Dehydroepiandrosterone Biosynthesis, Role, and Mechanism of Action in the Developing Neural Tube
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CORE Metadata, citation and similar papers at core.ac.uk ORIGINAL RESEARCH ARTICLE Provided by PubMed Central published: 07 February 2012 doi: 10.3389/fendo.2012.00016 Dehydroepiandrosterone biosynthesis, role, and mechanism of action in the developing neural tube Mark Galdo, Jennifer Gregonis, Christelle S. Fiore and Nathalie A. Compagnone* Laboratory for Spinal Cord Development and Regeneration, Department of Neurological Surgery, University of California, San Francisco, CA, USA Edited by: Dehydroepiandrosterone (DHEA) is synthesized from cholesterol by activity of P450scc Kazuyoshi Tsutsui, Waseda University, and P450c17, enzymes that we previously characterized in the developing nervous sys- Japan tem. We describe the localization of P450c17 in the differentiated field of the ventral spinal Reviewed by: Synthia H. Mellon, University of cord in different motor neuron subtypes. We show that, during organogenesis, P450c17 California San Francisco, USA activity is regulated along the antero/posterior axis of the spinal cord concomitantly with Ishwar Parhar, Monash University, the gradient of neurogenesis. To examine whether DHEA may modulate this process, Malaysia we measured proliferation and differentiation of ventral neural precursors in primary and *Correspondence: explant cultures. Our results showed that DHEA-induced the expression of class II pro- Nathalie A. Compagnone, Innovative Concepts in Drug Development, tein Nkx6.1, motor neuron precursor Olig-2, and definitive motor neuron marker Isl-1/2. Innovation Park Michel Caucik, BP2 DHEA also promoted proliferation of ventrally committed precursors in isolated spinal cord 100 rte des houilleres, 13590 precursor cultures and in whole spinal cord explants. Both the proliferative and inductive Meyreuil, France. effects of DHEA were dependent on sonic hedgehog signaling. The possibilities that the e-mail: [email protected] effects observed with DHEA were due to its metabolism into androgens or to activation of NMDA receptors were excluded. These results support the hypothesis that the tight regulation of DHEA biosynthesis may be a biologic clock restricting the period of ventral neuronal-precursor proliferation, thus controlling the number of pre-committed neurons in the developing neural tube. Keywords: DHEA, neurosteroid, spinal cord, sonic hedgehog, proliferation, induction, neural precursors INTRODUCTION DHEA is a secreted neurosteroid biosynthesized de novo in the The topography of the central nervous system (CNS) results developing CNS from the sequential action of two cytochrome from an orchestrated control of cell proliferation and motility, P450 enzymes, P450scc and P450c17 (Compagnone and Mellon, maturation of neuronal and glial cells, axonal growth, and the 2000). To determine whether DHEA is produced in the regions establishment of proper synapses. Neural specification and pat- where we have found P450c17 expressed and what its bioactivity terning of the CNS are controlled by the combinatory expression may be, we focused our study on the developing spinal cord where of genes in the neural tube during development, establishing a P450c17 expression is first observed. code for neuronal identity in restricted specific regions (Ruben- In this report, we establish a correlation between the expres- stein and Beachy, 1998). Sonic hedgehog (Shh) is one of the major sion and activity of P450c17 and show that P450c17 activity, morphogens controlling the expression of these homeodomain and thus most likely DHEA biosynthesis, are regulated along the proteins and plays a primordial role in dictating cell fate in neural antero/posterior (A/P) axis of the developing spinal cord con- tube precursors through a graded signaling activity along the comitantly with the gradient of neurogenesis. DHEA mimicked ventro-dorsal axis (Briscoe et al., 2000; Jessell, 2000; Lee and Pfaff, Shh signaling to promote the induction of motor neurons and 2001). sustained ventral progenitor proliferation in a Shh-dependent Patterning activity is not the only function of Shh. Mitogenic manner in vitro. The results of this work support the hypothesis activity of Shh has been observed in several tissues and organisms that the tightly regulated biosynthesis of DHEA may be a biologic during their development (reviewed in Ingham and McMahon, clock restricting the period of ventral neuronal-precursor prolifer- 2001; Brito et al., 2002; Marti and Bovolenta, 2002). In addition, ation and thus controlling the number of pre-committed neurons genetic alterations of Shh biogenesis or signal transduction lead to in the developing neural tube. CNS hyperplasia (reviewed in Litingtung and Chiang, 2000; Ruiz i Altaba et al., 2002; Wetmore, 2003). In the neural tube, prolifer- MATERIALS AND METHODS ative effects of Shh have been demonstrated in vitro and in vivo All experiments were reviewed and approved by the Institutional (Kalyani et al.,1998; Rowitch et al.,1999). However,environmental