<p>Heavy metals polluting our environment cause concern for developing organisms. Among them, cadmium with extremely slow elimination from the body, causes lower birth weight in humans but has not been classify as a human teratogen. Studies in different laboratory animals have shown that cadmium indeed is a potent teratogen. Exposure to cadmium during early mouse embryonic stages (e.g. day 7-8 post-coitus) interferes with the closure of the anterior neural pore producing exencephalic embryos. The underlying mechanisms are not understood, but the heavy accumulation of cadmium in extra- and intraembryonic endoderm and chorioallantoic placenta, however not in the neuroepithelium, suggests that the effects on neural tube closure is due to indirect mechanisms. In this thesis, the disruption in the mouse embryo at the time of neural tube closure of the hierarchies of some signalling pathways and gene regulatory networks that control embryonic development has been studied after cadmium exposure. Cadmium was shown to cause DNA damage as measured by Comet assay, and to activate genes and proteins in the apoptotic pathways (<i>p53, p21, Bcl-2, Bax</i>, and caspase-3), increasing the number of apoptotic cells mostly in areas of physiological cell death, especially in the neuroepithelium. Many of these effects could be reversed by zinc pre-treatment, known to counteract the teratogenic effect of cadmium. Cadmium was also shown to affect Zn-transport and –regulatory proteins in the embryo, but perhaps more importantly in yolk sac placenta, and in the decidua (ZnT-1, MT-I, and ZIP-4). Using gene arrays, cadmium was found to considerably affect gene expression of rather few genes, such as those of metallothioneins and stress-related proteins, supporting in principle an extraembryonic site of action of cadmium. In addition, a number of genes expressed in the anterior visceral endoderm (<i>Hesx1, HNF3β, Cerl, Otx2</i> and <i>Sox2</i>) where cadmium accumulates, and known to signal to the anterior neuroepithelium, was affected by cadmium. This finding may suggest a new principle for chemical teratogenesis.</p>

Heavy metals polluting our environment cause concern for developing organisms. Among them, cadmium with extremely slow elimination from the body, causes lower birth weight in humans but has not been classify as a human teratogen. Studies in different laboratory animals have shown that cadmium indeed is a potent teratogen. Exposure to cadmium during early mouse embryonic stages (e.g. day 7-8 post-coitus) interferes with the closure of the anterior neural pore producing exencephalic embryos. The underlying mechanisms are not understood, but the heavy accumulation of cadmium in extra- and intraembryonic endoderm and chorioallantoic placenta, however not in the neuroepithelium, suggests that the effects on neural tube closure is due to indirect mechanisms. In this thesis, the disruption in the mouse embryo at the time of neural tube closure of the hierarchies of some signalling pathways and gene regulatory networks that control embryonic development has been studied after cadmium exposure. Cadmium was shown to cause DNA damage as measured by Comet assay, and to activate genes and proteins in the apoptotic pathways (p53, p21, Bcl-2, Bax, and caspase-3), increasing the number of apoptotic cells mostly in areas of physiological cell death, especially in the neuroepithelium. Many of these effects could be reversed by zinc pre-treatment, known to counteract the teratogenic effect of cadmium. Cadmium was also shown to affect Zn-transport and –regulatory proteins in the embryo, but perhaps more importantly in yolk sac placenta, and in the decidua (ZnT-1, MT-I, and ZIP-4). Using gene arrays, cadmium was found to considerably affect gene expression of rather few genes, such as those of metallothioneins and stress-related proteins, supporting in principle an extraembryonic site of action of cadmium. In addition, a number of genes expressed in the anterior visceral endoderm (Hesx1, HNF3β, Cerl, Otx2 and Sox2) where cadmium accumulates, and known to signal to the anterior neuroepithelium, was affected by cadmium. This finding may suggest a new principle for chemical teratogenesis.

