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Synaptic connectivity is highly plastic in early development and undergoes extensive remodeling in response to changes in neuronal activity and sensory experience. Microglia, the resident central nervous system macrophages, participate in shaping mature neuronal circuits by dynamically surveying the brain parenchyma and pruning away less active synaptic connections. However, it is unknown how changes in neuronal activity regulates microglial pruning within circuits and whether this activity-dependent pruning is necessary to achieve plasticity. Using the rodent somatosensory circuit, I identified that microglia engulf and eliminate synapses in the cortex following early postnatal (P4) unilateral removal of mouse whiskers. I found this early life microglial synaptic remodeling requires specific chemokine signaling between neurons and microglia. Mice that lack expression of either the neuronal chemokine CX3CL1 (fractalkine), or its microglial receptor CX3CR1, have significantly reduced microglial synapse engulfment and fail to eliminate synapses following whisker removal. To gain more insight into how this signaling is regulated, I performed both single-cell RNA sequencing of the primary somatosensory cortex as well as microglia-specific Translating Ribosome Affinity Purification (TRAP) sequencing. I identified that the majority of central nervous system (CNS) cell populations in the somatosensory cortex, including microglia, undergo transcriptional changes following whisker removal. Further, the transcriptional changes in microglia after whisker cauterization require expression of the receptor CX3CR1. Importantly, I also found that Adam10, a gene encoding the metalloprotease known to post-translationally cleave CX3CL1 into a soluble chemokine, is upregulated in the deprived cortex after whisker ablation. Pharmacological inhibition of ADAM10 inhibits microglia-mediated removal of synapses in the deprived cortex. These data support a mechanism by which cleavage of membrane-bound CX3CL1 by ADAM10 is necessary for neuronal signaling to microglia via CX3CR1 to induce transcriptional changes within microglia upstream of synaptic engulfment and elimination following sensory deprivation.

