Fabio Piano et al. (2001) International C. elegans Meeting "RNAi analysis of 762 germline enriched genes."

Fabio Piano, Kenneth Kemphues, Aaron Schetter, Kris Gunsalus, Valerie Reinke, Stuart Kim, Diane Morton

[International C. elegans Meeting, 2001]

We are interested in identifying genes that play essential roles in early embryogenesis. Toward this goal, we have been studying the function of ovary expressed genes using RNAi. Here we report on the RNAi analysis of 762 genes selected by microarray studies to have high expression in the ovary. We inject dsRNA representing each gene into 9 worms and follow each worm and its progeny separately. We score for both embryonic and post-embryonic defects. For each gene giving embryonic lethality above 80%, we obtain time-lapse movies of the first 50 minutes of embryogenesis from embryos from three separate affected worms. Our protocol and results differ slightly from other large scale RNAi studies and we will present a comparison of the results. We have found that 31% of the germline enriched genes give rise to embryonic lethal phenotypes. Interestingly, although there is a strong correlation between a high degree of sequence conservation and embryonic lethality, the major lethal class is composed of proteins that are not annotated, thus these RNAi data provide the first functional data for these genes from any genome. Using the microarray data we are exploring the connection between expression patterns and phenotypes. Our analysis thus far allows us to extend a previous observation that genes required for embryogeneis are less likely to map on the X-chromosome. We are categorizing genes using defects seen in early embryogenesis. We expect that genes giving similar embryonic defects will identify components of biochemical pathways and protein complexes allowing us to predict functions for previously uncharacterized genes.

Fabio Piano et al. (2003) International Worm Meeting "Integration of functional genomic information for genes involved in C. elegans early embryogenesis"

Monica Martinez, Ning Li, Marc Vidal, David Hill, Debra Goldberg, Jackie Han, Tong Hao, Julie Ahringer, Frederick Roth, Fabio Piano

[International Worm Meeting, 2003]

Complete genome sequences of model organisms lead to large numbers of predicted genes, of which many are poorly or un-characterized functionally. In recent years various functional genomic projects have been carried out to characterize large numbers of genes at a time, and a large data sets emerging from these projects have been deposited into publicly available databases. However, due to the volume and error-prone property of such data sets, it is not obvious how they can help in elucidating biological processes of interest. Here we demonstrate an application of combined experimental and computational functional genomic approaches to C. elegans early embryogenesis. Proteins required for early embryogenesis were subjected to yeast two-hybrid analysis and putative protein-protein interactions were identified. For a significant number of these putative interactions, both proteins correspond to genes that gave rise to embryonic lethality phenotypes in high-throughput RNAi assays. In addition, gene pairs corresponding to some interactions have been found to exhibit very similar RNAi phenotypes during early embryogenesis, as recorded by differential interference contrast microscopy. In addition, we computationally generated a network of functional associations for these proteins by searching through publicly available data for genes that are either co-expressed across many different conditions, or share similar RNAi phenotypes, or both. Gene products that are specifically linked to proteins that are required for early embryogenesis were identified as potentially involved in this process. By examining the neighborhood cohesiveness of this network, we identified protein pairs of functional similarity for early embryogenesis. We propose that such integration of functional genomic information can be applied to other biological processes and to other organisms as well.

Marian Walhout et al. (2001) International C. elegans Meeting "Integration of functional genomics approaches: protein interaction mapping using genes enriched in the C. elegans germline"

Marian Walhout, Philippe Vaglio, Jerome Reboul, Marc Vidal, Olena Shtanko, Valerie Reinke, Stuart Kim

[International C. elegans Meeting, 2001]

