Hox as well as Meis proteins are known to bind DNA as heterodimers with members of the Pbx family, and it is believed that such complexes mediate the in vivo functions of Hox and Meis. To begin exploring the role of hoxb1b and meis3 in vertebrate development, we isolated and characterized a zebrafish pbx cDNA which encodes a novel member of the pbx family, which we called pbx4. In situ analysis revealed that pbx4 RNA is maternally deposited and is detected throughout the zebrafish embryo during blastula stages. It becomes excluded from ventroanterior structures at late gastrula stages and is detected within the developing central nervous system during segmentation stages. pbx4 expression overlaps with that of hoxb1b and meis3, in the region of the presumptive caudal hindbrain during gastrula stages. In vitro binding experiments revealed that Pbx4/Meis3 and Pbx4/Hoxb1b, as well as a novel trimeric complex containing Pbx4, Meis3 and Hoxb1b form in vitro. Thus, protein complexes of different combinations of Pbx4, Meis3 and Hoxb1b form in vitro and importantly pbx4, meis3 and hoxb1b are coexpressed in a domain at the level of the presumptive caudal hindbrain during zebrafish gastrula stages. These findings raised the possibility that similar complexes may exist in vivo and may be involved in the specification of distinct developmental fates. To address this possibility we overexpressed meis3,pbx4 and hoxb1b in zebrafish embryos and we tested for the effect on endogenous gene expression, morphology and neuronal specification. Our results demonstrate that Hoxb1b/Pbx4/Meis3-containing complexes induce extensive expression of several hindbrain genes (hoxb1a, hoxb2, krox20 and valentino) anterior to their normal expression domains, and mediate the transformation of anterior (forebrain and midbrain) fates to posterior (hindbrain) ones, including the formation of excess ectopic Mauthner neurons. Ectopic expression of Hoxb1b/Pbx4/Meis3-containing complexes also leads to truncation of the embryonic axis anteriorly. In contrast, Hoxb1b/Pbx4 expression induces ectopic expression of only hoxb1a (primarily in r2), but does not mediate axial truncations, and Hoxb1b (or its mouse homolog, HoxA1) has been reported to induce an ectopic pair of Mauthner neurons in r2 (Alexandre et al., 1996). Thus, binding of Meis3 to Hoxb1b/Pbx4 generates Hoxb1b/Pbx4/Meis3-containing complexes that have qualitatively (e.g. induction of hoxb2 expression) and quantitatively (e.g. larger number of ectopic Mauthner neurons) different effects than Hoxb1b/Pbx4-containing complexes. These results suggest that Meis3/Pbx4/Hoxb1b-containing complexes may be responsible for specification of hindbrain fates in vivo. In addition to meis3, three other members of the meis/prep gene family are expressed during early embryogenesis in zebrafish. Analysis of gene expression patterns revealed both common as well as unique spatial and temporal expression patterns for each of these genes. This finding raises the question of whether all family members are functionally similar to meis3 or meis3 performs unique functions. To address this question we overexpressed meis1.1, meis2.2 and prep1 in zebrafish embryos and we asked whether they are able to induce hindbrain fates like meis3 does. Overexpression of any Meis protein, or Prep, along with Pbx4 and Hoxb1b resulted in embryos that were truncated anteriorly and exhibited massive ectopic hoxb1a and hoxb2 expression anterior to their normal expression domains. Furthermore, in vitro analysis demonstrated that they are all able to form dimers with Pbx4 in vitro. In addition, analysis of their subcellular localization defined Pbx4 interaction as a prerequisite for nuclear localization of all Meis and Prep proteins. Thus, at least in the overexpression assay there are no functional differences among meis/prep genes. These results raise the question of what is exactly the function of Meis/Prep proteins. Is binding to Pbx proteins and to DNA their only function, or do they have additional roles? To address this question we performed a deletional analysis of Meis3 protein and we tested the requirement of each domain in the overexpression assay. Our experiments revealed that the domain N-terminal to the Pbx-Interaction-Domain (PID) as well as the domain C-terminal to the Homeodomain are not required for the function of Meis3, at least in the overexpression assay. Furthermore, the homeodomain and the domain between the PID and the homeodomain are not required. From our previous analysis (Vlachakis et al., 2001) we know that the PID is required for the Meis3/Pbx4/Hoxb1b synergistic induction of hindbrain fates. Our deletion analysis extended this fmding showing that the PID is also sufficient to provide the Meis3 function in vivo, at least in our overexpression assay. Furthermore, a mutant PID that does not bind Pbx, when fused to the Pbx4 homeodomain induced hindbrain fates upon overexpression along with Hoxb1b. This finding suggests that the PID (motifs M1, M2 and the domain in between them, ID) besides binding to Pbx may also bind another protein that is required for the Meis3/Pbx4/Hoxb1b synergistic induction of hindbrain fates. Taking all our results together, we propose the following roles for Meis proteins in the transcriptional activation complexes. First, they are involved in the nuclear localization of Pbx4. Second, they bind to DNA as heterodimers with Pbx4 facilitating binding of Hoxb1b to Pbx4, which occurs only on DNA. In doing so, they provide the specificity for DNA binding since the Meis3/Pbx4 dimer first recognizes the "hox response element" and then the Hox protein is recruited. Third, they stabilize the binding of Hoxb1/Pbx4 complex on DNA. Fourth, they are responsible for recruiting additional factors to DNA, necessary for activation of target genes. The complicate and dynamic spatial and temporal expression patterns of the meis/prep genes, suggest that they are involved in many different processes during embryogenesis, as well as in the adult organism. We believe that one or more members of the Meis/Prep family execute some of the functions listed above at different times and places during development, although all member are probably capable of executing all these functions.

