Cell Biology & Molecular Genetics Theses and Dissertations
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Item CHARACTERIZATION OF THE SHEDDASE-MEDIATED RELEASE OF A DROSOPHILA MEMBRANE-ANCHORED FGF THROUGH CYTONEMES(2023) Li, Yujia; Roy, Sougata; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)During tissue morphogenesis, cells must precisely coordinate their functions over distance by communicating with secreted paracrine signaling proteins, such as the fibroblast growth factors (FGFs). The spatiotemporal regulations of FGF signaling are critical for development and homeostasis in all organisms. However, the mechanisms that control the location, timing, and level of FGF secretion and ensure its target-specific delivery over distance are poorly understood. This thesis has addressed this fundamental question by using a Drosophila FGF Branchless (Bnl) during tracheal development. Bnl is produced in a small group of wing-disc cells. Bnl regulates the development of a disc-associated tracheal branch, the air-sac-primordium (ASP), a vertebrate lung bud analog. Although FGFs are secreted diffusible signals, previous work has shown that the inter-organ dispersion of Bnl from the source to the ASP is highly polarized, target-specific, and is mediated over distance via long polarized actin-based filopodia, named cytonemes. The contact-dependent cytoneme-mediated Bnl delivery is programmed by the glycosylphosphatidylinositol (GPI) anchoring of Bnl. A GPI anchor tethers Bnl to the outer leaflet of the source cell membrane and inhibits free/random Bnl secretion. Surprisingly, GPI anchoring also facilitates Bnl’s release, but only through the cytoneme contact sites, thereby activating MAPK signaling exclusively in those recipient cells that establish contact with its source. However, the molecular mechanisms that might spatiotemporally activate the release of the membrane-anchored Bnl at the cytoneme contact sites remained unknown. This work addresses this knowledge gap by using a combination of cell biological, biochemical, and genetic analyses. Visualization of the distribution of a fluorescently-tagged Bnl revealed that a proteolytic cleavage within the Bnl backbone is essential for its target-specific release. Phenotypic analyses of an uncleavable Bnl mutant generated by genome editing suggested that the proteolytic event that releases Bnl is essential for Bnl signaling and morphogenesis. An in vitro cell-culture-based screening for candidate sheddases identified five membrane-localized enzymes that specifically shed Bnl from the cell surface. These enzymes include two matrix metalloproteinases, MMP1 and MMP2, a pro-protein convertase, Furin1, and two ADAM family metalloproteases, Meltrin and Tace. An inherently disordered region within the Bnl backbone was identified as the substrate for the proteolytic cleavage. Serial mutagenesis in this region revealed four minimal zones required for enzymes to act on Bnl. Interestingly, one of the minimal regions at the juxta-membrane position harbors the Bnl cleavage site, and the other three zones apparently play regulatory roles. Further work on MMPs using mosaic gain-of-function analyses confirmed MMP-mediated Bnl shedding in vivo. However, MMPs are not expressed in the Bnl source. Consequently, Bnl is not freely released. In contrast, MMPs are expressed in the ASP and are localized on the recipient ASP cytonemes. in vitro cell culture experiments that reconstituted contact-dependent Btl-Bnl interactions between Bnl-source and recipient cells confirmed that MMPs are selectively recruited by the recipient cells to the signaling contact sites and activate Bnl shedding in trans. These results explained why and how Bnl is released only via the cytoneme contact sites. Notably, MMP1 and MMP2 are GPI-anchored proteins but are also catalytically active in secreted soluble forms. Therefore, how is Bnl prevented from non-specific shedding by the extracellular soluble MMPs? in vitro and in vivo experiments using GPI-modified and non-GPI-modified MMPs revealed that only the membrane-tethered MMPs that are retained on the cell surface could efficiently shed cell surface Bnl. These findings supported a working model suggesting that recipient ASP cells extend receptor-containing cytonemes and establish contact with the Bnl-source via the receptor-ligand interaction. Due to the GPI-anchoring, MMPs are presented on the surface of ASP cytonemes and are delivered via these cytonemes to their contact sites, leading to the contact-dependent target-specific Bnl trans-shedding. These results provide new insights into how contact-mediated signal dispersion via cytonemes can be modulated by cell surface sheddases. Additional work on Furin1 and ADAM proteases showed that Tace and Meltrin shed Bnl in a cell-non-autonomous manner, like MMPs. In contrast, Furin1 is required only in the source cells for Bnl shedding. How and under what contexts Furin1 and ADAM proteases are activated to shed Bnl needs to be explored in the future. Collectively, this work characterized a novel enzymatic Bnl shedding mechanism and provided insights into how Bnl sheddases might be controlled in space and time to ensure cytoneme-mediated Bnl exchange.Item Molecular and Biophysical Bases of Intracellular Electric Fields in Pollen Tubes(2022) Oliveira Nunes, Custódio; Feijό, José A.