Doctor of Philosophy

dissertationThe nervous system is comprised of an estimated 100 billion individual neurons, which are connected to one another to form a network that senses environmental stimuli and coordinates the organism's behavior. Because of the complexity of the nervous system, deciphering the developmental processes and adult wiring diagram has proved challenging. A number of axon guidance molecules have been identified; however, the means by which they guide billions of axons to their target cells in vivo remains poorly understood. Several axon guidance molecules have been found to be bifunctional, meaning they can elicit different growth cone responses depending on the presence or absence of other molecules, such as growth cone receptors, intracellular signal transduction molecules, or extracellular modulators. Axon sorting within axon tracts is perhaps a means by which axons are presorted to make a precise connection on their target cells. The zebrafish, Danio rerio, is an ideal model organism to study vertebrate axon guidance and axon sorting due to its external fertilization, optical transparency, amenability to forward genetics, and ease of making transgenic lines. In order to study axon guidance within the zebrafish retinotectal system, I developed a new method of misexpressing genes. Local misexpression can be induced by using a modified soldering iron in transgenic zebrafish in which a gene of interest is driven by a heat shock promoter. This method allowed me to examine the mechanisms by which Slit1a and Slit2 guide axons from the retina to the optic tectum. I determined the expression pattern of Slits in the zebrafish and used antisense morpholino technology to knock down Slit1a. The iv resultant axon guidance errors indicated that Slit1a acts to guide retinal axons through the optic tract. I then misexpressed Slit1a and Slit2 near the optic tract to observe their effect on axons. I found that both proteins appeared to attract retinal axons. Additionally, I saw that Slit2 seems to attract retinal axons earlier in the retinotectal pathway, at the optic chiasm. I also report on a new method, to whose development I contributed, for automated tracking of axons through electron microscopy datasets. Taken together, my results add new methods to the endeavor of mapping neural connectivity and development, and suggest a new role for Slits in axon guidance

Arx acts as a regional key selector gene in the ventral telencephalon mainly through its transcriptional repression activity

AbstractThe homeobox-containing gene Arx is expressed during ventral telencephalon development and required for correct GABAergic interneuron tangential migration from the ganglionic eminences to the olfactory bulbs, cerebral cortex and striatum. Its human ortholog is associated with a variety of neurological clinical manifestations whose symptoms are compatible with the loss of cortical interneurons and altered basal ganglia-related activities. Herein, we report the identification of a number of genes whose expression is consistently altered in Arx mutant ganglionic eminences. Our analyses revealed a striking ectopic expression in the ganglionic eminences of several of these genes normally at most marginally expressed in the ventral telencephalon. Among them, Ebf3 was functionally analyzed. Thus, its ectopic expression in ventral telencephalon was found to prevent neuronal tangential migration. Further, we showed that Arx is sufficient to repress Ebf3 endogenous expression and that its silencing in Arx mutant tissues partially rescues tangential cell movement. Together, these data provide new insights into the molecular pathways regulated by Arx during telencephalon development

Cadherin-Dependent Cell Morphology in an Epithelium: Constructing a Quantitative Dynamical Model

Cells in the Drosophila retina have well-defined morphologies that are attained during tissue morphogenesis. We present a computer simulation of the epithelial tissue in which the global interfacial energy between cells is minimized. Experimental data for both normal cells and mutant cells either lacking or misexpressing the adhesion protein N-cadherin can be explained by a simple model incorporating salient features of morphogenesis that include the timing of N-cadherin expression in cells and its temporal relationship to the remodeling of cell-cell contacts. The simulations reproduce the geometries of wild-type and mutant cells, distinguish features of cadherin dynamics, and emphasize the importance of adhesion protein biogenesis and its timing with respect to cell remodeling. The simulations also indicate that N-cadherin protein is recycled from inactive interfaces to active interfaces, thereby modulating adhesion strengths between cells

Modelling the emergence of whisker barrels

Brain development relies on an interplay between genetic specification and self-organization. Striking examples of this relationship can be found in the somatosensory brainstem, thalamus, and cortex of rats and mice, where the arrangement of the facial whiskers is preserved in the arrangement of cell aggregates to form precise somatotopic maps. We show in simulation how realistic whisker maps can self-organize, by assuming that information is exchanged between adjacent cells only, under the guidance of gene expression gradients. The resulting model provides a simple account of how patterns of gene expression can constrain spontaneous pattern formation to faithfully reproduce functional maps in subsequent brain structures

Roles of Presynaptic NMDA Receptors in Neurotransmission and Plasticity

Presynaptic NMDA receptors (preNMDARs) play pivotal roles in excitatory neurotransmission and synaptic plasticity. They facilitate presynaptic neurotransmitter release and modulate mechanisms controlling synaptic maturation and plasticity during formative periods of brain development. There is an increasing understanding of the roles of preNMDARs in experience-dependent synaptic and circuit-specific computation. In this review, we summarize the latest understanding of compartment-specific expression and function of preNMDARs, and how they contribute to synapse-specific and circuit-level information processing

