The migration of neurons from their place of birth to their place of function is an important process during neurodevelopment. Two adaptor proteins, Crk and Crkl, are known to be important factors for neuronal migration. In the neocortex, both molecules play a critical role in the well studied Reelin signaling pathway, guiding newly born neurons to their correct cell layer to create the laminated structure of this tissue. These two adaptor proteins are implicated in human disease of the nervous system. Heterozygous compound deletion of human chromosome 17p13, which includes CRK, occurs in Miller-Dieker syndrome, a severe type of lissencephaly (smooth brain syndrome). Autosomal dominant compound deletion of human chromosome 22q11.2, which includes CRKL, causes DiGeorge Syndrome, a neural crest migratory disease that effects the heart, kidneys, ears, immune system, and face. Danio rerio, or zebrafish, are a great model to study the developing nervous system, and are used in our studies. We characterized expression of crk and crkl at various stages of zebrafish embryo development and determined that both are expressed in the developing eye. We aim to determine if Crk and Crkl have a role in eye development, as the eye and neocortex are very similar in the way they are patterned, and little is known about the signaling mechanisms that guide lamination of the retina. Using mutant knockout lines, we have determined gross retinal phenotypes of Crk deficient, Crkl deficient, and Crk and Crkl compound deficient embryos. Crk and Crkl are both required for proper eye development, as combinations of Crk and Crkl deficiency lead to impaired formation of this tissue. Crk seems to be particularly important for proper eye size, and Crkl is required for proper lamination. This preliminary research is critical to further elucidating the role Crk and Crkl are playing in the retina.

No description available.

Thesis (Ph. D.)--University of Hong Kong, 2006.

The cerebral cortex develops through the coordinated generation of dozens of neuronal subtypes, but the mechanisms involved remain unclear. Here we show that mouse embryonic stem cells, cultured without any morphogen but in the presence of a sonic hedgehog inhibitor, recapitulate in vitro the major milestones of cortical development, leading to the sequential generation of a diverse repertoire of neurons that display most salient features of genuine cortical pyramidal neurons. When grafted into the cerebral cortex, these neurons develop patterns of axonal projections corresponding to a wide range of cortical layers, but also to highly specific cortical areas, in particular visual and limbic areas, thereby demonstrating that the identity of a cortical area can be specified without any influence from the brain. The discovery of intrinsic corticogenesis sheds new light on the mechanisms of neuronal specification, and opens new avenues for the modelling and treatment of brain diseases. In a further attempt to prove the validity of this model, we have initiated the study of the mechanism of action of FoxG1, a forkhead box transcription factor involved in the control of cell fate decision in the developing cortex.

During development of the vertebrate embryo, a highly conserved tissue called the organizer forms during gastrulation, and is required for establishment of the basic body plan. In mouse, the organizer gives rise to the node and notochord, which are both transient signaling centres involved in patterning the body axes. The genetic regulation and morphogenesis of these tissues, particularly in the mouse, is not well understood. To follow the formation of these tissues we used time-lapse live imaging together with conventional cell lineage tracking. This showed that the notochord has distinct morphogenetic origins along the anterior-posterior axis: anterior head process forms by condensation of dispersed midline organizer cells; trunk forms by convergent extension of node cells; tail forms from posteriorly migrating node cells—this challenges the previously accepted model that tail notochord forms by node regression. We have also found there are distinct genetic requirements within these different regions. Previous mouse mutant analysis showed that conserved transcription factors Foxa2 and Noto are required for either all notochord regions or just tail notochord, respectively. We found a novel genetic interaction between the two demonstrated Foxa2 compensates for Noto specifically in the trunk notochord. Furthermore, we found Noto has a conserved role in regulating axial (notochord) versus paraxial (somite) cell fate. Therefore, we proposed there are three distinct regions within the mouse notochord, each with its own unique morphogenetic origins and genetic control. We have also conducted two microarray-based screens to identify novel gene expression patterns in the node and notochord. First, we compared Foxa2 mutant and wild type gastrula embryos. Second, we isolated notochord progenitors from early somite stage embryos. Extensive in situ hybridization screening based on both data sets revealed over 50 node and notochord expression patterns. Lastly, we screened Foxa2-bound chromatin regions near these notochord-specific genes using a transient zebrafish expression assay, and identified two novel notochord cis-regulatory modules. Together, we found a combination of classical genetics, embryology, and novel imaging techniques, has given us a better understanding of the morphogenesis and genetic regulation of pattern formation in the developing mouse embryo.