Since the complete genome sequence of C. elegans became available at the end of 1998, several functional genomics projects have been initiated to functionally annotate the many predicted genes that had remained previously uncharacterized. The data generated by these projects can be viewed as hypotheses until validated further. We have proposed that the likelihood of such hypotheses to be biologically meaningful might increase as the data from different projects are integrated. For example, genes that cluster in expression profiling, protein interaction maps and phenotypic analysis might have a relatively high likelihood to functionally interact in the same biological process. Recently, microarray analysis has led to the identification of a set of genes that show increased expression in the germline (Reinke et al, Mol. Cell. 6, 605-616, 2000). We have cloned these ~750 open reading frames and transferred them into yeast two-hybrid vectors using Gateway cloning. Subsequently, we have generated a 750 X 750 protein interaction map. The data obtained and its integration with the RNAi data obtained by Fabio Piano and colleagues will be discussed.

Richard Allan et al. (2006) Development & Evolution Meeting "Genome-scale High-content Phenotypic Analysis of Sterility in C. elegans"

Fabio Piano, Kristin Gunsalus, Krishna Vijayendran, E. Jane Hubbard, Philip MacMenamin, Richard Allan

[Development & Evolution Meeting, 2006]

Genes with similar loss-of-function phenotypes often act in similar developmental processes. Coupled with genome-scale RNAi studies, this premise suggests that starting from RNAi-induced phenotypes, previously uncharacterized genes required for specific processes can be identified by grouping them with genes of known function that confer similar RNAi phenotypes. Successfully using this strategy for analysis of the early embryo, Piano et al. (2002) defined 47 discrete characters to characterize complex cell biological and developmental phenotypes resulting from RNAi of genes required for embryogenesis. Phenotypic signatures - combinations of explicit outcomes for each character - were generated for each RNAi experiment, and genes were clustered by phenotypic similarity (phenoclusters). These data provided hypotheses about the function of previously uncharacterized genes. Primary data, scoring criteria and results are publicly available in RNAiDB (http://www.RNAi.org) and can be analyzed and queried using additional tools (e.g. PhenoBlast; Gunsalus et al., 2004). We have applied the same approach to RNAi-induced sterility. In previous large-scale screens, over 800 genes conferred a sterile (Ste) or sterile progeny (Stp) phenotype, based on scoring of brood size (Gonczy et al., 2000; Fraser et al. 2001; Piano et al., 2000; Maeda et al., 2001; Hanazawa et al. 2001; Piano et al., 2002; Kamath et al., 2003; Simmer et al., 2003). To characterize specific mophological defects underlying the observed sterility, we have developed within RNAiDB a scoring system for ~300 discrete phenotypic features visible at high-magnification in adult hermaphrodites, providing data amenable to phenoclustering and PhenoBlast. As a pilot study, we have chosen a set of genes previously identified as Stp and/or Ste by RNAi (Fraser et al. 2001; Kamath et al., 2003) that confer a wide range of RNAi-induced defects. Analysis of phenotypic signatures (~100 characters) and phenoclustering are in progress. Data (Z-plane stacks) will be available at RNAiDB, together with the phenotypic scoring results. The scoring system and analysis tools will also be made available for general use.

Richard S Allan et al. (2005) International Worm Meeting "High-resolution, digitally-encoded phenotypic analysis of gonadogenesis in C. elegans"

E Jane Albert Hubbard, Richard S Allan, Philip MacMenamin, Kristin C Gunsalus, Fabio Piano, Krishna Vijayendran

[International Worm Meeting, 2005]

