In mammals, the most frequently observed hybrid dysgenesis effects are growth disturbances and male sterility. Profound defects in placental development have been described and our work on hybrids in genus Mus has demonstrated putative hybrid dysgenesis effects that lead to defects in lipid homeostasis and maternal behavior. Interestingly, mammalian interspecies hybrids exhibit strong parent-of-origin effects in that offspring of reciprocal matings, even though genetically identical, frequently exhibit reciprocal phenotypes. Recent studies have provided strong link between epigenetic regulation and growth, behavior and placental development. Widespread disruption of genomic imprinting has been described in hybrids between closely related species of the genus Peromyscus. The studies presented in this thesis aim to investigate the effects of disrupted epigenetics states on altered growth, female infanticide and placental dysplasia observed in Mus hybrids. We showed that loss-of-imprinting (LOI) of a paternally expressed gene, Peg1, was correlated with increased body weight of F1 hybrids. Furthermore, we investigated whether LOI of Peg1 in F1 females would interfere with maternal behavior. A subset of F1 females indeed exhibited highly abnormal maternal behavior in that they rapidly attacked and killed the pups. By microarray hybridization, a large number of differentially expressed genes in the infanticidal females as compared to normally behaving females were identified. In addtion to Peg1 LOI, we studied allelic expression of numerous imprinted genes in adult Mus interspecies hybrids. In contrast to the study from Peromyscus, patterns of LOI were not consistent with a direct influence of altered expression levels of imprinted genes on growth. Finally, we investigated the allelic interaction between an X-linked locus and a paternally expressed gene, Peg3, in placental defects in Mus hybrids. This study further strengthened the notion that divergent genetic and epigenetic mechanisms may be involved in hybrid dysgenesis in diverse groups of mammals.

The development of a new individual from the fertilized oocyte can ultimately be seen as the consequence of the establishment and maintenance of specific patterns of gene expression. Although regulation of gene activity occurs at different levels, cellular specialization and differentiation are the results of developmental cues that essentially take place at the transcriptional level. The involvement of epigenetics in this process has become increasingly clear during the last decade. Imprinted genes constitute an excellent example as monoallelic expression seems to reflect differential epigenetic marks on the two alleles. This is the case of the imprinted H19 and Igf2 genes were the monoallelic expression is coordinated through a differentially methylated region (hypermethylated on the paternal allele), known as ICR (imprinted control region). In the mouse the ICR harbours four binding sites for the methylation sensitive insulator protein CTCF. Previous studies with episomal constructs had shown that this region behaved as an insulator and that CTCF is required for the insulator activity of the H19 ICR This thesis establish a clear link between the insulator function and the chromatin structure at the H19 ICR and indicates that the precise allocation of the CTCF target sites in the linker regions can play a critical role in this process. The importance of the CTCF interaction at the ICR was also confirmed in vivo using a mouse model that showed how intact CTCF target sites are needed to manifest insulator activity and methylation protection. We have investigated the role of CTCF and a related protein BORIS in establishing the maternal to paternal imprint transition in chromatin structure at the H19/Igf2 locus in the male germline. This thesis also describe the development of a new technique for the localization of chromatin associated factors and modifications with higher sensitivity and resolution compared to existing approaches.

Placental development can be disturbed by various factors, such as mutation of specific genes or maternal diabetes. Our previous work on interspecies hybrid placental dysplasia (IHPD) and two additional models of placental hyperplasia, cloned mice and Esx1 mutants, showed that many genes are deregulated in placental dysplasia. Two of these candidate placentation genes, Cpe and Lhx3, were further studied. We performed in situ hybridization to determine their spatio-temporal expression in the placentas and placental phenotypes were analyzed in mutant mice. Our results showed that the placental phenotype in Cpe mutant mice mimics some IHPD phenotypes. Deregulated expression of Cpe and Cpd, a functionally equivalent gene, prior to the manifestation of the IHPD phenotype, indicated that Cpe and Cpd are potentially causative genes in IHPD. Lhx3 mutants lacked any placental phenotype. Deletion of Lhx3 and Lhx4 together caused an inconsistent placental phenotype which did not affect placental lipid transport function or expression of Lhx3/Lhx4 target genes. Down regulation of Lhx3/Lhx4 did not rescue the placental phenotype of AT24 mice and hence could be excluded as causative genes in IHPD. Analysis of placental development in diabetic mice showed that severe maternal diabetes resulted in fetal intrauterine growth restriction (IUGR) without any change in placental weight and lipid transport function. The diabetic placentas however exhibited abnormal morphology. Gene expression profiling identified some genes that might contribute to diabetic pathology. In another study, it was found that the heterochromatin protein CBX1 is required for normal placentation, as deletion of the gene caused consistent spongiotrophoblast and labyrinthine phenotypes. Gene expression profiling and spatio-temporal expression analysis showed that several genes with known function in placental development were deregulated in the Cbx1 null placenta.

Thesis (Ph. D.)--Duke University, 2008. / Includes bibliographical references.

