1 Department of Craniofacial Development, GKT Dental Institute, Kings College London, Floor 28 Guys Hospital, London Bridge, London SE1 9RT, UK
2 Department of Oral and Maxillofacial Surgery, Institute of Dentistry, University of Turku, FIN-20520 Turku, Finland
3 Cardiovascular Research Centre, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, Charlestown, MA 02129, USA
* Present address: MRC Centre for Developmental Neurobiology, 4th Floor, New Hunts House, Guys Campus, Kings College London, London Bridge, London SE1 1UL, UK
Author for correspondence (e-mail: paul.sharpe{at}kcl.ac.uk)
Accepted August 21, 2001
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SUMMARY |
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Key words: Activin, Activin receptors, Smad2, Irx1, Tooth development, Mouse
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INTRODUCTION |
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Despite a wealth of evidence implicating activins in mesoderm formation during gastrulation, targeted inactivation of either the activin ßA or ßB genes or both in mice does not affect mesoderm formation but does have profound effects on craniofacial development (Matzuk et al., 1995b). The most striking aspect of the craniofacial phenotype is in the activin ßA mutant mice, where development of teeth is differentially affected (Ferguson et al., 1998). Incisors and mandibular molar teeth fail to develop beyond a rudimentary bud in activin ßA mutant mice, whereas maxillary molar teeth develop normally. Expression of activin ßA is first detected at E10.5 in the pre-odontogenic mesenchyme at the sites of future tooth formation. It subsequently becomes expressed in condensing mesenchyme at the bud stage. The expression of activin ßA during early tooth development is the same in all tooth types, yet maxillary molar teeth are unaffected in the absence of activin. Activin is required before E11.5 for incisors and mandibular molar teeth to progress beyond bud formation, as was shown by the ability of exogenous activin protein to rescue tooth development in mutant tooth explants (Ferguson et al., 1998).
There are two possibilities that could account for the activin ßA tooth phenotype: either another TGFß molecule is acting to activate signalling via activin receptors or downstream effectors such as Smads, or completely different signalling pathways are responsible for maxillary molar development. We have investigated the possibility that an alternative TGFß signalling cascade is used redundantly to allow for maxillary molar development in these mutants. We also report a newly identified downstream target gene revealed by in situ hybridisation analysis of gene expression patterns in mutant tooth buds.
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MATERIALS AND METHODS |
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Explants cultured with soluble activin receptors
Mice of the CD-1 strain were used. Timed matings were set up such that noon of the day on which vaginal plugs were detected was considered to be E0.5. Mandibles and maxillae from embryos at E10.5 were dissected in D-MEM with glutamax 1 (Gibco BRL; see Fig. 4). To accurately assess the age of embryos, somite pairs were counted and the stage confirmed using morphological criteria (e.g. relative sizes of maxillary and mandibular primordia, extent of nasal placode invagination, and the size of limb buds). The explants were cultured as previously described (Ferguson et al., 1998) on membrane filters supported by metal grids following the Trowel technique as modified by Saxén (Trowell, 1959; Saxén, 1966). In the first set of experiments, COS cell supernatant containing soluble activin RIIB was a gift from Olli Ritvos (Department of Bacteriology and Immunology, Haartman Institute, Helsinki, Finland). Between 450-900 µl of supernatant was diluted in 1ml DMEM containing 1% foetal calf serum (FCS) and20 IU penicillin/streptomycin. In the second set of experiments, a mixture of recombinant human activin receptor IIA and IIB (R&D Systems) was used. The lyophilized protein was reconstituted in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA). Stock solutions of 2 mg/ml were aliquoted and stored at 70°C. The cultures were set up in four-well dishes, and 150 µl medium was added per well. This contained 1% FCS, 20 IU penicillin/streptomycin, and either 12.5, 25, 35 or 45 µg/ml each of reconstituted receptor IIA and IIB. After 2 days in culture, molar tooth germs were dissected from two thirds of the explants and transferred to kidney capsules. The remaining explants were fixed and prepared for radioactive section in situ hybridisation. The grafted explant tissue was cultured in host kidneys for 12 days to allow for full development of molar teeth.