During development of the vertebrate embryo, a highly conserved tissue called the organizer forms during gastrulation, and is required for establishment of the basic body plan. In mouse, the organizer gives rise to the node and notochord, which are both transient signaling centres involved in patterning the body axes. The genetic regulation and morphogenesis of these tissues, particularly in the mouse, is not well understood. To follow the formation of these tissues we used time-lapse live imaging together with conventional cell lineage tracking. This showed that the notochord has distinct morphogenetic origins along the anterior-posterior axis: anterior head process forms by condensation of dispersed midline organizer cells; trunk forms by convergent extension of node cells; tail forms from posteriorly migrating node cells—this challenges the previously accepted model that tail notochord forms by node regression. We have also found there are distinct genetic requirements within these different regions. Previous mouse mutant analysis showed that conserved transcription factors Foxa2 and Noto are required for either all notochord regions or just tail notochord, respectively. We found a novel genetic interaction between the two demonstrated Foxa2 compensates for Noto specifically in the trunk notochord. Furthermore, we found Noto has a conserved role in regulating axial (notochord) versus paraxial (somite) cell fate. Therefore, we proposed there are three distinct regions within the mouse notochord, each with its own unique morphogenetic origins and genetic control. We have also conducted two microarray-based screens to identify novel gene expression patterns in the node and notochord. First, we compared Foxa2 mutant and wild type gastrula embryos. Second, we isolated notochord progenitors from early somite stage embryos. Extensive in situ hybridization screening based on both data sets revealed over 50 node and notochord expression patterns. Lastly, we screened Foxa2-bound chromatin regions near these notochord-specific genes using a transient zebrafish expression assay, and identified two novel notochord cis-regulatory modules. Together, we found a combination of classical genetics, embryology, and novel imaging techniques, has given us a better understanding of the morphogenesis and genetic regulation of pattern formation in the developing mouse embryo.

The avian embryo is a popular animal model because it is widely available (Antin et al., 2004), it is easily manipulated, and it can provide important insights into normal and abnormal embryo development (Kulesa, 2004). While in vivo and in vitro cultures of chick embryos are common, in ovo cultures are rarer, and none have been designed where the egg did not have to be resealed afterwards. The present study aimed to develop a set-up in which the egg would be windowed without resealing the egg so that the embryo would remain accessible for experimental manipulation. As well, this study aimed to track cell movement during neurulation by microinjecting points of dye along the embryo. Two prototypes were developed based on the concept that temperature and moisture controlled air passing over the windowed egg would serve as a blanket. When these prototypes were unable to keep the embryo alive, a protocol developed by Kulesa and Fraser (2004) was adapted for the study. This protocol involved the construction of a Teflon window which was placed in the windowed egg and sealed with beeswax. Initial microinjection tests with Fast Green FCF showed that the dye dissipated quickly after injection, most likely because of the hydrophilicity of the dye. Therefore, a list of non-fluorescent, hydrophobic dyes were chosen and tested for suitability to cell tracking. Time restrictions prevented the actual cell tracking experiments from taking place, but it was found that Oil Red O fulfilled the criteria. As Oil Red O is usually used to identify lipids in static experiments, it remains to be seen whether it would function as a vital dye. Future experiments include expanding the set-up for use with a confocal microscope for a 4-D rendering of cell movement, and taking advantage of the symmetrical nature of neurulation in the chick embryo to examine perturbations to the normal progress of development, via drugs such as valproic acid.

The avian embryo is a popular animal model because it is widely available (Antin et al., 2004), it is easily manipulated, and it can provide important insights into normal and abnormal embryo development (Kulesa, 2004). While in vivo and in vitro cultures of chick embryos are common, in ovo cultures are rarer, and none have been designed where the egg did not have to be resealed afterwards. The present study aimed to develop a set-up in which the egg would be windowed without resealing the egg so that the embryo would remain accessible for experimental manipulation. As well, this study aimed to track cell movement during neurulation by microinjecting points of dye along the embryo. Two prototypes were developed based on the concept that temperature and moisture controlled air passing over the windowed egg would serve as a blanket. When these prototypes were unable to keep the embryo alive, a protocol developed by Kulesa and Fraser (2004) was adapted for the study. This protocol involved the construction of a Teflon window which was placed in the windowed egg and sealed with beeswax. Initial microinjection tests with Fast Green FCF showed that the dye dissipated quickly after injection, most likely because of the hydrophilicity of the dye. Therefore, a list of non-fluorescent, hydrophobic dyes were chosen and tested for suitability to cell tracking. Time restrictions prevented the actual cell tracking experiments from taking place, but it was found that Oil Red O fulfilled the criteria. As Oil Red O is usually used to identify lipids in static experiments, it remains to be seen whether it would function as a vital dye. Future experiments include expanding the set-up for use with a confocal microscope for a 4-D rendering of cell movement, and taking advantage of the symmetrical nature of neurulation in the chick embryo to examine perturbations to the normal progress of development, via drugs such as valproic acid.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Computational Biology and Molecular Biophysics." Includes bibliographical references.

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 37-40).