Theses and Dissertations from UMD
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Item SEROTONIN REGULATES AN OLFACTORY CRITICAL PERIOD IN DROSOPHILA(2024) Mallick, Ahana; Araneda, Ricardo; Gaudry, Quentin; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Serotonin (5-HT) is known to modulate early development during critical periods when experience drives heightened levels of plasticity in sensory systems. Studies in the somatosensory and visual cortices implicate multiple target points of serotonergic modulation, yet the underlying cellular and molecular mechanisms of 5-HT modulation of critical period plasticity remain elusive. Here, we take advantage of the genetically tractable olfactory system of Drosophila to investigate how 5-HT modulates critical period plasticity (CPP) in the CO2 sensing circuit of fruit flies. During the critical period, chronic exposure to CO2 has been shown to increase the volume of the CO2 sensing V glomerulus. We found that 5-HT release by serotonergic neurons in the antennal lobe (AL) is required for increase in the volume of the V glomerulus. Furthermore, signaling via the 5-HT1B, 5-HT2B and 5-HT7 receptors in different neuronal populations is also required during the critical period. Olfactory CPP is known to involve local inhibitory networks and consistent with this we found that knocking down 5-HT7 receptors in a subset of GABAergic local interneurons was sufficient to block CPP, as was knocking down GABA receptors expressed by olfactory sensory neurons (OSNs). Additionally, 5-HT2B expression in the cognate OSNs sensing CO2 is also essential for CPP indicating that direct modulation of OSNs also contributes to the olfactory CPP. Furthermore, 5-HT1B expression by serotonergic neurons in the olfactory system is also required during the critical period. Our study reveals that 5HT modulation of multiple neuronal targets is necessary for experience-dependent structural changes in an odor processing circuit. Finally, we wanted to isolate the neuromodulatory effects of individual serotonergic neurons. To achieve this, we combined a state-of-the-art technique to sparsely label serotonergic neurons and a computer algorithm to search against 10,000 Gal4 promoter lines and identify candidate lines that would allow individual manipulation of the 110 serotonergic neurons.Item IDENTIFICATION OF KEY MOLECULES IN PLACODE-DERIVED NEURONS THAT COORDINATE CHICK TRIGEMINAL GANGLIOGENESIS(2024) Hines, Margaret; Taneyhill, Lisa; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The trigeminal nerve is the largest of the cranial nerves, possessing three main branches (ophthalmic, maxillary, and mandibular) and relaying sensations of pain, touch, and temperature from the face and head to the brain. Cell bodies of this nerve are positioned in the trigeminal ganglion, which arises from the coalescence of neural crest cells and placode cells. These progenitor cells give rise to trigeminal sensory neurons, with placode cell differentiation occurring first. While the dual cellular origin of the trigeminal ganglion has been known for decades, the molecular mechanisms controlling trigeminal ganglion development remain obscure. To elucidate molecules involved in this process, we performed RNAsequencing on the forming chick trigeminal ganglion when only placode cells contribute neurons and identified Neurogenin2 (Neurog2), Neuronal Differentiation 1 (NeuroD1), and Elongator acetyltransferase complex subunit 1 (Elp1) for further study. While Neurog2, NeuroD1, and Elp1 have established roles in neurogenesis in other systems, their functions in placode cells during trigeminal gangliogenesis had yet to be investigated. To address this, we used the chick embryo due to experimental advantages afforded by this model for the study of trigeminal placode cells and trigeminal ganglion development. Using morpholino antisense oligonucleotides, we depleted Neurog2, NeuroD1, or Elp1 from trigeminal placode cells and demonstrated each are essential for proper trigeminal ganglion development. Knockdown of Neurog2, NeuroD1, or Elp1 reduced trigeminal ganglion size and led to aberrant innervation of the eye by the ophthalmic branch. While depletion of Neurog2 and NeuroD1 had opposite effects on the width of the ophthalmic branch, Elp1 reduction appeared to have no effect. However, Elp1 knockdown led to less compact trigeminal ganglion nerve branches, decreased axon projections, and general disorganization of neurons and neural crest cells. Taken together with prior findings, our results suggest a novel interrelationship among Neurog2, NeuroD1, and Elp1 during trigeminal gangliogenesis. Our results have potential high significance for providing new insights into the function of