-
[
International Worm Meeting,
2009]
The nematode eggshell is a rigid, impermeable structure that protects the embryo during early development, and is required for the first embryonic cell division. The essential function of the eggshell makes it an attractive drug target to combat parasitic nematode infection, but little is known about the composition of the eggshell or the process by which it forms. We have identified two genes redundantly required for eggshell formation.
cpg-1/cej-1 and
cpg-2 encode chondroitin proteoglycans, a class of extracellular glycoproteins modified with chondroitin sugar chains. Live imaging of fluorescent strains shows that CPG-1 and CPG-2 are secreted from caveolin-enriched cortical granules during meiosis I, while immuno-electron microscopy experiments show they associate with the inner layer (the permeability barrier) of the eggshell. To better understand the function of chondroitin proteoglycans in eggshell assembly, we depleted CPG-1, CPG-2, and SQV-5 (the enzyme that synthesizes chondroitin) by RNAi. In utero, embryos co-depleted of CPG-1/CPG-2 or chondroitin have identical phenotypes, exhibiting defects in osmotic integrity and early cortical events dependent on the actomyosin contractile network, including polar body extrusion during meiosis I and II, membrane ruffling, pseudocleavage formation, polarity establishment, and cytokinesis. However, when osmotic support is provided to dissected embryos, early embryonic events (including cytokinesis and polarity establishment) are robustly rescued in chondroitin depletions, but only partially rescued in CPG-1/CPG-2 co-depletions. Ultra-structural analysis shows embryos depleted of chondroitin are able to form all three eggshell layers, while embryos co-depleted of CPG-1/CPG-2 fail to form the inner layer. The correlation of structural and functional data suggests that CPG protein cores have a function separable from that of the chondroitin chains during eggshell assembly, and provides evidence for the first structural proteins required for eggshell formation. To expand on this work, we are conducting an RNAi-based screen to identify additional genes required for the enigmatic process of eggshell assembly.
-
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Metazoan oocytes have an extracellular coating that governs fertilization. Following fertilization, this covering is altered to prevent polyspermy and protect the developing embryo. In C. elegans, a vitelline layer covers oocytes prior to fertilization. Fertilization initiates conversion of the vitelline layer into a trilaminar eggshell consisting of an outer vitelline layer, a middle chitin-containing layer, and an inner layer proposed to serve as a permeability barrier. Here, we characterize CPG-1 and CPG-2, functionally redundant chondroitin proteoglycans that are the first described protein eggshell components. We show that CPG-1 and CPG-2 are delivered to the extracellular space after formation of the chitin layer by cortical granule exocytosis during meiosis I. Although they contain multiple chitin binding domains, CPG-1 and CPG-2 localize to the inner eggshell layer, whereas chitin is confined to the middle eggshell layer. We show that the inner eggshell layer is not the permeability barrier for small molecular weight solutes. Instead, this function resides in a previously undescribed layer that resides between the eggshell and the plasma membrane. Disruption of the permeability barrier leads to solute permeability and osmotic stress. Disruption of the inner CPG-1/2 eggshell layer causes these phenotypes, as well as adhesion of the embryonic plasma membrane to the eggshell and cytokinesis failure. Interfering with chitin layer assembly results in the inner layer phenotypes, plus polyspermy and catastrophic eggshell rupture. We conclude that the eggshell layers and permeability barrier are laid down in a step-wise fashion from outermost to innermost, with later assembly events requiring successful completion of previous ones.
-
[
International Worm Meeting,
2007]
Chondroitin is a long unbranched sugar polymer synthesized on a protein core that is found either on the cell surface or in the extracellular matrix. Roles for chondroitin in C. elegans embryogenesis and vulval morphogenesis were established based on identification of the squashed vulva (sqv) genes as enzymes required for chondroitin biosynthesis. Subsequently, we reported the identification of nine novel chondroitin proteoglycan (CPG) core proteins that harbor chondroitin chains, none of which are homologous to vertebrate chondroitin sulfate proteoglycans. Two of these proteoglycans, CPG-1(CEJ-1) and CPG-2(B0280.5), are translationally regulated in the germline by GLD-1, which suggested a role in the early embryo. While RNAi depletions of either CPG-1 or CPG-2 alone showed no phenotype, double depletions resulted in penetrant embryonic lethality. Early cortical events dependent on the actomyosin contractile network, including polar body extrusion during meiosis I and II, membrane ruffling, pseudocleavage formation, polarity establishment, and cytokinesis, were absent in doubly depleted embryos. However, nuclear events and spindle dynamics appeared to occur with normal morphology and kinetics. CPG-1 and -2 both contain functional chitin binding domains, suggesting they interact with the chitinous eggshell. We are therefore using a number of approaches to more thoroughly understand the enigmatic process of how the C. elegans eggshell is formed, what role CPGs play in this process, and why it is required for actomyosin contraction. To visualize formation of the eggshell, we are performing live imaging of transgenic lines expressing GFP-tagged versions of CPG-1 and -2, and are developing an assay to monitor, in real time, extrusion of the chitinous layer following fertilization. We are currently conducting immunoprecipitation experiments with CPG-1 and -2 antibodies and using chondroitin affinity columns to identify interacting proteins that may be involved in eggshell formation. While the eggshell has been reported to be dispensable for embryogenesis between the 2-cell stage and hatching, our studies suggest that the eggshell is essential for the actomyosin-driven cortical events immediately following fertilization prior to the first cell division.
