The zebrafish visual system relies on positional information in the retina and optic tectum, so that the spatial fidelity of light signals that enter the eye are preserved for visual processing. This positional information is essential for ordered topographic mapping of retinal ganglion cell axons. Spatial information in the retina and tectum relies on discrete signaling pathways that regulate polarized expression of axon guidance molecules in distinct domains in both the retina and tectum, thereby ensuring that accurate topographic maps are created. In this thesis, I have investigated the function of two families of transcription factors, Pbx and Meis, as well as a growth factor of the Bmp family, Gdf6a, in specifying positional identity in the zebrafish visual system. I demonstrate that two partially redundant pbx genes, pbx2 and pbx4, along with members of the meis family, are required for patterning of the dorsal retina and tectum in zebrafish. Embryos lacking these critical transcription factors exhibit retinal ganglion cell axon outgrowth errors, which are likely the result of tectal mis-patterning. Bone morphogenetic protein (Bmp) growth factors regulate dorsal retinal identity in vertebrate models, but the developmental timing of this signaling remains unclear. In this thesis, I investigate the functions of two zebrafish Bmps, Gdf6a and Bmp4, during initiation of dorsal retinal identity. Knockdown of zebrafish Gdf6a blocks initiation of dorsal marker expression, while knockdown of Bmp4 produces no discernable retinal phenotype. These data, combined with analyses of embryos ectopically expressing Bmps, demonstrate that Gdf6a is necessary and sufficient for initiation of dorsal retinal identity, and loss of such identity leads to errors in retinal ganglion cell topographic mapping. Finally, I demonstrate that gdf6a is required for numerous embryonic processes in addition to dorsal retina specification. Gdf6a in required for eye growth, as loss of Gdf6a function leads to microphthalmia. I have obtained preliminary evidence that this growth factor is also required for development of the lens and axial skeleton. Furthermore, many of these phenotypes are similar to those seen in human patients with mutations in GDF6, highlighting the importance of understanding the function of this growth factor in model organisms. / Molecular Biology and Genetics

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A cor da pele, olhos e cabelos são alguns dos traços fenotípicos mais aparentes quando nos referimos à identificação de características individuais. Essas características frequentemente são utilizadas na descrição de indivíduos em retratos falados usados em investigações policiais. Porém, em muitos casos as vítimas ou testemunhas não reconhecem o agressor, tornando inviável a produção desses retratos. Contudo, os vestígios biológicos deixados pelo criminoso poderiam ser utilizados na predição de suas características físicas, suprindo a falta ou complementando o retrato falado. Para que isso seja possível, é preciso conhecer as variáveis responsáveis pela formação desses fenótipos. No caso dos fenótipos de pigmentação há tanto um fator genético, quanto ambiental. Diversos genes participam da formação desses fenótipos, dentre eles está o gene MITF (Melanogenesis-associated transcription factor), um dos principais regulador da biossíntese de melanina nos melanócitos. Esse gene está fortemente associado às síndromes de Waadenburg e Tietz, as quais causam pigmentação anormal, principalmente na pele, e ao melanoma. No entanto, apesar do claro envolvimento do gene MITF na melanogênese, ainda não são conhecidas associações significativas de polimorfismos nesse gene com fenótipos de pigmentação. À vista disso, esse trabalho avaliou a relação da variabilidade do gene MITF com os fenótipos de pigmentação encontrados em uma amostra populacional do estado de São Paulo, por meio de sequenciamento de nova geração. Foram identificados 133 pontos de variação em toda a extensão do gene e sua região promotora, dos quais 21 estão associadas a pelo menos um fenótipo de pigmentação de pele, olhos ou cabelo. Adicionalmente foram encontradas associações com ao menos um fenótipo de pigmentação para 3 dos 17 haplótipos da região promotora, 7 dos 50 haplótipos da extensão que engloba a região 5UTR e codificante, e um dos 18 haplótipos encontrados na região 3UTR. Considerando os haplótipos encontrados para