Ein sehr organisiertes vielschichtiges Gewebe im Gehirn von Säugetieren ist der Neokortex, der höhere kognitive Funktionen ausübt wie Erlernen einer Sprache, Denken und räumliches Vorstellungsvermögen. Während der Evolution hat sich der Neokortex vergrößert, um den komplexen kognitiven Bedarf von höher entwickelten Tieren zu bewältigen. Es wird angenommen, dass diese kortikale Expansion primär in der Balance zwischen Proliferation und Differenzierung von neuralen Stammzellen und deren Vorläuferzellen begründet ist. Diese Prozesse sind sehr stark programmiert in Ort und Zeit, was viele Fragen über die involvierten molekularen Netzwerke aufwirft. In den letzten zwei Jahrzehnten haben sich lange nicht-kodierende RNAs (lncRNAs) als attraktive Ziele in der Entwicklungsbiologie dargestellt. Diese Moleküle haben unsere Wahrnehmung von der funktionalen Einheit einer Zelle revolutioniert, da sie bekannt dafür sind, deren Funktion mittels ihrer RNA Struktur auszuüben. Im Gegensatz zu microRNAs, die ihre Funktion über die Regulation von protein-kodierenden Genen auf post-transkriptionaler Ebene ausführen, agieren lncRNAs unterschiedlicher. Vor allem im Gehirn, dem Organ, was die größte Anzahl von lncRNAs exprimiert und das höchste Verhältnis von Gewebe- und Zell-spezifischen lncRNAs besitzt, wurde nachgewiesen, dass sie in fast jedem Prozess während der Entwicklung und bei Erwachsenen involviert sind. Mit dem Ziel eines besseren Verständnisses der molekularen Mechanismen der Gehirnentwicklung, habe ich angestrebt neue Rollen für lncRNAs in der Neurogenese zu identifizieren. Dafür habe ich Gebrauch von einem wirksamen genetischen Instrument gemacht, das zuvor in unserem Labor erzeugt wurde: ein Transkriptom von einer Mauslinie, die es ermöglicht, proliferierende, differenzierende und ausdifferenzierte Zellen zu sortieren. Die Analyse der differentiellen Expression von lncRNAs in den drei Zelltypen enthüllte interessante Kandidaten, die möglicherweise eine Rolle in der Neurogenese spielen. Die Manipulation dieser Kandidaten wurde in vivo getestet durch in utero Elektroporation. In dieser Studie habe ich Casc15 als Regulator von neuraler Stammzellproliferation und neuronaler Migration identifiziert. Überexpression von Casc15 im entwickelnden Kortex verursachte eine Deregulierung von Genen, die in der Entwicklung des Nervensystems und Zellteil-Morphogenese involviert sind. Insbesondere herunterregulierte Gene nach Casc15 Überexpression sind physiologisch angereichert in Neuronen. Diese schließen Gene ein, die verantwortlich für neuronale Migration und Reifung verantwortlich sind. Es wurde gezeigt, dass Casc15 Tbr2, einen neurogenen Transkriptionsfaktor, auf Protein- aber nicht mRNA-Level verringert. Außerdem wurde mittels einer Serie von bioinformatischen Programmen herausgefunden, dass Casc15 eine differentielle Gen-Isoform Benutzung im entwickelnden Gehirn verursacht, was eine Interaktion von Casc15 mit Spleißfaktoren suggeriert. Die Effekte von Casc15 auf Gen- oder Transkript-Expression kann nicht völlig erklärt werden durch Casc15’s Rolle in Neurogenese. Besonders sein Effekt auf Proteintranslation und –stabilität muss adressiert werden. Alles in allem zeigen meine Daten, wenn auch mechanistisch nicht sehr eindeutig, dass Casc15 ein wichtiger Regulator in der Gehirnentwicklung ist. Weiterführende Experimente sind nötig, um die molekularen Aspekte der Casc15 Funktionen zu erörtern.:Introduction 1 1.1 Development of the mammalian neocortex 2 1.2 Neurogenesis in the neocortex 4 1.2.1 Neural stem cells 4 1.2.2 Transient amplifying cells 7 1.2.3 Neurogenesis 9 1.3 Molecular control of neurogenesis in neocortex 11 1.3.1 Signaling pathways influencing the onset and progression of neurogenesis 12 1.3.2 Transcriptional control of neurogenesis 13 1.3.3 Epigenetics, Post-­‐translational modifications and more 15 1.4 Long non-­‐coding RNAs 16 1.4.1 General characteristics of lncRNAs 17 1.4.2 Versatile mechanisms of lncRNAs 20 1.4.3 Expression patterns of lncRNAs 21 1.4.4 lncRNAs in neurogenesis 23 1.5 Btg2RFP/Tubb3GFP mouse line to study cortical development 25 1.6 Aim of the study 28 2 Materials and Methods 29 2.1 Materials 30 2.1.1 Chemicals, buffers and culture media 30 2.1.2 Antibodies 31 2.1.3 Primers 32 2.1.4 Mouse strains 34 2.1.5 Bacterial Strains 34 2.1.6 Vectors 34 2.1.7 Kits and enzymes 35 2.2 Methods 35 2.2.1 Generation of plasmid 35 2.2.2 In utero electroporation 35 2.2.3 Mouse sample collection and treatment 36 2.2.4 Immunohistochemistry 36 2.2.5 Image acquisition and processing 37 2.2.6 Reverse transcription 37 2.2.7 Library preparation and supplemental bioinformatic analyses 37 2.2.8 Quantitative-­‐Reverse Transcriptase-­‐PCRs 39 2.2.9 Bioinformatic analysis 39 2.2.10 Statistical analysis 40 3 Results 41 3.1 Selection of potential regulators of neurogenesis 42 3.1.1 Differential expression analysis for RNA seq data 42 3.1.2 LincRNAs for in vivo manipulation 44 3.2 In vivo manipulation of K13, K10 and Casc15 48 3.2.1 K13 overexpression does not alter progenitors/neurons distribution in the cortex 49 3.2.2 K10 might affect migration of neurons in the developing cortex 50 3.2.3 Casc15 disrupted the distribution of cells across the four cortical layers 50 3.3 Characterization of the cellular phenotype of Casc15 52 3.3.1 Casc15 delays neuronal migration 52 3.3.2 Casc15 does not alter progenitors migration 54 3.3.3 Casc15 does not induce direct neurogenesis 54 3.3.4 Casc15 causes subtle changes on cell distribution after 24 hours 56 3.3.5 Effect of Casc16 on progenitors fate 58 3.4 Molecular effects of Casc15 61 3.4.1 Casc15 minimally changes gene expression in the developing cortex 61 3.4.2 Casc15 changes gene exon usage 64 4 Discussion 68 4.1 Casc15 is a potential regulator of neurogenesis 69 4.1.1 Casc15 induces proliferation of progenitors in the developing cortex 70 4.1.2 Casc15 delays neuronal migration in the developing cortex 71 4.2 Molecular aspects of Casc15 in neurogenesis 72 4.2.1 Casc15 roles in neurogenesis cannot be explained in light of changes in gene expression 73 4.2.2 Casc15 changes the transcriptome at an isoform level 76 4.3 Concluding remarks 78 5 Appendix 79 6 Bibliography 87

