R Ciosk et al. (2002) European Worm Meeting "Maintenance of embryonic stem cell identity in the germline blastomeres of C. elegans"

M DePalma, R Ciosk, Jim Priess

[European Worm Meeting, 2002]

In the early C. elegans embryo, both the somatic blastomere EMS and the germline precursor P2 contain the maternally supplied transcription factor SKN-1 (Bowerman et al., 1993). SKN-1 is required for pharynx and gut development in the EMS descendants (Bowerman et al., 1992). In P2, SKN-1 activity appears to be inhibited by another maternal protein PIE-1. In pie-1 mutants, P2 responds to SKN-1 and the P2 descendants produce pharynx and gut (Mello et al., 1992). Several lines of evidence suggest that PIE-1 maintains germline identity by inhibiting accumulation of RNA polymerase II-dependent mRNAs. This phenomenon is referred to as transcriptional inhibition, though the underlying mechanism has not been determined (Seydoux et al., 1996). To identify potential targets of PIE-1 in maintaining germline identity, we purified PIE-1 from C.elegans extracts. We found that PIE-1 associated with three PIE-1 associated proteins (PAP- 1-3). pap-2 (RNAi) produced a gradient of phenotypes ranging from viable embryos, through dead embryos with well-differentiated tissues, to embryos that failed to undergo cell division. Among the embryos that die with well-differentiated tissues are many with pharyngeal defects. By looking at the expression of the pharyngeal marker ceh-22::GFP, we found that majority of embryos had either no or only a few CEH-22::GFP-positive cells. Although we detected gut cells in almost all pap-2 (RNAi) embryos, intensity of the gut-specific birefringence was often reduced compared to the wild-type embryos. Despite the pharyngeal and intestinal defects, SKN-1 was present in pap-2 (RNA-i) embryos. In wild-type embryogenesis, SKN-1 specifies pharynx and gut development by triggering zygotic expression of the med-1 and med-2 transcription factors (Maduro et al, 2001). We tested whether MED-1::GFP was present in pap-2 (RNAi) embryos and found that MED-1::GFP was not expressed. Thus, SKN-1-dependent expression of med-1/2 appears to require PAP-2. This finding raises a possibility that in the P2 blastomere, PIE-1 may directly inhibit PAP-2 activity and by doing so, prevent SKN-1 from triggering expression of med-1/2. However, PAP-2 is probably not the only target of PIE-1 in maintaining germline identity, as another early zygotic gene, pes-10, appears to be expressed in pap-2 (RNAi) embryos.

Pekec, T. et al. (2017) International Worm Meeting "Cold-induced suspended animation."

Pekec, T., Ciosk, R.

[International Worm Meeting, 2017]

Wild-type C. elegans are sensitive to a sudden temperature drop but, following a short adaptation at an intermediate temperature, the animals survive at 4 oC for many days (1, 2). While in cold, the animals stop feeding and moving, suggesting that they enter a hibernation-like, suspended animation state. To investigate molecular pathways involved in hibernation, we have performed an unbiased RNAi screen, searching for genes essential for cold survival. Among others, this screen identified a conserved ribonuclease, REGE-1. One target of REGE-1 is the ets-4 mRNA (2). ETS-4 is a member of the ETS transcription factor family, which regulates life span (3). We found that the loss of ets-4 rescues the cold sensitivity of rege-1 mutants, as well as other rege-1 phenotypes, suggesting that ETS-4 is a key effector of REGE-1. To identify genes functioning downstream from ETS-4, we perform a forward genetic screen, searching for mutants suppressing the cold sensitivity of rege-1 animals, despite high levels of ETS-4. I will present and discuss characterization of candidate mutants at the meeting. 1. Ohta, A. et al. Light and pheromone-sensing neurons regulates cold habituation through insulin signalling in Caenorhabditis elegans. Nature communications (2014). 2. Habacher, C. et al. Ribonuclease-Mediated Control of Body Fat. Developmental cell (2016). 3. Thyagarajan, B. et al. ETS-4 is a transcriptional regulator of life span in Caenorhabditis elegans. PLoS genetics (2010).

