# The Absence of Claudin-10 in the Enamel Organ Alters Its Integrity

T N Nguyen, S Ribes, C Andrique, M Requin, J Bouchet, N Obtel, L Slimani, N Brouilly, C Torrens, A Schmitt, et al.

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T N Nguyen, S Ribes, C Andrique, M Requin, J Bouchet, et al.. The Absence of Claudin-10 in the Enamel Organ Alters Its Integrity. Journal of Dental Research, In press, 10.1177/00220345251349109 . hal-05323532

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1 The Absence of Claudin-10 In the Enamel Organ Alters Its Integrity

2

3 The Nghia Nguyen¹†, Sandy Ribes ¹†, Caroline Andrique¹, Magali Requin², Jerome Bouchet¹,

4 Nicolas Obtel ¹,³, Lotfi Slimani¹, Nicolas Brouilly², Coralie Torrens¹, Alain Schmitt⁴, Thomas

5 Guilbert⁴, Maria Morawietz⁵, Andreas Kiesow⁵, Alain Brunelle⁶, Aline Percot⁷, Smail Hadj-

6 Rabia⁸, Céline Gaucher¹,³, André Le Bivic², Pascal Houillier¹⁰,¹¹, Claire Bardet¹, Dominik

7 Muller¹², Fernando Ramirez Rozzi¹,¹³, Thibaud Coradin¹⁴, Tilman Breiderhoff¹²,¹⁵, Catherine

8 Chaussain¹,³

9 ¹Université Paris Cité, INSERM U1333 Santé Orale, et Plateforme Imagerie du Vivant (PIV),

10 FHU-DDS-net, IHMOA, Dental School, Montrouge, France

11 ²Aix Marseille Université, CNRS, IBDM-UMR7288, Marseille, France.

12 ³AP-HP Services de médecine bucco-dentaire, Hôpitaux Universitaires Bretonneau (CRMR

13 phosphore et calcium, filière OSCAR et ERN BOND), and Henri Mondor and Charles Foix

14 (filière TETECOU et ERN CRANIO), FHU DDS-net, Ile de France, France

15 ⁴Université Paris Cité, Institut Cochin, INSERM U1016, CNRS UMR8104, Paris, France

16 ⁵Fraunhofer Institute for Microstructure of Materials and Systems IMWS, Walter-Huelse-

17 Straße 1, 06120 Halle, Germany

18 ⁶Sorbonne Université, CNRS, Laboratoire d'Archéologie Moléculaire et Structurale, 75005

19 Paris, France

20 ⁷Sorbonne Université, CNRS MONARIS UMR8233, 4 Place Jussieu, 75005 Paris, France.

21 ⁸Université Paris Cité, INSERM1163 Institut Imagine; APHP5, Hôpital Necker-Enfants

22 Malades Dpt of Dermatology, Reference Center for rare skin diseases, Paris, France

23 ¹⁰Université Paris Cité, Sorbonne Université, Centre de Recherche des Cordeliers, INSERM,

24 CNRS-ERL8228, F-75006 Paris, France.
25
11APHP, Service de Physiologie, Centre de Référence des Maladies Rénales Héréditaires de
l'Enfant et de l'Adulte (MARHEA), Hôpital Européen Georges Pompidou, F-75015 Paris,
26
France
27

28
12Charité Universitaets medizin Berlin, Department of Pediatrics, Division of Gastroenterology,
Nephrology and Metabolic Diseases, Augustenburger Platz 1, 13353 Berlin, Germany
29

30
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13Eco-anthropologie (EA), Muséum national d'Histoire naturelle, CNRS, Université de Paris,
Musée de l'Homme 17 place du Trocadéro 75016 Paris, France

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14Sorbonne Université, CNRS, Laboratoire de Chimie de la Matière Condensée de Paris, 75005
Paris, France.

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15present address: German Center for Neurodegenerative Diseases (DZNE), Munich, Germany

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† Contributed equally to this work

37
*Corresponding authors: Catherine Chaussain, INSERM U133, Université Paris Cité, 1 rue
Maurice Arnoux 92120 Montrouge, France; Tel and Fax: +33(0)158076724; email:
catherine.chaussain@u-paris.fr and Tilman Breiderhoff, Campus Virchow-Klinikum |
Augustenburger Platz 1 | 13353 Berlin, Germany tilman.breiderhoff@dzne.de

Abstract word count: 298

Total word count (Introduction to discussion): 3189

Total number of figures: 5

Total number of references: 39

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42
**Key words:** Tight junction; Stratum intermedium; ameloblast; renal dysfunction; xerostomia
43 Abstract

Rare disorders related to tight junction (TJ) proteins have been associated with *Amelogenesis Imperfecta*. Pathogenic variants of *CLDN10*, encoding claudin-10b, a cation transport pore, cause the autosomal recessive HELIX syndrome (Hypohidrosis, Electrolyte imbalance, hypoLacrymia, Ichthyosis, Xerostomia). Patients exhibit salivary dysfunction and rapid enamel wear after tooth eruption. Since *Cldn10* is expressed in the dental epithelium, this study explores the role of claudin-10b in amelogenesis. We analyzed amelogenesis in constitutive and conditional *Cldn10* knockout (KO) murine models, comparing the findings to human HELIX enamel. First, analysis of constitutive *Cldn10* KO mice, which die within a few hours after birth, showed that claudin-10 is present at the plasma membrane of the stratum intermedium but not at the TJs during the secretory stage. Its absence altered gene expression related to ion transport and pH control, although without major disturbance in cell polarization or enamel matrix synthesis. Examination of later stages of amelogenesis in epithelium-targeted conditional *Cldn10* KO mice showed that claudin-10 is present in the papillary layer at the maturation stage.

