Generating GBX2 antibodies: A useful tool in determining developmental mechanisms regulated by GBX2 [abstract]

Abstract only availableThe GBX class of homeobox genes is comprised of Gbx1 and Gbx2. Both loss-of-function and gain-of-function studies in mice have shown that Gbx2 is vital for normal anterior hindbrain development of mammalian organisms. To gain more insight into the developmental mechanisms regulated by GBX2, we are generating GBX2 antibodies. To accomplish this, we have subcloned Gbx2 into the pRSET A protein expression vector and transformed the construct into BL21(DE3)pLysS cells. Protein expression was induced by IPTG. The expressed protein was analyzed by SDS-PAGE as well as Western analysis. The purified protein will be used to elicit an immune response in chickens to generate the antibodies against GBX2. Preliminary results from the SDS-PAGE and Western analysis have suggested that the GBX2 protein is being expressed. However, further testing is necessary for confirmation. Currently, we are using Western analysis to specifically target the 6x His tagged GBX2 fusion protein in order to identify the protein for further analysis by mass spectroscopy. Generation of GBX2 antibodies will provide an important tool to enhance our knowledge of how GBX2 functions in development. Having these antibodies will allow for cellular localization. In addition, the antibodies will be used in chromatin immunoprecipitation assays, which will allow for the production of a library that contains genes directly regulated by GBX2. The identification of target genes will provide a way to enable the collection of valuable data that will be useful for more long-term research goals involving the specific signaling and genetic pathways in which this transcription factor is involved

Genome-wide association study identifies multiple susceptibility loci for craniofacial microsomia

Craniofacial microsomia (CFM) is a rare congenital anomaly that involves immature derivatives from the first and second pharyngeal arches. The genetic pathogenesis of CFM is still unclear. Here we interrogate 0.9 million genetic variants in 939 CFM cases and 2,012 controls from China. After genotyping of an additional 443 cases and 1,669 controls, we identify 8 significantly associated loci with the most significant SNP rs13089920 (logistic regression P 1�4 2.15 � 10 � 120) and 5 suggestive loci. The above 13 associated loci, harboured by candidates of ROBO1, GATA3, GBX2, FGF3, NRP2, EDNRB, SHROOM3, SEMA7A, PLCD3, KLF12 and EPAS1, are found to be enriched for genes involved in neural crest cell (NCC) development and vasculogenesis. We then perform whole-genome sequencing on 21 samples from the case cohort, and identify several novel loss-of-function mutations within the associated loci. Our results provide new insights into genetic background of craniofacial microsomia

Genome-wide exploration of direct Gastrulation Brain Homeobox 2 target genes and their contributions to mouse development

[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.Includes bibliographical references (pages 166-186)

Gbx2 Is Required for the Migration and Survival of a Subpopulation of Trigeminal Cranial Neural Crest Cells

The development of key structures within the mature vertebrate hindbrain requires the migration of neural crest (NC) cells and motor neurons to their appropriate target sites. Functional analyses in multiple species have revealed a requirement for the transcription factor gastrulation-brain-homeobox 2 (Gbx2) in NC cell migration and positioning of motor neurons in the developing hindbrain. In addition, loss of Gbx2 function studies in mutant mouse embryos, Gbx2neo, demonstrate a requirement for Gbx2 for the development of NC-derived sensory neurons and axons constituting the mandibular branch of the trigeminal nerve (CNV). Our recent GBX2 target gene identification study identified multiple genes required for the migration and survival of NC cells (e.g., Robo1, Slit3, Nrp1). In this report, we performed loss-of-function analyses using Gbx2neo mutant embryos, to improve our understanding of the molecular and genetic mechanisms regulated by Gbx2 during anterior hindbrain and CNV development. Analysis of Tbx20 expression in the hindbrain of Gbx2neo homozygotes revealed a severely truncated rhombomere (r)2. Our data also provide evidence demonstrating a requirement for Gbx2 in the temporal regulation of Krox20 expression in r3. Lastly, we show that Gbx2 is required for the expression of Nrp1 in a subpopulation of trigeminal NC cells, and correct migration and survival of cranial NC cells that populate the trigeminal ganglion. Taken together, these findings provide additional insight into molecular and genetic mechanisms regulated by Gbx2 that underlie NC migration, trigeminal ganglion assembly, and, more broadly, anterior hindbrain development

