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Light regulates many biological processes through light-sensitive proteins called opsins. Opsins are involved in vision, but they are also expressed in extraretinal tissue, where their roles are far less clear. Fish have large opsin repertoires, derived from a long history of gene duplication and divergence, making them useful models to study opsin diversity and function. I introduce the deep-sea sablefish (Anoplopoma fimbria) as a model for opsin research for three main reasons: i) the availability of a draft genome and transcriptome, simplifying the characterization of this species’ opsin repertoire, ii) the proximity of the only sablefish aquaculture facility in the world, providing exclusive access to a large number of individuals at all developmental stages, iii) the observation that sablefish occupy very different light environments during the course of development, ranging from well-lit shallow waters to the aphotic zone, which provides a light environment context for opsin gene expression data. My survey of the genome showed that sablefish have 36 distinct opsin genes (7 visual and 29 non-visual), even though they spend most of their lives in the dark. The sablefish opsin sequences and repertoire are similar to those of other teleost fish. To test the hypothesis that the sablefish opsin repertoire is being expressed/transcribed during the comparatively brief period of time when this species is exposed to light (the free-swimming larval stage through to the juvenile stage), I quantified the expression of five paralogous genes from a well-studied non-visual opsin family (OPN4’s) in the brain across life stages. Data show statistically stable expression of Opn4m1 and Opn4m3 among life stages, a rough association of Opn4x1 and Opn4m2 expression with age and light environment, and little-to-no expression of Opn4x2. I localized proteins encoded by the most highly expressed class of OPN4 genes in the brain, the Opn4m genes, to the surface of the optic tectum just below a cranial ‘window’; a zone that has been shown to express dozens of opsins in zebrafish (a distant relative, with their ancestor diverging more than 230 million years ago). Thus, in some cases, expression appears to be correlated with light exposure not only temporally, but also spatially. By studying non-visual opsins in sablefish, I have challenged and broadened the current understanding of opsin evolution and function in fish and provided the foundation for future studies to test brain regions for light-sensitivity, perform opsin gene knock-outs, and explore potential light-independent processes. / Graduate / 2023-09-06

The Hippo signaling pathway was first discovered in Drosophila melanogaster and is involved in organ size control by regulating cell proliferation and apoptosis. This well conserved pathway is activated by various signal inputs, including cell-cell contact, mechanotransduction, and G-protein coupled receptors, with signals converging on the downstream effector protein Yap and its homologue Taz, which are transcriptional co-activators. When the Hippo pathway is activated, Yap/Taz are phosphorylated, leading to cytoplasmic retention and degradation, and diminishing their transcriptional activity. Yap has also been recently implicated as a potential oncogene, as it is upregulated and transcriptionally active in several tumor types. Furthermore, inhibiting Yap activity in various cancer models has been shown to revert cancer cells to a normal phenotype. Although the role of Yap has been described in several organ systems, there is a paucity of information about the function of Yap in the central nervous system. I investigated the function of Yap/Taz in the murine cerebellum to determine its significance during normal development and a potential role for Yap/Taz in medulloblastoma, a tumor that arises in the cerebellum. In Chapter 2, I describe the expression pattern of Yap from embryonic through adult stages in mice, and demonstrate the functional significance of Yap/Taz in different cell populations using conditional knockout mouse models. I show that Yap plays a significant role in cell fate determination as well as in cerebellar foliation: Yap is highly expressed in the ventricular zone and is required for the proper formation of ependymal cells, and is also strongly expressed in Bergmann glia (BG) during early developmental stages, where Yap, together with Taz, plays a significant role in cerebellar foliation. Furthermore, Yap/Taz-deficient BG exhibit migrational defects, as their cell bodies can be found mislocalized to the molecular layer (ML), rather than remaining tightly associated with Purkinje Cells (PCs) in the PC layer. BG support the health of PCs, and severely defective BG positioning eventually leads to a loss of PCs. However, although Yap is highly expressed in granule neuron progenitors (GNPs) during the rapid postnatal expansion stage, it does not appear to play a major role in proliferation of these cells as conditionally knocking-out Yap/Taz in GNPs does not alter their proliferative capacity. Our observations demonstrate that in the cerebellum, Yap has a novel function in glia that is required for the development of normal foliation and organization, but plays a minimal role in GNP proliferation. Importantly, I also show that the reduction of sphingosine-1-phosphate G-protein-coupled receptor (S1P1) signal transduction activates the upstream kinase Lats with concomitant increases of phosphorylated Yap as well as a reduction of the known Yap target connective tissue growth factor (CTGF). This study identifies a novel function of Yap/Taz in cerebellar glia that is required for the development of normal foliation and laminar organization with sphingosine-1-phosphate (S1P) signaling as a potential extracellular cue regulating Yap activity during cerebellar development. In Chapter 3, I present further support for the finding that Yap/Taz are not required for GNP proliferation in vivo by discussing the failure of Yap/Taz loss to rescue the Sonic-hedgehog (Shh) mediated medulloblastoma phenotype, in which GNPs are considered to be the tumor cell of origin. Furthermore, I provide evidence suggestive of a tumor suppressive function of Yap/Taz in the cerebellum. Together, previously unknown functions of Yap in the developing and malignant cerebellum are described, providing a foundation for future studies of Yap in the central nervous system (CNS). / Biomedical Sciences

