Enamel development is an integral process of symphonic dimensions that is characterized by a continuous interplay between cells, matrices, minerals, proteins, and signals over the entire period of amelogenesis. Allegorically speaking, the key players in this symphony have been known for decades, including a mineral section that undergoes a transition from amorphous calcium phosphate and a protein section made up by classic enamel proteins such as amelogenins, ameloblastin, and enamelin, as they are further processed by enamel-related enzymes, including MMP20 and KLK4. As amelogenesis progresses, the volume percentage of proteins and water decreases, while the mineral content increases, resulting in a 96% mineral content in the mature enamel layer of adult mammals (Deakins, 1942; Stack, 1960; Robinson et al., 1978, 1979, 1988). Changes in the metastable enamel matrix that result from the loss of water and proteins have even been described as a “kind of crisis” (Eastoe, 1979), referring to the multiple effects of water and protein resorption on the interface between remaining proteins and maturing enamel crystals.

For decades, the effect of individual enamel proteins such as amelogenin on enamel crystal growth have been a most intriguing and rewarding subject of study (Lagerström et al., 1991; Diekwisch et al., 1993; Gibson et al., 2001; Iijima and Moradian–Oldak, 2004; Jin et al., 2009; Gopinathan et al., 2014). In addition to deciphering individual aspects of amelogenin function, much progress has been made elucidating the role of the less prominent enamel-related proteins ameloblastin and enamelin on enamel crystal growth and habit (Masuya et al., 2005; Lu et al., 2011; Hu et al., 2014). Moreover, it has been demonstrated that enamel proteins undergo posttranslational processing by the enamel proteinases MMP20 and KLK4 (Bartlett and Simmer, 1999; Simmer and Hu, 2002; Bartlett, 2013).

While much is known about the major proteins and minerals involved in tooth enamel formation, it has become increasingly obvious that amelogenesis is more complex than a mixture of an aqueous enamel protein solution with a combination of calcium and phosphate ions, subjected to enzymatic protein digestion and gradual removal of water over time. Recent studies have illustrated the importance of ion transport mechanisms for mineral transport (Hubbard, 2000; Paine et al., 2001; Lacruz, 2017), the effect of pH modulation through regulatory molecules (Takagi et al., 1998; Lacruz et al., 2010; Moradian-Oldak, 2012; Robinson, 2014) and the role of junctional proteins such as cadherins for ameloblast movement (Bartlett and Smith, 2013; Guan et al., 2014). These molecules and events are only one part of a process that ensures a gradual deposition of minerals at the dentin-enamel junction throughout amelogenesis and their step-wise conversion into hydroxyapatite crystals and alignment into enamel prisms into one of the most fascinating biomaterials found in nature.

Development of a synthetic or mimetic model of amelogenesis would greatly benefit from a temporo-spatial integration of the multitude of processes involved in mammalian amelogenesis. Such multi-level and multi-scale data mining commonly requires a systems biology approach. Systems biology of development seeks to integrate bioinformatic data analysis with other molecular, cellular, and tissue-related information to reach a higher-level, multifaceted, and integrative understanding of developmental processes (Bard, 2007; Edelman et al., 2009). Integrative approaches toward biological problems have become possible as a result of recent advances in bioinformatics and omics technologies, including proteomics, transcriptomics, and metabolomics (Mochida and Shinozaki, 2011). In mineralized tissue biology, systems biology would need to integrate genetic and proteomic data with mineralogic, structural, and spectroscopic data to develop a multi-dimensional understanding of a complex process such as amelogenesis.

In the present study we have employed first mandibular mouse molar amelogenesis as a model system to systematically map proteomic, spectroscopic, temporo-spatial, and mineralogic events during the first 8 days of postnatal enamel development. The benefit of a model based on teeth with limited growth is the synchronicity of developmental events leading up to maturation of the entire tooth surface by the time of tooth eruption and providing a homogeneous enamel matrix at each stage ideally suited for proteomic and spectroscopic analysis. During the course of this study we have generated sets of spectroscopic, proteomic, and mineralogic data and integrated related events through their common timescale of development. Our analysis provides timing of events, novel proteomic and spectroscopic data, and identification of novel non-hydrophobic groups of proteins and individual proteins that may contribute toward amelogenesis. Future studies will enhance our understanding of the interconnectedness between these processes during the progression of amelogenesis as they contribute to the formation of highly organized tooth enamel.

