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Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists
Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists Shihai Jia a,1,2, Hyuk-Jae Edward Kwon a,2, Yu Lan a,b, Jing Zhou a,1, Han Liu a, Rulang Jiang a,b,n a b
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA Division of Plastic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
art ic l e i nf o
a b s t r a c t
Article history: Received 15 June 2016 Received in revised form 1 October 2016 Accepted 2 October 2016
Mutations in MSX1 cause craniofacial developmental defects, including tooth agenesis, in humans and mice. Previous studies suggest that Msx1 activates Bmp4 expression in the developing tooth mesenchyme to drive early tooth organogenesis. Whereas Msx1 / mice exhibit developmental arrest of all tooth germs at the bud stage, mice with neural crest-specific inactivation of Bmp4 (Bmp4ncko/ncko), which lack Bmp4 expression in the developing tooth mesenchyme, showed developmental arrest of only mandibular molars. We recently demonstrated that deletion of Osr2, which encodes a zinc finger transcription factor expressed in a lingual-to-buccal gradient in the developing tooth bud mesenchyme, rescued molar tooth morphogenesis in both Msx1 / and Bmp4ncko/ncko mice. In this study, through RNA-seq analyses of the developing tooth mesenchyme in mutant and wildtype embryos, we found that Msx1 and Osr2 have opposite effects on expression of several secreted Wnt antagonists in the tooth bud mesenchyme. Remarkably, both Dkk2 and Sfrp2 exhibit Osr2-dependent preferential expression on the lingual side of the tooth bud mesenchyme and expression of both genes was up-regulated and expanded into the tooth bud mesenchyme in Msx1 / and Bmp4ncko/ncko mutant embryos. We show that pharmacological activation of canonical Wnt signaling by either lithium chloride (LiCl) treatment or by inhibition of DKKs in utero was sufficient to rescue mandibular molar tooth morphogenesis in Bmp4ncko/ncko mice. Furthermore, whereas inhibition of DKKs or inactivation of Sfrp2 alone was insufficient to rescue tooth morphogenesis in Msx1 / mice, pharmacological inhibition of DKKs in combination with genetic inactivation of Sfrp2 and Sfrp3 rescued maxillary molar morphogenesis in Msx1 / mice. Together, these data reveal a novel mechanism that the Bmp4-Msx1 pathway and Osr2 control tooth organogenesis through antagonistic regulation of expression of secreted Wnt antagonists. & 2016 Elsevier Inc. All rights reserved.
Keywords: Bmp4 Msx1 Dkk2 Osr2 Sfrp2 Wnt signaling Tooth development Organogenesis Mouse
1. Introduction Tooth development is controlled by sequential and reciprocal signaling interactions between the epithelium and mesenchyme and has provided an excellent model system for understanding the molecular mechanisms regulating mammalian organogenesis (Jernvall and Thesleff, 2000). Embryological studies using tissue recombination assays have demonstrated that the tooth inductive signals initially arise in the early embryonic oral epithelium, which n Corresponding author at: Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. E-mail address: [email protected] (R. Jiang). 1 Present address: University of Utah School of Dentistry, Salt Lake City, UT 84112, USA. 2 Shihai Jia and Hyuk-Jae Edward Kwon contributed equally to this work as cofirst authors.
thickens to form dental placodes along the prospective dental arch and invaginates into the underlying neural crest-derived mesenchyme to form the early tooth buds (Lumsden, 1988; Mina and Kollar, 1987; Jernvall and Thesleff, 2000). At the early bud stage, the odontogenic potential shifts to the tooth mesenchyme such that the mesenchyme from the bud and later stages of tooth germs could induce tooth organogenesis when recombined with embryonic non-dental epithelium (Kollar and Baird, 1970a, 1970b; Ruch et al., 1973). As tooth development proceeds, an epithelial signaling center, termed the primary enamel knot (PEK), forms in the distal region of the tooth bud and produces many signaling molecules, including members of the Bmp, Fgf, and Wnt families and Shh (reviewed by Jernvall and Thesleff (2000), Tucker and Sharpe (2004), Zhang et al. (2005), Lan et al. (2014)). The PEKderived signals act upon both the dental epithelium and mesenchyme and continued epithelial-mesenchymal signaling
http://dx.doi.org/10.1016/j.ydbio.2016.10.001 0012-1606/& 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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interactions drive tooth morphogenesis through the subsequent “cap” and “bell” stages (Jernvall and Thesleff, 2000; Thesleff, 2003; Tucker and Sharpe, 2004; Lan et al., 2014). The Wnt signaling pathway plays critical roles in tooth bud initiation and morphogenesis. The canonical Wnt signaling pathway involves stabilization and nuclear accumulation of β-catenin. In the absence of Wnt signaling, cytoplasmic β-catenin is phosphorylated by the serine/threonine kinase GSK-3β and targeted for degradation by the ubiquitination-proteosome pathway, whereas activation of Wnt signaling inhibits GSK3β activity, leading to stabilization of β-catenin and its accumulation in the cellular nuclei where it interacts with and converts the TCF/Lef family DNAbinding proteins from transcriptional repressors to activators (reviewed by Clevers and Nusse (2012)). Mice lacking Lef1 and mice with tissue-specific inactivation of β-catenin in either the epithelium or tooth mesenchyme all exhibit tooth developmental arrest at the bud stage (van Genderen et al., 1994; Kratochwil et al., 1996; Andl et al., 2002; Liu et al., 2008; Chen et al., 2009). A recent comprehensive gene expression profiling analysis of the developing mandibular molar tooth epithelium and mesenchyme tissues, and of their responses to activation of Bmp, Fgf, Shh, and Wnt signaling pathways in explant cultures, led to the identification of a Wnt-Bmp feedback circuit as the major regulator of epithelialmesenchymal intertissue signaling interactions in early tooth organogenesis (O’Connell et al., 2012). A major feature of this WntBmp circuit is its asymmetric configuration, with cross-regulation of canonical Wnt and Bmp4 expression in the dental epithelium and joint regulation of Bmp4 expression by both the Bmp and Wnt pathways in the tooth mesenchyme (O’Connell et al., 2012). The molecular mechanisms mediating the cross-regulation between the Wnt and Bmp pathways in tooth development, however, remain to be elucidated. Bmp4 exhibits an expression pattern that coincides with the odontogenic potential, shifting from the epithelium to the underlying mesenchyme during early tooth bud formation, and exogenous Bmp4 protein was able to induce expression of endogenous Bmp4 and Msx1, which encodes a homeodomain-containing DNA-binding transcription factor critical for early tooth development, in mandibular mesenchyme explants (Chen et al., 1996; Satokata and Maas, 1994; Tucker et al., 1998; Vainio et al., 1993). Mice deficient in Msx1 exhibit tooth developmental arrest at the bud stage, with reduced Bmp4 mRNA expression in the dental mesenchyme by E13.5 (Chen et al., 1996; Satokata and Mass, 1994; Tucker et al., 1998). Remarkably, addition of exogenous Bmp4 protein to tooth germ explant cultures was able to rescue development of the Msx1 / mutant tooth germs to the late bell stage (Bei et al., 2000; Chen et al., 1996). These data led to the conclusion that Bmp4 induces its own expression in the early tooth mesenchyme through Msx1-mediated activation and acts downstream of Msx1 to drive tooth morphogenesis through the bud-to-cap transition (Bei et al., 2000; Miletich et al., 2011; O’Connell et al., 2012). However, transgenic expression of Bmp4 mRNAs driven by an Msx1 gene promoter only partially rescued maxillary molar tooth germs to the early cap stage, but not further morphogenesis, in Msx1 / mice (Zhao et al., 2000). Furthermore, in contrast to Msx1 / mice, mice with neural crest-specific inactivation of Bmp4 (Bmp4ncko/ncko), which lack functional Bmp4 in the developing tooth mesenchyme, exhibit bud-stage developmental arrest of only the mandibular molars while maxillary molars and incisors developed to mineralized teeth (Jia et al., 2013). These results indicate that there are additional as yet unresolved molecular mechanisms acting together with Bmp4 to mediate Msx1 function in tooth development. We have shown previously that the Osr2 transcription factor antagonizes Msx1 function to pattern molar tooth formation in a single row (Zhang et al., 2009). Osr2 is expressed in a lingual-to-
buccal gradient, mirroring that of Bmp4 mRNA expression, in the developing tooth bud mesenchyme. Osr2 / mice develop supernumerary teeth lingual to their molar teeth, accompanied by expanded expression of Bmp4 mRNAs into the lingual mesenchyme (Zhang et al., 2009). Remarkably, inactivation of Osr2 rescued molar tooth morphogenesis in both Msx1 / and Bmp4ncko/ncko mutant mice (Jia et al., 2013; Zhang et al., 2009). To further elucidate the molecular mechanism underlying the antagonistic regulation of tooth development by Msx1 and Osr2, we carried out RNA-seq analyses of the developing tooth mesenchyme in Msx1 / and Osr2 / mutant embryos and their control littermates. We found that Msx1 and Osr2 exert opposite effects on the expression of several secreted Wnt antagonists in the tooth bud mesenchyme. Through a combination of genetic inactivation and pharmacological inhibition studies, we demonstrate that the Bmp4-Msx1 pathway drives early tooth morphogenesis through regulation of expression of secreted Wnt antagonists, in particular Dkk2 and Sfrp2, in the developing tooth mesenchyme.
