Characterization of the RhoGAP proteins RGA-3 and RGA-4 and the centrosomal protein SAS-5 in the early "Caenorhabditis elegans" embryo

The Caenorhabditis elegans embryo serves as a great tool to study cell biological processes like polarization and cell divison. The first cell division is unequal and cell polarization is dependant on the Rho GTPase regulated rearrangement of the cell cortex and the following localization of PAR proteins. The embryo serves also as a great model system to study centrosome duplication, which leads to the duplication of the centrioles provided by the sperm and their assembly into mature centrosomes during S phase of the one-cell stage embryo. In this thesis, two novel Rho GTPase activation proteins (RhoGAPs), RGA-3 and RGA-4, were identified, constituting an essential part for development. Concomitant RNAi of RGA-3 and RGA-4 (rga-3/4 (RNAi)) resulted in a hyper-contractility phenotype with extensive membrane ruffling and furrowing of the zygote. These two RhoGAPs act redundantly in regulating the small GTPase RHO-1, which is essential for regulating the acto-myosin network during contractile polarization of the early C. elegans embryo. Simultaneous knock-down of rho-1 (RNAi) and rga-3/4 (RNAi) rescued the rga-3/4 ruffling phenotype demonstrating that RHO-1 is the GTPase regulated by RGA-3/4. In contrast, triple knock-down of rga-3/4 together with another small GTPase, CDC-42, which is involved in polarity maintenance in the embryo, did no rescue the rga-3/4 (RNAi) ruffling phenotype. Increased membrane ruffling was mainly observed at the anterior cortex of rga3/4 (RNAi) embryos. Consistently, RHO-1 and its effector NMY-2 were enriched in these extra furrows in rga-3/4 (RNAi) embryos. Furthermore, the known Rho GEF ECT-2 and the Rho kinase LET-502 alleviated the membrane ruffling phenotype caused by rga-3/4 (RNAi). As opposed to the third known RhoGAP of the early embryo, CYK-4, which is essential for posterior polarization and central spindle assembly, RGA-3/4 regulate anterior contractility of the early embryo. The RGAs play a role in regulating the acto-myosin network during cortical polarization, yet the initial establishment of polarity is not heavily affected in rga-3/4 (RNAi) embryos as indicated by the correct localization of the PAR proteins. However, the size of the anterior PAR-6 domain fluctuated more in rga-3/4 (RNAi) than in wild type. Over-expression of RGA-3/4 appears to be lethal in C. elegans, and no stable GFP::RGA-3 expressing line could be obtained, neither by injection nor microparticle bombardment. RGA-3/4 do not only have a role in the one-cell stage embryo, they are also necessary for germ line development. Knock-down of RGA-3/4 in the background of the let502 (sb106) mutant impaired the germ line development, a phenotype not observed for this mutant by itself. This result indicates that both LET-502 and RGA-3/4 are required for gonadal function. The second part of the thesis concerned the characterization of the centrosomal protein SAS-5. sas-5 (RNAi) resulted in a high penetrance of embryonic lethality. SAS-5 is a centrosomal protein and essential for centrosome duplication in C. elegans. Upon sas-5 (RNAi) the first cell division appeared to be unaffected. The two centrioles provided by the sperm were not duplicated, yet separated during S phase allowing the establishment of a bipolar spindle in the P0 cell. The centrosome duplication defect was obvious in the subsequent mitotic cycles and only mono-polar spindles were formed. As a consequence, nuclear morphology was strongly affected, but did not induce apoptosis

On the role of molecular mechanisms and unequal cleavage during neurogenesis in the C. elegans C lineage

