Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3

Defining how the molecular constituents of the tight junction interact is a prerequisite to understanding tight junction physiology. We utilized in vitro binding assays with purified recombinant proteins and immunoprecipitation analyses to define interactions between ZO-1, ZO-2, ZO-3, occludin, and the actin cytoskeleton. Actin cosedimentation studies showed that ZO-2, ZO-3, and occludin all interact directly with F-actin in vitro, indicating that actin is engaged in multiple interactions at the tight junction. Low speed sedimentation analyses demonstrated that neither ZO-2, ZO-3, nor occludin act as F-actin cross-linking proteins, and further evidence indicates that these proteins do not bind to actin filament ends. The binding interactions of ZO-2, ZO-3, and occludin were corroborated in vivo by immunofluorescence colocalization experiments which showed that all three proteins colocalized with actin aggregates at cell borders in cytochalasin D-treated Madin-Darby canine kidney cells. Exploration of other tight junction protein interactions demonstrated that ZO-2 binds directly to both ZO-1 and occludin. Contrary to previous beliefs, our immunoprecipitation results indicate that ZO-1, ZO-2, and ZO-3 exist in situ primarily as independent ZO-1.ZO-2 and ZO-1.ZO-3 complexes rather than a trimeric ZO-1.ZO-2.ZO-3 grouping. These studies elucidate direct binding interactions among tight junction-associated proteins, giving insight into their organization as a multimolecular structure.     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.     

Transcription factor epiprofin is essential for tooth morphogenesis by regulating epithelial cell fate and tooth number

In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn-/-). Epfn-/- mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn-/- molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.     

Responsiveness of developing dental tissues to fibroblast growth factors: expression of splicing alternatives of FGFR1, -2, -3, and of FGFR4; and stimulation of cell proliferation by FGF-2, -4, -8, and -9

To elucidate the roles of fibroblast growth factors (FGF) in tooth development, we have analyzed the expression patterns of fibroblast growth factor receptors (FGFR) in mouse teeth by in situ hybridization and studied the effects of FGF-2, -4, -8, and -9 on cell proliferation in vitro by local application with beads on isolated dental mesenchymes. mRNAs of FGFR-1, -2, and -3 were localized by probes specific for the alternative splice variants IIIb and IIIc. The expression patterns of FGFR1, -2, and -3 were completely different, and the two splicing variants of FGFR1 and 2 exhibited different expression domains. FGFR4 was not expressed in the developing teeth. The IIIb splice forms of FGFR1 and -2 were expressed in the dental epithelium during morphogenesis. The IIIc splice form of FGFR1 was expressed both in epithelium and mesenchyme whereas FGFR2 IIIc was confined to the mesenchymal cells of the dental follicle. Both splice forms of FGFR3 were expressed in dental papilla mesenchyme. None of the FGF-receptors was detected in the primary enamel knot, the putative signaling center regulating tooth morphogenesis. This may explain the fact that enamel knot cells do not proliferate, although they express intensely mitogenic FGFs. Beads releasing FGF-2, -4, -8, or -9 proteins stimulated cell proliferation in cultured dental mesenchymes. These data, together with our earlier data on FGF expression [Kettunen and Thesleff (1998): Dev Dyn 211:256–268] suggest that FGF-8 and -9 mediate epithelial-mesenchymal interactions during tooth initiation. During advancing morphogenesis FGF-3, -4, and -9 may act both on mesenchyme and epithelium. Finally, the intense expression of FGFR1 in odontoblasts and ameloblasts, and FGFR2 IIIb in ameloblasts suggests that FGFs participate in regulation of their differentiation and/or secretory functions. Dev. Genet. 22:374–385, 1998. © 1998 Wiley-Liss, Inc.     

Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis.     

Cell surface proteoglycan expression correlates with epithelial-mesenchymal interaction during tooth morphogenesis

Tooth morphogenesis and differentiation of the dental cells are guided by interactions between epithelial and mesenchymal tissues. Because the extracellular matrix is involved in these interactions, the expression of matrix receptors located at the cell surface may change during this developmental sequence. We have examined the distribution of an epithelial cell surface proteoglycan antigen, known to behave as a receptor for interstitial matrix, during tooth morphogenesis. Intense staining was seen around the cells of the embryonic oral epithelium as well as the dental epithelium at the early bud stage. With development, expression was greatly reduced in the enamel organ. Differentiation of these cells into ameloblasts was associated with the loss of expression, while the epithelial cells remaining in the stratum intermedium and stellate reticulum regained intense staining. The PG antigen was weakly expressed in the loose neural crest-derived jaw mesenchyme but it became strongly reactive in the condensed dental papilla mesenchyme when extensive morphogenetic movements took place. With development, the PG antigen disappeared from the advanced dental papilla mesenchyme but persisted in the dental sac mesenchyme, which gives rise to periodontal tissues. The PG antigen was not expressed by odontoblasts. Hence, the expression of the PG antigen changes during the epithelial-mesenchymal interactions of tooth development and is lost during terminal cell differentiation. The expression follows morphogenetic rather than histologic boundaries. The acquisition and loss of expression in epithelial and mesenchymal tissues during tooth development suggest that this proteoglycan has specific functions in the epithelial-mesenchymal interactions that guide morphogenesis.     

