B Esmaeili et al. (1999) International C. elegans Meeting "vab-7 controls the DB neuron fate"

D Miller, J Ross, J Ahringer, C Neades, B Esmaeili

[International C. elegans Meeting, 1999]

The C.elegans even-skipped homologue, vab-7 , is required for posterior muscle and epidermal patterning. vab-7 mutants are also forward Unc. VAB-7 is expressed in DB motorneurons, which innervate the dorsal body muscles and are required for proper forward movement. To examine the morphology of DB neurons, we used an unc-129::gfp reporter gene, which labels DA and DB processes and cell bodies. Both DA and DB neurons send circumferential commissures to the dorsal nerve cord (DNC), but on reaching it, DAs send axons anteriorly and DBs posteriorly. We found that in vab-7 mutants DB neurons have the same axonal polarity as the DAs (anteriorly directed) in the DNC, suggesting that they have DA characteristics. To investigate this further, we examined the expression of UNC-4, a homeodomain protein expressed in the DAs; unc-4::gfp is ectopically expressed in the DBs in vab-7 mutants. unc-4 is responsible for the change in polarity of DBs in vab-7 mutants, since their polarity is restored to wild type in unc-4; vab-7 double mutants. Furthermore expression of the DB marker, acr-5::gfp (a nicotinic acetylcholine a-receptor subunit) is lost in vab-7 mutants, also because of ectopic unc-4 expression. These data indicate that vab-7 is required for the DB fate. To investigate the sufficiency of vab-7 for DB fate determination we ectopically expressed vab-7 under the control of the unc-3 promoter, which drives expression in the DAs and DBs and some postembryonic neurons (AS, VA, VB). This causes DAs to have DB characteristics: DA axonal polarity in the DNC is reversed, unc-4::gfp expression is repressed, and acr-5::gfp is ectopically expressed. Furthermore, ectopic expression of vab-7 induces ectopic unc-129::gfp expression in postembryonic neurons, suggesting a possible DB fate acquisition by these neurons. In support of this model, the postembryonic AS neurons, which have anteriorly directed axons in the DNC, have reversed (posterior) polarity when vab-7 is ectopically expressed. Therefore, vab-7 is sufficient to induce at least some aspect of DB fate in both embryonic and postembryonic neurons. Interestingly, even-skipped in Drosophila also controls the guidance of neurons that innervate dorsal muscles, so there is conservation in the function of these proteins.

Kerk, SY et al. (2015) International Worm Meeting "Motor Neuron Identity Diversification via Selective Repression of Terminal Selector Target Genes."

Kratsios, P, Hart, M, Hobert, O, Mourao, R, Kerk, SY

[International Worm Meeting, 2015]

Complex neural circuits controlling body musculature that underlie animal movement comprise of diverse motor neuron (MN) classes. To investigate how such class identities are established, we turned to the ventral nerve cord cholinergic motor nervous system of Caenorhabditis elegans that consists of 6 distinct classes (DA/DB/VA/VB/AS/VC). In addition to morphological criteria, each MN class is molecularly defined by a unique combination of class-specific effector genes while sharing a multitude of commonly expressed ones. UNC-3/EBF is the terminal selector that confers cholinergic identity on the DA/DB/VA/VB/AS classes. Interestingly, despite its expression in all 5 MN classes, UNC-3 also directly controls the expression of the effector genes that are only selectively expressed in subsets of MN classes. We hypothesized that repressor proteins are the factors restricting the ability of UNC-3 to activate these genes in a MN class-specific manner. Forward genetic screens were performed using fluorescent reporters of MN class-specific effector genes [e.g. the DA/DB-specific unc-129 (DA/DB)] to examine if such repressors do exist. Indeed, we discovered bnc-1, a novel gene previously uncharacterized in C. elegans or any nervous systems which is the sole C. elegans ortholog of the vertebrate paired C2H2 zinc finger transcription factor, BNC. Loss of bnc-1 function causes unc-129 to be derepressed in VA/VB in an unc-3-dependent manner. Further experiments showed that BNC-1 is expressed specifically in VA/VB and works as a repressor via a putative binding site near that of UNC-3 (COE motif) within the cis-regulatory region of unc-129. Additionally, reporters of other unc-3-dependent effector genes that are not expressed in VA/VB [unc-8 (DA/DB/AS), unc-53 (DA/AS) & acr-16 (DB)] are also repressed in VA/VB by BNC-1. This concept of selective repression extends to the expression control of other unc-3-dependent MN class-specific effector genes, as determined by additional mutant analyses. For example, del-1 (VA/VB) is repressed in DA/DB by mab-9/TBX20 (DA/DB/AS) and in AS redundantly by both mab-9 and unc-55/NR2F (AS) whereas glr-4 (SAB/DA9) is repressed in DA/DB by cfi-1/ARID (DA/DB). Similarly, the aforementioned unc-53 is repressed in DB by vab-7/EVX (DB) whereas acr-16 is repressed in DA by unc-4/UNCX (DA/VA). We hence propose a general principle of MN identity diversification whereby unc-3-dependent MN class-specific effector genes are selectively repressed by evolutionarily conserved MN class-specific repressors via direct binding to cognate sites adjacent to the COE motif, giving rise to unique combinations of MN class-specific effector genes that consequentially define individual MN classes.