No description available.

Stem cell biology is an exciting field that will lead to significant advancements in science and medicine. We hypothesize that inducing the expression of stem cell genes, using the embryonic stem cell gene nanog, will reprogram cells and dedifferentiate human mesenchymal stem cells into pluripotent stem cells capable of neural differentiation. The aims of initial studies are as follows: Aim 1: Demonstrate that forced expression of the embryonic stem cell gene nanog induces changes in human mesenchymal stem cells to an embryonic stem cell-like phenotype. Aim 2: Demonstrate that induced expression of nanog up-regulates the expression of multiple embryonic stem cell markers and expands the differentiation potential of the stem cells. Aim 3: Demonstrate that these nanog-expressing stem cells have the ability to differentiate along neural lineages in vitro and in vivo, while mock-transfected cells have an extremely limited capacity for transdifferentiation. Alternatively, we hypothesize that embryonic stem cell genes can become activated in malignant gliomas and differentially regulate the subpopulation of cancer stem cells. This study examines the role of embryonic stem cell genes in transformed cells, particularly cancer stem cells. These studies explore has the following objectives: Aim 1: Isolate different sub-populations of cells from tumors and characterize cells with stem cell-like properties. Aim 2: Characterize the expression of embryonic stem cell markers in the sub-population of cancer stem cells. Aim 3: Examine the effects of histone deacetylase inhibitors at inhibiting the growth and reducing the expression of stem cell markers. Our research has demonstrated the potential of the embryonic transcription factor, nanog, at inducing dedifferentiation of human bone marrow mesenchymal stem cells and allowing their recommitment to a neural lineage. Specifically, we used viral and non-viral vectors to induce expression of NANOG, which produced an embryonic stem cell-like morphology in transduced cells. We characterized these cells using real-time PCR and immunohistochemical staining and find an up-regulation of genes responsible for pluripotency and self-renewal. Embryonic stem cell markers including Sox2, Oct4 and TERT were up-regulated following delivery of nanog. The role of nanog in the expression of these markers was further demonstrated in our induced-differentiation method where we transfected embryonic stem cell-like cells, that have been transduced with nanog flanked by two loxP sites, with a vector containing Cre-recominase. We tested the ability of these nanog-transfected cells to undergo neural differentiation in vitro using a neural co-culture system or in vivo following intracranial transplantation. Our next study characterized patient-derived glioblastoma cancer stem cells. We found that cells isolated from serum-free stem cell cultures were enriched for stem cell markers and were more proliferative than the bulk population of cells grown in convention serum-supplemented media. These cancer stem cells expressed embryonic stem cell markers NANOG and OCT4 whereas non-tumor-derived neural stem cells do not. Moreover, the expression of stem cell markers was correlated with enhanced proliferation and could serve as a measure of drug effectiveness. We tested two different histone deacetylase inhibitors, trichostatin A and valproic acid, and found that both inhibited proliferation and significantly reduced expression of stem cell markers in our cancer stem cell lines. These data demonstrate the potential use of stem cell genes as therapeutic markers and supports the hypothesis that cancer stem cells are a major contributor to brain tumor malignancy.

No description available.

Eukaryotic organisms require the ability to respond to their environments. They do so by utilizing signal transduction pathways that allow for signals to effect final biological responses. Many times, these final responses require new gene expression events that have been stimulated or repressed within the nucleus. Thus, much of the understanding of signal transduction pathways converges on the understanding of how signaling affects gene expression alterations (Kumar et al., 2004). The regulation of gene expression involves the modification of chromatin between condensed (closed, silent) and expanded (open, active) states. Histone modifications, such as acetylation, can determine the open versus closed status of chromatin. The PHD (Plant HomeoDomain) finger is a structural domain primarily found in nuclear proteins across eukaryotes. This domain specifically recognizes the epigenetic marks H3K4me2 and H3K4me3, which are di- and tri-methylated lysine 4 residues of Histone H3 (Loewith et al., 2000; Kuzmichev et al., 2002; Vieyra et al. 2002; Shiseki et al., 2003; Pedeux et al., 2005, Doyon et al., 2006). It is estimated that there are ~150 proteins that contain the PHD finger in humans (Solimon and Riabowol, 2007). The PHD finger is conserved in yeast and plants, however an analysis of this domain has only been performed done in Arabidopsis thaliana (Lee et al., 2009). The work presented in this report aims to extend the analysis of this domain in plants by identifying the PHD fingers of the crop species Oryza sativa (rice). In addition, a phylogenetic analysis of all PHD fingers in Arabidopsis and rice was undertaken. From these analyses, it was determined that there are 78 PHD fingers in Arabidopsis and 70 in rice. In addition, these domains can be categorized into classes and groups by defining features within the conserved motif. In a separate study, I investigated the function of two of the PHD finger proteins from Arabidopsis, ING1 (INhibitor of Growth1) and ING2. In humans, these proteins can be found in complexes associated with both open and closed chromatin. They facilitate chromatin remodeling by recruiting histone acetyltransferases and histone deacetylases to chromatin (Doyon et al., 2006, Pena et al., 2006). In addition, these proteins recognize H3K4me2/3 marks and are believed to be "interpreters" of the histone code (Pena et al., 2006, Shi et al., 2006). To understand the function of ING proteins in plants, I took a reverse genetics approach and characterized ing1 and ing2 mutants. My analysis revealed that these mutants are altered in time of flowering, as well as their response to nutrient and stress conditions. Lastly, I was able to show that ING2 protein interacts in vitro with SnRK1.1, a nutrient/stress sensor (Baena-Gonzalez et al., 2007). These results indicate a novel function for PHD proteins in plant growth, development and stress response. / Master of Science

