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The Wnts, a family of secreted glycoprotein ligands, act through the frizzled (Fz) receptor, a family of seven-transmembrane (7TM) receptor proteins, to mediate intracellular signaling pathways that regulate cell fate determination, cell migration, or both. Whereas many molecular components of the Wnt signal transduction cascade have been identified, it remains unclear how the signal is transduced from the Fz receptors to the cytoplasm. To address this important question, a membrane-based yeast two-hybrid (MbY2H) screen was performed to identify potential Fz-interacting proteins. For this screen, the Frizzled7 (Fz7) protein was used as the bait and a mouse brain library was used the prey. This screen identified resistance to inhibitors of cholinesterase 8 homolog A (Ric8A), a 542–amino acid cytoplasmic protein, along with other proteins as putative Fz7-binding proteins. Ric8A had been studied previously in C. elegans and D. melanogaster for its function in regulating asymmetric cell division as a receptor-independent guanine nucleotide exchange factor (GEF) for Gα proteins. Additional studies in M. musculus and X. laevis further uncovered a role for this protein during gastrulation and neurulation; however, the mechanisms by which Ric8A regulated these processes remained unclear. In this thesis, I show Ric8A to be a bona fide binding partner for both Fz7; that Ric8A can also bind to the phosphoprotein Dishevelled (Dvl); and that both its interaction with Fz7 and Dvl is Wnt-regulated. The spatial and temporal mRNA expression pattern of the Xenopus homologue of Ric8A suggests a potential role in regulating Wnt signaling. The Xenopus homologue of Ric8A was cloned and gain-of-function and loss-of-function approaches in Xenopus uncovered a role for Ric8A in gastrulation and neural tube closure. Additionally, we found inhibition of Ric8A function mechanistically prevents activation of Rac1 which is required downstream of Wnt/Fz signaling during gastrulation. Overall, this study uncovers a novel regulator of Wnt signaling during early development / Biology

Neural tube defects (NTDs) are the second most common type of congenital birth defect that affect infants worldwide. There are several proposed mechanisms for NTDs, but no definitive mechanism has yet been described. One possible mechanism is oxidative disruption of normal developmental signaling. The purpose of this study is to culture whole mouse embryos with three common developmental toxicants, mono-2-ethylhexyl phthalate (MEHP, a plastic pollutant), Fumonisin B1 (FB1, a corn mold), and valproic acid (VPA, an anticonvulsant drug), all of which are known to cause NTDs, and compare their shifts in redox potential and changes to important signaling pathways through transcriptomic analysis. To determine if prevention of oxidative stress decreases the likelihood of developing an NTD, preventative measures were taken by pretreating pregnant dams with 3H-1,2-dithiole-3-thione (D3T), an effective nuclear factor erythroid 2-related factor 2 (NRF2) inducer. It was found that between the three chemicals, two genes were significantly dysregulated, Makorin-2 (MKRN2) and Microtubule Associated Protein-6 (MAP6), both of which are implicated in NTDs. After staining embryos with lysotracker red (for apoptosis) or DCP-Rho1 (for oxidative stress), it was found that D3T pretreatment generally lowers the amount of apoptosis and oxidative stress in toxicant treated embryos. However, it only decreased the prevalence of NTDs seen in embryos cultured with FB1 or VPA. These results demonstrate common mechanisms that are potentially related in the formation of NTD and will help to target possible preventative measures.

