Animal & Avian Sciences Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2741
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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 MODULATING KEY GENES INVOLVED IN PANCREAS FORMATION AND INSULIN SIGNALING USING CRISPR/CAS9 IN THE PIG(2019) Sheets, Timothy P; Telugu, Bhanu P; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Among the metabolic diseases, diabetes remains a “pressing problem” as recognized by World Health Organization, not only due to the impact on individuals’ lives, but also because of the rapid increase in newly diagnosed patients. To better understand the mechanisms of diabetes, this dissertation investigates the role of NGN3 in pancreas development using CRISPR/Cas9 gene targeting in the pig model. NGN3 was selected for study because of its critical role in endocrine pancreas formation. Our research demonstrates that the targeted ablation of NGN3 blocks development of the endocrine pancreas, a finding supported through gene expression analysis. Furthermore, follow-up studies show that clonal piglets derived from NGN3-ablated animals lack the major endocrine islet cell types and subsequent expression of key endocrine hormones. This porcine model provides valuable insights into the study of type 1 diabetes in early post-natal life and future applications of human-to-pig chimeric organ development for transplant surgery. Expanding upon this porcine model for diabetes, we sought to apply this approach to the study of type 2 diabetes using a novel pig model, thus bridging the gap between mouse and human. For this endeavor, we identified GRB10 as a potential critical mediator in insulin signaling, development, and growth potential following an extensive literature review. The potential for dual applications in both agriculture and medicine was also identified as an objective. Analysis of qPCR data from in vitro overexpression studies supports that GRB10 modulates insulin signaling through the canonical insulin pathway. Additional data from two in vivo gene editing trials targeting the GRB10 locus in both Ossabaw and domestic pig breeds show a supportive qualitative trend towards growth regulation in the Ossabaw pig breed. Further evidence is required to determine whether GRB10 plays the same role in the domestic pig, as a limited cohort size of mutants precluded an extensive analysis of phenotypes. Together, our assessment of NGN3 and GRB10 offer significant potential for modeling of both type 1 and type 2 diabetes as well as modeling of growth traits in the pig through application of advanced genome engineering technology.Item ROLE OF ANNEXIN A6 IN SENSORY NEURONS DURING EARLY CHICK CRANIAL GANGLIA DEVELOPMENT(2017) Shah, Ankita; Taneyhill, Lisa A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The cranial sensory ganglia are created from neural crest cells and placode cell-derived neurons. Defects in the migration and interaction of these cells can cause abnormalities in craniofacial development and the sensory nervous system. To this end, we are using the embryonic chick cranial ganglia to elucidate the signaling mechanisms underlying cellular interactions. The Annexin protein superfamily has an evolutionarily conserved role in the development of the sensory ganglia. Our lab previously identified a function for chick Annexin A6 in modulating early NCC migration, but a later role for Annexin A6 in cranial ganglia assembly has not been investigated. We hypothesize that Annexin A6 acts a core cytoskeletal scaffold in cranial ganglia neurons to facilitate cranial ganglia formation. In support of this, our results show that placode cell-derived neurons express Annexin A6 during cranial ganglia assembly, and that expression is maintained throughout cranial gangliogenesis. Annexin A6 is also observed in neurons within the dorsal root ganglia and ventral neural tube, suggesting that Annexin A6 may be a specific neuronal marker. To investigate the function of Annexin A6 within the placode cells of the assembling cranial ganglia, we used a gene perturbation approach. Annexin A6 depletion from developing placode cells does not affect placode cell-derived neurons’ position within the ganglionic anlage nor disturb the surrounding neural crest cell corridors. Annexin A6 knockdown in placode cells results in neurons that produce very few short and/or no axonal projections instead of the normal bipolar morphology observed in the presence of Annexin A6. Placode cell-derived neurons with reduced level of Annexin A6 still express mature neuronal markers, they do not possess two long processes, which are characteristic morphological features of mature neurons, and fail to innervate their designated targets due to the absence of this bipolar morphology. In keeping with these results, Annexin A6 overexpression causes some placode cell-derived neurons to form extra protrusions alongside these bipolar processes. These data demonstrate that the molecular program associated with neuronal maturation is distinct from that orchestrating changes in neuronal morphology, and, importantly, reveal Annexin A6 to be a key membrane scaffolding protein during neuron membrane biogenesis.Item MOLECULAR MECHANISMS UNDERLYING CADHERIN-6B INTERNALIZATION IN PREMIGRATORY CRANIAL NEURAL CREST CELLS DURING THEIR EPITHELIAL-TO-MESENCHYMAL TRANSITION(2015) Padmanabhan, Rangarajan; Taneyhill, Lisa A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The generation of migratory cells from immotile precursors occurs frequently throughout development and is crucial to the formation and maintenance of a functioning organism. This phenomenon, called an epithelial-to-mesenchymal transition (EMT), involves the disassembly of intercellular adhesions and cytoskeletal rearrangements in order to promote migration. Importantly, aberrant EMTs and cell migration can lead to devastating human conditions including cancer metastasis and fibrosis. How cells accomplish EMT to become migratory is still an unanswered question in the biomedical field. To this end, we use chick neural crest cells as an in vivo model to elucidate the molecules and pathways that regulate EMT and migration. Neural crest cells are a population of embryonic cells that are originally stationary within the dorsal neural tube but later migrate to form a variety of adult derivatives, such as the craniofacial skeleton, skin pigment cells and portions of the