Esmaeili B et al. (1998) European Worm Meeting "The DB motorneurones have DA Characteristics in vab-7 mutants"

Esmaeili B, Neades C, Ahringer JA

[European Worm Meeting, 1998]

The C. elegans even-skipped homologue, vab-7, is essential for posterior epidermal and muscle patterning. As well as having posterior defects, vab-7 mutants have uncoordinated forward movement. When moving backwards vab-7 mutants can flex both dorsally and ventrally. However, in response to tail touch they curl ventrally and retain this posture for several seconds. Since the same muscles are used for forward and backward movement, the forward movement defect could be of neuronal origin. Using antibodies to VAB-7, we found that it is expressed in the DB motorneurones as well as 2 unidentified neurones in the head. The DB motorneurones innervate dorsal body muscles and are required for proper forward movement. Therefore, the forward movement defect of vab-7 mutants might be due to lack of vab-7 expression in the DB motorneurones. DA motorneurones (which are used for backward movement) and DB neurones have a similar structure: both send circumferential commissures to the dorsal nerve cord where they form neuromuscular junctions with dorsal body muscles. Some commissures run around the left hand side of the body and some on the right, depending on the neurone. Once the commissures reach the dorsal nerve cord they extend their axons along the dorsal nerve cord. DA axons turn anteriorly whereas DB axons turn posteriorly. Therefore, DA and DB motorneurones can be distinguished by means of their axonal polarity in the dorsal nerve cord. We examined the morphology of DA and DB motorneurones using an unc-129::GFP integrated strain (expressed in DAs and DBs; thanks to J. Culotti). Examination of DA and DB motorneurones showed that all the DA and DB cell bodies were present and their commissures were on the correct sides of the body in vab-7 mutants. However, the DB neurones were found to have anteriorly directed axons like DAs, instead of posteriorly directed ones. Furthermore, the dorsal nerve cords of vab-7 mutants is defasciculated. Recently it has been shown that unc-4::GFP is ectopically expressed in the DBs in vab-7 mutants [1]. In wildtype unc-4 is expressed in SABs, AVFs, DAs and VAs, and specifies proper presynaptic input to VAs [2]. To see whether the reversal of DB axonal polarity from posterior to anterior was due to ectopic expression of unc-4 we examined the dorsal nerve cord of unc-129::GFP; unc-4 (e120); vab-7 (e1562) animals. We found that in these animals the axonal polarity of DBs was restored to wildtype (posterior), but they still had a forward movement defect and dorsal cord defasciculations. Therefore, vab-7 is probably performing other functions in the DBs in addition to repressing unc-4. These results suggest that vab-7 might determine the DB fate. To explore the functions of vab-7 and unc-4 in motorneurone fate further, we are currently testing whether ectopic expression of vab-7 or unc-4 are sufficient for determining axonal polarity. 1. Meir J.Y.J. et al., Early 1998 East Coast Worm Meeting 2. Milller D.M. et al., (1995). Development 121, 2877-2886.

Shin-i T et al. (1997) International C. elegans Meeting "EXPRESSION PATTERN MAP ON THE INTERNET"

Kohara Y, Shin-i T

[International C. elegans Meeting, 1997]

We are constructing a database to integrate all the information of our expression pattern map of the C.elegans genome (see the abstract by Kohara et al.). The database consists of three parts; (1) "Clone tag DB" treats the cDNA tag sequencing data automatically to map the sequences onto the genomic sequence and to cluster them into unique cDNA groups. The processed data will be released quickly to the public domain, such as our WWW server, public DB and ACEDB. (2) "In situ hybridization DB" functions as an annotation workbench which supports us to add various information, particularly about the positive cell(s) and cell lineages in the hybridization, to the individual images of in situ analysis. Once the annotation is made by us, the in situ images are arranged appropriately and are made available at the WWW server. (3) "Feedback info DB" accommodates a wide range of information provided by the users of the cDNA clones. We plan to have a demonstration of the database at the meeting.

