS mutagenesis, we isolated two mutants, ast-4 (rh311) and ast-6 (rh313), that cause navigation defects of the AVG pioneer itself and of follower axons. In both mutants AVG axons grow out wrongly in the left axon tract or switch from right to left axon tract. The penetrance of these defects is low but significant. Axons of followers like motoneurons and interneurons are affected in various ways and show e.g. ventral cord midline crossing defects or disturbances in commissure outgrowth. Therefore ast-4 and ast-6 possibly encode signals of a axon guidance system used by AVG itself as well as other axons. Using SNPs we mapped ast-4 to the left end and ast-6 close to the center of chromosome IV. Identification and characterization of these genes will provide further insights to the molecular basis of the AVG pioneer follower relationship. In an independent approach we try to identify cis-regulatory elements that mediate expression of genes specific for AVG. Therefore we are subfragmenting several AVG promoters and testing them for driving reporter gene expression in AVG. In this way we were already able to narrow down the odr-2 and inx-18 promoters to less than 500 bp. The minimal reporter fragments will be screened for consensus sequences that can be used further to find putative AVG-expressed genes. To finally identify genes important for the correct outgrowth of AVG followers, genes encoding potential cell surface proteins will be tested for their dependence on lin-11, a LIM-homeobox transcription factor known to be essential for expression of genes required for AVG regulated axon guidance.

Thomas Unsoeld et al. (2006) European Worm Meeting "Identification of Novel Genes Mediating Pathfinding of the AVG Pioneer Axon and its Followers"

Thomas Unsoeld, Harald Hutter, Irene Wacker

[European Worm Meeting, 2006]

In order to form a correct neuronal network outgrowing axons face a challenging navigational problem due to the large numbers of neurons involved. The complexity of this problem is reduced by sequential outgrowth. The first single or few early outgrowing axons, the so called pioneers path the way. Later outgrowing axons then frequently follow these pre-made pathways. In C. elegans the right ventral cord axon tract is pioneered by the axon of the AVG neuron. In a screen for axonal guidance defects, using EMS mutagenesis, we isolated two mutants, ast-4(rh311) and ast-6(rh313), that cause navigation defects of the AVG pioneer itself and of follower axons. In both mutants AVG axons grow out into the left axon tract or switch from right to left axon tract. The penetrance of these defects is low but significant. Axons of followers like motoneurons and interneurons are affected in various ways and show ventral cord midline crossing defects or disturbances in commissure outgrowth. Therefore ast-4 and ast-6 possibly encode signals of a axon guidance system used by AVG as well as other axons. Using SNPs we mapped ast-4 to the left end and ast-6 close to the center of chromosome IV. Identification and characterization of these genes will provide further insights to the molecular basis of the AVG pioneer follower relationship.. In an independent approach to identify novel AVG-expressed genes important for the guidance of follower axons we try to identify cis-regulatory elements that mediate expression in AVG. Therefore we are subfragmenting promoters of AVG-expressed genes like odr-2 and inx-18 to define the minimal region required for AVG expression. Minimized reporter fragments will be screened for a consensus sequence which will be used subsequently to find putative AVG-expressed genes. After validation of AVG expression interesting candidates e.g. genes encoding putative cell surface receptors will eventually be tested for a potential role in the guidance of AVG follower axons.

Thomas Unsoeld et al. (2007) International Worm Meeting "Identification and Characterization of Novel Genes Mediating Pathfinding of the AVG Pioneer Axon and its Followers."

Harald Hutter, Thomas Unsoeld, Irene Wacker

[International Worm Meeting, 2007]

In order to form a correct neural network outgrowing axons face a challenging navigational problem due to the large numbers of neurons involved. The complexity of this problem is reduced by sequential outgrowth. At first a single or few early outgrowing axons, so called pioneers path the way. Later outgrowing axons then frequently follow these pre-made pathways. In C. elegans the right ventral cord axon tract is pioneered by the axon of the AVG neuron. In a screen for axonal guidance defects, using EMS mutagenesis, we isolated two mutants, ast-4 (rh311) and ast-6 (rh313) with defects in pioneer axon navigation. In a small but significant number of both ast-4 and ast-6 mutants the AVG pioneer axon grows out wrongly in the left tract or switches between ipsilateral and contralateral sides. Additionally defects in axons of various followers like midline-crossing defects in interneurons of the motorcircuit or disturbances in commissure outgrowth of motoneurons can be observed. The penetrance of these defects exceeds those observed for the AVG pioneer axon, indicating that these defects are at least in part primary defects in the follower axons itself and not mere secondary consequences of an initial pioneer defect. Thus ast-4 and ast-6 supposedly encode components of an axon guidance system used by the AVG pioneer axon as well as the later outgrowing follower axons. Besides defects in axon guidance ast-6 mutants exhibit a rather pleiotropic phenotype comprising additional defects in cell migration, movement and egg laying. We mapped ast-4 to the left end and ast-6 close to the center of chromosome IV. Rescuing experiments, using fosmids, are in progress. The identification and subsequent characterization of these genes will provide further insights to the molecular basis of axon guidance in general and the AVG pioneer - follower relationship in particular.

