Cell migration and differentiation are fundamental aspects of embryogenesis, essential to the development of any complex multicellular organism. Like most biological processes, the directional migration of different cell types and their differentiation into various specified cells with unique functions are regulated by intricate mechanisms, many details of which remain unresolved. The sea urchin embryo, which is optically clear and amenable to a wide variety of experimental manipulations, is an excellent model system to study these processes. Of specific significance is the formation of the embryonic endoskeleton, in which early cell migration and differentiation events can be observed in vivo. The sea urchin embryonic endoskeleton is formed by the sequential ingression, directed migration, and fusion of the primary mesenchyme cells (PMCs). The fused PMCs then secrete a calcareous matrix, forming the characteristic rigid endoskeleton of the embryo. The mechanisms governing skeletogenesis have been of interest to researchers for decades. However, several aspects of its regulation are still unclear. The work described in this thesis details progress made in understanding cell migration and differentiation using skeletogenesis in the sea urchin embryo as a model. Skeletogenesis is regulated by a complex gene regulatory network (GRN) which is arguably the most complete developmental GRN presently available. The aim of this work was to build linkages between the components of this GRN and observable morphological events during skeletogenesis. Recent research into skeletogenesis has been mainly focused on deciphering the roles that upstream transcription factors play in the specification of PMCs. Hence, a significant gap exists in our knowledge of the functions of downstream morphoeffector genes regulated by these well-studied transcription factors. To this end, we have analyzed the roles of two novel morphoeffector genes, p58-a and p58-b, which encode similar type 1 transmembrane proteins. These two genes are expressed specifically in the PMCs throughout development. We find that the knockdown of either p58-a or p58-b results in defects in skeletogenesis, though PMC specification, migration and fusion occur unperturbed. We conclude that p58-a and p58-b most likely play a role in biomineralization. Additionally, we describe progress made in understanding the role that ectodermal cues play during skeletogenesis, another poorly understood aspect of this process. The precise and extremely replicable pattern of PMC migration to specific sites within the blastocoel during skeletogenesis has long been of interest to researchers. However, the molecular mechanisms controlling this process have remained mostly elusive. Recent studies have identified the fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) signaling pathways as playing significant roles in regulating cell migration and differentiation during skeletogenesis in the sea urchin species Paracentrotus lividus, though these studies provided few details on the specific roles each of these pathways play. The FGF and VEGF pathways have long been shown to play complex, sometimes interacting roles in cell migration during development, and our research aimed at revealing the fine details of their functions in the sea urchin embryo. We have found that in the sea urchin species Lytechinus variegatus, VEGF signaling plays a more significant role in regulating skeletogenesis than the FGF pathway. Blocking VEGF signaling leads to profound defects in skeletogenesis: all aspects of PMC migration are abolished in these morphants, and the extension of filopodia from the PMCs is compromised. We have also identified a separate role for VEGF signaling in the synthesis of the endoskeleton and in regulating the expression of several morphoeffector genes in the PMC gene regulatory network. Conversely, we observed that inhibiting FGF signaling does not lead to severe defects in skeletogenesis, as FGF morphant embryos form extensive skeletal elements. Lastly, we document the presence of reciprocal signals from the PMCs regulating gene expression in the ectoderm, a phenomenon not previously described. These findings significantly expand our understanding of the regulation of directional cell migration and differentiation during embryonic development.