-
[
International Worm Meeting,
2011]
Metazoan oocytes have an extracellular coating that governs fertilization. Following fertilization, this covering is altered to prevent polyspermy and protect the developing embryo. In C. elegans, a vitelline layer covers oocytes prior to fertilization. Fertilization initiates conversion of the vitelline layer into a trilaminar eggshell consisting of an outer vitelline layer, a middle chitin-containing layer, and an inner layer proposed to serve as a permeability barrier. Here, we characterize CPG-1 and CPG-2, functionally redundant chondroitin proteoglycans that are the first described protein eggshell components. We show that CPG-1 and CPG-2 are delivered to the extracellular space after formation of the chitin layer by cortical granule exocytosis during meiosis I. Although they contain multiple chitin binding domains, CPG-1 and CPG-2 localize to the inner eggshell layer, whereas chitin is confined to the middle layer. We show that the inner eggshell layer is not the permeability barrier for small molecular weight solutes. Instead, this function resides in a previously undescribed layer that assembles between the eggshell and the plasma membrane following meiosis II. Disruption of the permeability barrier leads to solute permeability and osmotic stress. Disruption of the inner CPG-1/2 eggshell layer causes these phenotypes, as well as adhesion of the embryonic plasma membrane to the eggshell and cytokinesis failure. Interfering with chitin layer assembly results in the inner layer phenotypes, plus polyspermy and catastrophic eggshell rupture. We conclude that the eggshell layers and permeability barrier are laid down in a step-wise and cell cycle-dependent fashion, with later assembly events requiring successful completion of previous ones. To build on this work, we also conducted an RNAi screen to identify additional genes that regulate eggshell and permeability barrier assembly. Several screen hits rendered the eggshell permeable, with minimal deleterious effects on early embryonic development. We therefore developed a reliable method to permeabilize and immobilize embryos to allow temporally-controlled and acute drug/inhibitor treatment to study early embryonic processes with live imaging.
-
Down, T., Chen, R., Egelhofer, T., Ahringer, J., Chen, Q., Hillier, L., Stempor, P., Jeffers, T., Zeiser, E.
[
International Worm Meeting,
2013]
RNA PolII transcription initiation sites are largely unknown in C. elegans. The initial 5' end of most protein-coding transcripts are removed by trans-splicing, and non-coding initiation sites have not been investigated. We identify 73,500 distinct clusters of initiation. Bidirectional transcription is frequent, with a peak of transcriptional pairing at 120 bp. We assign transcription initiation sites to 7691 protein-coding genes and find that they display features typical of eukaryotic promoters. Strikingly, the majority of initiation occurs in intergenic regions with enhancer-like chromatin signatures. Remarkably, productive transcription elongation across enhancers is predominantly in the same orientation as that of the nearest downstream gene. This oriented transcription at upstream enhancers could potentially deliver RNA Pol II to a downstream proximal promoter, or alternatively might function as a distal promoter. CG dinucleotides (CpG islands) are enriched in mammalian promoters. CpG density is thought to be irrelevant in invertebrates that lack DNA methylation such as C. elegans. We find that CpG enrichment at worm promoters shares features of mammalian CpG islands. CpG clusters are found at protein-coding promoters showing nucleosome depletion. In mammals, non-methylated CpGs are bound by Cfp1/CXXC1, which leads to H3K4me3 marking at promoters through recruitment of Set1. Interestingly, a worm Cfp-1 ortholog was reported to be required for global H3K4me3 levels. We found that the worm Cfp-1 is enriched at high H3K4me3 promoters containing high density of CpGs. Moreover, we find that highly occupied target (HOT) regions bound by multiple transcription factors are CpG-rich promoters in worm and human genomes, suggesting that the HOT regions may be caused by CpG-induced nucleosome depletion. Our results suggest that non-methylated CpG-dense sequence is a conserved genomic signal dictating an open chromatin state and marking by the H3K4me3 modification.
