BAC transgenic mouse intestinal organoids.

<p>(A) Tagging cassettes for BAC recombineering. Upper panel illustrates the C-terminal GFP-tagging cassette with a neomycin resistance gene downstream of an IRES sequence, which was used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076871#pone.0076871-Poser1" target="_blank">[7]</a> to tag histone H2A and TUBB5. Lower panel shows the C-terminal tagRFP tagging cassette with a neomycine resistance gene downstream of a PGK promoter and flanked by loxP sites, which was used to tag the lysozyme gene. gb3: bacterial promoter, PGK: phosphoglycerate kinase promoter, IRES: internal ribosome entry site. (<b>B</b>) FACS analysis of mouse intestinal organoids 48 h after transfection with the pmax-GFP plasmid. Middle panel shows cell viability using propidium iodide (PI), right panel shows the percentage of GFP transfected cells. (<b>C</b>) Section of a well with mouse organoids 48 h after H2A-GFP BAC transfection using lipofectamine. Arrow points to a successfully transfected cell. Fluorescence images of a (<b>D</b>) H2A-GFP BAC transgenic organoid, (<b>E</b>) TUBB5-GFP BAC transgenic organoid, and (<b>F</b>) Lysozyme-tagRFP BAC transgenic organoid.</p

Generation of BAC Transgenic Epithelial Organoids

<div><p>Under previously developed culture conditions, mouse and human intestinal epithelia can be cultured and expanded over long periods. These so-called organoids recapitulate the three-dimensional architecture of the gut epithelium, and consist of all major intestinal cell types. One key advantage of these ex vivo cultures is their accessibility to live imaging. So far the establishment of transgenic fluorescent reporter organoids has required the generation of transgenic mice, a laborious and time-consuming process, which cannot be extended to human cultures. Here we present a transfection protocol that enables the generation of recombinant mouse and human reporter organoids using BAC (bacterial artificial chromosome) technology.</p></div

BAC transgenic human intestinal organoids.

<p>(<b>A</b>) FACS analysis of human intestinal organoids 48 h after transfection with the pmax-GFP plasmid. Middle panel shows cell viability using propidium iodide (PI), right panel shows the percentage of GFP transfected cells. (<b>B</b>) Section of a well with human organoids 48 h after H2A-GFP BAC transfection with lipofectamine. Arrows point to successfully transfected cells. (<b>C</b>) Fluorescence image of a H2A-GFP BAC transgenic human organoid.</p

Loss of the Wnt receptor frizzled 7 in the mouse gastric epithelium is deleterious and triggers rapid repopulation in vivo

The gastric epithelium consists of tubular glandular units, each containing several differentiated cell types, and populations of stem cells, which enable the stomach to secrete the acid, mucus and various digestive enzymes required for its function. Very little is known about which cell signalling pathways are required for homeostasis of the gastric epithelium. Many diseases, such as cancer, arise as a result of deregulation of signalling pathways that regulate homeostasis of the diseased organ. Therefore, it is important to understand the biology of how normal conditions are maintained in a tissue to help inform the mechanisms driving disease in that same tissue, and to identify potential points of therapeutic intervention. Wnt signalling regulates several cell functions, including proliferation, differentiation and migration, and plays a crucial role during homeostasis of several tissues, including the intestinal epithelium. Wnt3a is required in the culture medium of gastric organoids, suggesting it is also important for the homeostasis of the gastric epithelium, but this has not been investigated in vivo. Here, we show that the Wnt receptor frizzled 7 (Fzd7), which is required for the homeostasis of the intestine, is expressed in the gastric epithelium and is required for gastric organoid growth. Gastric-specific loss of Fzd7 in the adult gastric epithelium of mice is deleterious and triggers rapid epithelial repopulation, which we believe is the first observation of this novel function for this tissue. Taken together, these data provide functional evidence of a crucial role for Wnt signalling, via the Fzd7 receptor, during homeostasis of the gastric epithelium

Flexible hinge in RSPO1 and binding of ZNRF3 to RSPO1 and LGR5-RSPO1.

<div><p>A.Overlay of four representative RSPO1 structures in two orientations with free RSPO1 (grey; PDB code 4BSO), RSPO1 in LGR5-RSPO1-RNF43 complex (blue, PDB code 4KNG) and RSPO1 in complex with ZNRF3 (orange and red).</p> <p>B.Representative SPR dose-response curve used to determine equilibrium binding affinity of LGR5-RSPO1 or RSPO1 to ZNRF3, as described in Material and Methods. Standard deviations are calculated from four experiments.</p></div

ZNRF3-RSPO1 binding site coincides with LGR5-RSPO1 ‘trans’ site.

