Myeloid-derived suppressor cells (MDSCs) play an important role in cancer progression. Elucidating the mechanisms involved in the expansion and function of these cells is important in the fight against cancer. A microarray comparing splenic Gr-1+CD11b+ cells from tumor-bearing mice and tumor-free mice revealed C/EBPα expression was reduced more than 4-fold in the tumor-derived cells. Based on this finding and published reports, we hypothesized that tumors induce MDSC production through down-regulation of C/EBPα in myeloid cells. In Chapter II, the role of C/EBPα as a negative regulator of MDSC expansion was investigated. Deletion of myeloid C/EBPα in mice yielded an increase in myeloid progenitors and a reduction in mature myeloid cells. Upon inoculation with tumor cells, MDSC production was enhanced nearly two-fold in mice lacking myeloid C/EBPα, while myeloid progenitors were reduced, perhaps because more progenitors became MDSCs in the absence of C/EBPα. In Chapter III, we sought to determine whether C/EBPα is a negative regulator of the immune suppressive and pro-angiogenic properties of MDSCs. When inoculated with tumor cells, MDSC infiltration and tumor vascularization was significantly greater in C/EBPα conditional null mice, resulting in markedly accelerated tumor growth. When MDSCs were injected with tumor cells into mice, C/EBPα ablation resulted in an enhancement in the pro-tumor MDSC phenotype: tumor growth and tumor angiogenesis was significantly greater. We then measured the expression of genes involved in MDSC-mediated immune suppression and angiogenesis and found that C/EBPα deletion resulted in MMP-9, VEGF and iNOS upregulation. Additionally, we observed increased NO production but no difference in arginase expression or immune suppression. Since NO also regulates angiogenesis, we concluded that C/EBPα inhibits the pro-angiogenic but not the immune-suppressive properties of MDSCs. Our findings reveal dual negative roles for C/EBPα in the expansion and pro-angiogenic gene expression in MDSCs, suggesting that overcoming these functions through C/EBPα inhibition may be a critical step in MDSC maturation. Our work indicates that therapy aimed at restoring C/EBP expression in MDSCs may be a viable weapon in the fight against cancer.

Thesis (Ph.D.) -- University of Texas at Arlington, 2009.

Proteins of the Bcl-2 family mediate apoptosis by altering mitochondrial structure, function, and integrity. However, in certain contexts, some members of the Bcl-2 family have additional, non-apoptotic functions. Bid, a BH3-only Bcl-2 family member, is a potent activator of mitochondrial apoptosis. However, Bid also preserves cell viability in conditions of replication stress by localizing to sites of DNA damage on the chromatin to facilitate signaling through the DNA damage response pathway. Using Bid deficient mice and cell lines, we investigated how the DNA damage response regulates the phosphorylation and subcellular trafficking of Bid. We demonstrate that Bid is shuttled between the nucleus and mitochondria in a time dependent manner following replication stress and that this shuttling is regulated by Crm1. We also provide evidence that chromatin localized Bid is phosphorylated. These data suggest that the DNA damage response influences the sub-cellular localization of Bid and that the phosphorylation of Bid may regulate its association with the chromatin. In addition, we explored additional, non-apoptotic functions of Bid on mitochondrial structure and physiology. Our findings indicate that Bid is essential for the maintenance of mitochondrial cristae structure, efficient respiration, cardiolipin composition, and cell viability in a manner that is independent of its apoptotic function. Furthermore, we show that Bid deficiency sensitizes mice to left ventricular cardiac dysfunction in conditions of increased cardiac stress. We also identify a significant association between BID SNPs and myocardial infarction in human patients. We also demonstrate that mitochondrial respiration is dependent on methionine 148 of Bid, a site that is mutated by one of the identified human Bid SNPs. Thus, Bid is essential for normal mitochondrial structure and physiology as well as for tolerance of the heart to acute stress.