When it comes to cell types, the cerebral cortex is one of the most diverse regions in the mammalian brain. Mouse cortical neurons are generated during development from radial glial cells (RGCs). But how these stem cells generate the different neuronal subtypes is still an open question. In the adult, transcription factors, specially homeobox genes, have been identified as determinants of neuronal types throughout the animal kingdom. Thus, in this study, we hypothesise that different subpopulations of neuronal progenitors (RGCs) give rise to subsequent subtypes of neurons in the cortex, and these populations can be defined by homeobox gene expression. Starting from a scRNA- seq analysis, we identified differentially expressed genes across different progenitor populations in the developing cortex: Adnp2, Homez and Hmbox1. We characterised their mRNA and protein expression across cortical layers in postnatal mice and found that these genes are also differentially expressed among layers. We also find discordances between scRNA-seq data, mRNA expression, and protein expression data that could indicate specific post-transcriptional regulation of these genes. Altogether, these results point to a role of homeobox genes in neuronal identity.

Metabolic coupling between glial cells and neurons is essential for neuronal function. It is a well-conserved and vital feature of the bilaterian nervous system as well. Under normal and adverse conditions, glial cells act as a major metabolic hub fueling neuronal oxidative metabolism by producing lactate or ketone bodies. I now ask the question of whether such metabolic coupling is only necessary for preserving brain homeostasis or if it could also have implications in decision making such as food choice behavior. Choosing an appropriate food source is key for the survival of an organism. Carbohydrates are the preferred source of energy and thus evaluation of their nutritive content is essential. Several studies have demonstrated that Drosophila melanogaster larvae and adults, as mammals, can distinguish between nutritious and non-nutritious carbohydrates independent of their taste. Two groups of neurons, Diuretic Hormone 44 (Dh44)-expressing neurons and gustatory receptor 43a (Gr43a)-expressing neurons, have been implicated in postprandial sugar sensing in adult flies. Gr43a- expressing neurons are narrowly fine-tuned for sensing fructose in both adults and larvae. Nonetheless, in the larva, central nervous system (CNS) Gr43a neurons have been shown to act as the main sugar sensor. This raises the question of how CNS fructose-sensing neurons are involved in sensing non-fructose dietary sugars. To decipher this post-ingestive mechanism, I used frustrated total internal reflection (FTIR) - based larval tracking to investigate larval food choice behavior. The results present in this thesis suggest that besides Gr43a-expressing neurons, glial cells are indispensable for sensing nutritive non-fructose dietary sugars such as glucose and sorbitol. I show that post-ingestive carbohydrate sensing involves carbohydrate conversion into fructose locally in the glial cells. Glia-derived fructose then enables Gr43a-dependent postprandial carbohydrate sensing in the CNS and drives carbohydrate preferences with the help of the downstream signalling peptide corazonin. Thus, in post-ingestive carbohydrate sensing, the glial cells act as a master metabolite sensor and provide a fructose stimulus to neurons to regulate behavior.:Table of Contents Abstract 1 Zusammenfassung 2 1. Introduction 3 1.1. External Nutrient Sensing in mammals and Drosophila melanogaster 4 1.1.1. Taste detection in mammals 4 1.1.2. Taste detection in Drosophila melanogaster 6 1.1.3 Sweet and Umami Taste in mammals 8 1.1.4. Sweet Sensing in Drosophila melanogaster 8 1.1.5. Bitter Taste in mammals 9 1.1.6. Bitter Sensing in Drosophila melanogaster 9 1.1.7. Sour, Carbonation, Fatty acid and Salty taste in mammals 9 1.1.8. Amino acid, Fatty acid and Salt Sensing in Drosophila melanogaster 10 1.2 Post-ingestive nutrient sensing in mammals and Drosophila melanogaster 11 1.2.1. Post-ingestive amino acid sensing in mammals 11 1.2.2. Post-ingestive amino acid sensing in Drosophila melanogaster 12 1.2.3. Post-ingestive lipid sensing in mammals 13 1.2.4. Post-ingestive carbohydrate sensing in mammals 13 1.2.4.1. The role of the nervous system in post-ingestive carbohydrate sensing mammals 16 1.2.5. Post-ingestive carbohydrate sensing in Drosophila melanogaster 16 1.2.5.1. The role of the nervous system in post-ingestive carbohydrate sensing in flies 18 1.2.5.2. The role of the nervous system in post-ingestive carbohydrate sensing in larvae 19 1.3 The cellular architecture of the larval nervous system 20 1.3.1 The Blood-brain barrier (BBB) and carbohydrate transport 23 1.4 Aim of study 25 2. Experimental Procedures 26 2.1 Materials 26 2.1.1 Chemicals 26 2.1.2 Media 27 2.1.3 Buffer and Solution 27 2.1.4 Antibodies 28 2.1.5 Flystocks 29 2.2 Methods 33 2.2.1 Fly genetics 33 2.2.1.1 Maintenance and crosses 33 2.2.2 Immunohistochemistry & Microscopy 33 2.2.2.1 Immunohistochemistry of larval filets 33 2.2.2.2 Confocal Microscopy 33 2.3 Experimental Design 34 2.3.1 Studying food choice in Drosophila larvae 34 2.3.1.1 Concept 34 2.3.1.2 Experimental setup 36 2.3.1.3 Analysis 36 2.3.1.4 MATLAB Script 38 3. Results 40 3.1. Frustrated total internal reflection based two-choice assay (TCA) 40 3.1.1 Investigating larval sugar preference 40 3.2 The role of diuretic hormone 44 (Dh44) neurons in post-ingestive glucose sensing in third instar larvae 47 3.2.1 Immunohistochemical analysis of Dh44 in third instar larval brains 47 3.2.2 Role of Dh44 in glucose sensing 49 3.3 The role of gustatory receptor 43a (Gr43a) neurons in post-ingestive nutritive carbohydrate sensing in third instar larvae 51 3.3.1 Immunohistochemical analysis of Gr43a in third instar larval brains 51 3.4. Investigating the role of polyol pathway in post-ingestive carbohydrate sensing 56 3.4.1 Identifying the key polyol pathway enzyme in Drosophila melanogaster 56 3.4.2 Examining RNAi mediated neuronal and glial knockdown of polyol pathway enzymes in postprandial sugar sensing 57 3.4.2.1 CG9436 57 3.4.2.2 CG6084 and CG10863 60 3.4.2.3 Sodh-2 66 3.5 Determining the glia subtypes vital for conducting polyol pathway 69 3.6 The role of the blood brain barrier (BBB) in post-ingestive carbohydrate sensing 74 4. Discussion 80 4.1. Gr43a is the only sugar sensor in Drosophila larvae 80 4.2. Polyol Pathway is crucial for glucose and sorbitol sensing 82 4.3. Glia, the master metabolic sensor 84 4 4. Transport over BBB: a prerequisite for postprandial carbohydrate sensing85 4.5. Proposed model for larval post-ingestive nutritive carbohydrate sensing... 86 5. References 89 6. Abbreviation List 100 7. Appendix 103

