The model angiosperm Arabidopsis thaliana produces viable seed through coordinated growth of three constituents; seed coat, embryo and endosperm. During development the embryo grows to fill the space defined by the seed coat. The growing embryo is surrounded by endosperm, an ephemeral, nutritive structure. The process of programmed cell death (PCD) is essential for endosperm consumption by the embryo however very little is known about developmental PCD in the endosperm. ZOU is a transcription factor expressed specifically in endosperm adjacent to the growing embryo in the Embryo Surrounding Region (ESR) (Yang et al., 2008). zou seed likely have reduced PCD resulting in abnormally persistent endosperm and a small embryo at seed maturity which results in seed shriveling. A second zou phenotype is an impairment of cuticle development in the embryonic leaves (cotyledons), suggesting that ZOU may mediate a signal from endosperm to embryo. The ESR expressed gene ALE1 is down-regulated in zou. When ALE1 is artificially expressed in zou ESR by the construct pSUC5::ALE1 the zou epidermal phenotype is rescued but not the seed shriveling phenotype of zou (Xing et al., 2013). Fixed and resin-embedded sections of zou and pSUC5::ALE1 lines herein confirm that zou-like endosperm is exhibited in pSUC5::ALE1 lines. This confirms that the two phenotypes of zou are genetically separable. The involvement of ZOU in epidermal processes is further confirmed through genetic studies showing that ZOU acts in the same pathway to impart embryonic cuticle as the embryo-expressed Receptor Like Kinases GSO1 and GSO2. In order to quantify PCD in the endosperm of wild-type and zou seed, PCD expression marker and TUNEL analysis were conducted. One PCD marker, pCEP:H2A-YFP is shown to be expressed in wild-type ESR, it is not clear if expression is lost in zou. To identify candidate genes under the control of ZOU active in endosperm PCD the results from several transcriptional profiling experiments were analysed and validated; this detailed gene expression in wild-type, ale1 and zou siliques which allowed for the identification of targets of ZOU but not of ALE1, targets predicted to be PCD effectors. In silico expression and ontology analysis confirmed likely roles for some candidates in endosperm PCD processes (particularly cell wall modification). Selected targets were cloned under pSUC5 and expressed in the ESR of zou seed as part of a molecular screen for the rescue of the zou endosperm phenotype. The ZOU target FRINGE-Like, a Glycosyl Transferase which shows strong endosperm expression is shown to partially rescue the zou phenotype but does not rescue the epidermal phenotype, suggesting that it may mediate PCD processes under ZOU control. The initial discovery that a Glycosyl Transferase may be active in a developmental PCD process in plants is exciting and novel and benefits understanding of developmental PCD and endosperm breakdown, two poorly characterized processes in plants.

Programmed cell death and cell corpse clearance are an essential part of an organism’s overall health and development. Cell corpses are often engulfed by professional phagocytes such as macrophages. However, in certain tissues, neighboring non-professional cells can also carry out phagocytic functions. Here, we use the Drosophila melanogaster ovary to investigate novel genes required for engulfment by non-professional phagocytes. In the Drosophila ovary, neighboring epithelial cells facilitate the clearance of dying germline cells. We performed an unbiased kinase screen to identify novel proteins and pathways involved in cell clearance in the nurse cell. Several genes identified in this screen were members of the phosphoinositide 3-kinase (PI3K) family. The class II and III PI3Ks are required for nurse cell clearance and acidification during late-stage oogenesis. Class I PI3K is required for progression during engulfment in mid-stage oogenesis. This kinase screen has revealed novel genes for further exploration and investigation. / 2019-02-28T00:00:00Z

The amnioserosa (AS) is an epithelium that plays major roles in two crucial morphogenetic processes during Drosophila embryogenesis: Germ Band Retraction (GBR) and Dorsal Closure (DC). The AS is extraembryonic and as such, it does not contribute to the mature embryo but is eliminated during development by programmed cell death. In this thesis, a comprehensive investigation of the timing and characteristics of the AS death and degeneration is performed. It is demonstrated that AS elimination occurs in two phases: “cell extrusion” during DC, embryonic stages 12 to 14, and “tissue dissociation” following DC, embryonic stages 15 to 16. Ten percent of AS cells are eliminated during phase one while the remaining ninety percent are removed during phase two. It is found that both cell extrusion and tissue dissociation are absent in apoptotic defective backgrounds, as well as in genetic backgrounds associated with increased class I phosphoinositide 3-kinase (PI3K) activity, a key regulator of autophagy. It is also found that extrusion is enhanced two-fold in embryos expressing the pro-apoptotic reaper gene product, and that tissue dissociation also accelerates in this background. Interestingly, our observations suggest that the activation of caspase cascade is not complete until AS cells have lost apical contacts with neighboring cells. Shortly after the loss of apical contact, an apoptotic morphology including membrane blebbing, cell fragmentation, and macrophage engulfment is readily observed. Measurements of the rate of DC demonstrate that this process is protracted in backgrounds lacking extrusion, leading to the conclusion that extrusion contributes towards generating adequate AS tension required for normal DC rates. Overall, our data suggest that phase one extrusion and phase two dissociation are manifestations of the same cellular event and that both are caspase dependent. It is also demonstrated that autophagy is a key component of AS death that acts upstream of apoptosis. Strikingly, our results lead to the suggestion that autophagy may function to trigger apoptosis during the programmed elimination of this extra-embryonic tissue.