The goal of my graduate study is to understand the role of endocytosis for signaling receptor activation during development, especially ligand endocytosis for Notch activation. Notch is a transmembrane receptor which is conserved in metazoans. I am using the Drosophila model system. Notch is required in almost every developmental context and abnormality in Notch signaling components is related to many human diseases. Delta, one of the Notch ligands, is also a transmembrane protein. To activate Notch, endocytosis of Delta in the signaling cells is essential. However, the exact mechanism of how Delta endocytosis regulates Notch activation is not known. Liquid facets (Lqf) is an endocytic protein, called epsin in vertebrates, which is required only in the signaling cells for Delta endocytosis and Notch activation. Overexpression of Lqf in the eyes results in malformed eyes. Using this phenotype as a background, an EMS-mutagenesis screen was performed and auxilin mutants were isolated as enhancers of the eye phenotype. Auxilin is a J-domain protein involved in fission and uncoating of clathrin-coated vesicles. Mosaic clonal analysis showed that auxilin functions in Notch activation and that auxilin is required only in the signaling cells. The auxilin mutant phenotype was suppressed by addition of a clathrin heavy chain transgene. This result suggests that the auxilin phenotype is at least partly caused by clathrin depletion and that auxilin generates a pool of free clathrin which is required for Delta endocytosis. Auxilin is a multi-domain protein. Two C-terminal domains, the clathrin-binding and the J domains, are sufficient to function as auxilin in Drosophila. One of the popular models to explain why Delta endocytosis is required in the signaling cells is the 'recycling model' in which inactive Delta is endocytosed and recycled to the plasma membrane in active form. Rab11 is a small GTPase that regulates recycling. If the recycling model is correct, rab11 mutants may show a phenotype similar to auxilin, lqf and Delta mutants. The rab11 hypomorphs or expression of rab11 dominant negative result in fewer photoreceptor cells and less Delta protein in the eye. These phenotypes are the opposite of typical mutant phenotypes of Notch components. The rab11 mutant phenotype argues against the recycling model.

<p> <i>Myo-inositol</i> is a six-carbon sugar alcohol. It is essential as a precursor of the phospholipid membrane component phosphatidylinositol (PI) and the phosphoinositide signaling pathway in all eukaryotes. It aids in cellular metabolism, osmoregulation, and plays an important role in fertilization and diseases such as diabetes, bipolar disorder, and Alzheimer&rsquo;s disease. <i> Myo</i>-inositol metabolism is comprised of synthesis, transport, catabolism, and recycling. <i>Myo</i>-inositol synthesis is catalyzed by myo-inositol-3-phosphate synthase (MIPS). Surprisingly, synthesis of <i>myo</i>-inositol and its role in fertilization has not yet been studied in the model organism <span style="text-decoration:overline"> Drosophila melanogaster</span> (fruit fly). We hypothesize that MIPS expression is essential for growth and development of <i>D. melanogaster.</i> In this study, a precise deletion of the entire MIPS gene was generated and confirmed through PCR amplification and sequencing of the resultant DNA fragments. The lack of the MIPS transcript in homozygous MIPS deletion flies was confirmed by RT-PCR. During that experiment, two additional isoforms of MIPS were identified in wild-type flies (CS). Supplementation of chemically defined food with 0.5mM inositol was required to sustain all homozygous MIPS deletion fly strains. Fully-grown homozygous deletion flies could live without additional inositol in the food, but newly emerged larvae only survived to the first instar larval stage. However, even while on rich media supplemented with 170mM inositol, a homozygous MIPS deletion stock was unable to produce viable offspring. Homozygous MIPS deletion strains were identified as male-sterile, incapable of producing offspring when mated to any strain of females (including wild-type). Homozygous female MIPS deletion flies were fertile and maintained a high fecundity rate when mated to any strain (with an exception of homozygous male MIPS deletion flies). The male-sterility was complemented with the addition of a wild-type MIPS gene to chromosome 3. Testes dissections of homozygous male MIPS deletion flies revealed improper progression of spermatogenesis, specifically during sperm individualization. These studies contribute to the understanding of the role of inositol synthesis in growth, development, and fertilization.</p>