RIC-8B é um fator trocador de nucleotídeo de guanina (GEF) predominantemente expresso em neurônios olfatórios maduros de camundongos adultos. Trabalhos desenvolvidos em nosso laboratório mostraram que RIC-8B interage com G&#945;olf e G&#947;13, duas subunidades de proteína G que estão enriquecidas nos cílios dos neurônios olfatórios, onde participam da transdução do sinal de odorantes. In vitro, RIC-8B é capaz de amplificar a sinalização de receptores olfatórios através de G&#945;olf, no entanto, seu papel fisiológico ainda é desconhecido. Para determinar a função desempenhada por essa proteína in vivo, nós utilizamos a tecnologia de Gene Trap com o objetivo de produzir um camundongo knockout para Ric-8B. Apesar de a expressão de Ric-8B ser restrita a poucos tecidos no camundongo adulto, descobrimos que homozigotos para a mutação em Ric-8B são inviáveis e morrem por volta do dia embrionário E10,5. Além disso, são menores e apresentam evidente falha no fechamento do tubo neural na região cranial (exencefalia). Utilizamos o gene repórter &#946;-galactosidase expresso pelo alelo mutado para determinar o padrão de expressão de Ric-8B em embriões durante o desenvolvimento. Observamos que, no estágio E8,5, Ric-8B é expresso nas pregas neurais da região cefálica e na notocorda. De E9,5 a E12,5, a expressão de Ric-8B é detectada predominante no assoalho da placa. Esse padrão de expressão se assemelha ao de outro gene importante para a embriogênese, Sonic hedgehog (Shh). SHH é um morfógeno diretamente responsável pela padronização dorsoventral do sistema nervoso central e sua sinalização depende de cílio primário. Cílio primário é uma organela baseada em microtúbulos que se projeta da superfície da maioria das células de mamíferos e funciona como um centro de sinalização intracelular. Nossos dados mostram que fibroblastos embrionários Ric-8B-/- formam cílios primários, assim como alguns tecidos do embrião. Além disso, não encontramos alterações na sinalização de Shh em embriões homozigotos mutantes. No entanto, observamos que esses embriões apresentam apoptose aumentada em células migratórias da crista neural cranial. Shh é importante para a sobrevivência de células da crista neural migratória, sugerindo um possível papel para Ric-8B a downstream da sinalização de SHH. / Ric-8B is a guanine nucleotide exchange factor (GEF) which is predominantly expressed in mature olfactory sensory neurons in adult mice. We have previously shown that RIC-8B interacts with both G&#945;olf and G&#947;13, two G protein subunits, which are enriched in olfactory cilia and are required for odorant signal transduction. In vitro, RIC-8B is able to amplify odorant receptor signaling through G&#945;olf, however, its physiological role remains unknown. To determine the role played by RIC-8B in vivo we used the Gene trap technology to generate a Ric-8B knockout mouse. We found that, despite the limited distribution of Ric-8B gene expression in adult mice, Ric-8B homozygous mutants are not viable and die around the E10,5 stage. Mutant embryos are also smaller and fail to close the neural tube at the cranial region (exencephaly). We used the activity of the &#946;-galactosidase reporter gene to determine the pattern of expression of the Ric-8B gene in heterozygous embryos. At E8,5 the Ric-8B gene is expressed in the notochord and neural folds of the cephalic regions. From E9,5 to E12,5 Ric-8B is predominantly expressed in the floor plate, in a pattern that strongly resembles the one shown by Sonic hedgehog (Shh). SHH is a morphogen directly responsible for the dorsoventral patterning of the central nervous system and its signaling depends on primary cilia. Primary cilia are microtubule-based organelles that protrude from the surface of mammalian cells and act as a signaling center. We show that Ric-8B-/- embryonic fibroblasts and some embryonic tissues grow primary cilia normally. In addition, we did not find alterations in the SHH signaling of homozygous mutants. Instead, we found an increased apopotosis in migratory cells of the cranial neural crest in these embryos. Shh is an important factor to survival of neural crest cells, suggesting a role for RIC-8B downstream of the SHH signaling.

