Iwen Fu Grigsby et al. (2007) International Worm Meeting "UNC-85 is an Asf1-Related Histone Chaperone Functioning in C. elegans Post-Embryonic Neuroblast Replication."

Iwen Fu Grigsby, Fern P. Finger

[International Worm Meeting, 2007]

Normal animal development requires accurate cell divisions, not only in the early stages of rapid cleavage, but also in later stages of development. The C. elegans unc-85 gene is implicated only in cell divisions that occur post-embryonically, primarily in terminal neuronal lineages (1, 2). Variable post-embryonic cell division failures in ventral cord motoneuron precursors result in uncoordinated locomotion of unc-85 mutant larvae by the second larval stage. These neuroblast cell division failures, thought to occur at cytokinesis or anaphase, often result in unequally sized daughter nuclei, and sometimes in nuclear fusions (1, 2). Using a combination of conventional mapping techniques and microarray analysis (3), we have cloned the unc-85 gene, and find that it encodes one of two C. elegans Anti-silencing function 1 (Asf1)-related histone chaperones. The unc-85 gene is expressed in replicating cells throughout development, and the protein is localized in nuclei. Examination of unc-85 null mutants confirms that embryonic neuroblast cell divisions occur normally, but post-embryonic neuroblast cell divisions fail. Analysis of the DNA content of the mutant neurons indicates that defective replication in post-embryonic neuroblasts gives rise to ventral cord neurons with an average DNA content of approximately 2.5n. Specific histone H3 acetylations associated with S phase are decreased in unc-85 mutants, suggesting that UNC-85 functions with histone acetyltransferases. We conclude that UNC-85 functions in post-embryonic replication in ventral cord motoneuron precursors, and has a conserved role in acetylation of histone H3. This is the first example of a specific developmental role for an Asf1 histone chaperone. 1. Sulston, J. E. and Horvitz, H. R. Abnormal cell lineages in mutants of the nematode Caenorhabditis elegans. Dev Biol, 82: 41-55, 1981. 2. White, J. G., Horvitz, H. R., and Sulston, J. E. Neurone differentiation in cell lineage mutants of Caenorhabditis elegans. Nature, 297: 584-587, 1982. 3. Dybbs, M., Ngai, J., and Kaplan, J. M. Using microarrays to facilitate positional cloning: identification of tomosyn as an inhibitor of neurosecretion. PLoS Genet, 1: e2, 2005.

Sun, HaoSheng et al. (2021) International Worm Meeting "Temporal Maturation of the C. elegans Post-Embryonic Nervous System"

Sun, HaoSheng, Hobert, Oliver

[International Worm Meeting, 2021]

In most animals, the majority of the nervous system is generated and assembled into neuronal circuits during embryonic development. However, during juvenile stages, nervous systems still undergo extensive anatomical and functional changes to eventually form a fully mature nervous system by the adult stage. The molecular changes in post-mitotic neurons across post-embryonic development and the genetic programs that control these temporal transitions are not well understood. Using the model organism C. elegans, we comprehensively characterized the distinct functional states (locomotor behavior) and corresponding distinct molecular states (transcriptome) of the post-mitotic nervous system across temporal transitions from early post-embryonic periods to adulthood. We observed pervasive changes in gene expression, many of which are controlled by the developmental upregulation of the conserved heterochronic miRNA lin-4/mir-125 and the subsequent promotion of a mature neuronal transcriptional program through the repression of its target, the transcription factor lin-14. The functional relevance of these molecular transitions are exemplified by a temporally regulated target gene of the lin-14 transcription factor, nlp-45, a neuropeptide-encoding gene. We found that nlp-45 is required for temporal transitions in exploratory activity across larval stages, across sexual maturation, and into a diapause arrest stage. Combined, these studies provide new insights into regulatory strategies that control neuron-type specific gene batteries to modulate distinct behaviors states across temporal, sex and environmental dimensions of post-embryonic development, and also provide a rich atlas of post-embryonic molecular changes to uncover additional regulatory mechanisms.

Naoko Sakumoto et al. (2003) International Worm Meeting "Systematic analysis of post-embryonic function of essential genes by RNAi-by-L1-soaking"

Naoko Sakumoto, Yumi Iida, Yuji KOHARA, Asako Sugimoto, Nobuko Uodome, Chie Hayakawa

[International Worm Meeting, 2003]