-
Gemma, Carolina, Dong, Yan, Janes, Jurgen, Stempor, Przemyslaw, Ahringer, Julie, Chen, Ron, Zeiser, Eva, Down, Thomas, Feuer, Sky
[
International Worm Meeting,
2015]
A hallmark of most vertebrate promoters is their association with non-methylated CG-dense sequences (CpG islands), while methylation of the more sparsely distributed CpGs in the remainder of the genome is thought to contribute to transcriptional repression. Non-methylated CG dinucleotides are recognized by the CXXC finger protein 1 (CXXC1/CFP1) which is part of the COMPASS complex. In the complex, the Set1 methyltransferase (SET-2 in C. elegans) generates H3K4me3, a mark of active promoters. Promoter CpGs were thought to be either absent or irrelevant in invertebrates that lack DNA methylation, such as C. elegans. However, a C. elegans CXXC1 ortholog (CFP-1) is present and required for the generation of H3K4me3 (1,2). We found that C. elegans CFP-1 targets promoters with high CpG density and that these promoters are marked by high levels of H3K4me3. Furthermore, as for mammalian promoters, high promoter CpG content in C. elegans is associated with nucleosome depletion irrespective of transcriptional activity. We further show that highly occupied target (HOT) regions identified by the binding of a large number of transcription factors are CpG-rich promoters in C. elegans and human genomes, suggesting that the unusually high factor association at HOT regions may be a consequence of CpG-linked chromatin accessibility. Our results indicate that non-methylated CpG-dense sequence is a conserved genomic signal that promotes an open chromatin state, and is targeted by a CXXC1 ortholog and H3K4me3 modification in both C. elegans and human genomes. To understand the function of CFP-1 and COMPASS/H3K4me3, we are studying a
cfp-1 deletion mutant. This mutant has nearly undetectable H3K4me3 and has defects in fertility and the repression of somatic genes in the germline, similar to the phenotype of
set-2 mutants (3). Profiling RNA and chromatin in
cfp-1 mutants uncovered alterations in histone modifications, chromatin accessibility, and patterns of gene expression. Our results shed light on the mechanism of action and role of CFP-1/H3K4me3 in chromatin regulation and function.References: (1) Simonet et al., 2007, Dev. Biol. 312, 367-83. (2) Li and Kelly, 2011, PLoS Genet. 7,
e1001349 (3) Robert et al., 2014, Cell Reports 9, 443-450.
-
[
International Worm Meeting,
2007]
The C. elegans genome encodes an 11-member family of non-chitinase, chitin binding domain-containing proteins (CBDs) with distinct expression profiles. Our lab is investigating the roles of chitin and CBDs in development. We have shown that chitin and two CBD family members, CEJ-1 and CPG-2, are required in the single-cell embryo for high fidelity segregation of meiotic chromosomes, for polar body extrusion and for polarization (Johnston et al., 2006). Interestingly, while CEJ-1 and CPG-2 are functionally redundant for embryonic development, localization of CEJ-1::mCherry and CPG-2::Venus is largely distinct. CEJ-1::mCherry has a punctate cortical localization in oocytes, and is non-punctate and gradually enriched in the eggshell. Conversely, CPG-2::Venus is detected in the zone between the eggshell and the embryo. These findings imply that meiotic chromosome segregation, polar body extrusion and polarization require a very small amount of CEJ-1 or CPG-2. We are also investigating the developmental role of another CBD family member, H02I12.1, which is embryonic lethal by RNAi. H02I12.1::GFP is expressed at boundaries between developing germline nuclei. Chitin is not detected in the germline, suggesting that H02I12.1 may bind to non-chitin glycoconjugates. H02I12.1(RNAi) embryos have chitin, but are fragile, and often pinch in two as they enter the uterus, suggesting somatic gonad contraction/dilation may be defective. Furthermore, the germline is also defective, with an extended zone of oocyte phosphohistone H3, and a Pro phenotype in which there is a zone of germline proliferation proximal to the region of gametogenesis. The Pro phenotype has previously been reported for
glp-1 alleles with mutations in the extracellular domain, and for mutant alleles of genes involved in ribosome biogenesis in the somatic gonad. Taken together, these results suggest that H02I12.1 interacting with non-chitin glycoconjugates may modulate signaling between germ cells and the somatic gonad.