2. Materials and methods 2.1. Animals The Bmp4f/f, Msx1 þ / , Osr2 þ / , Sfrp2 / , and Wnt1Cre transgenic mice and generation of Bmp4f/f;Wnt1Cre (Bmp4ncko/ncko) compound mutant mice have been described previously (Satokata and Maas, 1994; Chai et al., 2000; Lan et al., 2004; Liu et al., 2008; Morello et al., 2008; Jia et al., 2013). Sfrp3 / (Frzb / ) mice (JAX 008615) were obtained from Jackson Laboratories. All mice were maintained in a mixed CD1 background. For the lithium chloride (LiCl) treatment study, pregnant mice were injected intraperitoneally with LiCl (6–12 μmol/g body weight) once every 24 h from gestational day 11-14 as described previously (Song et al., 2009). For the DKK inhibitor treatment study, pregnant mice were injected intraperitoneally with IIIC3a (Millipore, 10–25 μg/g body weight) once every 24 h from gestational day 12-14 (Li et al., 2012). All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children's Hospital Medical Center. 2.2. Laser capture microdissection (LCM) and RNA-seq Embryos were microdissected at predetermined stages and embryo heads frozen immediately in Tissue-Tek OCT compound. The samples were cryosectioned and collected on Arcturus PEN membrane glass slides (Applied Biosystems) and the tooth mesenchyme tissues were isolated by laser capture microdissection (LCM) using the Veritas Laser Microdissection instrument model 704. Total RNAs were extracted by using the RNeasy Micro Kit (Qiagen) and cDNA templates were generated from total RNAs extracted from molar mesenchyme tissues of three embryos of the same genotype by using the NuGEN Ovation RNA-Seq v2 system. Sequencing libraries generated by using the Illumina Nextera DNA Sample Prep Kit were sequenced using the Illumina Hiseq 2000, and sequenced reads were mapped to the reference mouse genome (mm9) using the Bowtie version 0.12.7 as described previously (Jia et al., 2013). Tophat version 1.4.1 for Illumina was used to align single-end reads (Brunskill and Potter, 2012). 2.3. RNA-seq data analysis RNA-seq data were analyzed using the StrandNGS software, with the reads per kilobase exon per million mapped sequences (RPKM) value calculated for each RefSeq gene for relative gene expression. For differential expression analyses, the fold change
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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cut-off was set at 2.0-fold or higher for the Msx1 / mutants and or 1.5-fold or higher for the Osr2 / mutants, and P-values o0.01 from the Audic Claverie test were considered to be statistically significant, with Benjamini Hochberg FDR multiple testing correction (Brunskill and Potter, 2012). 2.4. Quantitative real-time RT-PCR Complementary DNAs were generated from the LCM-isolated tissues, which were pooled from 3 embryos of each genotype, and were analyzed using real-time PCR using the C1000 Touch Thermal Cycler (Bio-Rad) and the Advanced SYBR Green Supermix (BioRad), with the conditions recommended by the manufacturer. PCR reactions were carried out in duplicate and relative levels of mRNAs were normalized to Hprt using the standard curve method. Student's t-test was used to analyze pair-wise differential expression, a P-value o0.05 was considered to be statistically significant. 2.5. Histology and in situ hybridization assays Embryos were collected from timed pregnant females, fixed in 4% paraformaldehyde (PFA) at 4 oC overnight, dehydrated through graded ethanol series, embedded in paraffin, and sectioned at 7 mm thickness. For histological analysis, sections were stained with Hematoxylin and Eosin (H-E) as described previously (Chen et al., 2009). Section in situ hybridization was performed as described in Zhang et al. (1999). Specimens were examined and imaged by using the Nikon Eclipse 80i microscope.
3. Results 3.1. Opposing effects of Msx1 and Osr2 on expression of Wnt signaling pathway genes in the developing tooth germ To investigate the molecular mechanism underlying antagonistic actions of Msx1 and Osr2 on early tooth development, we isolated the molar tooth mesenchyme from Msx1 / and Osr2 / mutants and their control littermates by using LCM and analyzed the gene expression profiles by RNA-seq. Direct comparison of the RNA-seq data revealed that 46 genes up-regulated in the Osr2 / mutant tooth mesenchyme were down-regulated in the Msx1 / mutant tooth mesenchyme whereas 42 genes down-regulated in the Osr2 / mutant tooth mesenchyme were up-regulated in the Msx1 / mutant tooth mesenchyme (Fig. 1A). Among these, expression of two genes encoding secreted Wnt antagonsits, Dkk2 and Sfrp2, was significantly up-regulated in the Msx1 / tooth mesenchyme and down-regulated in the Osr2 / tooth mesenchyme. Conversely, expression of two Wnt target genes, Lef1 and Tcf7, was down-regulated in the Msx1 / mutant tooth mesenchyme and up-regulated in the Osr2 / mutant tooth mesenchyme. The opposite effects of Msx1 and Osr2 on expression of these Wnt pathway genes were further confirmed by quantitative real-time RT-PCR assays (Fig. 1B). In addition, RT-qPCR assays showed that expression of Sfrp1 is also significantly up-regulated in Msx1 / mutant tooth mesenchyme and down-regulated in Osr2 / tooth mesenchyme (Fig. 1B). We further compared the expression patterns of these Wnt pathway genes in tooth development in the Osr2 / , Msx1 / , and Bmp4ncko/ncko mutant embryos and their control littermates at E13.5 by using in situ hybridization assays. As shown in Fig. 2, both Dkk2 and Sfrp2 mRNAs were preferentially expressed in the oral mesenchyme lingual to the developing tooth buds but excluded from the condensing tooth mesenchyme surrounding the distal tooth bud epithelium in the control embryos (Fig. 2A, E). In
Fig. 1. Opposing effects of Msx1 and Osr2 on expression of Wnt signaling pathway genes in the E13.5 mandibular molar tooth mesenchyme. (A) Venn diagram comparing genes whose expression was changed by more than 2.0-fold in Msx1 / mutants and 1.5-fold in Osr2 / mutants compared with their wildtype littermates, respectively, in the RNA-seq datasets from the laser capture microdissected E13.5 mandibular molar tooth mesenchyme. (B) Quantitative real-time RT-PCR analysis of the levels of Dkk2, Sfrp1, Sfrp2, Lef1, and Tcf7 mRNAs in the E13.5 mandibular molar tooth mesenchyme of Msx1 / and Osr2 / mutant embryos in comparison with their wildtype littermates. Complementary DNAs were generated from pooled LCM tissue RNA samples from 3 embryos for each genotype. *, p o0.05. Error bars represent S.E.M.
Osr2 / mutant embryos, Dkk2 and Sfrp2 expression was downregulated in the lingual mesenchyme and tooth mesenchyme compared to the controls (Fig. 2B, F). In contrast, in Msx1 / and Bmp4ncko/ncko mutant embryos, expression of the Dkk2 and Sfrp2 expanded into the distal tooth mesenchyme (Fig. 2C, D, G, H). Differences in the expression pattern of Dkk2 and Sfrp2 mRNAs were more apparent in the mandibular than in the maxillary tooth germs. Since previous studies have shown that Msx1-mediated propagation of mesenchymal odontogenic activity drives supernumerary tooth formation lingual to the primary molars in the Osr2 / mice and that inactivation of Osr2 was able to rescue molar tooth morphogenesis in both Msx1 / and Bmp4ncko/ncko mice, these data suggest that Osr2 patterns the molar tooth mesenchyme at least in part through modulating the effects of the Bmp4-Msx1 pathway on the expression of the secreted Wnt antagonists. 3.2. Pharmacological activation of the canonical Wnt signaling rescues mandibular molar tooth morphogenesis in Bmp4ncko/ncko mice The finding that multiple secreted Wnt antagonists were down-regulated in the Osr2 / mutant tooth mesenchyme prompted us to test whether pharmacological activation of canonical Wnt signaling could rescue molar tooth morphogenesis in Bmp4ncko/ncko and Msx1 / mutant mice. We first tested
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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Fig. 2. Comparison of patterns of Dkk2 and Sfrp2 mRNA expression in the developing molar tooth mesenchyme in E13.5 Osr2 / , Msx1 / , Bmp4ncko/ncko mutant and control embryos. (A–D) Frontal sections showing expression patterns of Dkk2 mRNAs in and around the maxillary (upper) and mandibular (lower) molar tooth mesenchyme in the E13.5 control (A), Osr2 / (B), Msx1 / (C), and Bmp4ncko/ncko mutant (D) embryos (n ¼ 3 for each genotype). (E–H) Frontal sections showing expression patterns of Sfrp2 mRNAs in and around the maxillary and mandibular molar tooth mesenchyme in the E13.5 control (A), Osr2 / (B), Msx1 / (C), and Bmp4ncko/ncko mutant (D) embryos. A white line separates the pictures for maxillary and mandibular tooth germs in Panels C and G because the two pictures are taken from distinct sections of a series from the same embryo. Arrow points to the lingual side of the mandibular molar tooth germ. Red dashed line marks the boundary between the tooth bud epithelium and mesenchyme.
