Hindsight (HNT) is a zinc finger transcription factor that is required for morphogenesis of the Drosophila embryo, having roles in germ band retraction (GBR) as well as dorsal closure (DC). HNT expression is also found in sensory organ precursors (SOP) of the developing pupal peripheral nervous system, and muscle progenitor cells, but the role of HNT in neurogenesis and myogenesis during embryogenesis has not been investigated in any depth. Microarray analysis of embryos over-expressing HNT during GBR and DC identified 1290 genes with significant changes in expression. This data set included many potential HNT targets, including genes associated with myogensis, and a disruption of muscle development was observed in embryos over-expressing HNT. It is possible that HNT may function to repress muscle identity genes in muscle founder cells. In addition, HNT over expressing embryos were found to resemble the neurogenic class of mutants. Among the potential target genes, D-Pax2 (shaven, sparkling, CG11049) expression, which is known to be expressed in the developing peripheral nervous system, was confirmed to be up-regulated following HNT over-expression. Interestingly, D-Pax2 and HNT expression were found to co-localize at the onset of their expression at stages 10-12 in embryos, but were not co-localized in later stages of embryogenesis. The up-regulation of D-Pax2 by HNT over-expression was further characterized and was found to be associated with strong ectopic HNT expression. The relevance of HNT to the regulation of D-Pax2 during normal development remains to be determined, but it is possible that endogenous expression of HNT is involved in D-Pax2 repression.

New neurons continue to be added to the dentate gyrus (DG) throughout adulthood and enhancing neurogenesis in this region holds therapeutic potential. However, the molecular mechanisms underlying DG neurogenesis remain elusive. Since developmental and adult neurogenesis often share the same signaling pathways, understanding how the DG develops is crucial to understanding adult neurogenesis. This study aims to determine the role of the retinoblastoma (Rb) protein in DG development and to determine if modulation of this pathway holds potential for enhancing neurogenesis in an adult system. A FoxG1 driven Cre is used to delete Rb in the developing forebrain and the resulting effects are analyzed in in vitro and in vivo mouse models. We show that Rb deletion enhances DG neurogenesis by specifically increasing proliferation of immature neurons. Overall this study suggests that Rb pathway modulation could hold potential for enhancing neurogenesis in the adult.

One strategy to repair an injured or degenerated retina is to stimulate the replacement of damaged or dead neurons with cells derived from endogenous stem- or progenitor cells. A successful strategy requires knowledge about how the proliferation and differentiation of the endogenous cells are regulated. In particular, this knowledge will be important in the establishment of protocols that produce sufficient numbers of specific neurons. The main aim of this thesis was to find and characterise factors regulating the proliferation and differentiation of retinal progenitor cells (RPCs) and hence, contribute to the knowledge of how to use progenitor cells for retinal repair.    The major result in this thesis is that GABA contributes to and maintains RPC proliferation. Inhibition of GABAA receptors decreases the proliferation of non-pigmented ciliary epithelial (NPE) cells and RPCs in the intact retina. We propose that this effect is mediated through changes in the membrane potential and voltage-gated calcium channels, which in turn regulate components of the cell cycle. Furthermore, we show that one of the endogenous RPC sources, the Müller cells, consists of two subpopulations based on Pax2 expression. This is interesting because Pax2 may suppress the neurogenic potential, characterised by de-differentiation and proliferation, in Müller cells. Finally, we show that over-expression of FoxN4 induces differentiation-associated transcription factors in the developing chick retina. However, FoxN4 over-expression did not trigger differentiation of NPE cells. These results indicate that the intrinsic properties of the RPCs are determinant for FoxN4-induced differentiation. The results presented in this thesis advance our understanding of how specific cells may be generated from different sources of RPCs. Our results show that the different sources are highly diverse in their potential to proliferate and produce neurons. GABA, Pax2 and FoxN4 may be factors to consider when designing strategies for retinal repair. However, the results indicate that the specific responses to these factors are highly associated with the specific properties of the progenitor cells. / <p>Doctor of Philosophy <strong>(Faculty of Medicine)</strong></p>