In its absence, the pH of the enamel matrix was more basic during early maturation, suggesting that claudin-10 determines enamel matrix pH. However, at later stage of the maturation process, the pH was corrected and the resulting enamel did not show major structural or elemental alterations. These later findings were confirmed by exploring the enamel of *Cldn10* KO transplanted tooth germs, which have developed in a controlled mineral environment. Nevertheless, higher contents of aluminum were detected in the enamel of transplanted germs and in human HELIX enamel, suggesting that claudin-10 deficiency may lead to a loss of enamel organ integrity. These data suggest that while salivary dysfunction is the main cause of enamel wear in HELIX, claudin-10 plays a direct role in amelogenesis by determining pH and enamel organ integrity.
**Introduction**

Tight junctions (TJs) form the backbone of the paracellular pathway for epithelial ion transport. Recently, the paracellular pathway has been shown to be highly ion-specific, tightly regulated, and causally involved in human diseases (Meoli and Gunzel, 2023). The specific set of claudins expressed at each TJ is uniquely tailored to the tissue and is responsible for the permeability and selectivity properties of the TJ. Several rare disorders resulting from pathogenic variants in a gene encoding a claudin (CLDN) have been reported; most of which result in disturbances of the ion homeostasis in the kidney (Meoli and Gunzel, 2023). We have previously reported that patients with FHHNC (Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis) due to mutations in CLDN16 or CLDN19 displayed amelogenesis imperfecta (AI) (Bardet et al., 2016; Yamaguti et al., 2017). Recently, a novel claudinopathy caused by pathogenic variants in CLDN10 has been reported, resulting in the HELIX syndrome (Hypohidrosis, Electrolyte imbalance-renal loss of NaCl with secondary hyperaldosteronism and hypokalemia-hypoLacrymia, Ichthyosis, and Xerostomia) (Hadj-Rabia et al., 2018; Klar et al., 2017). HELIX patients complained of xerostomia (dry mouth) due to a 98% reduction in salivary flow rate compared to controls (Hadj-Rabia et al., 2018). Claudin-10 exists in two isoforms, resulting from two alternative exons, 1a and 1b (CLDN10A, CLDN10B) (Gunzel et al., 2009). CLDN10B encodes claudin-10b, an integral membrane-spanning protein of tight junctions expressed in the kidney thick ascending limb of Henle's loop (TAL), skin, and salivary glands (Breiderhoff et al., 2012; He et al., 2024; Milatz, 2019) and also in the tooth germ (Chiba et al., 2020; Ohazama and Sharpe, 2007; Wang et al., 2020). We diagnosed early tooth wear in the six HELIX patients reported in (Hadj-Rabia et al., 2018). The teeth formed normally, but the enamel eroded rapidly after tooth eruption, suggesting that the enamel wear was mainly due to salivary dysfunction (Obtel et al., 2022). Since claudin-10b has been shown to be expressed in the mouse enamel organ (Chiba et al., 2020), a direct effect of its impairment on amelogenesis cannot be excluded. Therefore, this study aims to investigate the role of claudin-10b in amelogenesis. In this
purpose, we studied constitutive and conditional *Cldn10* KO mouse models, kidney capsule tooth germ transplantation model (Jimenez-Rojo and Mitsiadis, 2023), and human teeth (Obtel et al., 2022).

97

## 98 **Materials and Methods**

The detailed methodology is described in the Appendix Materials and Methods.

100

101 **Results**

102 *Claudin-10 is expressed in the stratum intermedium at all the stages of amelogenesis*
103 Converging data have indicated that *Cldn10/claudin-10* is expressed in the enamel organ, at
104 different locations and stages (Chiba et al., 2020; Ohazama and Sharpe, 2007; Wang et al.,
105 2020). In wild-type (WT) mice, both frontal and sagittal sections of the continuously growing
106 incisor revealed claudin-10 labeling in the stratum intermedium (SI), throughout all stages of
107 amelogenesis, from the cervical loop to the tip of the incisor at day 1 (P1) after birth (Fig.1A,
108 Appendix Fig.1-2A). Similar labeling was observed in the secretory ameloblasts of the first
109 molar germ (Appendix Fig.2B). The specificity of claudin-10 immunostaining was validated
110 using constitutive *Cldn10* KO mice (Appendix Fig. 3A). Co-labeling with the TJ protein ZO-1
111 showed the expression of claudin-10 in the SI, but not at the TJs (Fig.1A, Appendix Fig.1). To
112 clarify this later point, we performed STED microscopy and confirmed that the expression of
113 claudin-10 was restricted to the SI without co-labelling with ZO-1 (Fig1.B and Appendix Fig.3).
114 Co-labeling with connexin-43, a marker for gap junctions that are abundant in the SI (Liu et al.,
115 2016), also revealed no co-labelling with claudin-10 (Appendix Fig.4). Next, we performed
116 claudin-10 immunogold labeling and observed a positive signal in the plasma membranes of
117 the SI, but not associated with TJs (Fig.1C; Appendix Fig.5). Next, we assessed claudin-10
118

expression in the dental epithelium at the maturation stage in adult mice and observed positive
labeling of the papillary layer (Fig.1D).

