<i>Tcof1</i> heterozygous mutant mice show small brain.

<p>(A) Dorsal view of wild-type (+/+) and <i>Tcof1</i> heterozygous mutant (+/−) brains of P14 mice. (B) Brain weight per body weight and body weight of wild-type (n = 6) and <i>Tcof1</i>^+/− mice (n = 6). (C) Coronal sections of wild-type (+/+) and <i>Tcof1</i> heterozygous (+/−) cerebrum stained with HE. The brain is much smaller, but tissue architecture and morphology is normal. Scale Bars: C, 200 µm.</p

Treacle interacts with PLK1 and mediates its localization.

<p>(A) Immunofluorescent localization of Treacle (green) and PLK1 (red) in mitotic HeLa cells. (B) Interaction between FLAG-tagged Treacle and HA-tagged Plk1 as evidenced by immunoprecipitation (IP) using an anti-FLAG antibody. Precipitated Plk1 and Treacle proteins were detected by western analysis using anti-HA and anti-FLAG antibodies, respectively. Full-length (lane Full) and C-terminal half (lane C) of Plk1 are detected in the immunoprecipitated fraction. PLK1 is not detected in the immunoprecipitated fraction of untransfected HeLa cells (lane -). (C) Interaction between FLAG-tagged Treacle and endogenous PLK1 was examined by IP using anti-FLAG antibody. Precipitated PLK1 and Treacle proteins were detected by western analysis using anti-PLK1 and anti-FLAG antibodies, respectively. Endogenous PLK1 binds to FLAG-tagged full length (lane Full) and N-terminal part (lane N) of Treacle in HeLa cells. PLK1 is not detected in the immunoprecipitated fraction of untransfected HeLa cells (lane -). (D) HeLa cells immunolabelled for PLK1 (white and red) and centrin (green) 24 hours after control (siGL2) or <i>Tcof1</i> (siTcof1) siRNA transfection, revealing a marked diminishment of PLK1 from the centrosome and kinetochore of <i>Tcof1</i> knock-down cells. Scale Bars: A and D, 10 µm.</p

Dynamic localization of Treacle in mitotic cells.

<p>(A) Immunofluorescence images of wild-type forebrain at E11.5 detecting Treacle (red), the centrosome (γ-tubulin; green) and nuclei (DAPI; blue). Arrowheads and arrows indicate Treacle localization at the centrosome in interphase cells and mitotic cells respectively. (B) Immunofluorescence images of HeLa cells showing the dynamic localization of Treacle (green) during mitosis, particularly at the centrosomes (arrowheads) and kinetochore in prophase, prometaphase and metaphase cells as well as at the midzone during anaphase (arrow) and at the midbody (arrow) in telophase. The microtubular networks are detected by immunostaining with an anti-α-tubulin antibody. (C) Treacle co-localizes with the kinetochore marker, CENP-E. Scale Bars: A and B, 10 µm.</p

Plk1 co-operates in controlling mitotic progression and mitotic spindle orientation.

<p>(A) Expression of <i>Plk1</i> mRNA detected by <i>in situ</i> hybridization on coronal sections of E10.5–E16.5 embryos. (B) E11.0 mouse embryos were cultured with 50–200 nM BI 2536 for 16 hours. Mitotic cells were analyzed by immunostaining with phospho-Histone H3 (green) and Centrin (red). (C) The number of total pH 3-positive cells and percentage of surface and non-surface mitotic cells were quantified. The nuclei were stained with DAPI (blue). Scale Bars: A, 200 µm; B, 50 µm.</p

Abnormal brain development resulting from Treacle deficiency.

<p>(A) Neurons in E12.5–16.5 embryonic forebrains were visualized by immunofluorescence with anti-MAP2 antibody (green). (B) Quantification of neurons in forebrain. MAP2-positive cells in the wild-type and <i>Tcof1</i>^+/− brain were counted in a unit section of 100 µm width. (C) Immunostaining of cortical layers with anti-Reelin (layer I), anti-Cux2 (layer II, III and IV; asterisk) and anti-FoxP2 (layer V and VI) antibodies and <i>in situ</i> hybridization for <i>Cux2</i> on coronal sections of E18.5 wild-type and <i>Tcof1</i>^+/− mice. To observe the cortical cortex, MAP2-positive neurons (red) are co-stained with Cux2 and FoxP2 (green). Scale Bars: A, 25 µm; C, 100 µm.</p

Neural progenitor cells in <i>Tcof1</i>^+/− embryos exhibit mitotic defects.

<p>(A) Immunostaining of E10.5–E16.5 embryonic forebrains using a pH 3 antibody (red). Arrowheads indicate scattered progenitor cells in the ventricular and subventricular zones of the telencephalon in E14.5 <i>Tcof1</i>^+/− embryos. The neuron layers were visualized by immunofluorescence of anti-MAP2 antibody (green). (B) Bar graph depicting average numbers of mitotic cells in the telencephalon of E10.5–E16.5 wild-type and <i>Tcof1</i>^+/− embryos, counted in a unit section of 100 µm width. (C) Bar graph depicting the percentage of apical neural progenitor cells and abnormal scattered neural progenitor cells in the telencephalon of E12.5 and E14.5 wild-type and <i>Tcof1</i>^+/− embryos. (D) Co-labeling of the telencehalon in wild-type and <i>Tcof1</i> mutant embryos with IdU (green) and BrdU (red). S-phase and total cell cycle length were estimated by the number of IdU- and BrdU-positive cells and revealed an increase in total cell cycle length in <i>Tcof1</i>^+/− embryos compared to wild-type littermates. Scale Bars: A and D, 50 µm.</p

<i>Tcof1</i> deficiency affects the number of Pax6-positive apical progenitors and Tbr2-positive basal progenitor cells in the telencephalon.

