20 research outputs found
Formation of high molecular weight <i>mip1</i> promoter- protein complexes is dependent on pitx3 presence.
<p><b>A.</b> 48-hpf wild-type and <i>pitx3</i> morphant embryos showing normal body length and morphology that were selected for EMSA experiments. <b>B.</b> Results of RT-PCR performed with RNA extracted from the pooled tail tissues from wild-type and <i>pitx3</i> morphant embryos shown in C. A sharp reduction in normal <i>pitx3</i> transcript (black arrowhead) and the presence of abnormally spliced product (red arrowhead) are evident in <i>pitx3</i> morphant embryos. <b>C.</b> Coomassie Blue R-250 stained polyacrilamide gel demonstrating equal protein concentration in nuclear extracts obtained from wild-type (lane 1) and <i>pitx3</i> morphant (lane 2) embryos that were used in EMSA experiments shown in A. <b>D.</b> Electrophoretic mobility shift assays (EMSA) show formation of a DNA-protein complex when an oligonucleotide corresponding to the −44/−76 region of zebrafish <i>mip1</i> promoter and nuclear extracts from 48-hpf wild-type zebrafish embryos are used. Please note a presence of a specific slow migrating complex, which is formed by wild-type <i>mip1</i> probe and proteins extracted from nuclei of 48-hpf wild-type zebrafish embryos (lane 2), absence of this complex in lane 3 when the same nuclear extracts were combined with a mutant <i>mip1</i> probe where the pitx3-binding <i>bicoid</i> site GGATTA was replaced by AAATTA, and sharp reduction of this complex in lane 4 containing a combination of a wild-type <i>mip1</i> probe and nuclear extracts obtained from <i>pitx3</i> morphants (red arrow).</p
Human <i>MIP/AQP0</i> genomic region and <i>bicoid</i> elements.
<p><b>A.</b> Schematic representation of the <i>MIP/AQP0</i> gene and promoter region; <i>bcd1</i> and <i>bcd2</i> sites are indicated. <b>B.</b> Multiple species alignment of genomic sequences surrounding the <i>bcd1</i> and <i>bcd2</i> sites (highlighted in grey). GenBank accession numbers are as follows: NT_029419.12 (<i>Homo sapiens</i>); NC_007868.1 (rhesus, <i>Macaca mulatta</i>), NT_039500.7 (mouse, <i>Mus musculus</i>), NC_005106.2 (rat, <i>Rattus Norvegicus</i>), NC_006592.2 (dog, <i>Canis lupus familiaris</i>), AAKN02014837.1 G.Pig, <i>Cavia porcellus</i>), NC_009149.2 (horse, <i>Equus caballus</i>), NC_007303.4 (cow, <i>Bos taurus</i>), NC_007134.4 (zebrafish, <i>Danio rerio</i>).</p
Analysis of <i>mip1</i> expression in <i>pitx3-mo</i> and control embryos via <i>in situ</i> hybridization and RT-PCR.
<p><b>A, D, F-H.</b> Normal <i>mip1</i> expression in control-injected embryos at 29-, 34- and 48-hpf. <b>B, C, E, I–K.</b> Altered <i>mip1</i> expression is observed in <i>pitx3</i> morphants at 29-hpf with 64% of embryos demonstrating a complete absence of <i>mip1</i> expression (B) and the remaining larvae showing markedly reduced <i>mip1</i> expression (C and I). Reduced <i>mip1</i> expression is also observed in 34- and 48-hpf embryos (E, J, K). Red arrows show sites of expected <i>mip1</i> expression. Scale bars: A–E: 100 µM; F–L: 20 µM. <b>L.</b> Results of semi-quantitative RT-PCR showing reduced expression of <i>mip1</i> in <i>pitx3</i> morphants at early stages of development (red arrow).</p
Injection of <i>pitx3</i> morpholino results in abnormal splicing of <i>pitx3</i> transcript and small lens phenotype.
<p><b>A.</b> Schematic drawing of <i>pitx3</i> gene; position of initiation codon (ATG) and RT-PCR primers (1F and 3R) are indicated, exons are numbered. <b>B.</b> Sequencing of the <i>pitx3-mo</i> transcript generated with primers located in the first and third <i>pitx3</i> exons demonstrates absence of exon 2 in the resultant product. <b>C.</b> RT-PCR results obtained with <i>pitx3</i> 1F/3R primers using RNA extracted from embryos injected with control or <i>pitx3</i> morpholino. Please note a strong decrease in normal 276-bp product (black arrowhead) and presence of abnormal 148-bp product (red arrowhead) in <i>pitx3-mo</i> samples; hpf- hours post fertilization of analyzed embryos. <b>D–G.</b> Morphological phenotypes of zebrafish <i>pitx3-mo</i> embryos. In comparison to control embryos, a smaller head can be observed in <i>pitx3</i> morphants (72-hpf embryo is shown; D) as well as an obvious reduction in lens size (black arrow) at later stages (96-hpf embryos are shown; E–G). <b>H</b> and <b>I.</b> Expression of <i>pitx3</i> in the developing lens in 24-hpf embryos. Please note robust expression in the lens vesicle (arrows) as demonstrated by both whole mount (H) and section (I) in situ.</p
<i>MIP/AQP0</i> is activated by PITX3 via interaction with the proximal <i>bicoid</i> site, <i>bcd1</i>.
