Herrera, R.A. et al. (2015) International Worm Meeting "LEP-2/Mkrn promotes degradation of LIN-28 to time the larval-to-adult transition."

Fitch, D., Herrera, R.A., Kiontke, K.

[International Worm Meeting, 2015]

C. elegans and mammals share a developmental milestone: the juvenile-to-adult (J/A) transition. In mammals, the timely onset of the J/A transition (puberty) requires two highly conserved genes, LIN28b and Makorin3 (Mkrn), and altering their levels results in early or delayed pubertal onset. The C. elegans larval-to-adult (L/A) transition is scheduled by heterochronic genes and lin-28 is a key regulator. Here we present LEP-2/Mkrn as a novel heterochronic regulator of the L/A transition in C. elegans.lep-2 was found in a screen for defects in male tail tip morphogenesis (TTM), a process whereby the tail tip changes shape from long and pointed to short and rounded. lep-2 mutants display several retarded phenotypes: 1 TTM is delayed and young adults have larval-like tail tips that will undergo TTM as the male ages. 2 The adults of both sexes lack the adult-specific collagen ROL-1. 3 Supernumerary molts occur. 4 lep-2(-) males do not perform wild-type mating behavior, although male tail morphology is unaffected. Surprisingly, seam cell number and lateral alae are normal in lep-2(-) mutants. This suggests that lep-2 acts redundantly or is not involved in regulating the timing of seam cell development.Epistasis analysis demonstrates that lep-2 represses lin-28: lin-28 RNAi is sufficient to suppress the lep-2(-) phenotype. In wild type, LIN-28 is down-regulated during the L2 stage. In lep-2(-) mutants this down-regulation does not occur, and LIN-28 protein perdures, yet lin-28 mRNA levels are unaffected. Thus LEP-2 regulates LIN-28 post-transcriptionally. As a Mkrn, lep-2, has putative nucleic acid binding and E3 ubiquitin ligase activities. This allows for two predictions of how lep-2 regulates LIN-28, by inhibiting translation, or promoting LIN-28 degradation. Using a LIN-28::Dendra2 fusion, we determined that LEP-2 acts on LIN-28 stability, consistent with its function as a predicted E3 ubiquitin ligase.Taken together, our findings suggest that LIN-28 and LEP-2/Mkrn are conserved functionally in controlling J/A transitions, but may also have a conserved molecular interaction. Based on this, we propose that the C. elegans tail tip is a valuable model to study the J/A transition in animals in general.

Kiontke, K.C. et al. (2019) International Worm Meeting "The long non-coding RNA lep-5 promotes the juvenile-to-adult transition by destabilizing LIN-28."

Schwarz, E.M., Portman, D.S., Luo, J., Kiontke, K.C., Vuong, E., Fitch, D.H.A., Herrera, R.A.

[International Worm Meeting, 2019]

Using forward genetics, we discovered lep-5, which produces a lncRNA that acts in the C. elegans heterochronic pathway. Loss of lep-5 delays maturation of the epidermis and male tail tip morphogenesis (TTM), hallmarks of the juvenile-to-adult transition. lep-5 mutations have no effect on the seam-cell lineage and only a weak effect on alae production, suggesting that seam cells, unlike other epidermal cells, largely bypass the requirement for lep-5. The lep-5 RNA is ca. 600 nt and localized to the cytoplasm. Evidence that the RNA itself provides the primary function comes from structure-function experiments and patterns of conservation. Specifically, the lncRNA possesses three essential secondary structure motifs that are conserved across the Caenorhabditis genus. In contrast, none of the potential ORFs in C. elegans lep-5 are conserved nor essential for lep-5 function. lep-5 expression is temporally controlled, peaking prior to TTM onset. Epistasis tests show that lep-5 acts downstream of lin-4 and lin-14 and upstream of lin-28. Like the Makorin LEP-2 (which has RNA-binding and E3 ubiquitin-ligase domains), lep-5 facilitates the degradation of LIN-28. CoIP experiments show that both LIN-28 and LEP-2 associate with lep-5 in vivo, suggesting that lep-5 directly regulates LIN-28 stability, probably by scaffolding these two proteins together. Even though LEP-2 is expressed earlier than lep-5, LIN-28 degradation does not occur until lep-5 is expressed, suggesting that lep-5 plays the instructive role in timing LIN-28 degradation. These studies identify a key biological role for a lncRNA: by regulating protein stability, it provides a temporal cue to promote the juvenile-to-adult transition. Because LIN-28 and Makorins are highly conserved, as are their roles in juvenile-to-adult transitions, it is possible that a lncRNA similarly scaffolds LIN-28/Makorin interactions in other metazoa.

