The tilapias are a group of African and Middle Eastern cichlid fish that are widely cultured in developed and developing countries. With many different species and sub-species, and extensive use of interspecies hybrids, identification of tilapia species is of importance in aquaculture and in wild populations where introductions occur. This research set out to distinguish between tilapia species and sub-species by retrieving species-specific nuclear DNA markers (SNPs) using two approaches: (i) sequencing of the coding regions of the ADA gene; and (ii) next-generation sequencing, both standard RADseq and double-digest RADseq (ddRADseq). The mitochondrial DNA (mtDNA) marker cytochrome c oxidase subunit I (COI) was used to verify tilapia species status. ADA gene sequence analysis was partially successful, generating SNP markers that distinguished some species pairs. Most species could also be discriminated using the COI sequence. Reference based analysis (RBA: using only markers found in the O. niloticus genome sequence) of standard RADseq data identified 1,613 SNPs in 1,002 shared RAD loci among seven species. De novo based analysis (DBA: based on the entire data set) identified 1,358 SNPs in 825 loci and RBA detected 938 SNPs in 571 shared RAD loci from ddRADseq among 10 species. Phylogenetic trees based on shared SNP markers indicated similar patterns to most prior phylogenies based on other characteristics. The standard RADseq detected 677 species-specific SNP markers from the entire data set (seven species), while the ddRADseq retrieved 38 (among ten species). Furthermore, 37 such SNP markers were identified from ddRADseq data from a subset of four economically important species which are often involved in hybridization in aquaculture, and larger numbers of SNP markers distinguished between species pairs in this group. In summary, these SNPs are a valuable resource in further investigating hybridization and introgression in a range of captive and wild stocks of tilapias.

Understanding the complex mechanisms to assemble a functional brain demands sophisticated experimental designs. Drosophila melanogaster, a model organism equipped with powerful genetic tools and evolutionarily conserved developmental programs, is ideal for such mechanistic studies. Valuable insights were learned from research in Drosophila ventral nerve cord, such as spatial patterning, temporal coding, and lineage diversification. However, the blueprint of Drosophila cerebrum development remains largely unknown. Neural progenitor cells, called neuroblasts (NBs), serially and stereotypically produce neurons and glia in the Drosophila cerebrum. Neuroblasts inherit specific sets of early patterning genes, which likely determine their individual identities when neuroblasts delaminate from neuroectoderm. Unique neuroblasts may hence acquire the abilities to differentially interpret the temporal codes and deposit characteristic progeny lineages. We believe resolving this age-old speculation requires a tracing system that links patterning genes to neuroblasts and corresponding lineages, and further allows specific manipulations. Using modern transgenic systems, one can immortalize transient NB gene expressions into continual labeling of their offspring. Having a collection of knockin drivers that capture endogenous gene expression patterns would open the door for tracing specific NBs and their progenies based on the combinatorial expression of various early patterning genes. Anticipating the need for a high throughput gene targeting system, we created Golic+ (gene targeting during oogenesis with lethality inhibitor and CRISPR/Cas “plus”), which features efficient homologous recombination in cystoblasts and a lethality selection for easy targeting candidate recovery. Using Golic+, we successfully generated T2AGal4 knock-ins for 6 representative early patterning genes, including lab, unpg, hkb, vnd, ind, and msh. They faithfully recapitulated the expression patterns of the targeted genes. After preserving initial NB expressions by triggering irreversible genetic labeling, we revealed the lineages founded by the NBs expressing a particular early patterning gene. Identifying the neuroblasts and lineages that express a particular early patterning gene should elucidate the genetic origin of neuroblast diversity. We believe such an effort will lead to a deeper understanding of brain development and evolution.