Neurog2, NeuroD1, and Elp1 in trigeminal ganglion development and the etiology of human and animal diseases arising from defects in neural crest cells and/or placode cells.Item Spatiotemporal proteomic approaches for investigating patterning during embryonic development(2024) Pade, Leena Rajendra; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Characterization of molecular events as embryonic cells give rise to tissues and organs raises a potential to better understand normal development and design remedies for diseases. In this work, I integrated bioanalytical chemistry with neurodevelopmental biology to uncover mechanisms underlying tissue induction in a developing embryo. Specifically, I developed ultrasensitive proteomic approaches to study the remodeling of the proteome as embryonic cells differentiate in space and time to induce tissue formation. This dissertation discusses the design and development of proteomic strategies to deepen proteomic coverage from limited embryonic tissues. A novel sample preparation workflow and detection strategy was developed to address the challenge of interference from abundant proteins such as yolk in Xenopus tissues which in turn boosts the sensitivity of detecting low abundant proteins from complex limited amounts of tissues. The refined analytical workflow was implemented to study the development of critical signaling centers and stem cell populations and the tissues they induce to form in developing embryos.Item CHARACTERIZING THE ROLES AND MECHANISMS OF CYTONEMES IN ASYMMETRIC SIGNALING AND ORGANIZATIONS IN THE DROSOPHILA MUSCLE PROGENITOR NICHE.(2024) Patel, Akshay Jitendrakumar; Roy, Sougata; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tissue development and homeostasis rely on the ability of embryonic or stem cells to efficiently determine whether to multiply for self-renewal or differentiate to generate a wide range of cell types that constitute an adult body. Stem cells determine these fates in the context of a specialized microenvironment or the niche that they occupy. All stem cell niches characterized to date are known to function using two key processes - adhesive interactions and asymmetric growth factor signaling between the niche and stem cells. While adhesion to the niche maintains niche occupancy and stemness, the loss of niche adhesion and occupancy initiates stem cell differentiation. Moreover, niche cells produce secreted growth factors to support stem cell self-renewal. Despite the ability of secreted growth factors to disperse across tissues over a long range, only the niche-adhering stem cells receive the self-renewal signals. The genetically identical daughter cells that lack adhesion to the niche fail to receive self-renewal signals, even when located within one or two cell diameters away, leading to the activation of their post-mitotic fates. Therefore, understanding how asymmetric signal distribution and adhesive interactions are produced and coordinated within the niche is critical to understanding how stem cells determine their identity and prime differentiation to generate or regenerate tissues. This thesis investigated and characterized a new mechanism of asymmetric signaling and cell organization in the Drosophila Adult Muscle Progenitor (AMP) niche. By employing genetic, cell-biological, and high-resolution microscopy techniques, this work discovered that AMPs extend thin polarized actin-based filopodia, called cytonemes, by orienting toward the wing disc niche. Cytonemes play a dual role. Cytonemes help AMPs to physically adhere to the wing disc niche and also directly receive a self-renewal Fibroblast Growth Factor (FGF) through the cytoneme-niche contact sites. AMP cytonemes localize the FGF-receptor (FGFR), called Heartless (Htl), and selectively adhere to the wing disc areas that express two different Htl ligands, Pyramus and Thisbe, both mammalian FGF8 homologs. Htl on these cytonemes directly receives Pyramus and Thisbe through the cytoneme-niche contact sites. Although FGFs are long-range secreted paracrine signals and Htl is the only receptor shared by Pyramus and Thisbe, these FGFs are received and restricted only to the niche-adhering AMPs due to the contact-dependent cytoneme-mediated asymmetric delivery of the signals. Moreover, despite employing a common FGF signal transduction pathway, Thisbe- and Pyramus-signaling initiates divergence of AMP fates into two distinct muscle-specific lineages. These experiments showed that cytoneme-mediated signal communication forms the basis of asymmetric signaling and organization within the AMP niche. We next asked how AMPs determine the niche-specific polarity and affinity of cytonemes. This research discovered that FGF reception and signaling