Kiontke, K.C. et al. (2019) International Worm Meeting "Elucidating the gene regulatory network controlling male tail tip morphogenesis in C. elegans via ChIP-seq and tissue-specific transcriptomics."

Mason, D.A., Woronik, A., Fitch, D.H.A., Mahmood, R., Kiontke, K.C., Gunasekera, S., Herrera, R.A., Fernandez, P.

[International Worm Meeting, 2019]

How gene networks regulate the development of sexually dimorphic adult structures remains largely unknown. We address this question in the male tail tip of Caenorhabditis elegans, a relatively simple developmental model. The L4 male tail undergoes a remodeling event known as Tail Tip Morphogenesis (TTM). During TTM, the four tail tip cells fuse, change shape, and migrate anteriorly, generating a rounded adult tail in males. In hermaphrodites, these same four cells do not undergo TTM and tails remain pointed. Prior work showed that the DM-domain transcription factor DMD-3 is required and sufficient for TTM. The aim of this work is to uncover "effector" genes that function downstream of DMD-3 to power TTM. To identify DMD-3 targets specifically in male tail tips, we used laser-capture microdissection to isolate tail tips from wild type males, hermaphrodites and dmd-3(-) larval males and conducted tissue-specific RNASeq on pooled tail tips. Differential expression analyses yielded 561 differentially expressed genes, many of which are likely to be direct and indirect targets of DMD-3. To identify bona fide DMD-3-regulated genes, we are analyzing the expression pattern of transcriptional reporters in wild type and mutant tail tips and determining the loss-of-function phenotypes for a subset of these candidate genes. To distinguish direct versus indirect DMD-3-regulated genes we are also conducting a ChIP-seq experiment with tagged DMD-3. Finally, we are incorporating known interactions for TTM candidate genes to develop a draft interaction network. Our results will provide a draft for the gene regulatory network governing a postembryonic, sexually dimorphic morphogenetic process.

Nelson, Matthew D. et al. (2011) International Worm Meeting "Genetic architecture of male tail tip morphogenesis."

Fitch, David H.A., Kiontke, Karin C., Nelson, Matthew D., Herrera, R. Antonio

[International Worm Meeting, 2011]

A major goal of research in morphogenesis is to delineate all steps that connect spatiotemporal regulation to the cellular machinery which affects changes in cell shape and migration. The four-celled tip of the C. elegans male tail is a simple but powerful model for studying morphogenesis. It also presents a model for sexual dimorphism at the cellular level. Through a genome-wide post-embryonic RNAi-feeding screen, we identified 211 genes that regulate or participate in male tail tip morphogenesis. Indeed, we found regulatory roles for well-characterized developmental pathways: the posterior Hox genes, the TGF-beta pathway, nuclear hormone receptors, the heterochronic pathway (for which we have also identified new components, see abstracts by Vuong et al. and Herrera, Kiontke et al.) and GATA transcription factors. Based on gene ontology terms and information mined form WormBase, we have clustered all tail tip morphogenesis genes into 24 developmental pathways or general cell biological processes. Using expression epistasis, we have constructed a genetic interaction network between representatives of these 24 clusters underlying morphogenesis. We have found these pathways to converge at the transcription factors DMD-3, MAB-3 and possibly NHR-25, which then appear to coordinate several modules of the cellular machinery, including cell fusion, endocytosis, and the cytoskeleton. Based on these data, we hypothesize that male tail tip morphogenesis is governed by a gene regulatory network that has a bow-tie architecture. We are currently testing for additional interactions within unresolved parts of this network.