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Pollen tubes are the male gametophyte of flowering plants. They are arguably one of the fastest-growing cells in nature and inherently an excellent model for studying cellular processes like apical growth, polarity, and chemotropism. Pollen tube development is associated with a unique choreography of ion fluxes and cytosolic ion gradients of Cl-, Ca2+, H+, and K+, creating a unique electrochemical environment, where alternating depolarizing ionic currents at their growing apex are spatially separated from hyperpolarizing currents in their shank. We hypothesize that these electrical differences generated by the opposite ionic patterns could sustain a standing membrane potential gradient at the growing apex. In agreement with evidence from other cellular electrotaxis phenomena, we further hypothesize that a standing electric field gradient could be mechanistic in terms of cell polarity and chemotropism of pollen tubes.Here we show, for the first time, the existence of a standing membrane potential gradient in pollen tubes, confirmed in three different species, thus suggesting a conserved role in apical growth. This conclusion was achieved using three complementary methods, two membrane potential dyes with opposite fluorescence kinetics, and a genetic probe for cytosolic potassium (K+). The K+ gradient is focused at the pollen tube tip, and is compatible with previous information on the individual ion features. Of relevance, K+ shows a negative gradient from the tip, the first ever described in a living cell, suggestive of K+ apical efflux that contributes to the depolarized state. Quantifications of the fluorescent dyes estimate an apical depolarization of approximately 30mV compared to the shank. Screening of ion-channel mutants inducing male-fertility phenotypes supports the hypothesis that this bioelectric oddity is mechanistic for pollen tube’s critical functions, fast invasive growth and chemotropism. Furthermore, we determined that anionic lipids determine the emergence of the pollen tube and correlate with the apical depolarization area, suggesting that they may act as physical determinants of the growing apex. These results open important questions in our understanding of the bioelectrical processes determining cell growth, polarity, morphogenesis, and chemotropic reactions.Item Genome-wide identification and analysis of imprinted genes in strawberry seed development(2022) Joldersma, Dirk; Liu, Zhongchi Anne; Taneyhill, Lisa Anne; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The activation of zygotic gene expression is of fundamental importance to reproductive biology, but its regulation remains poorly understood. Within Angiosperm plants, fertilization occurs simultaneously in two locations, the embryo and its genetic twin, the endosperm, a nutritive tissue that is a defining feature of Angiosperm reproduction. Auxin hormone synthesized in the endosperm is essential to seed and fruit development. In the diploid strawberry Fragaria vesca, that auxin synthesis is regulated by FveAGL62, which is expressed specifically after fertilization in endosperm. How fertilization activates FveAGL62 expression in the endosperm, however, is presently unknown. I investigated the hypothesis that epigenetically regulated maternally- and paternally expressed genes (MEGs and PEGs, and together, “imprinted genes”) regulate the expression of FveAGL62. I hybridized two F. vesca accessions, isolated the endosperm from the F1 seeds, and sequenced the transcriptome of the F1 endosperm—a result facilitated by strawberry’s uniquely accessible seed. To identify imprinted genes within the endosperm, I assembled and annotated the genome of the maternal parent, F. vesca accession “Yellow Wonder” (FvYW5AF7), a model for the commercial strawberry. The paternal parent genome was obtained from a collaborator. 