Amygdala kindling is commonly used to study the neural mechanisms of temporal lobe epilepsy and its behavioral consequences. The repetitive seizure activity that occurs during kindling is thought to induce an extensive array of structural and functional modifications within the brain, particularly in the hippocampus and dentate gyrus regions. Some of these changes include the growth or sprouting of new axonal connections as well as the birth and integration of new neurons into hippocampal circuits. Previous work has shown that these changes in structural and functional plasticity are not necessarily beneficial events. For instance, the growth and reorganization of synaptic terminals in the hippocampus and other brain regions might serve as a substrate that enhances hyperexcitability and seizure generation. In addition, although seizures induce the birth of new neurons, many of these newly generated cells migrate and function improperly within the hippocampal networks. Considering the prominent role of the hippocampus in a variety of behaviours, including learning, memory, and mood regulation, it would appear that alterations involving the structural and functional properties of both mature and newly born neurons in this region could impact these hippocampal-dependent functions. However, to date, the role of kindling-induced changes in hippocampal structural plasticity and neurogenesis on behaviour is incomplete, and the molecular mechanisms that govern these pathological events are poorly understood.<p/> The aim of this dissertation is to gain a better understanding of the changes in synaptic plasticity and neurogenesis within the hippocampus that occur after amygdala kindling. In chapter 2, we will examine if kindling alters the expression of synapsin I, a molecular marker of synaptic growth and activity, in both the hippocampus and other brain regions. In addition, we will also set out to determine if changes in synapsin I are related to the development of behavioural impairments associated with kindling. In chapter 3, the effect of kindling on hippocampal neurogenesis will be examined. In addition, we will also evaluate the effect of kindling on the expression of Reelin and Disrupted-in-Schizophrenia 1 (DISC1), two proteins instrumental for mediating proper neuronal migrational and maturation during development. In chapter 4, the effect of altered DISC1 expression in the dentate gyrus after kindling will be examined more extensively. We will examine whether altered DISC1 expression in the dentate contributes to some of the pathological features associated with seizure-induced hippocampal neurogenesis, such as ectopic cell migration and dentate granule cell layer dispersion. Finally, in chapter 5, the impact of aberrant seizure-induced neurogenesis on behaviour will be examined by determining if seizure-generated neurons functionally integrate and participate in hippocampal circuits related to memory processing. The results of this dissertation enhances our understanding of the functional consequences that altered hippocampal synaptic plasticity and neurogenesis may have on the development of epilepsy and emergence of cognitive impairments associated with chronic seizures.<p/>

Amygdala kindling is commonly used to study the neural mechanisms of temporal lobe epilepsy and its behavioral consequences. The repetitive seizure activity that occurs during kindling is thought to induce an extensive array of structural and functional modifications within the brain, particularly in the hippocampus and dentate gyrus regions. Some of these changes include the growth or sprouting of new axonal connections as well as the birth and integration of new neurons into hippocampal circuits. Previous work has shown that these changes in structural and functional plasticity are not necessarily beneficial events. For instance, the growth and reorganization of synaptic terminals in the hippocampus and other brain regions might serve as a substrate that enhances hyperexcitability and seizure generation. In addition, although seizures induce the birth of new neurons, many of these newly generated cells migrate and function improperly within the hippocampal networks. Considering the prominent role of the hippocampus in a variety of behaviours, including learning, memory, and mood regulation, it would appear that alterations involving the structural and functional properties of both mature and newly born neurons in this region could impact these hippocampal-dependent functions. However, to date, the role of kindling-induced changes in hippocampal structural plasticity and neurogenesis on behaviour is incomplete, and the molecular mechanisms that govern these pathological events are poorly understood.<p/> The aim of this dissertation is to gain a better understanding of the changes in synaptic plasticity and neurogenesis within the hippocampus that occur after amygdala kindling. In chapter 2, we will examine if kindling alters the expression of synapsin I, a molecular marker of synaptic growth and activity, in both the hippocampus and other brain regions. In addition, we will also set out to determine if changes in synapsin I are related to the development of behavioural impairments associated with kindling. In chapter 3, the effect of kindling on hippocampal neurogenesis will be examined. In addition, we will also evaluate the effect of kindling on the expression of Reelin and Disrupted-in-Schizophrenia 1 (DISC1), two proteins instrumental for mediating proper neuronal migrational and maturation during development. In chapter 4, the effect of altered DISC1 expression in the dentate gyrus after kindling will be examined more extensively. We will examine whether altered DISC1 expression in the dentate contributes to some of the pathological features associated with seizure-induced hippocampal neurogenesis, such as ectopic cell migration and dentate granule cell layer dispersion. Finally, in chapter 5, the impact of aberrant seizure-induced neurogenesis on behaviour will be examined by determining if seizure-generated neurons functionally integrate and participate in hippocampal circuits related to memory processing. The results of this dissertation enhances our understanding of the functional consequences that altered hippocampal synaptic plasticity and neurogenesis may have on the development of epilepsy and emergence of cognitive impairments associated with chronic seizures.<p/>

