No description available.

Retinoic acid (RA) is an active metabolite of vitamin A that is essential for embryonic development, and homeostasis of adult tissues. RA is a ligand for the nuclear retinoic acid receptor, and RA-mediated signaling is critical for regulation of cell proliferation, differentiation and apoptosis. There is a spatio-temporal distribution of RA in the developing embryo such that some tissues are rich in RA, while others are devoid. This patterned distribution of RA is tightly controlled through the coordinated expression of RA-synthesizing (retinaldehyde dehydrogenase) and RA-catabolizing (CYP26) enzymes. In this thesis, I describe the cloning of a mouse gene encoding one of the CYP26 proteins, Cyp26b1. Cyp26b1 was shown to be highly expressed in the embryo, with transcripts localized to the hindbrain, limb buds and branchial arches. We also used homologous recombination to generate a line of transgenic mice with a loss-of-function deletion in Cyp26b1. These mice die shortly after birth with severe malformations affecting the limbs, craniofacial structures and epidermis; phenotypes that are all reminiscent of RA teratogenesis. We present an extensive characterization of the craniofacial and epidermal abnormalities in Cyp26b1-/- animals, and examine several molecular pathways that may be deregulated. CYP26B1 null embryos exhibit a truncated mandible, lack numerous facial bones, and show reduced ossification of the calvaria. Molecular analysis of Cyp26b1-/- embryos indicates hindbrain and branchial arch patterning is largely unaffected in early to mid-gestational mutants. However, there appear to be some subtle abnormalities in neural crest cell migration, which may contribute to the development of some of the observed phenotypes. CYP26B1 null mutants also lack hair follicles, which appears to be due to a downregulation of -catenin mediated signaling. Thus, in addition to cloning and characterizing the expression of murine Cyp26b1, we have demonstrated in vivo, that regulation of RA distribution by CYP26B1 is essential for morphogenesis of the epidermis and craniofacial structures. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2007-09-28 13:49:35.17

Long ncRNAs (non-protein coding transcripts generally considered longer than 200 nucleotides to be distinguished from classes of small RNAs) are abundantly transcribed from the mammalian genome. Despite their abundance, little is known about these transcripts. Although several individual long ncRNAs have been well-characterised and ascribed important cellular functions, there remains considerable controversy as to whether long ncRNAs are, in the main, functional. Indeed, their abundance has prompted many people to argue that long ncRNAs are simply transcriptional ‘noise’ generated by spurious transcription initiation events resulting from low RNA polymerase II fidelity. This thesis demonstrates that large numbers of long ncRNAs are specifically expressed along both temporal and spatial axes of mouse development in a manner consistent with a biological function. Custom-designed microarrays were employed to analyse the expression profiles of large numbers of long ncRNAs, along with protein-coding genes, in two models of cellular differentiation; the differentiation of mouse embryonic stem (ES) cells from pluripotency to differentiation along a hemopoietic lineage; and the commitment and differentiation of neural stem cells to oligodendrocytes. The core networks that include gene expression, transcription factor binding sites and chromatin domains that regulate ES cell pluripotency and lineage specification have been the subject of considerable attention and provide a detailed context in which to analyse ncRNA expression. Of those ncRNAs examined, 945 (26% of total) ncRNAs were expressed during the differentiation of ES to embryoid body (EB), of which 174 were significantly differentially expressed. Many of these ncRNAs were transcribed from genomic locations that overlapped modified chromatin domains, and in two further studied cases directly engaged with epigenetic machinery. Similarly, 332 long ncRNAs (9% of those examined) were expressed during processes of neuronal-glial fate switching, neurogenesis and oligodendrocyte progressive differentiation and termination, of which around half were also significantly differentially expressed. Furthermore, many of these ncRNAs exhibited expression profiles that coincided with pivotal events during the commitment and differentiation of neural stem cells (NSC) to mature myelinating oligodendrocytes. Consideration of the genomic context revealed many long ncRNAs were expressed from diverse places including intergenic, intronic, and imprinted loci and may overlap with, or are transcribed antisense to, protein-coding genes with previously described roles in either ES or NSC pluripotency and differentiation. This association also extended to expression profiles, where a comparative analysis often showed complex relationships of expression between ncRNAs and associated protein coding genes, suggesting a potential role for ncRNAs in regulating the expression of associated gene loci. The complexity and specificity of the long ncRNAs expression was illustrated by analysis of the in situ hybridisation (ISH) data conducted in collaboration with the Allen Brain Atlas. Of 1328 long ncRNAs, 849 (64%) were expressed in the mouse brain, 623 (47%) of which exhibited specific expression profiles associated with distinct neuroanatomical regions, cell types, or subcellular compartments. Again, examination of their genomic context revealed long ncRNAs were often associated with protein-coding genes of neurological importance and this association often extended to include linked expression profiles in the mouse brain. The comparative analysis of protein-coding gene expression relative to associated noncoding transcription also revealed an additional level of complexity in gene structure and genomic architecture. Analysis of both microarray and ISH data show 3’UTRs can exhibit discordant expression profiles relative to their associated protein coding genes, often in a tissue- and developmentally-specific manner. Indeed, a genome-wide analysis showed that the independent expression of 3’UTR transcripts is prevalent throughout the mouse genome where they may function intrinsically as long ncRNAs during development. Together, these genome-wide analyses indicate a large proportion of long ncRNAs exhibit specific expression profiles that are inconsistent with the notion they are meaningless transcriptional noise. Taken together with numerous studies published in recent years, this thesis provides evidence to support the emergence of long ncRNAs as a major functional component of the regulatory network that underpins differentiation and development in mammals and other complex organisms.

