GOPAL, SANDEEP et al. (2015) International Worm Meeting "Role of proteoglycans in germline development ."

GOPAL, SANDEEP, Pocock, Roger

[International Worm Meeting, 2015]

Proteoglycans (PGs) are composed of a core protein and multiple glycosaminoglycan chains. At least 14 PGs were identified in mammals whereas C. elegans carry four. The PGs present in C. elegans include, a syndecan (sdn-1), a perlecan (unc-52) and two glypicans (lon-2, gpn-1). Besides being the signalling molecules themselves, PGs are involved in the activation of other signalling molecules such as Wnts (Munoz et al., 2006) or Notch (Pisconti et al. 2010). In C. elegans, PG deletion mutants are associated with different developmental defects. For instance, loss of sdn-1 causes defective neuronal development (Rhiner et al., 2005). In addition, loss of sdn-1, unc-52 or lon-2 leads to a reduced brood size (Rhiner et al., 2005; Ceron et al., 2007), which could suggest defective germline development. PGs have been found in stem cells (SC), where they interact with various mitogens and morphogens that could play roles in SC niche creation and signal transduction (Gasimli et al., 2012). In C. elegans, the germline acts as the only source for SCs where they generate oocytes and sperm. We hypothesise that PGs play a role in germline proliferation and differentiation, based on the reduced brood size in PG knockout strains. In C. elegans, the SCs originate from a single distal tip cell that signals to the germline to control a network of RNA regulators. The FBF-1 and FBF-2 RNA-binding proteins promote mitosis in germ cells located close to the distal tip cell, while the GLD-1, GLD-2, GLD-3 and NOS-3 RNA regulators promote entry into meiosis as germ cells leave the SC niche. These RNA regulators are promoted via the Notch signalling pathway (Austin and Kimble, 1987; Doyle et al., 2000; Lambie and Kimble, 1991; Petcherski and Kimble, 2000). Previous studies in mouse SCs implicated syndecan-3 along with Notch in cell differentiation (Pisconti et al. 2010), though the underlying mechanism needs further investigation. In addition, extracellular mechanical tension generated at the SC niche plays a major role in signalling the SC differentiation. It is well known that distal tip cell in C. elegans provide SC niche. Studies also suggest that PGs indirectly contribute to cellular response resulting from extracellular forces. Furthermore, we have identified a role for PGs in transient calcium changes in C. elegans (unpublished). Transient calcium is an important factor in SC development where calcium fluxes push the cell cycle forward. Our study currently focuses on the role of PGs in SC development and differentiation through various factors including notch signalling, calcium metabolism and extracellular mechanical forces. .

Cao, Wei et al. (2019) International Worm Meeting "Decoding Transcriptional Control of Germline Development in C. elegans."

Pocock, Roger, Gopal , Sandeep, Cao, Wei

[International Worm Meeting, 2019]

The C. elegans hermaphroditic germline efficiently generates approximately 300 progeny over 3 days. Such rapid progeny production is driven by molecular mechanisms controlling stem cell maintenance, proliferation, cell fate specification, sperm/oocyte development and maturation. However, little is known of the function of transcription factors in germline development. Our initial work used RNA sequencing to identify 91 transcription factors that are expressed in the germline. The majority of these transcription factors have not previously been implicated in germline development. Therefore, deciphering their role(s) in the germline may lead to interesting new findings and more comprehensive understanding of germline development, stem cell maintenance and differentiation. Using RNA-mediated interference, confocal imaging and automatic germline nuclei number analysis, we performed a screen to investigate the roles of these transcription factors in germline development. We performed RNAi by commencing knockdown from either the L1 or L4 larval stage and analysed young adults. This enabled us to distinguish between genes acting in germline development (L1-L4) or germline function (adult). Subsequently, we used the germline-specific RNAi strain rrf-1(pk1417) and somatic-specific RNAi strain ppw-1(pk1425) to dissect the broad focus-of-action for each transcription factor. In our ongoing screen, 60 out of 87 transcription factors are found to regulate germline development when knocked down from the L1 stage, 36 out of 50 transcription factors regulate adult germline when knockdown from the late L4 stage, and 7 out of 21 transcription factors function cell autonomously in the adult germline.