activation in AMPs are required to activate polarized cytoneme formation orienting toward the wing disc niche. Without FGF signaling, AMPs cytonemes fail to polarize and adhere to the FGF-producing niche, causing them to exit the niche and start to differentiate. Thus, while target-specific asymmetric FGF distribution relies on cytonemes, activation of FGF signaling feedback maintains the polarity and adhesion of the signaling cytonemes toward the FGF-producing niche. A consequence of this interdependent relationship between niche adhesion, polarized FGF-reception, and stimulation of FGF signaling feedback is the maintenance of the self-organized niche-specific asymmetric signaling and organization via cytonemes. We next investigated whether the niche-adhering cytonemes receive additional fate-specifying cues, particularly the mechanical cues from the niche. Recent evidence suggests a critical role of mechanical and physical cues in determining stem cell fates. This work discovered that the AMP cytonemes are enriched with a common mechano-transducer, named Talin. AMP-specific genetic manipulation of talin indicates that Talin is critical for cytoneme-mediated niche occupancy and FGF signaling. Using a Talin-based force probe expressed at the physiological levels and FLIM-FRET microscopy, we discovered that Talin experiences pN level force within the cytonemes. These findings suggest that AMPs employ cytonemes not only for receiving FGFs in a restricted polarized manner but also for a mechanosensory function. In conclusion, these results strongly suggest a critical role of cytonemes in coordinating asymmetric signaling and organization in the stem cell niche. In addition, the work provides evidence that the stem cell cytonemes are critical organelles for integrating the inputs and outputs of both growth factor signaling and mechanical cues to sculpt tissues.Item Using CRISPR/Cas9 to functionally dissect Blimp1, a newly identified pair-rule gene in the hemipteran Oncopeltus fasciatus(2024) Reding, Katie; Pick, Leslie; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Genetic screens in the fruit fly Drosophila melanogaster identified a class of mutants displaying half as many segments as seen in wild type fly larvae (Nüsslein-Volhard and Wieschaus 1980). Careful examination of the larval cuticle revealed that one out of every two segments were deleted across the anterior-posterior (AP) axis, an unexpected phenotype suggesting that segmentation in Drosophila follows a ‘pair rule’. Thanks in part to the many genetic tools available for this model species, we now have a clear picture of how the AP axis of the Drosophila embryo is polarized and subsequently divided into distinct segments, and how the pair-rule genes (PRGs) define alternate segments during this process. Since all insects share a similar body plan, it is reasonable to expect that the processes regulating establishment of this body plan would be conserved. However, studies of the Drosophila segmentation gene orthologs in non-model insects suggest that this is not always the case. While the use of model organisms enables an unmatched depth of understanding of the mechanisms underlying development, it comes at the expense of understanding the diversity of these mechanisms across taxa. The milkweed bug Oncopeltus fasciatus (Ofas) (Hemiptera) is a particularly useful insect to study in this regard, as none of the orthologs of the Drosophila PRGs have clear PR-function in this species (Liu and Kaufman 2005b; Auman and Chipman 2018; Reding et al. 2019), while the gene E75A, which has no role in segmentation in Drosophila, is expressed in a PR pattern and its knockdown yields PR segmentation defects (Erezyilmaz et al. 2009). These results suggested that PR-regulation of segmentation in Oncopeltus might require a different set of factors than those discovered in Drosophila. To identify other non-canonical PRGs in this species, I conducted an expression pattern-based screen of transcription factor-encoding genes that are co-expressed with E75A during embryogenesis, and have identified the gene Blimp1 as an Oncopeltus PRG. Like the Drosophila PR mutants, Ofas-Blimp1 mutants display loss of alternate segments across the AP axis. No roles of Blimp1 in insect segmentation had been identified prior to this finding. This result suggests that while insect segmentation may be constrained to follow a pair rule, the genes responsible for regulating PR-segmentation are evolutionarily labile. Further, a major barrier to studying gene function in non-models is the lack of genetic tools such as visible markers and established methods for gene editing. Here I will describe deployment of CRISPR/Cas9 technology in Oncopeltus for targeted mutagenesis. While mutation of the ABC transporter-encoding gene white proved to be recessive lethal, I was able to generate a viable visible marker line by disrupting the X-linked gene Ofas-vermilion (v). Of-v is required for production of dark brown eye pigments, thus Ofas-v mutants have bright red eyes, easily discernible from the black eyes of wild type bugs. I show that a co-CRISPR approach using Of-v as a marker of germline mutation is a helpful strategy to identify mutations of interest at unlinked loci, enabling many future genetic manipulations in this species.