A fundamental premise of genetic analysis is that genes with similar loss-of-function phenotypes likely act in similar developmental processes. Extending this concept to RNAi-based genome-scale functional characterization, Piano et al. (2002) defined 47 embryonic phenotypic characters in C. elegans and generated a digital phenotypic signature for each RNAi experiment. Genes conferring similar signatures were clustered (phenoclusters), placing previously uncharacterized genes in clusters with genes of known function. The primary data (high-magnification time-lapse movies), scoring criteria and results are publicly available and can be analyzed and queried using additional tools (e.g. Phenoblast) (Gunsalus et al., 2004; http://www.RNAi.org).We have applied this approach to gonadogenesis and germline development in C. elegans. In previous large-scale RNAi screens, over 800 genes are reported to confer a sterile (Ste) or sterile progeny (Stp) phenotype, based on low-magnification screening (Gonczy et al., 2000; Fraser et al. 2001; Piano et al., 2001; Maeda et al., 2001; Hanazawa et al. 2001; Piano et al., 2002; Kamath et al., 2003; Simmer et al., 2003). A subset of these have been examined under high magnification (Maeda et al., 2001; Hanazawa et al., 2001; Colaiacovo et al., 2002), but these data are not available in a systematic digital format amenable to analyses such as phenoclustering or Phenoblast, nor to phenotypic search criteria.We developed and are implementing a web-based digital scoring system (currently 262 characters) for high-magnification phenotypes in adult hermaphrodites. Characters include both quantitative and qualitative features of the gonad, germ line and gametes as well as several gonad-related characters. We generate a series of digital Z-plane images at high magnification from each RNAi experiment. All data will be made publicly available on the RNAi.org website. Our test set is 147 Stp genes and 260 Ste genes conferring a wide range of RNAi-induced defects.

Anita Fernandez et al. (2006) Development & Evolution Meeting "A Network Model of C. Elegans Early Embryogenesis Identifies a Novel AT-Hook Protein that Coordinates Nuclear Envelope Function and Chromatin Maintenance"

Anita Fernandez, Fabio Piano

[Development & Evolution Meeting, 2006]

Using combined network analysis of large-scale functional genomic data we mapped multi-protein modules required for distinct processes during early embryogenesis (Gunsalus et al. 2005). A basic question is how these molecular modules are coordinated through the mitotic cycle to ensure the proper unfolding of early developmental events. To identify proteins that could coordinate different modules we searched for proteins that bridged different modules. One such protein, C38D4.3, could be placed in either the nuclear pore complex module or in the chromosome maintenance module by a network clustering algorithm M-CODE (Bader et al. 2003). Consistent with its predicted roles at the nuclear pore and in chromosome segregation, GFP fusions and anti-C38D4.3 immmunolocalizations show that C38D4.3 shuttles between the nuclear envelope and the kinetochore during the cell cycle. Functionally, C38D4.3 is required for proper nuclear envelope and chromatin maintenance. C38D4.3 (RNAi) embryos, like embryos without nuclear pore components, are incapable of completely separating cytoplasm from nucleoplasm, failing to exclude microtubules and affecting the nuclear localization of PIE-1, a protein normally enriched in the P1 nucleus (Mello, 1996). Additionally, pronuclei fail to meet, and centrosomes do not remain attached to the paternal pronucleus and segregate prematurely. In these embryos, metaphase spindles are not established and chromatin neither condenses, congresses, nor segregates properly. These phenotypes are reminiscent of RNAi phenotypes of genes from the Ran GTPase cycle (Askjaer 2002). Looking for C38D4.3 (RNAi) phenotypic neighbors using PhenoBlast (Gunsalus et al 2004) or phenoclusters from large-scale RNAi analyses (Sonnichsen et al 2005; Gunsalus et al 2005) we identified ~25 other genes with similar defects when analyzed by time-lapse Nomarski microscopy. Of these, genes that are part of the RanGTPase pathway (ran-1, ran-2, and npp-9) were required for proper C38D4.3 localization. Thus C38D4.3 is critical for both mitotic and interphase cell functions and is a likely target of the Ran GTPase pathway.

Michael Brauchle et al. (2007) International Worm Meeting "Evolution of early embryonic developmental mechanisms in Rhabditidae."