Diabetes is a worldwide health issue. In both type I and late stage type II diabetes, significant beta-cell loss causes insulin deficiency and hyperglycemia. Replenishing beta cells is a promising therapy and it requires enhancing beta-cell replication, activating a beta-cell neogenesis program or transplanting exogenous beta cells. This thesis investigates the in vivo pancreatic endocrine cell differentiation process and the regulation of endocrine lineage allocation by key transcription factors, which will be tremendously informative for regenerating beta cells and creating cell-based therapy for diabetes. In this thesis research, we designed a novel bipartite Cre cell lineage tracing technique to reconstitute Cre activity only in a subset of the Ngn3+ pro-endocrine progenitors and discovered that the Ngn3+Myt1+ subset favors beta-cell fate over alpha-cell fate. Transcriptional and epigenetic analysis of the Ngn3+ progenitors from different embryonic stages revealed that gene expression and DNA methylation of endocrine genes, including Myt1, undergo dynamic changes along the developmental timeline, which is correlated with the temporal change of the Ngn3+ progenitors' differentiation competence. Meanwhile, I designed a tamoxifen-inducible bipartite CreERT2 construct and characterized its recombination properties in cell lines. This inducible bipartite CreERT2 can be used to generate mouse models to further dissect the differentiation potential of the Ngn3+Myt1+ progenitors at various embryonic stages. In addition, we observed the variation of Cre reporter sensitivity and reported non-parallel recombination of floxed alleles in the same cell here as an integral part of the Cre technique. We also investigated the regulation of Ngn3 expression by Notch signaling and miRNAs and discovered that Ngn3 augments its own expression by a miRNA-mediated inhibition of Notch signaling. We further explored the possibility that miRNAs can translocate across the plasma membrane via gap junctions and exert their functions non-cell-autonomously to counteract the Notch lateral inhibition effect. With a better understanding of the in vivo beta-cell differentiation process, our research will shed light upon the development of cell replacement therapies for diabetes.

<p> The construction and refinement of early neuronal circuits is fundamentally relevant to adult brain function, developmental disorders, and learning and plasticity, but only a small part of this process is well understood (Cang and Feldheim, 2013; Ebert and Greenberg, 2013). A significant and long-standing debate in the field concerns the relative contributions of "hardwired" genetic and molecular determinants versus "plastic" environment and activity-driven alterations (Cline, 2003). While circuits are largely hardwired in most insect and invertebrate species (Hiesinger, 2006), the mammalian nervous system appears to rely on a combination of early molecular cues and later periods of activity-driven plasticity to refine circuits. An interesting middle ground in this process is a period during which circuits have begun to form and propagate activity, but do not yet function in an adult state (Huberman, 2008; Kirkby, 2013; Wong, 1999). At this time, multiple sensory systems are believed to experience "spontaneous" activity patterns that may help to refine circuits, but the form, relevance, and even existence of this activity is under debate (Cang and Feldheim, 2013). Similarly, relatively little is known about the molecules that drive these later stages of synapse and neuronal arbor formation, and the relation that they might have to available activity patterns (Feldheim and O'Leary, 2010). </p><p> In this thesis, I first describe experiments using <i>in vivo</i> calcium imaging in both retinal ganglion cell (RGC) axons and visual midbrain and cortex neuronal cell bodies that confirm the existence of patterned spontaneous activity ("retinal waves") throughout the early postnatal mouse visual system. In a second series of experiments with a genetic knockout believed to disrupt retinal waves <i>in vivo</i>, I find that both the frequency and pattern of waves are drastically disrupted in krrodcout mice relative to controls. Interestingly, I also find that downstream "spontaneous" activity patterns are "de-coupled" from retinal wave activity in this knockout. Conditional knockout experiments further revealed that retinal waves are required in a region-specific manner to drive circuit refinement, thus confirming the necessity of spontaneous activity to development in the visual system. Subsequent rescue experiments demonstrated that the properties of retinal waves are differentially relevant to separate visual circuits, implying that normal wave activity is likely optimized to refine multiple circuits concurrently. A final set of experiments was designed to investigate the role of Down syndrome cell adhesion molecule (DSCAM) in visual circuit development at ages when activity-dependent refinement is now believed to predominate. These results revealed that DSCAM knockout disrupts multiple visual circuits at a surprisingly late age, and in surprising ways. A striking "barrel" phenotype in the retinotopic map of germline and retina-specific conditional knockout mice strongly implies that loss of this cell-adhesion molecule can act on both axon-specific and non cell-autonomous levels during later ages when axon, synapse, and dendrite elaboration is currently believed to be primarily driven by spontaneous activity. </p><p> Together, these results depict visual system development as a process that initially relies on graded molecular cues to establish rough circuit guidelines, and then uses finely tuned patterns of spontaneous activity along with synaptic adhesion molecules to induce synapse and arbor elaboration and refinement. While there are likely a great deal of redundant and homeostatic mechanisms to ensure correct formation of such fundamental circuits, the defects induced by our manipulations were highly penetrant and often persisted into late adulthood, implying the existence of critical periods that will likely prove relevant to future studies of plasticity and developmental disorders. Overall, this work describes a system that relies on a complex synchronization of all available information to ensure the correct development of evolutionarily-relevant circuits within a short period of time.</p>