Tang, Lois et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Asymmetric regulation of the homeobox gene ceh-5 in early embryogenesis of C. elegans"

Abou-Zied, Akram, Hench, Jurgen, Tang, Lois, Shlyueva, Daria, Cesnulevicius, Konstantin, Burglin, Thomas

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Homeobox genes are highly conserved transcription factors that play key roles in the development of humans and animals (1). From a 4D microscopy expression screen performed in the Brglin lab, a homeobox gene ceh-5 displaying a unique asymmetrical expression was revealed. This ortholog of the human VAX genes is expressed in three different groups of cells during gastrulation. An intriguing aspect of ceh-5 expression in the two bilaterally symmetric c ell groups is a left-right asymmetry in their expression levels. Little is understood about cell fate specification after the beginning of gastrulation, and also how left-right asymmetries are generated; ceh-5 , therefore, provides an excellent entry point to study these phenomena. We are dissecting the promoter region of ceh-5 to understand how the distinct spatio-temporal expression is generated. We created a series of deletions in the promoter region of ceh-5 fused to GFP that were used to make transgenic animals. The GFP expression patterns in the transgenic worms were monitored using 4D-imaging. This investigation revealed a 21bp in the promoter region containing an E-box motif essential for the regulation of ceh-5 . This motif is highly conserved between different Caenorhabditis species and is known to be a binding site for basic helix-loop-helix (bHLH) transcription factors (2). In order to look for transcription factors involved in regulating the expression of ceh-5 , we screen via RNAi knockdown, searching for candidates that would create an altered ceh-5 expression pattern. Although the screen is not yet saturated, putative bHLH transcriptional regulators of ceh-5 have been identified. 1) Burglin, T.R. 2005. Encyclopedia of Molecular Cell Biology and Molecular Medicine (ed. R.A. Meyers). 2) Yamada, K. and Miyamoto, K. 2005. Front Biosci.

Erich M Schwarz et al. (2003) International Worm Meeting "Comparative analysis of cis-regulatory sequences using four Caenorhabditis species."

Nora Mullaney, Erich M Schwarz, Tristan De Buysscher, Paul W Sternberg, Barbara J Wold, John A DeModena, Eunpyo Moon, Hiroaki Shizuya

[International Worm Meeting, 2003]

Comparing homologous cis-regulatory DNA sequences from three or more genomes has advantages over pairwise comparison of only two. Cis-regulatory sequences are short (6-20 bp) and tolerate substantial variation. Purely random pairing of unrelated 100-bp DNA segments is expected to yield two perfect 6 bp matches. Alignment of a third or fourth sequence should greatly lower the frequency of false positive regions, allowing small but real cis-regulatory sequences to be efficiently detected. This increased resolution should also allow direct comparison between phylogenetically conserved sequences and statistically overrepresented sequences, which may yield complementary views of regulatory elements. In the Caenorhabditis genus, C. remanei appears to be most closely related to C. briggsae; two other species, CB5161 and PS1010, comprise the two closest known and culturable Caenorhabditis species outside the elegans-briggsae group (Fitch, 2000). CB5161 is closest to C. elegans, and PS1010 the next most divergent; these two species thus provide an evolutionarily graded series. We have constructed fosmid libraries from CB5161 and PS1010, and begun sequencing individual fosmids for comparative analysis of genes involved in vulval or sensory neuron development. At the same time, we have devised the Mussa software package to adapt the algorithms of Davidson and coworkers (Brown et al., 2002) to multiple sequence analysis. At this writing, we have sequence data from the egl-30, lin-11, and mab-5 loci of both CB5161 and PS1010. Initial results of sequencing and comparative sequence analysis will be presented. References: Brown, C.T., Rust, A.G., Clarke, P.J., Pan, Z., Schilstra, M.J., De Buysscher, T., Griffin, G., Wold, B.J., Cameron, R.A., Davidson, E.H., and Bolouri, H. (2002). New computational approaches for analysis of cis-regulatory networks. Dev. Biol. 246, 86-102; Fitch, D.H.A. (2000). Evolution of Rhabditidae and the male tail. J. Nematol. 32, 235-244.

Ellis HM et al. (1998) East Coast Worm Meeting "C. elegans era, homolog of an E. coli cell cycle gene?"

