Journal of Structural Biology 213 (2021) 107805

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Journal of Structural Biology

journal homepage: www.elsevier.com/locate/yjsbi

Graphical Structural Biology Review

A genetic model for the secretory stage of dental enamel formation

James P. Simmer a, *, Jan C-C. Hu a, Yuanyuan Hu a, Shelly Zhang a, Tian Liang a,
Shih-Kai Wang b, c, Jung-Wook Kim d, e, Yasuo Yamakoshi f, Yong-Hee Chun g, John D. Bartlett h,
Charles E. Smith a, i
a
Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, 1011 North University, Ann Arbor, MI 48108, USA
b
Department of Dentistry, National Taiwan University School of Dentistry, No. 1, Changde St., Zhongzheng Dist., Taipei City 100, Taiwan
c
Department of Pediatric Dentistry, National Taiwan University Children’s Hospital, No. 8, Zhongshan S. Rd., Zhongzheng Dist., Taipei City 100, Taiwan
d
Department of Molecular Genetics, School of Dentistry & Dental Research Institute, Seoul National University, Seoul, Korea
e
Department of Pediatric Dentistry, School of Dentistry & Dental Research Institute, Seoul National University, Seoul, Korea
f
Department of Biochemistry and Molecular Biology, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan
g
Department of Periodontics, School of Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
h
Division of Biosciences, Ohio State University College of Dentistry, Columbus, OH, USA
i
Department of Anatomy & Cell Biology, Faculty of Medicine & Health Sciences, McGill University, Montreal, Quebec H3A 0C7, Canada

A R T I C L E I N F O A B S T R A C T

Edited by ’Elia Beniash’ The revolution in genetics has rapidly increased our knowledge of human and mouse genes that are critical for
the formation of dental enamel and helps us understand how enamel evolved. In this graphical review we focus
Keywords: on the roles of 41 genes that are essential for the secretory stage of amelogenesis when characteristic enamel
Biomineralization mineral ribbons initiate on dentin and elongate to expand the enamel layer to the future surface of the tooth.
Amelogenesis
Based upon ultrastructural analyses of genetically modified mice, we propose a molecular model explaining how
Evolution
a cell attachment apparatus including collagen 17, α6ß4 and αvß6 integrins, laminin 332, and secreted enamel
Basement membrane
SLC13A5 proteins could attach to individual enamel mineral ribbons and mold their cross-sectional dimensions as they
ACP4 simultaneously elongate and orient them in the direction of the retrograde movement of the ameloblast
membrane.

1. Introduction epithelial-mesenchymal interactions that were co-opted for the induc­

tion of multiple epithelial organs (Thesleff, 2006). This common
The gar is a fish that has enamel on the surface of its scales and teeth evolutionary history is a basis for genetic mutations in single genes
(Kawasaki et al., 2021; Sire, 1995). Enamel was formed by the common resulting in the ectodermal dysplasias and other syndromes that
ancestor of coelacanth and gar (>450 mya) and appeared first on scales combine tooth agenesis and/or dental malformations with aplasia of
that pre-date the development of enamel on teeth (Braasch et al., 2016). lacrimal and salivary glands, reductions in tongue fungiform and fili­
Teeth and scales arise from odontodes that evolved from a common form papillae, hyperkeratosis of the skin, skin fragility, etc (Adaimy
founder odontode and consequently their developmental processes rely et al., 2007).
upon an overlapping repertoire of genetic elements (Chen et al., 2020). Because of its evolutionary history, dental enamel formation (ame­
A striking demonstration of this relationship is the significant loss of logenesis) relies upon a variety of genetic elements that are critical for
both scales and teeth in a teleost (Medaka) caused by a splice junction the attachment of skin to its underlying basement membrane and
mutation in Edar (Atukorala et al., 2011). In humans, EDAR mutations mesenchymal tissues. Because of this, mutations in COL17A1, LAMA3,
cause hypohidrotic ectodermal dysplasia showing tooth agenesis and LAMB3, LAMC2, PLEC, ITGB4 and ITGA6 cause epidermolysis bullosa
abnormal morphogenesis of teeth, hair, and sweat glands. The founder with skin fragility and enamel malformations (Chung and Uitto, 2010;
odontode for teeth apparently depended upon a series of reciprocal Fine, 2010). It is curious that the skin attachment apparatus is conserved

