Inferences of glia-mediated control in Caenorhabditis elegans.

Astrocytes modulate synaptic transmission; yet, it remains unclear how glia influence complex behaviors. Here, we explore the effects of Caenorhabditis elegans astrocyte-like cephalic glia (CEPglia ) and the glia-specific bHLH transcription factor HLH-17 on mating behavior and the defecation motor program (DMP). In C. elegans, male mating has been explicitly described through the male tail circuit and is characterized by coordination of multiple independent behaviors to ensure that copulation is achieved. Furthermore, the sex-specific male mating circuitry shares similar components with the DMP, which is complex and rhythmic, and requires a fixed sequence of behaviors to be activated periodically. We found that loss of CEPglia reduced persistence in executing mating behaviors and hindered copulation, while males that lacked HLH-17 demonstrated repetitive prodding behavior that increased the time spent in mating but did not hinder copulation. During the DMP, we found that posterior body wall contractions (pBocs) and enteric muscle contractions (EMCs) were differentially affected by loss of HLH-17 or CEPglia in males and hermaphrodites. pBocs and EMCs required HLH-17 activity in both sexes, whereas loss of CEPglia alone did not affect DMP in males. Our data suggest that CEPglia mediate complex behaviors by signaling to the GABAergic DVB neuron, and that HLH-17 activity influences those discrete steps within those behaviors. Collectively, these data provide evidence of glia as a link in cooperative regulation of complex and rhythmic behavior that, in C. elegans links circuitry in the head and the tail.

HES-Mediated Repression of Pten in Caenorhabditis elegans.

The hairy/enhancer-of-split (HES) group of transcription factors controls embryonic development, often by acting downstream of the Notch signaling pathway; however, little is known about postembryonic roles of these proteins. In Caenorhabditis elegans, the six proteins that make up the REF-1 family are considered to be HES orthologs that act in both Notch-dependent and Notch-independent pathways to regulate embryonic events. To further our understanding of how the REF-1 family works to coordinate postembryonic cellular events, we performed a functional characterization of the REF-1 family member, HLH-25. We show that, after embryogenesis, hlh-25 expression persists throughout every developmental stage, including dauer, into adulthood. Like animals that carry loss-of-function alleles in genes required for normal cell-cycle progression, the phenotypes of hlh-25 animals include reduced brood size, unfertilized oocytes, and abnormal gonad morphology. Using gene expression microarray, we show that the HLH-25 transcriptional network correlates with the phenotypes of hlh-25 animals and that the C. elegans Pten ortholog, daf-18, is one major hub in the network. Finally, we show that HLH-25 regulates C. elegans lifespan and dauer recovery, which correlates with a role in the transcriptional repression of daf-18 activity. Collectively, these data provide the first genetic evidence that HLH-25 may be a functional ortholog of mammalian HES1, which represses PTEN activity in mice and human cells.

Dopamine signaling in C. elegans is mediated in part by HLH-17-dependent regulation of extracellular dopamine levels.

In Caenorhabditis elegans, the dopamine transporter DAT-1 regulates synaptic dopamine (DA) signaling by controlling extracellular DA levels. In dat-1(ok157) animals, DA is not taken back up presynaptically but instead reaches extrasynpatic sites, where it activates the dopamine receptor DOP-3 on choligeneric motor neurons and causes animals to become paralyzed in water. This phenotype is called swimming-induced paralysis (SWIP) and is dependent on dat-1 and dop-3. Upstream regulators of dat-1 and dop-3 have yet to be described in C. elegans. In our previous studies, we defined a role for HLH-17 during dopamine response through its regulation of the dopamine receptors. Here we continue our characterization of the effects of HLH-17 on dopamine signaling. Our results suggest that HLH-17 acts downstream of dopamine synthesis to regulate the expression of dop-3 and dat-1. First, we show that hlh-17 animals display a SWIP phenotype that is consistent with its regulation of dop-3 and dat-1. Second, we show that this behavior is enhanced by treatment with the dopamine reuptake inhibitor, bupropion, in both hlh-17 and dat-1 animals, a result suggesting that SWIP behavior is regulated via a mechanism that is both dependent on and independent of DAT-1. Third, and finally, we show that although the SWIP phenotype of hlh-17 animals is unresponsive to the dopamine agonist, reserpine, and to the antidepressant, fluoxetine, hlh-17 animals are not defective in acetylcholine signaling. Taken together, our work suggests that HLH-17 is required to maintain normal levels of dopamine in the synaptic cleft through its regulation of dop-3 and dat-1.