The amnioserosa (AS) is an epithelium that plays major roles in two crucial morphogenetic processes during Drosophila embryogenesis: Germ Band Retraction (GBR) and Dorsal Closure (DC). The AS is extraembryonic and as such, it does not contribute to the mature embryo but is eliminated during development by programmed cell death. In this thesis, a comprehensive investigation of the timing and characteristics of the AS death and degeneration is performed. It is demonstrated that AS elimination occurs in two phases: “cell extrusion” during DC, embryonic stages 12 to 14, and “tissue dissociation” following DC, embryonic stages 15 to 16. Ten percent of AS cells are eliminated during phase one while the remaining ninety percent are removed during phase two. It is found that both cell extrusion and tissue dissociation are absent in apoptotic defective backgrounds, as well as in genetic backgrounds associated with increased class I phosphoinositide 3-kinase (PI3K) activity, a key regulator of autophagy. It is also found that extrusion is enhanced two-fold in embryos expressing the pro-apoptotic reaper gene product, and that tissue dissociation also accelerates in this background. Interestingly, our observations suggest that the activation of caspase cascade is not complete until AS cells have lost apical contacts with neighboring cells. Shortly after the loss of apical contact, an apoptotic morphology including membrane blebbing, cell fragmentation, and macrophage engulfment is readily observed. Measurements of the rate of DC demonstrate that this process is protracted in backgrounds lacking extrusion, leading to the conclusion that extrusion contributes towards generating adequate AS tension required for normal DC rates. Overall, our data suggest that phase one extrusion and phase two dissociation are manifestations of the same cellular event and that both are caspase dependent. It is also demonstrated that autophagy is a key component of AS death that acts upstream of apoptosis. Strikingly, our results lead to the suggestion that autophagy may function to trigger apoptosis during the programmed elimination of this extra-embryonic tissue.

Programmed cell death (PCD) is pervasive in eukaryotes, playing a fundamental role in development. PCD in animals has been studied in detail, partly due to Caenorhabditis elegans, a worm whose anatomy allowed for the investigation of exactly 131 cells that die via PCD. Elucidating this complex pathway in this simple worm laid the foundation for further insights into mammalian PCD. Overall, less is known regarding PCD in plants, where cell death is broadly separated into developmentally regulated and environmentally induced. The lace plant (Aponogeton madagascariensis) undergoes developmentally regulated PCD to form perforations between longitudinal and transverse veins over its leaf surface. The optimization of protoplast isolation and induced cell death via heat shock (HS) in the lace plant is detailed here. Following HS, protoplasts displayed characteristics of PCD including: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive nuclei, increases in vesicles as well as Brownian motion, and plasma membrane blebbing. Additionally, mitochondrial dynamics were investigated, and a role for the mitochondrial permeability transition pore (MPTP) was indirectly established via cyclosporine A (CsA) experimentation. The main focus of this dissertation was to elucidate cellular dynamics during developmentally regulated PCD in the lace plant, which is visibly discernable during the window stage of leaf development. A single areole within a window stage leaf was further divided into three areas based on the progression of PCD; non-PCD (NPCD) cells, early PCD (EPCD) cells, and late PCD (LPCD) cells. Using this gradient, mitochondria were delineated into four stages based on distribution, motility, and membrane potential. Additionally, it was determined that the MPTP also played a role in developmental lace plant PCD, as inhibition of the pore with CsA not only reduced caspase-like proteases (CLPs) but also stopped perforation formation. Furthermore, the actin cytoskeleton was also investigated, with evidence suggesting it as a possible target for CLPs. The novel use of lace plant leaves for long-term live cell imaging allowed for the establishment of a timeline of cellular events that occur during developmental PCD. Major conclusions of this dissertation reveal various similarities between environmental induced and developmentally regulated PCD in this one plant species.