A critical step during gene expression is the directional export of nuclear messenger (m)RNA through nuclear pore complexes (NPCs) to the cytoplasm. During export, Gle1 in conjunction with inositol hexakisphosphate (IP6) spatially regulates the activity of the DEAD-box protein Dbp5 at the NPC cytoplasmic face. Dbp5 acts to remodel the protein composition of mRNA-protein complexes in a terminal export step. In the cytoplasm, Gle1, IP6 and Dbp5 are also required for efficient translation termination. Additionally, during translation initiation, Gle1 modulates the DEAD-box protein Ded1. GLE1 mutations are causally linked to the human disease Lethal-Congenital Contracture Syndrome 1 (LCCS-1). The main causative mutation (FinMajor) results in a three amino acid insertion (PFQ) within Gle1s essential coiled coil domain. To determine the molecular defects underlying gle1-FinMajor pathology, we analyzed the functional significance of the coiled-coil domain for human (h) and Saccharomyces cerevisiae (y) Gle1. Both yGle1 and hGle1 self-associate via their coiled coil domain in vitro to form higher order homo-oligomeric complexes. Strikingly, using electron microscopy, the hGle1 form disk-shaped structures that were malformed with the h-gle1-FinMajor protein. Because LCCS1 is a homozygous recessive condition, we established an RNAi knockdown and add-back system to test for functional defects. Reduction of GLE1 activity in HeLa cells resulted in nuclear accumulation of poly(A)+ RNA. Co-expressing siRNA-resistant wild-type hGLE1BR rescued the mRNA export defect. However, co-expression of hgle1BR-FinMajor did not. Live cell microscopy studies found that GFP-hgle1B-FinMajor had altered nucleocytoplasmic shuttling dynamics. A parallel series of genetic studies were conducted with y-gle1 loss-of-function mutants that mimic the h-gle1-FinMajor allele. Growth defects of yeast mRNA export mutants were exacerbated when combined with y-gle1-Fin alleles; whereas, translation initiation and termination mutants were not impacted. We conclude that proper Gle1 self-association is specifically required during mRNA export, revealing a new model for controlling rounds of Dbp5 activity at NPCs. This work also provides the first evidence for the molecular mechanism causing the human LCCS-1 disease, and impacts the global understanding of the role for altered mRNA transport and gene expression in other human diseases.

Sonic hedgehog (Shh) signaling regulates critical processes during embryonic development and in homeostasis of adult tissues. Deregulated pathway activity is a major factor underlying the etiology of numerous developmental disorders and cancers. In this dissertation, I investigated early neonatal cerebellar development, where I identified that the Purkinje neuron utilizes bidirectional distribution of Shh to centrally regulate neurogenesis, and to expand a previously unappreciated stem cell progenitor cell lineage in the white matter niche. Additionally, I established a novel mouse model for a soft tissue sarcoma, Ewings sarcoma. These findings provide new understanding of how the Purkinje neuron oversees cerebellar development, as well as key insight into the molecular underpinnings of a Shh-driven sarcoma variant.

The Notch signaling pathway is an essential cell-cell signaling pathway that is involved in cell fate decisions and cell differentiation during early metazoan development. In humans, the Notch pathway is misregulated in cancers such as T-cell acute lymphoblastic leukemia, breast cancer, lung cancer, and colorectal cancer. The key downstream effector of the Notch signaling pathway is the Notch Intracellular Domain (NICD), which is produced from a series of ligand-dependent proteolytic cleavages. Upon its liberation from the plasma membrane, NICD translocates into the nucleus and binds to the transcription factor CSL to activate a Notch-specific transcriptional program. Previous cultured cell studies suggest that regulated NICD turnover plays a critical role in regulating its steady-state intracellular levels and subsequent Notch pathway activation. Correspondingly, stabilizing mutations in NICD1 have been linked to multiple types of cancer. These results support the idea that regulation of NICD protein turnover is an essential process in regulation of Notch signaling. However, the mechanisms that control NICD degradation, and their effect on Notch signal transduction is a major unanswered question in the Notch field. We found that human NICD1 degrades robustly in a proteasome-dependent manner in Xenopus egg extract, and have identified a 35-amino acid degron (N-Box) at the N-terminus of NICD1. The N-Box is both sufficient and necessary for degradation of NICD1 in Xenopus egg extract, and, when attached to a heterologous protein in cultured human cells, promotes its turnover. Mutations in key residues within N-Box stabilize the NICD1 protein and lead to increased Notch transcriptional activity in cultured mammalian cells. We present a model as to how the N-Box may regulate NICD1 stability and transcriptional activity in the context of known stabilizing mutations of NICD1 in human cancers.