Yes / Engrailed-2 (EN2) is a homeodomain-containing transcription factor that has roles in boundary formation and neural guidance in early development, but which is also expressed in a range of cancers. In addition to transcriptional regulation, it is secreted by cells and taken up by others through a mechanism that is yet to be fully elucidated. In this study, the distribution of EN2 protein in cells was evaluated using immunofluorescence with a set of antibodies raised against overlapping epitopes across the protein, and through the use of an EN2-GFP construct. MX2 expression in primary prostate tumors was evaluated using immunohistochemistry. We showed that EN2 protein is present in the cell membrane and within microvesicles that can be secreted from the cell and taken up by others. When taken up by normal cells from the stroma EN2 induces the expression of MX2 (MxB), a protein that has a key role in the innate immune response to viruses. Our findings indicate that EN2 secretion by tumors may be a means of preventing viral-mediated immune invasion of tissue immediately adjacent to the tumor. / The Ringrose Family Trust supported this study through a studentship awarded to N.P.

No description available.

One of the major goals of developmental biology is to understand how specialized groups of cells arise from an initially unspecified cell population. The vertebrate hindbrain is transiently segmented along its anterior-posterior axis into lineage-restricted compartments called rhombomeres, making it an excellent model in which to study the genetic mechanisms of axial patterning. Hox homeodomain transcription factors (TF), in close partnership with the Pbx and Meis families of TALE-class homeodomain proteins, impart unique molecular identities to the hindbrain rhombomeres, thereby specifying functionally specialized neurons within each segment. The broad goals of this thesis are to clarify the roles of Meis1 and Tshz3b TFs in Hox-dependent hindbrain patterning, and to examine the Hox-independent roles of Pbx and Meis proteins in axial patterning of the visual system. While it is clear that Hox-Pbx-Meis complexes regulate hindbrain segmentation, the contributions of individual Meis proteins are not well understood. I have shown that Meis1-depleted embryos exhibit neuronal patterning defects, even though the hindbrain retains its segmental organization. This suggests that Meis1 is making important contributions to neuronal development downstream of rhombomeric specification. A zinc-finger TF called Teashirt (Tsh) cooperates with Hox-Pbx-Meis complexes to establish segmental identity in Drosophila, but this role not been tested in vertebrates. I found that overexpression of tshz3b produces segmentation defects reminiscent of Hox-Pbx-Meis loss of function phenotype, likely by acting as a transcriptional repressor. Thus, Tshz3b may be a negative regulator of Hox- dependent hindbrain patterning. Like the hindbrain, visual system function requires that positional information be correctly specified in the retina and midbrain. I found that zebrafish Pbx and Engrailed homeodomain TFs are biochemical DNA binding partners, and that this interaction is required to maintain the midbrain as a lineage- restricted compartment. Additionally, I show that Meis1 specifies positional information in both the retina and midbrain, thereby helping to organize the axonal connections between the eye and brain. Taken together, this thesis clarifies our understanding of Hox-dependent hindbrain patterning, and makes the claim that Pbx and Meis perform a general axial patterning function in anterior neural tissues such as the hindbrain, midbrain and retina. / Molecular Biology and Genetics

No description available.

Members of the Six4/5 family of homeobox transcription factors have been implicated in multiple human disorders, including type I mytonic dystrophy, branchio-oto-renal syndrome, and holoprosencephaly, suggesting a role for these factors in the nervous system development. Using a forward genetics approach, we identified unc-39, a C. elegans homologue of the human SIX5 gene, as a novel regulator of the development of a specific neuron, called RID. Our data support the role of unc-39 early in C. elegans development and suggest a possibility of complete absence of RID neuron in unc-39 mutants. unc-39 mutant has a similar locomotion phenotype to the RID-ablated animals, which provides further support to the hypothesis that the absence of RID contributes to the locomotion phenotype observed in the mutant. We show that unc-39 functions at multiple points in the lineage that gives rise to the RID neuron, and that its function is context-dependent.