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Adam10-dependent Notch signaling establishes dental epithelial cell boundaries required for enamel formation

Summary The disintegrin and metalloproteinase Adam10 is a membrane-bound sheddase that regulates Notch signaling and ensures epidermal integrity. To address the function of Adam10 in the continuously growing incisors, we used Keratin14Cre/+;Adam10fl/fl transgenic mice, in which Adam10 is conditionally deleted in the dental epithelium. Keratin14Cre/+;Adam10fl/fl mice exhibited severe abnormalities, including defective enamel formation reminiscent of human enamel pathologies. Histological analyses of mutant incisors revealed absence of stratum intermedium, and severe disorganization of enamel-secreting ameloblasts. In situ hybridization and immunostaining analyses in the Keratin14Cre/+;Adam10fl/fl incisors showed strong Notch1 downregulation in dental epithelium and ectopic distribution of enamel-specific molecules, including ameloblastin and amelogenin. Lineage tracing studies using Notch1CreERT2;R26mT/mG mice demonstrated that loss of the stratum intermedium cells was due to their fate switch toward the ameloblast lineage. Overall, our data reveal that in the continuously growing incisors the Adam10/Notch axis controls dental epithelial cell boundaries, cell fate switch and proper enamel formation.

Rodent teeth (i.e., molars and the continuously growing incisors) represent powerful biological models for studying the molecular mechanisms involved in cell fate specification and differentiation of various cell lineages (Mitsiadis and Graf, 2009). Reciprocal inductive interactions between the epithelial cells and the underlying neural crest-derived mesenchyme give rise to diverse cell types that progressively form the complex dental structures (Balic and Thesleff, 2015;Cobourne and Mitsiadis, 2006;Pagella et al., 2020;Yu and Klein, 2020). Specifically, the dental epithelium is composed of four distinct cell layers, including the outer dental epithelium, stratum intermedium, stellate reticulum and inner dental epithelium/ameloblasts (Mitsiadis and Graf, 2009). Ameloblast layer is responsible for the formation of enamel, which is the most mineralized tissue of the body (Bartlett, 2013;Smith and Nanci, 1995). Our previous studies have shown that during odontogenesis the expression patterns of Notch receptors and ligands are restricted and complementary in dental epithelium (Mitsiadis et al., 1995(Mitsiadis et al., , 1997(Mitsiadis et al., , 1998(Mitsiadis et al., , 2005(Mitsiadis et al., , 2010. Notch1 expression is confined to stratum intermedium (Mitsiadis et al., 1995), whereas Dll1 and Jag2 are both expressed in the adjacent inner enamel epithelium/ameloblasts (Mitsiadis et al., 1998(Mitsiadis et al., , 2010, thus reflecting the subdivision of the dental epithelium into ameloblast and non-ameloblast layers. These results suggest that Notch signaling controls amelogenesis and regulates dental epithelial cell fate specification and differentiation events. Conversely, it was recently shown that Notch1-expressing cells in the stratum intermedium represent a reservoir of dental epithelial progenitors that are activated upon tooth injury and contribute to the regeneration of the ameloblast layer (Sharir et al., 2019). Adam10 expression in ameloblasts was recently reported, and in vitro results suggested a role for this molecule in the regulation of enamel secretion (Ikeda et al., 2019).
To further address the function of Adam10 and its relevance in Notch-mediated signaling during amelogenesis, we used transgenic mouse models in which Adam10 is conditionally deleted in the dental epithelium (Weber et al., 2011), as well as GI254023X, an Adam10 inhibitor. We found that the loss of Adam10 activity in the Keratin14 Cre/+ ;Adam10 fl/fl mice affects tooth enamel formation. This phenotype is associated with the loss of the stratum intermedium cells and the concomitant downregulation of Notch1 expression. Using Notch1 CreERT2 ;R26 mT/mG mice (Fre et al., 2011), we demonstrated that epithelial Adam10 deletion induces a cell fate switch of stratum intermedium cells toward ameloblasts. Conclusively, our data show that the Adam10/Notch1 signaling axis is crucial for establishing dental epithelial cell fates and essential for accurate enamel formation.