Programmed cell death (PCD) is an active process by which organisms coordinate the controlled destruction of cells. In tomato, the protein kinase Adi3 (AvrPto-dependent Pto-interacting kinase 3), acts as a negative regulator of PCD and shares important functional homologies with the mammalian anti-apoptotic AGC kinase PBK/Akt. Adi3 was originally identified as an interactor of the complex formed by the tomato resistance protein Pto and the Pseudomonas syringae pv. tomato (Pst) effector protein AvrPto. The complex formed by AvrPto and Pto causes a resistance response characterized by a rapid form of PCD that limits the spread of Pst and prevents the onset of the tomato bacterial speck disease. In an effort to characterize the mechanisms by which Adi3 regulates PCD, we identified Adi3 interacting partners in a Y2H screen. Here, I describe the interaction of Adi3 with two interacting partners identified: the Sucrose Non-fermenting (SNF1) kinase complex (SnRK) which is a eukaryotic master regulator of energy homeostasis and the E3 RING Ubiquitin ligase AdBiL. Using a combination of in vitro and in vivo approaches I found that AdBiL is an active ubiquitin ligase that ubiquitinates Adi3. Interestingly, Adi3 was found to be degraded in a proteasome-dependent manner suggesting ubiquitination could play a role in its degradation. On the other hand, Adi3 was found to inhibit the SnRK complex by directly interacting with its catalytic subunit as well as by phosphorylating the regulatory subunit SlGal83 at Ser26. SlGal83 is phosphorylated at multiple sites in vivo, and this phosphorylation state, as well as its intracellular localization was found to depend on a myristoylation signal present at its N-terminus. Phosphorylation at Ser26 by Adi3 was found to alter the localization of this subunit in a myristoylation-dependent manner. The interactions studied in this dissertation provide additional evidence on the functional homologies shared by Adi3 and PKB. In addition, the regulatory control of SnRK activity and cellular localization offers a novel connection between pathways involved in energy homeostasis and pathogen-mediated PCD.

During Drosophila melanogaster pupation, groups of undifferentiated adult cells proliferate and undergo regulated cell shape changes, while larval tissues are eliminated by programmed cell death (PCD). PCD is most commonly associated with apoptosis; however, a growing body of evidence suggests that autophagic cell death represents an alternative form of PCD. In some contexts autophagy can induce caspase-dependent PCD, but the regulatory pathways that link autophagy and apoptosis remain poorly understood. The overexpression of Atg16B induces autophagy-dependent caspase activation in the larval epidermis, and presumptive adult tissues. Also, the overexpression of Atg16B, in tissues specified by pnrGAL4, results in an adult cuticular phenotype that cannot be directly attributed to caspase activity. However, altering the level of Atg16B expression can modulate the adult cuticular phenotype. Therefore, the adult cuticular phenotype was used in a broad based genome-wide screen for dose-dependent modifiers of the regulation of autophagy. To date, 399 chromosomal deficiencies, representing approximately 92% of the 2nd and 3rd chromosomes, and 25 duplications have been tested. Consequently, 42 deficiencies have been identified as dominant enhancers of the Atg16B overexpression phenotype, 24 deficiencies have been identified as dominant modifiers, and 13 deficiencies have been identified as dominant suppressors. Further genetic analysis of these interacting deficiencies was carried out to initiate the process of identifying individual loci associated with the dose-sensitive modification of the Atg16B overexpression phenotype. It is hoped that the identification of these genes will elucidate the genetic pathways regulating autophagy and the autophagic induction of caspase activity.

Programmed cell death (PCD) is a highly evolved mechanism of cellular suicide that is aberrantly activated following neural injury. Two fundamental PCD signaling pathways termed the extrinsic (caspase-8-mediated) and intrinsic (caspase-9-mediated) pathways, have been described. While each pathway is initiated by distinct cellular stimuli, both pathways culminate in the activation of downstream executioner caspases. Previous efforts to isolate the in vivo contribution of each pathway have been hindered by the embryonic lethality of casp8 and casp9 null mice. In the present study, I overcame this obstacle to directly assess the contribution of each pathway following two well-characterized forms of acute neural injury; excitotoxic destruction of CA1 pyramidal neurons, and the loss of motor neurons following facial nerve transection. To determine the role of caspase-8, I constructed several lines of mice in which caspase-8 was conditionally ablated within the relevant neuronal populations. The results obtained from these animals definitively demonstrate that caspase-8 is not required by either motor neurons or CA1 pyramidal neurons to undergo PCD following injury. Therefore, these findings have provided the first direct experimental evidence to counter the widely held dogma of caspase-8 as the central effector of death receptor-mediated signaling within neurons. With respect to the intrinsic pathway, several lines of evidence suggest that the apoptosome predominantly regulates the death of motor neurons. I tested this hypothesis by performing facial axotomies in mice containing a point mutation introduced (“knocked in”) into the genomic locus of cytochrome c which abolishes its ability to activate the intrinsic pathway. Homozygous cytochrome c knock-in mice displayed a significant enhancement in motor neuron survival in comparison to control littermates following injury. However, the level of motor neuron protection differed from that previously reported in mice either overexpressing anti-apoptotic or lacking pro-apoptotic members of the Bcl-2 family. Therefore, the results of this study directly demonstrate the influence of the apoptosome on injury-induced neuronal PCD isolated from upstream Bcl-2 family-mediated effects. In addition, my results have provided the first evidence that activation of the apoptosome is required for the release of apoptosis inducing factor (AIF) from the mitochondria of injured motor neurons in vivo.

