Soochin Cho et al. (2001) International C. elegans Meeting "Hermaphrodite or female? Specification of germ cell fates during nematode evolution"

Katherine A Tucker, Suk-Won Jin, Pei-Jiun Chen, Soochin Cho, Adam B Cohen, Meng Yao, Ronald E Ellis

[International C. elegans Meeting, 2001]

Most animal species consist of two different sexes, whose body plans and behaviors are specialized to help them find and mate with each other. Despite the importance of sexual reproduction, all aspects of this process evolve rapidly. To elucidate how these rapid change occur, we are studying the control of germ cell fate in the genus Caenorhabditis , which contains nematodes with two different types of reproductive systems. C. elegans and C. briggsae have male and hermaphrodite sexes, whereas C. remanei has male and female sexes. In particular, we want to understand why XX animals become self-fertile hermaphrodites in some species, but become females in closely related species. In, C. elegans , both FOG-1, a Cytoplasmic Polyadenylation Element Binding (CPEB) Protein, and FOG-3 are required for germ cells to become sperm instead of oocytes. C. elegans also produces three other CPEB proteins, CPB-1, which is required for spermatogenesis, and CPB-2 and CPB-3, which have no known function (Luitjens et al . 2000). To learn how the control of sexual fate in germ cells has changed during evolution of the Caenorhabditids, we cloned the homologs of these genes from C. briggsae and C. remanei . We find that each species has four CPEB genes, just like C. elegans . In each species, RNA-mediated interference (RNAi) directed against these genes caused the same germline phenotypes previously reported in C. elegans , with one exception. In C. briggsae , cpb-1(RNAi) also resulted in a second phenotype, in the male tail. These data suggest that the divergence and specialization of these CPEB proteins pre-dates the origin of the genus Caenorhabditis , but this process is still continuing. We also find that each species has a single fog-3 gene, and that the structures and sequences of these genes are conserved. RNAi experiments show that FOG-3 is required in each species for germ cells to become sperm rather than oocytes, just like in C. elegans . Furthermore, in each species, the level of fog-3 transcripts is correlated with spermatogenesis. This observation suggests that the control of fog-3 expression might be responsible for determining if XX animals become females or hermaphrodites. How might this work? Transgenic rescue experiments using chimeric constructs show that the C. elegans and C. briggsae FOG-3 proteins are functionally interchangeable, as are the promoters from all three species. Furthermore, these promoters each contain multiple TRA-1A binding sites. Thus, our results suggest that the underlying mechanisms for controlling germ cell fates are similar in each species, but that the regulation of fog-3 expression has changed during recent evolution. Since we have shown that the fog-1 and fog-3 promoters are interchangeable in C. elegans , we propose that changes in the transcriptional regulation of this pair of genes might be responsible for the rapid evolution of germ cell fates. These changes could be caused by the modification of one of several upstream regulatory factors.

Alexander, K. et al. (2017) International Worm Meeting "Investigating Molecular Mechanisms Underlying Cholinergic Synapse Remodeling in the C. elegans Motor Circuit."

Doitsidou, M., Oliver, D., Alexander, K., Francis, M.M., Benard, C., Lambert, C., Philbrook, A., Thackeray, A.

[International Worm Meeting, 2017]

The nervous system comprises complex neural circuits that require precise patterns of connectivity. To accomplish this, there is often considerable remodeling of connectivity during development, typically an initial over proliferation of synapses that is followed by activity-dependent refinement or remodeling. We have only a limited molecular understanding of how this is achieved. To address this question, we have been investigating synaptic remodeling of GABAergic Dorsal D class (DD) motor neurons. Initially, DD motor neurons form neuromuscular synapses ventrally and receive synaptic inputs dorsally. By late larval stages, the polarity of these connections is reversed. DD motor neurons receive synaptic inputs from cholinergic motor neurons in the ventral nerve cord and form neuromuscular synapses to dorsal body wall muscles. Prior work has focused on the remodeling of presynaptic components in DD neurons and identified several genes involved. My work is focused on the remodeling of postsynaptic components using a specific marker (ACR-12::GFP) to label synaptic inputs to DD neurons. Interestingly, only a subset of genes implicated in presynaptic remodeling also affect postsynaptic remodeling. For example, disruption of the heterochronic gene hbl-1 similarly affects both processes [1]. In contrast, the caspase ced-3, involved in the removal of pre-synaptic components during remodeling [2], is not required for removal of dorsal ACR-12::GFP clusters. We are carrying out a forward genetic screen to identify novel genes involved in the remodeling process. To date, we have obtained two candidate mutants in which dorsal ACR-12 receptor clusters persist into adulthood. Through our ongoing characterization of these candidates and additional mutants identified from the screen, we aim to uncover conserved molecular mechanisms underlying synaptic refinement and remodeling. 1. Thompson-Peer et al., Neuron (2012) 2. Meng et al., Cell Reports (2015).