Deletion of Adam10 leads to defective enamel formation
We first analyzed Adam10 expression using in situ hybridization in postnatal mouse incisors (P1-P2). Adam10 mRNA was mainly expressed in stratum intermedium, inner enamel epithelial cells, including preameloblasts and ameloblasts (Figures S1A and S1B). At the protein level, Adam10 was distributed in stratum intermedium cells, preameloblasts and ameloblasts in P1-P6 incisors (Figures S1C-S1F). As the ameloblast differentiation advances, Adam10 staining intensified in ameloblasts, whereas it decreased in the stratum intermedium (Figures S1D-S1F). Surprisingly, strong Adam10 labeling was also observed in the enamel (Figures S1E, S1G, S1H), which suggests that either fragments of Adam10 protein are trapped within newly forming enamel or non-specific immunostaining. To elucidate these unexpected findings, mass spectrometry analyses should be realized in the future.
Next, we studied the role of Adam10 in amelogenesis by assessing the enamel of incisors from K14 Cre/+ ;Adam10 fl/fl mice, in which Adam10 is conditionally knocked out from dental epithelium. The enamel of wild-type mouse incisors is characterized by a yellowish-reddish pigmentation due to iron deposits ( Figures 1A and 1B), which increase the hardness of enamel . In contrast, mutant incisors displayed a translucent/white enamel (Figures 1A', 1B 0 ) that indicates a crucial role for Adam10 in the enamel synthesis and composition. We therefore studied in detail the enamel defects observed in the K14 Cre/+ ;Adam10 fl/fl incisors by micro-CT (mCT), scanning electron microscopy (SEM) and histological examination.
Enamel situated at the labial side of the incisors is visualized in SEM and mCT as a bright white layer that indicates the elevated mineral content of this tissue. In wild-type mice, the enamel layer was clearly defined in both longitudinal and frontal orientations of the incisors ( Figure 1C). In contrast, the enamel was not visible in the mutant incisors, although a very thin white layer was observed sporadically at their anterior part ( Figure 1C'). Further SEM analysis indicated mineral and structural disorganization of the K14 Cre/+ ;Adam10 fl/fl enamel. Indeed, the enamel of mutant incisors was thinner, severely hypocalcified and disorganized (notably in the orientation, arrangement and thickness of the enamel rods) when compared to the wild-type enamel ( Figures 1D, 1D 0 ). The degree of hard tissue mineralization can be estimated upon staining with Toluidine Blue (TB), which is retained by the organic matrix of the tissue. The iScience Article enamel of wild-type incisors, due to its high mineral content, did not retain the dye and exhibited a white color ( Figure 1E). In contrast, the enamel layer of the mutant incisors acquired a blue appearance after TB staining ( Figure 1E'), indicating a less mineralized enamel rich in organic elements. Transmission electron microscopy (TEM) analyses revealed defective enamel maturation in mutant incisors. Compared to the wild-type enamel, broader intercrystallite spaces appeared in the mutant enamel, due to the incapacity of enamel crystals to increase in width and thickness ( Figures 1F, 1F 0 ).

Disorganized ameloblasts and ectopic enamel matrix deposition in Adam10 mutant incisors
To investigate the cellular mechanisms that underlie the enamel phenotype in the K14 Cre/+ ;Adam10 fl/fl incisors, we analyzed histological sections of wild-type and K14 Cre/+ ;Adam10 fl/fl incisors (Figures 2A-2B 0 and S2). Amelogenesis is a complex process that requires the differentiation of inner enamel epithelium cells  iScience Article into preameloblasts/ameloblasts and the orchestration of fine-tuned events involved in the secretion of enamel specific matrices and deposition of minerals in order to generate the unique and well-organized enamel tissue . Histological sections stained with hematoxylin and eosin (H&E) showed that the ameloblast layer is completely disordered in the K14 Cre/+ ;Adam10 fl/fl incisors when compared to the well-organized ameloblast layer of wild-type incisors (compare Figures  To further characterize the effects of Adam10 deletion in amelogenesis we performed comparative expression analyses of molecules fundamental for enamel formation (i.e., Ameloblastin and Amelogenin) in wildtype and K14 Cre/+ ;Adam10 fl/fl incisors. In situ hybridization showed the expression of the enamel-specific gene Ambn (coding for the Ameloblastin protein) in ameloblasts of the wild-type incisors ( Figure 2E), whereas in the K14 Cre/+ ;Adam10 fl/fl incisors Ambn was expressed in both ameloblasts and cell clusters that are formed outside of the ameloblast layer ( Figure 2E'). Similarly, immunofluorescent staining with Amelogenin in wild-type incisors clearly demonstrated the presence of this enamel specific protein in both ameloblasts and enamel ( Figure 2F), whereas in the K14 Cre/+ ;Adam10 fl/fl incisors the staining was detected in all dental epithelial cell populations and the enamel ( Figure 2F').