The teratogenic effects of valproic acid (VPA) are well known, though its teratogenic mechanism remains unknown. VPA induces oxidative stress, which may lead to double strand breaks (DSBs) in DNA. Though the cell may repair this damage via homologous recombination (HR) and non-homologous end joining (NHEJ), repair is not always error-free; genomic instability may arise from gene deletions, amplifications, rearrangements, and loss of heterozygosity. Such alterations may underpin VPAʼs teratogenicity. The present study evaluated VPAʼs ability to induce NHEJ and HR and characterized the changes in expression of two proteins key to HR (RAD51) and NHEJ (XRCC4). Using pKZ1 transgenic mice (C57BL/6 genetic background), we sought to measure NHEJ events via X-gal staining. Although consistent staining was observed in adult male brain (positive control), no staining was observed in embryos 12 or 24 hours after in utero exposure to a teratogenic dose of VPA (500 mg/kg, maternal subcutaneous dose) on gestational day 9 (GD9). To determine whether the lack of staining observed in embryos was due to low/absent expression of key DSB-repair proteins, we measured mRNA/protein expression of RAD51 and XRCC4 in C57BL/6, GD9-exposed embryos and maternal brain. One hour after treatment, XRCC4 was increased at the protein level in brain and embryo. RAD51 was not increased in embryos and not detected in adult brain. These data suggest that embryos do possess the protein mediators of NHEJ and HR and that VPA-induced changes in expression of XRCC4 may influence the type of repair pursued, potentially affecting DSB repair fidelity (accuracy). Determination of fidelity of VPA-induced HR was attempted with the Chinese hamster ovary cell line (CHO33) using DNA sequencing; low template concentration and purity precluded successful sequencing of DNA from recombinant colonies and the assessment of fidelity. Overall, these data demonstrate that the lack of X-gal staining observed in pKZ1 embryos is not due to an underexpression of at least one key protein in the NHEJ pathway. Furthermore, a VPA-induced change in the the type of repair pathway pursued by the embryo may have teratological implications. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-10-15 11:06:30.613

Scratch2 (Scrt2) é um fator de transcrição (FT) expresso em células neurais recém-pós-mitóticas. O mecanismo de regulação de sua transcrição permanece desconhecido. Nós buscamos por potenciais elementos cis-regulatórios (CR) e sítios de ligação para FT na região genômica de Scrt2. Nós testamos o efeito in vivo da região CR intrônica através de eletroporação em embrião de galinha. Esta CR intrônica levou à expressão de eGFP tubo neural, mas não gerou um padrão semelhante ao Scrt2 endógeno. Estes dados sugerem que esta região CR pode contribuir com o controle da expressão de Scrt2, mas requer regiões regulatórias adicionais. Nós também verificamos o papel de mAsh1, cPax6, cNgn2 e Brn na regulação da expressão de Scrt2 através da superexpressão destes no tubo neural embrionário. As análises foram realizadas por qPCR e hibridação in situ. A superexpressão de mAsh1 e cNgn2 levou a um leve aumento na expressão de cScrt2 visto através da hibridação in situ. Já a superexpressão de cPax6 e mBrn-Eng reduziu os níveis de cScrt2, com Brn apresentando efeitos mais severos. / Scratch2 (Scrt2) is a transcription factor expressed in early post-mitotic neural cells. The mechanisms that control its transcription remain unknown. We searched for potential cis-regulatory elements and putative transcription factors binding sites in the genomic region of cScrt2. Then, we tested the function of a candidate cis-regulatory intronic region through electroporation in chick embryos. This fragment drove eGFP expression in the neural tube of chick embryo, but its expression did not resemble the cScrt2 endogenous pattern. Thus, this cis-regulatory region likely requires the presence of other regulatory regions. We also evaluated the role of mAsh1, cPax6, cNgn2 e Brn in regulating cScrt2 expression through overexpression of these factors in chick neural tubes and subsequent qPCR and in situ hybridization. Overexpression of mAsh1 and cNgn2 increased slightly cScrt2 expression level as seen by in situ hybridization. Overexpression of cPax6 and mBrn-Eng, reduced cScrt2 levels, with more severe effects with Brn.

General toxicity evaluation and risk assessment for human exposure is essential when developing new pharmaceuticals and chemicals. Developmental toxicology is an important part of this risk assessment which consumes large resources and many laboratory animals. The prediction of developmental toxicity could potentially be assessed in vitro using embryo-derived pluripotent stem cells for lead characterization and optimization. This thesis explored the potential of short-time assays with pluripotent stem cells of embryonic origin using toxicogenomics. Three established pluripotent stem cell lines; P19 mouse embryonal carcinoma (EC) cells, R1 mouse embryonic stem (mES) cells, and SA002 human embryonic stem (hES) cells were used in the studies. Valproic acid (VPA), an antiepileptic drug which can cause the neural tube defects spina bifida in human and exencephaly in mouse, was used together with microarrays to investigate the global transcriptional response in pluripotent stem cells using short-time exposures (1.5 - 24 h). In addition to VPA, three closely related VPA analogs were tested, one of which was not teratogenic in mice. These analogs also differed in their ability to inhibit histone deacetylase (HDAC) allowing this potential mechanism of VPA teratogenicity to be investigated. The results in EC cells indicated a large number of genes to be putative VPA targets, many of which are known to be involved in neural tube morphogenesis. When compared with data generated in mouse embryos, a number of genes emerged as candidate in vitro markers of VPA-induced teratogenicity. VPA and its teratogenic HDAC inhibiting analog induced major and often overlapping deregulation of genes in mES cells and hES cells. On the other hand, the two non-HDAC inhibiting analogs (one teratogenic and one not) had only minor effects on gene expression. This indicated that HDAC inhibition is likely to be the major mechanism of gene deregulation induced by VPA. In addition, a comparison between human and mouse ES cells revealed an overlap of deregulated genes as well as species specific deregulated genes.

