Ctip2/Bcl11b controls ameloblast formation during mammalian odontogenesis Olga Golonzhkaa,1, Daniel Metzgerb, Jean-Marc Bornertb, Brian K. Bayc, Michael K. Grossd, Chrissa Kioussia,2, and Mark Leida,2 Departments of aPharmaceutical Sciences, cMechanical Engineering, and dBiochemistry and Biophysics, Oregon State University, Corvallis, OR 97331 USA; and bInstitut de Ge´ne´ tique et de Biologie Mole´culaire et Cellulaire, Department of Functional Genomics, INSERM U596 and CNRS UMR 7104, Illkirch, F-67400 France Communicated by Michael G. Rosenfeld, University of California, San Diego at La Jolla, CA, January 16, 2009 (received for review December 28, 2008) The transcription factor Ctip2/Bcl11b plays essential roles in de- labial side of the developing incisor and promote enamel formation velopmental processes of the immune and central nervous systems on this aspect of the tooth. In contrast, odontoblasts are found on and skin. Here we show that Ctip2 also plays a key role in tooth both sides of the developing incisor (11). development. Ctip2 is highly expressed in the ectodermal compo- Several transcription factors have been implicated in tooth nents of the developing tooth, including inner and outer enamel development, including Pax9, Pitx2, Runx2, Msx1, Msx2, and epithelia, stellate reticulum, stratum intermedium, and the amelo- others (reviewed in ref. 12). Mutations in Pax9 and Msx1 cause blast cell lineage. In Ctip2؊/؊ mice, tooth morphogenesis appeared oligodontia in humans (13, 14), and Msx1-mutant mice exhibit to proceed normally through the cap stage but developed multiple arrest of tooth development at early stages (15). Msx2 regulates defects at the bell stage. Mutant incisors and molars were reduced proliferation of the enamel organ and cusp morphogenesis, and in size and exhibited hypoplasticity of the stellate reticulum. An terminal differentiation of ameloblasts (16). ameloblast-like cell population developed ectopically on the lin- Ctip2/Bcl11b is a transcriptional repressor (17–19) that plays gual aspect of mutant lower incisors, and the morphology, polar- critical roles in the development and function of several organ ization, and adhesion properties of ameloblasts on the labial side systems, including the central nervous (20, 21), immune (22, 23), of these teeth were severely disrupted. Perturbations of gene and cutaneous (24) systems. Germline deletion of Ctip2 is -expression were also observed in the mandible of Ctip2؊/؊ mice: associated with perinatal lethality (22), demonstrating the es expression of the ameloblast markers amelogenin, ameloblastin, sentiality of Ctip2 for life. and enamelin was down-regulated, as was expression of Msx2 and Although a tooth developmental defect has not been reported Ϫ/Ϫ epiprofin, transcription factors implicated in the tooth develop- in Ctip2 mice, the epidermal defects associated with loss of ment and ameloblast differentiation. These results suggest that Ctip2 (24), together with a high level of Ctip2 expression in Ctip2 functions as a critical regulator of epithelial cell fate and dental tissues of ectodermal origin (Fig. 1) prompted us to differentiation during tooth morphogenesis. investigate this possibility. We report herein that lack of Ctip2 compromises tooth development. Ctip2Ϫ/Ϫ molars were charac- amelogenesis ͉ cellular differentiation ͉ enamel ͉ tooth development ͉ terized by a hypoplastic stellate reticulum and poorly developed transcription factor cusps at later stages. Ameloblast-like cells developed inappro- priately on the lingual side of lower incisors in Ctip2Ϫ/Ϫ mice, while ameloblasts on the labial side were smaller, disorganized, ooth development is a model system for study of coordinated nonpolarized, and exhibited low levels of expression of amelo- Tmolecular interactions between ectoderm and the underly- blast-specific genes, such as amelogenin, ameloblastin, and enam- ing, neural crest-derived mesenchyme. Tooth development is elin. Expression of Msx2 was also downregulated in Ctip2Ϫ/Ϫ initiated with a thickening of the oral ectoderm at embryonic day mice, and ChIP studies revealed that Msx2, and other genes 10.5 (E10.5) that gives rise to the dental lamina, which in turn involved in ameloblast development and function, are likely expands into underlying mesenchyme, forming the tooth bud direct targets of Ctip2. Collectively, these data suggest that Ctip2 (bud stage, E12.5). The cap stage (E14.5) follows and includes is essential for the terminal differentiation of ameloblasts and folding of the dental ectoderm in a process that is regulated by proper tooth formation. a transient signaling center, the primary enamel knot. The enamel knot expresses signaling molecules that stimulate pro- Results liferation of surrounding epithelium and mesenchyme. The early Ctip2 Expression in Developing Jaw and Tooth. Antibody staining bell stage of tooth development (E15.5–E16.5) is characterized revealed high levels of Ctip2 expression in the oral ectoderm