Required for neurogenesis is a family of evolutionarily conserved bHLH transcription factors known as proneural genes. However, regulation of their initial expression remains a poorly understood aspect of neurodevelopment in any model, particularly Caenorhabditis elegans. A key mechanism by which cells acquire different fates is asymmetric division and in neuronal lineages these often generate unequally sized daughters. Whether this unequal size directly affects cell fate regulation is often unknown. Indeed, the question of how control of cell size intersects with fate decisions is poorly understood in biology more generally. Taking advantage of the single-cell resolution provided by the invariant cell lineage of C. elegans, I interrogate these two fundamental biological questions in the C lineage. Expression of the proneural gene hlh-14/Ascl1 in a single branch of the lineage is required for neurogenesis of the DVC and PVR neurons and is immediately preceded by unequal cleavages. Addressing both molecular and cellular regulators I perform a 4D-lineage based genetic screen for upstream regulators of hlh-14/Ascl1 and address the effect of unequal cleavage and daughter cell size. I find that a regulator of other neuronal lineage cleavages, PIG-1/MELK, is also required in the C lineage, yet equalisation does not affect the initiation of hlh-14/Ascl1 expression. Conversely, I demonstrate that unequal cleavage and acquisition of neuronal fate in separate successive divisions are controlled by the same key regulators. The first by an upstream regulator of hlh-14, the Mediator complex kinase module let-19/Mdt-13 and the second by hlh-14 itself. Taken together the results described in this thesis suggest that rather than acting to correctly segregate initial proneural gene expression, unequal cleavages are instead co-regulated by the same factors regulating neuronal fate acquisition. This co-regulation at successive divisions thus coordinates two separable aspects of fate; acquisition of neuronal identity and correct post-mitotic embryonic cell size

Identification of Factors That Establish Asymmetry and Cell-death Fate in the NSM lineage in Caenorhabditis elegans

During the development of a C. elegans hermaphrodite, 131 of the 1090 cells generated die due to programmed cell death, an important process conserved throughout the animal kingdom. Although a genetic pathway for programmed cell death has been established in C. elegans, not much is known about the signals that trigger cell death in cells destined to die. One particular cell-death event, the death of the NSM sister cell, occurs about 430 min after the first division of the zygote, just 20 min after its progenitor cell has undergone an asymmetric cell division. The sister of the NSM sister cell, the NSM, however, survives and differentiates into a serotonergic neuron located in the pharynx. Here, I show that the cell-death activator egl-1 is expressed in the NSM sister cell, which is destined to die, but not in the surviving NSM. In addition, using a candidate gene approach, I found that in hlh-2(bx108lf); hlh-3(bc248lf) animals, 30% of the NSM sister cells survive. This observation suggests that the NSM sister cell death is at least partially dependent on the activity of hlh-2 and hlh-3, which code for bHLH transcription factors. These and additional results suggest that egl-1 expression is directly activated in the NSM sister cell by a heterodimer composed of HLH-2 and HLH 3, which binds to a specific cis-regulatory region of the egl-1 locus. In order to identify additional factors that contribute to the NSM sister cell death, I performed a forward genetic screen. In particular, I screened for mutations that enhance the NSM sister cell survival caused by hlh-2(bx108). This screen resulted in the identification of mutations in at least six genes not previously implicated in this cell-death event. One of these mutations, bc212, is a loss-of-function mutation in the gene dnj-11. dnj-11 codes for a protein with a J domain, which is found in chaperones, as well as two SANT domains, which are implicated in transcriptional regulation. dnj-11 is an essential gene expressed in most if not all cells. Furthermore, it acts in the NSM sister cell death pathway by negatively regulating the activity of the snail-like gene ces-1. dnj-11 is required for the ability of the NSM mother cell to divide asymmetrically. I propose that dnj-11 promotes the death of the NSM sister cell by establishing polarity in the NSM mother cell. Moreover, I present evidence that the snail-like ces-1 gene is involved in establishing polarity in the NSM mother cell as well, revealing a new function of ces-1 in C. elegans

pig-1 MELK and ced-3 Caspase cooperate to control cell polarity in the C. elegans NSM neuroblast

