Kelly A Harradine et al. (2004) West Coast Worm Meeting "Analysis of pel-2, a gene required for coordination of pharynx and epidermal development."

Renaud LEGOUIS, Joel H Rothman, Kelly A Harradine

[West Coast Worm Meeting, 2004]

Organ development can be divided into two phases known as the restrictive phase, when cells become determined to adopt an organ-specific fate, and the expressive phase, when cells comprising the organ acquire their cell-specific identities. Accordingly, among the genes known to control pharynx development in C. elegans are those required to establish organ identity and those involved in pharynx differentiation. The regulatory mechanisms that coordinate development between different organs are not well understood. To illuminate how late organ development is achieved and to understand the coordination of regulatory mechanisms involved in development of the pharynx and epidermis, we have isolated and characterized a zygotic embryonic lethal mutant, p harynx and el ongation defective ( pel-2 ). pel-2 mutations result in a block in terminal differentiation of the pharynx, as well as a failure to express the pharynx differentiation-promoting factor PHA-1 and to maintain expression of the pharynx identity factor, PHA-4. These findings imply that pel-2 acts within the pathway that regulates pharynx organ identity and differentiation. pel-2 mutants also fail to express several epidermal genes in the epidermis and show ectopic expression of the same genes in the pharynx, suggesting that PEL-2 is required reciprocally to activate epidermal genes in the epidermis and to repress them in the pharynx. We mapped pel-2 to the right end of chromosome IV and found that YAC Y73F8A rescued the embryonic lethality associated with the pel-2 mutation. Single gene rescue experiments revealed that the gene Y73F8A.34, defined by expressed sequence tags, also rescues embryonic lethality of different pel-2 alleles. RNAi of two non-overlapping regions of Y73F8A.34 phenocopies the pel-2 mutation, lending further support that this gene is pel-2 . However, efforts to identify the molecular lesion in this gene from pel-2 mutants have not yet been successful. Y73F8A.34 encodes a novel cysteine-rich domain, C6HC or DRIL motif, flanked by two Zn-binding RING fingers of the TRIAD (two RING fingers and DRIL) class of proteins, suggesting that it might perform a novel function in pharynx development (Van der Reijden et al., 1999). To determine the spatial and temporal expression pattern of Y73F8A.34, we analyzed expression pattern of three integrated transcriptional reporters. Expression is first detected at about 200 min. after fertilization, when there are 27 pharyngeal precursors. This observation is consistent with our finding that pel-2 is required for normal expression of pha-1 and pha-4 at this early stage. Expression of this putative pel-2 gene in pharyngeal cells continues throughout embryogenesis and appears to increase as development proceeds. Expression in the pharynx persists into adulthood and is also detected in the gut. The 5'regulatory region of the Y73F8A.34 gene contains five putative PHA-4 binding sites (Gaudet and Mango, 2002), suggesting that Y73F8A.34 is likely to be regulated by PHA-4. Further studies are directed at understanding the role of this gene in the regulatory pathway for pharynx development and epidermal differentiation. B. A. Van der Reijden , C. A. J. Erpelinck-Verschueren, B.Lowenberg and J.H. Jansen Protein Science 8 :1557-1561 (1999). J. Gaudet and S. Mango Science 295 :821-825 (2002).

Kelly A Harradine et al. (2002) West Coast Worm Meeting "Analysis of pel-2, a gene required for embryonic pharyngeal differentiation and body morphogenesis"

Joel H Rothman, Kelly A Harradine

[West Coast Worm Meeting, 2002]

Organogenesis is a complex process requiring the coordinated specification, morphogenesis, and differentiation of disparate cell types. To better understand how metazoan organ development is achieved, we are investigating a gene required for embryonic pharynx development, pel-2 (pharynx and elongation defective). The pel-2 mutant was isolated in a screen designed to identify zygotic genes essential for embryonic development.