Abstract All-trans retinoic acid (ATRA) is a derivative of vitamin A. It is able to stimulate neutrophilic differentiation of normal progenitors and acute promyelocytic leukemia (APL) cells. Although ATRA-induced differentiation is not observed in any other acute myeloblastic leukemia (AML) subtypes, ATRA is known to be able to inhibit AML blast cell proliferation. The present in vitro study using AML cell lines representing subtypes other than APL focuses on the following questions: (1) Is the inhibitory effect of ATRA on AML cell growth related to apoptosis of cells? (2) Are the effects of ATRA dependent on two important regulators of apoptosis, p53 and Bcl-2? (3) Do mitochondria have any role in mediating the effects of ATRA? ATRA-induced apoptosis in AML cells was observed by morphology, DNA fragmentation, phosphatidylserine externalization, and poly(ADPribose)polymerase (PARP) cleavage. It was a slow event, manifested as DNA cleavage after 48 hours exposure and as morphological apoptosis after 72 hours exposure. The AML cells expressed constitutively p53 as determined by immunohistochemistry, Western blotting and flow cytometry. However, no mutation of TP53 was observed in exons 5 to 8 as analysed with a single strand conformation polymorphism technique. As the flow cytometer analysis showed, most of p53 was in a aberrant conformation, which was not changed into a wild type conformation by ATRA. Two of the cell lines were analysed more specifically in relation to Bcl-2 and mitochondral function: ATRA-induced apoptosis of the cell lines was associated with down-regulation of Bcl-2. Western blotting showed ATRA-induced apoptosis also to be related to the release of cytochrome c from mitochondria into cytosol, resulting in the activation of caspase-3, an apoptotic effector, which was manifested as a cleavage of its substrate PARP. The process was also accompanied by disruption of the mitochondrial membrane potential as determined fluoricytometrically. These results show that ATRA is able to induce apoptosis in AML cells other than APL, and ATRA-induced apoptosis in the AML cells studied is related to the down-regulation of Bcl-2 and the disruption of mitochondrial function, but is independent of the p53 pathway.

Programmed cell death (PCD) is an essential biological process in animal development and tissue homeostasis that is necessary to ensure the physiological well-being of the organism. During PCD, phagocytes facilitate the selective removal of excess, damaged, and potentially deleterious cells, in a multi-step engulfment process. Genetic studies in Drosophila melanogaster, Caenorhabditis elegans, and mammals have identified two evolutionarily conserved signal transduction pathways that act redundantly to regulate engulfment: the CED-1/-6/-7 and CED-2/-5/-12 pathways. Of these cell death (CED) proteins, the ABC transporter CED-7 is the only protein reported to be required in both the engulfing cell and the dying cell. However, its function in the cell death process remains the most enigmatic and the ced-7 ortholog previously has not been identified in Drosophila. Homology searches revealed a family of putative ced-7 orthologs that encode transporters of the ABCA family in Drosophila. To determine which of these genes functions similarly to ced-7/ABCA1 in PCD, we analyzed their engulfment function in oogenesis, during which 15 germ cells in each egg chamber undergo programmed cell death and are removed by neighboring phagocytic follicle cells. It has been shown that genetically knocking down individual engulfment genes results in inefficient clearance of the germ cells, which then persist in late-stage egg chambers. Only two of the putative ced-7/ABCA1 genes are expressed significantly in the ovary, CG31731 and CG1718, and we have characterized these genes using transposon insertions, deficiencies, and RNAi knockdowns. Our genetic analysis reveals that CG31731 is necessary for germ cell clearance in the Drosophila ovary. Immunostaining shows that genetically knocking down CG31731 results in uncleared germ cells which persist in late-stage egg chambers. Altogether, our findings suggest that CED-7/ABCA1/CG31731 play evolutionarily conserved roles during engulfment.