Biology Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2749
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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 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 MECHANISMS OF SEXUAL MODE EVOLUTION IN CAENORHABDITIS ELEGANS(2022) Skelly, Lauren E; Haag, Eric S; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACT Title of dissertation: MECHANISMS OF SEXUAL MODE EVOLUTION IN CAENORHABDITIS ELEGANS Lauren Skelly, Doctor of Philosophy, 2022 Dissertation directed by: Professor Eric S. Haag, Department of Biology The evolution of phenotypic novelties is a broad biological phenomenon, and how organisms evolve new traits is dependent on the molecular mechanisms that underlie those traits. Transcriptional regulation is often the focus of phenotype evolution, but post-transcriptional mechanisms such as mRNA splicing, stability, and translational control are also important components. Germ cells are particularly influenced by post-transcriptional mechanisms and are therefore a good system to study how these mechanisms lead to phenotypic novelties. This topic can be studied especially well in model systems that contain closely related species with recently evolved traits, such as self-fertility in Caenorhabditis elegans hermaphrodites. C. elegans hermaphrodites are essentially XX females that evolved the ability to produce sperm in an ovary. There are many known components necessary for sperm development, including a protein-protein-mRNA complex consisting of GLD-1, FOG-2 and tra-2. GLD-1 and FOG-2 dimerize and bind the 3’ UTR of tra-2 mRNA to repress its activity during spermatogenesis. In this work, I show FOG-2 directly interacts with TRA-2 protein, leading to the model that FOG-2 targets TRA-2 protein for degradation during its translation allowing sperm to form. I also show the expression pattern of tra-2 mRNA in the germline, and that GLD-1 binding on the 3’ UTR does not influence its localization. Another method for the study of novel traits is through hybridization of closely related species. In this work, I attempt to hybridize two closely related species of Caenorhabditis, another self-fertile species C. briggsae and an outcrossing species C. nigoni, to map the genetic loci underlying self-fertility. These hybrid crosses are unable to map genetic loci because males are inviable. These results agree with previous studies. This work contributes to the study of phenotypic evolution by adding an underlying molecular mechanism to Caenorhabditis sex determination.Item Development of the translaminar circuits in the mouse cortex(2020) Deng, Rongkang; Kanold, Patrick O; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The elaborated connections among cortical neurons form the cortical circuits, which are essential mechanisms underlying various cortical functions such as sensory perception, motor control, and other cognitive functions. The cortical circuits are composed of excitatory neurons and GABAergic interneurons. Excitatory neurons send excitatory connections to cortical neurons, while inhibitory neurons send inhibitory connections. Building the neural circuits is no easy task involving complex genetic programs and the influence of the environment through sensation. Malformation of the cortical circuits during development is implicated in causing neurological disorders, but our knowledge about the developmental process is scarce. The work in this dissertation uses in vitro electrophysiology in brain slices from transgenic mice to investigate how the excitatory connections onto GABAergic interneurons in the primary auditory cortex develop during the first two postnatal weeks. Furthermore, this dissertation explores the mechanisms that could regulate the early development of the cortical circuits by testing the requirement of sensory epithelium and N-methyl-D-aspartate receptors (NMDARs) in the early postnatal development of the neural circuits in the primary sensory cortex and temporal association cortex (TeA), respectively. Results from these studies fill crucial gaps in our understanding of how GABAergic interneurons are integrated into the cortical circuits and highlight the importance of sensory epithelium in the normal development of excitatory connections onto cortical GABAergic interneurons. My results also showed impaired development of GABAergic connections onto excitatory neurons lacking functional NMDARs in the TeA, suggesting an essential role of NMDARs for the early development of inhibitory circuits in the cortex.Item CONSERVED ROLE OF EMX2 IN ESTABLISHING POLARITY OF SENSORY HAIR CELLS(2016) Jiang, Tao; Carr, Catherine E; Wu, Doris K; Neuroscience and Cognitive Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sensory hair cells in the inner ear are