intraperitoneal injection of LiCl, a simple salt that is known to activate Wnt signaling through inhibition of GSK3β⋅(Klein and Melton, 1996; Song et al., 2009; Zeilbeck et al., 2014), in the pregnant mice bearing Bmp4ncko/ncko mutant embryos. As control, we treated mice with NaCl, a structurally similar salt but without an effect on GSK3β activity. NaCl treatment did not significantly affect the embryonic tooth developmental processes in control and Bmp4ncko/ncko mutant embryos, with Bmp4ncko/ncko mutant embryos exhibiting bud-stage arrest of the mandibular molar tooth germs (Fig. 3A, B, n ¼ 20). In contrast, LiCl treatment rescued mandibular first molar tooth development in Bmp4ncko/ncko mutant embryos to the cap stage by E16.5 and to the bell stage by E18.5 in 14 out of 30 samples (Fig. 3C–F). However, high doses of LiCl treatment caused lethality of the pregnant mice and the tolerable doses of LiCl treatment did not rescue molar tooth morphogenesis in Msx1 / mutant embryos. Although LiCl is known to activate β-catenin-mediated canonical Wnt signaling through inhibition of GSK3β (Klein and Melton, 1996; Song et al., 2009; Zeilbeck et al., 2014), the effect of LiCl treatment in utero is likely not limited to Wnt signaling since GSK3β has many endogenous substrates, in addition to β-catenin, and affects multiple signaling pathways and transcription factors (for recent reviews, Beurel et al., 2015; Takahashi-Yanaga, 2013). To directly test whether increased expression of secreted Wnt antagonists in the developing tooth mesenchyme is responsible for the mandibular molar developmental arrest in the Bmp4ncko/ncko mutant embryos, we injected IIIC3a, a small molecule that binds to the Wnt co-receptors Lrp5/6 to block inhibition of canonical Wnt signaling by Dkk1 or Dkk2, but itself does not cause activation of Wnt signaling in the absence of Wnt ligands (Li et al., 2012), into the pregnant mice bearing Bmp4ncko/ncko mutant embryos from gestational days 12-14. As control, we treated mice with equal
amount of DMSO used as a solvent in the IIIC3a solution. DMSO treatment did not exhibit any significant affect on the embryonic tooth developmental processes in control and Bmp4ncko/ncko mutant embryos, with Bmp4ncko/ncko mutant embryos showing budstage arrest of the mandibular molar tooth germs (Fig. 4A, B, n¼ 20). Remarkably, in IIIC3a-treated Bmp4ncko/ncko mutant embryos, 8 out of 12 mandibular first molar tooth germs progressed to the bell stage by E18.5 (Fig. 4C, D). We examined whether the IIIC3a treatment rescued mandibular molar morphogenesis in the Bmp4ncko/ncko mutant embryos through restoring Wnt signaling activity in the developing tooth germs. Lef1 and Axin2 are two known direct target genes of canonical Wnt signaling (Filali et al., 2002; Jho et al., 2002). During the bud-to-cap transition in molar tooth development, expression of both Lef1 and Axin2 mRNAs are activated in the distal tooth bud epithelium cells for PEK formation in the control embryos (Fig. 5A, E). In addition, Lef1 mRNAs are strongly expressed in the condensing tooth mesenchyme surrounding the molar tooth bud (Fig. 5A). In Bmp4ncko/ncko mutant embryos without IIIC3a treatment, expression of both Lef1 and Axin2 mRNAs is significantly reduced in the maxillary molar tooth germs and almost completely lost in the mandibular molar buds (Fig. 5B, F, n ¼4). In IIIC3a treated embryos, expression of both Lef1 and Axin2 mRNAs in maxillary and mandibular molar tooth germs is restored in 3 out of 5 Bmp4ncko/ncko mutant embryos to comparable levels as in the control littermates (Fig. 5C, D, G, H). Analysis of PEK formation using both Shh and p21 mRNA expression as markers show that, whereas Bmp4ncko/ncko mutant embryos fail to form PEK in the mandibular molar tooth buds, IIIC3a treatment rescued PEK formation in and bud-to-cap transition of the mandibular tooth germs in 3 of 5 Bmp4ncko/ncko mutant embryos (Fig. 5I–P). These data indicate that mandibular molar developmental arrest in the
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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Fig. 3. LiCl treatment rescues mandibular molar tooth development in Bmp4ncko/ncko embryos. (A–F) H-E-stained frontal sections through the maxillary and mandibular first molar tooth germs in control (A, C, E) and Bmp4ncko/ncko (B, D, F) embryos treated with NaCl (A, B), or LiCl (C-F). Arrowhead points to the mandibular first molar tooth germ in Bmp4ncko/ncko mutant embryos in B, D, and F. n¼ 6 for NaCl-treated control; n¼ 6 for LiCl-treated control; n¼ 20 for NaCl-treated Bmp4ncko/ncko; n¼ 14/30 for LiCl-treated Bmp4ncko/ncko mandibular molar tooth germs.
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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Fig. 4. IIIC3a treatment in utero rescues mandibular molar tooth morphogenesis in Bmp4ncko/ncko mutant embryos. (A–D) H-E-stained frontal sections through the maxillary and mandibular first molar tooth germs in E18.5 control (A, C) and Bmp4ncko/ncko mutant (B, D) embryos treated with DMSO (A, B) or IIIC3a (C, D). Arrowhead points to the mandibular first molar tooth germ in Bmp4ncko/ncko mutant embryos in B and D. n¼ 6 for DMSO or IIIC3a-treated control; n¼ 10 for DMSO-treated Bmp4ncko/ncko; n¼ 8/12 for IIIC3a-treated Bmp4ncko/ncko mandibular molar tooth germs.
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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Fig. 5. IIIC3a treatment in utero rescues Wnt signaling activity in and bud-to-cap transition of mandibular molar tooth germs in Bmp4ncko/ncko mutant embryos. (A-P) Comparison of Lef1 (A-D), Axin2 (E–H), Shh (I-L), and p21 (M-P) mRNA expression, respectively, in the maxillary and mandibular molar tooth germs in E14 control (A, C, E, G, I, K, M, O) and Bmp4ncko/ncko (B, D, F, H, J, L, N, P) embryos without (A, B, E, F, I, J, M, N) or with (C, D, G, H, K, L, O, P) IIIC3a treatment. mRNA signals are detected in blue color. A white line separates the pictures for maxillary and mandibular tooth germs in Panels C, D, G, H, K, L, O, and P because the two pictures are taken from distinct sections of a series from the same embryo. Arrowhead points to the mandibular first molar tooth germ in each panel. Red dashed line marks the boundary between the tooth bud epithelium and mesenchyme.
Bmp4ncko/ncko mutant mice resulted from reduced Wnt signaling activity at least in part due to the increased level of Dkk2 expression in the tooth bud mesenchyme.
3.3. Combination of pharmacological inhibition of DKK and genetic inactivation of Sfrp2/Sfrp3 rescues maxillary molar tooth
morphogenesis in Msx1 / mice Whereas IIIC3a treatment was able to rescue mandibular molar morphogenesis in Bmp4ncko/ncko mice, we did not detect any apparent rescue of tooth morphogenesis in Msx1 / mutant embryos using the same or higher doses of IIIC3a (Fig. 6A–D, n ¼34). Since our RNA-seq and real-time RT-PCR analyses showed that expression of Sfrp1 and Sfrp2 was significantly up-regulated in the
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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Msx1 / mutant molar tooth mesenchyme and expression of both was also significantly reduced in the Osr2 / mutant molar mesenchyme, we examined whether genetic inactivation of Sfrp2 affects molar tooth morphogenesis in Msx1 / mutant embryos. However, we found that Msx1 / Sfrp2 / double mutant mice (n 420) exhibit tooth developmental arrest similar to Msx1 / mice. We further tested whether inactivation of both Sfrp2 and Sfrp3, which is also highly expressed in the tooth bud mesenchyme (Sarkar and Sharpe, 1999), could rescue molar tooth morphogenesis in Msx1 / embryos and found that / / Msx1 Sfrp2 Sfrp3 / compound mutant mice (n 420) still exhibit tooth developmental arrest similar to Msx1 / mice. We then set up timed mating of Msx1 þ / Sfrp2 / Sfrp3 / males and females and treated the pregnant females with IIIC3a from gestational day 12 through 14. We found that 3 out of 10 Msx1 / Sfrp2 / Sfrp3 / embryos treated with IIIC3a showed rescued maxillary molar tooth germs that progressed to the bell stage by E18.5 although their mandiublar molar tooth germs remained arrested at the bud stage (Fig. 6E, F). These data confirm that Msx1 controls tooth morphogenesis at least in part through regulation of expression of the secreted Wnt antagonists in the developing tooth mesenchyme.
