Shen, Y et al. (2011) International Worm Meeting "Regulation of Hermaphrodite Development by the F-box Protein SHE-1."

Shen, Y, Ellis, Ronald E

[International Worm Meeting, 2011]

In two androdioecious species of Caenorhabditis, F-box proteins play critical roles in hermaphrodite development. In C. elegans, FOG-2 interacts with GLD-1 to repress the translation of tra-2 mRNAs. Although C. briggsae lacks an ortholog of FOG-2, the F-box protein SHE-1 acts independently of GLD-1 to down-regulate TRA-2. Because a missense mutation in the F-box domain inactivates SHE-1, it might function as a classical F-box protein, bringing a target to the E3 ubiquitin ligase complex, where it is marked for degradation. To identify possible SHE-1 targets, we are using the yeast two-hybrid system. From a screen of about 340,000 cDNAs, we identified three genes that gave multiple, independent clones. One of these clones passed two further tests. First, PQN-94 interacts with SHE-1 when used as bait in the yeast two-hybrid system. Second, using RNA interference to knock down pqn-94 partially suppresses the she-1 phenotype. At 25 deg C, she-1(v49); control(RNAi) mothers never produced hermaphrodites, but she-1(v49); pqn-94(RNAi) mothers produced 7% hermaphrodites. Additional tests with she-1(v35) showed the same pattern of suppression. However, knocking down pqn-94 on its own had no phenotype. To see if SHE-1 controls germ cell fates by targeting PQN-94 for degradation, we are carrying out three sets of experiments. (1) We are testing both proteins for interaction when expressed in HEK 293 cells. (2) We are preparing antibodies to each protein, to test their in vivo activities and distributions. And (3) we are looking for other proteins that interact with PQN-94, to learn how it might influence germ cell fates.

Shen Y et al. (2014) J Neurosci "EOL-1, the homolog of the mammalian Dom3Z, regulates olfactory learning in C. elegans."

Shen Y, Zhang J, Calarco JA, Zhang Y

[J Neurosci, 2014]

Learning is an essential function of the nervous system. However, our understanding of molecular underpinnings of learning remains incomplete. Here, we characterize a conserved protein EOL-1 that regulates olfactory learning in Caenorhabditis elegans. A recessive allele of eol-1 (enhanced olfactory learning) learns better to adjust its olfactory preference for bacteria foods and eol-1 acts in the URX sensory neurons to regulate learning. The mammalian homolog of EOL-1, Dom3Z, which regulates quality control of pre-mRNAs, can substitute the function of EOL-1 in learning regulation, demonstrating functional conservation between these homologs. Mutating the residues of Dom3Z that are critical for its enzymatic activity, and the equivalent residues in EOL-1, abolishes the function of these proteins in learning. Together, our results provide insights into the function of EOL-1/Dom3Z and suggest that its activity in pre-mRNA quality control is involved in neural plasticity.

Shen Y et al. (2016) Elife "An extrasynaptic GABAergic signal modulates a pattern of forward movement in Caenorhabditis elegans."

Qin Y, Kawano T, Liu H, Zhong C, Samuel AD, Wu M, Xu T, Harris G, Zhang Y, Shen Y, Wen Q

[Elife, 2016]

As a common neurotransmitter in the nervous system, -aminobutyric acid (GABA) modulates locomotory patterns in both vertebrates and invertebrates. However, the signaling mechanisms underlying the behavioral effects of GABAergic modulation are not completely understood. Here, we demonstrate that a GABAergic signal in C. elegans modulates the amplitude of undulatory head bending through extrasynaptic neurotransmission and conserved metabotropic receptors. We show that the GABAergic RME head motor neurons generate undulatory activity patterns that correlate with head bending and the activity of RME causally links with head bending amplitude. The undulatory activity of RME is regulated by a pair of cholinergic head motor neurons SMD, which facilitate head bending, and inhibits SMD to limit head bending. The extrasynaptic neurotransmission between SMD and RME provides a gain control system to set head bending amplitude to a value correlated with optimal efficiency of forward movement.

Shen Y et al. (2024) Mol Biol Evol "Rewiring the Sex-Determination Pathway During the Evolution of Self-Fertility."

Vassalotti A, Amin R, Romanowski J, Tierney A, Shen Y, Lin SY, Ellis RE, Schmidt E, Harbin J

[Mol Biol Evol, 2024]

Although evolution is driven by changes in how regulatory pathways control development, we know little about the molecular details underlying these transitions. The TRA-2 domain that mediates contact with TRA-1 is conserved in Caenorhabditis. By comparing the interaction of these proteins in two species, we identified a striking change in how sexual development is controlled. Identical mutations in this domain promote oogenesis in Caenorhabditis elegans but promote spermatogenesis in Caenorhabditis briggsae. Furthermore, the effects of these mutations involve the male-promoting gene fem-3 in C. elegans but are independent of fem-3 in C. briggsae. Finally, reciprocal mutations in these genes show that C. briggsae TRA-2 binds TRA-1 to prevent expression of spermatogenesis regulators. By contrast, in C. elegans TRA-1 sequesters TRA-2 in the germ line, allowing FEM-3 to initiate spermatogenesis. Thus, we propose that the flow of information within the sex determination pathway has switched directions during evolution. This result has important implications for how evolutionary change can occur.

