Eric L Smith et al. (2002) East Coast Worm Meeting "COORDINATE REGULATION OF GLUTAMATE RECEPTORS AND ncs-1 IN INTERNEURONS BY fax-1 AND unc-42"

Bruce Wightman, Rebecca Lombel, Rebecca Haviland, Eric L Smith, Sheila Mathieson

[East Coast Worm Meeting, 2002]

The C. elegans fax-1 gene encodes a nuclear receptor homolog of the human PNR gene. PNR has been implicated in the specification of photoreceptor identities in the human retina. In C. elegans, fax-1 functions in regulating aspects of neuron identity, including expression of flp-1 and axon pathfinding decisions in the AVK interneurons. The paired-like homeobox gene unc-42 is similarly required for specification of neuron identity, including expression of flp-1 and axon pathfinding in the AVK neurons, and axon pathfinding and expression of glutamate receptors glr-1, glr-4 and glr-5 in the AVA, AVD and AVE interneurons. We determined the expression pattern of fax-1 through immunofluorescence studies using anti-FAX-1 antisera. fax-1 is expressed in 18 neurons, the distal tip cells, and 4 "secondary" vulval cells. Expression in non-neuronal cells is limited to those stages during which these cells are undergoing migration or morphogenetic movements; the dtc's from L2 through L4, and the vulval cells only during L4. However, fax-1 deletion mutants do not have obvious gonadal or vulval defects. Expression in the nervous system appears to be very consistent from mid-embryogenesis through adulthood. FAX-1 protein is detected in the AVA, AVB, AVE, AIY, AVK, DVA, MI, and RIC neurons, plus two pairs of anterior neurons that have not yet been positively identified.

Katherine Weber et al. (2006) Development & Evolution Meeting "Domain Functions of NR2E Nuclear Receptors"

Danielle Snowflack, Stephen DeMeo, Melissa Cronin, Katherine Weber, Bruce Wightman, Rebecca Lombel, Sheila Clever

[Development & Evolution Meeting, 2006]

The nuclear receptors of the NR2E class play important roles in pattern formation and nervous system development. We are studying the function of NR2E DNA-binding domains (DBD) and ligand-binding domains (LBD) in C. elegans. We define two conserved groups of NR2E genes: the NR2E1 subclass, which includes C. elegans nhr-67, Drosophila tailless and dissatisfaction, and vertebrate Tlx (NR2E2, NR2E4, NR2E1), and the NR2E3 subclass, which includes C. elegans fax-1 and vertebrate PNR (NR2E5, NR2E3). PNR and Tll nuclear receptors have been shown to bind the hexamer half-site AAGTCA, instead of the hexamer AGGTCA recognized by most other nuclear receptors, suggesting unique DNA-binding properties for NR2E class members. FAX-1 (NR2E5), unlike NHR-67 and other NR2E1 subclass members, binds to hexamers with relaxed specificity: it will bind hexamers with the sequence ANGTCA, although it prefers a purine to a pyrimidine at the second position. We used site-directed mutagenesis to demonstrate that the difference between FAX-1 and NHR-67 binding preference is partially mediated by a conserved Asn residue at position 19 of the DNA-binding domain. This amino acid position is part of the “P box” that plays a critical role in defining binding site specificity and has been shown to make hydrogen-bond contacts to the second position of the hexamer in co-crystal structures for other nuclear receptors. We are currently examining the non-NR2E receptors NHR-4 and NHR-34 (provided by M. Van Gilst), which also have an Asn-19, to test whether Asn-19 is sufficient to confer the same relaxed specificity of DNA binding. The relaxed specificity allows FAX-1 to bind a large repertoire of potential binding sites. These findings define subclass-specific DNA-binding specificities for the NR2E1 and NR2E3 subclasses, and suggest that the relaxed specificity of binding observed for the NR2E3 subclass is partially mediated by a conserved Asn-19 in the P box. We are evaluating the specificity of LBD function by swapping the LBD"s from various NR2E receptors, including C. briggsae FAX-1, NHR-67, NHR-111, and human PNR, for the FAX-1 LBD, and testing the fusion genes for function in C. elegans by rescue of fax-1 mutants.