Here we have used the developing mouse mandibular molar as a model system to track changes in protein and mineral composition during postnatal amelogenesis and to correlate findings from individual protein and mineral analyses to synthesize an integrated systems perspective of enamel formation in the mouse molar. The key benefit of the postnatal mouse molar model was the suitability of the distal slopes of first molar cusps to harvest sufficient quantities of fresh enamel matrix for proteomic and mineral composition analysis in daily increments. Moreover, the mouse mandibular molar has a long history as a model for morphogenesis, cytodifferentiation, and tissue specific biomineralization (Gaunt, 1955; Slavkin et al., 1976, 1988), allowing for cross-referencing between classic developmental biology data, mouse genetics models of amelogenesis, proteomics analysis databases, and the present systems biological study. The mouse molar model owes its popularity to its resemblance to the human molar in terms of completion of enamel formation following eruption and to the ubiquitous availability of mice and mouse genetic tools for experimental studies. In general, the mouse incisor would be equally attractive for studies of mammalian amelogenesis. However, for historical reasons and because of its larger size, the continuously erupting rat incisor has provided an alternative model for the study of enamel matrix and mineral composition during development (Schour and Massler, 1949; Robinson et al., 1979, 1981; Smith and Nanci, 1989). The rat incisor model relies on the preparation of sequential segments along the rat incisor labial surface representative of the entire sequence of amelogenesis from the youngest enamel at the root apex to the oldest enamel at the incisal edge (Robinson et al., 1997). We expect that amelogenesis in the continuously erupting rat incisor and in mouse molars of limited growth are highly similar because of the known similarities in the rat and mouse genomes, overall developmental patterns, and enamel mineral composition. In the present study, the mouse molar amelogenesis model was chosen because of the availability of mouse proteomics analysis tools and the feasibility of fresh enamel matrix preparation on successive days of postnatal amelogenesis.

We began our study by documenting the daily incremental increase in the thickness of enamel covering the distal slopes of the molar cusps between 1 dpn until 8 dpn. During this time, the semi-transparent enamel layer increased in thickness from 1 μm covering to 75 μm. Throughout those 8 days, the matrix was pliable and ideally suited for further biochemical analysis as it allowed for harvesting of the matrix and the attached enamel organ in bulk and in daily increments. The pliable nature of the developing enamel matrix has impressed naturalists and early biochemists since John Hunter's time (Hunter, 1778; Eastoe, 1979; Termine et al., 1980), and as a result has been termed cheesy or cheese-like by the early enamel biochemists (Alan Fincham, personal communication). The mouse molar enamel matrix retained its opaque, soft, and cheese-like consistency throughout postnatal development, with the exception of postnatal day 8, when the hardening of the matrix began. At that stage, matrix birefringence was at its peak, causing the enamel matrix to appear in alternating polarization colors depending on the angle between polarizing and analyzing filters. Earlier studies have focused on the anisotropy of the enamel matrix during the secretory stage (Keil, 1935; Schmidt, 1959; Spears et al., 1993; Do Espirito Santo et al., 2006). The highly polarized structure of the enamel matrix is evidence of the highly ordered enamel matrix structure as it provides a template for the highly ordered enamel mineral layer as a biomechanical buffer to occlusal loads and stresses.

X-ray diffraction analysis unambiguously identified enamel matrix diffraction patterns from day 4 to day 8 postnatal as partially fluoride substituted hydroxyapatite, and apatite electron diffraction patterns of 6 and 7 dpn mouse molar enamel were confirmed in the present study and in earlier electron diffraction studies (Diekwisch et al., 1995; Diekwisch, 1998). In contrast, X-ray diffraction patters from 1 to 2 dpn samples contained less prominent peaks that nevertheless matched the same overall partially fluoride substituted hydroxyapatite pattern of the samples from older enamel, and similarly, our earlier electron diffractions studies revealed fewer and less pronounced diffraction rings, suggesting that these earlier stages of enamel matrix maturation contain precursor phases of apatite maturation, such as octacalcium phosphate or tricalcium phosphate (Diekwisch et al., 1995; Aoba et al., 1998; Diekwisch, 1998). There were several non-identified peaks present in the 2 dpn sample, which were neither found in the 1 dpn sample nor in the 4–8 dpn samples, suggesting the presence of unusual intermediate phases during the transition from apatite precursors to fully mature hydroxyapatite. Together, these data demonstrate that the bulk of the distal slope first mandibular mouse molar enamel matrix converts from a lesser order of crystallinity into crystalline partially fluoride substituted hydroxyapatite between 2 and 4 dpn.