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120

*The ameloblast layer is disturbed in constitutive Cldn10 KO pups.*

121

We next investigated constitutive Cldn10 KO mice to study the impact of claudin-10 deficiency
on the early stages of amelogenesis, considering that pups die a few hours after birth
(Breiderhoff et al., 2012) (Fig.2). Staining of sagittal sections from the continuously growing
incisor revealed disorganization in both the secretory ameloblast layer and the SI in constitutive
Cldn10 KO mice (Fig. 2A, right panels). In contrast, littermate WT pups showed well-aligned
ameloblasts (Fig.2A, left panels). Amelogenin-positive forming enamel matrix was observed
in both genotypes (Fig.2A insets; Fig.2B). Fluorescent labeling for markers of the cell
cytoskeleton and the cell polarity confirmed that Cldn10 KO ameloblasts exhibited an irregular
alignment but did not reveal loss of polarity or evident disorganization of the cortical actin
compared to WT, with ZO-1-positive TJs at both poles (Fig.2B; Appendix Fig.6-7). We
investigated the basal TJs at the ultrastructural scale as they are adjacent to the SI cells in which
Cldn10 is deleted. Measurement of their interspace thickness revealed enlarged paracellular
spaces between Cldn10 KO ameloblasts (p≤ 0.001) (Fig.2C). TEM analysis of the SI showed
no differences between KO and WT cells, with desmosomes and gap junctions between the
cells (Fig.2D).

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*Genes involved in ion transport are changed in the Cldn10 KO enamel organ.*

137

We next investigated the consequences of *Cldn10* deletion on amelogenesis by performing RT-
qPCR analysis on laser micro-dissected ameloblasts and SI cells (Fig.3A). In ameloblasts, we
explored genes related to ion transport and showed that *Slc4a4*, which encodes NBCe1, a
sodium bicarbonate cotransporter (Bronckers, 2017; Jalali et al., 2014), was significantly
decreased in KO ameloblasts. In contrast, the expressions of *Slc20a2* and *Slc27a4*, which

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encode the inorganic phosphate transporter Pit2 (Merametdjian et al., 2018) and a fatty acid transporter, respectively, were increased in KO ameloblasts compared to WT (Fig.3A, left panel). We investigated genes related to enamel synthesis by ameloblasts (*Amelx*, *Ambn*, *Mmp20*), which were unchanged, as confirmed at the protein level for amelogenin (Fig.2B). We also explored the expression of *Cldn1* and *Cldn3*, which encode two claudins essential for ameloblasts TJs integrity (Bardet et al., 2017). We found a significant decrease in *Cldn3* expression, confirmed at the protein level by immunofluorescence (Appendix Fig.8).

In the SI, we showed that neither the gene expression related to the non-canonical WNT signaling pathway nor the Notch pathway were changed as a direct consequence of *Cldn10* deletion in SI cells (Fig.3A, right panel). We investigated the genes related to SI activity, showing that the expression of *Slc4a4* and *Slc20a2*, which encode NBCe1 and Pit2, respectively, were significantly increased in *Cldn10* KO SI cells, while *TrpC6*, which encodes the canonical transient receptor potential 6 that is important for enamel formation (Yang et al., 2017), was decreased. No changes were found for *Alpl*, a marker of the SI (Wang et al., 2020), or for the Sonic Hedgehog pathway. At the protein level, NBCe1 immunolabeling was stronger in the SI and weaker in ameloblasts of KO pups compared to WT (Fig.3B), whereas ALP activity was similar (Appendix Fig.9).