-
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
The C. elegans eggshell is essential for multiple aspects of zygotic development, and deficiency of chitin or the chitin-binding proteins, CEJ-1 and CPG-2, results in defects in high fidelity segregation of meiotic chromosomes, polar body extrusion, polarization and the osmotic barrier. In addition to bearing multiple Peritrophin A-type chitin-binding domains, CEJ-1 and CPG-2 are modified with the glycosaminoglycan, chondroitin. Like depletion of chitin or CEJ-1/CPG-2, chondroitin deficiency results in disruption of the osmotic barrier. Ultrastructural analysis of the eggshell shows a multilamellate structure with an outer protein-rich layer, a middle chitin-containing layer and an inner lipid-rich layer. However, details of eggshell molecular organization have not been elucidated. We generated an integrated stra in carrying CEJ-1::mCherry and examined the distribution of eggshell CEJ-1 and chitin. CEJ-1::mCherry and chitin were closely associated, with CEJ-1::mCherry in distinct ring-like structures (CEJios) surrounding button-like foci of chitin. Depleting chitin by
chs-1(RNAi) did not prevent secretion of CEJ-1::mCherry, but the CEJio structure was disrupted and the protein was homogeneously distributed in the zone between the eggshell and the embryonic plasma membrane (marked by hPLCIII 1PH domain::GFP). CEJ-1::mCherry distribution, and its close association with chitin, was also disrupted by
sqv-4+
sqv-8(coRNAi), suggesting that chondroitin chains on CEJ-1 may be essential for chitin/CEJio architecture. These results reveal that eggshell chitin and CEJ-1 show a high degree of structural organization that depends not only on chitin, but also on glycosaminoglycan modification. Future studies will be aimed at understanding molecular details of this structural organization i n development of the C. elegans zygote.
-
[
Development & Evolution Meeting,
2008]
In C. elegans, one of the earliest events at fertilization is the secretion of a multilayered chitinous eggshell. Our lab has shown that the hexosamine pathway, chitin and the chitin-binding proteins, CEJ-1 and/or CPG-2, are developmentally essential at the one-cell stage for multiple actin-dependent events, including high fidelity meiosis, polar body extrusion and embryonic polarization. (Johnston et al., 2006, BMC Biology, 4:35), Consistent with these embryonic roles, chitin, CEJ-1::mCherry and CPG-2::Venus are localized to the eggshell and the extraembryonic matrix between the developing embryo and the eggshell. A third chitin-binding protein, H02I12.1, is also essential for embryonic development. However, distinct from chitin, CEJ-1:mCherry and CPG-2:Venus, H02I12.1::GFP is localized at or near the surface of developing oocytes. This localization suggests that in addition to, or instead of, an embryonic role, H02I12.1 in the extracellular matrix may be required for oocyte development. The proximal germline of H02I12.1(RNAi) hermaphrodites had an increased number of oocytes expressing phosphohistone H3. Furthermore, the number of oocytes in the proximal germline was increased and oocytes displayed significant physical overlapping. Additionally, embryos were often pinched in two, resulting in the production of anucleate cytoplasts. Together, these results suggest that H02I12.1 in the oocyte extracellular matrix inhibits oocyte maturation and may play a role in oocyte/sheath or oocyte/oocyte interaction. H02I12.1(RNAi) oocytes also failed to bind a number of GalNAc-recognising lectins, including SBA, MPA and GS-I, indicating that one or more transmembrane or extracellular matrix glycoconjugates are absent or defective. Our results show that the glycopolymer, chitin, and the chitin-binding proteins, CEJ-1, CPG-2 and H02I12.1, are required for multiple developmental events just preceding and just following fertilization. We are currently testing the hypothesis that chitin and its binding partners function as a scaffold that organizes specific transmembrane glycoproteins for intracellular signaling and polarity in the maturing oocyte and the newly fertilized embryo.
-
[
International Worm Meeting,
2017]
The CPG underlies several diverse and essential functions, functionally streamlined and localized for their non-divorceable utility. Until recently, however, the CPG has conventionally been understood unjustly in the confines of rhythmicity and motorized behavioral processes. Beginning and framing the C. elegan as a fundamental model for the incredible versatility of the basic CPG, we delve into the sensorimotor integration, maintanence of the locality and causality with a temporal-spatial model, and the conserved processes it demarcates in more complex behaviors, particularly human saccadic eye movement . Afterwards, we discuss the global trends and emergent properties across several organisms that characterize the manifestation and institution of a meaningful rhythmic neural pattern, premised initially by the highly elucidative and revealing neural system of the C. elegan. In finality, we demarcate the evolutionary morphology of the information code and neuromodulatory vehicles that biological pressures have enforced on the increasingly complex design of organisms, starring the C. elegan, once again, as the basis and transitional point for the adaption of an information code from an amplitude based phenomena (graded potential code) to a frequency based phenomena (action potential code). Ultimately, the primary motif of this article demonstrates the immense utility of understanding the 302 neuron system of the C. elegan towards unveiling unobserved homologies and information trends in the evolutionary ladder, across pattern generated behaviors. Our analyses incepts and extends from a compilation of personal lab discoveries and external literature interpretations of the C. elegan nervous system, and has been protracted to several homologies across various animalia and processes, including the aforementioned human saccades.