In animal development, some genes play roles at multiple developmental stages in multiple tissues. Although mutant analysis is a powerful method to study gene functions, in general, earlier mutant phenotypes prevent the analysis of later developmental events, which makes it difficult to characterize multiple gene functions. For example, conventional RNAi experiments in which dsRNA is introduced into adults, embryonic lethal phenotypes will mask later post-embryonic phenotypes. To circumvent this, RNAi-by-L1-soaking method can be used in which post-embryonic gene functions are specifically inactivated. In this method, L1 worms are soaked in dsRNA solution without food for 24-48 hours, and then recovered on a plate with food to examine their post-embryonic development. Because the RNAi mechanism appears to keep functioning even in a starved L1 worms, loss-of-function phenotypes can be observed from early larval development in a synchronized manner. We have been using this L1-soaking method to systematically examine the potential additional functions of embryonic lethal genes (Emb genes). As a set of Emb genes, we use a collection of cDNAs that caused embryonic lethality in our systematic RNAi analysis by the L4-soaking method. For each Emb cDNA, post-embryonic phenotypes by L1-soaking are characterized with both dissecting and Nomarski microscopes (For the embryonic phenotype analysis, see poster by Maruyama et al.). About a half of the Emb genes caused detectable defects in post-embryonic development, such as, growth, gonadogenesis, germline development, and vulval development. These results indicate that many essential genes function at multiple developmental processes. We have also initiated the RNAi-by-L1-soaking analysis for the genes that caused sterility of the soaked animals (P0 sterility) by L4-soaking. By examining the post-embryonic phenotypes of these P0 sterile genes, we are able to determine whether they are involved in both somatic and germline development, or germline development only. The post-embryonic RNAi phenotypes for each gene are recorded in a searchable database. In the future, the post-embryonic phenotype dataset will be integrated with the embryonic phenotype dataset. These phenotype profiles throughout development will be useful for predicting gene functions, and for identifying genetic networks in developmental processes

Naoko Iida et al. (2004) East Asia Worm Meeting "Profiling of post-embryonic phenotypes of essential genes by RNAi-by-L1-soaking"

Yuji KOHARA, Asako Sugimoto, Naoko Iida, Yumi Iida

[East Asia Worm Meeting, 2004]

In animal development, some genes play roles at multiple developmental stages in multiple tissues. However it is in general difficult to characterize multiple gene functions because earlier mutant phenotypes prevent the analysis of later developmental event. We have developed an approach for a large-scale characterization of post-embryonic gene functions of embryonic lethal genes, based on a stage-specific gene inactivation using "RNAi-by-L1-soaking" method. In this method, L1 worms are soaked in dsRNA solution without food for 24-48 hours, and then recovered on a plate with food to examine their post-embryonic development. The post-embryonic RNAi phenotypes for each gene are recorded using a combination of multiple independent criteria with a pre-determined set of vocabulary, so that it can be used for further computer-assisted analysis. As a set of Emb gene, we have been using the cDNAs that caused embryonic lethality in our systematic RNAi analysis by the L4-soaking method. Among the 377 Emb genes examined, a ~50% caused detectable defects in post-embryonic development, such as, growth, gonadogenesis, germline development, and vulval development. These results indicate that many gene essential for embryogenesis function at multiple developmental processes. We have also initiated the RNAi-by-L1-soaking analysis for the genes that caused sterility of the soaked animals (P0 Ste) by L4-soaking. 80% of the P0 Ste genes caused detectable defects in post-embryonic development; majority of them resulted in larval lethality, implicating these genes might play "house-keeping" function in evey cell. These data suggest that characterization of post-embryonic phenotypes by the L1-soaking method will unveil multiple gene functions that might have missed by previous analysis.

Loren Jack et al. (2004) West Coast Worm Meeting "Genetic Analysis of Gain of Function Acetylcholine Receptors"

Loren Jack, Mike Francis, A Villu Maricq

[West Coast Worm Meeting, 2004]

Communication between cells of the nervous system involves a diverse array of synaptic proteins whose expression must be temporally and spatially regulated. For example, many neurons express a variety of functionally distinct post-synaptic receptors that must be segregated to specific synapses and subdomains within synapses for proper signaling. How are these receptors differentially regulated? We have begun to examine this problem at the neuromuscular junction (NMJ) of the nematode Caenorhabditis elegans (C. elegans). The C. elegans NMJ is amenable to both electrophysiological and genetic analysis, and at least 3 distinct subtypes of post-synaptic receptors are expressed in C. elegans body wall muscle: a single subtype of GABA receptor and at least 2 subtypes of ionotropic acetylcholine receptor (AChR). Apart from the receptor subunits themselves, very few genes affecting the formation and organization of the post-synaptic receptor fields at the NMJ have been identified in C. elegans. One strategy for identifying novel genes that modify post-synaptic receptor function is to engineer mutations in post-synaptic receptors with the aim of providing a sensitized background in which to screen for suppressors of induced phenotypes. This general strategy has been used previously in the Maricq laboratory to identify genes affecting ionotropic glutamate receptors. We are currently engineering mutations in candidate acetylcholine receptors that we hope will produce easily scorable movement phenotypes in transgenic worms expressing these mutant receptors. Ultimately, we hope to identify novel genes that modify AChR function by screening for suppressors of these phenotypes.