responsible for relaying information such as sounds and head positions to the brain. Stereocilia, which are specialized microvilli, are arranged in a staircase-pattern with the longest row sitting adjacent to the kinocilium. These two structures together form the stereociliary bundle (hair bundle), which are polarized asymmetrically at the apical surface of the hair cell. Deflection of the stereocilia towards the kinocilium opens the mechanotransduction channels at the tip of the stereocilia, which enables ion influx to depolarize the hair cell and activates action potentials in the postsynaptic neurons. Deflection towards the opposite direction results in hyperpolarization. Thus, the stereociliary bundle polarity defines the directional sensitivity of a given hair cell. Each sensory hair cell organ displays a specific pattern of stereocilia polarity. In the maculae, which detect linear acceleration in all directions, HCs can be divided into two regions with opposite polarity by a line of polarity reversal (LPR). Similar LPR is also present in the neuromast of the zebrafish lateral line system that detect pressure change of surrounding water. My results show that the homeodomain transcription factor Emx2 is essential for establishing the LPR. Expression of Emx2 in the maculae and neuromasts determines the stereocilia polarity pattern in a cell-autonomous fashion. Gain- and loss-of-function in the sensory hair cell organs of mouse and zebrafish indicate that the role of Emx2 in polarity reversal is both necessary and sufficient. In addition, my results demonstrated that Emx2 mediates this polarity reversal via one of the heterotrimer G-proteins, Gαi. In summary, my results show that Emx2 has a conserved role in dictating stereociliary bundle polarity.Item Developmental Alterations in Inhibitory Neurotransmission in the Fragile X Syndrome Mouse Basolateral Amygdala(2012) Kratovac, Sebila; Corbin, Joshua G; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fragile X Syndrome, caused by Fmr1 gene inactivation, is characterized by symptoms including enhanced fear, hyperactivity, social anxiety, and autism, pointing to synaptic and neural circuit defects in the amygdala. Previous studies in Fmr1 knockout (KO) mice have demonstrated alterations in GABAA receptor (GABAAR) function in the basolateral amygdala during early postnatal development. In this study, we sought to determine whether these early defects in GABAAR function are accompanied by changes in protein expression of GABAAR alpha 1, 2, and 3 subunits, the pre-synaptic GABA-synthesizing proteins GAD65 and 67 (GAD65/67), and the post-synaptic GABAAR-clustering protein gephyrin. We found that the developmental trajectory of protein expression is altered in knockout mice for all tested proteins except GABAAR alpha 3 and GAD 65/67. Our results suggest that alterations in the timing of inhibitory synapse protein expression in early postnatal development could contribute to observed inhibitory neurotransmission deficits in the KO mouse basolateral amygdala.Item Regeneration, Fission and the Evolution of Developmental Novelty in Naid Annelids(2012) Zattara, Eduardo Enrique; Bely, Alexandra E; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Regeneration of lost structures and asexual reproduction by fission are post-embryonic trajectories related at the evolutionary and developmental levels. Their phylogenetic distribution within Metazoa has led to the hypothesis that fission can evolve by co-opting regenerative abilities. Fission has evolved multiple times within Annelida, including independent origins at the base of the Pristininae and Naidinae lineages of naid worms. Naids are thus a great system to study the evolution of developmental trajectories of regeneration and fission and their mutual physiological interactions. I made a comparative study of morphogenesis during regeneration and fission in a representative species, Pristina leidyi Smith (Pristininae), to test the hypothesis that both trajectories are closely linked by common origin, yet have undergone functional divergence; results show that regeneration and fission share numerous, sometimes exclusive developmental processes, but also present a number of differences spread out along their trajectories. I also examined cell proliferation and growth patterns in P. leidyi to characterize the resource allocation strategies it uses to integrate multiple developmental trajectories. I found evidence for a non-linear antero-posterior gradient in proliferation potential and clear interactions between regeneration and fission that strongly depend on fission stage and what body part is lost; similar interactions have been described for naidine annelids and turbellarian flatworms representing independent origins of fission, indicating