Genetic interaction between <i>Gata6</i> and <i>Gli3</i> in preaxial polydactyly development.

<p><b>A-J</b>: Alcian blue-stained autopod of indicated genotypes at E15.5. A-E: forelimbs, F-J: hindlimbs. Thin red arrows point to bifurcated d1 (<b>C</b>) and small projection (<b>H</b>) in fore- and hind-limbs, respectively in <i>Gli3</i>^<i>+/-</i> mutants. Thick red arrows in <b>D</b> and <b>I</b> point to anterior ectopic digits. Asterisks in E and J indicate digit tips of <i>Gli3</i>^<i>-/-</i> autopod. <b>K-O</b>: Expression pattern of <i>Shh</i> in hindlimb buds of indicated genotypes at E11.5. Black and red arrows point to normal and ectopic signals, respectively. <b>P-V</b>: <i>Sox9</i> in situ hybridization in hindlimbs of indicated genotypes at E12.5. Red arrows in <b>S</b> and <b>T</b> point to anterior ectopic digit condensation. Asterisks in <b>U</b> indicate distal tips of digit condensation. Red arrowheads in <b>V</b> point to distally-fused condensation.</p

GATA6 regulates subcellular localization of GLI3R.

<p><b>A</b>: Representative in vitro images of nuclear GATA6+nuclear GLI3R (upper), nuclear GATA6+cytosolic GLI3R (middle) and cytosolic GATA6+cytosolic GLI3R (bottom). <b>B</b>: Quantitation of subcellular localization of GATA6 and GLI3R. NC: predominantly nuclear localized. GATA6 mutants, indicated at the bottom, are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006138#pgen.1006138.g005" target="_blank">Fig 5E</a>. The number of cells examined for each set of transfection is indicated in the panel. <b>C-H</b>: Representative images of the anterior-proximal mesenchyme of hindlimb buds at E10.25. <b>C, E, G</b>: wild type, <b>D, F, H</b>: <i>Gata6</i> cKO. <b>I</b>: Quantitation of subcellular localization of GLI3R in the anterior-proximal mesenchyme of hindlimb buds at E10.25. Gray and black bars represent wild-type and <i>Gata6</i> cKO samples, respectively. The graph shows percentage of GLI3R localization patterns, such as predominantly nuclear (N>C), similarly in the nucleus and cytoplasm (N = C), or predominantly cytoplasmic (NGata6 cKO embryos were examined. * indicates P<0.05. <b>J</b>: Western blot of nuclear fractions from anterior part of wild-type and <i>Gata6</i> cKO hindlimb buds at E10.25–10.5. Histone H3 (H3) is included as a loading control.</p

Expression pattern of SHH targets and digit condensation in <i>Gata6</i> cKO; <i>Shh</i> allelic series.

<p>Expression pattern of <i>Gli1</i> (<b>A-D</b>), <i>Ptch1</i> (<b>E-H</b>) and <i>Sox9</i> (<b>I-L</b>) of wild type (<b>A, E, I</b>), <i>Gata6</i> cKO (<b>B, F, J</b>), <i>Gata6</i> cKO; <i>Shh</i>^<i>+/-</i> (<b>C, G, K</b>) and <i>Gata6</i> cKO; <i>Shh</i>^<i>-/-</i> (<b>D, H, L</b>) hindlimb buds. <b>A-H</b>: E11.5, <b>I-L</b>: E12.5. In <b>A</b>-<b>H</b>, black arrows and red arrows point to normal and ectopic signals, respectively. Blue arrowheads indicate loss of expression in <b>D</b> and <b>H</b>. In <b>I-L</b>, digit condensations are labeled as 1–5, and ectopic condensation is marked with red arrows.</p

Expression pattern of <i>Gli1</i>, <i>Ptch1</i> and <i>Pax9</i> in <i>Gata6</i>; <i>Gli3</i> allelic series.

<p>In situ hybridization of <i>Gli1</i> (<b>A-G</b>), <i>Ptch1</i> (<b>H-N</b>) and <i>Pax9</i> (<b>O-U</b>) of hindlimb buds of indicated genotypes at E11.5. Black and red arrows point to normal and ectopic signals, respectively. Blue arrows and arrowheads indicate reduced and loss of <i>Pax9</i> signals, respectively.</p

Loss of Gata6 causes preaxial polydactyly in hindlimbs.

<p><b>A-H</b>: Lateral views (<b>A, E</b>) of whole E14.5 embryos, and Alcian blue-stained cartilage (<b>B-D, F-H</b>) of wild type (<b>A-D</b>) and <i>Gata6</i> cKO (<b>F-H</b>) embryos at E14.5. <b>C</b> and <b>G</b> show hindlimb autopod, and <b>D</b> and <b>H</b> show tarsal and metatarsal elements. Red arrows in <b>E-G</b> point to the anterior ectopic digit. Yellow arrows point to hemorrhage in <i>Gata6</i> cKO embryos. Digits are numbered with 1–5 in <b>C</b> and <b>G</b>. Asterisks in <b>H</b> indicates ectopic elements. calc: calcaneus, cu: cuboid, fe: femur, fi: fibula, ic: intermediate cuneiform, lc: lateral cuneiform, mc: medial cuneiform, na: navicular ti: tibia. <b>I-X</b>: in situ hybridization of wild type (<b>I-P</b>) and <i>Gata6</i> cKO (<b>Q-X</b>) hindlimb buds at E10.5 with indicated probes. Black and red arrows point to normal and ectopic signals, respectively. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006138#pgen.1006138.s005" target="_blank">S1 Table</a>.</p

Physical and functional interaction between GATA6 and GLI3R.

<p><b>A</b>: GLI-specific luciferase reporter assay with indicated expression constructs. *: p<0.01, **: p<0.001. <b>B, C</b>: Co-immunoprecipitation assay of Flag-GATA6 and Myc-GLI3R. (B) Pulldown with anti-Myc, detection by anti-Flag. (C) Pulldown with anti-Flag, detection by anti-Myc. <b>D</b>: Co-immunoprecipitation of GATA6 and GLI3R from wild-type hindlimb buds. <b>E</b>: Schematic presentation of deletion mutants of GATA6. Binding with GLI3R in <b>F</b> and <b>G</b> is summarized in the right side of the panel. Orange bars represent transactivation domains. Red and blue bars represent zinc finger DNA binding domains and the nuclear localization signal, respectively. <b>F, G</b>: Co-immunoprecipitation assay of Flag-GLI3R and GATA6 mutants.</p

Additional file 2: of Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata

MS/MS analysis of Xac-2. (DOCX 157 kb