Shen Y et al. (2012) Science "A steroid receptor-microRNA switch regulates life span in response to signals from the gonad."

Shen Y, Magner D, Karalay O, Antebi A, Wollam J

[Science, 2012]

Although the gonad primarily functions in procreation, it also affects animal life span. Here, we show that removal of the Caenorhabditis elegans germ line triggers a switch in the regulatory state of the organism to promote longevity, co-opting components involved in larval developmental timing circuits. These components include the DAF-12 steroid receptor, which is involved in the larval stage two-to-stage three (L2-L3) transition and up-regulates members of the let-7 microRNA (miRNA) family. The miRNAs target an early larval nuclear factor lin-14 and akt-1/kinase, thereby stimulating DAF-16/FOXO signaling to extend life. Our studies suggest that metazoan life span is coupled to the gonad through elements of a developmental timer.

Shen, Y. et al. (2017) International Worm Meeting "Diverse types of regulatory mutations drive the evolution of sex in nematode germ cells."

Shen, Y., Lin, S.-Y., Ellis, R. E.

[International Worm Meeting, 2017]

In self-fertile nematodes, XX animals have been modified so that larvae make sperm. Since the XX soma remains female, these changes must be restricted to the germ line. Furthermore, they have to be precisely regulated so the animals can switch to oogenesis as adults. To identify the regulatory changes that underlie this trait, we are comparing the nematodes C. elegans and C. briggsae, and have started work with C. tropicalis. Each species evolved self-fertility independently. These nematodes share a conserved sex-determination pathway that acts through the transcription factor TRA-1, a homolog of the human Gli proteins. Males make HER-1, which binds to and inactivates the TRA- 2 receptor on each cell, allowing the FEM proteins to target TRA-1 for degradation. Females and hermaphrodites lack HER-1, allowing TRA-2 to inhibit the FEM complex. As a result, TRA-1 is cleaved, producing a repressor that turns off male genes. By studying evolutionary change in this pathway we show that: (1) each species has adopted independent and unique solutions for self-fertility, and (2) these regulatory changes can affect almost every step of the pathway upstream of TRA-1. Most of our data come from the direct comparison of similar mutations in different species, made through gene editing. First, the functions of chromatin regulatory complexes have been altered. The Tip60 HAT complex is essential for spermatogenesis in C. briggsae, but plays only a minor role in C. elegans. In addition, the NURF complex is needed for spermatogenesis only in C. briggsae. Finally, mutations that affect the WDR-5 proteins favor oogenesis in C. briggsae, but spermatogenesis in C. elegans. Second, translational regulation has changed dramatically. We analyzed translational control of fem-3, and found that 3'-UTR mutations that completely block oogenesis in C. elegans result in normal hermaphrodite development in C. briggsae. The Schedl lab showed that in C. elegans, the unique FOG-2 protein works with GLD-1 to block translation of tra-2 messages, allowing XX spermatogenesis. Finally, the Haag lab has shown that PUF proteins play unique roles in C. briggsae. Third, regulation through protein-protein interactions has been altered. An intracellular fragment of TRA-2 can bind TRA-1 in both C. elegans and C. briggsae. Mutations that prevent this interaction cause oogenesis in C. elegans, but orthologous mutations cause either constitutive spermatogenesis or hermaphrodite development in C. briggsae. Furthermore, C. briggsae relies on the novel SHE-1 protein for self-fertility, and SHE-1 works in part through its binding partner, PQN-94. Thus, selection can alter the sex-determination pathway at almost any point to promote self-fertility. We propose that pathways organized around a binary switch are particularly flexible during evolution, since any change to one of several competing factors should change the final decision.

Shen Y et al. (2016) PLoS One "C. elegans miro-1 Mutation Reduces the Amount of Mitochondria and Extends Life Span."

Inoue T, Gruber J, Shen Y, Low NP, Ng LF, Hagen T

[PLoS One, 2016]

Mitochondria play a critical role in aging, however, the underlying mechanism is not well understood. We found that a mutation disrupting the C. elegans homolog of Miro GTPase (miro-1) extends life span. This phenotype requires simultaneous loss of miro-1 from multiple tissues including muscles and neurons, and is dependent on daf-16/FOXO. Notably, the amount of mitochondria in the miro-1 mutant is reduced to approximately 50% of the wild-type. Despite this reduction, oxygen consumption is only weakly reduced, suggesting that mitochondria of miro-1 mutants are more active than wild-type mitochondria. The ROS damage is slightly reduced and the mitochondrial unfolded protein response pathway is weakly activated in miro-1 mutants. Unlike previously described long-lived mitochondrial electron transport chain mutants, miro-1 mutants have normal growth rate. These results suggest that the reduction in the amount of mitochondria can affect the life span of an organism through activation of stress pathways.

