Original Article Mol Syndromol 2012;3:158–168 Accepted: August 7, 2012 DOI: 10.1159/000342833 by M. Schmid Published online: September 27, 2012 From the Transcription of Genes Involved in Ectodermal Dysplasias to the Understanding of Associated Dental Anomalies a b a a a V. Laugel-Haushalter A. Langer J. Marrie V. Fraulob B. Schuhbaur a a a–c M. Koch-Phillips P. Dollé A. Bloch-Zupan a Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Centre National de la Recherche Scientifique (UMR 7104), Institut National de la Santé et de la Recherche Médicale (U 964), Université de b c Strasbourg, Illkirch , Faculty of Dentistry, University of Strasbourg, and Reference Centre for Orodental Manifestations of Rare Diseases, Pôle de Médecine et Chirurgie Bucco-Dentaires, Hôpitaux Universitaires de Strasbourg (HUS), Strasbourg , France Key Words of the corresponding mutant mouse models. Translational Dental anomalies ؒ Ectodermal dysplasia ؒ Gene approaches in development and medicine are relevant to -expression ؒ Mouse ؒ Tooth development gain understanding of the molecular events underlying clin ical manifestations. Copyright © 2012 S. Karger AG, Basel Abstract Orodental anomalies are one aspect of rare diseases and are increasingly identified as diagnostic and predictive traits. To Oral cavity and dental developmental anomalies are understand the rationale behind gene expression during one aspect of rare diseases or syndromes. These diseases, tooth or other ectodermal derivative development and the which encompass about 8,000 different entities, affect 4 disruption of odontogenesis or hair and salivary gland for- million people in France and almost 25 millions in Eu- mation in human syndromes we analyzed the expression rope. Per se and definition they affect less than a person patterns of a set of genes (Irf6, Nfkbia, Ercc3, Evc2, Map2k1) among 2,000 and 80% of them are genetically driven. involved in human ectodermal dysplasias in mouse by in situ Among more than 7,000 known syndromes, at least 900 hybridization. The expression patterns of Nfkbia , Ercc3 and have a dento/oro/facial phenotype and 750 display in Evc2 during odontogenesis had never been reported previ- their clinical synopsis a cleft lip/palate. This is under- ously. All genes were indeed transcribed in different tissues/ standable as the same genes and signaling pathways reg- organs of ectodermal origin. However, for Nfkbia, Ercc3, Evc2, ulate palate, tooth development and the organogenesis of and Map2k1 , signals were also present in the ectomesenchy- other systems. Considering the dual origin of teeth (the mal components of the tooth germs. These expression pat- oral ectoderm for the enamel organ and the derived am- terns were consistent in timing and localization with the eloblasts synthesizing the enamel matrix, and ectomes- known dental anomalies (tooth agenesis, microdontia, coni- enchyme originating from the cephalic neural crest cells cal shape, enamel hypoplasia) encountered in syndromes re- for the mesenchymal part of the tooth including pulp tis- sulting from mutations in those genes. They could also ex- sues, odontoblasts and the periodontium), orodental plain the similar orodental anomalies encountered in some anomalies are very often present in syndromes involving © 2012 S. Karger AG, Basel Agnès Bloch-Zupan 1661–8769/12/0034–0158$38.00/0 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) Fax +41 61 306 12 34 BP 10142 E-Mail [email protected] Accessible online at: 1 rue Laurent Fries, FR–67404 Illkirch Cedex (France) www.karger.com www.karger.com/msy E-Mail agnes.bloch-zupan @ unistra.fr ectodermal derivatives like ectodermal dysplasias (EDs) (Roche) in a 20- ␮ l volume. The following RNA polymerases (Sig- where abnormalities of tooth number (missing teeth), ma) were used: T7 polymerase (Ercc3 and Evc2 probes), T3 poly- shape (conical crown, taurodontic molars) or hard tissue merase (Irf6 and Map2k1 probes) and SP6 polymerase ( Nfkbia probe). The reaction was stopped with 2 ␮ l EDTA (0.2 M , pH 8), structures (enamel hypoplasia) are part of the phenotype. and RNA was precipitated with 1 ␮ l yeast tRNA (10 mg/ml), 2.5 These anomalies, associated to the clinical synopsis of ␮ l LiCl (4 M ) and 75 ␮ l absolute ethanol, followed by an incuba- these syndromes are increasingly identified as diagnostic tion for 30 min at –80 ° C and centrifugation at 12,000 rpm (30 min and predictive traits. at 4 ° C). The pellet was washed with 0.5 ml ethanol (70%) and re- The aim of this study is to increase the knowledge of centrifuged. The supernatant was discarded and the pellet was allowed to dry. The probe was then diluted in 20 ␮ l sterile H2 O. genes involved in tooth development and anomalies in a The quality of the probe was verified by electrophoresis in a 1% syndromic context within the group of EDs through the agarose gel. If