Cilia are microtubule-based organelles that project into the extracellular space and have various functions including transducing sensory information, regulating developmental signalling pathways, and generating directed fluid flow, making them important regulators of vertebrate development and homeostasis. Despite their importance, there are many aspects of cilia formation and function that remain poorly understood. The planar cell polarity (PCP) pathway is a branch of Wnt signalling that provides positional information to cells and is required for polarized morphogenic cell movements. Previous studies of PCP effector proteins suggested that PCP signalling was required for cilia formation. However, these proteins are not specific to the PCP pathway and are shared with other branches of Wnt signalling. To determine the role of a core and specific PCP regulator on ciliogenesis, I examined maternal-zygotic (MZ) vangl2 zebrafish mutants using an in vivo marker of cilia, Arl13b-GFP. Analysis of MZvangl2 mutants revealed that PCP is not required for cilia formation but is required for the posterior tilting and posterior positioning of motile cilia, essential for directed fluid flow. A parallel branch of studies suggested that cilia are actually required to regulate PCP signalling because defects in PCP-mediated morphogenic movements were observed with the knockdown of certain proteins that localize at or near cilia or basal bodies. To determine whether cilia were required to establish PCP, I generated MZ-intraflagellar transport-88 (IFT88) mutants, where ciliogenesis is completely abolished. Analysis of MZift88 mutants revealed that cilia are not directly required for PCP-mediated morphogenic movements. However, I observed that MZift88 mutants had defects in oriented cell divisions (OCD) occurring during gastrulation. Remarkably, these divisions occur prior to cilia formation, suggesting a cilia-independent role for IFT proteins in cell divisions, which may have important consequences on the interpretation of the role of cilia in disease.

“The scientist, by the very nature of his commitment, creates more and more questions, never fewer. Indeed the measure of our intellectual maturity, one philosopher suggests, is our capacity to feel less and less satisfied with our answers to better problems.” ~G.W. Allport, Becoming, 1955 It will be interesting to look back at this thesis in a few decades and reflect on how the questions and interpretation of data in the field of developmental biology have changed. Indeed, a biologist currently in their twilight years might reflect on their youth, before the discovery of hereditary material, and compare that bookend with the range of genome sequences and related knowledge currently available. How long will it take before this thesis reads like a debate about whether the male or female contributed the ‘homunculus,’ a miniature preformed human to the embryo that grows into an adult? In this thesis I asked three related questions: whether the role of Oct4 during embryogenesis provides insight into its contribution to pluripotency; how surfaceome changes contribute to functional maturation of neural stem cells and to what extent the murine genome is imprinted. Our data indicate that Oct4 is required for posterior expansion. We propose that the function of the protein is conserved, but that its expression has been coopted to yield different cell types based on its combination with different factors. We show that fundamental aspects of cell biology are altered during the maturation from pluripotent populations to neural stem cells, and identify mediators of proliferation, survival and adhesion that distinguish neural stem cell regulation from their precursors. Finally, we validated discovery of a dozen novel imprinted transcripts using a genomic approach. These discoveries will contribute to a holistic view of the causes and consequences of imprinting, but do not support a paradigm shift in the scale and consequences of imprinting.

“The scientist, by the very nature of his commitment, creates more and more questions, never fewer. Indeed the measure of our intellectual maturity, one philosopher suggests, is our capacity to feel less and less satisfied with our answers to better problems.” ~G.W. Allport, Becoming, 1955 It will be interesting to look back at this thesis in a few decades and reflect on how the questions and interpretation of data in the field of developmental biology have changed. Indeed, a biologist currently in their twilight years might reflect on their youth, before the discovery of hereditary material, and compare that bookend with the range of genome sequences and related knowledge currently available. How long will it take before this thesis reads like a debate about whether the male or female contributed the ‘homunculus,’ a miniature preformed human to the embryo that grows into an adult? In this thesis I asked three related questions: whether the role of Oct4 during embryogenesis provides insight into its contribution to pluripotency; how surfaceome changes contribute to functional maturation of neural stem cells and to what extent the murine genome is imprinted. Our data indicate that Oct4 is required for posterior expansion. We propose that the function of the protein is conserved, but that its expression has been coopted to yield different cell types based on its combination with different factors. We show that fundamental aspects of cell biology are altered during the maturation from pluripotent populations to neural stem cells, and identify mediators of proliferation, survival and adhesion that distinguish neural stem cell regulation from their precursors. Finally, we validated discovery of a dozen novel imprinted transcripts using a genomic approach. These discoveries will contribute to a holistic view of the causes and consequences of imprinting, but do not support a paradigm shift in the scale and consequences of imprinting.