809 PEGs and 825 MEGs were identified from RNA sequencing reads that align uniquely to the maternal or paternal genome. MEGs are enriched in genes catabolizing auxin and hence limit seed growth, while PEGs are enriched in genes involved in histone modification, thereby promoting cell differentiation and seed growth. The distinct roles of MEGs and PEGs supports and can be explained by parental conflict and kinship theories, which predict a maternal genome tends to restrict progeny consumption of maternal resources, while a paternal genome will encourage such consumption. In contrast to findings in other species, I find that the endosperm-specific auxin biosynthetic gene FveYUC10 is maternally expressed, but while its imprinting status has changed, it may still function as a fertilization sensor. The maternally expressed gene FveMYB98 contains a binding domain that targets motifs present in FveAGL62’s promoter and its homolog binds AtAGL62 promoter in Arabidopsis. With collaborators, I showed that overexpression and CRISPR knockout of FveMYB98 changes seed size. Transient expression, yeast one hybrid and quantitative PCR analyses suggest that FveMYB98 represses FveYUC10 expression directly and FveAGL62 expression indirectly. These results suggest that FveMYB98 expression is a vehicle for maternal regulation of the level of auxin in the endosperm and thereby endosperm proliferation and seed size. My dissertation research has produced a new genome assembly of a model strawberry, a transcriptome of strawberry endosperm, and identified imprinted genes at genomic scale. I find FveMYB98 regulates seed size—a function echoed broadly within MEG and PEG classes—providing supporting evidence for the parental conflict theory within the developing progeny. These results improve our understanding of zygotic expression in developing seeds, addressing a fundamental scientific gap and, more tangibly, may enable future production of fertilization-independent seeds and seedless fruits.Item MECHANISM OF DREAM COMPONENT TSO1 IN PLANT STEM CELL REGULATION(2022) Wang, Fuxi; Liu, Zhongchi ZL; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plants are important for human survival and the environment. They provide oxygen, food, medicine and fuel. Understanding the development of plants has been a fundamental research question. Among all the plant tissues, the most important ones are the meristems. Sitting at the tip of the shoot and the root are the shoot apical meristem (SAM) and the root apical meristem (RAM). The shoot apical meristem gives rise to the above-ground organs like leaves and flowers while the root apical meristem produces all the root tissues that help to anchor the plants and transport water and nutrients. As the meristem is capable of producing new organs throughout the lifespan of a plant, the study of meristem maintenance and development provides the key to the understanding of plant development.Arabidopsis transcription factor TSO1 plays an essential role for the proper development of shoot apical meristem and root apical meristem. TSO1 encodes a protein with a cysteine-rich repeats domain and TSO1 is a potential component of a cell cycle regulating complex, the DREAM complex. The tso1-1 mutant has fasciated SAM due to shoot meristem cell over-proliferation and complete sterility due to lack of differentiated female and male floral organs. Interestingly, the tso1-1 mutant also produces shorter root than the wild type, presumably caused by early differentiation of the cells in the RAM. A prior mutagenesis screen identified two major suppressors of tso1-1. Characterization of these tso1-1 suppressor mutations provides important insights to the understanding of TSO1-regulatory pathways. My dissertation project focuses on analyzing one of these suppressors that was shown to be a mutated type-A cyclin gene named CYCA3;4. Mutations in CYCA3;4 suppress the shoot phenotype but not the root phenotype of tso1-1. The suppressed plants can produce normal floral organs and become partially fertile. Using transgenic method, I showed that the expression of CYCA3;4 was increased in the tso1-1 SAM, and overexpression of CYCA3;4 in the tso1-3 mutant enhanced the fertility defect, suggesting that overexpression of CYCA3;4 partially mediates the tso1-1 shoot phenotype. In addition, I provided evidence supporting that TSO1 likely represses CYCA3;4 gene expression indirectly through MYB3R1, whose mutations also suppress tso1-1 mutants. My dissertation provides an important link between TSO1, a potential cell cycle regulatory complex component and meristem regulator, and cyclin A, a protein directly involved in cell cycle regulation. This link provides an important mechanistic insight into how plant meristems maintain their identity by limiting their cell division activity. To further investigate the mechanism of TSO1 action in the root, I collaborated with two other scientists to profile the gene expression in the tso1-1 root at single cell level. I compared the single cell RNA sequencing data of tso1-1 and wild type roots and identified molecular defects in the tso1-1 root vasculature. Correspondingly, the known regulators of vasculature development, the HD-ZIP III genes, are ectopically expressed in some of the vascular cells in the tso1-1 root. It suggests that the defects of root vasculature may be attributed to mis-expressed HD-ZIP III genes in the tso1-1 mutant. The HD-ZIPIII function was previously