Neural crest cells arise during neurulation, a process that occurs during the third week of embryogenesis. These diverse cells then divide into various subtypes including cranial neural crest cells and cardiac neural crest cells. Each of these subtypes gives rise to a wide range of features throughout the fetus. While these cells are extremely diverse, they are also incredibly sensitive to their surrounding environment. Many maternal conditions affect neural crest cell division and migration, but maternal alcohol consumption and hyperglycemia due to gestational diabetes will be discussed in detail, with special attention paid to tissues that derive from cranial neural crest cells. While the initial mechanisms of the pathology vary for both of these conditions, what is remarkable is that they ultimately cause effects in similar ways. Both mechanisms lead to the creation of reactive oxygen species, which in turn trigger apoptotic pathways. Neural crest cell death causes a variety of congenital anomalies in fetuses, including craniofacial defects and cardiac outflow tract defects. Treatment options that have been researched in both conditions also vary, but are based on similar principles. Antioxidant therapies reduce the production of reactive oxygen species, thus reducing the severity of the anomalies affecting the fetus during development. Both maternal alcohol consumption and gestational diabetes are important public health concerns, and their management is of utmost priority in society. By decreasing the rates of women who consume alcohol during pregnancy, and managing gestational diabetes in those at highest risk, the rates of fetal congenital defects could be decreased.

One in four known species of fruit flies inhabit the Hawaiian Islands. From a small number of colonizing flies, a wide range of species evolved, some of which managed to reverse-colonize other continental environments. In order to explore the developmental pathways, which separate the Hawaiian Drosophila proper and the Scaptomyza group that contains reverse-colonized species, the transcriptomes of two better-known species in each group, Scaptomyza anomala and Drosophila grimshawi, were analyzed to find changes in gene expression between the two groups. This study describes a novel transcriptome for S. anomala studies as well as unusual changes in gene expression in D. grimshawi relative to other species, revealing priorities of both species in early development.

No description available.

Yolk supplies the majority of embryonic calcium in snakes. Oviparous and viviparous snakes also receive calcium late in development from the eggshell and placenta, respectively. The pattern and mechanism of calcium transport are partly understood for oviparous snakes. I studied a viviparous snake, Virginia striatula, to determine the pattern of embryonic calcium accumulation as well as the ontogenetic expression of calcium transporting proteins in extraembryonic tissues. The pattern of embryonic calcium uptake of V. striatula occurs late in development, during the phase of highest embryonic growth. Calbindin-D28k, Ca2+ ATPase, and carbonic anhydrase II are expressed in chorioallantoic membrane, while yolk sac only expresses calbindin-D28k, coincident with the timing of calcium transport in embryos of V. striatula. Thus, the pattern of embryonic calcium accumulation in V. striatula is similar to that of oviparous snakes. Although calbindin-D28k and Ca2+ ATPase are likely active in embryonic calcium transport, the role of carbonic anhydrase II remains less clear.

<p>The Slit morphogen is a secreted glycoprotein that is naturally cleaved into two fragments. The amino fragment (N-Slit) contains Leucine Rich Repeats (LRR) that are recognised by Robo receptors, and is sufficient to mediate attractive or repellent signalling in <em>Drosophila </em>tissues, for example, during growth cone guidance at the midline of the nervous system. The carboxy fragment (C-Slit) is composed of EGF repeats and a Laminin-like globular domain. Although C-Slit expression does not restore repellent signalling, it does rescue other morphogenetic defects in <em>slit</em> mutants.</p> <p>Formation of the dorsal vessel (or heart) requires function of <em>slit</em> and <em>robo</em>. Slit is required for coordinated migration of heart cell precursors, cell polarisation and the formation of a lumen in the heart tube. We have characterised the morphogenetic activities of N- Slit and C-Slit during assembly of the heart.</p> <p>Our laboratory has shown that Slit transgenes lacking the LRR region fail to rescue the mutant phenotype in the nervous system. However, <em>slit</em> trangenes lacking the LRR results in a partial rescue phenotype in the heart suggesting that C-Slit might have functional significance in the heart. Therefore, Slit function in heart vasculogenesis has different requirements compared to the nervous system. For example, Slit –Robo2 interaction may have an adhesive function in addition to a signalling function during vasculogenesis.</p> <p>Our results indicate that C-Slit funtions as a heart morphogen. In <em>slit</em> mutants, over-expression of C-Slit results in a partial rescue phenotype with several features such as cell clumping, overlapping of cells and cells which are elongated. Together, these data suggest alternative functions for Slit during heart morphogenesis.</p> / Master of Science (MSc)

<p>RanBPM is a conserved putative scaffold protein of unknown function. Loss-of-function in <em>RanBPM</em> leads to pleiotropic phenotypes such as reduced locomotion, decreased size and larval lethality in the <em>Drosophila melanogaster</em>.</p> <p><em>dRanBPM</em> mutants have decreased branching and boutons at the neuromuscular junction, which may contribute to their locomotory defect. To investigate if dRanBPM is involved in controlling synaptic architecture at the neuromuscular junction, levels of two cytoskeletal proteins, Futsch and profilin, were assessed in <em>dRanBPM</em> mutants.</p> <p>Due to time constraints, immunoblots for Futsch were not fully optimized for protein measurement. Immunoblots for profilin, on the other hand, were successfully carried out. However, results from the reproduction of a blot demonstrating the negative regulation of <em>Drosophila</em> FMRP on profilin did not agree with that of the literature. In addition, results from an epistatic experiment demonstrated that profilin levels were not affected in FMRP deficient flies when compared to those with additional decrease in dRanBPM function.</p> <p>Targeted expression of <em>dRanBPM</em> to neurosecretory cells is able to rescue size and lethality of <em>dRanBPM</em> mutants, suggesting a common pathway through which both phenotypes operate is disrupted in these mutants. Activation of the insulin signaling pathway was indeed found to be downregulated in <em>dRanBPM</em> mutants. A longevity assay was alternatively carried out to demonstrate decreased insulin pathway activation in <em>dRanBPM</em> mutants. Unfortunately, due to inappropriate controls used for this experiment, no conclusive points can be made. Together, these findings contribute to the knowledge that RanBPM plays and to designing future experiments to test for RanBPM function.</p> / Master of Science (MSc)