Loss of the stratum intermedium layer in incisors of Adam10 mutant mice
Next, we analyzed the effects of Adam10 deletion on the other cell layers that compose the dental epithelium using histological sections of incisors stained with either H&E or TB. The most striking observation was the absence of the stratum intermedium layer in the K14 Cre/+ ;Adam10 fl/fl incisors (Figures S2A', S2B 0 and S3A', S3B 0 ). In wild-type incisors, stratum intermedium is composed of cuboidal-shaped cells (Figures S2A,S2B and S3A,S3B). Instead, in K14 Cre/+ ;Adam10 fl/fl incisors, a layer of flat, loose cells, which closely resemble the stellate reticulum cells, was occupying the space normally allocated to stratum intermedium (compare Figures 3A, 3B, to 3A', 3B 0 ). TEM analyses confirmed the absence of stratum intermedium cells and the existence of a loose epithelial tissue in K14 Cre/+ ;Adam10 fl/fl incisors (compare Figures 3C-3C').
Because Notch1 is specifically expressed by stratum intermedium cells in rodent teeth (Mitsiadis et al., 1995(Mitsiadis et al., , 1998, and Adam10 is a key modulator of Notch signaling activation, we wished to know if Notch1 expression persists in K14 Cre/+ ;Adam10 fl/fl incisors. Therefore, we analyzed and compared Notch1 expression in wild-type and K14 Cre/+ ;Adam10 fl/fl incisors using in situ hybridization. In the epithelium of wild-type   iScience Article 3E 0 ). We additionally used Alkaline Phosphate (ALP), which is a well-established marker of stratum intermedium cells (Larsson, 1976), to confirm their absence in K14 Cre/+ ;Adam10 fl/fl incisors. Although ALP activity strongly labeled the stratum intermedium cell layer in wild-type incisors ( Figure 3F), it was missing in the epithelium of K14 Cre/+ ;Adam10 fl/fl incisors ( Figure 3F).
In situ hybridization revealed that Adam10 deletion also affected Notch2 expression in the epithelium of K14 Cre/+ ;Adam10 fl/fl incisors. In wild-type incisors, strong Notch2 hybridization signal is exclusively detected in stellate reticulum ( Figures S4A, S4B). In contrast, diffused Notch2 expression is observed in all epithelial cell layers of the K14 Cre/+ ;Adam10 fl/fl incisors, with hotspots of reactivity in stratum intermedium and outer enamel epithelium cells ( Figures S4A', S4B 0 ).
In addition, using a specific anti-Notch1 antibody that exclusively detects the extracellular domain of this molecule (Mitsiadis et al., 1995), we analyzed its distribution in the wild-type and K14 Cre/+ ;Adam10 fl/fl incisors. In wild-type incisors, immunoreactivity for the Notch1 extracellular domain is localized exclusively at the basal end of ameloblasts ( Figure S4C), which indicates the internalization of this fragment released upon Notch1 cleavage (Hori et al., 2013). In contrast, Notch1 staining was absent in the epithelium of K14 Cre/+ ;Adam10 fl/fl incisors ( Figure S4C'). Similarly, strong Notch2 staining was detected in stellate reticulum and outer enamel epithelium cells of the wild-type incisors ( Figure S4D) whereas a weaker and diffused labeling was observed in these cell layers of K14 Cre/+ ;Adam10 fl/fl incisors ( Figure S4D').

