The vertebrate hindbrain is divided early in embryogenesis along its anterior-posterior axis into eight segments known as rhombomeres. This provides an excellent model for studying early segmentation and region-specific transcriptional domains. MafB, a basic domain leucine zipper transcription factor, is the first gene known to be expressed in the presumptive rhombomere 5 and 6 domain (r5-r6). MafB expression is directly activated by the homeodomain protein vHnf1. vHnf1 and MafB share an anterior expression limit at the r4/r5 boundary but vHnf1 expression extends beyond the posterior limit of MafB and, therefore, cannot establish the posterior expression boundary of MafB. Through the use of in situ hybridization, immunofluorescence, and chromatin immunoprecipitation analyses, I have determined that the caudal-related homeodomain protein Cdx1 establishes the posterior boundary of MafB by directly inhibiting expression beyond the r6/r7 boundary. My results indicate that MafB is one of the earliest direct targets of Cdx1.

The vertebrate hindbrain is divided early in embryogenesis along its anterior-posterior axis into eight segments known as rhombomeres. This provides an excellent model for studying early segmentation and region-specific transcriptional domains. MafB, a basic domain leucine zipper transcription factor, is the first gene known to be expressed in the presumptive rhombomere 5 and 6 domain (r5-r6). MafB expression is directly activated by the homeodomain protein vHnf1. vHnf1 and MafB share an anterior expression limit at the r4/r5 boundary but vHnf1 expression extends beyond the posterior limit of MafB and, therefore, cannot establish the posterior expression boundary of MafB. Through the use of in situ hybridization, immunofluorescence, and chromatin immunoprecipitation analyses, I have determined that the caudal-related homeodomain protein Cdx1 establishes the posterior boundary of MafB by directly inhibiting expression beyond the r6/r7 boundary. My results indicate that MafB is one of the earliest direct targets of Cdx1.

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To understand the gene regulatory network (GRN) governing caudal hindbrain formation in embryonic zebrafish, several early expressed factors have been manipulated, and multiple genetic mutants have been characterized. Such analyses have identified morphogens such as Retinoic Acid (RA) and Fibroblast growth factors (FGFs), as well as transcription factors like hoxb1b, hoxb1a, hnf1ba, and valentino as being required for rhombomere (r) r4-r6 formation in zebrafish. Considering that the caudal hindbrain is relatively complex – for instance, unique sets of neurons are formed in each rhombomere segment – it is likely that additional essential genes remain to be identified and integrated into the caudal hindbrain GRN. Our results reveal that r4 gene expression is unaffected by the individual loss of hoxb1b, hoxb1a or RA, but is under the combinatorial regulation of RA together with hoxb1b. In contrast, r5/r6 gene expression is dependent on RA, FGF, hnf1ba and valentino – as individual loss of these factors abolishes r5/r6 gene expression. Analysis of six mutant lines (gas6, gbx1, sall4, eglf6, celf2, and greb1l) did not reveal rhombomere or neuronal defects, but transcriptome analysis of one line (gas6 mutant) identified expression changes for genes involved in several developmental processes – suggesting that these genes may have subtle roles in hindbrain development. We conclude that r4-r6 formation is relatively robust, such that very few genes are absolutely required for this process. However, there are mechanistic differences in r4 versus r5/r6, such that no single factor is required for r4 development while several genes are individually required for r5/r6 formation.

In vertebrates, the embryonic hindbrain is transiently subdivided along its anterior-posterior (A-P) axis into 8 well defined segments termed rhombomeres (r1-8). Each rhombomere represents a true cellular compartment in transcriptional profile, lineage restriction and neuronal organization. Thus, the vertebrate hindbrain provides a beautiful model for studying mechanisms of anterior-posterior patterning, signal transduction and interpretation, initiation and maintenance of transcriptional profiles, cell sorting and border formation. The Kreisler/MafB gene, which encodes a basic leucine zipper (bZIP) transcription factor that regulates some Hox genes, is one of the first genes to be expressed segmentally in the hindbrain, and is subject to a dynamic and complex regulatory process. However, unlike the Hox genes, Kreisler/MafB is not located within a large cluster of genes and therefore provides a simple system for dissecting the molecular mechanisms involved in hindbrain compartmentalization. In dissecting the mechanisms that govern Kreisler/MafB regulation, we have identified the S5 regulatory element that directs early MafB expression in the future r5-r6 domain. We have found a binding site within S5 that is specific for the Variant Hepatocyte Nuclear Factor 1 (vHNF1) to be essential, but not sufficient for early induction of r5-r6-specific expression. Thus, early inductive events that initiate MafB expression are clearly distinct from later acting ones that modulate its expression levels. Using mouse mutants, we have shown that MafB is dependent on the M33 polycomb protein and other mechanisms of chromatin remodeling. We then utilized transgenic flies and mice as well as binding assays to identify and validate a PcG/trxG response element (PRE), PRE1 which acts to reorganize the surrounding chromatin, regulating S5-dependent expression. To our knowledge, PRE1 is the first validated vertebrate PcG/trxG response element. Thus, PRE1 provides a springboard for further exploration of the mechanisms governing chromatin remodeling.