Stephen DeMeo et al. (2004) East Coast Worm Meeting "Degenerate binding sites for the FAX-1 nuclear receptor predict potential downstream target genes"

Sheila Clever, Rebecca Lombel, Stephen DeMeo, Aaron Wagner, Eric Smith, Bruce Wightman, Danielle Snowflack

[East Coast Worm Meeting, 2004]

The fax-1 gene encodes a nuclear hormone receptor that regulates neuron specification during embryogenesis by orchestrating the coordinated transcription of neuron-specific gene products. The human ortholog of fax-1 , PNR, functions in the generation of photoreceptor cells in the retina. Mutations in human PNR are a cause of inherited retinal degeneration and blindness. The human diseases caused by PNR mutations include Enhanced S-cone Sensitivity, in which S-cones are over-represented in the retina at the expense of rods and other cone types. This defect in neuron specification is reminiscent of the incorrect specification of neurons in fax-1 mutants (see Wightman et al. abstract). We are studying the DNA-binding properties of FAX-1 towards the goal of predicting the candidate regulatory target genes that mediate neuron specification. Like other nuclear receptors, FAX-1 demonstrates selective binding to hexameric half-sites. Using yeast one-hybrid and gel-shift assay techniques, we have shown preferential binding of FAX-1 to two dimeric half sites, AAGTCA, a known target of PNR and also AGGTCA, a sequence not previously shown to be a target of the NR2E class of nuclear hormone receptors. Optimal binding occurs when the directly-repeated hexamers are separated by a single base-pair ( e.g., AAGTCANAAGTCA). FAX-1 does not bind monomeric hexamer sites, but the existence of a strong binding site allows dimerization and binding to an adjacent weak binding site. FAX-1 exhibits only marginal preference for purines at the second position of hexamers , suggesting that the protein is relatively indifferent to the bases at that position. There are approximately 130 sites in the C. elegans genome with the sequence ANGTCANANGTCA, the predicted FAX-1 binding site from our analysis. We are evaluating candidate downstream genes by creating promoter::GFP fusions and testing them for dependence on fax-1 . P> P> Our studies also have implications for amino acid-base pair contacts in DNA-binding by FAX-1. In crystal structures, the second base-pair of each hexamer DNA site is contacted by amino acid 19 of the nuclear receptor DNA-binding domain. Both FAX-1 and PNR have asparagines at this position, while Tailless and Tlx , two nuclear receptors that discriminate between A and G at hexamer second base positions have aspartates . Thus the asparagines at amino acid 19 may mediate relaxed DNA-binding specificity for FAX-1 and PNR. Because FAX-1 and PNR have identical P-boxes, the short a -helical region that directly contact the DNA base-pairs, we expect that core hexameric binding properties of both proteins will be very similar or identical. Therefore, the investigation of FAX-1 DNA-binding properties may be predictive for identifying candidate targets of FAX-1 in C. elegans and PNR in humans.

Sheila Clever et al. (2005) International Worm Meeting "The NR2E nuclear receptors: fax-1 and nhr-67"

Aaron Wagner, Bruce Wightman, Stephen DeMeo, Katie Weber, Danielle Snowflack, Angela Cenci, Sheila Clever, Michelle Seif, Rebecca Lombel, Melissa Cronin

[International Worm Meeting, 2005]