Loss of Adam10 affects cell proliferation but not apoptotic events in dental epithelium
The disappearance of stratum intermedium cells in the K14 Cre/+ ;Adam10 fl/fl incisors could be linked to apoptotic events. To test this hypothesis, we performed TUNEL assay that allows the detection of abnormal cell death in the dental epithelium of K14 Cre/+ ;Adam10 fl/fl incisors, and most specifically in the area attributed to the stratum intermedium layer in wild-type incisors ( Figures 4A-4B 0 ). However, we did not observe any apoptotic activity in the dental epithelium of either wild-type ( Figure 4D'). This increased number of cell divisions within the preameloblast layer in the K14 Cre/+ ;Adam10 fl/fl incisors could contribute to the expansion of the number of ameloblasts and the subsequent folding of the dental epithelium to compensate for the increased cell number. To test this hypothesis, whole mount imaging was performed in K14 Cre/+ ;R26 mT/mG (control; Figure 4E) and K14 Cre/+ ;Adam10 fl/fl ;R26 mT/mG (mutant; Figure 4E') incisors. The 3D imaging clearly demonstrated that the dental epithelium (green color) of K14 Cre/+ ;Adam10 fl/fl ; R26 mT/mG incisors acquired a jagged configuration upon Adam10 deletion (arrows in Figure 4E').
Alterations in dental epithelium in incisors of Amelx Cre/+ ;Adam10 fl/fl and K14 Cre/+ ;Notch1 fl/fl mutant mice To study whether the tooth phenotype is due to the loss of Adam10 function specifically in ameloblasts, we exploited Amelx Cre/+ ;Adam10 fl/fl mice, in which Adam10 is deleted exclusively in ameloblasts ( Figures S6A,  S6B). mCT analysis showed normal enamel formation in Amelx Cre/+ ;Adam10 fl/fl incisors ( Figure S6C). Similarly, histological sections stained with H&E showed that the dental epithelium of Amelx Cre/+ ;Adam10 fl/fl incisors did not present any alterations, neither in ameloblasts nor in stratum intermedium cells ( Figures S6D, S6E). These results indicate that enamel defects observed in K14 Cre/+ ;Adam10 fl/fl incisors are not due to the loss of Adam10 function in ameloblasts, but rather to the loss of Adam10 in progenitors and/or early precursors of preameloblast and stratum intermedium cell lineages.
We also studied whether epithelial Notch1 deletion is able to recapitulate the tooth phenotype observed in K14 Cre/+ ;Adam10 fl/fl mice. iScience Article incisors ( Figure S7D), as previously described (Jheon et al., 2016). However, the stratum intermedium in the K14 Cre/+ ;Notch1 fl/fl incisors was discontinuous, presenting both areas of ectopically formed cell clusters and territories of detachment from the ameloblast layer (arrows in Figures S7A-S7C). These results indicate a compensatory mechanism between the various Notch proteins, and thus explain the lack of severe phenotypic changes in K14 Cre/+ ;Notch1 fl/fl incisors.
Treatment with an Adam10 selective inhibitor recapitulates the tooth phenotype of Adam10 mutant mice GI254023X is a well-established Adam10 inhibitor (Hoettecke et al., 2010), and therefore we wanted to investigate if its application to wild-type incisors will recapitulate the phenotype observed in the  iScience Article K14 Cre/+ ;Adam10 fl/fl mutants. For this purpose, explants of isolated embryonic (E13.5) wild-type mouse incisors were cultured for 7 days in vitro in the presence of either DMSO (control) or the GI254023X inhibitor ( Figures 5A and 5B, 5B 0 ). Histological examination of H&E stained sections showed that all cell layers of the incisor epithelium were evident in explants cultured with DMSO ( Figure 5C). By contrast, the addition of the GI254023X inhibitor had a dramatic effect on the stratum intermedium layer ( Figure 5C'), mimicking the phenotype observed in the epithelium of K14 Cre/+ ;Adam10 fl/fl incisors. Moreover, ALP activity was severely reduced in the GI254023X treated incisors (compare Figures 5D-5D'), thus confirming the absence of stratum intermedium cells.
It is not clear whether Adam10 inhibition in the epithelium of incisors had an irreversible effect on the progenitors of stratum intermedium cells, or if it affected already established cell lines and was thus reversible.
To address this question, incisor explants were first cultured in vitro for one week in presence of either DMSO or GI254023X, followed by kidney capsule transplantation ( Figure 5A). Tissue was harvested one week after the transplantation and processed for histological analyses. H&E stained sections of control explants revealed that all dental epithelial cell layers were clearly defined, with no apparent cellular disorganization ( Figures 5E, 5F, 5G, 5H). In contrast, samples treated with GI254023X before transplantation demonstrated severe structural defects at the most anterior part of the incisors, where the whole epithelium was completely disordered and disorganized ( Figure 5E', blue asterisk, Figure 5G'). However, examination of the posterior and median parts of the GI254023X treated incisors clearly showed the reappearance of the stratum intermedium layer beneath a well-organized and ordered preameloblast/ameloblast layer (Figures 5F', 5H 0 ), thus indicating that the effects triggered by Adam10 inhibition are reversible. A detachment of these two layers was often observed in the incisors treated with GI254023X before transplantation ( Figure 5E'), possibly because the inhibitor was not entirely eliminated and continued to exert a mild effect on the transplanted incisors.