Shen Y et al. (2008) PLoS One "Comparing platforms for C. elegans mutant identification using high-throughput whole-genome sequencing."

Hobert O, Pe'er I, Shen Y, Sarin S, Liu Y

[PLoS One, 2008]

BACKGROUND: Whole-genome sequencing represents a promising approach to pinpoint chemically induced mutations in genetic model organisms, thereby short-cutting time-consuming genetic mapping efforts. PRINCIPAL FINDINGS: We compare here the ability of two leading high-throughput platforms for paired-end deep sequencing, SOLiD (ABI) and Genome Analyzer (Illumina; "Solexa"), to achieve the goal of mutant detection. As a test case we used a mutant C. elegans strain that harbors a mutation in the lsy-12 locus which we compare to the reference wild-type genome sequence. We analyzed the accuracy, sensitivity, and depth-coverage characteristics of the two platforms. Both platforms were able to identify the mutation that causes the phenotype of the mutant C. elegans strain, lsy-12. Based on a 4 MB genomic region in which individual variants were validated by Sanger sequencing, we observe tradeoffs between rates of false positives and false negatives when using both platforms under similar coverage and mapping criteria. SIGNIFICANCE: In conclusion, whole-genome sequencing conducted by either platform is a viable approach for the identification of single-nucleotide variations in the C. elegans genome.

Shen Y et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Identify and Characterize Novel Molecular Regulators of Olfactory Learning with Forward Genetic Screens"

Zhang Y, Shen Y, Ha H

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Neural plasticity is a remarkable feature of the nervous system because it enables organisms to modify their behavior by learning. One way to study neural plasticity is to identify and characterize conserved molecular and cellular mechanisms underlying learning and memory. To this end, we exploit Caenorhabditis elegans, because its nervous system is unusually simple and well-characterized and because it is accessible to genetic, molecular and imaging methods. Our previous work has shown that C. elegans learns to avoid the smell of bacteria that are pathogenic, such as Pseudomonas aeruginosa PA14, while remaining attracted to the smell of the ones that serve as food source, such as Escherichia coli OP50 (Zhang et al, 2005, Nature, 438:179). Our study on the functional assembly of the neuronal network for olfactory learning has identified a circuit that is specifically required for the learning to occur (Meeting abstract #18717). This learning circuit consists of a pair of serotonergic neurons and their downstream interneurons and motor neurons. Laser ablation of these neurons specifically compromised the ability of animals to learn to avoid pathogens without affecting their ability to recognize and distinguish different bacteria strains. We have conducted a genetic screen for novel molecular regulators that function in this learning circuit to generate aversive learning by screening for mutants whose phenotypes mimic the effects of killing the neurons in this circuit. We used EMS as the mutagen and screened about 1,000 haploid genomes with an automated learning assay. This screen has allowed us to identify 3 candidate mutants with different defects in their learning ability. One of the learning mutants appears to be learning defective while two other mutants showed increased learning ability. We are currently conducting backcrossing and plan to identify the molecular nature of the mutations with genome sequencing. Based on the efficiency of EMS and the size of the C. elegans genome, our screen is far from saturation. One immediate extension of the work is to continue the forward genetic screen to isolate additional mutants with learning defects. References:Zhang, Y., Lu, H. and Bargmann, C. I. Pathogenic bacteria induce aversive olfactory learning in C. elegans. Nature 438, 179-184 (2005).

Perreault J et al. (2007) Mol Biol Evol "Ro-associated Y RNAs in metazoans: evolution and diversification."

Perreault J, Perreault JP, Boire G

[Mol Biol Evol, 2007]

The Y genes encode small non-coding RNAs whose functions remain elusive, whose numbers vary between species, and whose major property is to be bound by the Ro60 protein (or its ortholog in other species). To better understand the evolution of the Y gene family, we performed a homology search in 27 different genomes along with a structural search using Y RNA specific motifs. These searches confirmed that Y RNAs are well conserved in the animal kingdom and resulted in the detection of several new Y RNA genes, including the first Y RNAs in insects and a second Y RNA detected in Caenorhabditis elegans. Unexpectedly, Y5 genes were retrieved almost as frequently as Y1 and Y3 genes, and, consequently are not the result of a relatively recent apparition as is generally believed. Investigation of the organization of the Y genes demonstrated that the synteny was conserved among species. Interestingly, it revealed the presence of six putative "fossil" Y genes, all of which were Y4 and Y5 related. Sequence analysis led to inference of the ancestral sequences for all Y RNAs. In addition, the evolution of existing Y RNAs was deduced for many families, orders and classes. Moreover, a consensus sequence and secondary structure for each Y species was determined. Further evolutionary insight was obtained from the analysis of several thousand Y retropseudogenes among various species. Taken together, these results confirm the rich and diversified evolution history of Y RNAs.