convergence of fission-associated allocation strategies. I then extended the fission-regeneration comparative study in P. leidyi to additional annelids, describing and comparing regeneration and fission in another pristinine, seven naidine and one outgroup species, and found very similar regeneration trajectories among all of them, along with striking levels of convergence of paratomic fission trajectories. Despite similarities, the two paratomic clades presented a distinctive mode of central nervous system development. Finally, I developed novel protocols for dynamic studies of the cellular basis of regeneration, laying groundwork for future comparisons at that level. Altogether, these results strongly support that fission originated multiple times by co-option of regenerative abilities; furthermore, convergence of fission trajectories and resource allocation strategies suggests that similar developmental capabilities, functional constraints and ecophysiological contexts can channel evolutionary trajectories into parallel paths, both in close and distant lineages.Item Functional analysis of Smyd1 and Myomesin in sarcomere organization in zebrafish embryos(2012) Xu, Jin; Du, Shaojun; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Myofibrillogenesis, the process of sarcomere formation, requires close interaction of sarcomeric proteins and molecular chaperones. Smyd1 is a lysine methyltransferase that plays important roles in myofibrillogenesis in both skeletal and cardiac muscles. Knockdown of smyd1 results in complete disruption of sarcomere organization. The molecular mechanism by which Smyd1 controls myofibril assembly is not clear. In this study, we analyzed the sub-cellular localization of Smyd1, the effect of smyd1 knockdown on protein methylation, and the effect of myomesin knockdown on sarcomere organization. We demonstrated that Smyd1b_tv1 is localized to the M-lines of skeletal muscles in zebrafish embryos. Knockdown of myomesin-1b or myomesin-3 had no effect on the sarcomere organization. Western blot analysis revealed that knockdown of smyd1 reduced the overall protein methylation in zebrafish embryos. Together, these studies indicate that Smyd1 is required for M-line organization and Smyd1 may play a role in protein methylation and is involved in sarcomere assembly.Item The role of Sox2 in inner ear development(2011) Evsen, Lale; Popper, Arthur N; Neuroscience and Cognitive Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The vertebrate inner ear is a structurally complex sensory organ responsible for detecting sound and maintaining balance. These functions are mediated by specialized sensory epithelia comprised of a mosaic of mechano-transducing hair cells and supporting cells. The sensory hair cells are innervated by neurons of the cochleo-vestibular ganglion (CVG, the VIIIth cranial nerve). Both neuronal and sensory lineages are thought to be specified early in the neural-sensory competent domain (NSD) of the ear rudiment. First, neuroblasts delaminate from the NSD to form neurons of the CVG. Then, cells remaining in the NSD adopt a sensory fate and develop into various sensory organs. The molecular mechanisms that specify neuronal and sensory cell fates are unclear. The aim of this dissertation is to provide a better understanding by examining the roles of the HMG (high mobility group)-box containing transcription factors Sox2 and Sox3 in developing chicken inner ears using gain and loss-of-function approaches. Over-expression of Sox2 in ovo readily induces Neurogenin 1(Ngn1) expression, an important gene required for the neurogenic fate. Nevertheless, neurogenesis fails to proceed based on the lack of Neurod1 up-regulation and consequently the size of the CVG is reduced. In contrast, over-expression of Ngn1 is capable of up-regulating Neurod1 and causes increased neuroblast formation, as well as Sox2 down-regulation. Similar increases in neurogenesis are obtained with over-expression of Neurod1. I provide evidence that Ngn1 and Neurod1 inhibit Sox2 transcription via the E-box of the nasal-otic placode specific enhancer 1 (NOP-1) within the Sox2 promoter. On the other hand, loss of Sox2 function paradigms did not result in loss of Ngn1 expression, suggesting that other factors may be required to induce Ngn1 normally. Furthermore, while Sox3 has been proposed to be up-stream of Sox2, it does not induce Ngn1 in a similar manner as Sox2. Taken together, my results suggest that Sox2 and likely other factors are involved in initiating neurogenesis by up-regulating Ngn1. The up-regulated Ngn1, in turn, down-regulates Sox2 expression and up-regulates Neurod1 to mediate progression of neurogenesis. Finally, I show that Sox2 and the Notch signaling pathway interact to specify neuronal and sensory cell fate choices.