Snail-like genes encode zinc-finger transcription factors that play essential roles in development, and one of their well-known functions is the epithelial-mesenchymal transition (EMT) induction. Many studies performed in organisms ranging from Drosophila melanogaster to mammals have reported that Snail transcription factors regulate various aspects of stem cell development, such as cell polarity and cell cycle progression. However, the mechanisms through which Snail-like genes regulate these developmental processes are not completely understood. To uncover these mechanisms, I studied the neurosecretory motor neuron neuroblast (NSMnb) lineage during C. elegans embryogenesis. In the NSMnb lineage, we have previously found that CES-1 Snail controls cell cycle progression by regulating expression of the gene cdc-25.2 CDC25. However, the mechanism by which ces-1 controls the asymmetric division of the NSMnb is unknown. By analyzing CES-1 ChIP-seq data acquired from the modENCODE Project, we identified more than 3,000 potential targets of CES-1 Snail. From the potential candidates that are involved in regulating asymmetric cell division, pig-1 was found to play an essential role in asymmetric NSMnb division. pig-1 encodes the sole C. elegans ortholog of Maternal Embryonic Leucine-zipper kinase (MELK) kinase. Through genetic studies, I confirmed that pig-1 acts downstream of ces-1 to control the asymmetric positioning of the NSMnb cleavage plane. Furthermore, by using a single-copy transcriptional reporter of pig-1, I observed that loss of ces-1 increases the transcriptional level of pig-1, while gain of ces-1 activity decreases the level of pig-1. Therefore, I conclude that CES-1 Snail regulates asymmetric positioning of the NSMnb cleavage plane by repressing expression of the gene pig-1. In the NSMnb, CES-1 Snail coordinates the cell cycle through cdc-25.2 and asymmetric positioning of the cleavage plane through pig-1 to ensure asymmetric cell division and the generation of two daughter cells of different sizes and fates: the larger NSM, which survives, and the smaller NSM sister cell (NSMsc), which dies. Apart from influencing the positioning of the cleavage plane, ces-1 and pig-1 also play roles in controlling the orientation of the NSMnb cleavage plane and in specifying the fate of the daughter cell, NSMsc. On the other hand, I show that ced-3, which encodes a Caspase and which usually executes cell death in C. elegans, also plays a role in regulating the asymmetric positioning of the NSMnb cleavage plane. Loss of ced-3 alone did not affect the asymmetric positioning of the NSMnb cleavage plane at lateral-dorsal side, but loss of both ced-3 and pig-1 reversed the cleavage plane to the medial-ventral side and generated a small NSM and a large NSMsc. This indicates that in the NSMnb lineage, ced-3 may have other functions in addition to executing cell death in the smaller daughter (NSMsc). Furthermore, I confirmed that this function is dependent on the Caspase activity of CED-3 protein. Taken together, ces-1 Snail and pig-1 MELK are two key factors that coordinate cell polarity and cell fate in the NSMnb lineage during C. elegans embryogenesis. In addition, ced-3 Caspase acts in parallel to pig-1 and ces-1 to promote the correct positioning of the cleavage plane in the NSMnb

The genome of Romanomermis culicivorax:revealing fundamental changes in the core developmental genetic toolkit in Nematoda

Background: The genetics of development in the nematode Caenorhabditis elegans has been described in exquisite detail. The phylum Nematoda has two classes: Chromadorea (which includes C. elegans) and the Enoplea. While the development of many chromadorean species resembles closely that of C. elegans, enoplean nematodes show markedly different patterns of early cell division and cell fate assignment. Embryogenesis of the enoplean Romanomermis culicivorax has been studied in detail, but the genetic circuitry underpinning development in this species has not been explored. Results: We generated a draft genome for R. culicivorax and compared its gene content with that of C. elegans, a second enoplean, the vertebrate parasite Trichinella spiralis, and a representative arthropod, Tribolium castaneum. This comparison revealed that R. culicivorax has retained components of the conserved ecdysozoan developmental gene toolkit lost in C. elegans. T. spiralis has independently lost even more of this toolkit than has C. elegans. However, the C. elegans toolkit is not simply depauperate, as many novel genes essential for embryogenesis in C. elegans are not found in, or have only extremely divergent homologues in R. culicivorax and T. spiralis. Our data imply fundamental differences in the genetic programmes not only for early cell specification but also others such as vulva formation and sex determination. Conclusions: Despite the apparent morphological conservatism, major differences in the molecular logic of development have evolved within the phylum Nematoda. R. culicivorax serves as a tractable system to contrast C. elegans and understand how divergent genomic and thus regulatory backgrounds nevertheless generate a conserved phenotype. The R. culicivorax draft genome will promote use of this species as a research model

A Study of Cell Polarity and Fate Specification in Early \u3cem\u3eC. Elegans\u3c/em\u3e Embryos: A Dissertation