Our ATR data were interpreted according to previously published band assignments (Vignoles-Montrejaud, 1984; Elliot, 1994; Gadaleta et al., 1996; Boskey et al., 2006; Antonakos et al., 2007; Leventouri et al., 2009) and provide spectroscopic evidence for the structural conversion of the mouse molar enamel matrix from a mixed amorphous calcium phosphate/protein layer at 2 dpn to a crystalline hydroxyapatite structure featuring highly elongated crystals at 8 dpn. On a protein level, this change was accompanied by increased peak heights of the amide I and II bands, especially from postnatal day 6 to day 8, indicative of changes in protein secondary structure and increased rigidity of the peptide bonds between the carboxyl and the amino groups of two adjacent amino acids through an increase in the double bond character of the peptide bond, resulting in increased structural rigidity of the enamel protein matrix.

Our ATR data revealed dramatic changes in the phosphate region (800–1,100 cm⁻¹) in the postnatal mouse molar develop enamel matrix. Specifically, our data demonstrated a transition from a contoured plateau (1,000–1,100 cm⁻¹) indicative of amorphous calcium phosphate at 2 dpn to a single sharp and highly elevated peak (1,015 cm⁻¹) with shoulders at 1,105 and 958 cm⁻¹ at 8 dpn representative of high crystalline hydroxyapatite with crystals featuring long c-axis dimensions (Gadaleta et al., 1996). These findings are in congruence with our previous studies suggestive of a gradual transition from amorphous calcium phosphate to highly ordered and elongated apatite crystals during amelogenesis through a process called Ostwald ripening (Diekwisch et al., 1995; Aoba et al., 1998; Diekwisch, 1998).

Moreover, there was strong evidence for carbonate substitution in the enamel matrix between postnatal days 6 and 8, as the peaks at 880 cm⁻¹ (A-type carbonate) and 1,405 and 1,445 cm⁻¹ (B-type carbonate) indicated. Carbonate is known to replace phosphate in biological apatites (Zapanta-Legeros, 1965; Wopenka and Pasteris, 2005), affects its physical properties, including decreased crystallinity and increased c-axis length (Fleet et al., 2004; Wopenka and Pasteris, 2005; Boskey et al., 2006) and mechanical properties, such as decreased hardness and Young's modulus (Morgan et al., 1997; Xu et al., 2012).

Our proteomic analysis provided only low counts for classic enamel proteins, which are known to be of hydrophobic nature (Eastoe, 1965), even though its most prominent member, amelogenin, comprises 80–90% of the developing enamel matrix protein composition (Fincham et al., 1999). This result is characteristic for unmodified proteomic studies, which have a systematic bias against hydrophobic proteins and membrane proteins and favor hydrophilic components instead (Santoni et al., 2000; Chandramouli and Qian, 2009; Josic, 2014). To address the systematic scarcity of hydrophobic proteins in proteomic datasets, classic Western blot studies of enamel protein expression from days 1 to 8 postnatal were performed. These studies demonstrated a continuous increase in amelogenin from day 1 to 8, relatively high levels of ameloblastin from days 1 to 5, peak enamelin protein level peaks on day 1 and 2, relatively high levels of MMP20 from days 1 to 3, and relatively high levels of carbonic anhydrase between postnatal days 2 and 4. These data underscore the continuous presence of amelogenin during the secretory stages of amelogenesis (Termine et al., 1980), while ameloblastin, enamelin, and MMP20 are only prominent during early secretory stage enamel development, possibly establishing a patterning template for enamel crystal and prism growth (Bartlett, 2013; Pugach et al., 2013; Zhu et al., 2014; Prajapati et al., 2016). Our Western blot data provided evidence for relatively high levels of carbonic anhydrase during early enamel development (days 1–4), indicative of a role for pH adjustment during initial crystal nucleation, while other regulators may be involved in the pH regulation during the massive elevation of phosphate and carbonate during late-stage crystal growth. In addition, the continuous increase in amelogenin during postnatal enamel development was also verified by our proteomics study, albeit at a lower level of counts due to its hydrophobicity. Three of the proteins analyzed in Figure 4 (Calmodulin, Cofilin-1, and Peptidyl-prolyl cis-trans isomerase) featured higher levels of expression on days 2 and 6, and a substantial decline in protein counts on day 4. We interpret these data to indicate that such proteins may have dual functions during enamel ion transport and enamel crystal growth.