<p><b>A.</b> Promoter activities of the <i>MIP656</i> reporters in human embryonic kidney cells <b>B.</b> Transactivation of <i>MIP656</i> reporters by PITX3 and its mutants in human embryonic kidney cells. Constructs and positions of <i>bicoid</i> sites are indicated on the left side. Wild-type <i>bcd1</i> or <i>bcd2</i> sites are depicted as open circles. Mutations (TAAT<u>CC</u> to TAAT<u>TT</u> substitutions) in <i>bcd1</i> or <i>bcd2</i> sites are depicted as dark circles with a strike-through. Student's paired t-Test with a one-tailed distribution was utilized to compare values. Experiments marked with asterisk (*) demonstrated a significant difference (P<0.001) in comparison to experiments performed with <i>MIP656</i> wild-type promoter (A) or <i>MIP656</i> wild-type promoter with PITX3-WT (B).</p
<i>MIP/AQP0</i> region demonstrates enrichment in chromatin immunoprecipitation experiments with PITX3 or FLAG antibody.
<p><b>A.</b> Endogenous PITX3 is bound to the proximal <i>MIP/AQP0</i> promoter in human lens epithelial (HLE) cell cultures. Chromatin immunoprecipitation assays were performed using untransfected HLE cells and human PITX3 or IgG (control) antibodies. The samples were analyzed by semi-quantitative PCR using <i>MIP/AQP0</i> proximal promoter- specific primers and negative control primers. Please note robust amplification of <i>MIP/AQP0</i> promoter region from ChIP sample precipitated with PITX3 but not with control IgG antibody (red arrow) and equal levels of DNA amplification for negative control region in both samples (black arrowhead). <b>B.</b> PITX3_FLAG is bound to proximal <i>MIP/AQP0</i> promoter in HLE cell cultures following transfection with PITX3-FLAG expression plasmid. HLE cells were transfected with either PITX3-FLAG expression plasmid or control pcDNA3.1 expression vector. ChIP assays were performed with FLAG-M2 or control IgG antibody. The ChIP samples were analyzed by semi-quantitative PCR as described in A. Please note enrichment of <i>MIP/AQP0</i> promoter region in ChIP sample obtained from PITX3-FLAG transfected cells and precipitated with FLAG antibody in comparison to FLAG-precipitated ChIP sample obtained from pcDNA3.1 transfected cells as well as IgG-precipitated ChIP sample obtained using either PITX3-FLAG or pcDNA3.1 transfected cells (red arrow). In addition to this, amplification of negative control region demonstrated similar levels across all samples (black arrowhead). <b>C</b> and <b>D.</b> Statistical analysis of multiple semi-quantitative PCR/ChIP experiments performed in untransfected (C) and transfected HLE cells (D) as described in A and B, correspondingly. Presence of <i>MIP/AQP0</i> promoter or negative control region DNA in various ChIP samples was evaluated by semi-quantitative PCR followed by densitometric analysis and expressed as a percentage of input values; mean and standard deviation for at least 3 independent experiments were calculated and analyzed by Student's t test. Please note statistically significant enrichment for <i>MIP/AQP0</i> promoter region DNA precipitated with PITX3 (C) or FLAG (D) antibody in comparison to control IgG-precipitated chromatin in HLE untransfected (C) or transfected (D) cells. IgG = normal mouse IgG; PITX3 = PITX3 polyclonal antibody; FLAG = anti-FLAG monoclonal antibody.</p
Electrophoretic mobility shift assays (EMSA) demonstrate interaction between PITX3 and <i>bcd1</i> and <i>bcd2</i> sites.
<p>EMSA performed with <i>bcd1</i> and <i>bcd2</i> oligonucleotides. DNA-PITX3 complexes are indicated with a full arrow; supershifts are shown with an arrowhead. ab = antibody, NE = Nuclear extracts, wt = wild type.</p
Embryonic expression of zebrafish <i>b3glct</i> genes.
<p>(A) RT-PCR analysis of <i>b3glct</i> expression demonstrates robust expression of both <i>b3glcta</i> (left panel) and <i>b3glctb</i> (middle panel) at different stages of development in whole embryos as well as various embryonic tissues at 48-hpf (right panel). Controls included <i>pitx2c</i> as negative control for 0-hpf, <i>rhodopsin</i> as negative control for the lens, <i>beta-actin</i> as positive control for all tissues and H<sub>2</sub>O as negative contamination control for all reactions. (B) In-situ hybridization analysis of <i>b3glcta</i> and <i>b3glctb</i> expression demonstrates broad expression in 24-120-hpf embryos with enrichment in the developing eyes, fins, brain, craniofacial region and somites. aer–apical ectodermal ridge, ase–anterior segment of the eye, b–brain, cmz–ciliary marginal zone, crc–craniofacial cartilage, e–eye, f–fins, h–heart, le–lens, sm–skeletal muscles.</p
Exonic structure, genomic context and multiple species alignment of B3GLCT/b3glct.
<p>(A) The two zebrafish orthologs of B3GLCT show overall similar exonic arrangement. The number of each exon is located within each box and the size of the exon (in base pairs) is shown above each exon. The 5’ and 3’ UTRs are indicated preceding the first ATG and following the stop codon (TAA/TAG). White indicates the N-terminal signal sequence, light grey indicates the stem region and dark grey indicates the catalytic domain. The vertical black bar in exon 12 of each gene indicates the location of nucleotides encoding for the catalytic tri-aspartic acid residues. Horizontal lines underneath the zebrafish genes indicate previously annotated sequence and sequence identified in this study. (B) Schematic of genomic context for B3GLCT/b3glct. (C) Multiple species alignment of B3GLCT orthologs from human (NP_919299), mouse (NP_001074673), Xenopus (NP_001072551), and zebrafish. Blue bar indicates signal peptide, green indicates stem region and orange indicates catalytic core. Grey shading of amino acids indicates conservation. The DxD motif is boxed in red.</p
Summary of differentially regulated genes implicated in ER quality control, unfolded protein response or cell survival.
<p>Summary of differentially regulated genes implicated in ER quality control, unfolded protein response or cell survival.</p