4. Discussion Mutations in MSX1 have been associated with craniofacial disorders including tooth agenesis in humans (Vastardis et al., 1996; van den Boogaard et al., 2000; Lidral and Reising, 2002). Although recombinant Bmp4 protein was able to rescue development of Msx1 / mutant mouse molar tooth germs through the bud-to-cap transition in explant culture assays (Chen et al., 1996; Bei et al., 2000), the molecular mechanisms mediating Msx1 and Bmp4 function in tooth development are still incompletely understood. We recently showed that Bmp4ncko/ncko mutant mice exhibit bud stage arrest of only the mandibular molars but their maxillary molars and incisors developed to mineralized teeth (Jia et al., 2013), indicating that other factors in addition to Bmp4 are involved in Msx1-mediated regulation of early tooth organogenesis. In this study, taking advantage of our recent finding that the Osr2 transcription factor patterns the buccolingual axis of the developing molar tooth mesenchyme through antagonizing Msx1 function (Zhang et al., 2009), we identify Dkk2 and Sfrp2 as targets of Msx1- and Osr2-mediated odontogenic regulation. The patterns of Dkk2 and Sfrp2 expression in the tooth bud mesenchyme in wildtype, Osr2 / , Msx1 / , and Bmp4ncko/ncko mutant embryos suggest that the Bmp4-Msx1 pathway normally represses expression of both Dkk2 and Sfrp2, whereas Osr2 antagonizes this repression, in the developing tooth mesenchyme. Our functional assays using a combination of pharmacological inhibition and genetic inactivation demonstrate that these Wnt antagonists indeed participate in and mediate at least part of the functions of Msx1 and Osr2 in the regulation of molar tooth organogenesis. Several studies have shown that Wnt signaling pathway plays critical roles in tooth morphogenesis (van Genderen et al., 1994; Kratochwil et al., 1996; Andl et al., 2002; Liu et al., 2008; Chen et al., 2009). Through comprehensive gene expression profiling studies of instructive signaling interactions between the early developing tooth epithelium and mesenchyme combined with mathematical modeling of the inter-tissue signaling dynamics, O’Connell et al. (2012) proposed that a Wnt-Bmp feedback circuit acts as the major regulator of epithelial-mesenchymal interactions in early tooth organogenesis. A major component of this Wnt-Bmp circuit is Bmp4, which is initially expressed in the dental lamina epithelium and its expression shifts into the developing dental mesenchyme during tooth bud formation and is turned back on in
the primary enamel knot in the dental epithelium by the late bud stage (O’Connell et al., 2012). Remarkably, constitutive activation of Wnt signaling in the oral epithelium by conditionally inactivating the adenomatous polyposis coli (Apc) gene is able to induce formation of supernumerary teeth even in the absence of Msx1 or Pax9 function (Wang et al., 2009; O’Connell et al., 2012). However, the ectopic teeth in the K14-Cre;Apcf/f;Msx1 / mutant mice formed from differentiation of the dental epithelium and underlying mesenchyme without progressing through the sequential bud-to-cap-to-bell tooth germ morphogenetic processes (Wang et al., 2009). In contrast, we showed previously that deletion of Osr2 rescued molar tooth organogenesis, including the normal sequential bud-to-cap-to-bell tooth germ morphogenesis, in the Msx1 / and Bmp4ncko/ncko mutant mice (Zhang et al., 2009; Jia et al., 2013). In this study, we demonstrate for the first time that the Bmp4/Msx1-dependent tooth morphogenesis program could be restored in the absence of Msx1 function by inactivating the mesenchymal Wnt antagonists. The mandibular molar tooth germs in the Bmp4ncko/ncko mutant mice, as well as the maxillary molar tooth germs in the Msx1 / Sfrp2 / Sfrp3 / mutant mice, treated with the DKK inhibitor IIIC3a progressed through the normal sequential tooth morphogenesis to the bell stage by E18.5. Moreover, unlike the K14-Cre;Apcf/f;Msx1 / mutant mice, the IIIC3a-treated mutant mice and their control littermates did not develop ectopic teeth because inhibition of the secreted Wnt antagonists does not cause constitutive activation of Wnt signaling. The transient IIIC3a treatment from E12.5 to E14.5 did not have any obvious effect on development and survival of the control littermates. It is interesting that IIIC3a treatment was unable to restore molar tooth morphogenesis in Msx1 / mutant mice but did rescue maxillary molar morphogenesis in 3 of 10 Msx1 / Sfrp2 / Sfrp3 / mutant mice. This result suggests that, while expression of Dkk2 and Sfrp2 is similarly regulated by Msx1 and Osr2 in the developing tooth mesenchyme, Dkk2 and Sfrp2 may play distinct and complementary roles in regulating tooth morphogenesis. Whereas DKK family members are known to antagonize only the canonical Wnt signaling pathway by binding to Wnt co-receptors Lrp5/6, SFRP family members function as soluble molecules that bind to the secreted Wnt ligands and compete with membrane Frizzled receptors, resulting in regulation of both canonical and non-canonical Wnt signaling (reviewed by Cruciat and Niehrs (2013)). It is possible that both canonical and non-canonical Wnt signaling activities are required for normal sequential progression of bud-to-cap-to-bell stage tooth germ morphogenesis, which could be why mice with constitutive activation of canonical Wnt signaling develop ectopic teeth that do not undergo the normal bud-to-cap-to-bell morphogenesis. Thus, whereas many studies have previously emphasized the role of the β-catenin-dependent canonical Wnt signaling in embryonic tooth development, whether non-canonical Wnt signaling plays a crucial role in tooth morphogenesis requires further investigation. We showed previously that both maxillary and mandibular molar tooth germs progressed to the late bell stage in E18.5 Msx1 / Osr2 / compound mutant embryos (Zhang et al., 2009), but DKK inhibitor IIICa treatment was able to rescue maxillary, but not mandibular, molar morphogenesis in the Msx1 / Sfrp2 / Sfrp3 / mutant embryos. Since we found that expression of Sfrp1 mRNAs in the mandibular molar mesenchyme is also significantly increased in Msx1 / embryos and significantly down-regulated in Osr2 / embryos (Fig. 1B), it is possible that antagonistic regulation of Sfrp1 expression by Msx1and Osr2 is also important during the bud-to-cap transition, particularly for mandibular molar development. Since Sfrp1 and Sfrp2 are known to function redundantly during embryo development and since mouse embryos lacking both Sfrp1 and Sfrp2 die during
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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Fig. 6. IIIC3a treatment in utero rescued maxillary molar tooth organogenesis in Msx1 / Sfrp2 / Sfrp3 / embryos. (A–F) H-E-stained frontal sections through the maxillary and mandibular first molar tooth germs at E18.5 in the control (A, C, E), Msx1 / (B, D), and Msx1 / Sfrp2 / Sfrp3 / mutant (F) embryos treated with DMSO (A, B) or IIIC3a (C–F). A white line separates the pictures for maxillary and mandibular tooth germs in Panel C because the two pictures are taken from distinct sections of a series from the same embryo. Arrowhead points to the mandibular first molar tooth germ and asterisk labels the maxillary first molar tooth mesenchyme in the mutant embryos (B, D, F). n¼ 12 for DMSO-treated control and Msx1 / samples; n¼ 34 for IIIC3a-treated Msx1 / and control samples; n¼ 3/10 for IIIC3a-treated Msx1 / Sfrp2 / Sfrp3 / samples.
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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midgestation with severe craniofacial defects (Satoh et al., 2006), future investigation of the roles of Sfrp1 and Sfrp2 in tooth developmental arrest in Msx1 / mice can be accomplished using conditional gene knockout approaches.
Funding This work was supported by National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (NIDCR) grant R01DE018401 to RJ.
Competing interests The authors declare no competing or financial interests.
Acknowledgements We thank the Gene Expression Microarray Core and the Genetic Variation and Gene Discovery Core facilities at Cincinnati Children's Hospital Medical Center for RNA quality analysis, cDNA amplification, and next-generation sequencing services.