Kelly A Harradine et al. (2003) International Worm Meeting "pel-2 is required to activate the pharynx organ selector gene and reciprocally regulates gene expression in the epidermis and pharynx"

Kelly A Harradine, Joel H Rothman

[International Worm Meeting, 2003]

While a good deal is known about the molecules that drive specification of the pharynx primordium, comparatively less is known about the morphogenetic and differentiation events that subsequently transform this primordium into the bilobed pharynx. To learn more about these processes, we are investigating a mutant called pel-2(pharynx and elongation defective) that is blocked in pharynx development between the early and late stages. Unlike the pha-4/FoxA and pha-1/bZip mutants, which are completely defective in pharynx formation and pharynx differentiation/morphogenesis respectively, the pel-2 mutant is only partially blocked in these processes, and is also fails to undergo overall elongation of the animal during morphogenesis. To form the pharynx primordium, the organ-specific selector gene pha-4 acts on disparate cells types to activate transcription of pharynx-specific genes. We have found that pha-4 is expressed at greatly reduced levels at all stages of pharynx development in pel-2 animals, suggesting that PEL-2 is required for transcriptional activation of pha-4. Furthermore, expression of a myo-2 reporter driven by a pha-4-dependent enhancer is drastically reduced in pel-2 mutant embryos, supporting the hypothesis that high levels of pha-4 expression are required to activate late-acting pharyngeal genes such as myo-2. Consistent with this observation, we have found that differentiated pharynx gland and marginal cells are diminished in number, or eliminated, while early muscle markers are unaffected. These findings support a model in which pel-2 acts on both pha-4 and pha-1 to complete pharyngeal differentiation and morphogenesis. When expression of a selector protein such as PHA-4 is altered, cell fate transformations often occur. Indeed, analysis of epidermal markers, including reporters for mel-11 and let-502, suggest that pharyngeal cells in pel-2 mutants are partially transformed into an ectodermal cell fate. Curiously, the normal expression of these genes in the epidermis is greatly reduced or abolished, indicating a reciprocal effect of pel-2 on activation and repression of their expression in the epidermis and pharynx respectively. The failure to express these genes in the epidermis may explain the elongation defect; indeed, terminal pel-2 embryos arrest at a similar stage in elongation to that of let-502 and mel-11 mutants. We have rescued the pel-2 mutant with a YAC clone and are currently identifying a minimal rescuing gene.

Kelly Harradine et al. (2001) International C. elegans Meeting "Molecular analysis of pel-2 and pel-3, two genes required for embryonic pharynx differentiation and morphogenesis"

Joel H Rothman, Kelly Harradine

[International C. elegans Meeting, 2001]

The pharynx is comprised of five different cell types, all of which derive from two blastomeres in the early embryo, ABa and MS. How are cells that arise from disparate lineages specified and patterned to form this single, coherently functioning organ? While some of the genes that impart pharynx-specific identity to the five pharynx cell types have been characterized, the mechanisms that regulate pharynx morphogenesis and differentiation are largely unknown. To address these problems, we are investigating two genes required for embryonic pharyngeal development, pel-2 IV and pel-3 II (p harynx and el ongation defective) . Both genes were identified from mutations isolated in a genome-wide screen for zygotic embryonic lethal mutants (JH Rothman, 8th International C. elegans Meeting). The pel-2 mutant shows a block in embryonic elongation, with arrest occurring at the comma to 1.5-fold stage. The pharynx neither elongates nor displays any signs of differentiation or morphogenesis (e.g., a lumen, grinder, buccal capsule, or muscle striations). The pel-3 mutant arrests at the two- to three-fold stage with a partially elongated pharynx that never attaches to the buccal opening and with variable defects in differentiation and morphogenesis (e.g., the grinder, lumen, and buccal capsule are variably absent). We are analyzing the expression of pharynx markers and the behavior of pharyngeal cells in an effort to assess whether the defects we observe arise from defective differentiation or morphogenesis of the pharynx. To test whether the pel genes act within the known pathway of regulators for pharynx development (e.g., pha-1, pha-4 , and ceh-22 ), we are examining expression of these genes in pel mutants and are investigating genetic interactions between them. We have narrowed the chromosomal positions of both pel genes using SNIP-SNP mapping in an effort to clone them. By dissecting the function of pel-2 and pel-3 genetically and molecularly, we hope to gain further insights into how organ development and assembly are molecularly orchestrated.

Kow, R.L. et al. (2019) International Worm Meeting "Polyadenylation nucleases modulate tau-induced toxicity in tau transgenic Caenorhabditis elegans."