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Mercury (Hg) is a ubiquitous environmental contaminant, which is capable of global atmospheric transport. As a result, even the most pristine aquatic ecosystems are affected by atmospheric Hg deposition, following which microbial transformation yield organic Hg forms, the most concerning of which is methylmercury (MeHg). Methylmercury is capable of bioaccumulation and biomagnification in food webs, resulting in potentially toxic body burdens due to regular dietary exposure in long-lived organisms at higher trophic levels. It is also a molecular mimic of some endogenous amino acids, providing a route of transfer from mother to offspring via large amino acid transporters. Exposure during neurodevelopment can lead to serious, irreversible neurological dysfunction, associated with a variety of cognitive and motor abnormalities across species. The present studies evaluate the effects of maternally-transferred dietary MeHg, at environmentally relevant concentrations on early life stage fathead minnows (Pimephales promelas). Embryos were collected from adult fatheads exposed to one of three diets with varying concentrations of MeHg for 30 days. Adult reproductive metrics were also monitored over the course of the study, with results indicating no effects on spawning frequency, clutch size, or total egg output. In embryos, Hg concentration was a function of female diet and the duration (number of days) of female exposure. Offspring spawned in tanks administered the low Hg diet displayed altered embryonic movement patterns (hyperactivity), decreased time to hatch, decreased mean larval size, and alterations to several metabolite abundances when compared with controls. Significantly altered metabolites include those associated with cellular energetics, fatty acid metabolism, and polyamine synthesis, indicating current environmental exposure scenarios are sufficient to disrupt important cellular pathways. Dysregulation of the dopaminergic system of embryos is also characterized, and may be a possible mechanism by which hyperactive behaviors are observed in these embryos. Offspring from tanks administered the high Hg diet exhibited delayed hatching, increased mortality, and physiological abnormalities. Brain tissue of exposed adults from the low diet were dissected into regions, and also evaluated for alterations in dopamine cycling. Collectively, these results indicate current exposure scenarios in North American lakes and rivers are sufficient to cause reductions in fitness and survival of early life stage fish. The potential for community structure impacts exists, as sensitive individuals and species become disproportionately affected by chronic, low-level MeHg exposure.

Neurodevelopmental risk factors have assumed a critical role in prevailing notions concerning the etiopathogenesis of neuropsychiatric disorders. Staging, diagnostic elements at which phase of disease is determined, provides a means of conceptualizing the degree and extent of factors affecting brain development trajectories, but is concurrently specified through the particular interactions of genes and environment unique to each individual case. For present purposes, staging perspectives in neurodevelopmental aspects of the disease processes are considered from conditions giving rise to neurodevelopmental staging in affective states, adolescence, dopamine disease states, and autism spectrum disorders. Three major aspects influencing the eventual course of individual developmental trajectories appear to possess an essential determinant influence upon outcome: (i) the type of agent that interferes with brain development, whether chemical, immune system activating or absent (anoxia/hypoxia), (ii) the phase of brain development at which the agent exerts disruption, whether prenatal, postnatal, or adolescent, and (iii) the age of expression of structural and functional abnormalities. Clinical staging may be assumed at any or each developmental phase. The present perspective offers both a challenge to bring further order to diagnosis, intervention, and prognosis and a statement regarding the extreme complexities and interwoven intricacies of epigenetic factors, biomarkers, and neurobehavioral entities that aggravate currents notions of the neuropsychiatric disorders.

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Due to their underlying genetic complexity, chromosomal disorders such as Down syndrome (DS), which is caused by trisomy 21, have long been understudied and continue to lack effective treatments. With over 200 genes on the extra chromosome, even the specific cell pathologies and pathways impacted in DS are not known, and it has not been considered a viable target for the burgeoning field of gene therapy. Recently, our lab demonstrated that the natural mechanism of dosage compensation can be harnessed to silence the trisomic chromosome in pluripotent cells. Using an inducible XIST transgene allows us to study the effects of trisomy in a tightly controlled system by comparing the same cells with either two or three active copies of chromosome 21. In addition, it raises the prospect that insertion of a single gene into a trisomic chromosome could potentially be developed in the future for “chromosome therapy”. This thesis aims to utilize this inducible system for dosage compensation to study the neurodevelopmental effects of trisomy 21 in vitro, and to answer basic epigenetic questions critical to the viability of chromosome silencing as a therapeutic approach. Foremost, for XIST to have any prospect as a therapeutic, and to strengthen its experimental utility, it must be able to initiate chromosome silencing beyond its natural context of pluripotency. Here I demonstrate that, contrary to the current literature, XIST is capable of initiating chromosome silencing in differentiated cells and producing fully dosage compensated DS neurons. Additionally, I show that silencing of the trisomic chromosome in neural stem cells enhances their terminal differentiation to neurons, and transcriptome analysis provides evidence of a specific pathway involved. Separate experiments utilize novel three-dimensional organoid technology and transcriptome analysis to model DS neurodevelopment in relation to isogenic euploid cells. Overall, this work demonstrates that dosage compensation provides a powerful experimental tool to examine early DS neurodevelopment, and establishes that XIST function does not require pluripotency, thereby overcoming a perceived obstacle to the potential of XIST as a therapeutic strategy for trisomy.

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