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Whole-mount in situ hybridisation was carried out using digoxigenin (DIG)-labelled Irx1 probes (Bosse et al., 1997).
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RESULTS |
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Tooth development in activin receptor and Smad2 mutants
To genetically test the possibility that another TGFß ligand compensates for activin in maxillary molar development by activating the activin signalling pathway, we examined tooth development in activin receptor and Smad2 mutant mice.
Targeted mutations in individual activin receptor (ActR) genes have been produced (Matzuk et al., 1995a; Oh, 1997; Gu et al., 1998; Gu et al., 1999). ActRIIA/ mice, which have a more severe head phenotype than ActRIIB/ mice, show anterior head defects such as a hypoplastic mandible (22% penetrance), which can result in secondary defects such as a lack of incisors (Matzuk et al., 1995a). ActRIIB/ mice have no mandibular or tooth defects but exhibit cleft palate at low penetrance, which varies according to the genetic background (Oh, 1997). Comparison of these phenotypes with that of activin ßA mutants indicates that there is functional compensation between the two type II receptors.
Because the activin signal is transduced via dimers of ActRI with ActRIIA or ActRIIB, a knockout of both type II receptor genes should be sufficient to disrupt signalling by activin as well as by any potential compensatory TGFß molecules binding to these receptors. We have recently generated mice carrying mutations of both ActRIIA and ActRIIB genes (Song et al., 1999). The compound mutants, ActR[IIA/IIB/] and ActR[IIA/IIB+/] all die in utero before E10.5, before the onset of activin ßA expression in tooth development. However, ActR[IIA+/IIB/] mice survive until birth and tooth development could thus be analysed in these animals.
A total of seven ActR[IIA+/IIB/] newborn mice were examined by serial sectioning of the heads. We found that two out of the seven had an obvious tooth phenotype where incisors and mandibular molars were absent and maxillary molars were present (Fig. 3A,D), while the other five had apparently normal molars. Six of the seven had cleft palates. This tooth phenotype was thus the same as that observed in activin ßA mutants.
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Soluble activin receptors block activin signalling and phenocopy the activin A mutants
The fact that ActR[IIA+/IIB/] and Smad2+/ tooth phenotypes are the same as the activin ßA/ phenotype, is consistent with the idea that the pathway is not active in maxillary molar tooth development. However, because in both these mutants the tooth phenotype was variably penetrant and there was always one functional allele present in the animals, there is the possibility that another TGFß molecule might be activating the pathway. It has been shown for example, that bone morphogenetic proteins (BMPs) can bind to activin receptors (Yamashita et al., 1995) and that truncated activin receptors can block BMP signalling in Xenopus embryos (Chang et al., 1997; New et al., 1997). In order to eliminate this possibility entirely, we used soluble forms of activin RIIA and RIIB receptors to sequester all ligands present in early tooth germs that might bind to these endogenous receptors and activate the pathway.
Two sets of experiments were performed using different sources of soluble receptors. In the first set, a cell supernatant from RIIB-expressing COS cells was used containing an unknown concentration of soluble receptors (Table 1). In the second set of experiments, commercially available (R&D Systems), purified RIIA and RIIB of known concentration were used (Table 2).
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In the second set of experiments, radioactive in situ hybridisation analysis was carried out on one third of the explants treated for 2 days with commercially purified soluble receptors (Fig. 5, Fig. 6). Each explant was treated with a combination of soluble RIIA and RIIB at a concentration of either 0, 12.5 or 25 µg/ml. For each sample, adjacent sections through tooth germs were analysed for the expression of different genes: follistatin (Fst) (Ferguson et al., 1998) and Irx1 expression were analysed to assess the effects of this treatment on known downstream targets of activin signalling; Msx1 expression, a homeobox gene that is expressed in the oral mesenchyme around the epithelial tooth buds (Vainio et al., 1993) was used to show viability of the explants after 2 days treatment, and also to indicate the position of the tooth germs within the explants (Fig. 5B,F,J, Fig. 6B,G,L). Msx1 expression is unaffected in the activin ßA mutants (Ferguson et al., 1998).