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Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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Developmental Biology journal homepage: www.elsevier.com/locate/developmentalbiology
Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists Shihai Jia a,1,2, Hyuk-Jae Edward Kwon a,2, Yu Lan a,b, Jing Zhou a,1, Han Liu a, Rulang Jiang a,b,n a b
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA Division of Plastic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
art ic l e i nf o
a b s t r a c t
Article history: Received 15 June 2016 Received in revised form 1 October 2016 Accepted 2 October 2016
Mutations in MSX1 cause craniofacial developmental defects, including tooth agenesis, in humans and mice. Previous studies suggest that Msx1 activates Bmp4 expression in the developing tooth mesenchyme to drive early tooth organogenesis. Whereas Msx1 / mice exhibit developmental arrest of all tooth germs at the bud stage, mice with neural crest-specific inactivation of Bmp4 (Bmp4ncko/ncko), which lack Bmp4 expression in the developing tooth mesenchyme, showed developmental arrest of only mandibular molars. We recently demonstrated that deletion of Osr2, which encodes a zinc finger transcription factor expressed in a lingual-to-buccal gradient in the developing tooth bud mesenchyme, rescued molar tooth morphogenesis in both Msx1 / and Bmp4ncko/ncko mice. In this study, through RNA-seq analyses of the developing tooth mesenchyme in mutant and wildtype embryos, we found that Msx1 and Osr2 have opposite effects on expression of several secreted Wnt antagonists in the tooth bud mesenchyme. Remarkably, both Dkk2 and Sfrp2 exhibit Osr2-dependent preferential expression on the lingual side of the tooth bud mesenchyme and expression of both genes was up-regulated and expanded into the tooth bud mesenchyme in Msx1 / and Bmp4ncko/ncko mutant embryos. We show that pharmacological activation of canonical Wnt signaling by either lithium chloride (LiCl) treatment or by inhibition of DKKs in utero was sufficient to rescue mandibular molar tooth morphogenesis in Bmp4ncko/ncko mice. Furthermore, whereas inhibition of DKKs or inactivation of Sfrp2 alone was insufficient to rescue tooth morphogenesis in Msx1 / mice, pharmacological inhibition of DKKs in combination with genetic inactivation of Sfrp2 and Sfrp3 rescued maxillary molar morphogenesis in Msx1 / mice. Together, these data reveal a novel mechanism that the Bmp4-Msx1 pathway and Osr2 control tooth organogenesis through antagonistic regulation of expression of secreted Wnt antagonists. & 2016 Elsevier Inc. All rights reserved.
Keywords: Bmp4 Msx1 Dkk2 Osr2 Sfrp2 Wnt signaling Tooth development Organogenesis Mouse
1. Introduction Tooth development is controlled by sequential and reciprocal signaling interactions between the epithelium and mesenchyme and has provided an excellent model system for understanding the molecular mechanisms regulating mammalian organogenesis (Jernvall and Thesleff, 2000). Embryological studies using tissue recombination assays have demonstrated that the tooth inductive signals initially arise in the early embryonic oral epithelium, which n Corresponding author at: Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. E-mail address: [email protected] (R. Jiang). 1 Present address: University of Utah School of Dentistry, Salt Lake City, UT 84112, USA. 2 Shihai Jia and Hyuk-Jae Edward Kwon contributed equally to this work as cofirst authors.
thickens to form dental placodes along the prospective dental arch and invaginates into the underlying neural crest-derived mesenchyme to form the early tooth buds (Lumsden, 1988; Mina and Kollar, 1987; Jernvall and Thesleff, 2000). At the early bud stage, the odontogenic potential shifts to the tooth mesenchyme such that the mesenchyme from the bud and later stages of tooth germs could induce tooth organogenesis when recombined with embryonic non-dental epithelium (Kollar and Baird, 1970a, 1970b; Ruch et al., 1973). As tooth development proceeds, an epithelial signaling center, termed the primary enamel knot (PEK), forms in the distal region of the tooth bud and produces many signaling molecules, including members of the Bmp, Fgf, and Wnt families and Shh (reviewed by Jernvall and Thesleff (2000), Tucker and Sharpe (2004), Zhang et al. (2005), Lan et al. (2014)). The PEKderived signals act upon both the dental epithelium and mesenchyme and continued epithelial-mesenchymal signaling
http://dx.doi.org/10.1016/j.ydbio.2016.10.001 0012-1606/& 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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interactions drive tooth morphogenesis through the subsequent “cap” and “bell” stages (Jernvall and Thesleff, 2000; Thesleff, 2003; Tucker and Sharpe, 2004; Lan et al., 2014). The Wnt signaling pathway plays critical roles in tooth bud initiation and morphogenesis. The canonical Wnt signaling pathway involves stabilization and nuclear accumulation of β-catenin. In the absence of Wnt signaling, cytoplasmic β-catenin is phosphorylated by the serine/threonine kinase GSK-3β and targeted for degradation by the ubiquitination-proteosome pathway, whereas activation of Wnt signaling inhibits GSK3β activity, leading to stabilization of β-catenin and its accumulation in the cellular nuclei where it interacts with and converts the TCF/Lef family DNAbinding proteins from transcriptional repressors to activators (reviewed by Clevers and Nusse (2012)). Mice lacking Lef1 and mice with tissue-specific inactivation of β-catenin in either the epithelium or tooth mesenchyme all exhibit tooth developmental arrest at the bud stage (van Genderen et al., 1994; Kratochwil et al., 1996; Andl et al., 2002; Liu et al., 2008; Chen et al., 2009). A recent comprehensive gene expression profiling analysis of the developing mandibular molar tooth epithelium and mesenchyme tissues, and of their responses to activation of Bmp, Fgf, Shh, and Wnt signaling pathways in explant cultures, led to the identification of a Wnt-Bmp feedback circuit as the major regulator of epithelialmesenchymal intertissue signaling interactions in early tooth organogenesis (O’Connell et al., 2012). A major feature of this WntBmp circuit is its asymmetric configuration, with cross-regulation of canonical Wnt and Bmp4 expression in the dental epithelium and joint regulation of Bmp4 expression by both the Bmp and Wnt pathways in the tooth mesenchyme (O’Connell et al., 2012). The molecular mechanisms mediating the cross-regulation between the Wnt and Bmp pathways in tooth development, however, remain to be elucidated. Bmp4 exhibits an expression pattern that coincides with the odontogenic potential, shifting from the epithelium to the underlying mesenchyme during early tooth bud formation, and exogenous Bmp4 protein was able to induce expression of endogenous Bmp4 and Msx1, which encodes a homeodomain-containing DNA-binding transcription factor critical for early tooth development, in mandibular mesenchyme explants (Chen et al., 1996; Satokata and Maas, 1994; Tucker et al., 1998; Vainio et al., 1993). Mice deficient in Msx1 exhibit tooth developmental arrest at the bud stage, with reduced Bmp4 mRNA expression in the dental mesenchyme by E13.5 (Chen et al., 1996; Satokata and Mass, 1994; Tucker et al., 1998). Remarkably, addition of exogenous Bmp4 protein to tooth germ explant cultures was able to rescue development of the Msx1 / mutant tooth germs to the late bell stage (Bei et al., 2000; Chen et al., 1996). These data led to the conclusion that Bmp4 induces its own expression in the early tooth mesenchyme through Msx1-mediated activation and acts downstream of Msx1 to drive tooth morphogenesis through the bud-to-cap transition (Bei et al., 2000; Miletich et al., 2011; O’Connell et al., 2012). However, transgenic expression of Bmp4 mRNAs driven by an Msx1 gene promoter only partially rescued maxillary molar tooth germs to the early cap stage, but not further morphogenesis, in Msx1 / mice (Zhao et al., 2000). Furthermore, in contrast to Msx1 / mice, mice with neural crest-specific inactivation of Bmp4 (Bmp4ncko/ncko), which lack functional Bmp4 in the developing tooth mesenchyme, exhibit bud-stage developmental arrest of only the mandibular molars while maxillary molars and incisors developed to mineralized teeth (Jia et al., 2013). These results indicate that there are additional as yet unresolved molecular mechanisms acting together with Bmp4 to mediate Msx1 function in tooth development. We have shown previously that the Osr2 transcription factor antagonizes Msx1 function to pattern molar tooth formation in a single row (Zhang et al., 2009). Osr2 is expressed in a lingual-to-
buccal gradient, mirroring that of Bmp4 mRNA expression, in the developing tooth bud mesenchyme. Osr2 / mice develop supernumerary teeth lingual to their molar teeth, accompanied by expanded expression of Bmp4 mRNAs into the lingual mesenchyme (Zhang et al., 2009). Remarkably, inactivation of Osr2 rescued molar tooth morphogenesis in both Msx1 / and Bmp4ncko/ncko mutant mice (Jia et al., 2013; Zhang et al., 2009). To further elucidate the molecular mechanism underlying the antagonistic regulation of tooth development by Msx1 and Osr2, we carried out RNA-seq analyses of the developing tooth mesenchyme in Msx1 / and Osr2 / mutant embryos and their control littermates. We found that Msx1 and Osr2 exert opposite effects on the expression of several secreted Wnt antagonists in the tooth bud mesenchyme. Through a combination of genetic inactivation and pharmacological inhibition studies, we demonstrate that the Bmp4-Msx1 pathway drives early tooth morphogenesis through regulation of expression of secreted Wnt antagonists, in particular Dkk2 and Sfrp2, in the developing tooth mesenchyme.