* Corresponding author.
E-mail addresses: jsimmer@umich.edu (J.P. Simmer), janhu@umich.edu (J.C.-C. Hu), yyhu@umich.edu (Y. Hu), shellyhuzhang@gmail.com (S. Zhang), tianl@
umich.edu (T. Liang), shihkaiw@ntu.edu.tw (S.-K. Wang), pedoman@snu.ac.kr (J.-W. Kim), yamakoshi-y@tsurumi-u.ac.jp (Y. Yamakoshi), chuny@uthscsa.edu
(Y.-H. Chun), bartlett.196@osu.edu (J.D. Bartlett), charles.smith@mcgill.ca (C.E. Smith).

https://doi.org/10.1016/j.jsb.2021.107805
Received 25 August 2021; Received in revised form 18 October 2021; Accepted 20 October 2021
Available online 27 October 2021
1047-8477/© 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
J.P. Simmer et al. Journal of Structural Biology 213 (2021) 107805

Fig. 1. Visualization of enamel formation by focused ion beam-backscattered scanning electron microscopy (FIB-bSEM) of a continuously growing 7-week mouse
mandibular incisor. A) Ameloblasts are separated from the unmineralized predentin matrix by a well-defined basement membrane (BM). The predentin matrix
contains odontoblastic processes, matrix vesicles released by odontoblasts, and numerous collagen fibrils/fibers that are oriented toward the ameloblasts where they
appear to attach to the BM. B) The BM thins as the ameloblast membrane becomes less linear. C) Ameloblast finger-like processes penetrate the BM and extend into
predentin between the collagen fibrils, as remnants of the BM accumulate along the ameloblast membrane that is reabsorbing it. D) Mineral nuclei appear in
predentin some distance from the ameloblast membrane (arrowheads). E) Dentin mineral coalesces into a continuous mineral layer that extends to near the
ameloblast membrane. F) Enamel ribbons initiate in patches on dentin mineral associated with the ends of mineralized collagen fibers. G) A continuous segment of
mouse incisor completing the formation of initial enamel (above the G) and showing the progressive formation of Tomes processes as the segment continues in a
second row below the G. The ends of Tomes’ processes form by the rapid extension of “prongs” of mineral ribbons at interrod growth sites along the peripheral part of
the distal ameloblast membrane near the cell junctions. H) High magnification of initial enamel ribbons forming on the ends of collagen fibers and extending at
different angles back to the ameloblast membrane, following the finger-like process as it retreated into the cell membrane. I) Rod and interrod enamel after formation
of the Tomes process [asterisk indicates a space of Weber (Bartlett et al., 2021)]. J) Tomes’ process showing the positions of rod (r) and interrod (ir) growth sites in
the mineralization front. K) Maturation stage enamel has a basal lamina comprised of SCPP proteins bound to the surface enamel. Key: Am, ameloblast; d, dentin, e,
enamel, ir, interrod growth site; pd, predentin; r, rod growth site; tp, Tomes’ process; white scale bars (under figure letters) = 500 nm.