Golden A, Powell BS, Court DL, Ellis HM

[East Coast Worm Meeting, 1998]

Members of the GTPase superfamily are found in organisms ranging from bacteria to humans. These proteins function as molecular switches, regulating a variety of cellular processes including signal transduction, cell cycle regulation and protein translocation. The E. coli GTPase, ERA (E.coli ras-like) is an essential protein required for normal progression through the cell cycle1. Reduction of era function results in cell cycle arrest after chromosome partitioning, but prior to cytokinesis. Sequence comparisons suggest that ERA and its homologs in other bacteria, C.elegans, mouse and human form a new subfamily of GTPases. These proteins show extensive homology both in the GTPase domain and in a C-terminal region of unknown function. In searches of the C.elegans database with the conserved carboxy-terminal region of E. coli ERA we identified a single homolog, E02H1.2, which we will refer to as Ce-ERA-1. Ce-ERA-1, is 31% identical (49% similar) to E.coli ERA in the GTPase domain and 19% identical (43% similar) to E.coli ERA in the C-terminal region. RT-PCR experiments generated an SL2 spliced cDNA with an exon structure matching that predicted by the genome project. The function of Ce-era-1 is unknown. Attempts to disrupt Ce -era-1 function by RNA-mediated interference did not generate any obvious phenotype. We are currently taking two approaches to the analysis of Ce-era-1 function: 1) Expression of transgenes carrying mutations predicted to produce dominant activated or dominant negative phenotypes. 2) Isolation of null mutations in a PCR based screen for deletions. In addition we have generated polyclonal antibodies against Ce-ERA peptides and will examine the temporal and spatial patterns of Ce-ERA expression. 1. Britton, R.A., Powell, B.S., Dasgupta, S., Sun, Q., Margolin, W., Lupski, J.R., and Court, D.L.(1998) Cell cycle arrest in Era GTPase mutants: a potential growth regulated checkpoint in Escherichia coli. Molecular Microbiology 27(4), 739-750. This work was sponsored by the National Cancer Institute, DHHS, under contract to ABL.

Evan Z Macosko et al. (2007) International Worm Meeting "Regulation of C. elegans roaming behavior by a multi-component pheromone."

Jon Clardy, Rebecca Butcher, Jim Thomas, Evan Z Macosko, Cori Bargmann

[International Worm Meeting, 2007]

Overall movement during animal foraging can take two general strategies: exploration of a wide area (roaming) and restriction to a local area (dwelling). C. elegans, when feeding on a bacterial lawn, has been shown to alternate between roaming and dwelling states.i Recordings of worm movements on food indicate that the behavioral state changes are arrhythmic and can be modified by food quality. We found that a multi-component pheromone released from incised or lysed worms dramatically inhibits C. elegans roaming behavior. The same worm lysate also acts as a potent repellent, suggesting that this mixture could be analogous to an alarm pheromone. Our chemical analysis indicates that there are at least two important components of the worm lysate that inhibit roaming: (1) a derivative of the sugar ascarylose, which is also a component of dauer pheromoneii and (2) a small indole-containing compound. We are currently asking if the same compounds also induce acute avoidance behavior. To determine the neuronal circuitry that regulates roaming and dwelling, we killed individual cells by laser ablation. Loss of either ADL, ASK, or AWC increased roaming in the presence of pheromone. In contrast, ablation of ASI resulted in a constitutive dwelling phenotype. The interneurons AIZ and AIY are important for proper roaming and dwelling behavior, while AIB and AIA are less important. Finally, though AVA is a crucial command interneuron for many locomotory behaviors, we found that AVA-ablated animals roam relatively normally. We also tested candidate mutants for sensitivity to pheromone. Mutations in egl-4, a gene previously shown to antagonize roaming,i reduce pheromone sensitivity. Rescue of egl-4 using heterologous promoters indicates that the EGL-4 gene product functions in chemosensory neurons to promote dwelling. iFujiwara M., P. Sengupta and S.L. McIntire, 2002 Regulation of body size and behavioral state of C. elegans by sensory perception and the EGL-4 cGMP-Dependent Protein Kinase. Neuron 36: 1091-1102. iiButcher R.A., M. Fujita, F.C. Schroeder, J. Clardy, Small Molecule Signaling of Dauer Formation in C. elegans, 2007 International Worm Meeting.

Powell BS et al. (2000) East Coast Worm Meeting "C. elegans era, homolog of an E. coli cell cycle gene?"