Tax FE et al. (1994) Worm Breeder's Gazette "Some Notes on Mapping Contigs Using Molecular Analysis of Deficiences"

Thomas JH, Tax FE

[Worm Breeder's Gazette, 1994]

Some notes on mapping contigs using molecular analysis of deficiencies Frans Tax and Jim Thomas, Dept. of Genetics, University of Washington, Seattle WA 98195

Thomas JH et al. (1994) Worm Breeder's Gazette "lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein"

Thomas JH, Horvitz HR

[Worm Breeder's Gazette, 1994]

lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein Jeffrey H. Thomas and H. Robert Horvitz, HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA

Taylor, Jesse et al. (2015) International Worm Meeting "Genetic Screens Reveal Genes Regulating Ventral Nerve Cord Asymmetry in C. elegans."

Taylor, Jesse, Unsoeld, Thomas, Hutter, Harald

[International Worm Meeting, 2015]

The ventral nerve cord (VNC) is the major longitudinal axon tract of the nematode C.elegans. The two fascicles of the VNC extend to the left and right of the ventral midline over a basement membrane. The majority of axons are found in the right tract resulting in a noticeable asymmetry between the two tracts. Interestingly the anterior most portion of the VNC is actually symmetrical in appearance, with all but four axons decussating into the right VNC tract just posterior to the nerve ring. Known guidance cues have little or no effect on the navigation at this point of decussation suggesting the existence of additional cues. Forward genetic screens in our lab identified four mutants displaying mildly penetrant symmetrical VNCs and midline crossing defects of axons extending in the VNC. Two of the four mutants, hd131 and hd133, have undergone preliminary mapping and whole genome sequencing and likely define previously uncharacterized axon guidance genes. A third allele, hd132, was identified as a loss of function mutation in unc-34 the enabled/VASP homolog. The fourth mutant from the screens was identified as an allele of a previously uncharacterized transmembrane collagen, col-99. col-99 encodes a type II transmembrane collagen consisting of a small intracellular domain and a large extracellular domain containing several collagen domain repeats. Sequencing revealed our allele (hd130) as an early nonsense mutation likely resulting in complete loss-of-function. Further characterization of axonal trajectories in col-99 mutant animals identified significant axonal defects in most VNC neurons. Axonal navigation defects were also observed in other longitudinal axon tracts including the dorsal nerve cord and the two dorsal sublateral tracts. During axonal outgrowth in the embryo, col-99::GFP reporter expression is prominent in the hypodermis but not the nervous system suggesting a non cell-autonomous role for COL-99. Sequence comparison with vertebrate homologs revealed a conserved cleavage site indicating that COL-99 may be shed from the cell surface for function. Presently we explore the idea that the ectodomain of COL-99 is shed by proprotein convertases and localizes to the basement membrane. Current evidence suggests COL-99 is likely acting as a guidance cue for outgrowing axons potentially through interactions with basement membrane components and discoidin domain receptors on extending axons.

Burglin TR et al. (1993) Worm Breeder's Gazette "ceh-6 Expression and Generation of a Knock-Out"

Plasterk RHA, Ruvkun GB, Burglin TR

[Worm Breeder's Gazette, 1993]

ceh 6 expression and generation of a knock-out Thomas Burglin, Ron Plasterk*, Gary Ruvkun Dept. of Molecular Biology, Wellman 8, Massachusetts General Hospital, Boston, MA, 02114, USA, and *Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.

Reilly, Molly et al. MicroPubl Biol

Hobert, Oliver, Reilly, Molly

[MicroPubl Biol, 2020]

We have previously described the expression patterns of all but one of the 102 homeobox genes present in the C. elegans genome, using either fosmid-based reporter transgenes or CRISPR/Cas9-engineered gfp reporter alleles (Reilly et al., 2020). The last remaining homeobox gene for which we were initially not able to generate a reporter reagent for is ceh-84 (Reilly et al., 2020). As previously recognized by Thomas Brglin (Hench et al., 2015), ceh-84 codes for an unusual homeodomain protein with two divergent homeodomains (Fig.1D). It likely is a tandem duplicate of the ceh-85 gene, which has been classified as a pseudogene (Fig.1B). ceh-84 has no recognizable homologs in other Caenorhabditis species (Hench et al., 2015). Together with its duplicate, ceh-85, localizes to a large cluster of extensively duplicated genes on chromosome II, described by James Thomas (Thomas, 2006). Most of the duplicated genes code for MATH and BTB type proteins (Fig.1A). The entire cluster seems to be C. elegans-specific (Thomas, 2006) and, apart from a number of pseudogenes, it also contains the C. elegans-specific ceh-81, ceh-82 and ceh-83 homeobox genes (Fig.1A).

Tissenbaum HA et al. (1995) International C. elegans Meeting "GENETIC AND MOLECULAR ANALYSIS OF daf-2 AND daf-16."

Gottlieb S, Lee L, Tissenbaum HA, Ruvkun GB, Lam F

[International C. elegans Meeting, 1995]

We have been studying the genetic interactions between the genes daf-2, daf-23 and daf-16 (Gottlieb and Ruvkun 1994). These genes either act in a branch separate from the daf-11/daf-21 and daf-7 group of the genetic epistasis pathway for dauer formation or further downstream in the pathway ( Riddle et al. 1981; Vowels and Thomas 1992; Thomas et al. 1993; Gottlieb and Ruvkun 1994). Molecular analysis of daf-2, daf-16 as well as daf-23 (see abstract by Morris et al. this meeting) will help to elucidate function (e.g. neurons vs. target tissues) as well as shed light on the complex genetic interactions of these genes.