[
International C. elegans Meeting,
1999]
Our lab is studying the evolution of developmental processes by comparing vulva development between C. elegans and Pristionchus pacificus . This cellular, genetic and molecular comparison links changes in cell fate specification to differences in the molecular mechanisms during development. Ultimately, comparative developmental biology has to consider not only the mechanistic but also the phylogenetic perspective. The identification of genes that have changed their function over long evolutionary timescales, such as C. elegans and P. pacificus can only be the first step. More detailed comparison at the phylogenetic level have to follow by studying more closely related species. Fortunately, molecular and morphological analyses provide us with detailed phylogenetic trees of nematodes. Based on the current phylogeny, several species can be selected for functional analysis. Here we describe our initial genetic and molecular work in Diplogasteroides berwigi , an ancestral member of the Diplogastridae. This hermaphroditic strain has a 5 day generation time and is accessible to genetic analysis. One of the aims of comparative genetics in this species is the functional comparison of
mab-5 , a homeotic transcription factor, that has changed its function dramatically between P. pacificus and C. elegans .
mab-5 cloning from D. berwigi and first mutant analysis is reported.
[
International Worm Meeting,
2007]
Hydrogen sulfide (H<sub>2</sub>S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H<sub>2</sub>S to begin to elucidate the molecular mechanisms of H<sub>2</sub>S action. We show that nematodes exposed to H<sub>2</sub>S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H<sub>2</sub>S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H<sub>2</sub>S. Increased thermotolerance and lifespan is not observed in the
sir-2.1(
ok434) deletion mutant exposed to H<sub>2</sub>S. However, mutants in the insulin signaling pathway (both
daf-2 and
daf-16), animals with mitochondrial dysfunction (
isp-1 and
clk-1) and a genetic model of caloric restriction (
eat-2) all exhibit H<sub>2</sub>S-induced increased thermotolerance. These data suggest that H<sub>2</sub>S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H<sub>2</sub>S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H<sub>2</sub>S.
Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.
[
International Worm Meeting,
2017]
Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.
[
International Worm Meeting,
2017]
Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.
[
International Worm Meeting,
2013]
Entomopathogenic nematodes of the genus Heterorhabditis are insect killers that live in mutually beneficial symbiosis with pathogenic Photorhabdus bacteria. Photorhabdus is rapidly lethal to insects and to other nematodes, including C. elegans, but is required for Heterorhabditis growth in culture and for the insect-killing that defines the entomopathogenic lifestyle. The symbiosis between Heterorhabditis and Photorhabdus offers the potential to study the molecular genetic basis of their cooperative relationship. We developing tools to make such studies more feasible: we have been studying multiple nematodes of the genus Heterorhabditis and developing tools for the molecular genetic analysis of Heterorhabditis bacteriophora.
Many species of Heterorhabditis and variants of Photorhabdus have been isolated; some pairings show specificity in their ability to establish a symbiotic relationship. To better understand these interactions and other variations in the lifestyles of Heterorhabditis, we have sequenced H. indica, H. megidis, H. sonorensis, and H. zealandica; a H. bacteriophora genome sequence is available. A comparison of these closely related species may help us to identify mechanisms that regulate the response to bacterial interactions and to find variations that correlate with differences in lifestyle or bacterial compatibility.
In addition to genomics, we are developing H. bacteriophora as a laboratory organism. H. bacteriophora grows well on plates, has been reported to be susceptible to RNAi and transgenesis, and can develop as a selfing hermaphrodite, and so should be a powerful system for the molecular genetic study of the aspects of biology to which it is uniquely well suited, most prominently symbiosis. This potential is severely diminished by inconvenient sex determination: the self-progeny of hermaphrodites are mostly females with some males; at low density, their progeny are almost exclusively females. We have screened for and isolated a constitutively hermaphroditic mutant for use in molecular genetic studies of symbiosis. This mutant also offers the opportunity to explore the basis of hermaphrodite sex determination in H. bacteriophora.
