Circadian control of eclosion: interaction between a central and peripheral clock in Drosophila melanogaster.

Drosophila melanogaster display overt circadian rhythms in rest:activity behavior and eclosion. These rhythms have an endogenous period of approximately 24 hr and can adjust or "entrain" to environmental inputs such as light. Circadian rhythms depend upon a functioning molecular clock that includes the core clock genes period and timeless (reviewed in and ). Although we know that a clock in the lateral neurons (LNs) of the brain controls rest:activity rhythms, the cellular basis of eclosion rhythms is less well understood. We show that the LN clock is insufficient to drive eclosion rhythms. We establish that the prothoracic gland (PG), a tissue required for fly development, contains a functional clock at the time of eclosion. This clock is required for normal eclosion rhythms. However, both the PG clock function and eclosion rhythms require the presence of LNs. In addition, we demonstrate that pigment-dispersing factor (PDF), a neuropeptide secreted from LNs, is necessary for the PG clock and eclosion rhythms. Unlike other clocks in the fly periphery, the PG is similar to mammalian peripheral oscillators because it depends upon input, including PDF, from central pacemaker cells. This is the first report of a peripheral clock necessary for a circadian event.

The circadian control of eclosion.

Eclosion is the stage in development when the adult insect emerges from the shell of its old cuticle. The sequence of behaviors necessary for eclosion is coordinated by an integrated system of hormones and is activated by hormones that relay developmental readiness. The circadian clock, which controls the timing of behaviors such as the rest: activity rhythm of adult insects, also controls eclosion timing. A number of groups are actively investigating the mechanisms by which the circadian clock restricts or gates eclosion to a particular time of day. Data from these studies are beginning to reveal details of the molecular and physiological basis of the eclosion rhythm.

Mapping a mutation in Caenorhabditis elegans using a polymerase chain reaction-based approach.

Many single nucleotide polymorphisms (SNPs) have been identified within the Caenorhabditis elegans genome. SNPs present in the genomes of two isogenic C. elegans strains have been routinely used as a tool in forward genetics to map a mutation to a particular chromosome. This article describes a laboratory exercise in which undergraduate students use molecular biological techniques to map a mutation to a chromosome using a set of SNPs. Through this multi-week exercise, students perform genetic crosses, DNA extraction, polymerase chain reaction, restriction enzyme digests, agarose gel electrophoresis, and analysis of restriction fragment length polymorphisms. Students then analyze their results to deduce the chromosomal location of the mutation. Students also use bioinformatics websites to develop hypotheses that link the genotype to the phenotype.

Measuring the effects of bacteria on C. elegans behavior using an egg retention assay.

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity and vibration. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior. EIW assays involve counting the number of eggs retained in the uterus of C. elegans. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine.

Gαo and Gαq regulate the expression of daf-7, a TGFß-like gene, in Caenorhabditis elegans.

Caenorhabditis elegans enter an alternate developmental stage called dauer in unfavorable conditions such as starvation, overcrowding, or high temperature. Several evolutionarily conserved signaling pathways control dauer formation. DAF-7/TGFß and serotonin, important ligands in these signaling pathways, affect not only dauer formation, but also the expression of one another. The heterotrimeric G proteins GOA-1 (Gα(o)) and EGL-30 (Gα(q)) mediate serotonin signaling as well as serotonin biosynthesis in C. elegans. It is not known whether GOA-1 or EGL-30 also affect dauer formation and/or daf-7 expression, which are both modulated in part by serotonin. The purpose of this study is to better understand the relationship between proteins important for neuronal signaling and developmental plasticity in both C. elegans and humans. Using promoter-GFP transgenic worms, it was determined that both goa-1 and egl-30 regulate daf-7 expression during larval development. In addition, the normal daf-7 response to high temperature or starvation was altered in goa-1 and egl-30 mutants. Despite the effect of goa-1 and egl-30 mutations on daf-7 expression in various environmental conditions, there was no effect of the mutations on dauer formation. This paper provides evidence that while goa-1 and egl-30 are important for normal daf-7 expression, mutations in these genes are not sufficient to disrupt dauer formation.