Lineage tracing of Notch1-expressing cells show that Adam10 deletion induces cell fate changes within the stratum intermedium cell layer We next studied the fate of Notch1-expressing cells upon Adam10 abrogation. For this purpose, we used the Notch1 CreERT2/+ ;R26 mT/mG transgenic mice in which, after 4-OHT induction, GFP expression is activated in the Notch1-expressing cells. The ability to conditionally mark Notch1-expressing cells allows following of the dental epithelial cell lineages associated with Notch1 expression and establishing its relationship to the continuous growth and regeneration of rodent incisors. Upon 4-OHT induction, Notch1/GFP-positive cells were localized in the inner dental epithelial cells, in agreement with in situ hybridization results. Notch1 is also expressed in the endothelial cells of the dental papilla and dental follicle. One week after 4-OHT induction, GPF staining was localized mainly in the cells of the stratum intermedium ( Figures 6A-6D). Interestingly, GFP staining was also sporadically detected in cells of the stellate reticulum and outer enamel epithelium, as well as in ameloblasts ( Figure 6D).
We then analyzed the fate of Notch1 expressing stratum intermedium cells upon blocking Adam10 function. After one day of treatment, we observed single cells from the stratum intermedium invading the ameloblasts layer (Figures 6F-6H). By the fourth day of treatment, we observed large invaginations of the dental epithelium into the mesenchyme ( Figures 6E, 6I, 6J). Within these invaginations, numerous Notch1/GFP-positive cells left the stratum intermedium layer and entered the ameloblast layer ( Figures 6K-6N). dersson et al., 2011;Artavanis-Tsakonas et al., 1999;Ho and Artavanis-Tsakonas, 2016;Kopan, 2012). In teeth, Notch signaling is essential for proper enamel formation, a process that requires the close cooperation of the four specific dental epithelial cell types. Indeed, components of the Notch pathway are specifically expressed in these four dental epithelial lineages and their deletion in mice affects both tooth morphology and enamel formation (Mitsiadis et al., 1995(Mitsiadis et al., , 1998(Mitsiadis et al., , 2005(Mitsiadis et al., , 2010. Amelogenesis requires a tightly controlled sequence of cell proliferation, differentiation, extracellular matrix secretion and mineral deposition (Bartlett, 2013;Zheng et al., 2013). Minor alterations in these processes lead to functionally relevant modifications of the enamel structure (Cantu et al., 2017). Enamel is the highest mineralized tissue in vertebrates and is characterized by a tightly organized structure that  iScience Article provides its exceptional hardness . Here we demonstrate that inhibition of Adam10/ Notch activity severely affects both the arrangement of the dental epithelium and the structure of enamel. The white and glossy appearance of enamel in the K14 Cre/+ ;Adam10 fl/fl incisors is indicative of important enamel alterations such as enamel hypoplasia, hypomaturation and hypomineralization (Mitsiadis and Luder, 2011).

Cell fate specification and differentiation is regulated by the Notch signaling pathway that mediates interactions between neighboring cells via the physical interaction of Notch ligands with Notch receptors (An
Detailed histological analyses of the K14 Cre/+ ;Adam10 fl/fl incisors show disappearance of a stratum intermedium as a distinct layer of cuboidal cells and a widespread disorder of the ameloblast and stellate reticulum layers that occurs concomitantly with initiation of mineral matrix deposition. Absence of stratum  iScience Article intermedium cells is not associated with apoptotic events, which suggests that inactivation of Adam10 affects their fate and misroutes these cells into alternative developmental pathways, either as ameloblasts or stellate reticulum cells (Figure 7). Increasing the number of one specialized cell type at the expense of another inevitably leads to changes in cell organization and composition, as well as in the enamel structure.  The apparent enamel defects and enamel deposition in ectopic sites in the Adam10 mutants are linked with extreme disorganization and disorientation of ameloblasts. Similar deformations, including defects in bristle shaft, irregular bristle polarity and abnormal deposition of lens material, have been observed in Drosophila eyes upon Notch signaling deregulation (Das et al., 2013;Fortini and Artavanis-Tsakonas, 1994).
