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

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 (IP3)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 IP3 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 IP3 levels during the reproductive cycle

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

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 post-embryonic 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

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

HLH-29 Regulates <i>ftn-1</i> Expression.

<p>(A) Schematic representation of the <i>ftn-1</i> transgenes used in this study. Empty triangle, predicted HLH-30 binding site (CACGTG); filled triangle, predicted HLH-29 binding sites (CATGCG or CACGCG) with arrows indicating the corresponding orientations; striped triangle, predicted IDE region containing binding sites for GATA and HRE types of transcription factors. B) Relative intensity of the expression from integrated P_EcoRV<i>ftn-1::GFP</i> in the indicated intestinal rings of wild-type animals (dark bars, N = 61) and <i>hlh-29</i> mutants (light bars, N = 72). C) Relative intensities of the indicated <i>ftn-1</i> reporters in L4 stage, wild type animals subjected to control RNAi (dark bars) or <i>hlh-29</i> RNAi (light bars) RNAi. D) Relative intensities of the indicated <i>ftn-1</i> reporters in L4 stage, wild type animals subjected to control RNAi (dark bars) or <i>hlh-30</i> RNAi (light bars) RNAi. E) RT-qPCR measurements of <i>ftn-1</i> mRNA in wild-type animals and <i>hlh-29</i> mutant subjected to control RNAi (dark bars) or <i>hlh-29</i> RNAi (light bars). F) Relative intensities of the full length <i>ftn-1</i> reporter in L4 stage, <i>hlh-29</i> mutants subjected to control RNAi (dark bars) or <i>hlh-30</i> RNAi (light bars) RNAi. Error bars represent SEM, *P-value <0.05, **P-value <0.005, *** P-value <0.0005).</p