<p>(A) Immunofluorescence detection of Pax6-positive progenitor cells (green) and anti-phospho-Histone H3 antibody (red) mitotic cells in the telencephalon of E14.5 wild-type and <i>Tcof1</i>^+/− embryos. Tissue sections were counterstained with DAPI (blue). (B) Bar graph depicting the number of Pax6-positive cell in the wild-type and <i>Tcof1</i>^+/− brain in a unit section of 125 µm width. (C) Co-immunostaining of the neuroepithelium of E14.5 wild-type and <i>Tcof1</i>^+/− embryos for Tbr2-positive neurons (green) and pH 3-positive (red) mitotic cells (D). Bar graph quantifying the number of Tbr2-positive cells in <i>Tcof1</i>^+/− embryos and their wild-type littermates in a unit section of 100 µm width. Scale Bars: A and C, 20 µm.</p

Essential function of Treacle in mitotic spindle formation and mitotic progression.

<p>(A) PCR analysis of efficacy of <i>TCOF1</i> knockdown in HeLa cells 48 hours after control (siGL2) or <i>TCOF1</i> (si TCOF1) siRNA transfection. (B) Immunostaining of mitotic HeLa cells in control knock-down (siGL2) and <i>TCOF1</i> knock-down (si TCOF1) cultures analyzed with anti-α-tubulin (red) and anti-Treacle (green) antibodies. (C) Mitotic cells in <i>TCOF1</i>knock-down (siTCOF1) and control (siGL2) cultures immunostained with anti-α-tubulin (red) and anti-centrin (green) antibodies. Arrowheads indicate abnormal mitotic spindle and chromosome alignment in <i>TCOF1</i> knock-down cultures. (D) Graph depicting the frequency of mitotic cells labeled via immunostaining with a pH 3 antibody at 6–16 hours post siGL2 or siTCOF1 transfection and the marked delay in mitotic exit exhibited by <i>TCOF1</i> knock-down cells. Scale Bars: B and C, 5 µm.</p

Growth of explanted embryonic ureters.

<p>E14 wild type mouse ureters were explanted into organ culture and maintained for up to six days. <b>A</b> and <b>B</b>. Phase contrast images of whole explants on day six of culture. Ureters cultured in the presence of FG7 (<b>B</b>) appeared bulkier than controls (Ctrl in <b>A</b>). <b>C</b>. Groups of explants cultured with FGF7 were significantly (#; p<0.05, as assessed by t-tests) longer than controls at three and six days of culture (each dot represents one ureter). <b>D</b>. Whole mount CK15 immunostaining of ureteric explant on day six of culture showed positive cells in the central, urothelial, core of the organ; the dotted line indicates the perimeter of the ureter and the top/proximal end is on the left of the frame. <b>E</b>-<b>J</b>. Cross sectional images of explanted ureters fed for six days with control media (<b>E</b>-<b>G</b>) or media supplemented with FGF7 (<b>H</b>-<b>J</b>). In each frame, the dotted line marks the border between the urothelium and the surrounding differentiating smooth muscle, and an asterisk has been placed in the lumen. Images E and H demonstrate that CK15+ (green) urothelial cells were present in both experimental groups. Images F and I show BrdU+ cell nuclei in each experimental group. Most of them were in the developing muscle layer but some were present in the urothelium. In the merged images (G and J, where DAPI/blue nuclear staining is also shown), it is apparent the the BrdU+ cells in the urothelium usually do not correspond to the CK15+ cells. In each of the frames <b>E-G</b>, the locations of the same two CK15+ cells are arrowed; the upper cell is BrdU- while the lower one is BrdU+. In <b>H</b>-<b>J</b>, there is a cluster of BrdU+ epithelial nuclei at ‘6-9 o’clock’ and they are separate from CK15+ cells. Bars are 50 μm. </p

Double immunostaining for CK15/P63 and CK15/CK19.

<p>Fluorescence images of cross sections of wild type E17 mouse ureters. In each frame, the asterisk indicates the ureteric lumen and the dotted lines indicate the border between the urothelium and differentiating smooth muscle. <b>A</b>-<b>C</b>. Double immunostaining for CK15 (green in <b>A</b>) and P63 (red in <b>B</b>), with the merged images shown in <b>C</b> where nuclei are stained blue with DAPI. The same two CK15+ cells are arrowed in A and C; note the presence of P63 in their nuclei. <b>D</b>-<b>F</b>. Double immunostaining for CK15 (red in <b>D</b>) and CK19 (green in <b>E</b>), with the merged images shown in <b>F</b> where nuclei have been stained blue with DAPI. The same two CK15+ cells are arrowed in <b>D</b> and <b>F</b>. Note that CK19 has an overlapping but more extensive distribution than CK15. Bars are 100 μm.</p