Aric Rogers et al. (2007) International Worm Meeting "Post-transcriptional regulation downstream of the insulin-like signaling pathway in C. elegans."

Gordon Lithgow, Pankaj Kapahi, Di Chen, Aric Rogers, Gawain McColl, Alan Hubbard

[International Worm Meeting, 2007]

The insulin-like signaling (ILS) pathway plays a major role in the regulation of lifespan, metabolism, and neurodegenerative phenotypes in C. elegans. These phenotypes are modulated by gene expression downstream of the forkhead transcription factor DAF-16. A number of studies have used CHIP, SAGE, and microarrays to identify downstream mediators of DAF-16 at the genome-wide level. These studies have begun to resolve the complicated transcriptional network regulated by DAF-16 that determines certain phenotypes associated with ILS mutants. Additional elucidation may involve a better understanding of post-transcriptional regulation, as some studies in yeast and mammalian cells have reported a lack of correlation between the transcriptome and the proteome (Gygi et al. 1999, Ideker et al. 2001). Furthermore, deregulation of post-transcriptional processing and mRNA translation is now known as a crucial determinant in aging and age-related diseases including cancer, diabetes, and neurodegeneration. Despite intense study of ILS by numerous researchers, little is known about how DAF-16 and its direct as well as indirect targets affect post-transcriptional regulation of gene expression. We examined post-transcriptional changes in gene expression at the genome-wide level using a method referred to as translational state array analysis (TSAA). To measure the translation state of the mRNA, transcripts bound by ribosomes (polysomes) were separated using sucrose density gradient centrifugation. Resolved polysomes were fractionated according to low or high numbers of ribosomes bound per transcript. Unbound (untranslated) mRNA was also collected and all three fractions were analyzed using standard microarray techniques. We report that a number of genes previously shown to regulate lifespan and fat storage exhibit differences in post-transcriptional processing in a comparison of daf-2 and daf-2:daf-16 C. elegans mutants. Furthermore, the expression of numerous RNA-binding and mRNA translation regulating genes involved in post-transcriptional processing show changes in gene expression as assessed by mRNA ribosomal load. Preliminary lifespan analyses using RNAi-mediated gene suppression suggests that this method has been highly successful in identifying novel genes that determine lifespan in this pathway. Together, these results are the first to demonstrate vast post-transcriptional control by the ILS pathway in C. elegans. A better understanding of post-transcriptional processing within the ILS pathway will provide more insight into the basic mechanisms that determine longevity and metabolism.

Burnaevskiy, N. et al. (2017) International Worm Meeting "Quantitative and functional analyses of the organs and cells of post adult reproductive diapause animals."

Mendenhall, A., Burnaevskiy, N., Kaeberlein, M.

[International Worm Meeting, 2017]

C. elegans have had a fantastical impact on our understanding of the aging process. In addition to ability to extend lifespan upon many pro-longevity treatments, C. elegans will also live a normal, wild-type lifespan after prolonged (weeks) periods in diapuased juvenile states (L1 arrest, dauer). The mechanisms that preserve the soma of these animals during diapaus are therefore attractive targets for understanding and modulating the aging process. Remarkably, it was found that upon starvation even adult animals are able to enter into diapause state, known as adult reproductive diapause (ARD) (Giana Angelo and Marc R. Van Gilst, 2009). Like diapauses in juvenile animals, upon refeeding and exit of the adult reproductive diapause, their lifespan is fully preserved. After prolonged periods in the ARD state, animals show dramatic morphological improvements upon refeeding, and their post-diapause lifespan is indistinguishable from the animals that never experienced starvation. However, it is unclear whether post-diapause animals follow the exact same physiological trajectory as their counterparts that were never starved. To gain a better understanding of the post-diapaused animals we applied quantitative measures of physiology including detailed electrophysiological analysis of pharynx function, 3-dimensional mitochondrial structure examination and measurement of gene expression capacity. Gene expression capacity, the ability to express genes into functional proteins, is a fundamental cellular property that is highly variable between the intestine cells of individual animals, and may affect many aspects of cellular physiology. Our in-depth quantitative analysis provides some mechanistic insights into the cellular processes underlying longevity and healthspan of post-diapause animals.