Asymmetric cell divisions constitute a basic foundation of animal development, providing a mechanism for placing specific cell types at defined positions in a developing organism. In a 4-cell stage embryo in Caenorhabditis elegansthe EMS cell divides asymmetrically to specify intestinal cells, which requires a polarizing signal from the neighboring P2 cell. Here we describe how the extracellular signal from P2 is transmitted from the membrane to the nucleus during asymmetric EMS cell division, and present the identification of additional components in the pathways that accomplish this signaling. P2/EMS signaling involves multiple inputs, which impinge on the Wnt, MAPK-like, and Src pathways. Transcriptional outputs downstream of these pathways depend on a homolog of β-catenin, WRM-1. Here we analyze the regulation of WRM-1, and show that the MAPK-like pathway maintains WRM-1 at the membrane, while its release and nuclear translocation depend on Wnt/Src signaling and sequential phosphorylation events by the major cell-cycle regulator CDK-1 and by the membrane-bound GSK-3 during EMS cell division. Our results provide novel mechanistic insights into how the signaling events at the cortex are coupled to the asymmetric EMS cell division through WRM-1. To identify additional regulators in the pathways governing gut specification, we performed suppressor genetic screens using temperature-sensitive alleles of the gutless mutant mom-2/Wnt, and extra-gut mutant cks-1. Five intragenic suppressors and three semi-dominant suppressors were isolated in mom-2 suppressor screens. One extragenic suppressor was mapped to the locus ifg-1, eukaryotic translation initiation factor eIF4G. From the suppressor screen using cks-1(ne549), an allele of the self-cleaving nucleopore protein npp-10 was identified as a suppressor of cks-1(ne549)and other extra-gut mutants. Taken together, these results help us better understand how the fate of intestinal cells are specified and regulated in early C. elegans embryos and broaden our knowledge of cell polarity and fate specification

Identification of transcription factor targets, gene expression profiles and accessible chromatin regions in the Caenorhabditis elegans epidermis using targeted DamID

Development is an exceptionally complex process that is performed with exquisite control. A series of developmental programmes allow the orchestrated and tightly-regulated deployment of the genomic information, governing events like cell division, cell fate maintenance and differentiation. Understanding the complete regulatory states that instruct a selective decoding of the genome capable of bringing about morphogenetic events is central to developmental biology. Among all cells, stem cells maintain the potential to produce cells that undergo transitions in developmental trajectories and thus are particularly interesting. In this study, I have used the postembryonic development of the Caenorhabditis elegans epidermis driven by the stem cell-like seam cells, to begin exploring the gene regulatory network, transcriptional states and epigenomic regulation involved in cell fate patterning. To that end, I have adapted and present here the first application of the targeted DamID (TaDa) methodology in C. elegans, for assaying protein-DNA interactions, to use as a single technique in approaching all of the above objectives. I show that TaDa requires little starting material, is reproducible and tissue-specific. Using TaDa I identify targets for the transcription factors LIN-22 and NHR-25 that propose new biological functions for these regulators in epidermal development. I acquire gene expression profiles for the seam cells and hypodermis that lead to the discovery of novel transcription and chromatin factors, as well as new miRNAs. Finally, I produce the first cell-type-specific chromatin accessibility maps in C. elegans for the seam cells and hypodermis and use them to identify tissue-specific enhancers. These findings expand our knowledge of the mechanisms underlying fate decisions in epidermal patterning and provide a proof-of-concept for the application of TaDa in C. elegans.Open Acces

A ced-3 caspase – ect-2 RhoGEF axis coordinates functional interactions between the apoptotic pathway and cell size in Caenorhabditis elegans

