Min Sung Choi et al. (2007) International Worm Meeting "Genes that act in specification of the vulval secondary (2) fate."

Iva Greenwald, Min Sung Choi

[International Worm Meeting, 2007]

LIN-12/Notch signaling specifies Vulval Precursor Cells (VPCs) to adopt the 2 fate. Upon ligand binding, the intracellular domain of LIN-12 is released and translocates to the nucleus, where it forms a transcriptional activation complex with the sequence-specific DNA binding protein LAG-1. Direct target genes of LIN-12 in VPCs have been identified by searching for genes that contain LAG-1 binding sites in their 5 flanking regions, using various search criteria (1,2). We have used a computational approach to predict novel LIN-12 target genes, using specific criteria based on the nature and arrangement of LAG-1 binding sites in the promoter of the LIN-12 target gene mir-61 (2). We will present the results of transcriptional expression pattern screening of the set of genes obtained in our search. We will also present our work in progress on a set of genes that share the 5 flanking region and are expressed in presumptive 2 VPCs, the pattern expected of LIN-12 target genes. Analysis of mir-61 and its target, vav-1, suggested that they constitute a positive autoregulatory loop in which LIN-12 activation leads to expression of a negative regulator (mir-61) of a negative regulator (vav-1) of lin-12 activity (2). Thus, vav-1 is an indirect target of LIN-12 that influences the 2 fate. We will present our ongoing work on another microRNA gene that appears to be a direct target of LIN-12; the protein-encoding gene that is its likely target behaves genetically as a negative regulator of lin-12 activity, and hence these genes together also appear to function in a positive autoregulatory loop. We will also discuss the potential intersection of these two microRNA-mediated loops. (1) Yoo et al., Science 303:663-6(2004). (2) Yoo and Greenwald, Science 310:1330-3 (2005).

Kershner, Aaron M. et al. (2011) International Worm Meeting "Two likely GLP-1/Notch targets are essential for germline stem sell maintenance in both larvae and adults."

Shin, Heaji J., Kimble, Judith, Kershner, Aaron M.

[International Worm Meeting, 2011]

C. elegans germline stem cells (GSCs) are maintained by GLP-1/Notch signaling and two redundant PUF family mRNA binding proteins, FBF-1 and FBF-2 (collectively, FBF). Notch signaling is essential for GSC maintenance in both larvae and adults, while FBF is essential only in late L4s and adults. The fbf-2 gene is the only Notch target gene known to date with a key role in GSC maintenance (1). Therefore, we sought additional Notch targets crucial for germline self-renewal. Two genes, lst-1 and T27F6.4, were identified as likely FBF targets (2) and also putative Notch targets (3). To ask whether lst-1 and T27F6.4 control GSC maintenance, we analyzed an lst-1 deletion mutant and T27F6.4 (RNAi) animals (progeny of L4s fed T27F6.4 RNAi bacteria). No effect was seen when either gene was removed singly, but depletion of the two together had a phenotype indistinguishable from that of a null glp-1 mutant: germ cell number was reduced from ~2000 to 8-10 total in L3 stage and these few germ cells differentiated as sperm. We conclude that lst-1 and T27F6.4 are redundant in their control of larval GSC proliferation and therefore dub T27F6.4 sygl-1, for synthetic germline proliferation defective. A different RNAi regimen, which depleted lst-1 and sygl-1 specifically in adults, revealed that these two regulators also control adult germline self-renewal. We suggest that the lst-1 and sygl-1 genes are pivotal for GSC maintenance throughout postembryonic germline development. References: (1) Lamont and Kimble (2004) Dev Cell 7(5):697-707; (2) Kershner and Kimble (2010) PNAS 107(8):3936-41; (3) Yoo and Greenwald (2004) Science 303(5658):637-8.

Escobar Restrepo, Juan et al. (2015) International Worm Meeting "Lipid asymmetry and signaling regulation during vulva development."

