Genome-wide exploration of direct GBX2 target genes and their contributions to mouse development
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The cells that comprise the vertebrate nervous system require molecular cues to determine their cellular position, identity and their ability to make appropriate connections with their target tissues. These requirements largely rely on the precise spatial-temporal regulation of gene expression and can be controlled by a variety of intracellular mechanisms. However, a critical regulatory step in controlling gene expression is the initial transcription of the gene from an organism's genome. Transcription factors are proteins that primarily function by recognizing direct target gene sequences and modulating their expression. As such, the investigation of genes directly targeted and regulated by transcription factors provides a fundamental link in addressing their contributions to vertebrate nervous system development. There are many known classes of transcription factors encoded by the genomes of vertebrate species. One class critical for vertebrate development is the homeobox transcription factors, which encode a highly conserved DNAbinding homeodomain. The Gastrulation brain homeobox (Gbx) genes consists of two known family members, Gbx1 and Gbx2. To date, previous Gbx2 misexpression studies in the mouse have largely ascribed requirements for Gbx2 in development of the hindbrain, spinal cord, ear and heart. However, prior to the work presented in this dissertation, the direct targets and molecular pathways mediated by GBX2 were largely unknown. In order to dramatically expand our knowledge of Gbx2 function, we conducted the first genome-wide investigation of direct GBX2 target genes. Our study identified over 1,000 target genes. Applying stringent selection criteria to the identified targets, we reduced the number to 286 target genes for subsequent analyses. Interestingly, 51% of GBX2 targets identified are expressed in the nervous system. Our studies have revealed that GBX2 binds to the promoter or intronic sequences of several targets including EEF1A1, NRP1, PLXNA4, ROBO1, PCDH15, USH2A and NOTCH2. The target genes PCDH15, USH2A and NOTCH2 are involved with inner ear development. PCDH15 and USH2A are also associated with the congenital disease Usher syndrome, a condition that is characterized by the progressive loss of hearing and sight. We further demonstrated that GBX2 interacts with the EEF1A1 core promoter and functions as a transcriptional activator within this region. A primary function of EEF1A1 is the delivery of aminoacyl-tRNA to the ribosome during protein synthesis. The impact of GBX2 regulating EEF1A1 may suggest a novel GBX2-mediated mechanism for regulating protein expression during vertebrate development. The development of the anterior hindbrain is dependent on the ability of divergent motor neuron and multipotent cranial neural crest (NC) cells to migrate to their target locations. During development, the hindbrain is transiently segmented into eight compartments known as rhombomeres. How Gbx2 impacts motor neuron, cranial NC cell development and gene expression within the hindbrain is not well understood. Loss-of-function studies in zebrafish and mouse have demonstrated a requirement of Gbx2 in motor neuron development in the anterior hindbrain and spinal cord. Here we show that a loss of Gbx2 and anterior hindbrain tissue results in a disruption of r2 motor neuron development and suggest a novel requirement of Gbx2 in the correct temporal repression of the transcription factor Krox20 in r3. Cranial NC cells originating in the hindbrain are highly motile and require coordinated inputs from divergent signaling pathways to ensure their appropriate positions in the developing embryo. Many of the defects in Gbx2 mutants are observed in NC-derived structures. Furthermore, the cardiac and craniofacial phenotypes observed in Gbx2 mutants are reminiscent of defects reported in individuals with the congenital disease DiGeorge syndrome. Interestingly, GBX2 target genes ROBO1 and NRP1 are involved with NC cell migration. In mice, loss of Robo1 or Nrp1 results in the disrupted migration of NC cell subpopulations within the developing heart and anterior hindbrain. Previous studies in Gbx2-/- mice have demonstrated that a loss of Robo1 disrupts cardiac NC cell migration and contributes to the observed arterial defects. Here we provide evidence that GBX2 functions directly upstream of ROBO1 and NRP1. Data presented in this dissertation further show a loss of Robo1 and a reduction of migrating cranial NC cells from r4 in Gbx2-/- mice. Additionally, studies in Gbx2 mutant mice revealed a loss of Nrp1 and an increase in apoptosis in a subpopulation of cranial NC cells from r2. Loss of Robo1 and Nrp1 are now thought to contribute to the NC cell migratory defects and subsequently the disrupted NC-derived structures observed in Gbx2 mutant mice. These findings have led to new insights into Gbx2 function during vertebrate development. Studies discussed in this dissertation have resulted in a dramatic increase in the number of known direct GBX2 target genes and molecular pathways potentially regulated by GBX2. The subset of GBX2 targets investigated thus far suggests that they may contribute to the development of tissues and structures impacted by the loss of Gbx2 in the mouse heart, ear and hindbrain. The involvement of GBX2 and direct target genes with multiple congenital diseases further illustrates the biological and clinical importance of the findings presented in the following body of work. Future studies aimed at elucidating the biological impact of GBX2 through direct target genes will reveal the precise molecular pathways impacted by GBX2 during vertebrate development.
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