The vertebrate inner ear is a complex structure responsible for hearing and balance. The inner ear houses sensory epithelia composed of mechanosensory hair cells and non-sensory support cells. Hair cells synapse with neurons of the VIIIth cranial ganglion, the statoacoustic ganglion (SAG), and transmit sensory information to the hindbrain. This dissertation focuses on the development and regulation of both sensory and neuronal cell populations. The sensory epithelium is established by the basic helixloop- helix transcription factor Atoh1. Misexpression of atoh1a in zebrafish results in induction of ectopic sensory epithelia albeit in limited regions of the inner ear. We show that sensory competence of the inner ear can be enhanced by co-activation of fgf8/3 or sox2, genes that normally act in concert with atoh1a. The developing sensory epithelia express several factors that regulate differentiation and maintenance of hair cells. We show that pax5 is differentially expressed in the anterior utricular macula (sensory epithelium). Knockdown of pax5 function results in utricular hair cell death and subsequent loss of vestibular (balance) but not auditory (hearing) defects. SAG neurons are formed normally in these embryos but show disorganized dendrites in the utricle following loss of hair cells. Lastly, we examine the development of SAG. SAG precursors (neuroblasts) are formed in the floor of the ear by another basic helix-loophelix transcription factor neurogenin1 (neurog1). We show that Fgf emanating from the utricular macula specifies neuroblasts, that later delaminate from the otic floor and undergo a phase of proliferation. Neuroblasts then differentiate into bipolar neurons that extend processes to hair cells and targets in the hindbrain. We show evidence that differentiating neurons express fgf5 and regulate further development of the SAG. As more differentiated neurons accumulate, increasing level of Fgf terminates the phase of neuroblast specification. Later on, elevated Fgf stabilizes the transit-amplifying phase and inhibits terminal differentiation. Thus, Fgf signaling regulates SAG development at various stages to ensure that proper number of neurons is generated.

Among mammals, primates are exceptional for their large brain size relative to body size. Relative brain size, or encephalization, is particularly striking among humans and their direct ancestors. Since the human-chimp split 5 to 7 million years ago, brain size has tripled in the human lineage (Wood &amp;amp; Collard 1999). The focus of this doctoral work is to investigate some of the cell biological mechanisms responsible for this increase in relative brain size. In particular, the processes that regulate symmetric cell division (ultimately generating more progenitors), the constraints on progenitor proliferation, and how neural progenitors have overcome these constraints in the process of primate encephalization are the primary questions of interest. Both functionally analyses in the mouse model system and comparative neurobiology of rodents and primates are used here to address these questions. Using the mouse model system, the cell biological role of the Aspm (abnormal spindle-like microcephaly associated) protein in regulating brain size was investigated. Specifically, Aspm function in symmetric, proliferative divisions of neuroepithelial (NE) cells was analyzed. It was found that Aspm expression in the mouse neuroepithelium correlates in time and space with symmetric, proliferating divisions. The Aspm protein localizes to NE cell spindle poles during all phases of mitosis, and is down-regulated in cells that undergo asymmetric (neurogenic) cell divisions. Aspm RNAi alters the division plane in NE cells, increasing the likelihood of premature asymmetric division resulting in an increase in non-NE progeny. At least some of the non-NE progeny generated by Aspm RNAi migrate to the neuronal layer and express neuronal markers. Importantly, whatever the fate of the non-NE progeny, their generation comes at the expense of the expansion of the proliferative pool of NE progenitor cells. These data have contributed to the generation of an hypothesis regarding evolutionary changes in the regulation of spindle orientation in vertebrate and mammalian neural progenitors and their impact on brain size. Specifically, in contrast to invertebrates that regulate the switch from symmetric to asymmetric division through a rotation of the spindle (horizontal versus vertical cleavage), asymmetric NE cell division in vertebrates is accomplished by only a slight deviation in the cleavage plane away from the vertical, apical-basal axis. The requirement for the precise alignment of the spindle along the apical-basal axis in symmetric cell divisions may have contributed to selection on spindle “precision” proteins, thus increasing the number of symmetric NE cell division, and contributing to brain size increases during mammalian evolution. Previous comparative neurobiological analyses have revealed an increase in basally dividing NE cells in the brain regions of highest proliferation and in species with the largest brains (Smart 1972a,b; Martinez-Cerdeno et al. 2006). The cell biological characteristics of these basally dividing cells are still largely unknown. We found that primate basal progenitors, similar to rodent apical progenitors, are Pax6+. This suggests that primate basal progenitors may share other properties with rodent apical progenitors, such as maintenance of apical contact. Our previous finding that artificial alteration of cleavage plane in NE cells affects their ability to continue proliferating supports the hypothesis that the apical membrane and junctional complexes are cell fate determinants (Huttner &amp;amp; Kosodo 2005). As such, the need to maintain apical membrane contact appears to be a constraint on proliferation (Smart 1972a,b; Smart et al. 2002). Together, these data favor the hypothesis that primate basally dividing cells maintain apical contact and are epithelial in nature.