Kyani-Rogers, Travis (2021) International Worm Meeting "Molecular and neuronal mechanisms underlying early experience-dependent chemosensory plasticity in C. elegans"

Kyani-Rogers, Travis

[International Worm Meeting, 2021]

Responses of animals to food or chemical stimuli can vary based on their internal state and experience. Work from our lab and others previously showed that adult animals which transiently experienced stress-induced dauer arrest during development (post-dauer) exhibit distinct gene expression profiles and behavioral responses compared to control adults which bypassed the dauer stage. We show that post-dauer adults exhibit significantly enhanced responses to volatile attractive odors, including odors sensed by the AWA and AWC olfactory neurons, as compared to control adults that bypassed the dauer stage. In contrast, responses of post-dauer adults to repellents is unaffected. We find that dauer larvae and post-dauer adults upregulate the diacetyl olfactory receptor ODR-10 in the AWA chemosensory neuron pair. Expression of this receptor in AWA neurons is known to drive food searching behavior in adult worms and is regulated in a sexual dimorphic manner. The dauer-induced upregulation of this receptor in AWA neurons requires the stress response transcription factor DAF-16/FOXO. Via spatiotemporally regulated depletion of DAF-16 in AWA, we have established that DAF-16 acts early in development prior to dauer entry to transcriptionally upregulate odr-10 expression, and that it is dispensable for the maintenance of higher expression levels in post-dauer adults. Our data suggest a model in which early developmental stress is encoded at the level of regulation of olfactory receptor gene expression, and may modulate behavioral differences between adult animals based on their respective developmental experiences. To determine whether additional sensory genes are also subject to similar experience-dependent regulation, we have isolated AWA neurons via FACS from control and post-dauer L4s and are performing differential expression analyses. Future goals will be to determine the contributions of these differentially expressed genes to sensory behavior, and to define the cis- and trans-regulatory mechanisms that underlie this plasticity in gene expression and behavior.

Chris Merritt et al. (2006) Development & Evolution Meeting "Post-transcriptional regulation: A common mode of gene regulation in the germline?"

Geraldine Seydoux, Chris Merritt, Dominique Rasoloson

[Development & Evolution Meeting, 2006]

Gene expression can be regulated at the transcriptional and post-transcription levels. RNA-binding proteins and 3?UTR sequences have been implicated in the regulation of several genes expressed in the germline, suggesting that post-transcriptional regulation is common in this tissue (e.g. Kimble and Crittenden, Lee and Schedl, WormBook). To begin to explore the extent to which germline genes depend on post-transcriptional mechanisms, we selected 8 genes with known protein expression patterns in the adult germline (antibody staining), representing a broad range of germ cell types, including all germ cells (tbb-2), mitotic germ cells (fbf-1 and fbf-2), pachytene germ cells (gld-1), post-pachytene cells (pgl-3), distal oocytes (pal-1), proximal oocytes (mex-5), and sperm (spe-11). ORF and 3? sequences from each gene were cloned downstream of the pie-1 promoter fused to GFP, and introduced into worms by biolistic transformation. We found that in all cases, we could reproduce the expression reported for the endogenous protein. In one case, the GFP reporter was also expressed at high levels in additional cells: pie-1promoter:GFP:SPE-11 was expressed in sperm but also in all adult germ cells, including oocytes. We conclude that, among our gene set, promoter sequences are not essential to drive cell-type specific expression, with the exception of sperm. Our results support the view that post-transcriptional mechanisms are commonly used to specify protein expression patterns in the germline. We are currently repeating this analysis using only 3? sequences to test whether the observed patterns depend on sequences in the ORF, the 3?UTR or a combination of both.

Park M et al. (1998) East Coast Worm Meeting "A cyclin D is required for larval, but not embryonic, cell divisions in C. elegans."

Krause MW, Park M

[East Coast Worm Meeting, 1998]

In multicellular organisms, the proper timing of cell divisions requires the coordination of developmental signals and factors that control the cell cycle. Cell cycle progression is controlled by the action of cyclin dependent kinases (CDKs). Several factors have been shown to regulate the activity of CDKs; these include cyclins, cyclin-dependent kinase inhibitors (CKIs), phosphorylation, and degradation. Specific cyclins in association with the appropriate CDK act during different stages of the cell cycle. Mitosis is controlled by the mitotic cyclins A and B, whereas G1 cyclins (D and E) function to control the decision to either enter S-phase or arrest in G1. In C. elegans, the post-embryonic blast cells respond to temporal controls and proliferate at specific times during larval development, indicating that the cell cycle in these cells is under developmental regulation. We have identified a C. elegans homolog of the G1-specific cell cycle regulator cyclin D (cyd-1). We will show that cyd-1 is necessary for the division of the post-embryonic blast cells. No embryonic defects are associated with the loss of this cyclin, suggesting that this cyclin function is specific for post-embryonic development, perhaps due to the lack of a G1 phase in embryonic cell cycles. Our model is that cyd-1 is essential to transduce signals for blast cell to exit G1 arrest throughout post-embryonic development.