Esmaeili B et al. (1997) International C. elegans Meeting "VAB-7 IS EXPRESSED IN DB NEURONS AND REQUIRED FOR FORWARD MOVEMENT"

Esmaeili B, Neades C, Ahringer JA

[International C. elegans Meeting, 1997]

We previously showed that vab-7 encodes an even-skipped-like homeodomain containing protein required for posterior epidermal and muscle patterning. In addition to posterior defects, vab-7 mutants also have abnormal forward movement. When backing in response to head touch, vab-7 mutants can flex both dorsally and ventrally. However, when moving forward in response to tail touch, they curl ventrally and become locked in this position for several seconds. This difference in behaviour when moving forward versus backward suggests that at least part of the movement defect has a neuronal origin, since the same muscles are used in both directions. However, our vab-7::lacZ reporter gene has no expression in the nervous system. We have now made mouse monoclonal antibodies to VAB-7. In wild-type embryos VAB-7 is first expressed in muscle and epidermal precursors in a pattern identical to that seen for the reporter gene, but reporter expression purdures longer than endogenous VAB-7. From early elongation VAB-7 is also expressed in some ventral cord neurons. By doing double staining with anti-VAB-7 and unc-25::lacZ (as a marker for D-type neurons; thanks to Y. Jin), we found that VAB-7 is expressed in the 7 DB neurons plus 2 additional unidentified neurons in the head. As DB neurons innervate dorsal muscles and function in forward movement, the behavioural defect described above is could be due to a failure of DB neurons to stimulate dorsal muscles when moving forward. We are now investigating the fate of the DB neurons in vab-7 mutants. To look at their axonal morphology, we are trying to construct a vab-7 reporter gene expressed in the DB neurons. In addition, we are examining vab-7 expression in a variety of unc mutants to look for possible regulators.

Muneesh Tewari et al. (2001) International C. elegans Meeting "Searching for new proteins involved in dauer formation by two-hybrid protein interaction mapping."

Muneesh Tewari, Patrick Hu, Marc Vidal, Gary Ruvkun, Svetlana Busiguina

[International C. elegans Meeting, 2001]

This project aims to identify new proteins involved in dauer formation by large-scale yeast two-hybrid screens using all the known dauer gene products as baits. Open reading frames encoding each of 34 proteins implicated in dauer formation (daORFs) were cloned into yeast two-hybrid bait (DB-daORFs) and prey (AD-daORFs) vectors by Gateway recombinational cloning. First all pairwise DB-daORF/AD-daORF combinations were tested. This matrix experiment recapitulated an interaction between the DAF-3 (an ortholog of SMAD4) and DAF-5 (a relative of the SNO oncoprotein; Garth Patterson, personal communication) gene products. Individual yeast two-hybrid screens of a C. elegans AD-cDNA library were then carried out with each DB-daORF. This large-scale mapping experiment identified a total of 58 putative interactors. Among others, an interaction between AGE-1 (an ortholog of PI-3-kinase) and the worm ortholog of the p85 adaptor protein was detected. Most of the interactors identified correspond to previously uncharacterized gene products. They will be subjected to functional analysis by systematic RNA-mediated interference to test their involvement in dauer formation.

Stephanie S. Sybingco et al. (2007) International Worm Meeting "Isolation and characterization of novel mutants exhibiting defects in pioneer axon guidance in Caenorhabditis elegans."

Marie T. Killeen, Matthew Bueno de Mesquita, Sang-Hyeon Park, Stephanie S. Sybingco

[International Worm Meeting, 2007]

During neuron development, axons are directed to their specific targets by extracellular guidance cues that bind to receptors on the growth cones of migrating axons. The cholinergic motor neurons of the DA and DB classes are known to be partially guided by the netrin signaling pathway (Hedgecock et al., 1990). They can be detected using unc-129::gfp which expresses in these neurons. In wild-type animals, the DA/DB motor neurons can be seen to leave the ventral cord and migrate circumferentially along the body wall to the dorsal cord. However, in an unc-5(e53) null genetic background, DA/DBs leave the ventral cord migrating about two-thirds of the way towards the dorsal cord, but then show aberrant axon migrations and fail to reach their dorsal targets. This suggests that other signaling cues must be responsible for the initial stage of migration, in addition to those in the netrin signaling pathway. Forward genetic screens were used to find mutants that had defects in this initial axon migration of DA/DB using unc-129::gfp in an unc-5(e53) background. 2100 haploid genomes were mutagenized using EMS and screened using epifluorescence to visualize the DA/DB motor neurons. Three mutants were recovered from this enhancer screen named rq1, rq2 and rq3. These mutants are slow growing and ataxic. Using snip-SNP mapping, rq1 was mapped to III and rq2 to X. No currently known members of the netrin pathway map to these regions.

