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.

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.