Retinoic Acid-Dependent Signaling Pathways and Lineage Events in the Developing Mouse Spinal Cord

Studies in avian models have demonstrated an involvement of retinoid signaling in early neural tube patterning. The roles of this signaling pathway at later stages of spinal cord development are only partly characterized. Here we use Raldh2-null mouse mutants rescued from early embryonic lethality to study the consequences of lack of endogenous retinoic acid (RA) in the differentiating spinal cord. Mid-gestation RA deficiency produces prominent structural and molecular deficiencies in dorsal regions of the spinal cord. While targets of Wnt signaling in the dorsal neuronal lineage are unaltered, reductions in Fibroblast Growth Factor (FGF) and Notch signaling are clearly observed. We further provide evidence that endogenous RA is capable of driving stem cell differentiation. Raldh2 deficiency results in a decreased number of spinal cord derived neurospheres, which exhibit a reduced differentiation potential. Raldh2-null neurospheres have a decreased number of cells expressing the neuronal marker β-III-tubulin, while the nestin-positive cell population is increased. Hence, in vivo retinoid deficiency impaired neural stem cell growth. We propose that RA has separable functions in the developing spinal cord to (i) maintain high levels of FGF and Notch signaling and (ii) drive stem cell differentiation, thus restricting both the numbers and the pluripotent character of neural stem cells

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

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

Retinoic acid deficiency alters gene expression profiles in spinal cord SP cells.

<p>Quantitative RT-PCR analysis of <i>Sox2</i>, <i>Pax6</i>, <i>Olig2</i>, <i>Msi1</i>, <i>Nestin</i>, <i>Abcg2</i>, <i>Mdr1</i>, <i>Blbp</i>, and <i>Glast</i> mRNA levels in SP fractions from WT and <i>Raldh2^−/−</i> brachial spinal cords (black and red bars, respectively) collected at E14.5 after a short-term (E7.5–8.5) RA-rescue. Data are represented as relative mRNA levels with respect to <i>Gapdh</i> levels.</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

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