Hajnal, Alex, Haag, Andrea, Escobar Restrepo, Juan

[International Worm Meeting, 2015]

C. elegans let-23 is the homologue of the EGFR family of receptor tyrosine kinases and is required for the formation of the hermaphrodite vulva. LET-23 in the Vulva Precursor Cell (VPC) P6.p interacts with LIN-3 and activates the LET-60/MPK-1 pathway to acquire a 1deg cell fate. This triggers lateral LIN-12 NOTCH signaling in the adjacent P5.p and P7.p VPCs which acquire a 2deg cell fate (Sternberg et.al, Shaye et.al and Yoo et. al). In addition, several components of the LET-60/MPK-1 pathway are down-regulated in 2deg fated cells, for example let-23 (Berset. et al). We performed an in vivo RNAi screen for defects in receptor localization and/or expression using a functional LET-23::GFP reporter (Haag et.al). RNAi against tat-5 caused increased expression of LET-23::GFP in the daughters of P5.p and P7.p as compared to empty vector RNAi. A mutant of tat-5 displays a Protruding vulva phenotype and in combination with a mutant of the close homologue tat-6, shows a low penetrant Adjacent Primary Fate (APF) phenotype. The APF phenotype is enhanced when combined with a let-60(gf) mutation and is accompanied by an increase in the number of induced cells. Thus tat-5/tat-6 may act as negative regulators of the LET-60/MPK-1 pathway. tat-5 encodes a P4-type ATPase that functions as a flippase to inhibit phosphatidylethanolamine (PE) exposure on the outer plasma membrane. This activity is required to regulate the release extracellular vesicles (ECVs) in the worm (Wehman et.al) and vesicular protein trafficking in lower eukaryotes (Reviewed in Paulusma C. et. al). Here we discuss possible roles of lipid asymmetry in the acquisition of cell fates during vulval development.

Ji Li et al. (2007) International Worm Meeting "A systematic analysis of the roles of heterochronic genes in VPC fate specification."

Iva Greenwald, Ji Li

[International Worm Meeting, 2007]

We plan a systematic investigation of the roles of heterochronic genes in vulval precursor cell (VPC) competence and fate patterning. We have a particular interest in a potential role of heterochronic genes in LIN-12-mediated lateral signaling, which promotes the 2o fate, because vulval bursting, a phenotype associated with many heterochronic mutants, may be a symptom of an underlying problem in lateral signaling (cf. Grishok et al., 2001; Yoo and Greenwald, 2005). Furthermore, elegant work by Ambros (1999) showed that LIN-12-mediated specification of the 2o fate appears to be "gated" by the cell cycle, which in turn is controlled by heterochronic gene activity (Euling and Ambros, 1996). As a first step, we are examining vulval cell fates in heterochronic mutants using 1o and 2o fate markers. In addition, we will use a tissue-specific rescue or depletion approach to determine the cellular focus of heterochronic genes for vulval phenotypes to gain insight into whether these genes function in the signaling or receiving end of events involved in VPC competence and patterning. Finally, we will look at the potential cell-cycle gating of LIN-12 target gene expression. Genes expressed specifically in P5.p and P7.p may promote the 2o fate, whether they are direct targets of LIN-12 or not. The let-7 family member mir-48, a heterochronic gene (Abbott et al., 2005; Li et al., 2005), has been reported to display a 2o cell specific expression pattern (Esquela-Kerscher et al., 2005). We are investigating the role of mir-48 in 2o fate specification. Preliminary results suggest that ectopic expression of mir-48 in the presumptive 1o VPC causes the expression of a 2o cell fate marker, and also causes a timing defect consistent with a heterochronic phenotype (Li et al., 2005). Our progress in these areas will be presented.

Agnieszka Trzebiatowska et al. (2006) European Worm Meeting "TEN-1 is required for the Formation of the Somatic Gonad in Caenorhabditis elegans"

Krzysztof Drabikowski, Agnieszka Trzebiatowska, Ruth Chiquet-Ehrismann

[European Worm Meeting, 2006]

