The mammalian forebrain, which consists of the telencephalon and the diencephalon, is responsible for many higher cognitive functions such as thinking, learning and memory. The cerebral cortex, which is important for language and processing information, is located in the dorsal portion of the telencephalon. The basal ganglia, which are important for movement, are located in the ventral telencephalon. Many genes are involved in patterning and the development of the forebrain. One gene that appears to be crucial for forebrain development is Gli3. Gli3 has been shown to work as both a transcriptional activator and a repressor of the Sonic Hedgehog (Shh) signalling pathway in the developing spinal cord and limb buds. In the telencephalon, Shh has been shown to be important for induction of ventral cell fate, but the exact function of Gli3 in the forebrain and the interactions between Gli3 and Shh are still obscure. Previous studies have shown that Gli3 is required for the formation of the cortical hem area of the telencephalon, which does not form in Gli3Xt/Xt mutant mice lacking functional Gli3. The residual dorsal telencephalon of the Gli3Xt/Xt mutants is partially ‘ventralized’. The main aim of this study was to re-examine the developing forebrain of Gli3Xt/Xt mouse mutants to gain insight into the function of Gli3 during forebrain development. In this thesis, the expression of Gli3 mRNA and protein was examined in the E12.5 and E14.5 wild type telencephalon. The highdorsal-to-lowventral expression pattern of Gli3 corresponds to severedorsal-to-mildventral defects observed in the Gli3Xt/Xt mutants. The ratios between the levels of the cleaved and full length isoforms of Gli3 in dorsal and ventral telencephalon resemble those described in dorsal and ventral spinal cord and in the anterior and posterior limb bud, respectively, suggesting Gli3 in the dorsal telencephalon may act as a repressor of the Shh signalling pathway. The total amount and the ratios of the two isoforms of Gli3 protein were examined in Shh and Foxg1 null mice, which lack ventral telencephalon. The results obtained agree with a role of Gli3 as a repressor of the Shh pathway in the dorsal telencephalon. The forebrains of Gli3Xt/Xt mutants were analysed systematically both anatomically and by molecular markers in this thesis. The border between the telencephalon and the diencephalon was delineated in the Gli3Xt/Xt mutants by using a combination of markers expressed in different areas within the forebrain. This lead to the observation that the previously reported ‘ventralization’ only occurred in the very rostral telencephalic sections of the Gli3Xt/Xt mutant embryos, suggesting a possible shape change of the Gli3Xt/Xt telencephalon. To examine the possible causes of the significant size reduction of Gli3Xt/Xt mutant telencephalon compared to wild type telencephalon from E10.5, cell proliferation and cell death properties studies were undertaken. The changes observed were not sufficient to explain the phenotypic differences between the Gli3Xt/Xt mutant and the wild type embryos indicating that they might be the result of an early patterning defect. The dorsal telencephalon is severely reduced in volume at both E12.5 and E10.5, containing cells from adjacent eminentia thalami, probably due to the loss of the dorso-medial telencephalon. Large clusters of eminentia thalami cells were observed at later developmental stages, when the neocortex becomes highly disorganized, forming rosettes comprising mainly neural progenitors. These results suggest Gli3 is important for the formation of an intact telencephalic-diencephalic boundary and for preventing the abnormal location of diencephalic cells in the dorsal telencephalon. The volume of Gli3Xt/Xt ventral telencephalon was increased compared to that of the wild types at E10.5, but became smaller than that of the wild type littermates at E12.5. This might have been the result of a combination of more cells exiting the cell cycle and increased cell death observed in the Gli3Xt/Xt ventral telencephalon at E10.5, suggesting Gli3 regulates cell differentiation and cell death properties at this age and brain region. The significant expansion of rostro-ventral telencephalon observed in the Gli3Xt/Xt mutant might correlate with the expansion of Fgf8 expression and this hypothesis has been tested in this thesis.

