Abnormal development of neural stem cell-derived neurospheres from <i>Raldh2^−/−</i> spinal cords.

<p>Neurospheres were derived from dorsal cervical/brachial spinal cord from E12.5 WT and <i>Raldh2^−/−</i> embryos. (A,B) After growth of dissociated progenitor cells in suspension, in 6-well plates, for 10 days in a defined selective medium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032447#s4" target="_blank">Materials and Methods</a>), the number of spheres developing from <i>Raldh2^−/−</i> embryos spinal cords is decreased by 25% compared to WT (E: 11.1±0.88 spheres/well for WT; 8.40±1.50 for mutants; n = 9; T- test: P value = 0,0009). (C,D) To further study progenitor cell differentiation, spheres were plated onto laminin-coated coverslips for 3 days, after 10 days of growth in suspension. <i>Raldh2^−/−</i> derived spheres exhibited reduced number of cells compared to WT (F: one quadrant of a plated WT sphere is composed of 105.4±16.6 cells, against 40.9±6,82 cells in a <i>Raldh2^−/−</i> sphere; DAPI positive nuclei were counted using ImageJ software; n = 9; P = 1.6×10⁻⁵). (G–O) After growth onto laminin coated coverslips for 3 days, cells were processed for immunocytochemistry. Nestin+ cells (I,M) are increased by 20% in the <i>Raldh2^−/−</i>derived spheres, while TuJ1+ cells (J,N) are decreased by half. (H,L, DAPI staining; K,O, merged images). (G) After counting and normalization for total cell numbers, assessed by DAPI positive cell nuclei, one quadrant of a WT sphere contains 41.6±14 Nestin+ and 42.0±9.76 TuJ1+ cells, against 57.9±9.21 and 25.9±6,66 in a <i>Raldh2^−/−</i> sphere (n = 9; P = 0,001 and 0,007, respectively).</p

Short-term RA-rescue of <i>Raldh2^−/−</i> embryos reveals abnormal dorsal spinal cord development.

<p>(A,B) Transverse paraffin sections of the brachial spinal cord of E12.5 WT (A) and <i>Raldh2^−/−</i> (B) embryos harboring the RARE-<i>lacZ</i> reporter transgene, collected after short-term RA-rescue (E7.5–8.5). Embryos were X-gal stained prior to sectioning, and sections counterstained with eosin. Black arrows show defective roof plate development in the mutant. (C–F) Transverse vibratome sections of WT and <i>Raldh2^−/−</i> embryos harboring the RARE-<i>lacZ</i> transgene, collected at E12.5 after short-term (C,D) or extended (E7.5–10.5; E,F) rescue, were processed for X-gal staining. These experiments confirmed the lack of RARE-<i>lacZ</i> activity in the dorsal spinal cord of short-term rescued mutants (B,D), whereas dorsally-restricted transgene activity was not properly restored upon extended rescue (E,F). A region of ectopic <i>lacZ</i> activity is seen in the middle region of the spinal cord ventricular layer, particularly under short-term rescue (white arrowheads in B,D). (G,H) ISH analysis of <i>Rarb</i> expression in WT and <i>Raldh2^−/−</i> embryos collected at E11.5 after short-term RA-rescue (vibratome sections of the cervical/brachial spinal cord). (I–L) ISH analysis of <i>Wnt1</i> (I,J, insets), <i>Wnt3a</i> (K,L, insets), <i>Math1</i> (I,J, main panels), and <i>Lbx1</i> (K,L, main panels) in E12.5 WT and <i>Raldh2^−/−</i> embryos after short-term rescue. Both <i>Wnts</i> are expressed in cells on each side of the dorsal midline, and the pattern observed in mutants (J,L) reveals that both sides of the neuroepithelium did not properly merge during neural tube closure. (M–P) ISH analysis of <i>Olig2</i> (M,N, main panels), <i>Shh</i> (M,N, insets), <i>Pax6</i> (O,P, main panels) and <i>Dbx1</i> (O,P, insets) in E12.5 WT and <i>Raldh2^−/−</i> embryos after short-term rescue. Brackets and arrowheads point to abnormal downregulations in developing interneuron and motor neuron populations, respectively.</p

<i>Raldh2^−/−</i> mutants exhibit severe reduction of developing brachial motor neuron pools.

<p>Whole-mount ISH analysis of <i>Isl1</i> (A,B), <i>Pea3</i> (C,D), <i>Hoxc6</i> (E,F) and <i>Hoxc8</i> (G,H) on dissected cervical/brachial regions of the spinal cords of WT and <i>Raldh2^−/−</i> embryos, analyzed at E12.5 after short-term rescue (genotypes as indicated). All spinal cords are viewed as flat-mounts after cutting the dorsal midline, and only half-sides are shown in E-H. An inset in D shows the <i>Pea3</i> labelling observed in the brachial spinal cord of a <i>Raldh2^−/−</i> embryos collected after an extended (E7.5–10.5) RA-rescue.</p

Decreased Notch signaling in the retinoid-deficient spinal cord.