No description available.

Vitamin A and its derivatives, collectively termed retinoids, are essential for proper growth and development as well as maintenance of homeostasis in the adult. Retinoic acid (RA), the major biologically active vitamin A metabolite, is well characterized for its crucial roles in gene activation during embryogenesis. Our lab had previously performed a microarray analysis to identify genes induced by exogenous RA in the tailbud of early mouse embryos. Three genes were chosen from the microarray results for further investigation; Germ Cell Nuclear Factor (GCNF/NR6A1), Prickle1 (Pk1) and Ror2, the latter of which are known members of the planar cell polarity (PCP) pathway. These genes were further examined for RA regulation by embryo culture and RT-PCR, which strongly supported a direct regulatory mechanism of NR6A1 by RA. Further analysis aiming to identify a functional response element in the promoter of the targets was attempted, including chromatin immunoprecipitation (ChIP), made possible by the generation and characterization of a highly specific antibody against RARγ. This antibody was used in a ChIP promoter walk, which identified regions on target gene promoters that are occupied by RARγ in vivo, and therefore likely harbor RA response elements.

Vitamin A and its derivatives, collectively termed retinoids, are essential for proper growth and development as well as maintenance of homeostasis in the adult. Retinoic acid (RA), the major biologically active vitamin A metabolite, is well characterized for its crucial roles in gene activation during embryogenesis. Our lab had previously performed a microarray analysis to identify genes induced by exogenous RA in the tailbud of early mouse embryos. Three genes were chosen from the microarray results for further investigation; Germ Cell Nuclear Factor (GCNF/NR6A1), Prickle1 (Pk1) and Ror2, the latter of which are known members of the planar cell polarity (PCP) pathway. These genes were further examined for RA regulation by embryo culture and RT-PCR, which strongly supported a direct regulatory mechanism of NR6A1 by RA. Further analysis aiming to identify a functional response element in the promoter of the targets was attempted, including chromatin immunoprecipitation (ChIP), made possible by the generation and characterization of a highly specific antibody against RARγ. This antibody was used in a ChIP promoter walk, which identified regions on target gene promoters that are occupied by RARγ in vivo, and therefore likely harbor RA response elements.