<em>Elongation Factor 1 alpha1</em> and Genes Associated with Usher Syndromes Are Downstream Targets of GBX2

<div><p><em>Gbx2</em> encodes a DNA-binding transcription factor that plays pivotal roles during embryogenesis. Gain-and loss-of-function studies in several vertebrate species have demonstrated a requirement for <em>Gbx2</em> in development of the anterior hindbrain, spinal cord, inner ear, heart, and neural crest cells. However, the target genes through which GBX2 exerts its effects remain obscure. Using chromatin immunoprecipitation coupled with direct sequencing (ChIP-Seq) analysis in a human prostate cancer cell line, we identified cis-regulatory elements bound by GBX2 to provide insight into its direct downstream targets. The analysis revealed more than 286 highly significant candidate target genes, falling into various functional groups, of which 51% are expressed in the nervous system. Several of the top candidate genes include <em>EEF1A1</em>, <em>ROBO1</em>, <em>PLXNA4</em>, <em>SLIT3</em>, <em>NRP1</em>, and <em>NOTCH2</em>, as well as genes associated with the Usher syndrome, <em>PCDH15</em> and <em>USH2A</em>, and are plausible candidates contributing to the developmental defects in <em>Gbx2^−/−</em> mice. We show through gel shift analyses that sequences within the promoter or introns of <em>EEF1A1</em>, <em>ROBO1</em>, <em>PCDH15</em>, <em>USH2A</em> and <em>NOTCH2</em>, are directly bound by GBX2. Consistent with these in vitro results, analyses of <em>Gbx2^−/−</em> embryos indicate that <em>Gbx2</em> function is required for migration of <em>Robo1</em>-expressing neural crest cells out of the hindbrain. Furthermore, we show that GBX2 activates transcriptional activity through the promoter of <em>EEF1A1</em>, suggesting that GBX2 could also regulate gene expression indirectly via EEF1A. Taken together, our studies show that GBX2 plays a dynamic role in development and diseases.</p> </div

GBX2 overexpression and ChIP in human PC-3 cells.

<p>(A) Schematic representation of the HA-GBX2 and HA-GBX2ΔHD recombinant proteins containing the proline-rich region (PR), DNA-binding homeodomain (HD), and the HA epitope tag located at the amino terminus. Immunoflouresence of transiently transfected human PC-3 cells with <i>HA-GBX2</i> (C, D), and, <i>HA-Gbx2</i>Δ<i>HD</i> (F, G). Blue channel identifies DAPI staining in the nucleus (B, E). Green channel identifies GFP-GBX2 fusion proteins. (D, G) Merge displays nuclear localization of GFP-GBX2 fusion proteins. Western blots of total lysates (H) and HA-immunoprecipitated samples (I) from mock, <i>HA-Gbx2</i>, and <i>HA-Gbx2</i>Δ<i>HD</i> transfected PC-3 cells.</p

ChIP-Seq identified GBX2 targets.

<p>Genes targeted by GBX2 in human PC-3 cells based on ChIP-Seq fragments aligned to the hg18 build of the human genome on the UCSC Human Genome Browser. Bold text indicates GBX2 targets expressed in the nervous system.</p

Confirmation of murine GBX2 by Western blot and mass spectral analysis.

<p>(A) Western analysis of recombinant GBX2 proteins. The amino acid sequences for GBX2 (B) and GBX2Δ<i>HD</i> (B') recombinant proteins. Bold type indicates matched peptides identified by mass spectrometry. (C) MS/MS fragmentation data table includes: precursor mass (peptide chosen for MS/MS), approximate weight of the band analyzed by mass spectrometry, peptide sequence, location of the peptide in the protein sequence, the Mascot ion score, and the mass error for each peptide sequence. The low mass error score in parts per million (ppm = {[observed mass – theoretical mass]/theoretical mass}×10⁶) suggests that the observed mass matches the theoretical mass.</p