The Distal-less (Dlx) homeobox genes of mammals are expressed in many tissues of the developing organism including the limbs, craniofacial skeleton and the forebrain. In the forebrain, Dlx1, Dlx2, Dlx5 and Dlx6 play a critical role in driving tangential migration of GABAergic progenitors from the ventral telencephalon to their final destinations, notably the neocortex and the striatum. These Dlx genes are organised into convergently transcribed clusters with short intergenic regions that contain notable cis regulators elements (CREs) that drive Dlx expression in unique subdomains of the developing ventral telencephalon. Previous studies have characterised Dlx regulation including but not limited to the direct activation of these CREs by effector proteins. However, to date very little work has been done to examine how the forebrain Dlx genes may be regulated at the level of the chromatin. To explore this, I used in silico and in vivo methods to examine some key histone modifications of the Dlx1/2 and Dlx5/6 bigene clusters in the developing forebrain; namely H3K27Ac, H3K4me3, H3K4me1 and H3K27me3. I found that within the Dlx expressing ganglionic eminences (GE), at midgestation, the Dlx loci are marked by bivalent chromatin which is enriched in both permissive H3K4me3 and repressive H3K27me3 marks. By performing ChIP-qPCR on the GE tissue of embryonic mice with targeted deletions of enhancer CREs, I found that these CREs play unique roles in shaping the chromatin. Removal of one of these CREs has widespread effects on the chromatin at both loci. Since these changes in chromatin signatures do not accompany significant changes in expression of histone modifying genes, we believe these CREs play yet-to-be determined roles in recruiting the modifying proteins to the loci, thereby establishing bivalent chromatin to fine-tune Dlx expression.

The temporal and spatial patterning that underlies morphogenetic events is controlled by gene regulatory networks (GRNs). These operate through a combinatorial code of DNA – binding transcription factor proteins, and non – coding DNA sequences (cis-regulatory elements, or CREs), that specifically bind transcription factors and regulate nearby genes. By comparatively studying the development of different species, we can illuminate lineage – specific changes in gene regulation that account for morphological evolution. The central nervous system of vertebrates is composed of diverse neural cells that undergo highly coordinated programs of specialization, migration and differentiation during development. Approximately 20% of neurons in the cerebral cortex are GABAergic inhibitory interneurons, which release the neurotransmitter gamma-aminobutyric acid (GABA). Diseases such as autism, schizophrenia and epilepsy are associated with defects in GABAergic interneuron function. Several members of the distal-less homeobox (Dlx) transcription factor family are implicated in a GRN underlying early GABAergic interneuron development in the forebrain. I examined the role played by orthologous dlx genes in the development of GABAergic interneurons in the zebrafish forebrain. I found that when ascl1a transcription factor is down-regulated through the micro-injection of translation – blocking morpholino oligonucleotides, Dlx gene transcription is decreased in the diencephalon, but not the telencephalon. Similarly, gad1a transcription is also decreased in this region for these morphants. As gad1a encodes an enzyme necessary for the production of GABA, these genes are implicated in a cascade underlying GABAergic interneuron development in the diencephalon.

The term “stem cell” is often broadly applied to a range of cell types that are relatively undifferentiated and have some capacity for proliferation. In this thesis, I employ a strict definition of stem cells as cells that are capable of both self-renewal and multilineage differentiation. These properties are tested in single precursor cells from the forebrain and its derivative, the retina, using clonal assays. Poor survival is a common problem in single cell cultures, and I show that low oxygen dramatically improves viability in neural stem cells clonally derived from mouse embryonic stem cells, as well as in cultured forebrain neural stem cells. Caspase-dependent and apoptosis-inducing factor-dependent cell death pathways were found to be differentially influenced in low oxygen culture of early, primitive and later, definitive neural stem cells. I isolate precursors from 2 separate regions of the adult mouse forebrain, the lateral ventricle and the hippocampus and argue that only cells resident in the lateral ventricle can be classified as stem cells while the hippocampus contains restricted progenitor cells. Unlike neural stem cells, the very existence of retinal precursors in the adult mammal is controversial. I investigate methods to prospectively identify a rare stem cell population in the pigmented ciliary epithelium of the adult mouse eye and show that, although this population intrinsically gives rise to all retinal cell types, cells can be directed specifically towards a photoreceptor fate by the addition of exogenous factors to the culture media. Pigmentation of retinal stem cells is used as a convenient marker to isolate a retinal stem cell from human embryonic stem cells differentiating under conditions known to promote neural differentiation. Retinal stem cells derived from human embryonic stem cells have highly similar properties to those directly isolated from the eye, and their progeny can similarly be driven to differentiate into photoreceptors. The findings presented in this thesis help to define intrinsic properties of adult neural and retinal precursors and provide a basis for manipulating these cells, potentially for future use in clinical applications.