Bjoern Biedermann et al. (2006) European Worm Meeting "Germline Totipotency and the worm teratoma"

Bjoern Biedermann, Rafal Ciosk, Mathias Senften

[European Worm Meeting, 2006]

Bjoern Biedermann, Matthias Senften and Rafal Ciosk. Teratomas (derived from teraton, the Greek word for monster) are germ cell tumors that contain a mixture of somatic tissue such as hair or bone. These tumors are thought to result from abnormal somatic differentiation of germ cells, but very little is known about the molecular mechanisms that normally maintain germ cell totipotency. Two conserved translational repressors, MEX-3 and GLD-1, have been recently found to maintain totipotency in the C. elegans germline (Ciosk et al., Science 311, 851, 2006). In mex-3 gld-1 mutants, germ cells transdifferentiate into various somatic cell types such as muscles or neurons. This worm teratoma requires the entry into meiosis and coincides with the disappearance of germline-specific P granules. To understand MEX-3/GLD-1 role in maintaining totipotency, we want to identify the proteins that normally need to be repressed by MEX-3 and GLD-1. Genetic and biochemical strategies will be discussed at the meeting.

Pekec, T. et al. (2019) International Worm Meeting "Cold-induced suspended animation."

Ciosk, R., Pekec, T., Guo, Y., Komur, A.A.

[International Worm Meeting, 2019]

Many animals, upon experiencing severe cold, enter a state of suspended animation known as hibernation. Also C. elegans adults can survive spells of near-freezing temperature (here simply "cold"), and our observations have suggested that they do so by entering a hibernation-like state (Habacher et al., 2016). Among the factors promoting cold survival is the conserved endoribonuclease REGE-1/Regnase 1. A key mRNA target of REGE-1 encodes a transcription factor, ETS-4. Previously, we reported that, by preventing ETS-4 accumulation, REGE-1 promotes the accumulation of body fat (Habacher et al., 2016). We find now that the REGE-1 mediated repression of ETS-4 is also important for cold survival. In fact, ets-4 mutants survive cold even better than wild-type animals. Interestingly, inhibiting insulin signaling, resulting in the activation of the transcription factor DAF-16/FOXO, was shown to enhance cold resistance (Savory et al., 2011; Ohta et al., 2014), and ETS-4 was suggested to synergize with insulin signaling in antagonizing the function of DAF-16 (Thyagarajan et al., 2010). In line with those studies, our genetic analysis suggests that the enhanced cold survival of ets-4 mutants reflects the activation of DAF-16 mediated transcription. Surprisingly, the cold survival-enhancing role of DAF-16 also requires the transcription factor PQM-1, which in other physiological contexts has been shown to antagonize DAF-16 (Tepper et al., 2013). Thus, the functional relationship between DAF-16 and PQM-1 appears to be context-dependent, and we are interested in understanding how these two transcription factors collaborate to facilitate worm "hibernation".

Hargitai, B. et al. (2013) International Worm Meeting "Epigenetic reprograming during germ line development."

Ciosk, R., Kalchhauser, I., Gutnik, S., Hargitai, B.

[International Worm Meeting, 2013]

Cell fate reprograming is critical for development. A profound reprograming takes place during germ cell development, which is why germ cells, and the various pluripotent cell lines derived from them, have been invaluable for dissecting the mechanisms controlling pluripotency. One conserved mechanism for restricting pluripotency is the formation of facultative heterochromatin by the Polycomb Repressive Complex 2 (PRC2). In mammals, subsets of PRC2-repressed genes become activated at different times during germ cell development, and thus, the PRC2-mediated repression of specific targets can be reversed. However, the mechanisms regulating both the reactivation and the specificity for particular targets remain unresolved. Similarly to mammals, the C. elegans PRC2 (consisting of the MES-2, -3, and -6 proteins) is required during germ cell development. Using this model to understand epigenetic reprograming, we find that the reactivation of a subset of PRC2-repressed genes is mediated by the Notch signaling pathway. GLP-1Notch signaling appears to directly relay information to chromatin, as the putative Notch-response elements in the promoter region of Notch-regulated genes are required for the reactivation. Because a number of the identified Notch-activated genes impact self-renewal and/or differentiation in the germ line, we propose that Notch signaling affects these cell fate events, at least in part, by inducing epigenetic remodeling.

Habacher, C. et al. (2019) International Worm Meeting "Characterizing the roles of the ABC transporter MRP-1 in fat metabolism."

Guo, Y., Ciosk, R., Bilnicka, A.A., Habacher, C., Miki, T.