in ameloblasts, because ameloblasts degrade their basement membrane 1 (SPARCL1), itself duplicated from secreted protein acidic cysteine-rich
prior to the onset of amelogenesis, then abruptly lose contact with a (SPARC), a gene that encodes a basement membrane protein (Jayadev
mesenchymal matrix once enamel starts to form. While the genes noted and Sherwood, 2017). There are 24 SCPP genes in humans (Kawasaki
above are associated with hemidesmosomes in skin, hemidesmosomes and Amemiya, 2014). The enamel-associated SCPP genes are ameloge­
are not observed in ameloblasts before the maturation stage (Sahlberg nin (AMEL), enamelin (ENAM), ameloblastin (AMBN), amelotin
et al., 1998). Nevertheless, ameloblasts detach from underlying mineral (AMTN), odontogenic, ameloblast associated (ODAM), secretory
specifically at the onset of enamel formation in Lama3− /− mice, con­ calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1)
firming that laminin 332 (comprised of LAMA3, LAMB3, and LAMC2) and dentin sialophosphoprotein (DSPP). DSPP is a dentin protein that is
strengthens the anchorage of secretory ameloblasts to the underlying transiently expressed by pre-ameloblasts, immediately prior to the onset
enamel matrix (Ryan et al., 1999; Sahlberg et al., 1998). Laminin 332 of enamel mineralization. Three SCPP genes are expressed by secretory
likely binds to secreted enamel proteins that bind directly to the enamel ameloblasts: AMEL, ENAM, and AMBN (Fincham et al., 1999).
mineral ribbons. Strong concepts of how dental enamel forms were formulated long
Secreted enamel proteins are encoded by genes belonging to the before the genetics revolution and centered attention on amelogenin,
secretory calcium-binding phosphoprotein (SCPP) gene family (Kawa­ the most abundant secreted enamel protein (Simmer and Fincham,
saki and Weiss, 2003), which was spawned by duplication of SPARC-like 1995; Termine et al., 1980). Amelogenin was the first enamel protein to

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Fig. 2. Graphical Illustration of ameloblasts showing proteins critical for the secretory stage. CLDN1, CLDN10, CLDN16, and CLDN19 encode tight junction proteins
restricting entry of intercellular ions and molecules into the enamel space and also function in cell motility. GJA1 encodes a gap junction protein important for
intercellular communication with adjacent ameloblasts. FAM20A and FAM20C encode components of the Golgi casein kinase complex that phosphorylates secreted
enamel proteins. FAM83H encodes a cytosolic protein associated with the trans-Golgi network (TGN) and the keratin cytoskeleton. GALNS encodes a lysosomal
hydrolase necessary for the degradation of glycosaminoglycans. PEX1, PEX6, and PEX26 are necessary for peroxisome biogenesis. SLC13A5 encodes a membrane
citrate channel providing citrate influx proximally and efflux (into the enamel matrix) distally. Small secretory vesicles (magenta) in the proximal and distal parts of
the Tomes’ process (distally) secrete AMELX, AMBN, ENAM, and MMP20 at the rod and interrod growth sites. Thin lines in the extracellular matrix (bottom)
represent the general direction of enamel mineral ribbons. Not shown: CACNA1C encodes an L-type (voltage-dependent calcium channel) thought to provide
regulated Ca2+ influx. SLC10A7 encodes a transmembrane transporter of unknown specificity that causes severe enamel defects when mutated. Graphic by
Shelly Zhang.

have its cDNA cloned and to be expressed as a recombinant protein. In enamel hypoplasia, or thin enamel, which is caused by deficiencies
vitro studies of recombinant amelogenin under different conditions affecting the secretory stage of amelogenesis (when the enamel layer
spawned competing theories of amelogenin’s role in amelogenesis (Bai achieves its final thickness). A table of 41 human genes that encode
et al., 2020; Fang et al., 2011; Fincham et al., 1994) and encouraged proteins necessary for normal formation of the full thickness of the
hope for the production of synthetic enamel as a dental restorative. The enamel layer is provided (Table S1). Ten of these genes regulate gene
genetics of human conditions associated with enamel malformations, expression or cease expression prior to the onset of enamel mineraliza­
however, suggests that amelogenesis is more complex than previously tion and are not discussed further.
realized.
A search in 2015 of Online Mendelian Inheritance in Man (OMIM) for 2. Ultrastructure of developmental changes along the
hereditary conditions that include an enamel phenotype revealed 91 ameloblast distal membrane
such conditions, 71 with a known molecular etiology or linked genetic
loci (Wright et al., 2015). The most common enamel phenotype is The fundamental feature of forming dental enamel is the growth of