Genome-wide microarrray analysis reveals roles for the REF-1 family member HLH-29 in ferritin synthesis and peroxide stress response.

In Caenorhabditis elegans, the six proteins that make up the REF-1 family have been identified as functional homologs of the Hairy/Enhancer of Split (HES) proteins. These transcription factors act in both Notch dependent and Notch-independent pathways to regulate embryonic events during development; however, their post-embryonic functions are not well defined. As a first step toward understanding how the REF-1 family works together to coordinate post-embryonic events, we used gene expression microarray analysis to identify transcriptional targets of HLH-29 in L4/young adult stage animals. Here we show that HLH-29 targets are genes needed for the regulation of growth and lifespan, including genes required for oxidative stress response and fatty acid metabolism, and the ferritin genes, ftn-1 and ftn-2. We show that HLH-29 regulates ftn-1 expression via promoter sequences upstream of the iron-dependent element that is recognized by the hypoxia inducible factor, HIF-1. Additionally, hlh-29 mutants are more resistant to peroxide stress than wild-type animals and ftn-1(RNAi) animals, even in the presence of excess iron. Finally we show that HLH-29 acts parallel to DAF-16 but upstream of the microphthalmia transcription factor ortholog, HLH-30, to regulate ftn-1 expression under normal growth conditions.

HLH-29 regulates ovulation in C. elegans by targeting genes in the inositol triphosphate signaling pathway.

The reproductive cycle in the nematode Caenorhabditis elegans depends in part on the ability of the mature oocyte to ovulate into the spermatheca, fuse with the sperm during fertilization, and then exit the spermatheca as a fertilized egg. This cycle requires the integration of signals between the germ cells and the somatic gonad and relies heavily on the precise control of inositol 1,4,5 triphosphate (IP(3))levels. The HLH-29 protein, one of five Hairy/Enhancer of Split (HES) homologs in C. elegans, was previously shown to affect development of the somatic gonad. Here we show that HLH-29 expression in the adult spermatheca is strongly localized to the distal spermatheca valve and to the spermatheca-uterine valve, and that loss of hlh-29 activity interferes with oocyte entry into and egg exit from the spermatheca. We show that HLH-29 can regulate the transcriptional activity of the IP(3) signaling pathway genes ppk-1, ipp-5, and plc-1 and provide evidence that hlh-29 acts in a genetic pathway with each of these genes. We propose that the HES-like protein HLH-29 acts in the spermatheca of larval and adult animals to effectively increase IP(3) levels during the reproductive cycle.

Modulation of dopamine-dependent behaviors by the Caenorhabditis elegans Olig homolog HLH-17.

In vertebrates and invertebrates, dopamine signaling modulates a wide variety of physical and behavioral functions and exerts these effects through heterotrimeric G proteins. The soil nematode Caenorhabditis elegans has been used to model dopamine signaling and reacts reproducibly to alterations in dopamine levels through eight well-characterized dopaminergic neurons located in the head. In C. elegans, the basic helix-loop-helix transcription factor HLH-17 is strongly and constitutively expressed in the glia cells that ensheath four of the dopaminergic neurons, yet it is not required for specification or development of either the glia or the neurons. In this study, we sought to determine whether HLH-17 functions in dopamine signaling. We found that, unlike wild-type animals, hlh-17 animals are resistant to the effects of exogenous dopamine on egg laying and mobility. hlh-17 animals are also defective in the basal slowing and gustatory plasticity behaviors that require functional dopamine signaling. We also found that the expression of the dopamine receptor genes dop-1, dop-2, and dop-3 and the RGS protein gene egl-10 is significantly reduced in hlh-17 animals. Together these results point to a role for HLH-17 in dopamine signaling in C. elegans.