During mouse development, the primordia of the posterior body including the trunk and tail tissues of the embryo forms largely from a bipotential cell population that resides in the posterior growth zone in vertebrate embryos. This bipotential cell population contains neuromesodermal progenitors (NMP) which are found in the tail bud which replaces the primitive streak after gastrulation and contributes to axial elongation by the formation of both the spinal cord and paraxial mesoderm derivatives. The three vertebrate Cdx genes, Cdx1, Cdx2 and Cdx4, encode transcription factors that play important roles in axial elongation since the triple Cdx mutant embryos fail to generate any tissue posterior to the occipital primordia. A comparison of Cdx mutant phenotypes suggests that Cdx2 is the most important contributor to axial elongation since Cdx2 heterozygous mutants exhibit foreshortened tails and Cdx2 conditional mutants exhibit axial truncation and complete loss of tail bud structures. Cdx2 target genes, such as Wnt3a, Cyp26a1 and T, are also essential for axial elongation. Cdx1 null mutants are viable and exhibit homeosis of cervical and anterior thoracic vertebrae, while Cdx4 null mutants are phenotypically normal. In addition, it has been shown that simultaneous loss of multiple copies of Cdx alleles disrupts axial elongation more severely than each single mutation which suggests there is overlapping function among the Cdx family. The genetic network underlying regulation of axial elongation by the Cdx family is not fully understood due in part to this functional overlap. In this thesis, I employed a conditional Cre-loxP system to derive conditional mutants lacking all Cdx functions. Additionally, Pax2-GFP transgenic mice where GFP is expressed under the control of Pax2 locus were used to enrich tail bud NMP cells for RNA-seq and ChIP-seq analysis for Cdx2. Using this approach, I revealed new target genes and pathways that are regulated by Cdx members and likely involved in axial elongation.

No description available.

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.

Imprinted genes are a subset of loci, positioned on autosomes and the X-chromosome, which are expressed monoallelically in a parent-of-origin specific manner. The influence of such genes on the regulation of embryonic growth and postnatal energy homeostasis is well established. The parental conflict hypothesis predicts that, in utero, paternally-expressed genes will promote maternal resource acquisition and thus growth, whereas maternally-expressed genes will oppose this action, restricting resource investment in a single brood in the interests of the lifetime reproductive success of the mother. Grb10 is an imprinted gene which encodes the cytoplasmic adaptor protein Growth factor receptor bound protein 10. In the majority of tissues, Grb10 is expressed from the maternally-derived chromosome. Consistent with conflict theory, transgenic mice inheriting a disrupted Grb10 allele through the maternal line (Grb10Δ2-4m/+) exhibit embryonic overgrowth, although the mechanisms and signalling pathways responsible for this effect are unclear. Grb10Δ2-4m/+ mice also demonstrate enhanced insulin signalling and improved whole body glucose clearance, consistent with the established role of imprinted genes in the regulation of postnatal metabolism. An integrated LacZ gene-trap in the Grb10Δ2-4 allele failed to fully recapitulate endogenous Grb10 expression, notably within the central nervous system. To address this issue, a second transgenic mouse line, Grb10KO, was created. This allele produced strong LacZ reporter expression in the central nervous system, but only when transmitted through the paternal line (Grb10KO+/p), establishing Grb10 as the only known imprinted gene with a reciprocal imprinting profile between the central nervous system and peripheral tissues. Grb10KO+/p mice exhibit a social dominance phenotype, suggesting distinct roles for maternally- and paternally-expressed Grb10, consistent with their respective sites of expression. The current study characterised the Grb10KO allele at the genetic level, and in doing so, revealed a phenotypic difference between Grb10KOm/p and Grb10Δ2-4m/p mice for which a possible explanation was provided. Importantly, with this knowledge, the current study elucidated the genetic and molecular basis for inconsistencies in reporter expression between the two transgenic lines, identifying a novel tissue-specific enhancer element at the locus. In addition to the central nervous system, this enhancer appeared to be active in the mammary epithelium, identifying a novel site of Grb10 expression, which was pregnancy-dependent and specifically from the maternally-inherited chromosome. Characterisation of the functional significance of expression in this tissue revealed that maternally-expressed Grb10 mediates a supply/demand system between lactating mother and suckling pup, acting as a supply promoter and demand suppressor. This role is inconsistent with conflict theory, but suggests the maintenance of the Grb10 imprint in the mammary epithelium might be associated with improved coadaptiveness between mother and offspring. Intriguingly, in utero, Grb10 is both a demand and supply suppressor. When considered together, these findings suggest a wider role for maternally-expressed Grb10 in the homeostatic control of growth and achievement of optimal fitness.