Developmental Determinants of Neuronal Identity in the Drosophila Embryo

The complex function of the nervous system is dependent on precise connectionsbetween hundreds of thousands of diverse neurons. During development, a small pool of neural progenitors is tasked with quickly generating this diverse set of molecularly and morphologically distinct neuronal subtypes. These neurons are then required to navigate a complex environment to locate the appropriate synaptic partners, and establish the circuitry required for behavior. For this reason, identifying the mechanisms used by neural progenitors to generate the correct neural subtypes is critical to understanding circuit formation, and behavior itself. During Drosophila development, each neural progenitor cell, or neuroblast (NB), generates a characteristic set of diverse neuronal progeny over time. This is accomplished through the process of temporal patterning, in which each NB sequentially expresses a cascade of temporal transcription factors (tTFs), giving rise to molecularly distinct neuronal progeny in each expression window. These tTFs are only transiently expressed; little is known about their downstream effectors and how they specify and maintain the unique molecular and morphological properties of each neuronal subtype throughout larval life. Our central hypothesis, is that each tTF induces or represses a combinatorial set of downstream identity transcription factors (iTFs), which in turn drive the expression of mature neuronal genes such as those encoding neurotransmitter machinery, ion channels, cell-surface protein expression and higher-order morphological features. Investigating the downstream targets of tTFs in a distinct embryonic lineage through single-cell sequencing will resolve this gap in understanding. This dissertation includes previously published, co-authored material

Supracellular organization confers directionality and mechanical potency to migrating pairs of cardiopharyngeal progenitor cells

Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that multicellular arrangements confer biochemical and biomechanical properties contributing to tissue-scale organization. The Ciona cardiopharyngeal progenitors provide the simplest model of collective cell migration, with cohesive bilateral cell pairs polarized along the leader-trailer migration path while moving between the ventral epidermis and trunk endoderm. We use the Cellular Potts Model to computationally probe the distributions of forces consistent with shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we identify cardiopharyngeal progenitors as the simplest cell collective maintaining supracellular polarity with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy to align collective polarity with the direction of migration, as observed with three or more cells in silico and in vivo. Our approach reveals emerging properties of the migrating collective: cell pairs are more persistent, migrating longer distances, and presumably with higher accuracy. Simulations suggest that cell pairs can overcome mechanical resistance of the trunk endoderm more effectively when they are polarized collectively. We propose that polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis.publishedVersio

Mechanisms of Immune Activation and Suppression by Parasitic Wasps of \u3cem\u3eDrosophila\u3c/em\u3e

