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.

Christina Schmid et al. (2001) International C. elegans Meeting "ast-1 and ast-2, novel genes important for fasciculation of axons in the ventral cord"

Harald Hutter, Edward M Hedgecock, Christina Schmid

[International C. elegans Meeting, 2001]

One of the major problems of developmental biology is how axons build their specific pattern of synaptic connections, which is crucial for the function of every nervous system. The mechanisms through which axons respond to guidance cues are not well understood. In the final stage of axon guidance the selection of synaptic partners within the target area takes place. Since synapses in C. elegans are made with 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 recognition mechanism between axons, a screen for mutants exhibiting fasciculation defects was designed. Animals that express GFP under the control of the glr-1 (glutamate receptor) promoter were used to label interneuron axons that are part of the motor circuit. In wildtype animals GFP labelled axons form a tightly fasciculated subbundle within the right ventral cord. After mutagenesis with EMS F2 progeny were scored under the fluorescence microscope for defects in fasciculation. This direct screen for axonal outgrowth defects is very sensitive for obtaining mutants with minor defects that do not lead to gross behavioural defects. Two novel genes identified in these screens are currently studied in more detail. ast-1 and ast-2 mutants are characterized by defasciculated axons in the ventral cord, where axons cross from the right to the left bundle. In a small number of animals one single interneuron axon is misguided and does not reach the ventral cord, but runs in lateral position. The penetrance of these defects is about 45% and the mutations are slightly temperature sensitive. There are no obvious defects in the outgrowth of other axons as judged by various GFP markers. Other tissues and cell migrations appear normal. The axonal phenotypes of both mutants are similar, but ast-2 mutants are somewhat lethargic and slightly Egl, whereas ast-1 mutants only show subtle changes in exploratory behaviour. Two alleles of ast-1 called rh300 and hd1 were isolated independently. The phenotypes are very similar and the mutants do not complement. One allele of ast-2 was isolated so far. ast-2 maps close to unc-13 on chromosome I. ast-1 maps to chromosome II four map units left of dpy-10 and near vab-1 . ast-1(hd1) complements vab-1(dx31) . Fine mapping was based on single nucleotide polymorphisms that can be detected by RFLP as a consequence of the modification of a restriction enzyme recognition site. The gene locus has been narrowed to a 400 kb region enclosed by cosmids ZK430 and C33F10.

Christina S Schmid et al. (2003) International Worm Meeting "Ast-1, an ETS Transcription Factor, controls Axon Fasciculation in the Ventral Cord"

Harald Hutter, Christina S Schmid, Edward M Hedgecock

[International Worm Meeting, 2003]

The correct outgrowth of axons is crucial for the establishment of neuronal networks during embryonic development. To identify novel genes involved in axon guidance and fasciculation we performed a direct visual screen for mutants with outgrowth defects in glr-1(glutamate receptor)::GFP expressing interneurons. Two alleles of ast-1(axonal steereing) called rh300 and hd1 were recovered independently from these screens. Their phenotypes are characterized by defasciculated interneuron axons in the ventral cord, where axons cross from the right to the left bundle. Occasionally individual interneuron axons do not reach the ventral cord but run in a lateral position instead. The penetrance of these defects combined is about 40%. The mutants are not significantly uncoordinated despite these defects in the command interneurons of the motorcircuit. There are no obvious defects in the navigation of other axons. Non-neuronal tissues and cell migration appear unaffected in ast-1 mutant animals. Rescue of the mutant phenotype was obtained by injection of cosmid H37P12 and with a PCR fragment containing the predicted open reading frame T08H4.3. This gene encodes a transcription factor with an ETS DNA-binding domain. Sequencing the DNA of the two mutant alleles revealed point mutations in exon six causing amino acid exchanges in the highly conserved region of the ETS domain. ETS proteins are targets of the Ras-MAPK signaling pathway and interact with other transcription factors that promote the binding of ETS proteins to composite Ras responsive elements. The C. elegans genome contains ten ETS genes. Ast-1 and C42D8.4 are closely related to the mammalian fli-1 subfamily. The ventral cord midline crossing defects characteristic for ast-1 mutants are also seen in several other mutants in genes controlling aspects of neuronal differentiation. Using double mutant analysis we found that ast-1 genetically interacts with the transcription factors zag-1 and lin-11. In addition, AST-1 shows synergistic effects with the netrin/UNC-6 receptor UNC-40 and the actin-regulating protein UNC-34. Currently we are trying to further elucidate the function of ast-1 in the regulatory network of axon guidance genes.