The human brain is a network of ninety billion neurons that allows for many of the behavioral adaptations considered unique to our species. One-fifth of these neurons are layered in an epithelial sheet known as the cerebral cortex, which is exquisitely folded into convolutions called gyri. Defects in neuronal number clinically present with microcephaly (Greek for “small head”), and in inherited cases these defects can be linked to mutations that identify genes essential for neural progenitor proliferation. Most microcephaly genes are characterized to play a role in the centrosome, however rarer presentations of microcephaly have identified different mechanisms. Charged multivesicular body protein/Chromatin modifying protein 1A (CHMP1A) is a member of the ESCRT-III endosomal sorting complex, but is also suggested to localize to the nuclear matrix and regulate chromatin. We show that loss-of-function mutations to human CHMP1A cause a rare microcephaly syndrome with reduced cerebellar volume. CHMP1A mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation, and loss of enrichment of INK4A promoter DNA in chromatin immunoprecipitations performed against BMI1, indicating a loss of the normal repression of INK4A by BMI1. Defects in zebrafish produced by morpholino-based knockdown of the CHMP1A orthologue resembled those seen after bmi1 knockdown, and were partially rescued by INK4A orthologue knockdown. Chmp1a is expressed in dividing cells in the developing cerebral cortex and cerebellar external germinal layer, and in vitro knockdown assays using short hairpin RNA implicate a role in Wnt- and Shh-pathway signal transduction. Altogether, this suggests that CHMP1A serves as a critical link between cytoplasmic and nuclear signals that regulate neural progenitor proliferation. Compared to microcephaly, polymicrogyria is a more heterogeneous brain malformation that has been suggested to implicate molecular mechanisms involved in pattern formation in the cortex. Many cases of polymicrogyria show an asymmetric distribution, and we demonstrate that these cases are strongly biased towards a right-predominant pattern. Using whole-exome sequencing in patients with polymicrogyria, we identify rare mutations in two primary microcephaly genes, ASPM and WDR62. Interestingly, some of these patients lack profound microcephaly, suggesting heretofore underappreciated pleiotropic effects of these centrosomal genes.