The term “stem cell” is often broadly applied to a range of cell types that are relatively undifferentiated and have some capacity for proliferation. In this thesis, I employ a strict definition of stem cells as cells that are capable of both self-renewal and multilineage differentiation. These properties are tested in single precursor cells from the forebrain and its derivative, the retina, using clonal assays. Poor survival is a common problem in single cell cultures, and I show that low oxygen dramatically improves viability in neural stem cells clonally derived from mouse embryonic stem cells, as well as in cultured forebrain neural stem cells. Caspase-dependent and apoptosis-inducing factor-dependent cell death pathways were found to be differentially influenced in low oxygen culture of early, primitive and later, definitive neural stem cells. I isolate precursors from 2 separate regions of the adult mouse forebrain, the lateral ventricle and the hippocampus and argue that only cells resident in the lateral ventricle can be classified as stem cells while the hippocampus contains restricted progenitor cells. Unlike neural stem cells, the very existence of retinal precursors in the adult mammal is controversial. I investigate methods to prospectively identify a rare stem cell population in the pigmented ciliary epithelium of the adult mouse eye and show that, although this population intrinsically gives rise to all retinal cell types, cells can be directed specifically towards a photoreceptor fate by the addition of exogenous factors to the culture media. Pigmentation of retinal stem cells is used as a convenient marker to isolate a retinal stem cell from human embryonic stem cells differentiating under conditions known to promote neural differentiation. Retinal stem cells derived from human embryonic stem cells have highly similar properties to those directly isolated from the eye, and their progeny can similarly be driven to differentiate into photoreceptors. The findings presented in this thesis help to define intrinsic properties of adult neural and retinal precursors and provide a basis for manipulating these cells, potentially for future use in clinical applications.

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pharmacology and Toxicology. Title from title-page of electronic thesis. Bibliography: leaves 84-94.

The temporal and spatial patterning that underlies morphogenetic events is controlled by gene regulatory networks (GRNs). These operate through a combinatorial code of DNA – binding transcription factor proteins, and non – coding DNA sequences (cis-regulatory elements, or CREs), that specifically bind transcription factors and regulate nearby genes. By comparatively studying the development of different species, we can illuminate lineage – specific changes in gene regulation that account for morphological evolution. The central nervous system of vertebrates is composed of diverse neural cells that undergo highly coordinated programs of specialization, migration and differentiation during development. Approximately 20% of neurons in the cerebral cortex are GABAergic inhibitory interneurons, which release the neurotransmitter gamma-aminobutyric acid (GABA). Diseases such as autism, schizophrenia and epilepsy are associated with defects in GABAergic interneuron function. Several members of the distal-less homeobox (Dlx) transcription factor family are implicated in a GRN underlying early GABAergic interneuron development in the forebrain. I examined the role played by orthologous dlx genes in the development of GABAergic interneurons in the zebrafish forebrain. I found that when ascl1a transcription factor is down-regulated through the micro-injection of translation – blocking morpholino oligonucleotides, Dlx gene transcription is decreased in the diencephalon, but not the telencephalon. Similarly, gad1a transcription is also decreased in this region for these morphants. As gad1a encodes an enzyme necessary for the production of GABA, these genes are implicated in a cascade underlying GABAergic interneuron development in the diencephalon.