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The lack of tooth development in treated mandibles was associated with the following gene expression profiles: on the addition of 12.5 µg/ml of each receptor, Fst was detectable but downregulated in tooth epithelium (compare Fig. 5H with 5D). At 25 µg/ml and higher concentrations of soluble receptors, Fst was not detectable in the dental epithelium of the explants (Fig. 5L). Irx1 expression, was also considerably reduced in dental epithelium after addition of soluble receptors at 12.5 and 25 µg/ml (Fig. 5C,G,K) but was not completely lost until concentrations of 35 µg/ml were used (data not shown). In maxillary explants, the expression of Fst and Irx1 showed similar downregulation after addition of soluble receptors (Fig. 6). The resulting tooth buds in all explants exhibited the expected morphology: the treated tooth buds in maxillary explants grew to the same size as controls, compared with the mandibular tooth buds in treated mandibles that do not seem to develop beyond the epithelial thickening stage after 2 days in culture.
In order to test the possibility that maxillary molar development may require TGFß signalling independent of the activin pathway, we treated mandible and maxilla explants with higher concentrations of soluble receptors to encourage nonspecific binding of ligands to the receptors. Explants treated with 35 µg/ml of soluble receptors produced five maxillary molars out of six transferred, whereas only three mandibular molars were recovered from eight transferred (Table 2). At 45 µg/ml of soluble receptors there was some evidence of toxicity, as the numbers of teeth recovered were low, with one maxillary tooth from four transferred and no mandibular molars from six transferred (data not shown). After 2 days in culture, explants treated with 45 µg/ml soluble receptors appeared smaller than controls, suggesting that may be slightly toxic and possibly responsible for the diminished survival of the renal capsule grafts. A proportion of the explants were kept aside for analysis by in situ hybridisation. In those treated explants that expressed Msx1, Irx1 expression was found to be abolished, as expected (data not shown). Thus, the explants that survived treatment with 45 µg/ml of soluble receptors resulted in the development of maxillary and not mandibular molar teeth.
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DISCUSSION |
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In vertebrates, Iroquois-related homeobox genes are expressed in the distinct patterns in the developing nervous system and heart and are thought to be involved in pattern formation and tissue specification in these systems (Bellefroid et al., 1998; Bosse et al., 2000; Bosse et al., 1997; Christoffels et al., 2000; Cohen et al., 2000; Gomez-Skarmeta et al., 1998; Goriely et al., 1999; Tan et al., 1999). Six murine Iroquois-related homeobox genes have been identified, Irx1 to Irx6 (Bosse et al., 2000; Bosse et al., 1997; Bruneau et al., 2000; Cohen et al., 2000), of which Irx1 to Irx3 and Irx6 are expressed in the branchial arch epithelium. However, no knockouts of these genes have been reported to date, and thus the role of Irx genes in mammalian development is not understood.
Odontogenesis involves a series of epithelial-mesenchymal interactions. Activin has been implicated as an essential early mesenchymal signal that we believe, through data collected from recombination experiments, has a crucial role in activating other mesenchymally expressed genes (Ferguson et al., 1998). To date, however, no mesenchymal targets have been identified. The fact that the epithelially expressed targets Fst and Irx1 are missing from maxillary molars suggests that their role is to modify the levels of activin signalling, rather than have a direct role in odontogenesis per se. A modulatory role has been demonstrated for follistatin, which has been reported as a specific antagonist of activin in other systems (Michel et al., 1993; de Winter et al., 1996). Moreover, we showed previously that Fst, which is expressed in the dental epithelium immediately overlying activin-expressing mesenchyme, can be induced directly by activin, and possibly acts to set up a sink to regulate the levels of activin protein/site of activin activity in the developing tooth germ (Ferguson et al., 1998). In the absence of activin ßA it would not be required in maxillary molars. In the experiments where we treated explants with soluble activin type II receptors, the effects of concentration on gene expression patterns showed that Fst expression was more sensitive than Irx1 to levels of activin signalling, as it was downregulated at a lower concentration of receptors than that required for loss of Irx1. This suggests that unlike Fst, Irx1 may not be a direct target of activin signalling. Furthermore, explant cultures of mandibular epithelium that was separated from mesenchyme at E10.5-E11, did not result in significant loss of Irx1 expression, suggesting that once established, Irx1 expression rapidly becomes independent of mesenchymal signals, including activin A (data not shown).