a extensão total do gene MITF e sua promotora, 20 dos 132 haplótipos encontrados estão associados a algum fenótipo de pigmentação. A maior parte das associações encontradas, tanto para alelos e genótipos quanto para haplótipos, são referentes a fenótipos mais escuros como cabelos castanhos escuros e pretos e pele escura. Associações com fenótipos mais claros, tais como olhos azuis e verdes e cabelos loiros e ruivos, também foram encontradas, porém envolvendo variantes e haplótipos de frequência baixa na população amostrada; tais associações, entretanto, representam achados falsos positivos. Os resultados confirmam a hipótese de que a variabilidade do gene MITF pode contribuir para a formação dos fenótipos de pigmentação de pele, olhos e cabelos dos indivíduos da população brasileira / Skin, eye and hair colors are some of the most noticeable phenotypes when referring to the identification of individual characteristics. These characteristics are often used to describe individuals in police sketches used in investigations. However, in many cases the victims or witnesses are unable to recognize the assaulter, making this sketches unfeasible. Nonetheless, biological traces left by the assaulter could be used to predict their physical characteristics, compensating or complementing these sketches. To make this possible, its necessary to know the variables responsible for the development of these traits. Pigmentation phenotype development relies on genetic and environmental aspects. A variety of genes contribute to the development of these phenotypes, among them MITF (Melanogenesis-associated transcription factor), one of the main regulators of melanin synthesis in melanocytes. This gene is strongly associated with Waardenburg and Tietz syndrome, which cause abnormal pigmentation, mostly in skin, and melanoma. Although MITFs clear involvement in melanogenesis, significant associations between this genes polymorphisms and pigmentation phenotypes are still unknown. Thus, this study evaluated the relation between MITF genetic variability and pigmentation phenotypes found in a population sample from the state of São Paulo, through next generation sequencing. There were identified 133 variation points through the whole gene and its promoter, from which 21 were associated with at least one skin, eye or hair pigmentation phenotype. Additionally, 3 of the 17 promoter haplotypes, 7 of the 50 haplotypes comprising the 5UTR and coding regions and one of the 18 3UTR haplotypes were associated with at least one pigmentation phenotype. Considering the haplotypes found for the whole gene and its promoter, 20 of the 132 haplotypes found were associated with at least one phenotype. The majority of the associations found for alleles, genotypes and haplotypes were related to darker phenotypes, like dark brown and black hair and dark skin. Associations with lighter phenotypes, like blue and green eyes and blonde and red hair, were also found, although involving variants and haplotypes with low frequencies in the studied population; these associations, however, represent false positives. The results corroborate the hypothesis that the MITF variability can contribute to the formation of pigmentation phenotypes in skin, eye and hair in the Brazilian population

A cor da pele, olhos e cabelos são alguns dos traços fenotípicos mais aparentes quando nos referimos à identificação de características individuais. Essas características frequentemente são utilizadas na descrição de indivíduos em retratos falados usados em investigações policiais. Porém, em muitos casos as vítimas ou testemunhas não reconhecem o agressor, tornando inviável a produção desses retratos. Contudo, os vestígios biológicos deixados pelo criminoso poderiam ser utilizados na predição de suas características físicas, suprindo a falta ou complementando o retrato falado. Para que isso seja possível, é preciso conhecer as variáveis responsáveis pela formação desses fenótipos. No caso dos fenótipos de pigmentação há tanto um fator genético, quanto ambiental. Diversos genes participam da formação desses fenótipos, dentre eles está o gene MITF (Melanogenesis-associated transcription factor), um dos principais regulador da biossíntese de melanina nos melanócitos. Esse gene está fortemente associado às síndromes de Waadenburg e Tietz, as quais causam pigmentação anormal, principalmente na pele, e ao melanoma. No entanto, apesar do claro envolvimento