Drosophila melanogaster has served as an excellent model organism to study the molecular processes of innate immunity. Flies essentially lack adaptive immunity and the innate immune system is often divided into the humoral and cellular responses (Lemaitre and Hoffmann 2007). The humoral arm involves the production of antimicrobial peptides, secreted from the fat body, to combat bacterial and fungal infections. The cellular response involves the production of hemocytes (blood cells: crystal cells, plasmatocytes, and lamellocytes) in the larval lymph gland, in the sessile pools, and in circulation (Gold and Bruckner 2014). Microbial pathogens are phagocytosed by plasmatocytes whereas larger parasites such as parasitic wasp eggs are neutralized by egg encapsulation, principally by lamellocytes. The innate immune response is vital for survival against the abundant pathogens and parasites in their natural habitats. The range of microbial as well as Hymenoptera species that attack their Diptera hosts is vast. These pathogens and parasitoids have evolved strategies to either evade or suppress host immune responses (Keebaugh 2013). This thesis contains two chapters. In Chapter 1, we focused on the mechanisms underlying host defense in response to specialist wasps of D. melanogaster, Leptopilina boulardi. Chapter 1 is already published (Small et al. 2014) and I shared first authorship with Dr. Small. Previous experiments demonstrated that Notch (N) signaling is essential for crystal cell specification and differentiation (Duvic et al. 2002; Lebestky et al. 2003), and also promotes lamellocyte differentiation (Duvic et al. 2002). The N ligand, Serrate is expressed in the posterior signaling center (PSC), a non-hematopoietic cell population, also called the niche. Through direct contact, the PSC activates N signaling in the developing hematopoietic cells and instructs them to become crystal cells (Lebestky et al. 2003). L. boulardi infection promotes lamellocyte but inhibits crystal cell differentiation (Krzemien et al. 2010). ROS production is also activated in the PSC upon wasp infection (Sinenko et al. 2011). In Chapter 1, we demonstrate a second function for N signaling: L. boulardi parasitization inactivates N signaling in the developing lymph gland lobes; reduction of N signaling correlates with lamellocyte differentiation. We also demonstrate an unexpected link between N signaling and ROS in restricting differentiation of hematopoietic progenitors (Small et al. 2014). In chapter 2, we focused on strategies that the generalist parasitic wasp L. heterotoma employs to actively suppress the hosts’ immune responses. Building on previous work that showed that L. boulardi infection activates NF-κB signaling in the PSC (Gueguen et al. 2013), we examined changes in the PSC and hematopoietic progenitors after L. heterotoma infection and found reduction in gene expression in the PSC, presence of VLPs around (but not within) PSC cells, and significant reduction in the progenitor population. Consistent with previous results (Chiu 2002), this reduction correlates with Caspase activation in plasmatocytes and lysis of lamellocytes, within the lymph gland and circulating hemocyte populations. These responses are mediated by virus-like particles (VLPs) produced in the L. heterotoma venom. L. heterotoma VLPs have 4-8 spikes and the spike-to-spike distance is roughly 300 nm (Rizki and Rizki 1990). A mouse polyclonal antibody against VLPs was generated previously in our lab and immuno-electron microscopy (EM) experiments localized this protein’s origin to secretory cells of the venom gland (Chiu et al. 2006). The p40 protein is also present in large amounts in the lumen of the venom gland where VLPs undergo biogenesis and assembly (Morales et al. 2005)(Chiu et al. 2006). VLPs are ultimately deposited into the host hemocoel during the egg laying process (Chiu et al. 2006). Immuno-EM of purified mature VLPs localizes p40 to the VLP spike surface and spike termini. p40 is also present in plasmatocytes and lamellocytes of host cells. Proteomic analyses of L. heterotoma VLPs reveal more than 150 proteins, some of which are not expressed in L. boulardi (Govind lab, unpub. results). In Chapter 2, we show (1) differential effects of L. boulardi (lamellocyte differentiation, activation of gene expression in the PSC) and L. heterotoma (cell death, repression of gene expression in PSC) on lymph gland homeostasis; (2) the subcellular localization of p40 (punctate and vesicular in plasmatocytes, nuclear in lamellocytes); (3) Rab5-dependent entry into plasmatocytes but not in lamellocytes; (4) an immune function for the PSC. Molecular characterization of p40 revealed a protein with signal sequence, a central helical domain, and C-terminal transmembrane domain. The central helical domain share structural similarity with proteins of the SipD/IpaD family, normally present on tips of Gram negative bacterial type three secretion system needles. Incubation of bacterial extracts with live lamellocytes resulted in alteration in cell morphology. We hypothesize a direct role for p40 in mediating VLP entry into lamellocytes. These studies constitute the first detailed investigation of any VLP protein and begin to uncover mechanisms of active immune suppression by VLPs. They also contribute to our understanding of the biotic nature of VLPs

Elucidating regulators and biomarkers of synaptic stability during neurodegeneration

Synapses are an early pathological target in a wide range of neurodegenerative conditions including adult-onset Alzheimer’s and Parkinson’s, and diseases of childhood such as spinal muscular atrophy and neuronal ceroid lipofuscinoses (NCLs). However, our understanding of the mechanisms regulating the stability of synapses and their exceptional vulnerability to neurodegenerative stimuli remains in its infancy. To address this, we have used the NCLs to model the molecular alterations underpinning synaptic vulnerability. Our primary objective is to identify novel regulators of synaptic stability as well as highlight novel therapeutic targets which may prove effective across multiple neurodegenerative conditions where synapses are an early pathological target. The NCLs, are the most frequent autosomal-recessive disease of childhood. There are currently 14 individual genes whose mutations result in similar phenotypes including blindness, cognitive/motor deficits, seizures and premature death. This suggests that despite the difference in the initiating mutation and the degenerative processes across this collective group are likely to impact on overlapping pathways. Focusing on two murine models of NCL; one with an infantile onset - CLN1 disease (Ppt1-/-) and one with a juvenile onset - CLN3 disease (Cln3-/-) we made use of the temporo-spatial synaptic vulnerability pattern in these mice to plan proteomic and in silico analyses. This pipeline was utilised to identify perturbed protein candidates and pathways correlating with differential regional synaptic vulnerability. This ultimately allowed the generation of a list of candidate proteins, some of which were relevant to human NCL as they were altered in post mortem brain samples. Interestingly, many of the correlative candidates also appear to show conserved alterations in both NCL forms examined and other neurodegenerative diseases. Next, candidates were genetically and/or pharmacologically targeted to study their modulatory effects on neuronal stability in vivo. This was done using CLN3 Drosophila as a rapid screening assay and led to the successful characterisation of a subset of candidates as either enhancers or suppressors of the CLN3-induced phenotype in vivo. As well as identifying regulators of neuronal stability, following a similar pipeline, we identified a set of putative biomarkers of disease progression in muscle and blood in the Ppt1- /- mice, a subset of which appeared conserved in Cln3-/- mice. One of these conserved candidates presented the same directionality of change in human post mortem brain samples, indicating its relevance to the human NCL. Following this workflow from spatio-temporal profiling of murine synaptic populations, to in silico analyses and in vivo phenotypic assessment, we demonstrate that we can identify multiple protein candidates capable of modulating neuronal stability in vivo and identified putative biomarkers that tracked disease progression