Gene-specific trinucleotide repeat expansions are the cause of an ever-growing number of disorders, including myotonic dystrophy type 1 (DM1) and spinocerebellar ataxia type 7 (SCA7). Both DM1, and SCA7 are characterized by large differences in repeat numbers between tissues that are differentially affected, indicating tissue-specific mechanisms of repeat instability. The mechanism(s) of both somatic as well as germline instability are complex and still poorly understood, with evidence supporting the contribution of cis-elements, trans factors, and DNA metabolic processes that are hypothesized to involve alternative structure formation within the DNA tract. This thesis involves investigations into the role of a particular cis-element (CTCF) on instability, as well as the detection of slipped-DNAs in patient tissues and the presence of rare mutations within those same tissues. Here I identify the first endogenous cis-element reported to show regulation of instability at a trinucleotide repeat disease locus, the DNA binding site for the insulator protein CCCTC- binding factor (CTCF) downstream of the SCA7 repeat. Using a mouse model with a mutation in the CTCF binding domain, I show that the loss of CTCF binding stimulates germline and somatic instability in a tissue-specific and age-dependent manner. The binding of CTCF likely protects the repeat tract from expansion by shielding it from other elements that may contribute to expansion. DNA metabolic processes such as replication, repair, and transcription likely play a role in repeat expansion at disease loci, with the general mechanism hypothesized to be the extrusion and aberrant repair of slipped-DNA structures during the unwinding process for each. While characterizing DM1 patient tissues in order to isolate slipped-DNA structures, I characterized two non-CTG repeat insertion mutations that had completely replaced the repeat tract in a small subset of cells in only two tissues in one patient. Given the hypermutable nature of expanded repeat tracts, it is possible that these types of mutations are more common than suspected. Finally, I report on the detection and isolation of slipped-DNA structures from the endogenous DM1 locus from patient tissues. The slip-outs appear as clusters along a length of DNA, rather than single isolated slip-outs, and more unstable tissues contain greater amounts of slipped-DNA compared to more stable tissues. This detection implies that slipped-DNA structures are not merely transient intermediates in the mutation and expansion process as has long been assumed, but remain within the DNA at detectable levels. The data reported herein both furthers our understanding of trinucleotide repeat instability, and additionally confirms the decades-long hypothesis that slipped-DNAs are in fact forming in patient tissues in a tissue-specific manner.

In vertebrates, the heart is the first organ to form and function. The basic steps and molecular pathways involved in heart development are highly conserved. Myocardial progenitor-fated cells are among the first cells to migrate during gastrulation away from the primitive streak. These cells move bilaterally to populate the heart-forming region (HFR) in the anterior lateral plate mesoderm (ALPM). Once cells have reached the HFR, they receive the signals necessary to differentiate into myocardial progenitor cells. It is clear that the development of myocardial progenitor cells entails the migration of cells from the lateral embryonic margin to the ALPM. However, it is unclear whether cells are specified for a myocardial progenitor fate early in embryogenesis, a step that may promote their migration specifically to the ALPM, or whether the migration of cells to the ALPM alone is sufficient for differentiation into myocardial progenitor cells. A zebrafish mutant, grinch (grn), was indentified in which there is a defect in the development of myocardial progenitor cells. The mutation resulting in the grn phenotype was mapped to the gene encoding the G protein-coupled receptor Apelin receptor b (Aplnrb). I have used the aplnrb mutant embryo, as well as morpholino-mediated knockdown (morphant embryos) of aplnrb, and its paralog aplnra, to determine the function of Aplnr signaling in myocardial progenitor development. Results demonstrate that Aplnr signaling is necessary for the migration of cells from the lateral embryonic margin of the zebrafish embryo to the heart-forming region. Interestingly, this entails a novel cell-non-autonomous function for Aplnr signaling. Furthermore, both the only identified ligand for the receptor, Apelin, and the canonical mediators of Aplnr signaling, Gαi/o proteins, appear to be dispensable for this process. Loss of Aplnr signaling also appears to affect embryonic patterning of the early embryo through subtle perturbations of Nodal, Wnt, and Bmp signaling and attenuation of Nodal signaling can partially recapitulate the aplnr morphant cardiac phenotype. Taken together, my results suggest that Aplnr signaling plays a role in creating an environment that allows for the migration of cells to the heart-forming region, possibly through the regulation of early embryonic patterning.