Kow, R.L., Strovas, T.J., Chickering, K., Kraemer, B.C., Saxton, A.

[International Worm Meeting, 2019]

The microtubule-associated protein tau accumulates into toxic aggregates in multiple human neurodegenerative diseases such as Alzheimer's disease. To identify novel genes that modulate human tau-induced toxicity, we have used a Caenorhabditis elegans model in which human tau protein is overexpressed in all C. elegans neurons. This causes significant motor dysfunction, progressive loss of neurons, shortened lifespan, and the accumulation of hyperphosphorylated and insoluble tau protein. Using chemical mutagenesis, we identified and then verified that loss of sut-2 strongly suppresses tau-induced toxic phenotypes in tau transgenic C. elegans. The mammalian homolog of sut-2, MSUT2/ZC3H14, was shown to regulate polyadenylation (poly(A)); loss of MSUT2 leads to longer poly(A) tails (Kelly et al (2014) RNA 20:681-688). We examined whether genes encoding poly(A) nucleases, which regulate poly(A) tail length by removing poly(A), also modulated tau-induced toxicity. We found that loss of function alleles in ccr-4, panl-2, and parn-1 enhanced tau-induced toxicity while loss of parn-2 suppressed tau-induced toxicity. This suggests that although poly(A) nucleases can modulate tau-induced toxicity, they may do so via different mechanisms.

Ming Ming Huang et al. (2001) International C. elegans Meeting "Construction of GFP Tagged Chromosomes"

Don Moerman, Ming Ming Huang, Robert Barstead

[International C. elegans Meeting, 2001]

We have constructed 17 C elegans strains carrying GFP or YFP expressing transgenes integrated randomly in wild type chromosomes. The fluorescent transgenes will be useful as benign dominant markers in crosses to replace chromosomes and as weak balancers for mutations in closely linked essential genes. The integrated transgenes began life as an extrachromosomal array, with GFP fused to promoters for myo-2 and pes-10 or a gut-specific enhancer from F22B7.9. The arrays were integrated using standard methods (1). The resulting strains were outcrossed four times to wild type. Presently, we are mapping the location of each transgene. We will describe their map positions and phenotypes. 1. Mark Edgley, Jun Kelly Liu, Don Riddle, Andy Fire. Worm Breeder's Gazette 15(5): 20 (February 1, 1999).

Katherine M Walstrom et al. (2005) International Worm Meeting "Transcription regulation of endogenous genes by C. elegans RNA Helicase A"

Deborah Schmidt, Christopher R Bauer, Katherine M Walstrom

[International Worm Meeting, 2005]

Transcriptional silencing is an important process required for early germline development in some organisms such as C. elegans and Drosophila (Leatherman and Jongens (2003) BioEssays 25, 326-335). RNA helicase A (RHA) is a conserved protein with roles in transcription regulation and histone modification in numerous organisms such as flies and humans. We are studying the role of C. elegans RHA (RHA-1) in transcription regulation and germline development. A null mutant, rha-1(tm329), has a temperature-dependent defect in germline transcriptional silencing of transgene arrays (Walstrom, Schmidt, Bean, and Kelly (2005) Mech. Dev. In press.). In addition, histone modifications are mis-localized on the transgene arrays, on autosomes, and on the single male X chromosome. These defects in gene regulation lead to a sterile phenotype in hermaphrodite and male worms. We are using real-time RT-PCR to determine if endogenous genes have altered expression in the mutants, and we are focusing on the X chromosome in the mutant males. The rha-1 mutant also has a temperature-dependent sterile phenotype, and we hope to identify genes with altered expression that could lead to the sterile phenotype. Recent results will be presented at the meeting.