## Enamel characterization in epithelium-targeted adult inducible *Cldn10* KO mice

Since i) the death of *Cldn10* KO pups after a few hours prevents the exploration of the later stages of amelogenesis and enamel, and ii) claudin-10 is expressed in the SI throughout all the stages of amelogenesis and in the papillary layer at the maturation stage, we generated inducible *Krt14 Cre<sup>+/−</sup>-Cldn10 iKO* (Cre<sup>+</sup>) mice. This Cre model is used to target epithelial cells (Vasioukhin et al., 1999) and Keratin 14 is expressed in the dental epithelium (Li et al., 2015) (Appendix Fig.10). At 2 months after tamoxifen or vehicle injections induction, we examined the enamel of the lower continuously growing incisor, which formed after *Cldn10* deletion, and
of molars, which had formed prior to *Cldn10* deletion. Visual examination showed no striking
difference in color and morphology of the incisor between tamoxifen- and vehicle-injected
females. However, tamoxifen-injected males exhibited marked white discolored incisors
(Fig.4A). In males, sagittal reconstructions of Micro-CT revealed a delayed onset of enamel
mineralization, resulting in a significantly lower enamel volume in iKO incisors (Fig. 4B).
Consistently, enamel thickness was significantly lower in tamoxifen-induced mice under the
distal aspect of second molar compared to vehicle (Fig. 4D). In addition, enamel density was
significantly lower in tamoxifen-induced males compared to vehicle (Fig.4B). SEM analysis
showed well-formed enamel rods in both groups (Fig. 4C and Appendix Fig.11A). Using EDX,
no significant difference was detected in the calcium-to-phosphorus ratio (Appendix Fig.11B).
Nanoindentation showed no difference in Young's modulus and micro-hardness between
animals (Appendix Fig.11C). Similar investigations were carried out on molars revealing a
significant lower enamel volume in tamoxifen-induced male mice compared to vehicle with no
change in density (Appendix Fig.12). In contrast, no differences were found between Cre⁺
female mice (Appendix Fig.13), or similarly treated Cre⁻ animals (Appendix Fig.14-15). We
next evaluated the pH of enamel matrix at the maturation stage. GBHA staining showed two
red bands in vehicle-treated mice, reflecting the position of smooth-ended maturation
ameloblasts (SA) (Takano et al., 1982). These bands appeared broader in the *Cldn10* iKO mice,
suggesting a more basic pH (Fig. 4E). Consistently, pH measurement across these bands using
BCECF fluorescence (Damkier et al., 2014) showed that the pH was more basic in the *Cldn10*
iKO enamel at the proximal part of the maturation zone (proximal band) (Fig.4E-F). We next
investigated the expression of Carbonic anhydrase 2 (CA2), ATP6V1b subunit of the V-ATPase
proton pump, and NBCe1, known to be involved in pH regulation during maturation (Lacruz et
al., 2017). ATP6V1b and CA2 labeling were increased at the apical border of ameloblasts, as
well as NBCe1 labeling in the papillary layer in tamoxifen-induced *Cldn10* iKO animals
compared to vehicle (Fig.4G; Appendix Fig.16-17).

## Enamel characterization in transplanted *Cldn10* KO molar germs

We next transplanted molar germs of newborn *Cldn10* KO pups into the kidney capsule of adult WT mice (Fig.5A). In this model (Jimenez-Rojo and Mitsiadis, 2023), the direct effect of the gene deletion on amelogenesis can be studied because the germs develop under normal mineral conditions. Three months upon implantation, a normal tooth with a periodontium formed in both genotypes (Fig.5B). Enamel examination by SEM (Fig.5C), EDX (Fig.5D) and Raman (Fig.5E) showed no difference between WT and KO germs, except for a slightly lower value of Cl content in the KO germs (Appendix Figure 18). However, TOF-SIMS, a highly sensitive method for chemical analysis and imaging of biological tissues (Van Nuffel and Brunelle, 2022), revealed a higher level of aluminum species in KO germs compared to WT (x 9.4). Ultimately, we assessed this finding in a HELIX tooth collected before tooth eruption (Obtel et al., 2022) and found a larger amount of this element in the enamel compared to a gender and age-matched control tooth (x 6.6) (Fig.5F). Other elements, such as calcium, strontium and magnesium were increased in the murine KO germs compared to WT, but no difference was observed in the human enamel (Appendix Table 1).

## Discussion

Accumulating data have shown that *Cldn10/claudin-10* is expressed in the enamel organ since the early stages of amelogenesis, specifically in the SI, where it has been proposed to be a marker of dental epithelial cells (Chiba et al., 2020; Ohazama and Sharpe, 2007; Wang et al., 2020). Our data demonstrate that claudin-10 is indeed located in the SI, in the cell membrane but not at the TJs. In the kidney, claudin-10 has been shown to be present in the TJs of a subset
215 of medullary and cortical TAL cells where it acts as a selective cation channel, but also in other
216 non-TJ-associated membrane domains (Meoli and Gunzel, 2023; Prot-Bertoye et al., 2021).
217 This latter observation is consistent with our findings in the SI; however, in both systems, the
218 exact role of non-TJ-associated claudin-10 remains to be elucidated. Furthermore, our data
219 show that claudin-10 is expressed in the papillary layer at the maturation stage.

220 At the secretion stage, the expression of several molecules involved in the ion transport and pH
221 regulation of the enamel matrix was altered in both the *Cldn10* KO SI cells and ameloblasts.
222 Since claudin-10 is expressed in SI but not in ameloblasts, our data show that *Cldn10* deletion
223 has direct effects on the SI and indirect effects on ameloblasts. In addition, at the maturation
224 stage, proteins involved in pH regulation were changed in ameloblasts and in the papillary layer,
225 also indicating direct and indirect effects of *Cldn10* deletion.