Golden A, Ellis HM, Powell BS, Court DL

[East Coast Worm Meeting, 2000]

Members of the GTPase superfamily are found in organisms ranging from bacteria to humans. These proteins function as molecular switches, regulating a variety of cellular processes including signal transduction, cell cycle regulation and protein translocation. The E. coli GTPase, ERA is an essential protein required for normal progression through the cell cycle1. Reduction of era function results in cell cycle arrest after chromosome partitioning, but prior to cytokinesis. Sequence comparisons suggest that ERA and its homologs in other bacteria, C.elegans, mouse and human form a new subfamily of GTPases. These proteins show extensive homology both in the GTPase domain and in a C-terminal RNA binding domain. In searches of the C.elegans database with the conserved carboxy-terminal region of E. coli ERA we identified a single homolog, E02H1.2, which we will refer to as Ce-ERA-1. Ce-ERA-1, is 31% identical (49% similar) to E.coli ERA in the GTPase domain and 19% identical (43% similar) to E.coli ERA in the C-terminal region. RT-PCR experiments generated an SL2 spliced cDNA with an exon structure matching that predicted by the genome project. The first gene in this operon E02H1.1 is homologous to the bacterial rRNA methyltransferase ksgA, which interacts genetically with E. coli era. Thus there might be a functional relationship between the two genes in the C. elegans operon. RNA-mediated interference of E02H1.1 results in a severe developmental delay and larval lethality. The function of Ce-era-1 is unknown. Attempts to disrupt Ce-era-1 function by RNA-mediated interference did not generate any obvious phenotype. A null allele of Ce-era-1 was recently identified in a PCR screen (R. Ranganathan and H.R. Horvitz, personal communication). Characterization of this allele will be presented. 1. Britton, R.A., Powell, B.S., Dasgupta, S., Sun, Q., Margolin, W., Lupski, J.R., and Court, D.L.(1998) Cell cycle arrest in Era GTPase mutants: a potential growth regulated checkpoint in Escherichia coli. Molecular Microbiology 27(4), 739-750.

Szentgyorgyi, Erik et al. (2015) International Worm Meeting "Polar body extrusion in the early C. elegans embryo."

Muller-Reichert, Thomas, Koenig, Julia, Szentgyorgyi, Erik

[International Worm Meeting, 2015]

Polar body extrusion (PBE) plays an essential role in genome haploidization during female meiosis. The underlying mechanism, however, is poorly understood. The process of extruding a small polar body out of the large oocyte can be considered as an extreme case of asymmetric cytokinesis. In mammalian cytokinesis, actomyosin-driven membrane constriction leads to a compression of the central spindle. ESCRT-III components then cause a secondary constriction at the intercellular bridge, which is accompanied with a severing of microtubules caused by spastin. Importantly, microtubule severing is the rate-limiting step in this process [1].We aim to unravel the mechanism of PBE in the early C. elegans embryo. How is the polar body extruded, and how are the constriction of the contractile ring and the disassembly of the central spindle coordinated in this system?To answer these questions we have started to combine live-cell imaging and electron tomography. Live-cell imaging of wild-type embryos indicated a difference in spindle disassembly during extrusion of the first and second polar body. In the first meiotic division, the central spindle appears to be disassembled in the middle between the separating homologous chromosomes. In contrast, spindle disassembly in meiosis II appears to take place adjacent to the chromatin involved in the formation of the female pronucleus. In addition, we aim to deplete components of the chromosomal passenger complex (CPC) and the centralspindlin complex by RNAi [2]. Since both complexes are likely to be involved in contractile ring formation and constriction during PBE we want to observe the influence of contractile ring constriction on central spindle disassembly. Furthermore, we have started to analyze taxol- and nocodazole-treated embryos to investigate the influence of microtubule stability on polar body extrusion. In parallel, we compare the ultrastructure of spindles in late anaphase I and II by electron tomography.Interestingly, we found no indication for the formation of helical structures involved in membrane constriction, indicating that PBE in C. elegans is most likely achieved through an ESCRT filament-independent mechanism.[1] Guizetti, J. et al.; Cortical Constriction During Abscission Involves Helices of ESCRT-III-Dependent Filaments; Science 331, 1616 (2011)[2] Green, R.A. et al.; Cytokinesis in Animal Cells; Annu. Rev. Cell Dev. Biol. 28:29-58 (2012).