[International Worm Meeting, 2019]

Obesity is a global problem associated with numerous diseases, therefore understanding the molecular mechanisms controlling fat is important. A ribonuclease REGE-1 inhibits the expression of a transcription factor, ETS-4, to maintain normal fat levels. To understand the underlying mechanism, we performed forward genetic screening and identified loss-of-function alleles of mrp-1 that suppressed fat loss caused by rege-1 deletion. The mrp-1 gene encodes an ATP-binding cassette (ABC) transporter homologous to mammalian multidrug resistance-associated proteins (MRPs). Staining with Oil red O showed that fat levels of rege-1; mrp-1 double mutant animals were higher than rege-1 null animals, while those of mrp-1 single mutant animals were comparable to wild-type animals. Quantifications of mRNA levels revealed that rege-1; mrp-1 double mutant animals displayed a moderate reduction in ets-4 mRNA levels, compared to rege-1 null animals. Whether this reduction in ets-4 explains MRP-1 effect on body fat, or MRP-1 functions via a different mechanism, is currently being investigated.

Nate Dudley et al. (2007) International Worm Meeting "An RNAi-based screen identifies genes that affect pluripotency in the C. elegans germline."

Joel Rothman, Nate Dudley

[International Worm Meeting, 2007]

Developmental constraints prevent most cells from changing their differentiated states. However, germ cells remain pluripotent, though they do not differentiate into somatic tissue until after gametogenesis. We are investigating the molecular mechanisms that constrain the somatic differentiation potential of the developing germline. The endoderm-determining transcription factor END-3 is sufficient to redirect early non-endodermal embryonic cells into the endoderm differentiation pathway. We established an RNAi-based screening strategy to assay for mutants in which hs-end-3 can activate expression of a target gene, elt-2::gfp, in the germline. Candidate genes include those encoding translational regulators, chromatin/DNA/RNA binding proteins, and signal transduction components. Ciosk et al recently reported the redundant requirement of mex-3 and gld-1 in preventing somatic differentiation in the germline: loss of gld-1 and mex-3 together resulted in dramatic transformations of germline cells into somatic tissue within the developing germline (1). We report here that RNAi of either gld-1 or mex-3 alone is sufficient to result in substantial gut differentiation in response to END-3 expression in the germline: 14.9% (n=269, sd=5.88) of gld-1 (RNAi) and 16.8% (n=307, sd=5.46) of mex-3 (RNAi) animals alone show frequent ectopic GFP expression in the germline, whereas wild-type worms almost never showed expression (1.3%, n=1315, sd=1.31). In addition, we found that loss of glp-1 function by RNAi results in germline expression of the endoderm marker (7.8%, n=161, sd=2.5). Our glp-1 results are consistent with results obtained by Ciosk et al , showing that GLP-1 activity can prevent somatic differentiation in proliferating germline cells. Interestingly, we found that activation of the endoderm marker in the germline was frequent when worms were reared at 25C but not at 20C. Since our results have features of transgene-induced silencing (e.g., temperature-sensitivity) one possible explanation may be that rearing worms at 25C at least partially de-silences germline expression of the hs-END-3 extra-chromosomal array. This approach sets the stage for analysis of mechanisms that restrict developmental competency for reprogramming, allowing us to study the comprehensive set of genes that function to restrict developmental potential in the C. elegans germline. Analysis of candidates obtained from the screen will be reported. 1.Ciosk et al , Science vol311pg.851 2006.

Pagano, John M et al. (2009) International Worm Meeting "RNA recognition by the cell fate determinant MEX-3."

Pagano, John M, Ryder, Sean P

[International Worm Meeting, 2009]