226 We found that the expression of *Slc4a4*, which encodes NBCe1, a bicarbonate sodium
227 cotransporter, was significantly increased in the SI whereas it was decreased in the ameloblasts,
228 in KO mice, compared to WT. It was also increased in the papillary layer at the maturation
229 stage in adult conditional mice. NBCe1 is important for enamel matrix pH regulation (Jalali et
230 al., 2014), and pathogenic variants of *SLC4A4* have been linked to *amelogenesis imperfecta* in
231 patients with proximal renal tubular acidosis (Kantaputra et al., 2022). Follow-up of incisor
232 growth in adult male mice with tamoxifen-induced *Cldn10* silencing showed that enamel
233 volume was reduced, concomitantly with an increase of pH in the enamel matrix at the early
234 stages of maturation. This finding contrasts sharp with our report on claudin-16, a cation-
235 selective intercellular pore located in the apical TJs of secretory ameloblasts, which expression
236 is required for a high paracellular permeability to Ca²⁺ and Mg²⁺, and which deletion is
237 associated with an acidic pH of enamel matrix (Bardet et al., 2016). These data suggest that
238 both claudin-16 and claudin-10 may contribute to pH regulation during amelogenesis with
opposite roles. Noteworthy, it has been shown that, in the TAL, claudin-10 expression at tight junctions almost never overlapped with that of claudin-16 and claudin-19 (Brideau et al., 2024).

The exploration of amelogenesis in constitutive *Cldn10* KO mice was partially hampered by the premature death of the pups a couple of hours after birth (Breiderhoff et al., 2012). In these pups, the enamel organ appeared disorganized but the enamel matrix was still forming and secretory ameloblasts maintained their organization (cytoskeleton, polarity, junctions). Consistent with this observation, the exploration of enamel in the molar germ transplantation model and in the tamoxifen-induced *Cldn10* KO adult male mice did not reveal significant disturbances, as evidenced by a well distinguishable decussating prism pattern and normal Ca/P ratio. However, in tamoxifen-induced KO male mice, the enamel of the continuously growing incisors was discolored, suggesting a loss of iron uptake, which may result in a less resistant enamel to post-eruptive acidic challenges (Srot et al., 2024). Noteworthy, HELIX syndrome affects both genders in a similar manner (Hadj-Rabia et al., 2018), and the differences observed here between male and female Cre mice may be due to the construction of this CreERT model (Vasioukhin et al., 1999).

It has been previously reported that *Adam10* deletion in the dental epithelium induced downregulation of Notch1 expression, leading to loss of the SI layer and ALP activity, and disorganization of ameloblasts (Mitsiadis et al., 2022). Here, our RT-qPCR analysis showed no change in the Notch pathway in the *Cldn10* KO SI. In addition, ALP activity remained robust in *Cldn10* KO SI. Therefore, in contrast to *Adam10* (Mitsiadis et al., 2022), *Cldn10* deletion does not significantly alter the Notch pathway, resulting in the maintenance of the SI layer. However, it seems relevant in the future to generate conditional models that target more specifically the various development stages of the dental epithelium (Yu and Klein, 2020), for example by generating a *Notch1*-CRE mouse for the SI (Mitsiadis et al., 2022).
We found a lower enamel volume in the molars of the activated *Krt14-Cldn10* iKO male mice compared to the vehicle. Since *Cldn10* deletion occurred long after molar eruption, this indicates exacerbated enamel wear due to salivary deficiency. The discoloration of the erupted enamel of the lower incisors in these mice may also result from hyposalivation. Our data are consistent with the decreased claudin-10 expression in NOD mice, a model of Sjögren's syndrome, associated with hyposalivation (He et al., 2024; Scuron et al., 2019). Progressive enamel erosion has been shown in the molars of NOD mice (Tulek et al., 2020), which is consistent with our findings in the erupted enamel of the conditional KO mice and in HELIX patients (Hadj-Rabia et al., 2018).

Our recent investigation of a HELIX third molar, which had not yet erupted, revealed no significant abnormality in the enamel structure (Obtel et al., 2022), consistent with our present findings in the pre-eruptive incisor of the Cre-Lox model and in the transplanted germs. These data suggest that salivary dysfunction is the primary cause of the rapid enamel wear associated with HELIX.

TOF-SIMS imaging (Bich et al., 2013) revealed high aluminum content in the enamel of HELIX tooth and *Cldn10* KO germs that had mineralized upon implantation in WT mice. Although widely distributed in body fluids including blood, aluminum is considered as an adventitious element present in food, water, with no known biological function (Exley and Mold, 2015). In France, tap water contains about 16 µg/ml of aluminum (Le Bot et al., 2016), meaning that both mice and patients reported here were chronically exposed to aluminum during amelogenesis. Our data show that diffusion of aluminum through the enamel organ was greater in the *Cldn10*-deficient implanted germs and patient, suggesting a loss of enamel organ integrity in the absence of claudin-10.

Aluminum ions originating from the blood vessels adjacent to the outer layer of the enamel organ must go across its cell layers to reach the enamel matrix. Little is known about aluminum
288 ion transport, which appears to occur both by transcellular and paracellular routes (Exley and
289 Mold, 2015). Enlargement of the basal TJs in *Cldn10* KO ameloblasts together with the
290 decreased expression of claudin-3, a sealing claudin, may be consistent with an increased
291 paracellular transport of aluminum. Furthermore, the presence of aluminum in enamel, either
292 integrated to the hydroxyapatite crystals or not (Chappard and Hubert, 2016), may not increase
293 its solubility (Kleber and Putt, 1985) and is therefore unlikely to contribute to its fragility. It
294 will be important to measure the levels of aluminum in other HELIX/ *Cldn10* KO tissues as for
295 example the brain, where it has been shown to be neurotoxic (Kumar and Gill, 2009).