Claudia Walser et al. (2006) European Worm Meeting "Distinct Roles of the Pumilio and FBF Translational Repressors during C. elegans Vulval Development"

Gopal Battu, ALEX HAJNAL, Claudia Walser, Erika Frohli Hoier

[European Worm Meeting, 2006]

Claudia Walser, Gopal Battu, Erika Frhli Hoier and Alex Hajnal. Members of the conserved PUF (Pumilio and FBF repeat) protein family regulate various aspects of germ line development by selectively binding to the 3 untranslated region of their target mRNAs and repressing translation. FBF-1 and FBF-2 regulate the sperm/oocyte switch and mitosis versus meiosis decision by repressing fem-3 and gld-1 translation, respectively (Zhang et al., 1997; Crittenden et al., 2002). We found that puf-8, fbf-1 and fbf-2 also act in the soma where they negatively regulate vulval development.. Loss-of-function mutations in puf-8 cause excess vulval differentiation when combined with mutations in negative regulators of the EGFR/RAS/MAPK pathway, and they suppress the Vulvaless phenotype caused by mutations that reduce EGFR/RAS/MAPK signaling. A PUF-8::GFP translational reporter is initially expressed in all six vulval precursor cells (the VPCs P3.p through P8.p) but gets restricted to the distal, uninduced (3) VPCs after vulval fate specification. Moreover, the fusion of the distal VPCs to the hypodermal syncytium is retarded in puf-8(lf) mutants. Thus, PUF-8 acts cell-autonomously to limit the temporal competence of the vulval precursor cells to respond to the extrinsic patterning signals. fbf-1 and fbf-2, on the other hand, function as redundant inhibitors of vulval cell fate specification in a distinct pathway that involves the repression of gld-1 translation. fbf-1 fbf-2 double mutants show ectopic 1 cell fate marker expression in adjacent VPCs. We conclude that the FBFs ensure that only one VPC (P6.p) is selected for the 1 cell fate.. Therefore, the PUF family translational repressors regulate various aspects of cell fate specification in the soma, and they may play a conserved role in modulating signal transduction during animal development.

Stefano Canevascini et al. (2001) International C. elegans Meeting "Cloning of negative regulators of the RAS/MAPK pathway in C. elegans."

Stefano Canevascini, ALEX HAJNAL, Erika Froehli, Gopal Battu, Amit Dutt, Thomas Berset

[International C. elegans Meeting, 2001]

The EGF/RTK/RAS/MAPK signal transduction pathway has been conserved during evolution. It controls cell differentiation in organisms as different as insects, worms and vertebrates. Its activity must be tightly controlled for the correct development of the organism and its incorrect activation can lead to tumour formation in vertebrates. In C. elegans, an EGF/RTK/RAS/MAPK signalling pathway is involved in vulval development. The anchor cell expresses the lin-3 ligand, a TGF-a homolog that activates the RTK/RAS/MAPK pathway in the P6.p cell, one of the six equivalent vulva precursor cells (VPC). In turn, the P6.p cell recruits the two neighbouring VPCs P5.p and P7.p through a lateral signal. The three cells then differentiate and form a functional vulva. Constitutive activation of the RTK/RAS/MAPK causes all six VPCs to be activated resulting in a multivulva (Muv) phenotype. Mutations that inactivate the RTK/RAS/MAPK pathway cause a vulvaless (Vul) phenotype because the VPCs remain uninduced and fuse with the hypodermis. We are cloning new factors involved in the negative regulation of the EGF/RTK/RAS/MAPK pathway in C. elegans. We carried out an EMS mutagen screen in a gap-1(0) background and selected for multivulva animals in the F2 generation. Gap-1 is a negative regulator of the RAS pathway that does not have a phenotype as single mutant. The new mutants look normal in a wild type background and show the multivulva phenotype only in a gap-1(0) background. For this reason we named them "multivulva enhancer of gap-1O(meg). We isolated six mutations that fall into four complementation groups, ie meg-1, meg-2, meg-3, meg-5 and meg-4 (see also presentation of Gopal Battu). We will report about the cloning of meg-3 that maps on chromosome II between the unc-4 and unc-53 markers.