Developmental expression patterns of Bcl-2, Bcl-x, Bax, and Bak in teeth

The ontogenic profile of expression of four members of the Bcl-2 family (Bcl-2, Bcl-x, Bax and Bak) was examined in the mouse by immunohistochemistry using paraffin sections. All four members were expressed in changing patterns during critical stages of tooth morphogenesis. Expression was detected in epithelial cell populations including the dental lamina, internal dental epithelium (IDE; differentiating ameloblasts), stratum intermedium and stellate reticulum cells, as well as in the condensed dental mesenchyme. The temporo-spatial localization of the various members of the Bcl-2 family in dental epithelium and mesenchyme showed striking overlapping areas but often their expression patterns differed. In general, contemporaneous co-expression of the Bcl-2 and Bax proteins, and of the Bcl-x and Bak proteins was noted in various types of cells during the developmental process, with the intensity of Bcl-2>Bax and of Bak>Bcl-x. Expression was pronounced at sites where interaction between surface ectoderm and induced mesenchyme takes place, and at the enamel knot, which is regarded as organization/regulating center for tooth development. Around birth, after the structural maturation was accomplished, the expression was down-regulated. The absence of elevated expression of each of these four members of the Bcl-2 family after birth in the teeth suggests that these proteins are relevant during the accomplishment of the basic architecture but not once the structure of the tooth is established.     

Truncation mutants of the tight junction protein ZO-1 disrupt corneal epithelial cell morphology

The tight junction is the most apical intercellular junction of epithelial cells and regulates transepithelial permeability through the paracellular pathway. To examine possible functions for the tight junction-associated protein ZO-1, C-terminally truncated mutants and a deletion mutant of ZO-1 were epitope tagged and stably expressed in corneal epithelial cell lines. Only full-length ZO-1 and one N-terminal truncation mutant targeted to cell borders; other mutants showed variable cytoplasmic distributions. None of the mutants initially disrupted the localization of endogenous ZO-1. However, long-term stable expression of two of the N-terminal mutants resulted in a dramatic change in cell shape and patterns of gene expression. An elongated fibroblast-like shape replaced characteristic epithelial cobblestone morphology. In addition, vimentin and smooth muscle actin expression were up-regulated, although variable cytokeratin expression remained, suggesting a partial transformation to a mesenchymal cell type. Concomitant with the morphological change, the expression of the integral membrane tight junction protein occludin was significantly down-regulated. The localizations of endogenous ZO-1 and another family member, ZO-2, were disrupted. These findings suggest that ZO-1 may participate in regulation of cellular differentiation.     

The epithelial genotype controls the pattern of extracellular enamel prism formation

Abstract. Enamel formation in the developing tooth organ is the product of epithelial-mesenchymal interactions which result in the differentiation of ameloblasts, the secretion of enamel proteins, and the production of a highly organized extracellular matrix. The three-dimensional organization of enamel prisms is species-specific: irregular polygonshaped in rabbit and rectangular-shaped in mouse. We designed experiments to test the hypothesis that three-dimensional organization of enamel prism formation is genetically determined by epithelium; the prediction being that speciesspecific enamel prism pattern formation is expressed independent of mesenchymal instructions. Our strategy employs scanning electron microscopy to examine enamel prism patterns formed during rabbit and mouse tooth morphogenesis in situ and in vitro, and to then determine the specific tissue type required for regulating these patterns using heterotypic tissue recombinations. Morphometric analyses demonstrated that cap stage tooth organs cultured on the chick chorioallantoic membrane (CAM) formed enamel prisms equivalent to prism patterns observed for in situ controls. Heterotypic tissue recombinations, using cap stage molar organs, formed rabbit-like prisms with rabbit epithelium/mouse mesenchyme, and mouse-like prisms with mouse epithelium/rabbit mesenchyme. These results indicate that dental papilla mesenchyme has no apparent influence on enamel prism pattern formation. Enamel prism pattern appears to be genetically regulated by the inner enamel epithelium.     