Indiana University-Purdue University Indianapolis (IUPUI) / The young field of gene therapy offers the promises of significant progress towards the treatment of many different types of human diseases. Gene therapy has been proposed as an innovative way to treat Acute Kidney Injury (AKI). Through proteomic analysis, the upregulation of two enzymes, IDH2 and SULT1C2, within the mitochondrial fraction has been identified following ischemic preconditioning, a treatment by which rat kidneys are protected from ischemia. Using the hydrodynamic fluid gene delivery technique, we were able to upregulate the expression of IDH2 and SULT1C2 in the kidney. We found that the delivery of IDH2 plasmid through hydrodynamic fluid delivery to the kidney resulted in increased mitochondrial oxygen respiration compared with injured kidneys without gene delivery. We also found that renal ischemic preconditioning altered the membrane fluidity of mitochondria. In conclusion, our study supports the idea that upregulated expression of IDH2 in mitochondria can protect the kidney against AKI, while the protective function of upregulated SULT1C2 needs to be further studied.

Indiana University-Purdue University Indianapolis (IUPUI) / The Cre/loxP system is a tool for targeted recombination of DNA. For applying Cre recombinase-mediated genome modifications, there is a requirement for reliable, high-fidelity, and specific transgenic expression of the Cre recombinase. This study focuses on the reliability of different bone cell specific Cre models in the Cre/loxP system. In this study, DMP1-Cre transgenic mouse which has a transgene driven by DMP1 promotor that allows Cre-expression only in late stage osteoblasts and osteocytes was used. Ctsk-Cre mouse with a driven by Ctsk promoter was used so that only osteoclasts would undergo Cre-mediated recombination. E2A-Cre mouse where the Cre recombinase is driven by a global promoter E2A was also included in this study as a control line to test the Cre reporter line Ai9. Dmp1-Cre, Ctsk-Cre and E2A-Cre mice were crossed to the fluorescent Cre-reporter line—Ai9, which harbors a floxed stop codon, followed by the fluorophoremTomato, inserted into the Rosa26 locus. This construct is expected to give red fluorescence when it recombines with Cre-expressing mouse cells and no fluorescence in non-recombinant mouse cells. Double positive (Ai9+/Cre+) offspring selected by PCR were perfused, and 5mu-m thick section of bone and soft tissues were examined for red fluorescent expression. Cre positive cells were quantitated using ‘ImageJ’ software program. The DMP1-vi Cre mouse results showed significant expression in the targeted osteocytes and osteoblasts. In addition, skeletal muscle tissue also showed significant Cre- expression. Ctsk-Cre mice showed significant expression in targeted osteoclasts. But brain tissue was positive in Cre-expression. Bone-Cre mouse models are expected to express Cre recombinase only in their respective bone cells and they have been used for gene deletion studies in bone cells. However, this study has revealed that the bone cell specific Cre mouse models DMP1-Cre and Ctsk-Cre have unexpected expression in muscle and brain respectively. In order to use these models for targeted gene deletion in bone cells, further testing and studies have to be conducted.

<p>The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated.</p><p>This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.</p>

The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated. This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.