<p>ISH analysis of <i>Delta1</i> (A–D), <i>Hes1</i> (E–H) and <i>Hes5</i> (I–L) was performed on transverse vibratome sections of brachial spinal cords from WT and <i>Raldh2^−/−</i> embryos collected at E12.5 (A,B,E,F) or E11.5 (I,J) after short-term RA-rescue, and on whole-mount E8.5 unrescued embryos (C,D,G,H,K,L). Genotypes are indicated in each panel.</p

Retinoid deficiency affects dorsal root gangliogenesis.

<p>(A–D) Whole-mount ISH analysis of <i>Sox10</i> (A,B), <i>Isl1</i> (C,D, main panels) and <i>Eya2</i> (C,D insets) in the prospective dorsal root ganglia (drg) of E10.5 WT and <i>Raldh2^−/−</i> embryos, collected after short-term rescue (genotypes as indicated). (E,J) ISH analysis of <i>Sox10</i> (E,F, main panels) and <i>Hrt2</i> (E,F, insets), anti-neurofilament (NF) staining (G,H) and activated-Caspase 3 immunodetection (I,J) on transverse vibratome sections of brachial spinal cords of E12.5 WT and <i>Raldh2^−/−</i> embryos after short-term rescue (genotypes as indicated). Insets in H, I and J show details of a dorsal nerve root (H) and dorsal ganglia (I,J) of embryos collected at the same stage after an extended (E7.5–10.5) rescue. Red arrowheads indicate the dorsal nerve exit points, which are sites of excess apoptosis in the short-term rescued mutant (J).</p

Decreased FGF activity in the spinal cord of RA-rescued <i>Raldh2^−/−</i> mutants.

<p>Immunodetection of FGF2 (A,B, alkaline phosphatase staining) and p-ERK1/2 (C,D, peroxidase staining) on transverse vibratome section of the brachial spinal cord from E12.5 WT and <i>Raldh2^−/−</i> embryos, collected after short-term rescue.</p

Retinoic acid deficiency increases the surrogate stem cell SP fraction in fetal spinal cord.

<p>A–D: Hoechst 33342 FACS profiles (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032447#s4" target="_blank">Materials and Methods</a>) of cell suspensions from dissected spinal cords of E12.5 WT (A) and <i>Raldh2^−/−</i> mutants (B), and from WT and mutant samples preincubated in the channel blocker verapamil prior to dye addition (C,D). Percentages of cells within the SP fraction (red) are indicated. E: Mean percentages of SP cells in WT and mutant E12.5 spinal cords, respectively (n = 9 cell-sorting experiments performed on independent pools of 10–20 WT or mutant samples, respectively).</p

Cadherin-11 in Renal Cell Carcinoma Bone Metastasis

<div><p>Bone is one of the common sites of metastases from renal cell carcinoma (RCC), however the mechanism by which RCC preferentially metastasize to bone is poorly understood. Homing/retention of RCC cells to bone and subsequent proliferation are necessary steps for RCC cells to colonize bone. To explore possible mechanisms by which these processes occur, we used an <i>in vivo</i> metastasis model in which 786-O RCC cells were injected into SCID mice intracardially, and organotropic cell lines from bone, liver, and lymph node were selected. The expression of molecules affecting cell adhesion, angiogenesis, and osteolysis were then examined in these selected cells. Cadherin-11, a mesenchymal cadherin mainly expressed in osteoblasts, was significantly increased on the cell surface in bone metastasis-derived 786-O cells (Bo-786-O) compared to parental, liver, or lymph node-derived cells. In contrast, the homing receptor CXCR4 was equivalently expressed in cells derived from all organs. No significant difference was observed in the expression of angiogenic factors, including HIF-1α, VEGF, angiopoeitin-1, Tie2, c-MET, and osteolytic factors, including PTHrP, IL-6 and RANKL. While the parental and Bo-786-O cells have similar proliferation rates, Bo-786-O cells showed an increase in migration compared to the parental 786-O cells. Knockdown of Cadherin-11 using shRNA reduced the rate of migration in Bo-786-O cells, suggesting that Cadherin-11 contributes to the increased migration observed in bone-derived cells. Immunohistochemical analysis of cadherin-11 expression in a human renal carcinoma tissue array showed that the number of human specimens with positive cadherin-11 activity was significantly higher in tumors that metastasized to bone than that in primary tumors. Together, these results suggest that Cadherin-11 may play a role in RCC bone metastasis.</p></div

Generation of organ-tropic 786-O RCC cells.

<p>(A) Parental 786-O RCC cells were labeled with luciferase and GFP. (B) Images of bioluminescence of mice at indicated time point after intracardiac injection with parental 786-O cells. (C) 786-O cells derived from liver, lymph node and bone, were GFP-positive.</p