The K14 Cre/+ ;Adam10 fl/fl incisors demonstrate increased number of ameloblasts, due to increased proliferative activity of preameloblasts and the fate change of stratum intermedium cells. A strict balance between the number of ameloblasts and the space they occupy exists in the wild-type incisors. In K14 Cre/+ ;Adam10 fl/fl incisors, however, this equilibrium is disturbed by the increased number of ameloblasts, thus resulting in the folding and further disorganization of the ameloblast layer. Consequently, the ameloblasts in K14 Cre/+ ;Adam10 fl/fl incisors are characterized by loss of polarity, as indicated by the ectopic secretion of enamel at their basal end, and defective differentiation, characterized by the ectopic expression of Notch2 and K14. Of importance, these changes are not caused by a loss of Adam10 activity in ameloblasts because exclusive deletion of Adam10 in ameloblasts, and consequently enamel, of Amelx Cre/+ ;Adam10 fl/fl incisors had no effect neither on organization and morphology of ameloblast layer nor enamel structure.
Although the function and the roles of ameloblasts in the process of enamel formation in the continuously growing incisors are well established (Bartlett, 2013;Mitsiadis and Graf, 2009), fewer information exists for the other three dental epithelial cell types. For example, it is not yet known when the fate of these various cell types is established. A number of studies suggested that the ameloblasts and stratum intermedium share a common progenitor in transit-amplifying cells (Harada et al., 1999(Harada et al., , 2006Sharir et al., 2019), and recently outer dental epithelium progenitors were identified by scRNA-seq (Krivanek et al., 2020). The current notion is that the stem cells responsible for the generation of all dental epithelial cell lineages reside in the cervical loop of the incisor (Juuri et al., 2012;Krivanek et al., 2020;Li et al., 2012;Mitsiadis et al., 2007;Sharir et al., 2019). Our findings suggest that the fate determination of these cells is more flexible and is regulated by the Adam10/Notch axis. Previous studies suggested that Notch signaling induces the divergence of the stratum intermedium from the ameloblast lineage (Harada et al., 2006). Recently, we showed that Notch1 expressing cells of the stratum intermedium represent progenitor cells that are activated upon injury and regenerate the ameloblast layer (Sharir et al., 2019). The persisting Notch1 expression in the posterior part of K14 Cre/+ ; Adam10 fl/fl incisors suggests the presence of stratum intermedium precursors in these teeth.
Here we show that the absence of stratum intermedium generates a less refined and organized enamel structure resembling to the enameloid of lower vertebrates such as sharks (Fraser et al., 2013;Kawasaki et al., 2021;Smith et al., 2015;Wilmers et al., 2021). We have previously shown that K14 Cre/+ ;Jagged2 fl/fl mice display aberrant tooth morphology, disorganization of the epithelium and altered enamel formation and structure (Mitsiadis et al., 2005(Mitsiadis et al., , 2010. Other studies, where Notch signaling was pharmacologically inhibited postnatally, demonstrated absence of a direct effect on ameloblasts (Jheon et al., 2016). However, contacts between ameloblasts and stratum intermedium cells were disrupted, which eventually led to enamel defects (Jheon et al., 2016). Similarly, our analyses of K14 Cre/+ ;Adam10 fl/fl incisors showed an irregular stratum intermedium, with territories of both detachment from the ameloblast layer and ectopic formation of cell clusters whereas the overall organization of the ameloblast layer was not affected. Therefore, Notch signaling deregulation within the dental epithelium does not allow cell lineages to maintain their specific molecular print, which is important for their cellular specificity and function. The tooth enamel phenotype shows a direct effect of Adam10 on the ability to generate signals linked to Notch1 activation for the maintenance of stratum intermedium and overall dental epithelial architecture. Our results nevertheless indicate that Adam10 activity is necessary to maintain Notch1-expressing cells identity as stratum intermedium cells. These cells are multipotent and can acquire the ameloblast fate upon injury, as we recently demonstrated (Sharir et al., 2019). This injury-dependent cell fate switch is homologous to the one induced by Adam10 inhibition, which thus suggests that modulation of Adam10 activity could be a mechanism underlying the recruitment of stratum intermedium cells upon injury. Hence, Adam10 deletion likely causes switch of early Notch1 expressing progenitors to cells of the inner enamel epithelium/preameloblast layer instead of the stratum intermedium layer. However, we cannot exclude the possibility that Adam10 deletion might induce the conversion of some stratum intermedium cells to preameloblasts via inhibition of the Notch signaling pathway, although our data do not provide a direct evidence of this conversion. Although the phenotype clearly highlights the role of Adam10 in Notch1 signaling, we cannot exclude the possibility that the reduced shedding of other Adam10 targets, which include important adhesion molecules (Wetzel et al., 2017), could partly contribute to these severe abnormalities.