Yes / Expression of the transmembrane NG2 chondroitin sulphate proteoglycan (CSPG) defines a distinct population of NG2-glia. NG2-glia serve as a regenerative pool of oligodendrocyte progenitor cells in the adult central nervous system (CNS), which is important for demyelinating diseases such as multiple sclerosis, and are a major component of the glial scar that inhibits axon regeneration after CNS injury. In addition, NG2-glia form unique neuron–glial synapses with unresolved functions. However, to date it has proven difficult to study the importance of NG2-glia in any of these functions using conventional transgenic NG2 ‘knockout’ mice. To overcome this, we aimed to determine whether NG2-glia can be targeted using an immunotoxin approach. We demonstrate that incubation in primary anti-NG2 antibody in combination with secondary saporin-conjugated antibody selectively kills NG2-expressing cells in vitro. In addition, we provide evidence that the same protocol induces the loss of NG2-glia without affecting astrocyte or neuronal numbers in cerebellar brain slices from postnatal mice. This study shows that targeting the NG2 CSPG with immunotoxins is an effective and selective means for killing NG2-glia, which has important implications for studying the functions of these enigmatic cells both in the normal CNS, and in demyelination and degeneration.

Yes / Protocols which permit the extraction of primary astrocytes from either embryonic or postnatal mice are well established however astrocytes in culture are different to those in the mature CNS. Three dimensional (3D) cultures, using a variety of scaffolds may enable better phenotypic properties to be developed in culture. We present data from embryonic (E15) and postnatal (P4) murine primary cortical astrocytes grown on coated coverslips or a 3D polystyrene scaffold, Alvetex. Growth of both embryonic and postnatal primary astrocytes in the 3D scaffold changed astrocyte morphology to a mature, protoplasmic phenotype. Embryonic-derived astrocytes in 3D expressed markers of mature astrocytes, namely the glutamate transporter GLT-1 with low levels of the chondroitin sulphate proteoglycans, NG2 and SMC3. Embroynic astrocytes derived in 3D show lower levels of markers of reactive astrocytes, namely GFAP and mRNA levels of LCN2, PTX3, Serpina3n and Cx43. Postnatal-derived astrocytes show few protein changes between 2D and 3D conditions. Our data shows that Alvetex is a suitable scaffold for growth of astrocytes, and with appropriate choice of cells allows the maintenance of astrocytes with the properties of mature cells and a non-reactive phenotype. / BBSRC