<p>Shown in cartoon with transparent surface representation are the crystal structure of the 2:2 LGR5-RSPO1 complex (PDB code 4BSR) and the structure of ZNRF3-RSPO1 superimposed on RSPO1 on the right-hand side (orange), only the Cα trace of ZNRF3 (red) is shown for clarity. The overlapping LGR5 chain is shown in blue. The remaining part of the 2:2 LGR5-RSPO1, i.e. left-hand side RSPO1 and right-hand side LGR5, is shown in grey.</p

Crystal structure of the ZNRF3-RSPO1 complex.

<div><p>A.Cα trace with transparent surface representation of ZNRF3 (green) and RSPO1 (orange). ZNRF3 makes contacts to the β-hairpins 1-2 of the Fu1 domain of ZNRF3.</p> <p>B.Two views of the binding sites with interface residues indicated.</p> <p>C.Identical (red) and conserved (orange) residues between ZNRF3 and RNF43 are shown in surface representation; two views of ZNRF3 are shown. Identical residues are labeled.</p></div

β-catenin-dependent H3K79 methylation and MLLT10/AF10, DOT1L recruitment to human <i>AXIN2</i> gene.

<p>Cell lysates from Ls174T CRCs were immunoprecipitated with antibodies against endogenous MLLT10/AF10 (A) and DOT1L (B) complexes and analyzed by Western blotting with the indicated antibodies. (C) MLLT10/AF10 interaction with TCF4 is mediated by β-catenin. Western blot analysis of β-catenin depletion in Ls174T cells expressing doxycycline (Dox)-inducible β-catenin shRNA. Immunoprecipitated TCF4-protein complexes from untreated or Dox-treated cells were resolved by SDS-PAGE followed by Western blotting with the indicated antibodies. (D) Schematic representation of the human <i>AXIN2</i> locus and amplicons scanned in Chromatin immunoprecipitation experiments by qPCR. ChIP in Ls174T CRCs uninduced or induced with Dox using antibodies specific for TCF4 (E), β-catenin (F), MLLT10/AF10 (G), DOT1L (H), H3K79 dimethyl (I), and H3K79 trimethyl (J). The immunoprecipitated DNA was analyzed by qPCR using primers specific for the <i>AXIN2</i> locus as indicated. Results are presented as percent immunoprecipitated over input and are representative of three independent experiments.</p

MLLT10/AF10 and DOT1L are essential and dedicated to the Wnt-induced transcriptional program in HEK293T cells.

<p>Wnt-induced association of MLLT10/AF10-DOT1L with and regulation of Wnt target genes in HEK293T cells. (A–F) ChIP assays in HEK293T cells uninduced or induced with Wnt3A conditioned media at 2 and 12 h using antibodies specific for TCF4 (A), β-catenin (B), MLLT10/AF10 (C), DOT1L (D), H3K79 di-methyl (E), and H3K79 tri-methyl (F). The immunoprecipitated DNA was analyzed by qPCR using primers specific for the <i>c-MYC</i> and <i>ZCCHC12</i> loci as indicated. Results are presented as percent immunoprecipitated over input and are representative of three independent experiments. (G) Comparison of the corresponding expression pattern after siRNA suppression of MLLT10/AF10, DOT1L, BRG1, and p300 in the Wnt induced condition. Heatmap showing 1988 Wnt regulated transcripts after 9 h Wnt-induction (relative to uninduced sample (no Wnt)) in HEK293T cells with greater than 1.5-fold variation, and the comparison of the corresponding expression pattern after siRNA suppression of MLLT10/AF10, DOT1L, BRG1, and p300 in Wnt-induced condition (relative to 9 h Wnt induction). Red, upregulated after Wnt; green, downregulated after Wnt induction; grey, missing data. Western blot analysis of MLLT10/AF10, DOT1L, BRG1, p300, and Tubulin upon siRNA depletion of each gene as indicated. (H) Venn diagram depicting the comparison of Wnt-induced genes and genes downregulated after MLLT10/AF10, DOT1L, BRG1, or p300 suppression in HEK293T cells after Wnt induction.</p

Polyguanine genotyping data

This archive contains polyguanine genotyping data, distance matrices and phylogenetic trees