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J.P. Simmer et al. Journal of Structural Biology 213 (2021) 107805

Fig. 3. bSEM and FIB-bSEM visualization of initial enamel forming in 7-week mouse mandibular incisors in Amelx− /− , Enam− /− , Ambn− /− , Amelx− /− Ambn− /− ,
Mmp20− /− , Acp4R110C/R110C, and Slc13a5R337*/R337* mice. Top: bSEM sections of incisor cross-sections at the level of the labial alveolar crest (near the point of
eruption). All of the knockouts showed severe enamel hypoplasia, many with ectopic mineral deposition within the overlying enamel organ. Below: FIB-bSEM images
of where enamel should be forming in 8 different mouse incisors all at the same magnification. Characteristic initial enamel mineral ribbons form in Mmp20− /− ,
Amelx− /− , and Acp4R110C/R110C mice, while no enamel ribbons form in Enam− /− and Ambn− /− mice. Ambn− /− nulls show accumulations of organic material
comprised mainly of amelogenin that are greatly reduced in the Amelx− /− Ambn− /− double null. Key: Am, ameloblast; b, bone; d, dentin; e, enamel.

characteristic thin mineral ribbons that start on the surface of dentin ameloblast membrane. In humans, the cross-sectional dimension of the
(Smith et al., 2016) and extend to a mineralization front 250–300 Å from ribbons at the mineralization front is about 15 Å × 150 Å (Kerebel et al.,
the ameloblast plasma membrane (Ronnholm, 1962b) where the min­ 1979). Near the mineralization front where the ribbons elongate, they
eral ribbons elongate, apparently by the addition of ions or mineral to are comprised of amorphous calcium phosphate, but crystalize with
their tips. The mineralization front is only slightly more distant from the depth where they produce diffraction patterns characteristic of apatite
ameloblast membrane as the basement membrane was prior to its earlier (Beniash et al., 2009). The morphology and organization of the mineral
degradation and reabsorption (Ronnholm, 1962a). in enamel is determined prior to its crystallization, suggesting that the
Enamel formation is a biological process that is accomplished by the consistent dimensions of the cross-sections at the mineralization front is
ameloblast distal membrane as it progresses sequentially through a se­ accomplished by molding, since non-crystalline minerals such as
ries of modifications that can be readily observed at the ultrastructural amorphous calcium phosphate that don’t have crystalline faces cannot
level, as shown in Fig. 1. All vertebrates yet characterized that form be shaped by proteins binding to and selectively inhibiting the growth of
“ancestral” or “true” enamel (Kawasaki et al., 2021) do so by ameloblast- specific crystal faces. The smooth increase in cross-sectional dimensions
like cells depositing characteristically thin mineral ribbons on mineral­ of the crystallites with depth (Nylen et al., 1963; Warshawsky, 1989) is
ized collagen and extending those ribbons in close proximity to the accomplished by a relatively slow, but steady rate of ions depositing

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J.P. Simmer et al. Journal of Structural Biology 213 (2021) 107805

Fig. 4. FIB-bSEM visualization of initial enamel forming in 7-week mouse mandibular incisors in in Slc13a5R337*/R337* and wild-type mice. A) Although delayed,
patches of enamel ribbons form on the surface of mineralized dentin in Slc13a5R337*/R337* mice. These patches do not appear to be stable and were not observed
further incisally (shown in B). This phenotype suggests that tribasic citrate (C6H5O73− ) is required to stabilize the thin mineral ribbons. After the initial ribbons
disappear or fail to form, the mineral surface on dentin appears “spiky”. These spikes are possibly mineral associated with the frayed ends of collagen fibrils. In some
places the ends of these spikes are associated with the ameloblast membrane, suggesting they possibly elongate as enamel mineral ribbons in wild type mice. A spiky
dentin surface is also observed in Enam− /− , Ambn− /− , and Ambn− /− Amelx− /− mice after initial enamel deposition fails (Liang et al., 2019). C) Enamel ribbon for­
mation in a wild type incisor at a similar position as the one shown in A above. Note that at this level of development, enamel has already progressed beyond initial
enamel formation and is forming rod and interrod enamel. Also note that the wild type enamel mineral ribbons show flexibility and bend without breaking. This
flexibility of enamel mineral ribbons may be imparted by their association with enamel proteins and/or citrate. Key: Am, ameloblast; d, dentin; e, enamel.