Im Rahmen dieser Arbeit wurde die Rolle des Rezeptors VEGFR-2, Flk-1, im neuronalen Kontext untersucht. In einem ersten Schritt wurde in embryonalen Stammzellen der Maus das fluoreszierende Protein eGFP unter der Kontrolle regulatorischer Sequenzen des flk-1-Promotors, -Enhancers exprimiert. Nach der Differenzierung zu Sphäroiden wurden Endothelzellen nachgewiesen, die sowohl eGFP als auch das zelltypspezifische Oberflächenantigen CD31 ausprägen. Ebenso wurden nach der neuronalen Differenzierung in Gegenwart von Stromazellen eGFP-exprimierende Zellen identifiziert. Diese standen mit Zellen, die das für neuronale Vorläuferzellen charakteristische Protein Nestin ausprägten, in einem räumlichen Zusammenhang. Die Vorgehensweise, die Inaktivierung des flk-1-Gens mit der Differenzierung embryonaler Stammzellen in vitro zu kombinieren, sollte hier die Interpretation des Phänotyps des flk-1-defizienten Mausmodells ermöglichen. Der Rezeptor war während der neuronalen Differenzierung der Stammzellen auf Stromazellen in vitro für die Regulation der Anzahl der Vorläuferzellen essentiell. Ferner spielte der Rezeptor im Rahmen eines weiteren Differenzierungsmodells, das auf der Zugabe relevanter Wachstumsfaktoren beruht, eine instruktive Rolle im Hinblick auf die Identität der Neuronen. Kriterium war hier die differentielle Expression Homeobox-enthaltender Transkriptionsfaktoren. In einem zweiten Schritt wurden mit Hilfe dieses Modells differentiell-exprimierte Gene von Stammzellen des Wildtyps sowie Zellen mit einer Inaktivierung des flk-1-Gens nach der neuronalen Differenzierung durch subtraktive Hybridisierung in Verbindung mit der PCR identifiziert. Tatsächlich wurde das Protein PEA-15 nicht nur differentiell exprimiert sondern auch als Bestandteil des VEGFR-2-vermittelten Signalwegs identifiziert. Die biologischen Funktionen des Proteins PEA-15 wurden durch VEGF-vermittelte Phosphorylierung reguliert. Die Stimulation durch VEGF führte zunächst zu einer Aktivierung des Proteinkinase B-, Akt-Signalwegs. Für die Stimulation des Akt-Signalwegs war die Phosphorylierung der intrazellulären Tyrosinreste Y1052 und Y1057 des Rezeptors essentiell. Damit einhergehend wurde PEA-15 gegenüber der proteasomalen Degradation stabilisiert. Es wurde gezeigt, daß das Protein PEA-15 die Teilungsaktivität von Zellen beeinflusst. Die VEGF- vermittelte Stimulation führte zur Phosphorylierung der Mitogen-aktivierten Proteinkinasen ERK1 und ERK2. Die weitere Phosphorylierung der Substrate dieser Kinasen im Zellkern wurde durch Interaktion mit PEA-15 unterdrückt. Die Regulation des c-fos-Promotors war zugleich Indikator der Inhibition der Phosphorylierung betreffender Substrate sowie der proliferativen Aktivität. Auf diese Weise ist die Phosphorylierung von PEA-15 nach Stimulation durch VEGF für die Selektivität des Flk-1-vermittelten Signalwegs von unmittelbarer Bedeutung. Die Regulation der biologischen Funktion von PEA-15 erklärt die differentielle Ausprägung im Rahmen der neuronalen Differenzierung embryonaler Stammzellen in vitro. So war die Anzahl GFAP- beziehungsweise PEA-15-exprimierender Zellen nach Differenzierung muriner Stammzellen mit einer Inaktivierung des flk-1-Gens deutlich geringer. Die differentielle Expression identifizierter Gene wurde im Mausmodell nach konditionaler Inaktivierung des flk-1-Gens überprüft. Tatsächlich wurde Vimentin in verschiedenen Arealen des Gehirns differentiell ausgeprägt. Ein Zusammenhang zwischen der differentiellen Expression des Proteins PEA-15, der Anzahl GFAP-exprimierender Zellen und der Ausprägung des Rezeptors Flk-1 ergab sich aus der Identifikation einer Zellpopulation in der subgranulären Zone des Gyrus Dentatus. Dort wurde in flk-1-defizienten, adulten Mäusen eine geringere Anzahl GFAP-exprimierender Zellen nachgewiesen. Schließlich wurden sowohl im Cerebellum als auch im Cortex histologische Unterschiede deutlich, die sich im adulten Organismus aus der Inaktivierung des Rezeptors Flk-1 ergeben. Die vorliegende Arbeit zeigt, daß der Rezeptor VEGFR-2, Flk-1, im neuronalen Kontext eine Rolle spielt, die sich nicht ausschließlich auf die Vermittlung eines Schutzmechanismus gegenüber der neuronalen Apoptose beschränkt, sondern auch auf eine Beteiligung an der Neurogenese hinweist. Die Vorgehensweise, mit Hilfe der subtraktiven Hybridisierung Bestandteile Rezeptor-vermittelter Signalwege vor dem Hintergrund der Differenzierung embryonaler Stammzellen zu identifizieren, verdeutlicht die Eignung der Methode auch bei komplexen Zellpopulationen.