linked to their regulation of cytokinin biosynthesis genes, which were ectopically expressed in tso1-1 roots as revealed by our scRNA-seq data. Together, our data suggest that the over-production of cytokinin might be the cause of tso1-1 short root phenotype. In summary, my dissertation research revealed previously unknown links between TSO1 and cell cycle regulation in the shoot and root meristems as well as the molecular mechanisms of TSO1 function in the root vascular development at single cell level. These findings have furthered our understanding of how cell cycle regulation is integrated with plant development.Item THE MECHANISMS AND ROLES OF POST-TRANSLATIONAL PROCESSING OF THE DROSOPHILA FIBROBLAST GROWTH FACTOR BRANCHLESS DURING DEVELOPMENT(2019) Sohr, Alexander Ronayne; Roy, Sougata; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)During embryonic development, cells communicate with each other to cooperate to form organized tissues. Cells spatiotemporally coordinate with each other by communicating with signaling proteins such as Fibroblast Growth Factor (FGF) that travel from source to target cells to activate various functions. To better understand cell communication during tissue morphogenesis, this study aimed to address a fundamental question: how different cellular and molecular mechanisms in signal-producing cells prepare and release signals at the correct time and location and at an appropriate level. This research focuses on the intercellular communication of the Drosophila FGF Branchless (Bnl) to elucidate this question. Bnl is dynamically produced in restricted groups of cells to induce morphogenesis of tracheal airway epithelial tubes. Tracheal cells receive the signal over distance by extending long receptor-containing filopodia, or cytonemes, to dynamically contact the Bnl-source. This work discovered two post-translational modifications of Bnl that regulate its polarized intracellular trafficking and cytoneme-mediated intercellular dispersal. During intracellular trafficking through the source cell Golgi network, Bnl is endo-proteolytically cleaved at a single site by the protease Furin-1. This cleavage activates polarized intracellular trafficking of the truncated signal exclusively to the surface of the source cells that faces the recipient tracheal cells. Thus, the intracellular cleavage acts as a switch to catalyze the efficient trafficking of the signal to the correct location from where cytonemes can subsequently receive it. Secondly, in the endoplasmic reticulum of source cells, Bnl is modified with a glycosylphosphatidylinositol (GPI) moiety at its C-terminus. This lipid moiety tethers Bnl molecules to the outer leaflet of the cell membrane, inhibiting its free release and ensuring signal exchange solely by direct physical contacts established by cytonemes. Therefore, this study discovered how Bnl is prepared by the source cells to ensure its subsequent target-specific intercellular dispersion through cytonemes. Conserved FGF family proteins are essential for regulating a broad spectrum of biological functions and defects in spatiotemporal levels of FGF signaling leads to severe diseases. Given the conservation of developmental signaling mechanisms in all organisms, the discovery of new regulatory mechanisms of FGF signaling has fundamental implications for understanding development and disease in humans.Item Novel Models for Studying Trophoblast Development and Placental Pathologies(2019) Pence, Laramie; Telugu, Bhanu; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Placental development begins in the mammalian blastocyst, when the first lineage specification event commits one cell population to making extraembryonic tissues, including the placenta, and commits another cell population to making the embryo proper. The mouse is an excellent animal model to study these early events and how the resulting placenta organ supports normal fetal development and a healthy pregnancy in the mother. The studies included in this Dissertation use the mouse to understand the role of long non-coding RNAs during early placental development, and to create a lineage biasing model that takes advantage of what is known about the first lineage specification event in mammals. Using expression analysis and the CRISPR/Cas9 system to create a knockout mouse strain, a placental-specific lncRNA was discovered and shown to be expressed in derivatives of the ectoplacental cone. Additionally, using the line age bias model to cause biased ablation of Hif1α in the placenta has revealed a role for fetal vs. placental contribution of resulting phenotypes.Item A Network Approach to Identify Key Regulators of Fruit Development in Fragaria vesca, a Diploid Strawberry(2018) Shahan, Rachel; Liu, Zhongchi; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Post-embryonic organogenesis is a feature unique to plants, an example of