Activin ßA, activin receptor and Smad2 mutants have a common tooth phenotype
We have shown that ActR[IIA+/;IIB/] and Smad2+/ mice exhibit the activin ßA/ tooth phenotype, in which the incisors and mandibular molars are missing but the maxillary molars develop normally. Although this phenotype occurs with low penetrance, it is consistent with previous reports that these receptors and Smad2 do indeed mediate activin signalling (Mathews and Vale, 1991; Attisano et al., 1992; Graff et al., 1996; Macias-Silva et al., 1996; Baker and Harland, 1996; Zhang et al., 1996). Furthermore, given that the maxillary molars are not affected, it is consistent with the idea that there is no redundancy between activin A and another TGFß-like molecule that may use this pathway and allow development of these molars. Despite the consistency of the tooth phenotype in the three mutants, the presence of one functional allele in these receptor and Smad2 mutants, may be sufficient to allow the activin signalling pathway to be activated. As stated previously, early embryonic lethality of homozygous mutants prevents analysis of tooth formation in the absence of these receptors and Smad2. This, together with the problem of low penetrance, may only be overcome by the production of chimaeric mice with ActR[IIA/;IIB/] embryonic stem cells.
Soluble receptors phenocopy the activin ßA mutants
The addition of soluble forms of activin type II receptors to developing tooth germs provided a non-genetic way of inhibiting activin signalling. Our results show that treatment of explants produced the same results as the activin ßA/ mutation, i.e. the numbers of mandibular molars are drastically reduced, whereas maxillary molars are comparatively unaffected. By using a combination of soluble activin type IIA and IIB receptors at various concentrations, we set out to sequester and inhibit the action of all possible ligands that might trigger the activin signalling pathway. Surprisingly, even at high concentrations (35-45 µg/ml), we found that maxillary molars still developed, indicating that the inhibition of mandibular molar development was unlikely to be due to toxicity of the soluble receptors. From these results there appears to be no compensatory TGFß-like ligand in the maxilla that can act via the activin signalling pathway to maintain the development of maxillary molars in the activin ßA mutants.
We have previously shown that Act[ßA/ßB/] mutant mice have normal maxillary molars, but lack other teeth (Ferguson et al., 1998). Thus, there is no functional redundancy between the two activins for maxillary molar development. Moreover, the expression patterns of other TGFß family members that have been found in odontogenic tissues to date (e.g. TGFß1-TGFß3, and Bmp2, Bmp4 and Bmp7 (http://bite-it.helsinki.fi/)) (Ferguson et al., 1998) show no spatial restriction to maxillary tissues that would suggest a compensatory role. The two downstream genes, Fst and Irx1, the expression of which we have shown to be downregulated in the absence of functional activin ßA, are presumably targets of Smad2-mediated transcription activation. The fact that these genes are downregulated in maxillary molars is consistent with the entire activin signalling pathway being lost in ActßA/ maxillary molars.
These results suggest a parallel independent genetic pathway for maxillary molars, one that may possibly involve Dlx genes. The Dlx-1/Dlx2 double mutants also have a tooth phenotype, but remarkably, one that is the reciprocal phenotype of ActßA/ mutants, where only the development of maxillary molars is lost (Thomas et al., 1997; Qiu et al., 1997). A common factor between these two pathways, apart from the fact that they are involved in odontogenesis, is that they share an upstream activator: FGF8 has been shown to be upstream of Dlx and activin ßA genes, in that these genes can be induced in mesenchyme surrounding an FGF8-soaked bead (Ferguson et al., 1998; Ferguson et al., 2000; Thomas et al., 2000). Quite why development of what are grossly very similar structures, molar teeth, is controlled by different genetic mechanisms on the upper and lower jaws remains to be addressed. One obvious possibility, however, is that this may reflect the different responses to FGF8 of the cranial neural crest cells that populate the mandibular and maxillary arches (Ferguson et al., 2000).
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ACKNOWLEDGMENTS |
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REFERENCES |
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