Im Rahmen dieser Arbeit wurde die Rolle des Rezeptors VEGFR-2, Flk-1, im neuronalen Kontext untersucht. In einem ersten Schritt wurde in embryonalen Stammzellen der Maus das fluoreszierende Protein eGFP unter der Kontrolle regulatorischer Sequenzen des flk-1-Promotors, -Enhancers exprimiert. Nach der Differenzierung zu Sphäroiden wurden Endothelzellen nachgewiesen, die sowohl eGFP als auch das zelltypspezifische Oberflächenantigen CD31 ausprägen. Ebenso wurden nach der neuronalen Differenzierung in Gegenwart von Stromazellen eGFP-exprimierende Zellen identifiziert. Diese standen mit Zellen, die das für neuronale Vorläuferzellen charakteristische Protein Nestin ausprägten, in einem räumlichen Zusammenhang. Die Vorgehensweise, die Inaktivierung des flk-1-Gens mit der Differenzierung embryonaler Stammzellen in vitro zu kombinieren, sollte hier die Interpretation des Phänotyps des flk-1-defizienten Mausmodells ermöglichen. Der Rezeptor war während der neuronalen Differenzierung der Stammzellen auf Stromazellen in vitro für die Regulation der Anzahl der Vorläuferzellen essentiell. Ferner spielte der Rezeptor im Rahmen eines weiteren Differenzierungsmodells, das auf der Zugabe relevanter Wachstumsfaktoren beruht, eine instruktive Rolle im Hinblick auf die Identität der Neuronen. Kriterium war hier die differentielle Expression Homeobox-enthaltender Transkriptionsfaktoren. In einem zweiten Schritt wurden mit Hilfe dieses Modells differentiell-exprimierte Gene von Stammzellen des Wildtyps sowie Zellen mit einer Inaktivierung des flk-1-Gens nach der neuronalen Differenzierung durch subtraktive Hybridisierung in Verbindung mit der PCR identifiziert. Tatsächlich wurde das Protein PEA-15 nicht nur differentiell exprimiert sondern auch als Bestandteil des VEGFR-2-vermittelten Signalwegs identifiziert. Die biologischen Funktionen des Proteins PEA-15 wurden durch VEGF-vermittelte Phosphorylierung reguliert. Die Stimulation durch VEGF führte zunächst zu einer Aktivierung des Proteinkinase B-, Akt-Signalwegs. Für die Stimulation des Akt-Signalwegs war die Phosphorylierung der intrazellulären Tyrosinreste Y1052 und Y1057 des Rezeptors essentiell. Damit einhergehend wurde PEA-15 gegenüber der proteasomalen Degradation stabilisiert. Es wurde gezeigt, daß das Protein PEA-15 die Teilungsaktivität von Zellen beeinflusst. Die VEGF- vermittelte Stimulation führte zur Phosphorylierung der Mitogen-aktivierten Proteinkinasen ERK1 und ERK2. Die weitere Phosphorylierung der Substrate dieser Kinasen im Zellkern wurde durch Interaktion mit PEA-15 unterdrückt. Die Regulation des c-fos-Promotors war zugleich Indikator der Inhibition der Phosphorylierung betreffender Substrate sowie der proliferativen Aktivität. Auf diese Weise ist die Phosphorylierung von PEA-15 nach Stimulation durch VEGF für die Selektivität des Flk-1-vermittelten Signalwegs von unmittelbarer Bedeutung. Die Regulation der biologischen Funktion von PEA-15 erklärt die differentielle Ausprägung im Rahmen der neuronalen Differenzierung embryonaler Stammzellen in vitro. So war die Anzahl GFAP- beziehungsweise PEA-15-exprimierender Zellen nach Differenzierung muriner Stammzellen mit einer Inaktivierung des flk-1-Gens deutlich geringer. Die differentielle Expression identifizierter Gene wurde im Mausmodell nach konditionaler Inaktivierung des flk-1-Gens überprüft. Tatsächlich wurde Vimentin in verschiedenen Arealen des Gehirns differentiell ausgeprägt. Ein Zusammenhang zwischen der differentiellen Expression des Proteins PEA-15, der Anzahl GFAP-exprimierender Zellen und der Ausprägung des Rezeptors Flk-1 ergab sich aus der Identifikation einer Zellpopulation in der subgranulären Zone des Gyrus Dentatus. Dort wurde in flk-1-defizienten, adulten Mäusen eine geringere Anzahl GFAP-exprimierender Zellen nachgewiesen. Schließlich wurden sowohl im Cerebellum als auch im Cortex histologische Unterschiede deutlich, die sich im adulten Organismus aus der Inaktivierung des Rezeptors Flk-1 ergeben. Die vorliegende Arbeit zeigt, daß der Rezeptor VEGFR-2, Flk-1, im neuronalen Kontext eine Rolle spielt, die sich nicht ausschließlich auf die Vermittlung eines Schutzmechanismus gegenüber der neuronalen Apoptose beschränkt, sondern auch auf eine Beteiligung an der Neurogenese hinweist. Die Vorgehensweise, mit Hilfe der subtraktiven Hybridisierung Bestandteile Rezeptor-vermittelter Signalwege vor dem Hintergrund der Differenzierung embryonaler Stammzellen zu identifizieren, verdeutlicht die Eignung der Methode auch bei komplexen Zellpopulationen.