Yee C et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "ENU-3 is a novel putative transmembrane protein affecting both outgrowth and guidance of the DA and DB motor neuron axons in C. elegans"

Sybingco S S, Park S, Naqvi Z, Kholkina G, Bueno de Mesquita M, Yee C, Lackraj T, Serdetchnaia V, Killeen M T

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Axon outgrowth and guidance of the DA and DB motor neurons in C. elegans is strongly dependent on the ligand UNC-6/netrin and its two receptors UNC-5 and UNC-40/DCC/Frazzled. To identify genes in a parallel pathway required for motor axon outgrowth, we conducted a genetic enhancer screen in a genetic background lacking UNC-5. We treated unc-5(e53) animals with EMS and screened for F2 animals with an increased number of DA and DB motor neuron axon outgrowth defects. Five mutants were identified in the screen and enu-3 has been extensively characterized. The outgrowth defects of the DB axons in both an enu-3;unc-5(e53) and enu-3;unc-6(ev400) double mutant backgrounds were enhanced relative to the single mutants. This suggests that ENU-3 functions in a pathway parallel to the netrin pathway proteins. DA and DB motor neuron axon migrations were also affected in the enu-3 mutant. enu-3 defects were rescued by a single gene that encodes a novel putative trans-membrane protein mapped to chromosome III. The gene is highly homologous to several other proteins in C. elegans and has only weak homologies with several human proteins including mucin 13 and hydin. Expression of transcriptional and translational reporters indicate that ENU-3 is expressed in a dynamic expression pattern. The function of the protein remains to be determined.

Hwai-Jong Cheng et al. (2001) International C. elegans Meeting "A GENETIC SCREEN FOR DA/DB AXON GUIDANCE MUTANTS IN C. ELEGANS"

Marc Tessier-Lavigne, Hwai-Jong Cheng, Xun Huang, Yishi Jin

[International C. elegans Meeting, 2001]

In order to identify novel factors involved in axonal guidance, we have taken a genetic approach and used C. elegans to identify mutant animals that have defects in axonal projections in a subset of motoneurons which normally project their axons dorsally, the DA and DB neurons. These axons are already known to be repelled by the netrin UNC-6 (Ishii et al., Neuron 9:873-881, 1992), and also require the TGF-beta family member UNC-129 derived from dorsal muscle for proper guidance (Colavita et al., Science 281:706-709, 1998). These neurons actually also express unc-129, so that their axons can be visualized by driving GFP expression from the unc-129 promoter. We have mutagenized unc-129::gfp worms with EMS and screened for mutants that have defects in projections of DA and DB axons. Reasoning that most of severely uncoordinated mutants with global axon guidance defects might have been isolated in previous screens, we focused our screen on mutants with minimal uncoordinated phenotypes and less severe axon guidance defects. We have isolated mutants in this screen, and named these mutants "max" for motor axon guidance defect. We have characterized and mapped one of the mutants, max-1.

Walhout AJM et al. (1998) East Coast Worm Meeting "Towards a comprehensive C. elegans protein interaction map: identification of "Interaction-Sequence Tags""

Kreutzer MA, Vidal MI, van den Heuvel SJL, Walhout AJM, Wong WL

[East Coast Worm Meeting, 1998]

The complete genome sequence of C. elegans should be available by the end of this year. It is estimated that the sequence will reveal approximately 13,000 ORFs, most of which correspond to predicted gene-products whose functions have yet to be determined. Hence, in addition to the powerful genetic and biochemical approaches used conventionally, it will be important to develop high-throughput strategies to help addressing the biological role of these gene-products. Since many cellular processes depend on protein-protein interactions, one approach used to elucidate functions consists in identifying physical interactions between proteins. Thus, our long-term goal is to generate a comprehensive protein interaction map for C. elegans, as a way to help characterizing regulatory networks and protein complexes in this model organism (see the abstract by Vidal et al.). We have selected the yeast two-hybrid system to identify potential interactions since on the one hand, its effectiveness has been demonstrated widely, and on the other, it is readily automatable. In this system, interaction between X and Y proteins, expressed as fusions with a DNA binding domain (DB) and an activation domain (AD) respectively, reconstitutes a functional transcription factor which in turn leads to a selectable phenotype. Each potential interaction is referred to as an Interaction Sequence Tag (IST), defined as a pair of short sequences obtained from cDNAs encoding interacting proteins in the two-hybrid system. We intend to release ISTs into AceDB in a format similar to the introduction of ESTs. We will describe the following aspects of the project: -the choice of the first subset of DB-X hybrid proteins used as "baits", -the semi-automated method used to generate the DB-X fusions, -the design and construction of an AD-cDNA library, -the semi-automated two-hybrid selections used to identify ISTs, and -the potential significance of the initial ISTs identified. Since ISTs merely represent hypotheses of interactions, we are also developing high throughput strategies to assess the biological significance of the potential interactions directly in worms (see the abstract by Vidal et al.). The combination of both the IST information available in AceDB and the functional assessment in vivo should be helpful in generating a comprehensive interaction map for C. elegans.