Disregarding the moderate counts for classic enamel proteins such as amelogenin, ameloblastin, and enamelin, our proteomics analysis detected a number of other proteins relevant for enamel mineralization during our 2–6 days postnatal mouse molar enamel matrix sampling window. Among these was the protein with the highest number of counts in our analysis, alkaline phosphatase, which peaked at day 2 during the early secretory stage. At the onset of amelogenesis, alkaline phosphatase may be involved in transporting phosphate from blood vessels near the stratum intermedium into the enamel organ by increasing local phosphate concentrations in the stratum intermedium via hydrolysis of phosphorylated substrates. Phosphatase mediated hydrolysis of pyrophosphate may also be involved in the generation of other phosphorylated macromolecules (Woltgens et al., 1995; Liu et al., 2016). The other mineralization-related protein with an early peak at 2 days postnatal was the protein folding chaperone calnexin, which may play a role in the prevention of endoplasmic reticulum stress due to misfolded enamel proteins or in calcium transport (Wang et al., 2005; Brookes et al., 2014). Calreticulin, which peaked at postnatal day 4, is another enamel protein with a dual function misfolded protein processing and calcium transport (Somogyi et al., 2003) that was detected in our proteomics analysis. Two other calcium transport proteins, calmodulin and reticulocalbin-3, peaked on day 6 (maturation stage, calmodulin) or on day 4 (late secretory stage, reticulocalbin-3). Calmodulin and reticulocalbin are capable of binding calcium using an EF-hand motif, and this function may be related to the protein-mediated transport of calcium ions through the ameloblast cell layer. The most prominent enzyme in our proteomics analysis of mouse molar enamel organs was the lysosomal cysteine protease cathepsin B, which peaked on day 4 postnatal. Cathepsin B is an important lysosomal enzyme of the enamel matrix (Al Kawas et al., 1996; Tye et al., 2009) that has been shown to enhance the activity of other proteases such as metalloproteinases and cathepsin D (Hammarström et al., 1971; Blair et al., 1989). As such, cathepsin B may promote the proteolytic degradation of the enamel matrix by enhancing the activity of MMP20 and cathepsin D.

Four of the six high-scoring cytoskeletal proteins in the enamel organ, including actin, tubulin, tropomyosin, and vimentin isoforms, peaked at postnatal day 4 during the late secretory stage. Increased presence of cytoskeletal proteins during the late secretory stage is likely indicative of their role in cell polarization and vesicular secretion (Manneville et al., 2003; Neco et al., 2003; De Lisle, 2015). Highly specific actin and tubulin immunoreactivity in secretory ameloblasts has been reported earlier (Diekwisch, 1988; Kero et al., 2014). Cytoskeleton associated protein 4 (CLIMP63) is actually a transmembrane protein that is instrumental in anchoring the endoplasmic reticulum to microtubules (Vedrenne et al., 2005), and its early secretory stage expression matches that of other transmembrane proteins reported here (cadherin, catenin). In contrast, Cofilin-1 levels only became elevated during the resorptive maturation stage (6 dpn), which may be explained by its role in actin depolymerization (Maciver and Hussey, 2002; Morita et al., 2016). Vimentin has long been hailed as an intermediate filament protein marker of mesenchymal tissues (Kidd et al., 2014). However, our present study reports strong evidence for vimentin signals in the enamel organ, lending support to earlier studies about transitory vimentin expression in the stellate reticulum (Kasper et al., 1989; Kero et al., 2014). Together, our proteomic data provide strong evidence for high levels of cytoskeletal proteins at the late secretory stage, likely related to their role in ameloblast polarization and enamel matrix secretion.