Brown AL et al. (2007) J Gen Physiol "Gain-of-function mutations in the MEC-4 DEG/ENaC sensory mechanotransduction channel alter gating and drug blockade."

Brown AL, Goodman MB, Liao Z, Fernandez-Illescas SM

[J Gen Physiol, 2007]

MEC-4 and MEC-10 are the pore-forming subunits of the sensory mechanotransduction complex that mediates touch sensation in Caenorhabditis elegans (O''Hagan, R., M. Chalfie, and M.B. Goodman. 2005. Nat. Neurosci. 8:43-50). They are members of a large family of ion channel proteins, collectively termed DEG/ENaCs, which are expressed in epithelial cells and neurons. In Xenopus oocytes, MEC-4 can assemble into homomeric channels and coassemble with MEC-10 into heteromeric channels (Goodman, M.B., G.G. Ernstrom, D.S. Chelur, R. O''Hagan, C.A. Yao, and M. Chalfie. 2002. Nature. 415:1039-1042). To gain insight into the structure-function principles that govern gating and drug block, we analyzed the effect of gain-of-function mutations using a combination of two-electrode voltage clamp, single-channel recording, and outside-out macropatches. We found that mutation of A713, the d or degeneration position, to residues larger than cysteine increased macroscopic current, open probability, and open times in homomeric channels, suggesting that bulky residues at this position stabilize open states. Wild-type MEC-10 partially suppressed the effect of such mutations on macroscopic current, suggesting that subunit-subunit interactions regulate open probability. Additional support for this idea is derived from an analysis of macroscopic currents carried by single-mutant and double-mutant heteromeric channels. We also examined blockade by the diuretic amiloride and two related compounds. We found that mutation of A713 to threonine, glycine, or aspartate decreased the affinity of homomeric channels for amiloride. Unlike the increase in open probability, this effect was not related to size of the amino acid side chain, indicating that mutation at this site alters antagonist binding by an independent mechanism. Finally, we present evidence that amiloride block is diffusion limited in DEG/ENaC channels, suggesting that variations in amiloride affinity result from variations in binding energy as opposed to accessibility. We conclude that the d position is part of a key region in the channel functionally and structurally, possibly representing the beginning of a pore-forming domain.

Sagulo, Seth et al. (2015) International Worm Meeting "Investigating the Role of Host Cell Factor 1 and Ribosomal S6 Kinase in Regulation of Longevity."

Sagulo, Seth, Lee, Siu Sylvia

[International Worm Meeting, 2015]

The Lee lab has identified Host Cell Factor 1 (HCF-1) as a transcriptional corepressor of DAF-16/FOXO, a major downstream effector of the highly conserved insulin/insulin-like (IIS) signaling pathway. C. elegans that are mutant in hcf-1 have an extended lifespan that is dependent on daf-16. Host Cell Factor 1 is a nuclear expressed protein that acts as a scaffolding protein as it does not have a clear DNA binding domain. Thus it likely plays a role in various protein-protein interactions, some of which may be important for regulating lifespan. Since many of the proteins that interact with HCF-1 have not yet been identified immunoprecipitation-mass spectrometry (IP-MS) was performed in collaboration with the Ruvkun lab and Ming Zhu, Meng-Qiu Dong of the Dong lab. This was done using a strain of C. elegans expressing HCF-1 tagged with GFP. Several chromatin related factors including the histone deacetylase SIN-3 which has been previously shown to interact with HCF-1 in addition to many metabolism proteins were found to interact with HCF-1. Surprisingly, the downstream kinase of the highly conserved target of rapamycin (TOR) pathway, ribosomal S6 kinase (RSKS-1/S6K1) was also identified which suggests it may play a role in the regulation of HCF-1 through phosphorylation. Interestingly, previously published data shows that rsks-1 mutants have an extended lifespan that is dependent on daf-16 which is similar to hcf-1 mutants. To support the IP-MS data, the genetic relationship of rsks-1 and hcf-1 was tested. The rsks-1;hcf-1 double mutant does not show an additive extension of lifespan compared to either of the rsks-1 or hcf-1 single mutants providing evidence that HCF-1 and RSKS-1 may be working through the same pathway to regulate longevity in C. elegans. Since HCF-1 and RSKS-1 are both highly conserved proteins understanding the novel biochemical and genetic connection between them will be relevant to aging in mammals.