RIC-8B é um fator trocador de nucleotídeo de guanina (GEF) predominantemente expresso em neurônios olfatórios maduros de camundongos adultos. Trabalhos desenvolvidos em nosso laboratório mostraram que RIC-8B interage com G&#945;olf e G&#947;13, duas subunidades de proteína G que estão enriquecidas nos cílios dos neurônios olfatórios, onde participam da transdução do sinal de odorantes. In vitro, RIC-8B é capaz de amplificar a sinalização de receptores olfatórios através de G&#945;olf, no entanto, seu papel fisiológico ainda é desconhecido. Para determinar a função desempenhada por essa proteína in vivo, nós utilizamos a tecnologia de Gene Trap com o objetivo de produzir um camundongo knockout para Ric-8B. Apesar de a expressão de Ric-8B ser restrita a poucos tecidos no camundongo adulto, descobrimos que homozigotos para a mutação em Ric-8B são inviáveis e morrem por volta do dia embrionário E10,5. Além disso, são menores e apresentam evidente falha no fechamento do tubo neural na região cranial (exencefalia). Utilizamos o gene repórter &#946;-galactosidase expresso pelo alelo mutado para determinar o padrão de expressão de Ric-8B em embriões durante o desenvolvimento. Observamos que, no estágio E8,5, Ric-8B é expresso nas pregas neurais da região cefálica e na notocorda. De E9,5 a E12,5, a expressão de Ric-8B é detectada predominante no assoalho da placa. Esse padrão de expressão se assemelha ao de outro gene importante para a embriogênese, Sonic hedgehog (Shh). SHH é um morfógeno diretamente responsável pela padronização dorsoventral do sistema nervoso central e sua sinalização depende de cílio primário. Cílio primário é uma organela baseada em microtúbulos que se projeta da superfície da maioria das células de mamíferos e funciona como um centro de sinalização intracelular. Nossos dados mostram que fibroblastos embrionários Ric-8B-/- formam cílios primários, assim como alguns tecidos do embrião. Além disso, não encontramos alterações na sinalização de Shh em embriões homozigotos mutantes. No entanto, observamos que esses embriões apresentam apoptose aumentada em células migratórias da crista neural cranial. Shh é importante para a sobrevivência de células da crista neural migratória, sugerindo um possível papel para Ric-8B a downstream da sinalização de SHH. / Ric-8B is a guanine nucleotide exchange factor (GEF) which is predominantly expressed in mature olfactory sensory neurons in adult mice. We have previously shown that RIC-8B interacts with both G&#945;olf and G&#947;13, two G protein subunits, which are enriched in olfactory cilia and are required for odorant signal transduction. In vitro, RIC-8B is able to amplify odorant receptor signaling through G&#945;olf, however, its physiological role remains unknown. To determine the role played by RIC-8B in vivo we used the Gene trap technology to generate a Ric-8B knockout mouse. We found that, despite the limited distribution of Ric-8B gene expression in adult mice, Ric-8B homozygous mutants are not viable and die around the E10,5 stage. Mutant embryos are also smaller and fail to close the neural tube at the cranial region (exencephaly). We used the activity of the &#946;-galactosidase reporter gene to determine the pattern of expression of the Ric-8B gene in heterozygous embryos. At E8,5 the Ric-8B gene is expressed in the notochord and neural folds of the cephalic regions. From E9,5 to E12,5 Ric-8B is predominantly expressed in the floor plate, in a pattern that strongly resembles the one shown by Sonic hedgehog (Shh). SHH is a morphogen directly responsible for the dorsoventral patterning of the central nervous system and its signaling depends on primary cilia. Primary cilia are microtubule-based organelles that protrude from the surface of mammalian cells and act as a signaling center. We show that Ric-8B-/- embryonic fibroblasts and some embryonic tissues grow primary cilia normally. In addition, we did not find alterations in the SHH signaling of homozygous mutants. Instead, we found an increased apopotosis in migratory cells of the cranial neural crest in these embryos. Shh is an important factor to survival of neural crest cells, suggesting a role for RIC-8B downstream of the SHH signaling.