which is flower and fruit production. Previous work on strawberry fruit development has focused primarily on the latter stages, including ripening. Comparatively little is known about the molecular events underpinning fruit set, the pivotal stage at which fruit development proceeds or terminates. This thesis investigates early fruit development using Fragaria vesca, a diploid strawberry, as a model. In collaboration with a bioinformatician, I generated gene co-expression networks from 92 previously generated RNA-Seq libraries profiling multiple tissues and stages of strawberry flower and fruit development. I demonstrate the utility of co-expression networks in illuminating molecular processes underlying fruit development. Experimental validation of the networks includes demonstration of increased iron transport soon after fertilization and identification of FveUFO1 as an important regulator of floral meristem determinacy and floral organ identity. Using the co-expression networks, I discovered the surprising expression of FvFT1, a homolog of FLOWERING LOCUS T (FT), in the fleshy fruit immediately post-fertilization. In many plant species, the FT peptide is a non-cell autonomous signal that initiates flowering in response to inductive photoperiod. I found that FvFT1 expression is responsive to temperature, but not photoperiod, in strawberry fruit. Further, transcriptional activation is detectable in the vascular bundles connecting the fruit to the seeds, raising the possibility that FvFT1 may facilitate cross-tissue communication. Signal from an FvFT1-GFP translational fusion protein is visible in seed nuclei despite its localized transcription in the vasculature. However, analysis of FvFT1 RNAi plants failed to identify a fruit phenotype, possibly due to redundancy among three FvFT paralogs. Finally, to develop additional research tools for F. vesca, I isolated and tested fruit tissue-specific promoters based on genes identified with differential expression analyses. These analyses revealed genes strongly expressed in the receptacle fruit, thereby identifying potential regulators of early fruit development and attractive candidates for future study. Together, this work advances the systems-level infrastructure for studying molecular regulation of F. vesca fruit development, points to a novel role for FT distinct from its known function in floral initiation, and provides molecular tools useful to the F. vesca community.Item Development and the Early Animal Fossil Record: Evolution and Phylogenetic Applications(2016) Tweedt, Sarah Maureen; Delwiche, Charles F; Erwin, Douglas H; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Although evolutionary developmental biology and paleontology are linked by the study of morphology, the application of development to paleontological questions has only recently become more prominent. The growth of a robust developmental genetic framework for studying the origin and evolution of morphological features, however, holds great promise for understanding ancient animal life. As paleontology provides the historical record as well as the temporal and environmental context of past morphological evolution, uniting knowledge of developmental genetic systems with this historical record will form a key synthetic approach to understanding the early evolution of developmental processes. Ultimately unraveling the sequence of ancient animal developmental evolution will require combining analysis of comparative developmental data, critical assessment of fossil morphology within a developmental framework, and the targeted exploration of specific geologic periods to fill in the missing record of key times in animal developmental evolution. This study addresses each of these three approaches. First, I provide a new compilation and evaluation of recent comparative and experimental developmental biology data to review the nature of developmental ‘toolkits’ at the origin of the most basal animal clades. I reconstruct early animal developmental capacities and integrate these data within a temporal framework to better understand the context of earliest animal development. Second, I assess longstanding evolutionary hypotheses about the origin of the panarthropod clade and the phylogenetic position of Cambrian ‘lobopod’ fossils by examining signal present within current morphological datasets. I apply new methods to fossil panarthropod phylogeny estimation and suggest strategies for developmentally-informed phylogenetic coding of morphological data. Third, I present the discovery of the oldest spicule-bearing fossil sponges in the rock record, which co-occur in latest Ediacaran strata with classic enigmatic Ediacaran fauna. I provide a formal systematic description of fossil material from localities in both Nevada and southern Namibia. The combined approaches presented herein are a first step towards a deeper integration