Our proteomics data indicated that six high-scoring extracellular matrix/cell surface molecules identified in the present study all peaked on day 2 postnatal at the onset of the secretory stage. This group included the classic extracellular matrix molecule laminin, the integrin β1 cell surface receptor, the β-integrin binding extracellular matrix adhesion molecule vinculin, and three cell adhesion molecules of the catenin/cadherin complex. The presence of laminin as part of the ameloblast basal lamina at the dentin-enamel junction has been demonstrated to play a role in terminal odontoblast differentiation (Lesot et al., 1981), and this basal lamina is removed during further ameloblast differentiation, presumably facilitating enamel deposition in sarcopterygian vertebrates (Diekwisch et al., 2002). Based on previous studies, we speculate that both the adherens junction protein vinculin and its binding partner β-integrin function to establish the sliding interface between secretory ameloblasts that allows for ameloblast cell movements during prism formation (Kubler et al., 1988; Nishikawa et al., 1988, 1990; Xu et al., 1998; Saito et al., 2015). Three of the six significantly upregulated proteins belonged to the cadherin-based adherens junction complex, namely catenin α1 and cadherins 1 and 2. Catenins are known to interact with cadherins (Rangarajan and Izard, 2013) and provide a link between adherens junctions and the actin cytoskeleton (McCrea and Gu, 2010). In ameloblasts, E-cadherin appears to be involved in ameloblast polarization (Terling et al., 1998) and β-catenin is essential for ameloblast movement (Guan et al., 2016). A switch between E-cadherin and N-cadherin has been documented in ameloblasts that slide by each other to form decussating enamel rod patterns (Guan et al., 2014). Together, the high levels of adherens junction and matrix proteins in the early enamel organ are indicative of the involvements of these proteins at the early secretory stage of amelogenins.

Three heat shock/stress proteins also were among the high-scoring proteins that peaked at the onset of the secretory stage. These proteins included the 60 kDa heat shock protein (Hspd1), the 78 kDa glucose regulated protein (Grp-78), and the chaperone stress 70 protein (Hsp70). All three of these proteins are involved in macromolecular assembly, the prevention of misfolding, as well as the prevention of aggregation. One of the major challenges of amelogenesis is the transport of amelogenin, a protein prone to self-assembly (Zhang et al., 2011), through the ameloblast cells. It is likely that both Grp-78 and Hsp70 may be involved in the prevention of amelogenin self-assembly inside of the ameloblast cell body, as both proteins are known to prevent protein aggregation (Wegele et al., 2004; Mayer and Bukau, 2005), while Hspd1 might rather function to prevent amelogenin misfolding (Xu et al., 2006).

Three isomerases joined the list of high-scoring enamel proteins, including the protein disulfide isomerases A3 and A6, and the peptidyl-prolyl cis-trans isomerase. Disulfide isomerases (PDIs) catalyze protein folding by facilitating disulfide bond formation and arrangement (Kersteen and Raines, 2003), a process that apparently takes place during the entire secretory stage, as PDI peaks in our proteomics data indicate. In contrast, peptidyl-prolyl cis-trans isomerase interconverts the trans-isomers of newly synthesized peptide bonds into cis-isomers of higher biological activity (Herzberg and Moult, 1991; Pal and Chakrabarti, 1999; Balbach and Schmid, 2000; Shaw, 2002). Peptidyl-prolyl cis-trans isomerase function may play a role in the conformational modification of amelogenin and enable its role as a molecular hinge in the promotion of crystal growth (Delak et al., 2009).

In conclusion, this integrative proteomic/cell biological analysis of postnatal mouse molar enamel development identifies many of the substantial changes in the enamel matrix protein and mineral phase that take place during postnatal mouse molar amelogenesis, including (i) relatively high levels of matrix protein secretion during the early secretory stage on postnatal day 2, (ii) conversion of calcium phosphates to apatite, peak protein folding and stress protein counts, and increased cytoskeletal protein levels such as actin and tubulin on day 4, as well as (iii) secondary structure changes, isomerase activity, highest amelogenin levels, and peak phosphate/carbonate incorporation between postnatal days 6 and 8 (Figure 5C).

All animal experiments were approved by the IACUC committees at the University of Illinois, Chicago and Texas A&M College of Dentistry.

MP, WZ, and TD wrote the manuscript. SG, TK, XL and TD designed the experiments. MP, HL, SD, LL,WZ, SP, and RD performed the experiments. MA designed and performed the statistical analysis.