In vertebrates, the embryonic hindbrain is transiently subdivided along its anterior-posterior (A-P) axis into 8 well defined segments termed rhombomeres (r1-8). Each rhombomere represents a true cellular compartment in transcriptional profile, lineage restriction and neuronal organization. Thus, the vertebrate hindbrain provides a beautiful model for studying mechanisms of anterior-posterior patterning, signal transduction and interpretation, initiation and maintenance of transcriptional profiles, cell sorting and border formation. The Kreisler/MafB gene, which encodes a basic leucine zipper (bZIP) transcription factor that regulates some Hox genes, is one of the first genes to be expressed segmentally in the hindbrain, and is subject to a dynamic and complex regulatory process. However, unlike the Hox genes, Kreisler/MafB is not located within a large cluster of genes and therefore provides a simple system for dissecting the molecular mechanisms involved in hindbrain compartmentalization. In dissecting the mechanisms that govern Kreisler/MafB regulation, we have identified the S5 regulatory element that directs early MafB expression in the future r5-r6 domain. We have found a binding site within S5 that is specific for the Variant Hepatocyte Nuclear Factor 1 (vHNF1) to be essential, but not sufficient for early induction of r5-r6-specific expression. Thus, early inductive events that initiate MafB expression are clearly distinct from later acting ones that modulate its expression levels. Using mouse mutants, we have shown that MafB is dependent on the M33 polycomb protein and other mechanisms of chromatin remodeling. We then utilized transgenic flies and mice as well as binding assays to identify and validate a PcG/trxG response element (PRE), PRE1 which acts to reorganize the surrounding chromatin, regulating S5-dependent expression. To our knowledge, PRE1 is the first validated vertebrate PcG/trxG response element. Thus, PRE1 provides a springboard for further exploration of the mechanisms governing chromatin remodeling.

<p>Medulloblastoma is the most common malignant brain tumor in children. The understanding of the genetic alterations in this tumor is emergent, and many such genetic driver events have yet to be functionally-characterized. Our studies have sought to understand the causes and consequences of OTX2 dysregulation in established medulloblastomas and in its putative cellular origins. Using a tumor genetic approach, we have uncovered frequent OTX2 copy number gains driving expression of this oncogene in a subset of medulloblastomas. However, OTX2 is frequently expressed in medulloblastomas independent of genomic copy number gain, and we thus sought to understand the transcriptional regulation of this gene in these tumors. We have found that chromatin accessibility, promoter DNA methylation, and activity of a distal downstream enhancer is distinct between OTX2-expressing and -nonexpressing medulloblastomas. Notably, autoregulation serves to maintain OTX2 expression in some medulloblastomas, whereas DNA methylation actively suppresses OTX2 in tumors not expressing this gene. Finally, we describe the effect of expressing Otx2 (the mouse homolog of OTX2) aberrantly in the developing mouse hindbrain, revealing that Otx2 disrupts spatiotemporal restrictions of neuronal progenitor cell proliferation. The effect of Otx2 in vivo is transient, with ectopically-proliferating cells give way to differentiated neurons. We found that OTX2 expression was not able to give rise to high penetrance medulloblastoma when combined with P53 deletion or double heterozygosity for P53 and PTEN. Thus, although Otx2 alters migration and proliferation dynamics of hindbrain neuronal progenitor cells, further studies are needed to identify the genetic alterations that cooperate with this oncogene to give rise to medulloblastoma.</p> / Dissertation

Treatment of newborn rats with 6-hydroxydopa (6-OHDOPA, 60 μg/g IP) increased the levels of norepinephrine in the adult cerebellum and hindbrain. Concurrent treatment with morphine sulfate (20 μg/g IP) potentiated the response to 6-OHDOPA in the cerebellum and pons-medulla. In addition, increased noradrenergic neurite density in 4 week cerebellar cortex (as observed with histofluorescent staining by glyoxylic acid) suggests that neonatal morphine increased the sprouting of noradrenergic neurons in the 6-OHDOPA treated rats.

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