onto the sides of the ribbons. Many studies support the concept that sites (Skobe, 2006) and generate enamel rods. Parallel arrays of char­
secreted enamel proteins, particularly the abundant amelogenins, can acteristic enamel mineral ribbons in rods extend at different orientations
bind to the sides of the crystallites and inhibit mineral deposition and than the parallel arrays of identical ribbons extending at interrod growth
regulate the conversion of ACP to apatite (Bartlett et al., 2021; Hu et al., sites. Reproducible variations in the patterns of rod and interrod enamel
2016; Margolis et al., 2014; Shaw et al., 2020; Yamazaki et al., 2019). between different types of mammals and even at different depths within
Proteolytic cleavages and influxes of calcium, phosphate, and citrate a single tooth provide strong evidence for ameloblast membrane control
ions potentially regulate the attachment of proteins to mineral surfaces. over the oriented elongation of enamel mineral ribbons (Boyde, 1969).
Enamel mineral ribbons are oriented with respect to the ameloblast The hierarchical organization of enamel mineral ribbons in mammals
membrane extending them. The ribbons are typically oriented perpen­ appears to be unexplainable by models that form mineral in the presence
dicular to the membrane and in the direction of the membrane’s retro­ of amelogenin in vitro without cell participation.
grade movement that provides space for ribbon elongation. After
forming a thin initial enamel layer on dentin mineral with essentially all 3. Critical functions of ameloblasts
of the mineral ribbons oriented in a parallel array, mammalian amelo­
blasts develop a characteristic distal membrane specialization called a Secretory stage ameloblasts are tall, cylindrical, densely-packed cells
“Tomes process” by temporarily extending enamel ribbons only from forming a sheet that delineates the protected space covering the surface
“interrod growth sites” along the periphery of the ameloblast distal of dentin and forming enamel (Smith, 1979). Ameloblasts absorb ions
membrane near its borders with adjacent ameloblasts. This generates and nutrients while expelling waste products along their proximal
“prongs” of enamel ribbons that cause the central part of the distal membrane. They secrete and reabsorb ions and proteins along their
membrane to protrude into the forming enamel (Nanci and War­ distal membrane, while attaching to and elongating the enamel mineral
shawsky, 1984). “Rod growth sites” located on the protruding part of the ribbons. Groups of ameloblasts within the sheet move relative to other
Tomes extend enamel ribbons at a deeper level than the interrod growth groups and retreat from the enamel surface as the enamel layer expands.

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J.P. Simmer et al. Journal of Structural Biology 213 (2021) 107805

expression 21-fold and Enam expression 7.6-fold (Mohazab et al., 2013).

The ultrastructure of the initial enamel formed in wild-type, Amelx− /
−
, Enam− /− , Ambn− /− , Amelx− /− Ambn− /− , Mmp20− /− , Acp4R110C/R110C,
and Slc13a5R337*/R337*mice is shown in Fig. 3. While amelogenesis is
seriously disrupted in all of these mice, oriented initial enamel ribbons
form in Amelx− /− , Mmp20− /− , and Acp4R110C/R110C mice, whereas none
form at all in Enam− /− and Ambn− /− mice. Only isolated patches of
short, poorly organized enamel ribbons form in Slc13a5R337*/R337* mice
and appear to dissolve before they can extend further (Fig. 4).
The formation, organization, and orientation of enamel mineral
ribbons are fundamental characteristics that have so far proved to be
inseparable genetically. Long, characteristically-shaped enamel mineral
ribbons that are oriented haphazardly have never been observed in
knockout mice.