Robert J Johnston Jr et al. (2005) International Worm Meeting "Nuclear function of an actin-binding protein in neuronal fate determination"

Oliver Hobert, Robert J Johnston Jr

[International Worm Meeting, 2005]

The formin homology 2 (FH2) domain controls actin nucleation both in vivo and in vitro. Recent structural studies provided the mechanistic basis for this activity. In a screen for mutants that affect ASE cell fate determination (see abstract by Flowers et al.), we were surprised to identify an allele of K01B6.1, a predicted gene containing a FOrmin homology 2 domain and two DNA-binding ZInc finger motifs (hence called fozi-1). fozi-1 alleles were also retrieved in a screen by Kelly Lius lab for genes affecting mesoderm specification. We find that fozi-1 is expressed in a very restricted number of head neurons, including ASEL and ASER. The effect that fozi-1 has on ASEL and ASER cell fate specification is unusual. In contrast to many other left/right asymmetry-inducing genes that we have analyzed (see abstract by Johnston et al.), fozi-1 mutants display a "mixed" phenotype, in that ASEL fate is partially de-repressed in ASER, but ASER fate is unaffected. We find that a rescuing fozi-1::gfp transgene localizes to the nucleus consistent with a role of fozi-1 in gene regulation. The presence of an FH2 domain in fozi-1 suggests that an actin interacting protein, and therefore by interference, actin, has a role in the nucleus. Recent in vitro evidence indeed indicates that actin may play a role in RNA Pol I, II and III-mediated nuclear transcription events. fozi-1 may be the first DNA-binding protein identified to provide a direct, genetically verified link between actin and transcription.

Snyder, Matthew P. et al. (2013) International Worm Meeting "Turnover of the H3K9me2 Mark During Late Spermatogenesis."

Snyder, Matthew P., Xu, Xia, Maine, Eleanor

[International Worm Meeting, 2013]

Meiotic silencing is a conserved phenomenon targeting unpaired chromosomes and chromosomal regions during prophase of meiosis I. Meiotic silencing in animals typically occurs at the chromatin level and involves accumulation of histone modifications thought to promote a closed chromatin configuration. This chromatin structure may contribute to transcriptional repression and meiotic chromosomal events such as chromosome disjunction (Bean et al 2004, Jaramillo-Lambert and Engebrecht 2010). During meiosis in C. elegans, non-synapsed chromosomes are enriched for H3K9me2 relative to synapsed chromosomes (Kelly et al. 2002; Bean et al. 2004). Such non-synapsed chromosomes include the male X, homologous chromosomes that fail to synapse due to mutation, and chromosomal translocations/duplications. The pattern of H3K9me2 accumulation during meiosis depends on activity of the small RNA machinery, which may have a role in targeting the mark to unpaired chromosomes and ensuring that the mark does not persist abnormally (Maine et al. 2005; She et al. 2009). Taking a combined biochemical/genetic approach, we are identifying additional factors important for regulating H3K9me2 distribution during meiosis. Through this approach, we are identifying genes that appear to be important for turnover of the H3K9me2 mark during late spermatogenesis.

Guo, Yiqing et al. (2013) International Worm Meeting "Differential expression of germline genes in the presence/absence of H3K9me2."

Maine, Eleanor, Guo, Yiqing

[International Worm Meeting, 2013]

In C. elegans, indirect immunofluorescence experiments indicate a high level of histone H3 lysine 9 dimethylation (H3K9me2) on any unpaired/unsynapsed chromosomes during first meiotic prophase, particularly in pachytene stage nuclei (Kelly et al 2002; Bean et al 2004). In contrast, H3K9me2 marks are relatively low on paired chromosomes at this time. H3K9me2 marks are typically associated with transcriptional repression. Thus, the elevated level of H3K9me2 on unpaired chromosomes may play a role in meiotic silencing of unpaired chromosomes (MSUC). Germline H3K9me2 marks require activity of MET-2 (Bessler et al 2010), a SET domain protein that is the only C. elegans ortholog of human SETDB1 H3K9 methyltransferase (Schultz et al 2002). To begin to investigate the importance of H3K9me2 marks on unpaired chromosomes, we performed RNA-seq on mRNA isolated from him-8 vs met-2;him-8 adult hermaphrodite gonads. Our data identify genes that are expressed in the gonads and that differentially expressed in met-2(0) and met 2(+). In most cases, the differentially expressed transcripts are elevated in met-2(0), consistent with a role for H3K9me2 in limiting transcription. Although elevated transcripts are not preferentially X-linked, we observe a greater average fold-change for X-linked transcripts than for autosomal transcripts. To evaluate whether the unpaired status of the X contributes to this pattern, we are evaluating additional genotypes. As a complementary approach, we are also investigating the distribution of H3K9me2 on paired vs unpaired chromosomes using a ChIP-seq approach.