Au cours de ce projet de thèse, j’ai étudié le lien fonctionnel entre la morphogénèse épithéliale et la signalisation Delta-Notch, dans le cadre de la formation du tube neural chez le poisson-zèbre. La signalisation Delta-Notch est primordiale pour le développement embryonnaire et le maintien de l’homéostasie des tissus adultes. De façon inattendue, j’ai observé suite à la perte-de-fonction de Mib une perte de la polarité apico-basale dans le neuro-épithélium de la moelle épinière embryonnaire. L’analyse plus poussée de ce phénotype m’a ensuite permis de montrer que l’activité de l’intégralité de la signalisation Notch est requise pour l’établissement de la polarité apico-basale dans le tube neural de poisson-zèbre. En effet, l’inhibition des ligands de Notch et des activateurs transcriptionnels situés en aval, Rbpja et Rbpjb, résulte en l’interruption de la polarité apico-basale. De plus, l’activation ectopique de Notch entraîne un sauvetage complet de la polarité apico-basale dans les embryons déplétés pour Mib. Finalement, le mutant Mib échoue à activer la transcription de protéines de polarité apicale Crumbs1 et Crumbs2a au cours de la formation du tube neural, ce qui suggèrerait que la signalisation Notch agit en amont des complexes de polarité. De façon surprenante, nous avons également montré que le composant de la signalisation Notch, Mib, affecte les mouvements de convergence-extension et la division cellulaire orientée, appelée C-divisions, durant la neurulation et la gastrulation à travers la signalisation PCP. Cet effet de Mib sur la PCP est indépendant de son rôle sur la signalisation Notch. Généralement, cette étude révèle un double-rôle de Mib. / In this Ph.D. project, I study the functional link between epithelial polarity and Delta-Notch signaling in the context of zebrafish neural tube morphogenesis. Notch signaling, one of the major signaling pathways and of prime importance in neurogenesis, has been widely studied for its function in cell fate specifications. Surprisingly, I found that the Notch signaling component Mindbomb (Mib) loss-of-function led to a loss of apico-basal polarity in the neuroepithelium of the embryonic spinal cord. I further explored that the activity of the entire Notch signaling pathway is actually required for the earliest steps of establishment of apico-basal polarity in the zebrafish neural tube. Indeed, inhibition of Notch ligands and downstream transcriptional activators Rbpja and Rbpjb resulted in a disruption of apico-basal polarity. Moreover, ectopic activation of Notch ensued to a complete rescue of apico-basal polarity in Mib loss of function embryos. Furthermore, Mib mutant embryos fail to upregulate the transcription of the apical polarity proteins Crumbs1 and Crumbs2a in the course of neural tube formation, suggesting that Notch signalling might act upstream of polarity complexes. Moreover, I found that Mib affects convergent-extension movements and oriented cell divisions during neurulation and gastrulation through an effect on planar cell polarity. Remarkably, this effect of Mib on PCP is independent of its role in Notch signaling. These results indicate a novel role of Mib in the regulation of PCP signaling. Altogether, this study revealed a dual role of Mib in the epithelial morphogenesis of the zebrafish neural tube.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-03-29T18:19:17Z No. of bitstreams: 1 ricardocampelodaconceicao.pdf: 3005415 bytes, checksum: 93485f733d9f5ab38c981b4eca76fc2b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-03-30T11:24:14Z (GMT) No. of bitstreams: 1 ricardocampelodaconceicao.pdf: 3005415 bytes, checksum: 93485f733d9f5ab38c981b4eca76fc2b (MD5) / Made available in DSpace on 2017-03-30T11:24:14Z (GMT). No. of bitstreams: 1 ricardocampelodaconceicao.pdf: 3005415 bytes, checksum: 93485f733d9f5ab38c981b4eca76fc2b (MD5) Previous issue date: 2009-12-17 / Os defeitos do fechamento do tubo neural (DFTN) figuram como a segunda maior causa de anomalias congênitas, e a sua incidência situa-se em torno de 1/1.000 nascidos vivos. Possui etiologia multifatorial, tendo na deficiência do ácido fólico seu principal fator de risco ambiental identificado. A suplementação de ácido fólico pré e pós-concepcional pode reduzir em até 70% o número de ocorrência