2. Materials and methods 2.1. Animals The Bmp4f/f, Msx1 þ / , Osr2 þ / , Sfrp2 / , and Wnt1Cre transgenic mice and generation of Bmp4f/f;Wnt1Cre (Bmp4ncko/ncko) compound mutant mice have been described previously (Satokata and Maas, 1994; Chai et al., 2000; Lan et al., 2004; Liu et al., 2008; Morello et al., 2008; Jia et al., 2013). Sfrp3 / (Frzb / ) mice (JAX 008615) were obtained from Jackson Laboratories. All mice were maintained in a mixed CD1 background. For the lithium chloride (LiCl) treatment study, pregnant mice were injected intraperitoneally with LiCl (6–12 μmol/g body weight) once every 24 h from gestational day 11-14 as described previously (Song et al., 2009). For the DKK inhibitor treatment study, pregnant mice were injected intraperitoneally with IIIC3a (Millipore, 10–25 μg/g body weight) once every 24 h from gestational day 12-14 (Li et al., 2012). All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children's Hospital Medical Center. 2.2. Laser capture microdissection (LCM) and RNA-seq Embryos were microdissected at predetermined stages and embryo heads frozen immediately in Tissue-Tek OCT compound. The samples were cryosectioned and collected on Arcturus PEN membrane glass slides (Applied Biosystems) and the tooth mesenchyme tissues were isolated by laser capture microdissection (LCM) using the Veritas Laser Microdissection instrument model 704. Total RNAs were extracted by using the RNeasy Micro Kit (Qiagen) and cDNA templates were generated from total RNAs extracted from molar mesenchyme tissues of three embryos of the same genotype by using the NuGEN Ovation RNA-Seq v2 system. Sequencing libraries generated by using the Illumina Nextera DNA Sample Prep Kit were sequenced using the Illumina Hiseq 2000, and sequenced reads were mapped to the reference mouse genome (mm9) using the Bowtie version 0.12.7 as described previously (Jia et al., 2013). Tophat version 1.4.1 for Illumina was used to align single-end reads (Brunskill and Potter, 2012). 2.3. RNA-seq data analysis RNA-seq data were analyzed using the StrandNGS software, with the reads per kilobase exon per million mapped sequences (RPKM) value calculated for each RefSeq gene for relative gene expression. For differential expression analyses, the fold change
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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cut-off was set at 2.0-fold or higher for the Msx1 / mutants and or 1.5-fold or higher for the Osr2 / mutants, and P-values o0.01 from the Audic Claverie test were considered to be statistically significant, with Benjamini Hochberg FDR multiple testing correction (Brunskill and Potter, 2012). 2.4. Quantitative real-time RT-PCR Complementary DNAs were generated from the LCM-isolated tissues, which were pooled from 3 embryos of each genotype, and were analyzed using real-time PCR using the C1000 Touch Thermal Cycler (Bio-Rad) and the Advanced SYBR Green Supermix (BioRad), with the conditions recommended by the manufacturer. PCR reactions were carried out in duplicate and relative levels of mRNAs were normalized to Hprt using the standard curve method. Student's t-test was used to analyze pair-wise differential expression, a P-value o0.05 was considered to be statistically significant. 2.5. Histology and in situ hybridization assays Embryos were collected from timed pregnant females, fixed in 4% paraformaldehyde (PFA) at 4 oC overnight, dehydrated through graded ethanol series, embedded in paraffin, and sectioned at 7 mm thickness. For histological analysis, sections were stained with Hematoxylin and Eosin (H-E) as described previously (Chen et al., 2009). Section in situ hybridization was performed as described in Zhang et al. (1999). Specimens were examined and imaged by using the Nikon Eclipse 80i microscope.
3. Results 3.1. Opposing effects of Msx1 and Osr2 on expression of Wnt signaling pathway genes in the developing tooth germ To investigate the molecular mechanism underlying antagonistic actions of Msx1 and Osr2 on early tooth development, we isolated the molar tooth mesenchyme from Msx1 / and Osr2 / mutants and their control littermates by using LCM and analyzed the gene expression profiles by RNA-seq. Direct comparison of the RNA-seq data revealed that 46 genes up-regulated in the Osr2 / mutant tooth mesenchyme were down-regulated in the Msx1 / mutant tooth mesenchyme whereas 42 genes down-regulated in the Osr2 / mutant tooth mesenchyme were up-regulated in the Msx1 / mutant tooth mesenchyme (Fig. 1A). Among these, expression of two genes encoding secreted Wnt antagonsits, Dkk2 and Sfrp2, was significantly up-regulated in the Msx1 / tooth mesenchyme and down-regulated in the Osr2 / tooth mesenchyme. Conversely, expression of two Wnt target genes, Lef1 and Tcf7, was down-regulated in the Msx1 / mutant tooth mesenchyme and up-regulated in the Osr2 / mutant tooth mesenchyme. The opposite effects of Msx1 and Osr2 on expression of these Wnt pathway genes were further confirmed by quantitative real-time RT-PCR assays (Fig. 1B). In addition, RT-qPCR assays showed that expression of Sfrp1 is also significantly up-regulated in Msx1 / mutant tooth mesenchyme and down-regulated in Osr2 / tooth mesenchyme (Fig. 1B). We further compared the expression patterns of these Wnt pathway genes in tooth development in the Osr2 / , Msx1 / , and Bmp4ncko/ncko mutant embryos and their control littermates at E13.5 by using in situ hybridization assays. As shown in Fig. 2, both Dkk2 and Sfrp2 mRNAs were preferentially expressed in the oral mesenchyme lingual to the developing tooth buds but excluded from the condensing tooth mesenchyme surrounding the distal tooth bud epithelium in the control embryos (Fig. 2A, E). In
Fig. 1. Opposing effects of Msx1 and Osr2 on expression of Wnt signaling pathway genes in the E13.5 mandibular molar tooth mesenchyme. (A) Venn diagram comparing genes whose expression was changed by more than 2.0-fold in Msx1 / mutants and 1.5-fold in Osr2 / mutants compared with their wildtype littermates, respectively, in the RNA-seq datasets from the laser capture microdissected E13.5 mandibular molar tooth mesenchyme. (B) Quantitative real-time RT-PCR analysis of the levels of Dkk2, Sfrp1, Sfrp2, Lef1, and Tcf7 mRNAs in the E13.5 mandibular molar tooth mesenchyme of Msx1 / and Osr2 / mutant embryos in comparison with their wildtype littermates. Complementary DNAs were generated from pooled LCM tissue RNA samples from 3 embryos for each genotype. *, p o0.05. Error bars represent S.E.M.
Osr2 / mutant embryos, Dkk2 and Sfrp2 expression was downregulated in the lingual mesenchyme and tooth mesenchyme compared to the controls (Fig. 2B, F). In contrast, in Msx1 / and Bmp4ncko/ncko mutant embryos, expression of the Dkk2 and Sfrp2 expanded into the distal tooth mesenchyme (Fig. 2C, D, G, H). Differences in the expression pattern of Dkk2 and Sfrp2 mRNAs were more apparent in the mandibular than in the maxillary tooth germs. Since previous studies have shown that Msx1-mediated propagation of mesenchymal odontogenic activity drives supernumerary tooth formation lingual to the primary molars in the Osr2 / mice and that inactivation of Osr2 was able to rescue molar tooth morphogenesis in both Msx1 / and Bmp4ncko/ncko mice, these data suggest that Osr2 patterns the molar tooth mesenchyme at least in part through modulating the effects of the Bmp4-Msx1 pathway on the expression of the secreted Wnt antagonists. 3.2. Pharmacological activation of the canonical Wnt signaling rescues mandibular molar tooth morphogenesis in Bmp4ncko/ncko mice The finding that multiple secreted Wnt antagonists were down-regulated in the Osr2 / mutant tooth mesenchyme prompted us to test whether pharmacological activation of canonical Wnt signaling could rescue molar tooth morphogenesis in Bmp4ncko/ncko and Msx1 / mutant mice. We first tested
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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Fig. 2. Comparison of patterns of Dkk2 and Sfrp2 mRNA expression in the developing molar tooth mesenchyme in E13.5 Osr2 / , Msx1 / , Bmp4ncko/ncko mutant and control embryos. (A–D) Frontal sections showing expression patterns of Dkk2 mRNAs in and around the maxillary (upper) and mandibular (lower) molar tooth mesenchyme in the E13.5 control (A), Osr2 / (B), Msx1 / (C), and Bmp4ncko/ncko mutant (D) embryos (n ¼ 3 for each genotype). (E–H) Frontal sections showing expression patterns of Sfrp2 mRNAs in and around the maxillary and mandibular molar tooth mesenchyme in the E13.5 control (A), Osr2 / (B), Msx1 / (C), and Bmp4ncko/ncko mutant (D) embryos. A white line separates the pictures for maxillary and mandibular tooth germs in Panels C and G because the two pictures are taken from distinct sections of a series from the same embryo. Arrow points to the lingual side of the mandibular molar tooth germ. Red dashed line marks the boundary between the tooth bud epithelium and mesenchyme.