Neurons exhibit highly polarized structures, including two morphologically and functionally distinct domains, axons and dendrites. Dendrites and axons receive versus send information, and proper execution of each requires different sets of molecules. Differential distribution of membrane proteins in distinct neuronal compartments plays essential roles in neuronal functions. The major goal of my doctoral thesis was to study the molecular mechanisms that govern the differential distribution of membrane proteins in neurons, using the Drosophilalarval mushroom body (MB) as a model system. My work was initiated by an observation of differential distribution of distinct Dscam isoforms in neurons. Dscam stands for Down Syndrome Cell Adhesion Molecule, which is a Drosophila homolog of human DSCAM. According to genomic analysis, DrosophilaDscam gene can generate more than 38,000 isoforms through alternative splicing in its exons 4, 6, 9 and 17. All Dscam isoforms share similar domain structures, with 10 immunoglobulin domains and 6 fibronectin type III repeats in the ectodomain, a single transmembrane domain and a cytoplasmic endodomain. There are two alternative exons in exon 17 (17.1 and 17.2), which encodes Dscam’s transmembrane domain. Interestingly, in ectopic expression, Dscam isoforms carrying exon 17.1 (Dscam[TM1]) can be preferentially localized to dendrites and cell bodies, while Dscam isoforms carrying exon 17.2 (Dscam[TM2]) are distributed throughout the entire neuron including axons and dendrites. To unravel the mechanisms involved in the differential distribution of Dscam[TM1] versus Dscam[TM2], I conducted a mosaic genetic screening to identify the possible factors affecting dendritic distribution of Dscam[TM1], established an in vivoTARGET system to better distinguish the differential distribution of Dscam, identified the axonal and dendritic targeting motifs of Dscam molecules and further showed that Dscam’s differential roles in dendrites versus axons are correlated with its localization. Several mutants affecting dendritic distribution of Dscam[TM1] have been identified using a MARCM genetic screen. Three of these mutants (Dlis1, Dmn and p24) are components of the dynein/dynactin complex. Silencing of other dynein/dynactin subunits and blocking dynein function with a dominant-negative Glued mutant also resulted in mislocalization of Dscam[TM1] from dendrites to axons. However, microtubule polarity in the mutant axons was maintained. Taken together, this was the first demonstration that the dynein/dynactin complex is involved in the polarized distribution of membrane proteins in neurons. To further examine how dynein/dynactin is involved in the dendritic distribution of Dscam[TM1], I compromised dynenin/dynactin function with dominant-negative Glued and transiently induced Dscam[TM1] expression. The results suggested that dynein/dynactin may not be directly involved in the targeting of newly synthesized Dscam[TM1] to dendrites. Instead, it plays a role in maintaining dendritic restriction of Dscam[TM1]. Notably, dynein/dynactin dysfunction did not alter distribution of another dendritic transmembrane protein Rdl (Resistant to Dieldrin), supporting involvement of diverse mechanisms in distributing distinct molecules to the dendritic membrane. To identify the targeting motifs of Dscam, I incorporated the TARGET (Temporal and regional gene expression targeting) system into mushroom body (MB) neurons, and this