of developmental principles in the study and discovery of ancient animal life, and contribute to our understanding of the evolution of ancient animal developmental processes.Item KINESIN MOTOR PROTEINS ARE ESSENTIAL FOR MALE GAMETOPHYTE DEVELOPMENT IN MARSILEA VESTITA(2016) Tomei, Erika Jean; Wolniak, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The male gametophyte of the semi-aquatic fern, Marsilea vestita, produces multiciliated spermatozoids in a rapid developmental sequence that is controlled post-transcriptionally when dry microspores are placed in water. Development can be divided into two phases, mitosis and differentiation. During the mitotic phase, a series of nine successive division cycles produce 7 sterile cells and 32 spermatids in 4.5-5 hours. During the next 5-6 hours, each spermatid differentiates into a corkscrew-shaped motile spermatozoid with ~140 cilia. This document focuses on the role of motor proteins in the regulation of male gametophyte development and during ciliogenesis. In order to study the mechanisms that regulate spermatogenesis, RNAseq was used to generate a reference transcriptome that allowed us to assess the abundance of transcripts at different stages of development. Over 120 kinesin-like sequences were identified in the transcriptome that represent 56 unique kinesin transcripts. Members of the kinesin-2, -4, -5, -7, -8, -9, -12, -13, and -14 families, in addition to several plant specific and ‘orphan’ kinesins are present. Most (91%) of these kinesin transcripts change in abundance throughout gametophyte development, with 52% of kinesin mRNAs enriched during the mitotic phase and 39% enriched during differentiation. Functional analyses show that the temporal regulation of kinesin transcripts during gametogenesis directly correlates with kinesin protein function. Specifically, Marsilea makes one kinesin-2 (MvKinesin-2) and two kinesin-9 (MvKinesin-9A and MvKinesin-9B) transcripts, which are present during spermatid differentiation and ciliogenesis. Silencing experiments showed that MvKinesin-2 and MvKinesin-9A are required for ciliogenesis and motility in the Marsilea male gametophyte; however, these kinesins display atypical roles during these processes. In contrast, spermatozoids produced after the silencing of MvKinesin-9B exhibit normal morphology. MvKinesin-2 is necessary for cytokinesis as well as for regulating ciliary length and MvKinesin-9A is needed for the correct orientation of basal bodies, events not typically associated with these proteins. In addition, Marsilea makes motile, ciliated gametophytes without the help of IFT dynein, outer arm dynein, or the BBsome. These results are the first to investigate the kinesin-linked mechanisms that regulate ciliogenesis in a land plant.Item Evolution of Pair-rule genes(2015) Lu, Yong; Pick, Leslie; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)All insects have a segmented body. The genes controlling segment development have been well characterized in the fruit fly, Drosophila melanogaster. These genes were divided into three categories: gap genes specify several continuous segments over a broad region of the embryo; Pair-Rule Genes (PRG) are responsible for segment formation and are the first set of genes to be expressed in repetitive patterns in the embryo; Segment polarity genes define anterior and posterior polarities within each segment.To understand how PRGs evolve, I took a comparative approach in this thesis. First, I compared the function of the Drosophila PRG ftz-f1 to that of its mammalian orthologs by expressing them all in Drosophila embryos. I found that the molecular function of this family of nuclear receptors has been highly conserved during evolution. Next, I set out to establish new insect model systems to study PRG function. While, some PRGs have been studied in other insects, most of these studies focused on holometabolous insects. My work focused on the sister group to the holometabolous insects, the Hemipteroid Assemblage. I participated in the genome annotation of a hemipteras insect, Oncopeltus fasciatus. I annotated nuclear receptor super family, Hox and PRGs in Oncopeltus. I further studied the expression and function of four PRGs in Oncopeltus. Using in situ hybridization and RNAi, I found that, Of-ftz and Of-hairy do not have segmentation function, while Of-ftz-f1 has function in oogenesis and segmentation. Of-runt was found to induce cell death in oocytes, but its function in segmentation needs further analysis. Using the knowledge and expertise I gained from Oncopeltus, I successfully set up in situ hybridization, antibody staining and parental RNAi in an invasive hemipteran insect pest, the Brown Marmorated Stink Bug (BMSB) Halyomorpha halys. These studies show that the expression and function of PRGs varies extensively in diverse insects, despite the overall conservation of a segmented body plan.