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 Next-generation Mass Spectrometry With Multi-omics For Discoveries In Cell And Neurodevelopmental Biology(2022) Li, Jie; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Understanding tissue formation advances our understanding of the causes of disease and the obtained knowledge can be potentially applied to develop personalized interventions. However, to explore the underlying mechanisms that govern tissue formation, there is a high and unmet need to develop new technologies to characterize different types of biomolecules from early-stage embryonic precursor cells and their descendent cells during development. This dissertation discusses new technological advancements to facilitate multi-omic (proteomic and metabolomic) analysis to explore cell-to-cell differences and uncover mechanisms underlying tissue formation. The work presented herein illustrates the development of in vivo microsampling and single-cell mass spectrometry (MS) to uncover cell heterogeneity among embryonic cells. Additionally, this dissertation work studies the biological role of metabolites in cell fate determination by exploring the mechanisms underlying metabolite-induced cell fate change. Moreover, this work introduces a novel technique called MagCar developed to track and isolate tissue-specific cells at later stages, which enables studying temporal molecular changes to gain new information about tissue formation.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 Quantifying the relative contribution and furthering qualitative understanding of ftz cis-regulatory elements in Drosophila melanogaster(2022) Fischer, Matthew Douglas; Pick, Leslie; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Embryonic development is coordinated by interactions within gene regulatory networks. This process is orchestrated at the level of transcription through the regulatory properties of enhancers, which direct spatiotemporal expression patterns when bound by specific trans-acting factors. Though enhancers can act upon promoters located at great distances irrespective of orientation, the contributions from these cis-regulatory elements (CREs) are limited by insulators and/or tethering elements that organize chromatin architecture. Much research has been conducted towards understanding the coordination of the segmentation genes that pattern the basic body plan of the fruit fly, Drosophila melanogaster, during embryogenesis. The pair-rule genes (PRGs) of this pathway, such as fushi tarazu (ftz), are expressed in seven alternating stripes across the embryo. These PRGs are required for the development of body segments, and the mis-regulation of a single transcriptional domain can result in the loss of a segment. Here, I have investigated the ftz CREs to more precisely determine their sufficiency to direct expression within ftz stripe domains and their necessity for doing so in the native context of the gene. To investigate the sufficiency, I have generated 36 standardized reporter transgenes from 18 CREs, tested in both forward and reverse orientations. All CREs examined have been inserted into the same XbaI site of the reporter plasmid, and the transgenes have been inserted into the same genomic region. Through in situ hybridization experiments, I have determined that the qualitative patterns conferred by every CRE is orientation-dependent, and I have identified two putative insulators and/or tethering elements, proposed to explain this observation. To investigate their necessity, I targeted four genomic regulatory regions for precise deletion using the CRISPR/Cas9 system to generate seven deletion mutants. Though deletions were expected to cause lethality, most of the mutants are homozygous viable and fertile; only a mutant simultaneously removing two seven-stripe CREs was homozygous lethal. Quantitative gene expression analysis by fluorescent in situ hybridization chain reaction revealed that there is a critical threshold of ftz abundance required in each stripe for segmentation to proceed. In conclusion, I have determined that the ftz CREs are redundant and function together in a non-additive manner.