Ο σχηματισμός του νευρικού συστήματος επιτυγχάνεται από αναπτυξιακούς μηχανισμούς που ελέγχουν τις διαδικασίες του κυτταρικού πολλαπλασιασμού και της κυτταρικής διαφοροποίησης. Το χαρακτηριστικό αυτό υποδεικνύει ότι μόρια που συμμετέχουν και στις δύο παραπάνω διαδικασίες ενδεχομένως διαδραματίζουν σημαντικό ρόλο στην πορεία της νευρογένεσης. Η Geminin είναι μία πρωτεΐνη που εμπλέκεται στους δύο προαναφερόμενους μηχανισμούς. Η Geminin ελέγχει τον κυτταρικό πολλαπλασιασμό ρυθμίζοντας τον παράγοντα αδειοδότησης της αντιγραφής του DNA Cdt1. Επίσης, η Geminin εμφανίζει και νευροεπαγωγική δράση, καθώς προάγει το σχηματισμό νευρικού ιστού στα έμβρυα του Xenopus laevis. Επιπρόσθετα, η Geminin αλληλεπιδρά με μεταγραφικούς παράγοντες και παράγοντες αναδιαμόρφωσης της δομής της χρωματίνης, όπως είναι τα μόρια Six3, Hox, Brg1, Brahma και Polycomb, επηρεάζοντας με αυτόν τον τρόπο την ισορροπία μεταξύ πολλαπλασιασμού και διαφοροποίησης. Μελετήσαμε το πρότυπο έκφρασης των πρωτεϊνών Geminin και Cdt1 στο αναπτυσσόμενο κεντρικό νευρικό σύστημα (ΚΝΣ) εμβρύων ποντικού με τις τεχνικές του in situ υβριδισμού και της ανοσοϊστοχημείας. Η παρουσία της Geminin ανιχνεύτηκε στην κοιλιακή ζώνη του αναπτυσσόμενου ΚΝΣ, όπου εδρεύουν τα πρόδρομα νευρικά κύτταρα. Το πρότυπο έκφρασης της Cdt1 ήταν παρόμοιο με αυτό της Geminin. Τα κύτταρα που εκφράζουν τις Geminin και Cdt1 κατανέμονται μεταξύ των κυττάρων που εκφράζουν τον παράγοντα Sox2, έναν μοριακό δείκτη πρόδρομων νευρικών κυττάρων. Ανιχνεύσαμε ένα μερικώς αλληλεπικαλυπτόμενο πρότυπο έκφρασης των δύο πρωτεϊνών με το αντίστοιχο του μορίου Mash1, το οποίο καταδεικνύει πρόδρομα κύτταρα που έχουν αρχίσει να διαφοροποιούνται προς νευρώνες. Τέλος, η κατανομή των Geminin και Cdt1 είναι τελείως διακριτή από την κατανομή της πρωτεΐνης Τουμπουλίνη βΙΙΙ που εκφράζεται μόνο από τους ώριμους νευρώνες. Τα αποτελέσματά μας υποδεικνύουν ότι οι πρωτεΐνες Geminin και Cdt1 εκφράζονται από τα πρόδρομα αδιαφοροποίητα κύτταρα του αναπτυσσόμενου νευρικού συστήματος. / Nervous system formation is accomplished through developmental mechanisms which integrate tight control of proliferation and differentiation. This feature indicates that molecules participating in both of these processes may have prominent roles during neurogenesis. Geminin is a protein involved in both of these mechanisms. It controls proliferation by negatively regulating the DNA replication licensing factor Cdt1 and, in addition, it acts as a neuralizing factor during early gastrulation of Xenopus embryos, defining in dorsal ectoderm a future nervous territory. Moreover, Geminin’s interaction with transcription and chromatin remodeling factors such as Six3, Hox, Brg1, Brahma and Polycomb proteins affects the balance between proliferation and differentiation. We performed a detailed analysis of the expression patterns of Geminin and Cdt1 in the central nervous system of mouse embryos by in situ hybridization and immunohistochemistry. We detected Geminin at the ventricular zone (VZ) of the developing CNS, where the proliferating progenitors reside, and its expression pattern overlapped that of Cdt1. Geminin and Cdt1 expressing cells defined a subpopulation of the Sox2 positive cells, which is a marker of neural progenitor cells. A partial co-localization was seen with Mash1, a marker of precursor cells committed towards a neuronal phenotype, whereas no overlap was detected with the neuronal marker TubulinβIII. Our results indicate that Geminin and Cdt1 are localized in progenitors/stem cells of the developing CNS.