296 In *Cldn10* KO pups, we showed that the expression of NBCe1 was increased in the SI and
297 decreased in the ameloblasts, suggesting a role of claudin-10 in acid and base transport. This
298 finding is consistent with the basic pH measured in the proximal part (adjacent to secretion) of
299 the tamoxifen-induced *Cldn10* KO incisors at the maturation stage. However, we do not have
300 an explanation for the overexpression of ATP6V1B1 at the apical membrane of iKO
301 ameloblasts at the maturation stage. The exact mechanisms underlying this pH regulation need
302 to be further investigated by functional studies in other organs that are easier to investigate than
303 the enamel organ, such as the renal tissue, or by using acid and base transport inhibitors.

304 Conclusions

305 Our data demonstrate that claudin-10 is expressed in the cellular membrane of the SI where it
306 contributes to the integrity of the enamel organ, directly in the SI and indirectly in ameloblasts.
307 In its absence, enamel forms properly despite a basic pH of enamel matrix at early maturation
308 stage, suggesting a role of claudin-10 in acid-base-regulation of the enamel organ. Overall,
309 xerostomia appears to be the main culprit for enamel wear observed in HELIX patients.

310

311 Data availability
The data that support the findings of this study are available on request from the corresponding author.

## Fundings

This study received financial support from Université Paris Cité and from the T-JUST program (ANR-17CE14-0032-02). Thè Nghia Nguyen PhD was supported by the Ambassade de France in Vietnam. The IBDM imaging facility, member of the national infrastructure France-BioImaging is supported by the French National Research Agency (ANR-10-INBS-04 & ANR-10-INSB-04-01call "investissements d'Avenir"). MR and ALB salaries are supported by CNRS (Centre National de la Recherche Scientifique). IMAG'IC core facility (Institut Cochin) is part of the National Infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04). The electron microscopy experiments were performed on the PiCSL-FBI core facility (IBDM, AMU-Marseille), member of the France-BioImaging national research infrastructure (ANR-10-INBS-04) and member of the Marseille Imaging Institute, an Excellence Initiative of Aix Marseille University A*MIDEX, a French "Investissements d'Avenir" programme (AMX 19 IET 002).

## Acknowledgments

Authors do thank Dr Olivier Duverger (NIH, Bethesda, USA) for helpful discussion regarding the tooth germ transplantation model. Authors thank David Montero from the Institut des Matériaux de Paris Centre (IMPC FR2482) for servicing FEG-SEM & EDX instrumentation and Sorbonne Université, CNRS and C'Nano projects of the Région Ile-de-France for funding (Paris, France).
335 Author contributions

336 The Nghia Nguyen, Caroline Andrique, Magali Requin, Jerome Bouchet contributed to study
337 design, data acquisition, analysis and interpretation and drafted the manuscript. Sandy Ribes
338 contributed to study design, data acquisition, analysis and interpretation. Nicolas Obtel, Lotfi
339 Slimani, Nicolas Brouilly, Coralie Torrens, Alain Schmitt, Thomas Guilbert, Maria Morawietz,
340 Andreas Kiesow, Alain Brunelle Aline Percot contributed to data acquisition and analysis, and
341 data interpretation; Smail Hadj-Rabia, Céline Gaucher, Dominik Muller, Claire Bardet
342 contributed to study design and data interpretation. Pascal Houillier and André Le Bivic
343 contributed to study conception and design and to data acquisition, analysis and interpretation.
344 Fernando Ramirez Rozzi and Thibaud Coradin, contributed to study design and to data
345 acquisition, analysis and interpretation. Catherine Chaussain and Tilman Breiderhoff
346 contributed to study conception and design and to data analysis and interpretation and drafted
347 the manuscript. All listed authors critically revised the manuscript, gave their final approval
348 and agree to be accountable for all aspects of the work.

349

350 Competing interests:

351 "All other authors declare they have no competing interests.

352
FIGURE LEGENDS

Figure 1: Claudin-10 expression profiling and protein identification in the WT murine incisor germ

(A) Localization of claudin-10 in the stratum intermedium.

Top line: fluorescence microscopy shows the secretory ameloblasts (Am) alignment and below, stratum intermedium (SI) lines. The TJ-associated protein zonula occludens-1 (ZO-1) in green was localized both at the distal and proximal end of ameloblasts (arrows). Arrows indicate TJs. Claudin-10 labeling (in red) is present only on the SI cell membrane.

Bottom line: Inset of top line = proximal end of ameloblasts. Data show that there is no colocalization of claudin-10 and ZO-1 at the proximal TJ at this stage of post-natal day 1 (P1) WT murine germ.

Abbreviations: Am = ameloblasts; SI = stratum intermedium; dp = distal pole of ameloblast; pp= proximal pole of ameloblast

(B) Immunofluorescence analysis by STED microscopy at post-natal day 1 showing no co-labeling between claudin-10 and ZO-1 and reveals that claudin-10 labeling is restricted to the SI.