no smear was observed and the size was as expect- analysis of their expression patterns during mouse odon- ed, the probe was considered to be ready for use. The quantity of RNA was evaluated by a Nanodrop (ND-1000 Spectrophotome- togenesis, as well as in other ectodermal organs like sali- ␮ vary glands and vibrissae. Using a bioinformatic ap- ter, Labtech) and adjusted to 150 ng/ l in hybridization buffer, then stored at –20 ° C until use. proach, we selected known genes involved in EDs within online databases (Online Mendelian Inheritance in Man, In situ Hybridization Orphanet [Aymé et al., 1998; Aymé, 2003]) and the litera- Slides were allowed to thaw to room temperature (RT) for 2 h. ture ([Gorlin et al., 2001; Hennekam et al., 2010], PubMed Then they were post-fixed on ice in 4% paraformaldehyde (di- luted in PBS) for 10 min and rinsed in PBS. The hybridization (http://www.ncbi.nlm.nih.gov/pubmed/)) with limited buffer was composed of 50% deionized formamide, 10% dextran information about their expression pattern or role during sulfate, 1-mg/ml yeast tRNA, 1 ! Denhardt’s solution, and 1 ! salt ,odontogenesis. The online EURExpress in situ hybridiza- solution (0.195 M NaCl, 5 m M Tris pH 7.2, 5 m M NaH2 PO4 ؒ 1 H2 O ؒ tion atlas (http://www.eurexpress.org; http://www.gene- 5 m M Na2 HPO4 12 H2 O, 5 m M EDTA pH 8). The probe was di- ␮ paint.org/) [Diez-Roux et al., 2011] was then used to iden- luted in hybridization buffer at a concentration of 1 g/ml. The probe mix was denatured by a 10-min incubation at 70 ° C and tify genes showing detectable expression in tooth buds of placed on ice. An aliquot of 100 ␮ l was applied on each slide, embryonic day (E)14.5 mice. Five genes were thus select- which were covered by coverslips and allowed to hybridize over- ed, for which we performed a detailed analysis of their night at 65 ° C in humidified chambers. The slides were then ! expression patterns by in situ hybridization at various washed 2 times for 30 min at 65 ° C in 1 standard saline citrate stages of mouse development. (SSC), 50% formamide, 0.1% Tween-20, and 2 times for 30 min at RT in MABT buffer (1! MAB (Maleic acid buffer): 0.5 M maleic acid (Roche), 0.75 M NaCl, NaOH to ph 7.5 plus 0.1% Tween-20). Probe detection was performed using antibodies and reagents Materials and Methods from Roche. Slides were incubated for 1 h at RT with a blocking solution (20% goat serum, 2% blocking reagent in MABT). The Sample Preparation anti-DIG antibody was diluted 1: 2,500 in blocking solution, and Mouse embryos/fetuses were collected at E12.5, E14.5, E16.5, 200 ␮ l was added to each slide, which were covered by Parafilm and on the day of birth (hereafter referred to as E19.5), after natu- and incubated overnight at 4 ° C. Slides were washed 5 times in ral matings between C57BL6 mice. For E14.5 and older samples, MABT for 20 min and then 2 times for 10 min in NTMT buffer ,the whole head was embedded in OCT 4583 medium (Tissue- (100 m M NaCl, 100 m M Tris-HCl pH 9.5, 50 mM MgCl2 ؒ 6 H 2 O TEK, Sakura) and frozen on the surface of dry ice. E12.5 embryos 0.1% Tween-20). An aliquot of 200 ␮ l of freshly prepared staining were fixed overnight in 4% paraformaldehyde (pH 7.5, w/v) in solution (3.5 ␮ l nitro-blue tetrazolium chloride (Roche), 3.5 ␮ l (PBS, cryoprotected by overnight incubation in 20% sucrose (w/v) 5-bromo-4-chloro-3 ؅ -indolylphosphate p-toluidine salt (Roche in PBS, and cryoembedded as described above. Cryosections (Lei- in NTMT buffer) was placed on each slide, covered by a Parafilm ca CM3050S cryostat) at 10 ␮ m were collected on Superfrost plus and incubated overnight in the dark at RT. The staining solution slides and stored at –80 ° C until hybridization. E12.5 and E14.5 was changed every day and when signal was optimal the slides samples were sectioned in a frontal plane, whereas other stages were rinsed 2 times during 5 min in NTMT buffer. The slides were were sectioned sagittally. further rinsed by PBS and water, allowed to dry overnight, and mounted in Coverquick 2000 mounting medium (Labonord). Probe Synthesis All probes were synthesized from PCR-generated DNA tem- plates kindly provided by the EURExpress consortium (http:// www.eurexpress.org). The template sequences are given in online R e s u l t s suppl. fig. 1 (www.karger.com/doi/10.1159/000342833). DIG-labeled antisense riboprobes were transcribed in vitro by ␮ Selection of Candidate Genes incubation for 2 h at 37 ° C using 1 g of the PCR product, 20 U RNA polymerase, 5 ! transcription buffer (Promega), 10 ! DIG As described in the introduction section, 5 genes were RNA labeling Mix (Roche), 0.5 M DTT, 20 U RNAse inhibitor selected on the basis of their involvement in rare human Expression of Ectodermal Mol Syndromol 2012;3:158–168 159 Dysplasia-Causing Genes diseases and orodental anomalies (for an example, see fig.