e recorrência nos DFTN. O presente estudo tem o objetivo de avaliar o grau de conhecimento de médicos e enfermeiros obstetras sobre a prevenção dos DFTN. Foi realizado um estudo de natureza descritiva, do tipo transversal, em instituições públicas e privadas de saúde, maternidades e consultórios médicos da cidade de Juiz de Fora, MG, no período de dezembro de 2008 a junho de 2009. Participaram do estudo 118 voluntários (95 médicos obstetras e 23 enfermeiros obstetras), que responderam um questionário sem identificação, composto por 21 questões mistas, sobre a temática da prevenção dos DFTN. Foram analisadas as respostas em função das recomendações preconizadas pelo Ministério da Saúde em nosso país e das evidências científicas disponíveis. Verificou-se diferença entre as recomendações do Ministério da Saúde e o conhecimento baseado em evidências (p<0,001). Não foi encontrado nesse estudo, diferença entre o grau de conhecimento dos entrevistados quando comparado o nível de formação dos mesmos, o tempo que realizam atendimento pré-natal e entre as duas classes de profissionais avaliados. Houve diferença entre o grau de conhecimento relatado pelos profissionais e o grau de conhecimento avaliado pela pesquisa, baseado nas recomendações do Ministério da Saúde, e o que preconizam as evidências científicas (<0,001; p<0,001). Dos profissionais avaliados 94,1% relataram conhecer o papel do ácido fólico, embora apenas 11,9% afirmaram terem recebido capacitação sobre o assunto, e 64,2% relataram não saber ou erraram o período de início da suplementação do ácido fólico. Além disso, 68,6% responderam que a dose ideal deve ser de 5 mg/dia, e 68,6% também afirmaram que o período de suplementação deve ser o primeiro trimestre da gestação, conforme orientação do Ministério da Saúde. Os resultados demonstraram claramente a necessidade de melhor capacitação dos profissionais na formação acadêmica e na pós-graduação, além da manutenção permanente de programas de atualização para os envolvidos no atendimento à mulher. / Neural tube defects (NTD) ranks as the second leading cause of congenital anomalies and its incidence is situated around 1/1000 live births. It has a multifactorial etiology and the lack of folic acid is the main identified environmental risk factor. Supplementation of folic acid pre-and post-conception can reduces by up to 70% the number of occurrence and recurrence of NTD. Our study aimed to evaluate the knowledge level of physicians and obstetric nurses on the use of folic acid in the prevention of neural tube defects. We conducted a cross-sectional descriptive study, in public and private health facilities, in the city of Juiz de Fora, MG, from December 2008 to June 2009. The study included 118 volunteers (95 obstetricians and 23 obstetric nurses) who answered an anonymous questionnaire composed of 21 questions about the folic acid use in the prevention of NTD. We analyzed the responses according to the recommendations issued by the Ministry of Health and available scientific evidence. We found no difference between the knowledge levels of respondents when comparing the level of their training, experience in prenatal care and between the two classes of professionals evaluated. There was a difference between the reported knowledge level and the knowledge assessed level, based on the recommendations of the Ministry of Health (MOH), and the scientific evidence (p <0.001, p <0.001), respectively. It was also noticed the difference between the level of knowledge based on the recommendations of the MOH and the level of knowledge based on scientific evidences (p <0.001). The great majority of the study population (94.1%) has reported know the role of folic acid, while only 11.9% said they received training on the subject, and 64.2% has reported not know or had a incorrect answered about the period of early supplementation of folic acid. In addition, 68.6% answered that the optimal dose should be 5 mg / day and 68.6% also said that the period of supplementation should be the first trimester of pregnancy, as directed by MOH. The results clearly demonstrated the need for better training for professionals in academic and during graduate school, as well as ongoing maintenance of training programs for those involved in the women treatment.