do gene MITF na melanogênese, ainda não são conhecidas associações significativas de polimorfismos nesse gene com fenótipos de pigmentação. À vista disso, esse trabalho avaliou a relação da variabilidade do gene MITF com os fenótipos de pigmentação encontrados em uma amostra populacional do estado de São Paulo, por meio de sequenciamento de nova geração. Foram identificados 133 pontos de variação em toda a extensão do gene e sua região promotora, dos quais 21 estão associadas a pelo menos um fenótipo de pigmentação de pele, olhos ou cabelo. Adicionalmente foram encontradas associações com ao menos um fenótipo de pigmentação para 3 dos 17 haplótipos da região promotora, 7 dos 50 haplótipos da extensão que engloba a região 5UTR e codificante, e um dos 18 haplótipos encontrados na região 3UTR. Considerando os haplótipos encontrados para a extensão total do gene MITF e sua promotora, 20 dos 132 haplótipos encontrados estão associados a algum fenótipo de pigmentação. A maior parte das associações encontradas, tanto para alelos e genótipos quanto para haplótipos, são referentes a fenótipos mais escuros como cabelos castanhos escuros e pretos e pele escura. Associações com fenótipos mais claros, tais como olhos azuis e verdes e cabelos loiros e ruivos, também foram encontradas, porém envolvendo variantes e haplótipos de frequência baixa na população amostrada; tais associações, entretanto, representam achados falsos positivos. Os resultados confirmam a hipótese de que a variabilidade do gene MITF pode contribuir para a formação dos fenótipos de pigmentação de pele, olhos e cabelos dos indivíduos da população brasileira / Skin, eye and hair colors are some of the most noticeable phenotypes when referring to the identification of individual characteristics. These characteristics are often used to describe individuals in police sketches used in investigations. However, in many cases the victims or witnesses are unable to recognize the assaulter, making this sketches unfeasible. Nonetheless, biological traces left by the assaulter could be used to predict their physical characteristics, compensating or complementing these sketches. To make this possible, its necessary to know the variables responsible for the development of these traits. Pigmentation phenotype development relies on genetic and environmental aspects. A variety of genes contribute to the development of these phenotypes, among them MITF (Melanogenesis-associated transcription factor), one of the main regulators of melanin synthesis in melanocytes. This gene is strongly associated with Waardenburg and Tietz syndrome, which cause abnormal pigmentation, mostly in skin, and melanoma. Although MITFs clear involvement in melanogenesis, significant associations between this genes polymorphisms and pigmentation phenotypes are still unknown. Thus, this study evaluated the relation between MITF genetic variability and pigmentation phenotypes found in a population sample from the state of São Paulo, through next generation sequencing. There were identified 133 variation points through the whole gene and its promoter, from which 21 were associated with at least one skin, eye or hair pigmentation phenotype. Additionally, 3 of the 17 promoter haplotypes, 7 of the 50 haplotypes comprising the 5UTR and coding regions and one of the 18 3UTR haplotypes were associated with at least one pigmentation phenotype. Considering the haplotypes found for the whole gene and its promoter, 20 of the 132 haplotypes found were associated with at least one phenotype. The majority of the associations found for alleles, genotypes and haplotypes were related to darker phenotypes, like dark brown and black hair and dark skin. Associations with lighter phenotypes, like blue and green eyes and blonde and red hair, were also found, although involving variants and haplotypes with low frequencies in the studied population; these associations, however, represent false positives. The results corroborate the hypothesis that the MITF variability can contribute to the formation of pigmentation phenotypes in skin, eye and hair in the Brazilian population