Programmed cell death via apoptosis is a common cell fate during animal development and its mis-regulation can have serious implications in diseases and disorders. Therefore, it is of high importance to study to apoptosis. The highly conserved central apoptotic pathway was initially discovered in C. elegans and consists of four genes acting in a sequence – egl-1 BH3-only, ced-9 Bcl-2, ced-4 Apaf-1 and ced-3 caspase. The most downstream gene in the pathway, ced-3, encodes for a cysteine protease called caspase, and is essential in the execution of apoptosis. A previous study in our lab had uncovered a novel non-apoptotic role of ced-3 caspase in promoting asymmetric division of C. elegans neuroblasts (Mishra et al., 2018). However, the mechanism by which ced-3 caspase promotes asymmetric division still remained to be elucidated. Thus, in my study, I aimed to decipher the mechanism(s) by which the apoptotic gene, ced-3 caspase, promotes asymmetric cell division. To that end, I first demonstrated that CED-3 caspase protein physically and directly interacts with a regulator of actomyosin contractility, called ECT-2 RhoGEF (Rho guanine-nucleotide exchange factor). Furthermore, using the NSM (neurosecretory motor neuron) lineage in C. elegans, I found that ECT-2 RhoGEF is asymmetrically enriched in the NSM neuroblast, which is the mother of the apoptotic cell. I also found that the asymmetric enrichment of ECT-2 RhoGEF depends on ced-3 caspase activity. Next, by analysing the cell size ratios of the daughters of the NSM neuroblast, my colleagues and I found that genetically, ced-3 caspase acts upstream of ect-2 RhoGEF to promote the asymmetric division by size of the NSM neuroblast. We refer to this as the ced-3-ect-2 axis. Based on these findings, we propose that the ced-3-ect-2 axis promotes polar actomyosin contractility in the NSM neuroblast, which results in its asymmetric division by size and thereby the formation of its smaller apoptotic daughter cell called the NSMsc (NSM sister cell). Molecularly, we propose that CED-3 de-recruits ECT-2 from the dorsal cortex of the NSM neuroblast before metaphase, and that this de-recruitment of ECT-2 is important for the NSM neuroblast to divide asymmetrically. 6 Next, my colleagues and I investigated the effect of the size of the smaller daughter cell, the NSMsc, on its apoptotic fate. We found that increasing the size of the NSMsc by reducing ect-2 activity decreases its probability to undergo apoptosis. Conversely, for the first time, we showed that decreasing the size of the NSMsc by hyperactivation of ect-2 can increase its probability to undergo apoptosis. Thus, we propose that cell size and apoptosis are inversely corelated – larger cells are more prone to survive and smaller cells are more prone to die. Taken together, the findings from this study have found reciprocal interactions between the apoptotic pathway and cell size. In the NSM neuroblast, the apoptotic pathway acts upstream of cell size i.e. ced-3 promotes asymmetric division of the NSM neuroblast and the formation of a smaller NSMsc. Conversely, in the NSMsc, cell size acts upstream of the apoptotic pathway i.e. the small size of the NSMsc promotes the activation/activity of CED-3 and thereby its apoptosis

Investigating the role of the fusogen eff-1 and natural genetic variation in Caenorhabditis elegans seam cell development

Robustness is the ability of biological systems to produce invariant phenotypes despite perturbations. Development is especially robust to internal perturbations, like stochastic gene expression or mutations, and external perturbations, such as changes in environmental factors including temperature and nutrition. The highly invariant developmental patterning in Caenorhabditis elegans offers an ideal system to study the genetic and molecular mechanisms underlying developmental robustness. This work describes an experimental paradigm to discover the mechanistic basis and consequences of developmental robustness using the C. elegans seam cells as a model. Seam cells are lateral epidermal cells that are stem cell-like in their ability to produce differentiated cells and maintain proliferative potential. Through a forward genetic screen, I describe a novel role for the fusogen gene eff-1, which was previously known to drive cell fusion events, in the robustness of seam cell patterning. Furthermore, I show that eff-1 is not required for differentiation of seam cells, therefore I demonstrate that fusion is uncoupled from the differentiation programme. In another set of experiments, I show for the first time that the terminal number of seam cells in C. elegans is robust to standing genetic variation. A consequence of developmental robustness is the acquisition of cryptic genetic variation that does not modify the phenotype under normal conditions but manifests phenotypically upon perturbation. I demonstrate that the genetic background affects seam cell number at a higher developmental temperature of 25 C or upon mutations in the GATA transcription factor and target of the Wnt pathway, egl-18. CB4856 (Hawaii) suppressed the effect of temperature on the seam cell number compared to the lab reference N2 (United Kingdom), as well as lowered the expressivity of egl-18 mutations. Multiple regions of the genome were found to interact epistatically to modify egl-18 mutation expressivity, suggesting that a complex genetic architecture underlies seam cell development. Taken together, this work increases our knowledge on the robustness of seam cell patterning to various sources of variation.Open Acces