Previous studies of genes associated with retroviral-induced neoplasia have provided the foundation for much of our current knowledge of both tumor suppressor and oncogenes, and have contributed to our understanding of both gene function and malignant transformation. The study of Friend virus-induced erythroleukemia, a well-studied example of multistage malignancy, has led to the identification of several oncogenes, including the Ets transcription factor, fli-1. Fli-1 plays a vital role in hematopoiesis, and vasculogenesis through the transcriptional regulation of its target genes, some of which are critical for the control of cellular proliferation, differentiation, and survival. The aberrant regulation of Fli-1 is associated with a number of cancers and human diseases, including erythroleukemia, Ewing’s sarcoma, lupus, and Jacobsen or Paris Trousseau syndrome. The essential goal set out to be achieved by the research presented herein is to establish a better understanding of both the oncogenic and developmental roles of Fli-1 by investigating the molecular basis by which its deregulated expression leads to fundamental aberration in the fine balance between proliferation and differentiation.

Gene-specific trinucleotide repeat expansions are the cause of an ever-growing number of disorders, including myotonic dystrophy type 1 (DM1) and spinocerebellar ataxia type 7 (SCA7). Both DM1, and SCA7 are characterized by large differences in repeat numbers between tissues that are differentially affected, indicating tissue-specific mechanisms of repeat instability. The mechanism(s) of both somatic as well as germline instability are complex and still poorly understood, with evidence supporting the contribution of cis-elements, trans factors, and DNA metabolic processes that are hypothesized to involve alternative structure formation within the DNA tract. This thesis involves investigations into the role of a particular cis-element (CTCF) on instability, as well as the detection of slipped-DNAs in patient tissues and the presence of rare mutations within those same tissues. Here I identify the first endogenous cis-element reported to show regulation of instability at a trinucleotide repeat disease locus, the DNA binding site for the insulator protein CCCTC- binding factor (CTCF) downstream of the SCA7 repeat. Using a mouse model with a mutation in the CTCF binding domain, I show that the loss of CTCF binding stimulates germline and somatic instability in a tissue-specific and age-dependent manner. The binding of CTCF likely protects the repeat tract from expansion by shielding it from other elements that may contribute to expansion. DNA metabolic processes such as replication, repair, and transcription likely play a role in repeat expansion at disease loci, with the general mechanism hypothesized to be the extrusion and aberrant repair of slipped-DNA structures during the unwinding process for each. While characterizing DM1 patient tissues in order to isolate slipped-DNA structures, I characterized two non-CTG repeat insertion mutations that had completely replaced the repeat tract in a small subset of cells in only two tissues in one patient. Given the hypermutable nature of expanded repeat tracts, it is possible that these types of mutations are more common than suspected. Finally, I report on the detection and isolation of slipped-DNA structures from the endogenous DM1 locus from patient tissues. The slip-outs appear as clusters along a length of DNA, rather than single isolated slip-outs, and more unstable tissues contain greater amounts of slipped-DNA compared to more stable tissues. This detection implies that slipped-DNA structures are not merely transient intermediates in the mutation and expansion process as has long been assumed, but remain within the DNA at detectable levels. The data reported herein both furthers our understanding of trinucleotide repeat instability, and additionally confirms the decades-long hypothesis that slipped-DNAs are in fact forming in patient tissues in a tissue-specific manner.