Post-transcriptional regulation of gene expression governs the development of the Caenorhabditis elegans germline and early embryo. The putative RNA binding protein MEX-3 is essential for maintaining totipotency of germline stem cells and for specification of the anterior founder cell (AB) during embryogenesis. mex-3 mutants are maternal effect lethal with a terminal phenotype that includes posterior muscle proliferating into the anterior (1). In addition, worms that are deficient in MEX-3 and a second RNA binding protein, GLD-1, are sterile and form transdifferentiated germline tumors (2). Genetic studies reveal that two genes involved in early development, pal-1 and nos-2, are dependent upon mex-3 for their protein expression pattern (1, 3). MEX-3 contains two conserved KH domains and is thought to control mRNA stability, translation efficiency, and/or mRNA localization through specific interactions with target maternal mRNAs. However, the RNA binding specificity and network of regulatory targets have not been determined. Here, we used in vitro selection (SELEX) and quantitative biochemical methods to define the consensus MEX-3 recognition element (MRE). We demonstrate that MEX-3 binds specifically to a bipartite element consisting of two four-nucleotide elements with variable spacing between each half-site. The putative mRNA targets pal-1 and nos-2 each contain two MREs in their 3''UTR. Furthermore, the transcript nos-2 has an MRE within a critical cis-regulatory element required to repress NOS-2 expression in early embryos (4). Our results show that MEX-3 binds specifically to this element. The MRE is present in several other genes that are required for germline development and embryogenesis. Candidate regulatory targets include a number of RNA binding proteins (mex-1, gld-1, spn-4, puf-6, puf-7) and membrane proteins (glp-1, ooc-3). Target prediction based on the MRE will be used to further elucidate the role of MEX-3 in early development. 1. Draper BW, et al. (1996) Cell 87, 205-216 2. Ciosk R, et al. (2006) Science 311, 851-853 3. Jadhav S, et al. (2008) Development 135, 1803-1812 4. D''Agostino I, et al. (2006) Dev. Biol. 292, 244-252.

Ahmed M Elewa et al. (2005) International Worm Meeting "Understanding atx-2 and rethinking DNA primases: atx-2 and pri-1/2 suppress the mom-2/Wnt gutless phenotype."

Craig C Mello, Ahmed M Elewa, Masaki Shirayama

[International Worm Meeting, 2005]

In the 4 cell stage C. elegans embryo, Wnt signaling between the posterior and ventral blastomeres, P2 and EMS, is required to specify endoderm. In order to identify new components of this pathway, we conducted a suppressor genetic screen beginning with the most upstream component MOM-2/Wnt. We mutagenized mom-2(ne2626ts) (90% gutless) and screened for restored endoderm development at the non-permissive temperature. Among over 50 suppressors identified, 10 exhibited ts-lethal phenotypes on their own when crossed away from mom-2. These lethal mutants defined 4 loci, namely, pri-1, pri-2, atx-2 and cdl-1. cdl-1 encodes a protein that binds the 3 end of histone transcripts, stabilizing them and promoting their expression. This mutant appears to bypass Wnt signaling causing ectopic endoderm development in most lineages of early embryos, conceivably by reduced expression of histone proteins required for repressing endoderm genes. Mutations in the other 3 loci, restore endoderm differentiation within the E lineage specifically, suggesting that they may be more directly relevant to P2/EMS signaling. In humans, lesions in Ataxin-2/ATX2 have been shown to cause spinocerebellar ataxia type 2 (SCA2), a hereditary neurodegenerative disorder, and the DNA primases (pri-1, and pri-2) have well-established roles in DNA replication. But why would mutations in these proteins suppress mom-2? The primase mutants are non-null alleles with no obvious defects in cell division timing, and the atx-2 allele is a premature stop codon near the normal end of the gene. In yeast Pbp1 (yeasts likely functional homolog of ATX-2) interacts with proteins implicated in mRNA decay (Dhh1p), nuclear export (Crm1p), and translation (Pab1p) (1). Interestingly, yeast Pri2p interacts with some of same functional subsystems, including Pop2p (mRNA decay) and Crm1p (nuclear export). Finally, C. elegans interaction screens link both PRI-1 and ATX-2 to PAB-1 (poly A tail binding protein). These findings suggest that ATX-2 and the PRI proteins may be post-transcriptional regulators of mRNA expression. Consistent with this idea recent reports have shown that ATX-2 functions with GLD-1 & MEX-3 to regulate translation in the C. elegans germline (2). Therefore, our working model is that pri-1/2 and atx-2 suppressor alleles may increase the translation of endoderm determinants to render E more sensitive to impaired endoderm induction. Understanding these suppressors will lead to new insights into the functions of these highly conserved factors, and may link Wnt signaling to new modes of post-transcriptional regulation. 1) Ralser M. et al. J. Mol. Biol. 346, 203-214 2) Ciosk R. et al. Development 131, 4831-4841 *A.M.E & M.S. contributed equally

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.