The NR2E subfamily of nuclear receptors includes genes in vertebrates and invertebrates that function in nervous system development. For example, Tailless and its orthologs play critical roles in central nervous system development and PNR functions in photoreceptor differentiation in humans. In C. elegans, nhr-67 encodes the Tailless ortholog and fax-1 encodes the PNR ortholog. FAX-1 protein accumulates in the nuclei of eighteen neurons. Mutations in fax-1 cause an Unc phenotype, and we have previously described defects in glutamate receptor expression in the AVA and AVE neurons of fax-1 mutants. We are studying the DNA-binding properties of FAX-1 to identify target genes that mediate neuron specification. FAX-1 binds ANGTCA dimers that are separated by a single bp. Thus FAX-1 is much more permissive than Tailless, which preferentially binds AAGTCA. There are approximately 130 predicted FAX-1 binding sites in the genome. We evaluated one of these sites, upstream of the M05B5.6 gene, and saw no evidence for dependence on FAX-1. These studies have implications for amino acid-base pair contacts by FAX-1.The second base-pair of each hexamer DNA site is contacted by amino acid 19 of the nuclear receptor DNA-binding domain. Both FAX-1 and its orthologs have Asn at this position, while Tailless and its orthologs have Asp. Thus the Asn at amino acid 19 may mediate relaxed DNA-binding. We are investigating this possibility using site-directed mutagenesis. Deletion of nhr-67 results in L1 arrest. RNAi studies have shown a role for nhr-67 in movement and vulval morphogenesis. An nhr-67::gfp reporter is expressed in a few neurons in the head and transiently in the anchor cell during mid-larval development. We propose that nhr-67 functions in the anchor cell to regulate genes that participate in orchestrating vulval morphogenesis. Because fax-1 is expressed in four vulval cells during the L4 stage, it is possible that nhr-67 and fax-1 participate in a nuclear receptor conversation between AC and vulval cells. We are investigating ligand binding domain (LBD) function in nematodes by assaying for rescue of the fax-1 phenotype when various NR2E LBD's are fused to a fax-1 promoter and DNA-binding domain. We hope to learn how permissive LBD function is by evaluating which LBDs can substitute for the FAX-1 LBD.

Rebecca Hall et al. (2006) European Worm Meeting "Identification of pH Regulated Genes in the Nematode Caenorhabditis elegans: New Potential Drug Targets for the Treatment of Pathogenic Nematodes"

Fritz A. Muhlschlegel, Peter Klappa, Rebecca Hall

[European Worm Meeting, 2006]

Rebecca Hall, Peter Klappa and Fritz A. Mhlschlegel. Human pathogenic nematodes infect over 160 million people per year. Brugia malayi and Onchocerca volvulus are the causative agents of elephantiasis and river blindness, respectively. Treatment options are restricted and the most commonly used drug, Ivermection, is stage specific and has significant side effects. Furthermore, treatment failures due to drug resistance have been reported.. Brugia malayi and O. volvulus exhibit an indirect life cycle where an incubation period within an intermediate host vector is essential for development of the infectious stage. During host-vector transition pathogenic nematodes are exposed to extreme environmental changes including variations in pH. We decided to exploit the different environmental pH conditions encountered, in order to identify new therapeutic targets. Using C. elegans as a model we designed a survival assay and were able to show that the nematodes are extremely resistant to environmental pH changes. This result was shown to be independent of the cuticle by the use of collagen cuticle mutants. Microarray experiments identified a set of pH-regulated genes. These genes were up regulated at pH9, an environment encountered by pathogenic nematodes residing in the vector gut. The genes were silenced using RNA mediated interference to establish their requirement for survival. There were no significant phenotypic changes observed after five days of treatment, and exposure of the silenced nematodes to acidic and alkaline environments did not significantly reduce survival. However, in vivo studies are currently being carried out using carbonic anhydrase inhibitors to identify whether the candidate genes have potential for drug targeting.