Andreas Steimel et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Ast-2 and ast-7, novel genes involved in pioneer-follower axon guidance and establishment of ventral cord asymmetry"

Andreas Steimel, Irene Wacker, Harald Hutter

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

The asymmetrically organized ventral cord of C. elegans is the main nerve cord along the anterior-posterior body axis. The correct wiring of all axons in the ventral cord is a challenging task. Only a few genes are known to be involved in this complex process. In an EMS screen for axon outgrowth defects in command interneurons we isolated two novel genes involved in pioneer-follower axon guidance and establishment of ventral cord asymmetry. The mechanisms of establishing ventral cord asymmetry are largely unknown. The majority of ventral cord axons like the axons of command interneurons run in the right axon tract. A significant number of ast-7(hd11) animals shows a rather symmetric then asymmetric distribution of command interneuron axons in the ventral cord. Command interneuron axons fail to cross the midline after leaving the left side of the nerve ring and remain in the left axon tract. Ast-7 seems to play a role in the establishment of ventral cord asymmetry. Ast-7 maps to the centre of chromosome II. During nervous system development the left side of the ventral cord is pioneered by the PVPR axon from the posterior closely followed by the PVQL axon. Ablation of PVPR forces the PVQL axon to grow along the right axon tract (Durbin, PhD thesis, Cambridge 1987). Moreover in animals with defects in PVPR axon guidance the PVQL axon always follows the misguided PVPR axon. These results imply a tight pioneer-follower relationship between the PVPR and the PVQL axon. In ast-2(rh308) animals the pioneer-follower relationship between PVPR and PVQL is disturbed. The PVQL axon crosses the ventral midline, leaves the ventral cord or stops prematurely independent of the PVPR axon. Similar but less penetrant defects were observed in the right axon tract. To our knowledge ast-2(rh308) is the first mutant with PVP-independent PVQ axon outgrowth defects. Ast-2 might be involved in cell-cell communication or adhesion between the PVP and the PVQ axon. Ast-2 is located on the right arm of chromosome V. Currently rescue experiments for both mutants are in progress.

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.

Doitsidou, Maria et al. (2011) International Worm Meeting "A combinatorial code of transcription factors defines the terminal differentiation of dopaminergic neurons."

Doitsidou, Maria, Hobert, Oliver, Felton, Terry, Flames, Nuria

[International Worm Meeting, 2011]

Dopaminergic neurons control various aspects of behaviour and their loss leads to Parkinson's disease. There are 8 dopaminergic (DA) neurons in the C. elegans hermaphrodite that fall into 4 morphological classes. All 4 classes display very distinct developmental histories, but nevertheless express the core pathway genes responsible for the biosynthesis and transport of dopamine. We call these genes the dopamine pathway genes. It was previously shown (Flames, 2009) that ast-1, an ETS transcription factor, is necessary for the expression of all dopamine pathway genes in all dopaminergic cell types. AST-1 exerts its action through binding to the dopaminergic motif, present in the promoters of the dopamine pathway genes. However, neither AST-1 nor its corresponding binding sites are sufficient to activate the dopamine pathway genes. We used a combination of genetic and promoter analysis approaches to identify the transcription factors as well as the corresponding regulatory elements that act in concert with ast-1 for terminal differentiation of DA neurons. Extensive analysis of the dopaminergic motif revealed the presence of functional homeodomain and PBX/Hox binding sites in all DA pathway genes. Automated COPAS forward genetic screens combined with Whole Genome Sequencing, uncovered a homeodomain transcription factor of the Distal-less family, ceh-43, and a Hox domain/MEIS transcription factor, unc-62, that are necessary for terminal differentiation of DA neurons. We find that these transcription factors are required for proper expression of all dopamine pathway genes in all DA neuronal classes. Phenotypic analysis, ectopic expression studies and epistatic relationships between ceh-43, unc-62 and ast-1 will be presented. In addition, candidate approach revealed that a PBX transcription factor, ceh-20, is also involved in terminal differentiation of DA fate. In conclusion, we have uncovered a combinatorial code of transcription factors, as well as the corresponding cis-regulatory elements that control terminal differentiation of dopaminergic neurons.

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.

Flames, Nuria et al. (2009) International Worm Meeting "Regulatory logic of dopaminergic neuron differentiation."

Hobert, Oliver, Flames, Nuria

[International Worm Meeting, 2009]

Vertebrate dopaminergic neurons play a central role in the coordination of movement, attention, memory and reward. In addition, defects in the synthesis or function of the neurotransmitter dopamine underlie several pathological conditions such as Parkinson''s disease, schizophrenia and drug addiction. The dopamine synthesis pathway is extremely well conserved from worms to human. However, the factors that directly activate the expression of the dopamine synthesis pathway genes remain only poorly understood in both organisms. Caenorhabditis elegans hermaphrodite contains eight dopaminergic neurons that are involved in locomotory control and several aspects of neuronal plasticity. These eight neurons fall into 4 distinct classes, each composed of a pair of bilaterally symmetric neurons. All 4 classes display very distinct developmental histories, but nevertheless express the core pathway genes that define the dopaminergic cell fate (dat-1, cat-2, cat-1, cat-4 and bas-1). In this work, we show that in C. elegans, a simple cis-regulatory element controls the expression of all dopamine synthesis pathway genes in all dopaminergic cell types (including also the male dopaminergic neurons). We show that an ETS transcription factor, AST-1, is the trans-acting factor responsible for the activation of the dopaminergic neuron cis-regulatory element. Loss of the ast-1 gene results in loss of specification of all different dopaminergic neuron classes and ectopic expression of ast-1 is sufficient to activate the dopamine production pathway in several additional neurons. We also show that AST-1 is expressed in all the dopaminergic neurons throughout the life of the animal and its function is constantly required to maintain the dopaminergic fate. In conclusion, our studies reveal an astoundingly simple regulatory logic of dopaminergic neuron differentiation.