allowed the demonstration of the differential distribution of Dscam[TM1] and Dscam[TM2] with more clarity than conventional overexpression techniques. Using the TARGET system, I identified an axonal targeting motif located in the cytoplasmic juxtamemebrane domain of Dscam[TM2]. This axonal targeting motif is dominant over the dendritic targeting motif located in Dscam’s ectodomain. Scanning alanine mutagenesis demonstrated that two amino acids in the axonal targeting motif were essential for Dscam’s axonal distribution. Interestingly, swapping the cytoplasmic juxtamembrane portions between TM1 and TM2 not only reversed TM1’s and TM2’s differential distribution patterns but also their functional properties in dendrites versus axons. My thesis research also involved studying endodomain diversity of Dscam isoforms. Besides the diversity originally found in the ectodomain and transmembrane domain of Dscam, my colleagues and I further demonstrated the existence of four additional endodomain variants. These four variants are generated by skipping or retaining exon 19 or exon 23 through independent alternative splicing. Interestingly, different Dscam endodomain isoforms are expressed at different developmental stages and in different areas of the nervous system. Through isoform-specific RNA interference, we showed the differential involvement of distinct Dscam endodomains in specific neuronal morphogenetic processes. Analysis of the primary sequence of the Dscam endodomain indicated that endodomain variants may confer activation of different signaling pathways and functional roles in neuronal morphogenesis. In Summary, my thesis work identified and characterized several previously unknown mechanisms related to the differential distribution of membrane proteins in neurons. I showed that there may be a dynein/dynactin-independent mechanism for selective transport of dendritic membrane proteins to dendrites. Second, dynein/dynactin plays a maintenance role in dendritic restriction of Dscam[TM1]. Third, different membrane proteins may require distinct combinations of mechanisms to be properly targeted and maintained in certain neuronal compartments. Further analysis of the mutants indentified from my genetic screen will definitely help to resolve the missing pieces of the puzzle. These findings provide novel mechanistic insight into the differential distribution of membrane proteins in polarized neurons.

Indiana University-Purdue University Indianapolis (IUPUI) / LIM homeodomain 3 (LHX3) is an important regulator of pituitary and nervous system development. To date, twelve LHX3 gene mutations have been identified in patients with combined pituitary hormone deficiency disease (CPHD). Understanding the molecular mechanisms governing LHX3/Lhx3 gene regulation will provide critical insights into organ development pathways and associated diseases. DNA methylation has been implicated in gene regulation in multiple physiological systems. This dissertation examines the role of DNA methylation in regulating the murine Lhx3 gene. To determine if demethylation of the Lhx3 gene promoter would induce its expression, murine pre-somatotrope pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with the demethylating reagent, 5-Aza-2’-deoxycytidine. This treatment lead to activation of the Lhx3 gene and thus suggested that methylation contributes to Lhx3 gene regulation. Proteins that modify chromatin, such as histone deacetylases (HDACs) have also been shown to affect DNA methylation patterns and subsequent gene activation. Pit-1/0 pituitary cells treated with a combination of the demethylating reagent and the HDAC inhibitor, Trichostatin A led to activation of the Lhx3 gene, suggesting crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNA were subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the methyl-binding protein, MeCP2 and the Lhx3 promoter regions in the Pit-1/0 cell line. Overall, the study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.