zebrafish <i>pitx2</i> knockdown and associated phenotype.

<p><b>A</b>. Schematic drawing of <i>pitx2</i> genomic structure. Exons are shown as numbered boxes, sizes are indicated at the top (for exons) or at the bottom (for introns). The positions of primers to amplify <i>pitx2</i> transcripts are shown and numbered 1–3; primers 1 and 3 are used for <i>pitx2a</i> and 2 and 3 for <i>pitx2c</i>. The position of antisense morpholino oligonucleotides, <i>pitx2a^ATG</i>, <i>pitx2c^ATG</i> and <i>pitx2^ex4/5sp</i>, are shown with red lines. <b>B</b>. RT-PCR of <i>pitx2</i> expression in <i>pitx2^ex4/5</i> morphants. Please note a complete absence of normal <i>pitx2</i> transcripts (indicated with black arrows) and the presence of an abnormal large PCR product (indicated with red arrowheads) in mRNA extracted from <i>pitx2^ex4/5</i> embryos at 24–48-hpf, the presence of both normal (diminished) and abnormal products at 72–96-hpf <i>pitx2^ex4/5</i>, and normal levels of <i>pitx2</i> by 120-hpf due to weakening of morpholino effects. <b>C</b>. DNA sequencing of the abnormal PCR product observed in <i>pitx2^ex4/5</i> morphant embryos identified the presence of the 902-bp intron 4 in the <i>pitx2^ex4/5</i> transcript consistent with aberrant splicing (forward sequence is shown and the beginning of the intron is indicated with a red arrow; exon 4 sequence is shown in upper case while intron 4 is in lower case letters; the exon-intron junction sequence corresponding to the <i>pitx2^ex4/5</i> antisense oligomer is indicated in red). Therefore, the <i>pitx2^ex4/5</i> protein is predicted to contain partial <i>pitx2</i> sequence (lacking amino acids encoded by exon 5) followed by 10 erroneous amino acids (<i>pitx2^ex4/5</i> stop codon is indicated with red box). <b>D</b>. Representative images of <i>pitx2^ex4/5</i> morphants, control morpholino-injected embryos and larvae developed from uninjected eggs at 48-hpf, 96-hpf and 7-dpf. <b>E</b>. Bar graph showing the distribution of observed embryonic phenotypes following <i>pitx2^ex4/5</i> morpholino injections into <i>p53</i>−/− and wild-type zebrafish eggs. e- eye, j- jaw, pe- pericardial edema.</p

Deficiency of <i>pitx2</i> results in craniofacial and ocular defects.

<p><b>A–H</b>. <i>pitx2</i> knockdown disrupts development of the pharyngeal arches. Lateral (<b>A, B, E, F</b>) and ventral (<b>C, D, G, H</b>) views of 72-hpf (<b>A–D</b>) and 120-hpf (<b>E–H</b>) larvae stained with Alcian blue. Numerous defects in the development of the lower jaw (anterior arches) and ceratobranchial (posterior) arches can be observed. <b>I–T</b>. <i>pitx2</i> knockdown disrupts eye development. Representative images of histological sections of 72-hpf (<b>I,J,O,P</b>), 120-hpf (<b>K,L,Q,R</b>) and 8-dpf (<b>M,N,S,T</b>) control and <i>pitx2^ex4/5</i> morphants; head sections at the optic nerve level are shown (<b>I–N</b>) as well as high magnification images for the eye (<b>O–T</b>). Please note the accumulation of cells in the anterior segment in 72-hpf <i>pitx2^ex4/5</i> morphant embryos (arrowhead in <b>P</b>) and abnormal eye size/shape in 120-hpf and 8-dpf morphants (arrowheads in <b>R</b>, <b>T</b>). Abbreviations: <b>A–H</b>- bb,basibranchial; bh, basihyal; cb, ceratobranchial (P3–P7); ch, ceratohyal (P2); ep, ethmoid plate; hs, hyosympletic (P2); m, Meckel's cartilage (P1); pq, palatoquadrate (P1); <b>I–T</b>- ase, anterior segment of the eye; le, lens; on, optic nerve; re- retina.</p

In situ hybridization with <i>pitx2-exon 5</i> antisense riboprobe in control and morphant embryos.