Bronchopulmonary dysplasia is a life-threatening lung disease affecting low birth weight preterm infants. While the occurrence of BPD is correlated with chorioamnionitis, the origination and pathway of fetal lung inflammation is less clear. It is unknown which cell type in the fetal lung detect pathogens and initiate inflammation. We hypothesized that fetal lung macrophages drive development-inhibiting inflammation through NF-κB activation, and that NF-κB activation alters macrophage development. While LPS normally inhibits airway branching in fetal lung explants, depleting macrophages with clodronate or inhibiting NF-κB activation in macrophages protected fetal lung explants from the effects of LPS. Activating NF-κB in macrophages inhibited airway branching, lead to abnormal lung morphogenesis, and induced perinatal lethality. In addition to the effects of macrophage activation on lung morphogenesis, NF-κB signaling can alter normal macrophage maturation. Flow cytometry experiments show two macrophage populations in the fetal lung, CD11bhiF4/80lo and CD11bloF4/80hi, with most macrophages being CD11bhiF4/80lo. After NF-κB activation, there is an increase in the CD11bhiF4/80lo subpopulation, which expressed higher levels of CD204 and CD206. High levels of CD204 and CD206 are also found on mature, alveolar macrophages, indicating similarities in marker expression with the CD11bloF4/80hi subpopulation. Fetal lung macrophages are unique in that they do not follow the typical polarization paradigm, but rather a maturation pathway towards alveolar macrophages. Overall, macrophages have a primary role in the fetal lung inflammatory. NF-κB activation in macrophages inhibits lung development and influences fetal macrophage maturation.

In recent times, major advances have been made towards understanding sexual dimorphism in the brain on a molecular basis. This thesis summarises my modest contributions to these endeavours. Sexual dimorphisms are manifested throughout the spectrum of biological complexity, and can be studied by numerous approaches. The approach of this thesis is to explore sex-biased gene expression in mammalian somatic tissues. Paper I describes an evolutionarily conserved sexual gene expression pattern in the primate brain. Conserved sex-biased genes may underlie important sex differences in neurobiology. In Paper II, Y-chromosome genes expressed across several regions of the human male brain during mid-gestation are identified. Such genes may play male-specific roles during brain development. The studies of Papers III and IV explore sex-biased gene expression in several somatic tissues from mouse. The amount of genes with sex-biased expression varied in different brain regions. The striatum was particularly interesting, with an order of magnitude increase in the number of sex-biased genes as compared to the other included brain regions. Of potentially wider significance are my observations regarding the transcriptional regulation of domains that escape X-chromosome inactivation (XCI). Specifically, I provide the first evidence that long non-coding RNAs (lncRNAs) transcribe together with protein-coding genes in XCI-escaping domains. This raises the possibility that lncRNAs mediate the transcriptional regulation of XCI-escaping domains. I also present evidence that the mouse X-chromosome has undergone both feminisation and de-masculinisation during evolution, as indicated by the sex-skewed regulation of genes on this chromosome. This finding is relevant for understanding the selective forces that shaped the mammalian X-chromosome. In the final chapter, Paper V, the generation of a novel transgenic mouse line, Gpr101-Cre, is described. Its progeny can be used for functional studies of striatum, a brain structure with major sexual dimorphism, as is further demonstrated in the Papers of this thesis.

Thesis (M.S.) in Biochemistry--University of Maine, 2007. / Includes vita. Includes bibliographical references (leaves 96-97).

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph.D.) --University of Adelaide, School of Medical Sciences, Discipline of Pathology, 2006. / "August 2006" Bibliography: leaves 154-176. Also available in print form.

Thesis (M.S.)--Rutgers University, 2007. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 28-32).