(C) Immunolocalization of claudin-10 in the stratum intermedium at post-natal day 1. Immunogold staining using anti-claudin-10 antibodies and 6 nm gold beads conjugated secondary antibodies on ultrathin sections (see material and methods) of P1 WT mouse tooth. Beads (yellow stars) are only detected along the membranes of cells from the stratum intermedium (SI). Desmosomes and cell-cell junctions are indicated by green and orange arrowheads, respectively. Black arrows indicate membranes. (b) and (c) are enlarged views of (a). Scale bars are indicated.
377 (D) At the maturation stage, ZO-1 immunolabeling (in green) is observed in the tight junctions located at the apical end of ruffle-ended ameloblasts (RA) and is absent in smooth-ended ameloblasts (SA) as SA remove their apical tight junctions (Lacruz et al., 2017). Claudin-10 labeling (in red) is present in both cases in the papillary layer (3-month old adult mice).

381 Abbreviations: Am = ameloblasts; SI = stratum intermedium; SI N: Stratum Intermedium
382 Nucleus; PL: papillary layer

383

384 **Figure 2: Analysis of the incisor enamel organ in P1 Cldn10 KO mice.**

385 (A) Hematoxylin and eosin staining of sagittal sections of P1 incisor showing the organization
386 of secretory stage ameloblasts (Am) associated at their apical end with the forming enamel
387 matrix (e) stained in violet, in *Cldn10* KO and WT pups. Ameloblasts appeared well polarized
388 with a regular nucleus located at the basal end of the cells in WT pups compared to *Cldn10* KO.

389 (B) Immunofluorescence analysis of frontal sections of P1 incisor in *Cldn10* KO and WT mice, claudin-10 - ZO-1 co-labeling, phalloidin staining, P-ezrin – ZO1 co-labeling, β-catenin – E-cadherin and phalloidin co-labeling, and amelogenin staining. (C) Tight junction (red arrowhead) imaging by TEM at the basal end of secretory ameloblasts from *Cldn10* KO and WT mice. Tight junction (red arrowhead) imaging by TEM was defined by the actin-rich area (orange zone) at the basal end of the secretory ameloblasts. The intercellular spaces at the tight junction level appear to be narrower in WT than in *Cldn10* KO mice. Quantification analysis of tight junction thickness at the basal end of ameloblasts shows a significant increase in tight junction thickness in *Cldn10* KO in comparison to WT mice (n=9 in WT and n = 15 in *Cldn10* KO; p < 0.001). (D) Images of gap junctions and desmosomes in SI. Abbreviations Am = ameloblasts; SI = stratum intermedium; en = enamel; dp = distal pole of ameloblasts; pp = proximal pole of ameloblasts; red arrowhead: tight junctions; yellow arrowhead: gap junctions; green arrowhead: desmosomes.
402

**Figure 3: Consequences of Cldn10 deletion on amelogenesis in P1 Cldn10 KO mice.**

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413

(A) Gene expression of ion transporters (Slc family genes and TrpC6), Shh and Gli1 as stratum intermedium marker, and Wnt non-canonical pathway with Atf2, Camkd2 and Rock2, and AmelX, Ambn, Mmp20, KLK4 as ameloblasts markers, and Cldn1 and Cldn3 as tight junction markers. (B) Immunofluorescence analysis of frontal sections of P1 incisor in Cldn10 KO and WT mice with NBCel (Slc4a4). The right panels represent the mean fluorescence intensity profile plot of a surface projection with a width of 30 µm, perpendicular to the apical membrane of ameloblasts, including ameloblasts and stratum intermedium. In ameloblasts, the NBCel labeling is mainly observed at the apical end and is stronger in WT compared to KO ameloblasts. In contrast, the cell membrane of SI is labeled and the labeling is stronger in KO cells than in WT. Abbreviations Am = ameloblasts; SI = stratum intermedium; dp = distal pole of ameloblasts; pp = proximal pole of ameloblasts.

414

**Figure 4: Dental phenotype of adult epithelium- specific inducible male Cldn10 KO mice.**

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(A) Enamel incisor appearance of tamoxifen-injected compared with vehicle-injected Krt14-Cre +/− Cldn10 iKO mice (Cre+ mice). (B) Quantitative analyses of high-resolution micro-CT of mandibular samples showed a significantly lower enamel volume and density in incisors of activated iKO mice compared to vehicle control ones (n = 16 in tamoxifen group vs n = 20 in control group). In control mice, incisor mineralization is observed under the third molar (M3), whereas in activated iKO mice it is detected under the second molar (M2). (C) Scanning electron microscopy analysis of the enamel structure of incisor at mesial root of first molar. (D) Quantitative analyses of high-resolution micro-CT of mandibular samples showed a significantly lower incisor enamel surface area under distal root of M2 of activated Krt14-Cldn10 iKO mice compared to vehicle treated mice (n = 8 in control group vs n=10 in activated group), **p < 0.01. (E) Labial views of lower incisors stained with GBHA showing a proximal band (PB) and a distal band (DB) in the maturation zone of activated iKO mice and vehicle
mice. These bands correspond to the region of smooth-ended (SA) maturation ameloblasts. (F)
Graphs showing the distribution of pH values across the SA bands (n = 8 in control group vs
n=5 in activated group). In the proximal band (close to the secretory stage), significantly higher
pH values are measured in activated iKO incisors compared to vehicle ones. Data are expressed
as median± interquartile range (IQR). (F) ATP6V1B1 immunofluorescence (in red) on sagittal
sections of the enamel organ in *Krt14 Cre +/- Cldn10* iKO (Cre+) mice with tamoxifen (T) or
vehicle (V) injection. Representative pictures showing a stronger labeling at the apical
membrane of transition and maturation ameloblasts in tamoxifen-injected animals compared to
vehicle ones. (n = 3 mice per group).