<p>The <i>pitx2</i> antisense riboprobe comprising exon 5 sequence detects wild-type and abnormally spliced <i>pitx2^ex4/5</i> transcripts. Images of whole mount embryos (<b>A–F</b>, <b>J–M</b> and <b>Q–U</b>) and sections (<b>G–I</b>, <b>N–P</b> and <b>V, W</b>) are shown for embryonic stages of 24-hpf (<b>A–D</b>), 48-hpf (<b>E–I</b>), 72-hpf (<b>J–P</b>) and 120-hpf (<b>Q–W</b>). Please note abnormal <i>pitx2</i> transcripts around the developing eye (arrowheads in <b>B, D</b>) and pharyngeal arches (arrows in <b>B</b>) in morphants at 24-hpf, which is similar to <i>pitx2</i> expression in control embryos (<b>A</b>, <b>C</b>). Staining for <i>pitx2</i> positive cells in morphant embryos at 48-, 72- and 120-hpf identifies abnormal patterns during ocular and craniofacial development in comparison to <i>pitx2</i> expression in control embryos (<b>E–W</b>). In terms of ocular patterns, some 48- and 72-hpf morphants demonstrate an accumulation of <i>pitx2</i> transcriptionally active cells in the anterior segment of the eye (arrowheads; images for whole mount and sections from two different <i>pitx2^ex4/5</i> morphant embryos at 48-hpf (<b>F, H, I</b>) and 72-hpf (<b>K, M, O, P</b>) in comparison to control 48-hpf (<b>E,G</b>) and 72-hpf (<b>J, L, N</b>) are shown). In 120-hpf eyes, a disorganized pattern of <i>pitx2</i> positive cells continues to be observed in <i>pitx2^ex4/5</i> morphant embryos (arrowheads in <b>T</b>, <b>U</b>, two different morphant embryos are shown, and <b>W</b>) in comparison to <i>pitx2</i> expression in control embryos (<b>S, V</b>); in addition to the abnormal pattern in the anterior structures, an increased signal behind the lens corresponding to the hyaloid vasculature is also observed (asterisks in <b>W</b>). With regards to craniofacial development, in 72-hpf morphant embryos, strong staining is observed around the malformed oral cavity and arches with the level of expression similar to control <i>pitx2</i> expression (arrows in <b>J–M</b>); at 120-hpf, <i>pitx2</i> transcripts continue to be strongly expressed in the malformed pharyngeal arches (arrows in <b>R</b> and <b>W</b>) while expression of wild-type <i>pitx2</i> is downregulated in control 120-hpf embryos (arrows in <b>Q</b> and <b>V</b>).</p

Craniofacial development and gene expression in <i>pitx2^ex4/5</i> morphants.

<p>Developmental patterns of <i>foxd3</i>, <i>dlx2a</i> and <i>dlx4a</i>, and <i>Tg(fli1:gfp)</i> expression in <i>pitx2 ^ex4/5</i> embryos. In situ hybridization was performed with <i>foxd3</i>, <i>dlx2a</i>, <i>dlx4a</i> or <i>gfp</i>-specific antisense riboprobe in control (<b>A–P</b>) or morphant (<b>A′–P′</b>) zebrafish embryos. Please note similar expression patterns in migrating neural crest cells (<i>foxd3</i>) and pharyngeal arch primordial regions (<i>dlx2a</i>) at 24-hpf (arrows in control (<b>A–C</b>) and morphant (<b>A′–C′</b>) embryos). Starting at 32-hpf, expression of <i>dlx2a</i>, <i>dlx4a</i> and <i>Tg(fli1:gfp)</i> in the posterior arches appears to be reduced in <i>pitx2^ex4/5</i> fish (arrowheads in control (<b>D–G</b>) and morphant (<b>D′–G′</b>) embryos) while expression in the anterior arches (arrows in control (<b>D–G</b>) and morphant (<b>D′–G′</b>) embryos) is not significantly affected. In 48–72-hpf embryos, an increased expression of <i>dlx4a</i> in the anterior arches is detected (arrows in control (<b>H–N</b>) and morphant (<b>H′–N′</b>) embryos) while expression in the posterior arches remains reduced (arrowheads in control (<b>H–N</b>) and morphant (<b>H′–N′</b>) embryos); <i>dlx4a</i> expression around the oral cavity in 48-hpf embryos (arrow with asterisk in <b>K</b> and <b>K′</b>) as well as a frontward extension of the anterior arches at 72-hpf (arrows in <b>L–N</b> for controls and <b>L′–N′</b> for morphants) are not observed in <i>pitx2^ex4/5</i> fish. Analysis of sections at 72-hpf also shows broadened expression of <i>dlx4a</i> in the craniofacial region of morphant embryos (<b>O′, P′</b>) in comparison to controls (<b>O, P</b>).</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

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

<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

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