4. Enamel ribbon attachment, elongation and orientation

A molecular model of enamel mineral ribbon formation is proposed

in Fig. 5. We hypothesize that enamel ribbons are shaped by a molding
process that simultaneously lengthens and orients the mineral ribbons
Fig. 5. Molecular model for an ameloblast attachment apparatus that simul­
taneously shapes, elongates, and orients enamel mineral ribbons. The specifics by the incremental addition of ions or mineral to the tips of existing
of the model are hypothetical and are based upon a model for skin attachment enamel ribbons in the direction of the retrograde movement of the
(Chung and Uitto, 2010). This model proposes that enamel ribbon shape is ameloblast membrane. We propose that the molds are comprised of
determined by a molding process that determines the cross-sectional shape of secreted enamel proteins, most likely enamelin and ameloblastin, con­
the tips of the enamel ribbons as increments of mineral are added to extend the nected to integrin complexes (α6ß4 and/or αvß6 integrin) via laminin
ribbons in the direction of the retrograde movement of the ameloblast mem­ 332 and COL17A1. Maintaining a portion of the mold bound to hard­
brane (in light blue and spanned by the SLC13A5 citrate transporter on the left), ened mineral secures the attachment of ameloblasts to the ribbons.
thereby orienting the ribbons as they elongate. The ENAM/AMBN mold is Extending the molds proximally allows for daily increments of apposi­
thought to be extended proximally and disassembled distally, presumably
tional growth, while degrading the molds distally allows for the slow
assisted by MMP20 cleavages. Intact enamelin (containing its C-terminus) is
growth of the enamel ribbons in width and thickness. This model sug­
only detected at the mineralization front (Hu et al., 1997). Amelogenin is scant
at the mineralization front (Nanci et al., 1998; Uchida et al., 1989) and not
gests that each ribbon is associated with a specific membrane apparatus
critical for shaping or orienting enamel ribbons, but is more important in throughout the secretory stage, that the membrane assemblies can rotate
separating the ribbons and guiding their transition from amorphous calcium to allow for the random orientation of enamel ribbons with respect to
phosphate into apatite. The connection of the mineral-associated enamel pro­ their A and B axes and can slip past each other in the membrane so that
teins to integrins α6ß4, αVß6 and COL17A1 is mediated by laminin 332. Inside enamel ribbons associated at the dentino-enamel junction (DEJ) are not
the cell the integrins bind to keratin filaments via plectin. SLC13A5 channels fixed in their relationship to adjacent ribbons as they elongate.
tribasic citrate ions into the enamel matrix. ACP4 is a membrane-bound acid This model explains how the mammalian adaptation of the Tomes’
phosphatase associated with the ameloblast Tomes’ process that may function process (which establishes partially distinct rod and interrod growth
during endocytosis and dephosphorylates enamel proteins (EP), perhaps prior sites with retrograde movements in different orientations), can establish
to their reabsorption, providing phosphate in the matrix for mineralization.
rod and interrod organizations of identical crystals oriented in different
RELT is a tumor necrosis factor receptor and LTBP3 is co-secreted with TGFß
directions (Nanci and Warshawsky, 1984). In rodent incisors the
that may mediate matrix-cell interactions. Graphic by Shelly Zhang.
movement of groups of ameloblasts relative to other groups generates a
decussation (X-shaped) pattern of rods in the enamel. Our model pre­
Many of the critical genes and proteins that carry out these activities
dicts that to maintain the cellular connection to each ribbon elongating
have been identified by their contributions to the etiology of inherited
at the mineralization front, the interrod enamel ribbons (which extend
enamel defects (Fig. 2; Table S1). Identifying critical genes allows
near the cell junctions) must contribute to multiple interrod structures as
genetically modified mice to be generated and characterized to learn
the ameloblast progresses from the DEJ to the final surface of enamel
more about the function of each gene and its protein products. The
(Nanci and Warshawsky, 1984).
primary objective is to determine the mechanism of enamel mineral
Future genetic advances will likely lengthen the list of genes critical
ribbon formation, which requires ultrastructural characterization of the
for the secretory stage of amelogenesis. We haven’t identified the pro­
mineral generated in the modified mice.
teins that transport calcium and phosphate ions into the secretory stage
We have yet to explain the roles of RELT, LTBP3, SLC13A5, ACP4,
enamel matrix, which would be a welcome addition to future amelo­
PLEC, LAMA3, LAMB3, COL17A1, ITGA6, ITGB4, ITGAV, and ITGB6.
genesis models.
RELT is a tumor necrosis factor receptor expressed by secretory stage
ameloblasts specifically, but its role in amelogenesis is unknown (Kim
5. Conclusion
et al., 2019). LTBP3 is co-secreted with TGFß, functions early in tooth
formation, and is associated with tooth agenesis. Plectin (PLEC), Lami­
The model proposes that each mineral ribbon is attached to a
nin 332 (LAMA3, LAMB3, LAMC2), COL17A1, and α6ß4 (ITGA6, ITGB4)
COL17A1 and integrin based membrane complex via secreted proteins
integrin combine to form an attachment complex in skin (Chung and
(laminin 332, ENAM, and AMBN) that mold the cross-sectional di­
Uitto, 2010) that is also required for attachment of ameloblasts to the
mensions of mineral added to the tip of each ribbon. The density of
underlying mineral (Ryan et al., 1999; Sahlberg et al., 1998). Integrin
membrane complexes establishes the average distance separating the
αvß6 (ITGAV, ITGB6) also contributes to cell attachment (Mohazab
ribbons, which determines the crystallites’ final thicknesses during
et al., 2013). The α6ß4 and αvß6 integrin attachment complexes provide
enamel maturation when they interlock with adjacent crystals. ACP4
intracellular feedback concerning conditions outside the cell at the
may dephosphorylate enamel proteins prior to their reabsorption into
mineralization front that significantly affect the expression levels of
ameloblasts, and release their phosphate for mineralization. SLC13A5
critical enamel proteins. Itgb6− /− ameloblasts increased Amelx
transports citrate into the enamel matrix, which stabilizes the thin