Abbreviations: M1 = first molar, M2 = second molar, M3 = third molar; en = enamel; de =
dentin; V = Vehicle; T = Tamoxifen; Am = ameloblast; PL= papillary layer

<caption>Figure 5: Analyzing the enamel structure of *Cldn10* KO molars after transplantation in the kidney capsule.</caption>

(A) Molars germs from *Cldn10* KO and WT mice were harvested at P1 and transplanted in the renal capsule of 3-month-old WT male mice for 3 months. (B) 2D & 3D images reconstructed by micro-CT after 3 months of implantation show a complete molar with full morphology: crown with its five cusps, two full-length roots and surrounding alveolar bone. (C) SEM analyses were performed on the implanted germs and enamel prisms appeared normal in *Cldn10* KO molars compared to WT (n=4 per group). (D) EDX analysis of three enamel layers showed a similar calcium/phosphorus (Ca/P) ratio observed in *Cldn10* KO and WT implanted molars (n = 4 per group). (E) **a, d**: Optical microscopy images of sections of WT and KO samples. The white area delineates the surface mapped by Raman spectroscopy. **b, e**: Raman mapping of the WT and KO samples based on the full width at half maxima (FWHM) of the v(PO₄³⁻) vibration band at 960 cm⁻¹, indicative of the crystallinity of the mineral phase **c, f**: Raman mapping of WT and KO samples based on the ratio between the integrated area of the v(CO₃²⁻) and v(PO₄³⁻)
452 vibration bands at 1070 cm⁻¹ and 960 cm⁻¹, respectively, indicative of the carbonatation rate of
453 hydroxyapatite. Both parameters showed little variations over the whole enamel thickness down
454 to the DEJ and did not differ significantly between control and KO samples, indicating that the
455 mineral phase of enamel was of similar crystallinity and carbonatation degree in the two types
456 of teeth. (F) TOF-SIMS ion images of Al⁺ m/z 26.98 and of CaPO₂⁺ m/z 102.92 cations detected
457 at the surface of the sample surfaces. Field of view 500 µm × 500 µm, 256 × 256 pixels, pixel
458 size 1.95 µm. All images recorded with the same primary ion fluence of 5.1 × 10¹¹ ions·cm⁻².
459 Color scale bars, with amplitude in number of counts, are indicated to the right of each ion
460 image. The amplitude of the color scale corresponds to the maximum number of counts (MC)
461 and could be read as [0, MC]. TC is the total number of counts recorded for the specified m/z
462 (sum of counts in all the pixels). TC value for Al³⁺ are gathered on the graph, highlighting the
463 higher abundance of this element in (1) enamel (defined by a white line) from germs of KO
464 mice compared to WT, (2) enamel from the HELIX patient compared to control. Abbreviations:
465 en = Enamel; de = Dentin.
466
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610
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

Fig.1
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>Fig.2</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>B</figcaption></figure>

DAPI / NBCe1 (Slc4a4)

<figure><img src="image_11.png" /></figure>

Fig.3
A **Incisor overview**

<figure><img src="image_12.png" /></figure>

CRE+: Vehicle

<figure><img src="image_13.png" /></figure>

CRE+: Tamoxifen

C

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

B

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

D

M1-Mesial M1-Distal M2-Mesial M2-Distal

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

V

T

E

<figure><img src="image_20.png" /></figure>

G

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

Enamel incisor

volume

<figure><img src="image_23.png" /></figure>

density

Enamel density of incisor

<figure><img src="image_24.png" /></figure>

2 months after tamoxifen/vehicle induction

Mineralization delay

<figure><img src="image_25.png" /></figure>

F

<figure><img src="image_26.png" /></figure>

Proximal band

<figure><img src="image_27.png" /></figure>

Distal band
A

Pups
(Post natal D1)

WT adult mice

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

Implantation time:
3 months

B

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

C

WT

<figure><img src="image_34.png" /></figure>

Cldn10 KO

<figure><img src="image_35.png" /></figure>

D

Ratio Ca/P

<figure><img src="image_36.png" /></figure>

WT

Cldn10 KO

E

WT

<figure><img src="image_37.png" /></figure>

Cldn10
KO

F

Mouse germ

WT

<figure><img src="image_38.png" /></figure>

Cldn10 KO

<figure><img src="image_39.png" /></figure>

Human enamel

CTRL

<figure><img src="image_40.png" /></figure>

HELIX

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

X 6.6

X 9.4

Fig.5