The Distal-less homeobox (Dlx) genes encode homeodomain transcription factors found in all animals of the phylum Chordata. These genes are involved in early vertebrate development of limbs, sensory organs, branchial arches and the forebrain (telencephalon and diencephalon). The mouse and human genomes each have six Dlx genes organized into convergently transcribed bigene clusters (Dlx1/2, Dlx3/4 and Dlx5/6). In the forebrain, Dlx1/2 and Dlx5/6 genes play essential roles in GABAergic neuron proliferation, migration and survival. Each bigene cluster includes a short intergenic region (~3.5-16kb) harboring cis-regulatory elements (CREs) that control expression of the Dlx genes. The Dlx1/2 intergenic region harbors the I12b/I12a CREs, while Dlx5/6 includes I56i/I56ii. In determining the regulatory roles of the CREs on Dlx activity and forebrain development, I have characterized the phenotypic changes that occur in mice that have an I56i enhancer deletion. I have also characterized mice with double deletions of I56i and I12b as well as mice that harbored an I12b deletion and have a SNP in the I56i enhancer (vI56i). Mutant mice with a single targeted deletion of I56i are viable, fertile and do not show obvious developmental defects. These mice have significant decreases in Dlx5/6, Gad1/Gad2 and Evf-2 expression in the forebrain and have defects related to GABAergic neuron development. The ΔI56i mutants demonstrate a behavioral phenotype related to anxiety and learning deficits. Mice that lack the I12b enhancer and have the vI56i do not show morphological abnormalities but have severely disrupted Dlx expression. When mice are homozygous for the I56i and I12b enhancer deletion, they do not survive past post natal day 5 and exhibit a dwarfed body size. These mice look weak and seem to have limited motor ability. In characterizing mice with targeted deletions of highly conserved Dlx enhancers, we will have a better understanding of forebrain development.

The temporal and spatial patterning that underlies morphogenetic events is controlled by gene regulatory networks (GRNs). These operate through a combinatorial code of DNA – binding transcription factor proteins, and non – coding DNA sequences (cis-regulatory elements, or CREs), that specifically bind transcription factors and regulate nearby genes. By comparatively studying the development of different species, we can illuminate lineage – specific changes in gene regulation that account for morphological evolution. The central nervous system of vertebrates is composed of diverse neural cells that undergo highly coordinated programs of specialization, migration and differentiation during development. Approximately 20% of neurons in the cerebral cortex are GABAergic inhibitory interneurons, which release the neurotransmitter gamma-aminobutyric acid (GABA). Diseases such as autism, schizophrenia and epilepsy are associated with defects in GABAergic interneuron function. Several members of the distal-less homeobox (Dlx) transcription factor family are implicated in a GRN underlying early GABAergic interneuron development in the forebrain. I examined the role played by orthologous dlx genes in the development of GABAergic interneurons in the zebrafish forebrain. I found that when ascl1a transcription factor is down-regulated through the micro-injection of translation – blocking morpholino oligonucleotides, Dlx gene transcription is decreased in the diencephalon, but not the telencephalon. Similarly, gad1a transcription is also decreased in this region for these morphants. As gad1a encodes an enzyme necessary for the production of GABA, these genes are implicated in a cascade underlying GABAergic interneuron development in the diencephalon.

The Distal-less homeobox (Dlx) genes encode homeodomain transcription factors found in all animals of the phylum Chordata. These genes are involved in early vertebrate development of limbs, sensory organs, branchial arches and the forebrain (telencephalon and diencephalon). The mouse and human genomes each have six Dlx genes organized into convergently transcribed bigene clusters (Dlx1/2, Dlx3/4 and Dlx5/6). In the forebrain, Dlx1/2 and Dlx5/6 genes play essential roles in GABAergic neuron proliferation, migration and survival. Each bigene cluster includes a short intergenic region (~3.5-16kb) harboring cis-regulatory elements (CREs) that control expression of the Dlx genes. The Dlx1/2 intergenic region harbors the I12b/I12a CREs, while Dlx5/6 includes I56i/I56ii. In determining the regulatory roles of the CREs on Dlx activity and forebrain development, I have characterized the phenotypic changes that occur in mice that have an I56i enhancer deletion. I have also characterized mice with double deletions of I56i and I12b as well as mice that harbored an I12b deletion and have a SNP in the I56i enhancer (vI56i). Mutant mice with a single targeted deletion of I56i are viable, fertile and do not show obvious developmental defects. These mice have significant decreases in Dlx5/6, Gad1/Gad2 and Evf-2 expression in the forebrain and have defects related to GABAergic neuron development. The ΔI56i mutants demonstrate a behavioral phenotype related to anxiety and learning deficits. Mice that lack the I12b enhancer and have the vI56i do not show morphological abnormalities but have severely disrupted Dlx expression. When mice are homozygous for the I56i and I12b enhancer deletion, they do not survive past post natal day 5 and exhibit a dwarfed body size. These mice look weak and seem to have limited motor ability. In characterizing mice with targeted deletions of highly conserved Dlx enhancers, we will have a better understanding of forebrain development.