Agnieszka Trzebiatowska1, Krzysztof Drabikowski2, Ruth Chiquet-Ehrismann1. TEN-1 is a member of a novel family of transmembrane proteins named teneurins that have been characterized in Drosophila, zebrafish, chicken, mouse and man. Teneurins were shown to be mainly expressed in the developing and adult nervous system and to play a role in morphogenesis, cell migration and at sites of pattern formation. C. elegans TEN-1 was proposed to act as a receptor that signals directly to the nucleus. Its intracellular domain is cleaved and translocates to the nucleus where it may influence gene expression. Little is known, however, about the cleavage mechanism and possible target genes. C. elegans has a single teneurin orthologue, which is under control of alternative promoters. The upstream promoter is active in the somatic gonad, some muscle cells, the gut and in a number of neurons. The expression from the downstream promoter is mainly detected in the nervous system. TEN-1 is a type II transmembrane protein consisting of a short intracellular domain followed by a transmembrane domain and a long highly conserved extracellular part containing eight EGF-like repeats, a cysteine rich region and YD repeats.. In order to determine the function of TEN-1 in C.elegans, we investigated two loss-of-function mutants: ten-1(ok641) and ten-1(tm651). The former carries an in frame deletion of four EGF-like repeats and a part of the cysteine rich region; the latter has a deletion that removes the transmembrane domain and introduces a frameshift mutation resulting in the loss of the entire protein except for the first 194 aminoacids. In both mutants transcripts for the predicted truncated proteins could be detected on the level comparable to the wild type form. Both ten-1 mutants show a pleiotropic phenotype, similar to the phenotype observed after reduction of ten-1 expression by RNAi. Mutant worms are sterile due to various defects in the somatic gonad and ectopic germline formation in the proximity of the vulva. Time course analysis of germline development showed that these abnormalities may result from an early defect in the somatic gonad formation. We were able to partially rescue the phenotype of ten-1(ok641) mutant worms by injecting the cosmid F36A3 carrying the entire genomic region of the ten-1 gene. Since such an extrachromosomal array is likely to be silenced in the germline, gonadal defects appear to be the result of impaired signaling in the somatic gonad.

Agnieszka Trzebiatowska et al. (2005) International Worm Meeting "ten-1 is essential for gonad development in C. elegans."

Krzysztof Drabikowski, Agnieszka Trzebiatowska, Ruth Chiquet-Ehrismann

[International Worm Meeting, 2005]

ten-1 is the C. elegans orthologue of the Drosophila pair-rule gene ten-m. In the class of pair-rule genes ten-m is the first member identified, which does not encode a transcription factor but a transmembrane protein. Teneurins are vertebrate orthologues of ten-m and they are mainly expressed in the developing nervous system. The ten-1 gene is under control of alternative promoters resulting in two transcripts differing in their N-terminal sequences. They show complex but distinct expression patterns during embryogenesis and in the adult. During postembryonic development the upstream promoter is active in the somatic gonad, in the body wall muscle, pharynx, gut, coelemocytes and in a number of neurons. Expression from the downstream promoter is mainly detected in the nervous system. ten-1 is also expressed in the germline. Ten-1 consists of N-terminal intracellular domain followed by a transmembrane domain and a large extracellular part containing eight EGF-like repeats, a cysteine rich region and YD repeats. In order to determine the function of ten-1 in C. elegans, we investigated the ten-1(ok641) mutant from The C. elegans Gene Knockout Consortium. The strain carries an in-frame deletion (2130 bp) removing four EGF-like repeats and part of the cystein rich region. The ten-1(ok641) worms show a pleiotropic phenotype, similar to the phenotype observed after reduction of ten-1 expression by RNAi. It was shown that ten-1 is important for posterior body part development, neuronal migration, pathfinding and survival, somatic gonad and germline development. Most of the ten-1(ok641) worms are sterile due to ectopic germline forming in the proximity of the vulva, usually localized outside of the gonad. This mass of germ cells starts to grow already at the L3 stage and is present in both hermaphrodites and males. The ten-1 mutation also severely disrupts somatic gonad development. To visualize the gonadal sheath cells, the deletion mutants were crossed with lim-7::GFP worms. The ten-1(ok641) worms showing the ectopic germline proliferation have also sheath cells migration defects. Ten-1 may be one of the proteins essential for the function of the gonadal sheath cells as ten-1(ok641) hermaphrodites show ovulation defects, Emo and Fog phenotypes. We were able to partially rescue the ten-1(ok641) mutants by injecting the cosmid F36A3 carrying the entire genomic region of ten-1 gene. Since such an extrachromosomal array is likely to be silenced in the germline, germline defects appear to be a result of impaired signaling from the somatic gonad.