Function of the adult nervous system relies on the appropriate establishment of neural circuits during embryogenesis. In vertebrates, the neurons that make up motor circuits form in distinct domains along the dorsoventral (DV) axis of the neural tube. Each domain is characterized by a unique combination of transcription factors (TFs) that promote a specific fate, while repressing the fates of adjacent domains. The prdm12 TF is required for the expression of eng1b and the generation of V1 interneurons in the p1 domain, but the details of its function remain unclear. We used CRISPR/Cas9 genome editing technology to generate the first germline mutants for the prdm12 gene and used this resource, together with classical luciferase reporter assays and co-immunoprecipitation experiments, to study prdm12b function in zebrafish. We also generated germline mutants for bhlhe22 and nkx6.1 to examine how these TFs act with prdm12b to control p1 formation. We find that prdm12b mutants lack eng1b expression in the p1 domain and also possess an abnormal Mauthner cell-dependent escape response. Using cell culture-based luciferase reporter assays, we demonstrate that Prdm12b acts as transcriptional repressor, most likely by recruiting EHMT2/G9a. We also show that the Bhlhe22 TF binds to the Prdm12b zinc finger domain to form a Bhlhe22:Prdm12b complex. However, bhlhe22 mutants display normal eng1b expression in the p1 domain. While prdm12 has been proposed to promote p1 fates by repressing expression of the nkx6.1 TF, we do not observe an expansion of the nkx6.1 domain upon loss of prdm12b function, nor is eng1b expression restored upon simultaneous loss of prdm12b and nkx6.1. We conclude that prdm12b germline mutations produce a phenotype that is indistinguishable from that of morpholino-mediated loss of prdm12 function. In terms of prdm12b function, our results indicate that Prdm12b acts as transcriptional repressor and interacts with both EHMT2/G9a and Bhlhe22. However, bhlhe22 function is not required for eng1b expression in vivo, perhaps indicating that other bhlh genes can compensate for its loss during embryogenesis. Lastly, we do not find evidence for nkx6.1 and prdm12b acting as a repressive pair in the formation of the p1 domain – suggesting that prdm12b is not solely required to repress non-p1 fates, but is also needed to promote p1 fates.

One of the first embryonic structures generated during early human development is the neural tube. The embryonic process of neurulation, including neural tube closure, is necessary for proper brain and spinal cord development, whereas improper closure leads to neural tube defects including anencephaly, spina bifida, and craniorachischisis. The mechanism by which these defects occur is unknown, but some evidence suggest that redox disruption may play a role. Cellular redox state is important in regulating key processes during neural tube closure, including differentiation, proliferation, gene expression, and apoptosis. This study aims to determine whether redox potential shifts and these key processes are affected similarly or differentially after treatment with three neural tube defect-inducing developmental toxicants: ceramide (C2), valproic acid (VPA), and fumonisin (FB1). Using the P19 cell model of neurogenesis, in both undifferentiated and terminally differentiated cells, we analyzed glutathione (GSH) redox (Eh) potential to evaluate the effect of each toxicant over time. We show that in C2 and VPA treated cultures an oxidizing shift occurs, but interestingly, FB1 treatment results in a reducing shift in embryonic GSH Eh as compared to untreated cultures. Using the chick embryo model, comparable redox shifts were observed as were seen in P19 cells, supporting similarity between the models. To better understand how differential shifts in the redox state can result in similar defects, we then examined potential variances in neuronal differentiation and cellular proliferation, survival, metabolism, adhesion, and gene expression under each treatment. We report changes to cellular and embryonic endpoints that support dysmorphogenesis, likely the result of oxidizing or reducing stress that altered redox state. These results support the need for broad comparative analyses such as this to determine whether toxicants that cause the same types of defects, whether NTDs or others, act through similar or different mechanisms. This can better inform preventative measures used to reduce the risk and occurrence of birth defects.