Animal Care and Use Committee at the University of California, cues regulate the ability of precursor cells to proliferate in response San Francisco, CA, USA. to Shh (Rowitch et al., 1999; Barzi et al., 2010). We proposed that the neurosteroid dehydroepiandrosterone (DHEA) might be one REAGENTS AND CHEMICALS such cue controlling the proliferative response of precursors to DHEA, BrdU, poly-d-lysine, progesterone, and streptavidin– Shh. FITC were purchased from Sigma/RBI (St. Louis, MO, USA); www.frontiersin.org February 2012 | Volume 3 | Article 16 | 1 Galdo et al. DHEA and ventral spinal cord precursors Collagenase H, insulin–transferrin–sodium selenium supplement, from the center of the hindlimb bud to three somites rostrally, and the terminal deoxynucleotidyl transferase biotin d-UTP nick- and only the ventral and intermediate neural plates were retained. end labeling (TUNEL) “In situ Cell Death Detection Kit,”Fluores- Dissected segments were mechanically separated from the mes- cein from Roche Diagnostics (Indianapolis, IN, USA); and bFGF, enchyme and dissociated with collagenase H (0.045 U/ml). Iso- Leibovitz’s L15 medium, high-glucose formula of DMEM, and lated cells were cultured on poly-d-lysine-coated glass coverslips HAM-F12 medium from Invitrogen/Gibco (Carlsbad, CA, USA). at 43,000 cells/cm2 in a serum-free medium (DMEM/F12) con- [3H]pregnenolone was obtained from NEN/Perkin-Elmer Life taining 50 ng/ml basic fibroblast growth factor (bFGF), 5 μg/ml Sciences (Boston, MA, USA). The following antibodies were pur- insulin, 5 μg/ml transferrin, 3 × 10−8 M selenium, 2 × 10−8 M chased from the Developmental Studies Hybridoma Bank (DSHB, progesterone, and, when tested, DHEA at 10−10 M. When stated, University of Iowa, IA, USA): anti-Shh (5E1), Isl-1 (40.2D6), cells were grown in the presence of the 3 μg/ml 5E1 anti-Shh Lim1/2 (4F2), and Pax6. The anti-Shh (5E1), anti-Isl-1 (40.2D6), antibody raised against the N-terminal fragment of Shh. and anti-Lim1/2 (4F2) antibodies, developed by Dr. Thomas Jes- After various times in culture, ranging from 3 to 45 h, BrdU sell, the anti-Pax6 and the anti-Pax7 antibodies, developed by Dr. (10 μM) was incorporated for 1 h and cells were washed in PBS Atsushi Kawakami were fully characterized prior to their use in and fixed with glycine:ethanol (3:7, v:v) for 45 min at room tem- this paper, Hoechst, Alexa 546-coupled goat anti-mouse, Alexa perature. DNA was denatured by using 4 M HCl for 10 min. Cells 350-coupled goat anti-rabbit, neutravidin–Alexa 350, wheat-germ were washed and immunostained for BrdU (mouse anti-BrdU agglutinin, and the FluoroReporter Mini-biotin protein labeling antibody used at 1:50), for NeuN (biotinylated mouse anti-NeuN kit were obtained from Molecular probes (Eugene,OR,USA). Goat antibody used at 1:200), for Isl-1 (rabbit anti-Isl-1/2 antibody, gift anti-rabbit coupled to Phycoerythrin was bought from Chemi- from Dr. Pfaff, used at 1:2,000), for Nkx6.1 (rabbit anti-Nkx6.1, con (Temecula, CA, USA) and goat anti-mouse coupled to FITC gift from Dr. German, used at 1:4,000), and for Pax6 (DSHB, from Jackson ImmunoResearch Laboratories Inc., (West Grove, used at 1:20). Secondary antibodies were a goat anti-mouse-Alexa PA, USA). 546, a streptavidin–FITC, and a goat anti-rabbit-Alexa 350, which were used at 1:500, 1:200, and 1:500 respectively. TUNEL analy- ANALYSIS OF ENZYMATIC ACTIVITY sis was performed by using the “In situ Cell Death Detection Kit” Embryos were obtained from CD-1 mice (Charles River, Wilm- as described previously (Hagedorn et al., 2000). Total cell number ington, MA, USA) at different ages from embryonic day (E) 10 was determined on each slide after Hoechst or fluorescent-coupled to E16.5 (E0.5 = day of vaginal plug). Spinal cords were dissected wheat-germ agglutinin counter-staining. in L15 medium containing 5% heat-inactivated horse serum. The At least five independent experiments were performed for each spine was exposed after removal of the ventral organs. The noto- time point. Cells immunopositive for BrdU, NeuN, Isl-1, Nkx6.1, chord was peeled off and collected. Mesenchyme surrounding the Pax6, Chx10, Eng-1, or O4 were counted in 10 randomly selected spinal cord was carefully removed and collected, as were the dorsal 1mm2 fields/slide; three slides were counted per treatment and per root ganglia (DRG). The spinal cords were opened dorsally, flat- experiment. A general factorial multiple analysis of variance was tened, and then cut into dorsal (roof plate + alar plate) and ventral performed to determine the effects of the time in culture and of (floor plate + motor columns) segments. Dorsal and ventral halves the presence of DHEA in the culture medium, as well as a possible at all A/P levels were collected and analyzed separately. The geni- interaction between these two factors. tal ridges/gonads were collected as a positive control for P450c17 activity. Parts collected from three embryos were routinely pooled HISTOCHEMISTRY for the analyses and yielded reproducible and significant levels of In situ hybridization and immunochemistry in mouse embryos DHEA biosynthesis.