intraperitoneal injection of LiCl, a simple salt that is known to activate Wnt signaling through inhibition of GSK3β⋅(Klein and Melton, 1996; Song et al., 2009; Zeilbeck et al., 2014), in the pregnant mice bearing Bmp4ncko/ncko mutant embryos. As control, we treated mice with NaCl, a structurally similar salt but without an effect on GSK3β activity. NaCl treatment did not significantly affect the embryonic tooth developmental processes in control and Bmp4ncko/ncko mutant embryos, with Bmp4ncko/ncko mutant embryos exhibiting bud-stage arrest of the mandibular molar tooth germs (Fig. 3A, B, n ¼ 20). In contrast, LiCl treatment rescued mandibular first molar tooth development in Bmp4ncko/ncko mutant embryos to the cap stage by E16.5 and to the bell stage by E18.5 in 14 out of 30 samples (Fig. 3C–F). However, high doses of LiCl treatment caused lethality of the pregnant mice and the tolerable doses of LiCl treatment did not rescue molar tooth morphogenesis in Msx1 / mutant embryos. Although LiCl is known to activate β-catenin-mediated canonical Wnt signaling through inhibition of GSK3β (Klein and Melton, 1996; Song et al., 2009; Zeilbeck et al., 2014), the effect of LiCl treatment in utero is likely not limited to Wnt signaling since GSK3β has many endogenous substrates, in addition to β-catenin, and affects multiple signaling pathways and transcription factors (for recent reviews, Beurel et al., 2015; Takahashi-Yanaga, 2013). To directly test whether increased expression of secreted Wnt antagonists in the developing tooth mesenchyme is responsible for the mandibular molar developmental arrest in the Bmp4ncko/ncko mutant embryos, we injected IIIC3a, a small molecule that binds to the Wnt co-receptors Lrp5/6 to block inhibition of canonical Wnt signaling by Dkk1 or Dkk2, but itself does not cause activation of Wnt signaling in the absence of Wnt ligands (Li et al., 2012), into the pregnant mice bearing Bmp4ncko/ncko mutant embryos from gestational days 12-14. As control, we treated mice with equal
amount of DMSO used as a solvent in the IIIC3a solution. DMSO treatment did not exhibit any significant affect on the embryonic tooth developmental processes in control and Bmp4ncko/ncko mutant embryos, with Bmp4ncko/ncko mutant embryos showing budstage arrest of the mandibular molar tooth germs (Fig. 4A, B, n¼ 20). Remarkably, in IIIC3a-treated Bmp4ncko/ncko mutant embryos, 8 out of 12 mandibular first molar tooth germs progressed to the bell stage by E18.5 (Fig. 4C, D). We examined whether the IIIC3a treatment rescued mandibular molar morphogenesis in the Bmp4ncko/ncko mutant embryos through restoring Wnt signaling activity in the developing tooth germs. Lef1 and Axin2 are two known direct target genes of canonical Wnt signaling (Filali et al., 2002; Jho et al., 2002). During the bud-to-cap transition in molar tooth development, expression of both Lef1 and Axin2 mRNAs are activated in the distal tooth bud epithelium cells for PEK formation in the control embryos (Fig. 5A, E). In addition, Lef1 mRNAs are strongly expressed in the condensing tooth mesenchyme surrounding the molar tooth bud (Fig. 5A). In Bmp4ncko/ncko mutant embryos without IIIC3a treatment, expression of both Lef1 and Axin2 mRNAs is significantly reduced in the maxillary molar tooth germs and almost completely lost in the mandibular molar buds (Fig. 5B, F, n ¼4). In IIIC3a treated embryos, expression of both Lef1 and Axin2 mRNAs in maxillary and mandibular molar tooth germs is restored in 3 out of 5 Bmp4ncko/ncko mutant embryos to comparable levels as in the control littermates (Fig. 5C, D, G, H). Analysis of PEK formation using both Shh and p21 mRNA expression as markers show that, whereas Bmp4ncko/ncko mutant embryos fail to form PEK in the mandibular molar tooth buds, IIIC3a treatment rescued PEK formation in and bud-to-cap transition of the mandibular tooth germs in 3 of 5 Bmp4ncko/ncko mutant embryos (Fig. 5I–P). These data indicate that mandibular molar developmental arrest in the
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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Fig. 3. LiCl treatment rescues mandibular molar tooth development in Bmp4ncko/ncko embryos. (A–F) H-E-stained frontal sections through the maxillary and mandibular first molar tooth germs in control (A, C, E) and Bmp4ncko/ncko (B, D, F) embryos treated with NaCl (A, B), or LiCl (C-F). Arrowhead points to the mandibular first molar tooth germ in Bmp4ncko/ncko mutant embryos in B, D, and F. n¼ 6 for NaCl-treated control; n¼ 6 for LiCl-treated control; n¼ 20 for NaCl-treated Bmp4ncko/ncko; n¼ 14/30 for LiCl-treated Bmp4ncko/ncko mandibular molar tooth germs.
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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Fig. 4. IIIC3a treatment in utero rescues mandibular molar tooth morphogenesis in Bmp4ncko/ncko mutant embryos. (A–D) H-E-stained frontal sections through the maxillary and mandibular first molar tooth germs in E18.5 control (A, C) and Bmp4ncko/ncko mutant (B, D) embryos treated with DMSO (A, B) or IIIC3a (C, D). Arrowhead points to the mandibular first molar tooth germ in Bmp4ncko/ncko mutant embryos in B and D. n¼ 6 for DMSO or IIIC3a-treated control; n¼ 10 for DMSO-treated Bmp4ncko/ncko; n¼ 8/12 for IIIC3a-treated Bmp4ncko/ncko mandibular molar tooth germs.
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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Fig. 5. IIIC3a treatment in utero rescues Wnt signaling activity in and bud-to-cap transition of mandibular molar tooth germs in Bmp4ncko/ncko mutant embryos. (A-P) Comparison of Lef1 (A-D), Axin2 (E–H), Shh (I-L), and p21 (M-P) mRNA expression, respectively, in the maxillary and mandibular molar tooth germs in E14 control (A, C, E, G, I, K, M, O) and Bmp4ncko/ncko (B, D, F, H, J, L, N, P) embryos without (A, B, E, F, I, J, M, N) or with (C, D, G, H, K, L, O, P) IIIC3a treatment. mRNA signals are detected in blue color. A white line separates the pictures for maxillary and mandibular tooth germs in Panels C, D, G, H, K, L, O, and P because the two pictures are taken from distinct sections of a series from the same embryo. Arrowhead points to the mandibular first molar tooth germ in each panel. Red dashed line marks the boundary between the tooth bud epithelium and mesenchyme.
Bmp4ncko/ncko mutant mice resulted from reduced Wnt signaling activity at least in part due to the increased level of Dkk2 expression in the tooth bud mesenchyme.
3.3. Combination of pharmacological inhibition of DKK and genetic inactivation of Sfrp2/Sfrp3 rescues maxillary molar tooth
morphogenesis in Msx1 / mice Whereas IIIC3a treatment was able to rescue mandibular molar morphogenesis in Bmp4ncko/ncko mice, we did not detect any apparent rescue of tooth morphogenesis in Msx1 / mutant embryos using the same or higher doses of IIIC3a (Fig. 6A–D, n ¼34). Since our RNA-seq and real-time RT-PCR analyses showed that expression of Sfrp1 and Sfrp2 was significantly up-regulated in the
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Msx1 / mutant molar tooth mesenchyme and expression of both was also significantly reduced in the Osr2 / mutant molar mesenchyme, we examined whether genetic inactivation of Sfrp2 affects molar tooth morphogenesis in Msx1 / mutant embryos. However, we found that Msx1 / Sfrp2 / double mutant mice (n 420) exhibit tooth developmental arrest similar to Msx1 / mice. We further tested whether inactivation of both Sfrp2 and Sfrp3, which is also highly expressed in the tooth bud mesenchyme (Sarkar and Sharpe, 1999), could rescue molar tooth morphogenesis in Msx1 / embryos and found that / / Msx1 Sfrp2 Sfrp3 / compound mutant mice (n 420) still exhibit tooth developmental arrest similar to Msx1 / mice. We then set up timed mating of Msx1 þ / Sfrp2 / Sfrp3 / males and females and treated the pregnant females with IIIC3a from gestational day 12 through 14. We found that 3 out of 10 Msx1 / Sfrp2 / Sfrp3 / embryos treated with IIIC3a showed rescued maxillary molar tooth germs that progressed to the bell stage by E18.5 although their mandiublar molar tooth germs remained arrested at the bud stage (Fig. 6E, F). These data confirm that Msx1 controls tooth morphogenesis at least in part through regulation of expression of the secreted Wnt antagonists in the developing tooth mesenchyme.