Für die Bildung eines funktionalen Nervensystems in Vertebraten ist ein Gleichgewicht zwischen inhibitorischen und exzitatorischen Neuronen essentiell. Ein Schlüsselfaktor in der Regulation dieses Gleichgewichts ist der bHLH Transkriptionsfaktor Ptf1a, welcher GABAerge inhibitorische Neurone in der Retina, dem Hinterhirn und im Rückenmark von Vertebraten spezifiziert, zugleich jedoch glutamaterge exzitatorische Neurone unterdrückt. In diesem Zusammenhang benötigt die Aktivität von Ptf1a die Bildung eines trimeren Komplexes, in welchem Ptf1a an ein allgemein exprimiertes E-Protein und an ein Mitglied der Su(H)-Familie bindet. Ptf1a fördert ebenfalls generelle neuronale Differenzierung in X. laevis Embryonen und Explantaten, was darauf hinweist, dass Ptf1a proneurale Aktivität besitzt. In dieser Doktorarbeit wurde die Rolle von Ptf1a im Zusammenhang mit genereller Neurogenese (frühe Funktion) und neuronaler Subtypen-Spezifizierung (späte Funktion) untersucht. Durch eine zeitliche Expressionsanalyse bekannter Gene konnte gezeigt werden, dass Ptf1a durch die Aktivierung von nachgeschalteten Genen, ähnlich dem proneuralen Transkriptionsfaktor Ngn2, in animalen Kappen (naives Ektoderm) zu frühen Zeitpunkten Neurogenese induziert. In späteren Stadien hingegen aktivierte Ptf1a die Expression von Markergenen, die GABAerge Neurone kennzeichnen, während neuronale glutamaterge Markergene von Ngn2 induziert wurden. Eine mutierte Version von Ptf1a (Ptf1aW224A/W242A), welche nicht in der Lage ist, mit dem Kofaktor Su(H) zu interagieren, behielt die Fähigkeit, generelle Neurogenese zu induzieren, nicht aber GABAerge Markergene zu aktivieren. Diese Ergebnisse lassen darauf schließen, dass Ptf1a in der Entwicklung des Nervensystems kontext-spezifische Transkriptionskomplexe bildet: einen Su(H)-unabhängigen Komplex zur Aktivierung genereller Neuorgenese und einen Su(H)-abhängigen Komplex zur Spezifizierung GABAerger Neurone. Da die Zielgene von Ptf1a in der Entwicklung des Nervensystems nicht genau bestimmt sind, wurden zwei unabhängige Transkriptom-Analysen durchgeführt, um das Ptf1a nachgeschaltete genetische Netzwerk aufzuzeigen. In diesen Untersuchungen wurde eine zeitliche Analyse von Genen durchgeführt, die durch wildtyp Ptf1a, Ptf1aW224A/W242A und Ngn2 in X. laevis animalen Kappen aktiviert werden; direkte Zielgene für Ptf1a und Ptf1a/Su(H) wurden bestimmt durch die Aktivierung dieser Transkriptionsfaktoren unter Vorhandensein eines Proteinsyntheseinhibitors (CHX). Durch dieses Vorgehen konnten viele mutmaßlich neue frühe und späte Zielgene von Ptf1a identifiziert werden. Eine weitere Analyse dieser nachgeschalteten Zielgene dürfte darüber Aufschluss geben, wie Ptf1a generelle Neurogenese und neuronale Subtypen-Spezifizierung reguliert.

New neurons are continually produced throughout adult life in the dentate gyrus of the hippocampus, in a process termed adult neurogenesis. Although there is a significant effort in the literature to understand the functional significance of hippocampal neurogenesis, conflicting experimental reports have left the role of neurogenesis unclear. Recently, computational modelling studies have hypothesized that neurogenesis may play a role in allowing association between event and context to be formed in memory. By using a novel odour task and a raised plus maze task, our work demonstrates that the reduction of hippocampal neurogenesis using focal irradiation impairs the ability of animal subjects to utilize contextual information to learn interfering information. The result of this work provides experimental evidence of a unique role neurogenesis may play in learning and memory.