Fabio Piano, Michael Brauchle

[International Worm Meeting, 2007]

Natural diversity ultimately arises from the evolution of developmental programs. The molecular mechanisms underlying the evolution of developmental programs are still largely mysterious. A critical question is thus to discover mechanisms that enable evolution of developmental programs in light of the highly constrained mechanistic view that arises from developmental genetics, where altering important developmental genes is known to lead to lethality. We are using nematode early embryogenesis (EE) as a model to study these questions. When analyzed by time-lapse microscopy, nematode embryos show a remarkable array of different characteristics during EE. To analyze this diversity in more detail, we have developed a series of 40 discrete phenotypic characters describing the first two rounds of cell division among 35 Rhabditidae species. When all the characters are mapped onto a molecular phylogeny we find that they have evolved multiple times, showing a high rate of convergent evolution. This observation supports the idea that early development is plastic. The diversity in EE is surprising given the reproducible way in which each single species, like C. elegans, develop. One possible reason for this evolutionary plasticity is that early development is governed by highly modular molecular mechanisms that can direct specific events independently from one another. In support of this idea, we find almost no evidence for correlation (co-evolution) among characters across the 35 species. To obtain molecular clues that could underlie the diversity we observed, we compared the evolutionary characters with the phenotypes observed from RNAi data in C. elegans. Surprisingly, for almost every character we could find a matching RNAi phenotype that phenocopies the character of another species. One hypothesis that arose from these RNAi comparisons was that a rotation of the spindle in the AB cell seen in Protorhabditis and Diploscapter species was due to a change in the polarity network. The polarity network is defined by the regulatory interactions among the PAR proteins in C. elegans. These comparisons led us to postulate that the PAR-3/PAR-6/PKC-3 complex may be missing or inactive in Diploscapter or Protorhabditis one-cell embryos. We tested this hypothesis by localizing PAR-1 in these species and found a novel localization pattern that support the idea that the PAR network is evolving across these species.

Nathalie Velarde et al. (2005) International Worm Meeting "F-actin dynamics in early C. elegans development"

Nathalie Velarde, Fabio Piano

[International Worm Meeting, 2005]

Actin has been shown to play key roles during early development in the C. elegans embryo (Schneider and Bowerman, 2003). However, it has been difficult to faithfully reproduce F-actin dynamics in vivo during early embryogenesis. To visualize F-actin we have generated a transgenic line that uses the F-actin binding domain of Drosophila moesin to decorate endogenous actin filaments with GFP (GFP::Moe). The GFP::Moe fusion line appears to be specific for filamentous actin and allows the visualization of F-actin dynamics in C. elegans in embryos. Preliminary evidence shows that F-actin is very dynamic in many cellular processes from prior to fertilization through the first mitotic cellular division. During fertilization a posterior actin cap forms and then dissipates. Prior to the first cell division F-actin becomes enriched in the anterior, similar to the anterior PAR proteins. As seen in par-3 mutants (Kirby et. al., 1990), depleting the embryo of PAR-6 with RNAi prevents this enrichment, suggesting that PAR-6 and the other anterior PARs may be required for F-actin to accumulate in the anterior. We have also observed the presence of highly dynamic actin comets in the early embryo. Preliminary evidence suggests that these comets may be involved with endocytic processes. Using results from large-scale RNAi surveys, we are employing the GFP::Moe line to perform secondary screening of genes associated with pseudocleavage defects to further characterize this process. We will present our progress in using this GFP::Moe line to study genetic interactions involving actin dynamics in early development.

Nathalie Velarde et al. (2007) International Worm Meeting "Actin dynamics during C. elegans oogenesis and fertilization."