Andreas Steimel et al. (2005) International Worm Meeting "Axon Guidance Through Pioneers: Ast-2, a novel gene controlling pioneer dependent PVQ axon outgrowth in the ventral cord."

Irene Wacker, Andreas Steimel, Harald Hutter

[International Worm Meeting, 2005]

The ventral cord of C. elegans is divided into a right and a left axon bundle. During embryogenesis the left axon bundle is pioneered by the PVPR axon from the posterior end followed by the PVQL axon. The axon outgrowth of PVQ is an example of a strict pioneer-follower relationship as ablation of PVPR leads to loss of the left axon bundle and the PVQL axon joining the right side of the ventral cord (Durbin, PhD thesis, Cambridge, 1987). Moreover PVQ axons always follow PVP axons in animals with significant PVP axon guidance defects. In a visual screen for axon outgrowth defects, we found a mutant with perturbations in the correlation of PVP and PVQ axon outgrowth defining the novel gene ast-2(rh308). PVPR and PVQL axons show ventral midline crossing defects in about 80 % of all ast-2 animals, whereas this is the case for the PVQR axon in less than 30 %. No defects are observed for the PVPL axon. Interestingly PVQ axons frequently cross the ventral midline between the two axon bundles independent of the PVP axons. Ast-2 is the first identified gene involved in the pioneer-follower relationship of PVP and PVQ. Furthermore the PVQ axons often stop and do not reach the nerve ring a defect never observed for other axons in ast-2 including the PVP axons. Therefore it seems likely that the primary defect is in the PVQ axons themselves. Ast-2 is located on the right arm of chromosome V in an interval of about 3 map units containing around 800 putative genes. Additional mapping is necessary to narrow down the interval. Finally YACs and cosmids will be used to identify the affected gene.The molecular nature of the signals mediating follower axon navigation is unknown. Therefore we try to identify genes expressed in PVPs that might have a function in the guidance of the PVQ follower axons. We analyze promoters active in PVPs to identify promoter elements specific for gene expression in PVP. Through bioinformatical tools we will search for cell surface genes expressed in PVP. In a second approach we will use SAGE and microarray analysis to identify PVP expressed genes. To this end we generate strains where PVP is specifically labeled using the recGFP system. This allows isolation of PVP via FACS and cell specific isolation of RNA.We will analyze candidate genes for a potential function in axon guidance.

Sara Maxwell et al. (2002) European Worm Meeting "Functional analysis of the C. elegans T-box gene family"

Alison Woollard, Roger Pocock, Lizzie Clarke, Sara Maxwell

[European Worm Meeting, 2002]

T-box genes are a group of developmentally important transcription factors united by a common DNA binding domain. T-box genes are present in all metazoan species so far analysed but are absent from yeast. There are 20 T-box genes in C. elegans, more than twice the number found in Drosophila. Many of the C. elegans T-box genes are highly diverged from those found in other species while others have clear orthologues present throughout the metazoan kingdom. One highly conserved T-box gene is mab-9, a member of the tbx20 sub-family1. This was the first C. elegans T-box gene to be identified by mutation and is required for cell fate specification during hindgut and male tail development, and aspects of nervous system function. One other conserved T-box gene has recently been reported to be important for a particular muscle cell fate specification2. We have inactivated the remaining C. elegans T-box genes by RNAi and have found obvious phenotypes only in very few cases. These phenotypes include embryonic lethality, L1 lethality, and a Dpy phenotype with weakly penetrant male tail defects, and will be described in detail. The remaining T-box genes give no obvious phenotype by RNAi. Phylogenetic analysis reveals that several pairs of T-box genes are very similar to eachother and are therefore likely to be the result of recent duplications. This might suggest functional redundancy. Double RNAi experiments have revealed this to be the case with at least two of the T-box gene pairs (see also poster by Pocock et al). Study of the expression patterns of the whole T-box family may suggest other potential redundancy relationships which can be explored by RNAi. Comparison of the C. elegans T-box genes with the set of T-box genes now defined for C. briggsae is being used as a tool for defining potentially important regulatory regions present in orthologous genes.