(1975) Worm Breeder's Gazette "general description of Hirsh lab activities"

[Worm Breeder's Gazette, 1975]

As most people are probably aware by now, I first started by collecting a large number of temperature-sensitive mutants that are blocked in the reproductive life cycle. These mutants were then placed into six phenotypic categories. The zygote-defective mutants are those that lay fertilized eggs that fail to hatch. Gonadogenesis- defective mutants are those that when reared at restrictive temperature produce neither fertilized eggs nor progeny. Spermatogenesis mutants are those that when reared at restrictive temperature, can be rescued for progeny production by mating with wild type males at restrictive temperature. The accumulators are those that grow to an intermediate larval stage and either stop growing or die at an immature stage. The abnormal F1 are mutants that when placed at restrictive temperature grow up to be adults, produce progeny, but those progeny grow up to be sterile adults. In addition, a few temperature sensitive morphological mutants were isolated. Rebecca Vanderslice and I originally studied in greater detail three particular mutants in the zygote-defective category. They were interesting in two respects. (1) Not only did they show zygote- defective phenotype when the adults were placed in restrictive temperature, they also showed gonadogenesis-defective phenotype if they had been reared from L1 onward at high temperature. (2) Each of the three mutants is a maternal-effect mutant. At the present time, Becky Vanderslice and Bill Wood are re-examining the zygote-defective class and the gonadogenesis-defective class to find out which are and which are not maternal-effect mutants. One of the central questions we are asking is, what is the distribution of critical times of temperature sensitivity among the mutants and which of these are maternal effect mutants? We're asking how far into development do the mutants go with maternal contributions.

Jeb Gaudet et al. (2001) International C. elegans Meeting "Specification of Organ Identity by the C. elegans FOXA homologue PHA-4."

Jeb Gaudet, Michael Horner, Susan E Mango

[International C. elegans Meeting, 2001]

During development, cells acquire distinct cell fates in response to a host of regulators, many of which encode transcription factors. A longstanding issue has been the nature of the target genes controlled by these transcriptional regulators: do they activate a few genes at the top of a hierarchy, or control many genes that function at multiple stages throughout development? This question has been difficult to resolve because few direct targets have been identified. To address how transcription factors influence cell fate decisions, we are studying the role of the fork head box protein PHA-4 during pharynx development. In embryos that lack pha-4 activity, cells that would normally become part of the pharynx develop as ectoderm instead. Conversely, ectopic expression of pha-4 produces excess pharyngeal cells at the expense of other cell types. Because pha-4 normally functions in pharyngeal cells irrespective of the cells' lineage or cell type, we propose that pha-4 endows cells with pharyngeal organ identity. We have used a microarray chip approach to identify genes selectively expressed in the pharynx and have analyzed those genes to determine which are direct PHA-4 targets. We took advantage of maternal effect mutants that produce embryos with excess (par-1) or no (skn-1) pharyngeal cells to identify candidate pharyngeal genes. Included among our positive clones were most (>70%) genes known to be expressed in the pharynx, indicating that our approach worked well. Three lines of evidence suggest that PHA-4 directly activates most or all pharyngeally-expressed genes. First, we examined the pharynx-specific expression of a random set of microarray positives and found that the expression of each depended on one or more predicted PHA-4 binding sites. Second, PHA-4 is able to bind these sites in vitro. Third, temperature-shift studies indicate that PHA-4 is required throughout development, consistent with a model in which PHA-4 regulates both early and late acting genes. Our analysis also showed that the affinity of PHA-4 for different binding sites regulates the relative time of onset of the different target genes. When a PHA-4 binding site is converted by a single base-pair to a higher affinity site, expression initiates earlier. Conversely, a single base-pair mutation of a PHA-4 recognition sequence to one with lower affinity leads to a delay in the onset of expression. The affinity of PHA-4 binding in vitro matches the behavior of these reporter constructs in vivo. These findings suggest that modulating the affinity of PHA-4 binding sites within a promoter provides the embryo with a subtle mechanism to coordinate expression temporally throughout a developing organ. Since direct target genes of mammalian FOXA proteins include genes expressed early (e.g. endoderm) or late (e.g. liver) during vertebrate development, we suggest that global regulation of transcription within the foregut may be a common feature of this family of developmental regulators. We are indebted to Stuart Kim, Rebecca Begley, Carrie van Doren, Kyle Duke, and Min Jiang, who performed the microarray hybridization experiments.