The microphthalmia with linear skin defects syndrome (MLS) is an X-linked dominant disorder with male lethality. In the majority of the patients reported, the MLS syndrome is caused by segmental monosomy of the Xp22.3 region. To date, five male patients with MLS and 46,XX karyotype (“XX males”) have been described. Here we report on the first male case with MLS and an XY complement. The patient showed agenesis of the corpus callosum, histiocytoid cardiomyopathy, and lactic acidosis but no microphthalmia, and carried a mosaic subtle inversion of the short arm of the X chromosome in 15% of his peripheral blood lymphocytes, 46,Y,inv(X)(p22.13∼22.2p22.32∼22.33)[49]/46,XY[271]. By fluorescence in situ hybridization (FISH), we showed that YAC 225H10 spans the breakpoint in Xp22.3. End-sequencing and database analysis revealed a YAC insert of at least 416 kb containing the genes HCCS and AMELX, and exons 2–16 of ARHGAP6. Molecular cytogenetic data suggest that the Xp22.3 inversion breakpoint is located in intron 1 of ARHGAP6, the gene encoding the Rho GTPase activating protein 6. Future molecular studies in karyotypically normal female MLS patients to detect submicroscopic rearrangements including the ARHGAP6 gene as well as mutation screening of ARHGAP6 in patients with no obvious chromosomal rearrangements will clarify the role of this gene in MLS syndrome. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Le facteur de transcription Microphthalmia (Mitf) et la voie de signalisation des " Mitotic Activated Protein Kinase " (MAPK) sont des éléments déterminants pour la différentiation, la prolifération et la survie des melanocytes. L'altération des fonctions de l'un ou l'autre se manifeste par une perte totale ou partielle de ce type cellulaire. A l'inverse, le mélanome est associé très majoritairement à une activation constitutive des MAPK, et parfois, à un gain d'activité de MITF. Afin d'étudier les interaction entre les MAPK et l'activité de MITF, nous nous sommes intéressés aux modifications post transcriptionnelles qui permettent la génération d'isoformes multiples dotées d'activité différentes. MITF contient un site de phosphorylation, la serine 73 (S73), qui a été démontré par le passé comme jouant un rôle à la fois dans l'augmentation de l'activité et dans la réduction de la stabilité de MITF in vitro. Pour comprendre le rôle de cette serine in vivo, une tentative de mutation S73A de Mitf a été réalisée. Ce codon fait parti d'un " Exon splicing Enhencer " et sa mutation réduit l'affinité de fixation de la protéine SRp40 et l'exclusion de l'exon 2B dans lequel est situé ce site de phosphorylation. Pour dissocier l'exclusion de l'exon 2B et de sa phosphorylation, nous avons donc altéré la jonction de l'exon 2A-2B afin qu'elle ne soit plus reconnue par le spliceosome. En conséquence, l'exon 2B ne peut plus être exclu des transcrits Mitf quelque soit le statut du codon 73. La comparaison de 3 nouveaux allèles Mitf S-S73A S-S73D et S-S73S, où l'inclusion de l'exon 2B est forcée, nous a permis d'associer l'absence de phosphorylation à un gain d'activité de MITF.

Dissertation (Ph.D.)--University of Toledo, 2010. / "Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences." Title from title page of PDF document. "A Dissertation entitled"--at head of title. Bibliography: p. 63-71, 126-140.

The microphthalmia with linear skin defects syndrome (MLS) is an X-linked dominant disorder with male lethality. In the majority of the patients reported, the MLS syndrome is caused by segmental monosomy of the Xp22.3 region. To date, five male patients with MLS and 46,XX karyotype (“XX males”) have been described. Here we report on the first male case with MLS and an XY complement. The patient showed agenesis of the corpus callosum, histiocytoid cardiomyopathy, and lactic acidosis but no microphthalmia, and carried a mosaic subtle inversion of the short arm of the X chromosome in 15% of his peripheral blood lymphocytes, 46,Y,inv(X)(p22.13∼22.2p22.32∼22.33)[49]/46,XY[271]. By fluorescence in situ hybridization (FISH), we showed that YAC 225H10 spans the breakpoint in Xp22.3. End-sequencing and database analysis revealed a YAC insert of at least 416 kb containing the genes HCCS and AMELX, and exons 2–16 of ARHGAP6. Molecular cytogenetic data suggest that the Xp22.3 inversion breakpoint is located in intron 1 of ARHGAP6, the gene encoding the Rho GTPase activating protein 6. Future molecular studies in karyotypically normal female MLS patients to detect submicroscopic rearrangements including the ARHGAP6 gene as well as mutation screening of ARHGAP6 in patients with no obvious chromosomal rearrangements will clarify the role of this gene in MLS syndrome. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.