Analysis of transcription factors expressed at the anterior mouse limb bud

<div><p>Limb bud patterning, outgrowth, and differentiation are precisely regulated in a spatio-temporal manner through integrated networks of transcription factors, signaling molecules, and downstream genes. However, the exact mechanisms that orchestrate morphogenesis of the limb remain to be elucidated. Previously, we have established EMBRYS, a whole-mount <i>in situ</i> hybridization database of transcription factors. Based on the findings from EMBRYS, we focused our expression pattern analysis on a selection of transcription factor genes that exhibit spatially localized and temporally dynamic expression patterns with respect to the anterior-posterior axis in the E9.5–E11.5 limb bud. Among these genes, <i>Irx3</i> showed a posteriorly expanded expression domain in <i>Shh</i>^<i>-/-</i> limb buds and an anteriorly reduced expression domain in <i>Gli3</i>^<i>-/-</i> limb buds, suggesting their importance in anterior-posterior patterning. To assess the stepwise EMBRYS-based screening system for anterior regulators, we generated <i>Irx3</i> transgenic mice in which <i>Irx3</i> was expressed in the entire limb mesenchyme under the <i>Prrx1</i> regulatory element. The <i>Irx3</i> gain-of-function model displayed complex phenotypes in the autopods, including digit loss, radial flexion, and fusion of the metacarpal bones, suggesting that <i>Irx3</i> may contribute to the regulation of limb patterning, especially in the autopods. Our results demonstrate that gene expression analysis based on EMBRYS could contribute to the identification of genes that play a role in patterning of the limb mesenchyme.</p></div

Production of microRNA deficient mice with TALEN.

<p>(A) Schematic representation of TALENs that target microRNA. Wild type genomic sequence is shown with black line. miRNA-5p and -3p on the genomic sequence are with orange boxes. TALENs are indicated with green boxes and recognition sequences are indicated with light green boxes. Scissors show the location of cutting site of FokI endonuclease. Genomic sequence of mutated allele is indicated with black line at the bottom. Deleted sequence is shown with a dark orange box with a white waved line. (B) PCR genotyping of the miRNA mutants. Genomic sequence is shown at the top with the target sequences of a pair of TALEN colored in green. Blue lines and boxes indicate pre-miRNAs and mature-miRNAs sequences, respectively. Deleted and inserted nucleotides are indicated with red dashes and blue letters, respectively. The sizes of deletions and insertions are shown to the right of mutated alleles with ▵ and +, respectively. Alleles without mutation are indicated with wt (wild type). (C) Genotyping of F1 offspring of <i>mmu-mir-10a</i> KO (Mutant3) mouse by T7EI assay. Arrows indicate the digested PCR products containing mutation. DNA size marker is shown at left. Lane 1-9: F1 individuals; lane 10: founder; lane 11: Wt. (D) Genotyping of F1 offspring of <i>mmu-mir-10b</i> KO (Mutant1) mouse by T7EI assay. Arrows indicate the digested PCR products containing mutation. DNA size marker is shown at left. Lane 1-6: F1 individuals; lane 7: founder; lane 8: Wt.</p

Cell based assay of TALEN activities. Each sequence shows the result of PCR direct sequencing of cloned PCR products amplified from TALEN transfected cells.

<p>Green letters designate target sequences of TALENs. Red dashes and blue letters indicate deleted and inserted nucleotides, respectively. The sizes of deletions are shown to the right of mutated alleles with ▵ and/or +. Alleles without mutation are indicated with Wild type. The numbers in the parenthesis indicate the number of clones obtained.</p

Oligonucleotide primers and Taqman probes used for quantitative RT-PCR.

<p>Oligonucleotide primers and Taqman probes used for quantitative RT-PCR.</p

Real-time RT-PCR analysis of miR-10a*.

<p>Genotype of each animal is indicated at bottom. Mut indicates mutant allele. Expression level of the wt animal was adjusted as 1.</p

Gene targeting efficiencies.

*<p>TALENs for <i>mmu-mir-10a</i> and <i>mmu-mir-10b</i> were mixed and microinjected. †Genotype of <i>mmu-mir-10a</i> and <i>mmu-mir-10b</i> were assayed and results were indicated separately in a cell.</p

TGF-β and EGFR pathways cooperate to induce stemness in GIF-14 cells.

<p>(A) Changes in the expression of stemness- and EMT/mesenchymal-associated marker genes in response to TGF-β1 and EGF. GIF-14 cells were treated with murine EGF (10 ng/ml) or TGF-β1 (2.5 ng/ml) or in combination for 15 h. Quantitative PCR measurements of gene expression levels are normalized against <i>Gapdh</i> levels, and expressed relative to the control sample (means ± SEM, n = 4). Student's t-tests are performed in which single and double asterisks denote <i>p</i> value<0.05 and <i>p</i> value<0.01, respectively and n.s represents not significant (Black bracket: TGF-β1 responsiveness; Red bracket: cooperative induction by TGF-β1 and EGF). (B) Cooperative induction of stemness by EGF and TGF-β1. GIF-14 cells were pretreated with TGF-β1 (2.5 ng/ml) for varying periods at 15 h, 24 h, 48 h and 72 h before the addition of murine EGF (10 ng/ml) for another 15 h. Changes in the mRNA levels of stemness marker <i>Hmga2</i>, regulators of EGF signaling <i>EGFR</i> and <i>Lrig1</i>, EMT markers <i>Snai1</i> and <i>fibronectin1</i> (<i>Fn1</i>) were determined by qRT-PCR. The values are normalized against those of <i>Gapdh</i> and are expressed relative to that of the control (means ± SEM, n = 3). Student's t-tests are performed where indicated. Single and double asterisks represent <i>p</i> value<0.05 and <i>p</i> value<0.01, respectively while n.s denotes not significant (Black bracket: TGF-β1 responsiveness; Blue bracket: EGF responsiveness; Red bracket: cooperative induction by TGF-β1 and EGF). (C) TGF-β1 induction of <i>Hmga2</i> is abrogated by inhibitors of EGFR and MEK1/2. GIF-14 cells were treated with SB431542 (TGF-βRI inhibitor; 10 µM) or AG1478 (EGFR inhibitor; 10 µM) or U0126 (MEK1/2 inhibitor; 10 µM) or in combination with TGF-β1 (2.5 ng/ml) for 48 h. Changes in the mRNA levels of stemness marker <i>Hmga2</i> and EMT marker <i>Snai1</i> were ascertained by qRT-PCR and normalized values are expressed relative to the control values (means ± SEM, n = 4). Student's t-tests are performed where indicated. Single and double asterisks denote <i>p</i> value<0.05 and <i>p</i> value<0.01, respectively. (D) The effects of TGF-β1 and EGF on the phosphorylation states of Egfr and Erk. GIF-14 cells were treated with TGF-β1 (2.5 ng/ml; top panel) or murine EGF (10 ng/ml; bottom panel) for various short periods of time from 15 to 120 min. The expression levels of phosphorylated Egfr at tyrosine residues 1068 and 1092 and Erk1/2 were measured by Western blot analysis using pEgfr^Y1068/1092- and pErk1/2–specific antibodies Total Egfr expression was determined using anti-Egfr antibody #2 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070427#pone-0070427-g001" target="_blank">Figure 1F</a>. The expression level of α-tubulin serves as a control for the amount of proteins loaded.</p