Nathalie Velarde, Fabio Piano

[International Worm Meeting, 2007]

The actin cytoskeleton plays critical roles in early development of metazoans. How actin participates in a diverse number of early embryonic processes has been classically addressed using drugs and studying fixed samples. However, the role of actin during oocyte maturation, ovulation, and fertilization remains largely unknown. In vivo imaging of GFP::MOE, which decorates F-actin, in C. elegans has allowed us to begin to decipher the microfilament dynamics during these developmental times. We show that F-actin is asymmetrically enriched at the proximal end of maturing oocytes prior to ovulation. At fertilization, a discrete actin cap forms over the site of sperm entry which is then dispersed along the periphery of to the future posterior half of the fertilized oocyte. The actin cap requires sperm entry. In the absence of sperm entry, as in spe-9 mutants or with the depletion of sperm receptors, EGG-1 and EGG-2 by RNAi, the actin cap fails to form correctly, indicating that sperm entry directs proper actin cap formation. Sperm devoid of the cytoskeletal protein SPE-11, also failed to form the actin cap. To further characterize the role of the cap, we tested if genes in the oocyte integrity and meiosis module, defined by a combined analysis of genome-wide data (Gunsalus et. al., 2005), affected its formation or dynamics. Among the candidate genes we have identified that affect actin cap formation, so far, are two genes that encode putative tyrosine phosphatases, glutamine-fructose-6-phosphate amidotransferase, and other components of the chitin biosynthesis pathway. Additionally, we have found that depletion of POD-1 or CYK-4 by RNAi , genes known to be involved in establishing embryonic polarity, affect the asymmetric distribution of actin prior to ovulation and the polarized dispersal of the actin cap. Depleting these proteins, results in evenly distributed actin throughout the oocyte cortex, indicating that these proteins might be involved in establishing polarity in the oocyte. These analyses have allowed us to visualize fertilization and actin dynamics in vivo. Our results indicate that there is an inherent polarity of the actin cytoskeleton in the oocyte prior to ovulation that may be either reset or refined by the sperm. The inherent polarity in the oocyte may be crucial in setting embryonic polarity in related nematode species that lack sperm.

Nathalie Velarde et al. (2006) Development & Evolution Meeting "Actin Function During Early C. elegans Development"

Nathalie Velarde, Fabio Piano

[Development & Evolution Meeting, 2006]

The actin cytoskeleton plays critical roles in early development of metazoans. How actin participates in diverse early embryonic processes has been classically addressed using drugs and studying fixed samples. To further understand the complex roles of actin in early embryogenesis we have been using RNAi and in vivo imaging of F-actin dynamics. Time course analysis of act-1(RNAi) revealed that among the earliest, most sensitive processes showing actin requirements are polar body extrusion, pseudocleavage, and establishment of polarity. Progressively more severe depletions of actin protein showed reduced elongation of the central spindle, failure to displace the spindle toward the posterior, cytokinesis failure, improper coalescence of telophase chromosomes and eventually resulted in sterility. No defects were observed in centrosome duplication or separation, or in the rotation of the mitotic spindle of the one cell embryo. in vivo imaging shows that F-actin is enriched at the cortex of developing and maturing oocytes in the gonad. Around the time of fertilization, transient F-actin dynamics are observed. Following fertilization, two “actin caps” appear in succession on the presumptive posterior cortex. The first actin cap appears immediately upon contact with the spermatheca and dissipates within a minute, while the second actin cap appears about two minutes later. Preliminary data suggests that the first actin cap requires the sperm receptor EGG-1 and thus may be triggered by sperm entry. The second actin cap appears to be independent of sperm entry. During cortical contractions, a filamentous network of actin fibers is found throughout the cortex. However, by the time of pronuclear meeting the F-actin network has become asymmetrically enriched at the anterior cortex and appears as morphologically distinct foci. Both the proper redistribution and specific morphological transitions of F-actin are dependent on the activity of the PAR proteins as well as PFN-1 and NMY-2. As the cell cycle progresses, highly dynamic actin comets appear and peak in number at pro-metaphase. Disruption of F-actin regulators causes a significant decrease in the number of comets observed at this time. Preliminary evidence suggests that these comets are involved in vesicle or organelle trafficking in the cell. These analyses have revealed new insights into the roles of actin during early embryogenesis in C. elegans.