OPEN ACCESS
iScience 25, 105154, October 21, 2022 13 iScience Article Taken together, our results show that Adam10 exerts a fundamental role in preserving Notch-dependent cell identity in dental epithelial cells, and the establishment of the four distinct dental epithelial cell populations that orchestrate the generation and proper composition of enamel.

Limitations of the study
In this work, we demonstrate that Adam10 deletion leads to loss of stratum intermedium cells due to their switch toward the ameloblast lineage. We propose that this event induces depolarization of ameloblasts and overcrowding of the ameloblast layer, which in turn causes the formation of defective enamel. Although this would be the most logical and simple explanation, it is challenging to obtain a full, mechanistic dissection of these series of events. We further demonstrate that Adam10 deletion is accompanied by loss of Notch1-expressing stratum intermedium cells, and our lineage tracing experiments strongly support a fate switch of these cells toward the ameloblast lineage. Nevertheless, our data do not provide direct evidence of this conversion.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  All mice were maintained and handled according to the Swiss Animal Welfare Law and in compliance with the regulations of the Cantonal Veterinary office, Zurich (License for animal experimentation ZH15½014; ZH146/17; ZH018/17). The animal facility provided standardized housing conditions, with a mean room temperature of 21 G 1 C, 50% G 5% humidity, and 15 complete changes of filtered air per hour (HEPA H 14filter); air pressure was controlled at 50 Pa. The light/dark cycle in the animal rooms was set to a 12 h/12 h cycle (lights on at 07:00, lights off at 19:00) with artificial light of approximately 40 Lux in the cage. The animals had unrestricted access to sterilized drinking water, and ad libitum access to a pelleted and extruded mouse diet in the food hopper (Kliba No. 3436; Provimi Kliba/Granovit AG, Kaiseraugst, Switzerland). Mice were housed in a barrier-protected specific pathogen-free unit and were kept in groups of max. 5 adult mice per cage in standard IVC cages (Allentown Mouse 500; 194 3 181 3 398 mm, floor area 500 cm2; Allentown, NJ, USA) with autoclaved dust-free poplar bedding (JRS GmbH + Co KG, Rosenberg, Germany). A standard cardboard house (Ketchum Manufacturing, Brockville, Canada) served as a shelter, and tissue papers were provided as nesting material. In addition, crinklets (SAFE crinklets natural, JRS GmbH + Co KG, Rosenberg, Germany) were provided as enrichment and further nesting material. The specific pathogen-free status of the animals was monitored frequently and confirmed according to FELASA guidelines by a sentinel program. The mice were free of all viral, bacterial, and parasitic pathogens listed in FELASA recommendations (Felasa Working Group On Farm Animals Corina Mihaela Berset Convenor et al., 2021).
To study the effect of Adam10 deletion in the dental epithelium, Adam10 fl/fl (MGI: 4838238) mice (Jorissen et al., 2010) were crossed with Keratin14-Cre (K14 Cre/+ ) (MGI: 2445832) and R26 mT/mG (MGI: 3716464) mice (The Jackson Laboratory). Homozygous Adam10 fl/fl ;R26 mT/mG mice were crossed with K14 Cre/+ ;Adam10 fl/+ mice. Conditional K14 Cre/+ ;Adam10 fl/fl knockout mice were harvested for analysis at different developmental points, as detailed in the ''Method details'' section and in the figure legends. Littermates of Adam10 fl/+ mice were used as controls. To trace Notch1 expressing cells, Notch1-CreERT2 SAT (Notch1 CreERT2/+ ; MGI: 5304912) mice (Fre et al., 2011) were crossed with R26 mT/mG mice to obtain Notch1 CreERT2/+ ;R26 mT/mG mice. To assess the effects of the deletion of Adam10 specifically in ameloblasts, we crossed Amelx Cre/+ mice (Said et al., 2019) (a gift from Professor Petros Papagerakis) with Adam10 fl/fl mice and R26 mT/mG mice. At least 3 biological replicates were used for each experiment, unless differently indicated. Animals were allocated to experimental groups based on their genotype; animals of different sex were randomly allocated.