In vertebrates, the heart is the first organ to form and function. The basic steps and molecular pathways involved in heart development are highly conserved. Myocardial progenitor-fated cells are among the first cells to migrate during gastrulation away from the primitive streak. These cells move bilaterally to populate the heart-forming region (HFR) in the anterior lateral plate mesoderm (ALPM). Once cells have reached the HFR, they receive the signals necessary to differentiate into myocardial progenitor cells. It is clear that the development of myocardial progenitor cells entails the migration of cells from the lateral embryonic margin to the ALPM. However, it is unclear whether cells are specified for a myocardial progenitor fate early in embryogenesis, a step that may promote their migration specifically to the ALPM, or whether the migration of cells to the ALPM alone is sufficient for differentiation into myocardial progenitor cells. A zebrafish mutant, grinch (grn), was indentified in which there is a defect in the development of myocardial progenitor cells. The mutation resulting in the grn phenotype was mapped to the gene encoding the G protein-coupled receptor Apelin receptor b (Aplnrb). I have used the aplnrb mutant embryo, as well as morpholino-mediated knockdown (morphant embryos) of aplnrb, and its paralog aplnra, to determine the function of Aplnr signaling in myocardial progenitor development. Results demonstrate that Aplnr signaling is necessary for the migration of cells from the lateral embryonic margin of the zebrafish embryo to the heart-forming region. Interestingly, this entails a novel cell-non-autonomous function for Aplnr signaling. Furthermore, both the only identified ligand for the receptor, Apelin, and the canonical mediators of Aplnr signaling, Gαi/o proteins, appear to be dispensable for this process. Loss of Aplnr signaling also appears to affect embryonic patterning of the early embryo through subtle perturbations of Nodal, Wnt, and Bmp signaling and attenuation of Nodal signaling can partially recapitulate the aplnr morphant cardiac phenotype. Taken together, my results suggest that Aplnr signaling plays a role in creating an environment that allows for the migration of cells to the heart-forming region, possibly through the regulation of early embryonic patterning.

Cell migration and extension is essential for development. The ability of a cell or cell extension to reach its target is dependent on spatial cues and receptors that translate positional information into directed plasma membrane extension. For example, the UNC-40/DCC receptor is required to direct circumferential migrations towards the source of the ligand, UNC-6/Netrin, expressed at the ventral midline. To better understand the process of cell extension, I used a specialized process called muscle arms as a model system. In C. elegans, body wall muscles (BWMs) extend membrane projections called muscle arms to the nearest nerve cord at the midline. These muscle arms harbor the postsynaptic element of the neuromuscular junction and extend in a stereotypical and regulated manner. In a screen for muscle arm development defective (Madd) mutants, I isolated madd-2, a novel component of the UNC-40 pathway. MADD-2 is a C-1 TRIM protein and functions cell-autonomously to direct numerous muscle and axon extensions to the ventral midline of worms. In a striking analogy, mutations in a human homologue of MADD-2, MID1, cause numerous ventral midline defects that culminate as Opitz Syndrome. How MID1 regulates midline development is unclear. MADD-2 enhances UNC-40 pathway activity by facilitating the physical interaction between UNC-40 and the downstream Rho-GEF, UNC-73. It is possible that MID1 may mediate the function of a DCC-like pathway at the ventral midline of humans. This work provides the first indication that C1-TRIM proteins may have a conserved biological role of regulating midline-oriented development events and may provide key insights into the role of MID1 in the pathogenesis of Opitz syndrome.

One percent of children in North America may be affected by fetal alcohol spectrum disorder (FASD). FASD remains difficult to diagnose because confirmation of maternal alcohol use is a diagnostic criterion, and women consuming alcohol during pregnancy are reluctant to divulge this information for fear of stigmatization and losing custody of the child. Consequently, using a biomarker to assess alcohol exposure would provide a tremendous advantage. Recently, the measurement of fatty acid ethyl ester (FAEE) in hair has provided a powerful tool for assessing alcohol exposure. My thesis fills a translational gap of research between the development of the FAEE hair test and its application in the context of FASD. The guinea pig has been a critical model for FASD research, in which FAEE hair analysis has previously distinguished ethanol-exposed dams/offspring from controls. My first study, reports a positive dose-concentration relationship between alcohol exposure and hair FAEE, in the human, and the guinea pig. Humans also displayed over an order of magnitude higher FAEE incorporation per equivalent alcohol exporsure, suggesting that the test will be a sensitive clinical marker of fetal alcohol exposure. My second study utilized multi-coloured rats to investigate the potential of a hair-colour bias, as has been reported for other clinical hair assays; no evidence of bias is reported here. My third study is the first to examine the clinical use of the FAEE hair test in parents at high risk of having children with FASD. Over one third of parents tested positive for excessive alcohol use. Parents were investigated by social workers working for child protection services, and my fourth study reports that hair FAEE results agree with social worker reports. Individuals highly suspected of abusing alcohol were at a significantly greater risk of testing positive, whereas individuals tested based on other reasons (such as to cover all bases) were negatively associated with testing positive. The last study of my thesis, confirmed an association between alcohol and drug use by parents at high risk for having children with FASD, posing an added risk to children. This work helps bridge a gap in translational research, suggesting that the FAEE hair test has potential for use in FASD diagnosis and research.