Hough DM et al. (1993) Worm Breeder's Gazette "Search for RXR Homologs in Nematodes"

Hough DM, Maina CV, Richer J

[Worm Breeder's Gazette, 1993]

We are studying the genetic regulation of molting in the nematodes C. elegans and Dirofilaria Immitis through the use of ecdysone and its receptor, EcR (see abstract by Richer et al., this issue). EcR is a member of the nuclear hormone receptor (NHR) superfamily. It requires the presence of ecdysone and another Drosophila protein ultraspiracle, usp, in order to activate transcription. usp, also a member of the NHR superfamily (Yao et al. 1992, Cell 71:63-72), is a homolog of the vertebrate retinoid X receptor (RXR, Oro et al. 1990, Nature 347:298-301). To determine if a nematode EcR behaves as does the Drosophila EcR, we have initiated a search for nematode usp homologs. We have used the Drosophila usp to search for D. immitis and C. elegans homologs using several approaches, the most fruitful being PCR with degenerate primers to the DNA binding domain. Two degenerate primers (C1 and C3 )encoding the amino acids shown in the figure were used in PCR experiments with genomic DNA from both D. immitis and C. elegans. All PCR products were cloned into pUC19 and sequenced. The C1 /C3primer combination amplified 2 DNA fragments from C. elegans (crf-6 and crf-7 ,in keeping with the nomenclature for nematode NHR s established by Sluder et al. WBG 11, #3) and 1 from D. immitis (dirf-4), ranging in size from 137 to 190 bps. The predicted amino acid sequence for each of the 3 shows significant similarity to the NHR superfamily. 1 of the 3 contains a putative intron of 50 bps at the site indicated by the arrow. The sequence of dirf-4 shows 93% similarity at the amino acid level to the Drosophila usp. While the crf-4 NHR shows less similarity to usp (68%), we are pursuing it further because it shows cross hybridization to the D. immitis dirf-4 done and most of the matches in a TFASTA search of GenBank</EMBL are to RXR sequences. Neither of the C. elegans NHR s have been described previously and they have been mapped to the genome (see figure). crf-6 maps to chromosome III and cosmid DF12 .crf-7 shows hybridization to 2 noncontiguous regions of the genome, strong hybridization to chromosome II and weak hybridization to chromosome IV. We have obtained a cDNA for 1, crf-6 ,from a screen of a mixed stage cDNA library (Stratagene). The D. immitis RXR homolog is the first to be described for a nematode and we are presently screening an adult female D. immitis cDNA library (Grandea et al 1989, Mol Biochem Parasitol 35:31-42) for a cDNA copy of this gene.

Koelle MR (1998) East Coast Worm Meeting "G protein regulators in C. elegans: a survey of their expression patterns and functions"

Koelle MR

[East Coast Worm Meeting, 1998]

RGS proteins (regulators of G protein signaling) inhibit G protein signaling by binding directly to G protein alpha subunits and converting them from their active GTP-bound form to their inactive GDP-bound form. We have previously described the C. elegans RGS protein EGL-10 protein, which is expressed in most or all C. elegans neurons and inhibits signaling by the G protein GOA-1 to regulate several behaviors, including egg laying and locomotion. We found that both the mammalian and C. elegans genomes encode large families of proteins that share sequence similarity with EGL-10 in the ~120 amino acid "RGS domain", the part of these proteins that interacts with G protein alpha subunits. To help understand why there might be so many G protein regulators, we have analyzed the expression patterns and functions of all 11 RGS genes identified so far by the C. elegans genome sequencing project. We used GFP reporter transgenes to examine the expression patterns of the RGS genes. Our results suggest that about half of the RGS genes in C. elegans are expressed quite widely, and have expression patterns that overlap extensively. For example, egl-10, C05B5.7, C41G11.3, and the RGS genes on cosmids C16C2 and F45B8 each appear to be expressed in most or all neurons, and B0336.4 appears to be expressed in the neurons as well as most other cells in the animal. Because these genes do not all have the same functions (for example, egl-10 has a mutant phenotype, and thus is not completely redundant with the other RGS genes expressed in the same cells), we suggest that different RGS proteins may achieve specific functions by acting specifically on different G protein targets. Five RGS genes in C. elegans appear to be expressed in patterns that are highly spatially and/or temporally restricted. These RGS proteins may achieve specific functions in part by virtue of this fact. For example, GFP reporters for C29H12.3 are expressed only in seven amphid neurons and the two phasmid neurons. This suggests a specific role for this RGS gene in down-regulating or desensitizing responses to the chemosensory or mechanosensory stimuli detected by these cells through G protein signaling pathways. We have begun to examine the functions of the RGS genes by analyzing animals that overexpress them from multicopy transgenes. Overexpression of egl-10 in this manner gives a phenotype similar to that obtained by deleting the gene for its known G protein target, GOA-1. We hope that overexpression of the other RGS genes will similarly result in gain-of-function phenotypes for these genes. As described in abstracts for this meeting by Meng-Qiu Dong and Georgia Patikoglou, two additional RGS genes besides egl-10, when overexpressed, also stimulate egg laying, presumably by also inhibiting the G protein GOA-1. These RGS genes, C05B5.7 and F16H9.1, appear to be expressed in patterns that overlap the GOA-1 expression pattern in the nervous system, consistent with this idea. Another RGS gene, on cosmid C16C2, also appears to be widely expressed in the nervous system, but causes an opposite phenotype when overexpressed. This RGS gene could encode an inhibitor of EGL-30, a G protein that activates egg laying. We are further testing these ideas by examining loss-of-function mutations in the RGS genes.