Tissue isolation and preparation
Tissues used in this study were harvested at different stages ranging from embryonic day 13.5 (E13.5) to postnatal day 19 (P19). Experiments at postnatal stages involving the K14 Cre/+ ;Adam10 fl/fl mice were mainly performed on P1-P2 incisors because only very few mutant pups survived after this age, as previously reported (Weber et al., 2011). Mouse embryos were sacrificed by decapitation and heads were fixed in 4% paraformaldehyde (PFA) overnight at 4 C. Endocardial perfusion with 4% PFA was performed in pups, after which the heads were harvested and further fixed in 4% PFA overnight at 4 C. The heads were then processed for paraffin embedding or OCT embedding for microtome or cryostat sectioning, respectively.

Microcomputed tomography (mCT)
After fixation, heads from three P19 K14 Cre/+ ;Adam10 fl/fl and three wild-type mice were gradually dehydrated to 70% Ethanol and scanned at 70 kV and 114 mA with an isotropic resolution of 20 mm in a high-resolution mCT system (mCT 40, Scanco Medical). Three-dimensional (3D) reconstruction of the whole heads from the obtained slices was obtained using the software VGStudio MAX 2.0 (Volume Graphics GMBH).
Following the mCT evaluation, heads of P19 mice were divided in two-halves. One-half of each specimen was left fully mineralized and processed for examination in the scanning electron microscope (SEM). The other half was decalcified for 8 weeks in 10% ethylenediaminetetraacetic acid (EDTA) and then processed for paraffin embedding.

Histological evaluation
Dehydrated wild-type and mutant tissue samples from different stages were paraffin embedded and sectioned to 5 mm thick sections. Hematoxylin and eosin (H&E) staining was used for the histological analysis of these samples.

Orientation analysis of ameloblasts
Orientations of the symmetry axis of ameloblasts nuclei in wild-type and K14 Cre/+ ;Adam10 fl/fl incisors was measured by rotating a vector over the images and determining the angle between the basal and apical poles of the nuclei. Student's t-tests were used for statistical analyses.
In situ hybridization Notch1, Notch2 (Mitsiadis et al., 1995(Mitsiadis et al., , 1998, Adam10 (a gift from Prof. Yufang Zheng), Ameloblastin (Ambn; a gift from Dr Yoshihiko Yamada) riboprobes were used for in situ hybridization on 14 mm cryosections, as previously described (Mitsiadis et al., 1998). The hybridization signal was detected using the NBT/ BCIP substrate solution, sections were mounted with Glicergelâ mounting medium (Dako) and imaged with Leica DM6000 microscope equipped with the Leica DFC420C camera. Images were processed with the Leica Application Suite (LAS) software.
For whole mount staining, incisors were fixed in 1% PFA in PBS for 1 h at RT, followed by permeabilization and blocking with 0.3% Triton X-100/10% Normal Horse Serum (NHS)/0.2% BSA in PBS overnight. Samples were incubated with primary antibodies for four days at 4 C, followed by 10 h wash with PBS. The incisors were then incubated with Alexa488 secondary antibody for 2 days at 4 C, followed by incubation with DAPI overnight at 4 C. Samples were then cleared with the clearing agent FocusClearª (CelExplorer, Taiwan, FC-101) and mounted with 2.5% 1,4-Diazobicyclo-[2.2.2]-Octane (DABCO; Sigma-Aldrich, D2522) for analysis under a Leica SP5 confocal microscope.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) TUNEL assay was performed on paraffin sections using terminal deoxynucleotidyl transferase (TdT) enzyme (Roche, 1176730500) and biotinylated dUTP (Roche, 11388908910). Briefly, samples were deparaffinized, rehydrated, and treated with a mixture of TdT enzyme and biotinylated dUTP. Sections were incubated briefly with DAPI and mounted with 2.5% DABCO. Pictures were taken using the Leica DFC350FX camera and the LAS AF software.