RNA-binding proteins are key players in post-transcriptional regulation of gene expression by orchestrating RNA fate from synthesis to decay. Hundreds of proteins with RNA-binding capacity have been identified so far, yet only a small fraction has been functionally characterized and presumably many more RNA-binding proteins await discovery. The roles of RNA-binding proteins in the nervous system are of particular interest because accumulative evidence has linked RNA-based mechanisms to neural development, maintenance and repair. Here, the three RNA-binding proteins under study are IGF-II mRNA binding proteins IMP-1 and IMP-2, known to be involved in mRNA localization, translational control and stability, and adenomatous polyposis coli (APC), identified as a novel RNA-binding protein. To systematically identify their RNA binding profiles, a high-throughput approach combining protein-RNA crosslinking and immunoprecipitation with next-generation sequencing (HITS-CLIP) was applied in embryonic mouse brain. A nonparametric method was developed to computationally analyze the CLIP sequencing data, mapping transcriptome-wide protein-RNA interactions. The identified target mRNAs of IMP-1 and IMP-2 were highly enriched for functions related to neural development, especially neuron projection morphogenesis and axon guidance signaling. Moreover, these target mRNAs were associated with a variety of neurological diseases, including neurodevelopmental and neurodegenerative disorders. Supporting roles in axon development, knockdown of IMP-1 or IMP-2 caused aberrant trajectories of commissural axons in chicken spinal cord. APC mRNA targets were highly enriched for APC-related functions, including microtubule organization, cell and axon motility, Wnt signaling, cancer and neurological disease. Among the APC targets was Tubulin &#946;-2B (Tubb2b), previously known to be required for neuronal migration. It was found that Tubb2b was synthesized in axons, and localized preferentially to dynamic microtubules in the peripheral domain of the growth cone. Blocking the APC binding site in the Tubb2b mRNA 3'UTR caused reduction in its expression in axons and loss of the growth cone peripheral area, and impaired cortical neuron migration in vivo. These findings offer an informative snapshot of the protein-RNA interactome, which can provide a basis to better understand the roles of RNA-binding proteins in the nervous system.

My PhD thesis is focused on two fundamental aspects of stem cell metabolism: (1) the role of Lin28 in programming stem cell metabolism, and (2) how metabolism in turn fuels and governs pluripotency. Our studies led us to discover that the stem cell factor Lin28a promotes gigantism by enhancing glucose metabolism in mice, coinciding with discoveries that LIN28B polymorphisms influence height variation in human GWAS. Subsequently, we discovered that the Lin28/let-7 pathway controls glucose metabolism by orchestrating the upregulation of multiple insulin-PI3K-mTOR components, particularly in skeletal muscle progenitors. Since let-7 accumulates with aging, our discoveries suggest that let-7 could represent a new drug target for treating insulin resistance and type 2 diabetes during aging. During these studies, we also observed that Lin28a enhances tissue regeneration in adulthood. Regeneration capacity has long been known to decline with aging, but why juvenile organisms show enhanced tissue repair had remained unexplained. We found that Lin28a reactivation improved the regrowth of skin, hair, cartilage, bone and mesenchyme after injuries. Let-7 repression was necessary but insufficient to explain these phenotypes. In parallel, Lin28a bound to and enhanced the translation of mRNAs for several oxidative enzymes, thereby increasing OxPhos. Lin28a-mediated tissue repair was negated by OxPhos inhibition, whereas a pharmacologically-induced increase in OxPhos promoted wound repair. Thus, Lin28a enhanced tissue regeneration in adults by reprogramming cellular bioenergetics. My interest in the central principles of stem cell metabolism also led us to map the metabolic pathways associated with pluripotency during iPS reprogramming and Lin28/let-7 perturbation. Surprisingly, we found that Thr-Gly-S-adenosylmethionine (SAM) metabolism consistently showed the best correlation with pluripotency. 13Carbon isotope metabolomics further revealed that Thr was catabolized to generate Gly and acetyl-CoA, and ultimately SAM - essential for all methylation reactions. Thr is required for SAM and histone H3K4 methylation in mouse ESCs, thus regulating the open euchromatin and pluripotency of ESCs. Our study shed light on a novel amino acid pathway in stem cells, and demonstrated that metabolic conditions can direct cell fate. In summary, my work has helped us to understand how we can reprogram and manipulate metabolic networks to regulate stem cell homeostasis.