[
Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI,
2010]
Heterorhabditis bacteriophora is a species of insect-parasitic nematode that lives in mutually beneficial symbiosis with pathogenic Photorhabdus luminescens bacteria. P. luminescens bacteria are lethal to insects and to other nematodes, including the soil nematode Caenorhabditis elegans, but are required for H. bacteriophora growth. The symbiosis between H. bacteriophora and P. luminescens therefore offers the potential to study the molecular genetic basis of their cooperative relationship. We are interested in developing tools to make such studies more feasible; in particular, we propose to generate tools for genetic mapping in H. bacteriophora. We will test independent isolates of H. bacteriophora to ensure that the isolates are cross-fertile. We will then examine the abilities of these isolates to grow on and to become infected with different wild-type and mutant Photorhabdus bacteria. From these tests we will select an isolate on which we will use next-generation high-throughput sequencing technology for the purpose of refining the existing draft genome sequence and for the creation of a SNP map. We anticipate that this SNP map will enable us and the wider insect-parasitic nematode community to identify induced mutations and natural variations affecting the interactions between H. bacteriophora nematodes and pathogenic Photorhabdus bacteria.
[
International Worm Meeting,
2013]
MicroRNAs (miRNAs) are small non-coding regulatory RNAs that regulate gene expression at the post-transcriptional level and are involved in a broad spectrum of biological processes. Rearrangements of inter- or intra-molecular RNA structures and conformational changes of ribonucleoprotein complexes in the multiple processes of the miRNA pathway have led to the involvement of RNA helicase activities but little is known so far. In eukaryotes, RNA helicases generally belong to the superfamily 2 (SF2) in helicase classification, especially the DExD/H-box helicase family. The DExD/H-box proteins have been shown in association with many cellular processes involving RNA. To better understand the possible roles of DExD/H-box RNA helicases in miRNA function, we employed RNAi screen to identify genetic interaction between C. elegans DExD/H-box RNA helicases and the
let-7 miRNA, which controls the timing of cell cycle exit and terminal differentiation. In addition to the RNA helicase
p72, a component of Drosha Microprocessor complex, and CGH-1 that has been reported to facilitate the function of miRNA-induced silencing complex (miRISC), we found several DExD/H-box RNA helicases, which are involved in ribosomal RNA processing, pre-mRNA splicing and mRNA surveillance, may also take part in miRNA biogenesis and/or function. (Support: National Science Council, Taiwan. NSC 100-2311-B-002-006-MY3).
[
Development & Evolution Meeting,
2006]
Several Notch interactions occur in rapid succession during early C.elegans embryogenesis, each resulting in a distinct fate. We have previously shown that
ref-1, a gene distantly related to Drosophila E(spl), is a direct Notch target in all these interactions. We show here that
ref-1 expression is controlled by multiple, Notch-dependent enhancers that are specific for each interaction. We have identified a 150bp enhancer that is specific to Notch interactions in the endoderm, and found similar enhancers with the same activity in C.briggsae and C.remanei. The endoderm enhancer contains multiple, conserved binding sites for LAG-1/Su(H) and GATA transcription factors. We demonstrate that all LAG-1/Su(H) and GATA sites are required for full activity of this enhancer. We provide evidence that a GATA transcription factor called ELT-2 is a cooperative factor for Notch in the endoderm. Ectopic expression of ELT-2 in non-endodermal lineages caused activation of the endoderm enhancer only in Notch-signaled cells, suggesting that the presence of the cooperative factor dictates in which Notch interaction a Notch-dependent enhancer becomes responsive in vivo. Previous studies in Drosophila showed that synergy between Su(H) and the bHLH transcription factor Daughterless requires a specific configuration of Su(H) sites, known as a Su(H) paired site, SPS. The endoderm enhancer contains a putative SPS. However, we find that Notch-GATA synergy does not require a SPS, and instead requires a non-SPS configuration of oriented LAG-1/Su(H) sites. Thus, it appears that that each configuration couples Notch signaling with a specific class of transcription factor.