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mineral ribbons. AMEL and its MMP20 cleavage products bind, separate, Fang, P.-A., Conway, J.F., Margolis, H.C., Simmer, J.P., Beniash, E., 2011. Hierarchical
self-assembly of amelogenin and the regulation of biomineralization at the
and support the mineral ribbons, and guide their transition into apatite.
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editing. Yong-Hee Chun: Writing – review & editing. John D. Bartlett: Hu, Y., Smith, C.E., Cai, Z., Donnelly, L.A., Yang, J., Hu, J.C., Simmer, J.P., 2016. Enamel
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Declaration of Competing Interest secretory calcium-binding phosphoprotein gene cluster. PNAS 100 (7), 4060–4065.
Kawasaki, K., Amemiya, C.T., 2014. SCPP genes in the coelacanth: tissue mineralization
The authors declare that they have no known competing financial genes shared by sarcopterygians. J. Exp. Zool. B Mol. Dev. Evol. 322, 390–402.
Kawasaki, K., Keating, J.N., Nakatomi, M., Welten, M., Mikami, M., Sasagawa, I.,
interests or personal relationships that could have appeared to influence Puttick, M.N., Donoghue, P.C.J., Ishiyama, M., 2021. Coevolution of enamel, ganoin,
the work reported in this paper. enameloid, and their matrix SCPP genes in osteichthyans 24 (1), 102023. https://
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Kim, J.-W., Zhang, H., Seymen, F., Koruyucu, M., Hu, Y., Kang, J., Kim, Y.J., Ikeda, A.,
This study was supported by NIDCR/NIH research grants Kasimoglu, Y., Bayram, M., Zhang, C., Kawasaki, K., Bartlett, J.D., Saunders, T.L.,
Simmer, J.P., Hu, J.-C., 2019. Mutations in RELT cause autosomal recessive
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