Molecular characterization of the Caenorhabditis elegans REF-1 family member, hlh-29/hlh-28.

Members of the Caenorhabditis elegans REF-1 family of bHLH proteins are atypical in that each protein contains two bHLH domains. In this study we describe a functional and molecular characterization of the REF-1 family members, hlh-29/hlh-28. 5'-RACE results confirm the presence of two bHLH domain coding regions in a single transcript and quantitative PCR (qPCR) shows that hlh-29/hlh-28 mRNA is detected in wild-type animals throughout development. A promoter fusion of hlh-29 to the green fluorescent protein shows post-embryonic reporter activity in cells of the vulva, the somatic gonad, the intestine and in neuronal cells of the head and tail. Loss of hlh-29/hlh-28 function via RNA interference (RNAi) results in multiple phenotypes including late embryonic lethality, yolk protein accumulation, everted vulva, bordering behavior, and alter chemosensory responses.

Middle/high school students in the research laboratory: A summer internship program emphasizing the interdisciplinary nature of biology.

We describe an eight-week summer Young Scientist in Training (YSIT) internship program involving middle and high school students. This program exposed students to current basic research in molecular genetics, while introducing or reinforcing principles of the scientific method and demonstrating the uses of mathematics and chemistry in biology. For the laboratory-based program, selected students from Baltimore City Schools working in groups of three were teamed with undergraduate research assistants at Morgan State University. Teams were assigned a project that was indirectly related to our laboratory research on the characterization of gene expression in Caenorhabditis elegans. At the end of the program, teams prepared posters detailing their accomplishments, and presented their findings to parents and faculty members during a mini-symposium. The posters were also submitted to the respective schools and the interns were offered a presentation of their research at local high school science fairs.

Molecular characterization of HLH-17, a C. elegans bHLH protein required for normal larval development.

The basic helix-loop-helix (bHLH) transcription factor family regulates numerous developmental events in eukaryotic cells. In the model system, C. elegans, thirty-seven bHLH proteins have been identified via genome-wide sequence analysis and fourteen have been genetically characterized to date. These proteins influence cell fate specification of neural lineages and differentiation of myogenic lineages and have distinct roles in somatic gonadogenesis. We report here on the molecular characterization of HLH-17, a protein whose putative bHLH domain is homologous to the mammalian bHLH proteins BETA3 and bHLHB5. The gene hlh-17 is transcriptionally active at all developmental stages, with the highest steady state accumulation of hlh-17 mRNA during embryogenesis. An upstream hlh-17 sequence drives expression of GFP in the sheath cells of the cephalic sensilla. Finally, animals that are defective in HLH-17 via RNAi display egg-laying defects, while those carrying null mutations in hlh-17 do not develop beyond the L2 stage and are less attracted to potassium and sodium ions. We propose that hlh-17 affects the ability of C. elegans to respond to food cues, with possible downstream effects on insulin-signaling genes involved in the normal development and reproductive viability of the worm.

Identifying novel helix-loop-helix genes in Caenorhabditis elegans through a classroom demonstration of functional genomics.

A 14-week, undergraduate-level Genetics and Population Biology course at Morgan State University was modified to include a demonstration of functional genomics in the research laboratory. Students performed a rudimentary sequence analysis of the Caenorhabditis elegans genome and further characterized three sequences that were predicted to encode helix-loop-helix proteins. Students then used reverse transcription-polymerase chain reaction to determine which of the three genes is normally expressed in C. elegans. At the end of this laboratory activity, students were 1) to demonstrate a rudimentary knowledge of bioinformatics, including the ability to differentiate between "having" a gene and "expressing" a gene, and 2) to understand basic approaches to functional genomics, including one specific technique for assaying for gene expression. It was also anticipated that students would increase their skills at effectively communicating their research activities through written and/or oral presentation. This article describes the laboratory activity and the assessment of the effectiveness of the activity.