of by continued epithelial expansion and differentiation into the the first branchial arch at E9.5 (Fig. 1A). Ctip2 expression inner (IEE) and outer enamel epithelium (OEE), stratum continued to be detected in oral ectoderm, with lower levels in intermedium (SI), and stellate reticulum (1–6). the first branchial arch mesenchyme at E10.5 (Fig. 1B). At E12.5, During the mid- to late-bell stage of odontogenesis (E16.5– Ctip2 expression was detected in the thickening of the oral E19.5), 2 tooth-specific cell types are formed: ameloblasts, which ectoderm, the dental epithelium of future molars (Fig. 1C), differentiate from the IEE and secrete enamel, and odonto- blasts, which derive from dental mesenchyme and produce dentin (5, 7, 8). Ameloblasts also synthesize and secrete the Author contributions: O.G., D.M., C.K., and M.L. designed research; O.G., J.-M.B., B.K.B., and enamel matrix proteins, amelogenins, and nonamelogenins, C.K. performed research; O.G., B.K.B., M.K.G., C.K., and M.L. analyzed data; and O.G., C.K., which are assembled into a structural framework (9, 10). Amelo- and M.L. wrote the paper. blasts transport calcium and phosphate ions into the extracellular The authors declare no conflict of interest. matrix, which results in nucleation and growth of hydroxyapatite 1Present address: Nina Ireland Laboratory of Developmental Neurobiology, University of California, Rock Hall, Room RH 284C, UCSF MC 2611, 1550 4th Street, San Francisco, CA crystals. 94158-2324. Enamel formation on mouse incisors is an asymmetric process 2To whom correspondence may be addressed. E-mail: [email protected] or resulting from differential distribution of ameloblasts around these [email protected]. teeth during development. The lingual side of mouse incisors lacks This article contains supporting information online at www.pnas.org/cgi/content/full/ ameloblasts and is enamel-free, while ameloblasts localize on the 0900568106/DCSupplemental. 4278–4283 ͉ PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900568106 Downloaded by guest on September 26, 2021 A B C DE F G H I J Fig. 1. Ctip2 expression profile during tooth development. (A–J) IHC in coronal sections of mouse embryonic heads at indicated developmental ages. Ctip2 staining appears green in all panels. (A) Expression of Ctip2 in the ectoderm of the 1st BA and future oe at E9.5. (B) Expression of Ctip2 in de at E10.5 and across the 1st BA. (C and D) Ctip2 expression in the oe, de, and cmes of a developing lower molar (C) and upper incisor (D) at E12.5. (E–G) Ctip2 expression in the developing lower molar at E14.5 (E), E16.5 (F), and E18.5 (G). (H–J) Double-label IHC of coronal sections of a lower incisor at E18.5 using antibodies against Ctip2 and amelogenin and counterstained with DAPI. The IHC data presented in this figure have been reproduced 4–6 times over the course of Ϸ2 years. [Scale bars: (A, B, D, G) 200 ␮m; (C, E, F) 100 ␮m; (H)20␮m.] a, ameloblast; BA, branchial arch; cl, cervical loop; cmes, condensing mesenchyme; dc, dental cord; de, dental epilethium; iee, inner enamel epithelium; oe, oral epilethium; oee, outer enamel epithelium; p, papilla, si, stratum intermedium; sr, stellate reticulum. incisors (Fig. 1D), and surrounding mesenchyme (Fig. 1 C and blast-specific proteins on the lingual side of Ctip2Ϫ/Ϫ incisors D), and to a lesser extent in condensing dental mesenchyme (Fig. (white arrows in Fig. 3 D, J, and P). 1C). Substantial Ctip2 expression was noted in the dental An expansion of the epithelium was also present on the lingual epithelium and enamel knot at E14.5 (Fig. 1E), and at E16.5, side of the developing, mutant incisor (Fig. 2H, green asterisk), when the dental epithelium had differentiated into IEE and adjacent to the ectopic, ameloblast-like cells in Ctip2Ϫ/Ϫ mice OEE, Ctip2 expression was strongly detected in both structures (Fig. 2H, red asterisk). The stellate reticulum on the labial side and in the cervical loop and stellate reticulum (Fig. 1F). Ex- of the mutant incisors was indiscernible (Fig. 2 J, black asterisk), pression of Ctip2 in the ectoderm persisted at E18.5 (Fig. 1 G–J), which resulted in a close association between the OEE and IEE, but was not appreciably detected in mesenchyme-derived tissues, and stratum intermedium, hindering identification of the indi- including dental papilla. vidual structures of the developing tooth. The OEE of Ctip2Ϫ/Ϫ mice was hypoplastic and the odontoblasts appeared structurally Ctip2 Expression in Ameloblasts. Ctip2 was highly expressed in the disorganized, but this effect was less severe than the disruption IEE (Fig. 1 F and G), which gives rise to ameloblasts. Double- of cells of the ameloblast lineage (Fig. 2 D, F, and J). Thus, loss labeling immunohistochemistry (IHC) revealed colocalization of of Ctip2 appeared to affect the ameloblast lineage preferentially, Ctip2 and the ameloblast marker amelogenin in the late bell as the formation of ameloblasts, the boundary of the ameloblast stage (Fig. 1 H–J).