<i>Tcf3</i> deficiency suppresses serum-induced differentiation.

<p><b>A</b>. Parental NN97-5 cells differentiate after 4 days in serum without LIF while <i>Tcf3</i> gene trap mutant P1-2 cells remain undifferentiated and retain uniform Oct4 expression in serum. <b>B</b>. Flow cytometry analysis for Rex1-EGFP positive cells during monolayer differentiation in serum. P1-2, Tcf3 gene trap mutant; CreA12, heterozygous Tcf3 Cre-revertant; CreD10, homozygous Tcf3 Cre-revertant. Graph shown is a representative of two independent experiments. <b>C</b>. <i>Tcf3</i> mutant (P1-2) and the <i>Tcf3</i> reverted cells were plated at single cell density in serum with or without LIF for colony forming assay. Colonies were stained after 9 days for alkaline phosphatase (AP) activity and colony numbers were quantified manually. Undifferentiated colonies are showing in red in figure and partially differentiated showing in green and differentiated showing in yellow. <b>D</b>. Images show typical AP positive morphologically undifferentiated ES cell colonies generated by P1-2 cells in serum with or without LIF. The experiment has been repeated once.</p

<i>Tcf3</i> mutation has subtle molecular consequences.

<p><b>A</b>. Relative gene expression analysis by qRT-PCR in <i>Tcf3</i> mutant (P1-2) compared to Cre-reverted (CreD10)(Blue column) and wild type (NN97-5)(Red column). <b>B</b>. TOPFlash assay of Tcf-mediated transcriptional activation. None, N2B27 alone; Wnt, Wnt3A; Wnt+LIF, Wnt3A plus LIF. <b>C</b>. qRT-PCR analyses of Wnt target gene expression. S+L, Serum plus LIF. <b>D</b>. Immunoblotting analysis of Nanog and Oct4 protein expression in serum and LIF. <b>E</b>. NN97-5 and P1-2 cells cultured in serum and LIF immunostained for Oct4 and Nanog. Images show typical heterogeneous Nanog protein expression in NN97-5 cells compared to more uniform staining in P1-2 cells. <b>F</b>. Mean nuclear staining intensity of Oct4 and Nanog in individual cell was quantified using Cell profiler software and presented as a scatter plot using Microsoft Excel. 1600 cells were scored for NN97-5 and 2172 cells for P1-2. The experiment has been repeated three times.</p

Gene trap mutants from monolayer differentiation screen.

<p><b>A</b>. Images show typical differentiated and non-differentiated morphologies after 7 days monolayer neural differentiation assay. P1-1, P1-2, P1-4, P1-11, P1-12, P1-19 and P1-20 are clones carrying <i>Tcf3</i> gene trap mutation. <b>B</b>. Splinkerette-PCR amplified genome junctions flanking PB inserts. Gel images showing the genome junction flanking PB 5′ terminal repeat region (5′TR) and 3′ terminal repeat region (3′TR). Arrows indicate that a 500 bp 3′TR fragment and a 300 bp 5′TR fragment were amplified in multiple clones. Sequencing locates this band to <i>Tcf3</i> locus.</p

<i>piggyBac</i> mutagenesis and monolayer differentiation screen.

<p><b>A</b>. Binary <i>piggyBac</i> gene trap system composed of gene trap vector, <i>pGG85</i>, and transposase expressing helper plasmid, p<i>CAGG-PBase</i>. <b>B</b>. G418 resistant colonies produced by co-electroporation of 1 µg of <i>pGG85</i> and 3 µg of helper plasmid. <b>C</b>. Splinkerette PCR amplified genome junction flanking PB insertions indicating the number of PB inserts in each clone. <b>D</b>. Schematic representation of monolayer differentiation screen.</p

Monolayer neural differentiation of individual gene trap clones.

<p>Monolayer neural differentiation of twenty clones from gene trap mutation pool 1 is presented. Clones with <i>Tcf3</i> mutation are labelled with “*”. Two clones from mutant pool 2 were also included as a control for monolayer differentiation assay. “D” represents clones showing extensive neural differentiation. “Non-D” represents cells showing predominantly undifferentiated ES cell morphology. P1-8 cells differentiated to flat non-neural cells.</p

siRNA knockdown of Tcf3 in <i>Blm</i> wild type cells.

<p><b>A</b>. qRT-PCR analysis of <i>Tcf3</i> knockdown in <i>Tcf3</i> siRNA treated ES cells and control siRNA treated cells. <b>B</b>. Graph shows population of Rex1-EGFP positive cells in <i>Tcf3</i> siRNA and control siRNA treated cells after 2 days in monolayer differentiation with or without serum. <b>C</b>. qRT-PCR analysis of <i>Rex1</i> expression in <i>Tcf3</i> siRNA or control siRNA treated cells in monolayer differentiation with or without serum. <b>D</b>. Images showing a typical AP positive ES cell colony formed in <i>Tcf3</i> siRNA treated cells after 5 days in serum while only differentiated colonies formed from control siRNA treated cells. Error bar represents standard deviation from three individual plating.</p

<i>Tcf3</i> gene trap mutants.

<p><b>A</b>. <i>Tcf3</i> gene trap (<i>Tcf3^trp</i>) and Cre-reverted <i>(Tcf3^rev)</i> alleles. Cre recombination deletes the gene trap cassette to leave a reverted allele retaining the PB terminal repeats. <b>B</b>. RT-PCR analysis of <i>Tcf3</i> expression in gene trap mutants. <i>Tcf3</i> mRNA was not detected in clones P1-2, P1-12 and P1-19 but evident in clones P1-1, P1-11 and P1-20. <b>C</b>. Diagram showing generation of het or homozygous reverted cells. <b>D</b>. RT-PCR analysis of <i>Tcf3</i> gene trap (<i>Ex3-SA</i>) and <i>Tcf3</i> wild type (<i>Ex3-Ex7</i>) transcripts. CreA12-1 and CreD10-4 are subclones of CreA12 and CreD10. <b>E</b>. qRT-PCR analysis of <i>Tcf3</i> expression. F. After 9 days monolayer differentiation multiple ES cell colonies formed from <i>Tcf3</i> homozygote P1-2, but not from parental NN97-5 or revertant CreA12 or CreD10 cells.</p

Bone Mineral Density Assessment Technique.

<p>Bone Mineral Density Assessment Technique.</p

Pharmacological Intervention.

<p>Pharmacological Intervention.</p

Bone Mineral Density Assessment.

<p>Bone Mineral Density Assessment.</p