4. Discussion Mutations in MSX1 have been associated with craniofacial disorders including tooth agenesis in humans (Vastardis et al., 1996; van den Boogaard et al., 2000; Lidral and Reising, 2002). Although recombinant Bmp4 protein was able to rescue development of Msx1 / mutant mouse molar tooth germs through the bud-to-cap transition in explant culture assays (Chen et al., 1996; Bei et al., 2000), the molecular mechanisms mediating Msx1 and Bmp4 function in tooth development are still incompletely understood. We recently showed that Bmp4ncko/ncko mutant mice exhibit bud stage arrest of only the mandibular molars but their maxillary molars and incisors developed to mineralized teeth (Jia et al., 2013), indicating that other factors in addition to Bmp4 are involved in Msx1-mediated regulation of early tooth organogenesis. In this study, taking advantage of our recent finding that the Osr2 transcription factor patterns the buccolingual axis of the developing molar tooth mesenchyme through antagonizing Msx1 function (Zhang et al., 2009), we identify Dkk2 and Sfrp2 as targets of Msx1- and Osr2-mediated odontogenic regulation. The patterns of Dkk2 and Sfrp2 expression in the tooth bud mesenchyme in wildtype, Osr2 / , Msx1 / , and Bmp4ncko/ncko mutant embryos suggest that the Bmp4-Msx1 pathway normally represses expression of both Dkk2 and Sfrp2, whereas Osr2 antagonizes this repression, in the developing tooth mesenchyme. Our functional assays using a combination of pharmacological inhibition and genetic inactivation demonstrate that these Wnt antagonists indeed participate in and mediate at least part of the functions of Msx1 and Osr2 in the regulation of molar tooth organogenesis. Several studies have shown that Wnt signaling pathway plays critical roles in tooth morphogenesis (van Genderen et al., 1994; Kratochwil et al., 1996; Andl et al., 2002; Liu et al., 2008; Chen et al., 2009). Through comprehensive gene expression profiling studies of instructive signaling interactions between the early developing tooth epithelium and mesenchyme combined with mathematical modeling of the inter-tissue signaling dynamics, O’Connell et al. (2012) proposed that a Wnt-Bmp feedback circuit acts as the major regulator of epithelial-mesenchymal interactions in early tooth organogenesis. A major component of this Wnt-Bmp circuit is Bmp4, which is initially expressed in the dental lamina epithelium and its expression shifts into the developing dental mesenchyme during tooth bud formation and is turned back on in
the primary enamel knot in the dental epithelium by the late bud stage (O’Connell et al., 2012). Remarkably, constitutive activation of Wnt signaling in the oral epithelium by conditionally inactivating the adenomatous polyposis coli (Apc) gene is able to induce formation of supernumerary teeth even in the absence of Msx1 or Pax9 function (Wang et al., 2009; O’Connell et al., 2012). However, the ectopic teeth in the K14-Cre;Apcf/f;Msx1 / mutant mice formed from differentiation of the dental epithelium and underlying mesenchyme without progressing through the sequential bud-to-cap-to-bell tooth germ morphogenetic processes (Wang et al., 2009). In contrast, we showed previously that deletion of Osr2 rescued molar tooth organogenesis, including the normal sequential bud-to-cap-to-bell tooth germ morphogenesis, in the Msx1 / and Bmp4ncko/ncko mutant mice (Zhang et al., 2009; Jia et al., 2013). In this study, we demonstrate for the first time that the Bmp4/Msx1-dependent tooth morphogenesis program could be restored in the absence of Msx1 function by inactivating the mesenchymal Wnt antagonists. The mandibular molar tooth germs in the Bmp4ncko/ncko mutant mice, as well as the maxillary molar tooth germs in the Msx1 / Sfrp2 / Sfrp3 / mutant mice, treated with the DKK inhibitor IIIC3a progressed through the normal sequential tooth morphogenesis to the bell stage by E18.5. Moreover, unlike the K14-Cre;Apcf/f;Msx1 / mutant mice, the IIIC3a-treated mutant mice and their control littermates did not develop ectopic teeth because inhibition of the secreted Wnt antagonists does not cause constitutive activation of Wnt signaling. The transient IIIC3a treatment from E12.5 to E14.5 did not have any obvious effect on development and survival of the control littermates. It is interesting that IIIC3a treatment was unable to restore molar tooth morphogenesis in Msx1 / mutant mice but did rescue maxillary molar morphogenesis in 3 of 10 Msx1 / Sfrp2 / Sfrp3 / mutant mice. This result suggests that, while expression of Dkk2 and Sfrp2 is similarly regulated by Msx1 and Osr2 in the developing tooth mesenchyme, Dkk2 and Sfrp2 may play distinct and complementary roles in regulating tooth morphogenesis. Whereas DKK family members are known to antagonize only the canonical Wnt signaling pathway by binding to Wnt co-receptors Lrp5/6, SFRP family members function as soluble molecules that bind to the secreted Wnt ligands and compete with membrane Frizzled receptors, resulting in regulation of both canonical and non-canonical Wnt signaling (reviewed by Cruciat and Niehrs (2013)). It is possible that both canonical and non-canonical Wnt signaling activities are required for normal sequential progression of bud-to-cap-to-bell stage tooth germ morphogenesis, which could be why mice with constitutive activation of canonical Wnt signaling develop ectopic teeth that do not undergo the normal bud-to-cap-to-bell morphogenesis. Thus, whereas many studies have previously emphasized the role of the β-catenin-dependent canonical Wnt signaling in embryonic tooth development, whether non-canonical Wnt signaling plays a crucial role in tooth morphogenesis requires further investigation. We showed previously that both maxillary and mandibular molar tooth germs progressed to the late bell stage in E18.5 Msx1 / Osr2 / compound mutant embryos (Zhang et al., 2009), but DKK inhibitor IIICa treatment was able to rescue maxillary, but not mandibular, molar morphogenesis in the Msx1 / Sfrp2 / Sfrp3 / mutant embryos. Since we found that expression of Sfrp1 mRNAs in the mandibular molar mesenchyme is also significantly increased in Msx1 / embryos and significantly down-regulated in Osr2 / embryos (Fig. 1B), it is possible that antagonistic regulation of Sfrp1 expression by Msx1and Osr2 is also important during the bud-to-cap transition, particularly for mandibular molar development. Since Sfrp1 and Sfrp2 are known to function redundantly during embryo development and since mouse embryos lacking both Sfrp1 and Sfrp2 die during
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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Fig. 6. IIIC3a treatment in utero rescued maxillary molar tooth organogenesis in Msx1 / Sfrp2 / Sfrp3 / embryos. (A–F) H-E-stained frontal sections through the maxillary and mandibular first molar tooth germs at E18.5 in the control (A, C, E), Msx1 / (B, D), and Msx1 / Sfrp2 / Sfrp3 / mutant (F) embryos treated with DMSO (A, B) or IIIC3a (C–F). A white line separates the pictures for maxillary and mandibular tooth germs in Panel C because the two pictures are taken from distinct sections of a series from the same embryo. Arrowhead points to the mandibular first molar tooth germ and asterisk labels the maxillary first molar tooth mesenchyme in the mutant embryos (B, D, F). n¼ 12 for DMSO-treated control and Msx1 / samples; n¼ 34 for IIIC3a-treated Msx1 / and control samples; n¼ 3/10 for IIIC3a-treated Msx1 / Sfrp2 / Sfrp3 / samples.
Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i
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midgestation with severe craniofacial defects (Satoh et al., 2006), future investigation of the roles of Sfrp1 and Sfrp2 in tooth developmental arrest in Msx1 / mice can be accomplished using conditional gene knockout approaches.
Funding This work was supported by National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (NIDCR) grant R01DE018401 to RJ.
Competing interests The authors declare no competing or financial interests.
Acknowledgements We thank the Gene Expression Microarray Core and the Genetic Variation and Gene Discovery Core facilities at Cincinnati Children's Hospital Medical Center for RNA quality analysis, cDNA amplification, and next-generation sequencing services.
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Please cite this article as: Jia, S., et al., Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists. Dev. Biol. (2016), http://dx.doi.org/10.1016/j.ydbio.2016.10.001i