heart. To facilitate EMT, chick premigratory neural crest cells lose intercellular contacts mediated, in part, by the transmembrane cell adhesion protein Cadherin-6B (Cad6B). While Cad6B mRNA is transcriptionally repressed in premigratory neural crest cells, loss of Cad6B protein does not directly follow and instead occurs ~90 minutes later, just prior to migration. This rapid depletion of Cad6B is all the more striking given that the half-life of most cadherins, including Cad6B, is ~6-8 hours in vitro. As such, unique post- translational mechanisms must exist to remove Cad6B from premigratory neural crest cell plasma membranes to facilitate neural crest EMT. Since cadherins are known to be downregulated through internalization mechanisms (e.g., endocytosis, macropinocytosis) in other in vitro systems, the hypothesis of this dissertation is that Cad6B is internalized, and that this process plays a critical function to enable neural crest EMT. To this end, we document the existence of Cad6B cytoplasmic puncta in cultured cells, cultured neural crest cells and transverse sections of chick embryos. We subsequently identified a p120-catenin binding motif in the Cad6B cytoplasmic tail and demonstrated its functionality through site-directed mutagenesis, revealing a role in enhancing Cad6B internalization and reducing the stability of membrane-bound Cad6B. Furthermore, we uncover for the first time that Cad6B is removed from premigratory cranial neural crest cells through cell surface internalization events that include clathrin-mediated endocytosis and macropinocytosis. Both of these processes are dependent upon the function of dynamin, and inhibition of Cad6B internalization abrogates neural crest cell EMT and migration. Collectively, our findings provide a molecular blueprint for how cadherins are dynamically regulated during the formation of migratory cell types required for normal embryonic development and tissue repair as well as those generated during human diseases and cancers. Importantly, our research is multi-disciplinary, integrating cell biology and physiology to reveal how a cellular event, the active downregulation of a membrane protein, results in a physiological event, neural crest EMT and migration.Item CELLULAR PATHWAYS INVOLVED IN EPITHELIAL-TO-MESENCHYMAL TRANSITIONS IN NEURAL CREST CELLS(2013) Li, Shen; Taneyhill, Lisa A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neural crest cells are a population of multi-potent progenitor cells in the developing vertebrate embryo that undergo an epithelial-to-mesenchymal transition (EMT) and migrate extensively to generate diverse derivatives. As such, abnormal development of neural crest cells can lead to human congenital and hereditary malformations, diseases and cancers. Both internal molecular signals and external mechanical factors play essential roles in facilitating neural crest cell EMT. How cells modulate their adhesion machinery and dynamically reorganize their actin cytoskeleton to respond to the mechanical features of their external environment during EMT is not well understood. To evaluate the role of the actomyosin cytoskeleton during neural crest cell EMT and migration, midbrain neural folds that contain premigratory neural crest cells were dissected out from chick embryos, explanted into chamber slides, and incubated to allow for the formation of migratory neural crest cells. Time-lapse imaging technique was used to record cell behaviors. To elucidate cellular pathways controlling EMT and migration, chemical inhibitors (blebbistatin, Y-27632, latrunculin-A, and nocodazole) that perturb molecular cascades regulating cellular structures were employed. Effects of these perturbations on neural crest cell EMT and migration were quantified in terms of the spreading rate of the explants, and vorticity of collectively moving cell groups. We observed that blebbistatin treatment reduced the overall velocity of migratory neural crest cells to negligible levels. Moreover, migratory neural cells developed rounder cell bodies, and lamellipodia were transformed into filopodia at the periphery of the extract. Y-27632 treatment led to more neural crest cells coming out from these explants within a shorter time period compared to control. Nocodazole treatment blocked neural crest cell EMT and the resumption was dose-dependent. Latrunculin-A caused cell death at a very low concentration. These results implicate roles for non-muscle myosin II, the target of blebbistatin, and ROCK, the target of Y-27632, as well as microtubules and actin filaments, in chick midbrain neural crest cell EMT and migration. Actin crosslinkers such as α-actinin and actin-associated proteins like palladin also participate in pathways affected by these cytoskeletal inhibitors through their regulation of focal adhesion formation and cytoskeletal organization, thereby modulating cell stiffness and migration. We are also documented the distribution of α-actinin and palladin in migratory neural crest cells in vivo. Collectively, our studies have provided insight into specific cellular pathways regulating neural crest cell EMT and migration and the impact on various biophysical parameters upon perturbation of these pathways.Item The role of the adherens junction protein alphaN-catenin in neural crest-derived trigeminal ganglia formation(2012) Hooper, Rachel; Taneyhill, Lisa; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neural crest cells (NCCs), a transient, multipotent population of cells that arise during neurulation, are a class of cells crucial to normal vertebrate development. NCCs must be tightly regulated by molecular and structural cues to de-adhere from the neural tube, migrate to their final destinations in the developing embryo, and differentiate to contribute to a variety of structures throughout the adult body. αN-catenin is the neural subtype of an adherens junction protein found in the apical region of premigratory NCCs, and plays an important role in controlling early phases of NCC migration. Although down-regulation of αN-catenin is critical for initial stages of NCC migration, the functional role of αN-catenin in later NCC migration and differentiation remains elusive. In this study, we investigate the spatio-temporal expression pattern of αN-catenin and elucidate effects on NCC movement and contribution to the trigeminal ganglia after perturbation of αN-catenin in the premigratory NCC population.