Sonic hedgehog regulates growth and morphogenesis of the tooth

During mammalian tooth development, the oral ectoderm and mesenchyme coordinate their growth and differentiation to give rise to organs with precise shapes, sizes and functions. The initial ingrowth of the dental epithelium and its associated dental mesenchyme gives rise to the tooth bud. Next, the epithelial component folds to give the tooth its shape. Coincident with this process, adjacent epithelial and mesenchymal cells differentiate into enamel-secreting ameloblasts and dentin-secreting odontoblasts, respectively. Growth, morphogenesis and differentiation of the epithelium and mesenchyme are coordinated by secreted signaling proteins. Sonic hedgehog (Shh) encodes a signaling peptide which is present in the oral epithelium prior to invagination and in the tooth epithelium throughout its development. We have addressed the role of Shh in the developing tooth in mouse by using a conditional allele to remove Shh activity shortly after ingrowth of the dental epithelium. Reduction and then loss of Shh function results in a cap stage tooth rudiment in which the morphology is severely disrupted. The overall size of the tooth is reduced and both the lingual epithelial invagination and the dental cord are absent. However, the enamel knot, a putative organizer of crown formation, is present and expresses Fgf4, Wnt10b, Bmp2 and Lef1, as in the wild type. At birth, the size and the shape of the teeth are severely affected and the polarity and organization of the ameloblast and odontoblast layers is disrupted. However, both dentin- and enamel-specific markers are expressed and a large amount of tooth-specific extracellular matrix is produced. This observation was confirmed by grafting studies in which tooth rudiments were cultured for several days under kidney capsules. Under these conditions, both enamel and dentin were deposited even though the enamel and dentin layers remained disorganized. These studies demonstrate that Shh regulates growth and determines the shape of the tooth. However, Shh signaling is not essential for differentiation of ameloblasts or odontoblasts.     

Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.     

Localization of occludin, ZO-1, and pan-cadherin in rabbit ciliary epithelium and iris vascular endothelium

Previous studies have used conventional electron microscopy and freeze fracture to identify the morphological equivalents of the blood-aqueous barrier in the mammalian eye. These equivalents are the tight junctions that form a part of the apicolateral junctional complex between adjacent non-pigmented ciliary epithelial cells and the tight junctions present between endothelial cells of the iris vasculature. Recent investigations have begun to unravel the molecular assembly of the tight junction and some variability has been found. Our goal in the present study was to probe the ciliary epithelium and iris vascular endothelium of the rabbit eye to determine if certain molecular constituents associated with tight junctions in other tissues are also present as parts of the blood-aqueous barrier. The selected constituents were occludin, ZO-1, and a representative, adherens junction-related cadherin. Immunohistochemical and immunoelectron microscopic methods were used. The results showed that occludin was distributed exclusively at known locations of tight junctions. ZO-1 was also expressed at these locations but its distribution extended beyond that of occludin, along the adjacent membranes. Pan-cadherin was expressed ubiquitously within the ciliary epithelium and negligibly in iris vascular endothelium. Our results demonstrate that occludin and ZO-1 are integral components of the blood-aqueous barrier of the normal rabbit eye.     

Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.     

Shigella flexneri regulates tight junction-associated proteins in human intestinal epithelial cells

Shigella spp. are a group of Gram-negative enteric bacilli that cause acute dysentery in humans. We demonstrate that Shigella flexneri has evolved the ability to regulate functional components of tight junctions after interaction at the apical and basolateral pole of model intestinal epithelia. In the regulation of tight junctional protein assemblies, S. flexneri can engage serotype-specific mechanisms, which targets not only expression, but also cellular distribution and membrane association of components of tight junctions. Distinct mechanisms resulting in the regulation of tight junction-associated proteins are initiated after either apical or basolateral interactions. S. flexneri serotype 2a has the ability to remove claudin-1 from Triton X-insoluble protein fractions upon apical exposure to T-84 cell monolayers. S. flexneri serotype 2a and 5, but not the non-invasive Escherichia coli strain F-18, share the ability to regulate expression of ZO-1, ZO-2, E-cadherin and to dephosphorylate occludin. The disruption of tight junctions is dependent on direct interaction of living Shigella with intestinal epithelial cells and is supported by heat-stable secreted bacterial products. Intestinal epithelial cells have the ability to compensate in part for S. flexneri induced regulation of tight junction-associated proteins.     

Ring1a/b polycomb proteins regulate the mesenchymal stem cell niche in continuously growing incisors

Rodent incisors are capable of growing continuously and the renewal of dental epithelium giving rise to enamel-forming ameloblasts and dental mesenchyme giving rise to dentin-forming odontoblasts and pulp cells is achieved by stem cells residing at their proximal ends. Although the dental epithelial stem cell niche (cervical loop) is well characterized, little is known about the dental mesenchymal stem cell niche. Ring1a/b are the core Polycomb repressive complex1 (PRC1) components that have recently also been found in a protein complex with BcoR (Bcl-6 interacting corepressor) and Fbxl10. During mouse incisor development, we found that genes encoding members of the PRC1 complex are strongly expressed in the incisor apical mesenchyme in an area that contains the cells with the highest proliferation rate in the tooth pulp, consistent with a location for transit amplifying cells. Analysis of Ring1a(-/-);Ring1b(cko/cko) mice showed that loss of Ring1a/b postnatally results in defective cervical loops and disturbances of enamel and dentin formation in continuously growing incisors. To further characterize the defect found in Ring1a(-/-);Ring1b(cko/cko) mice, we demonstrated that cell proliferation is dramatically reduced in the apical mesenchyme and cervical loop epithelium of Ring1a(-/-);Ring1b(cko/cko) incisors in comparison to Ring1a(-/-);Ring1b(fl/fl)cre- incisors. Fgf signaling and downstream targets that have been previously shown to be important in the maintenance of the dental epithelial stem cell compartment in the cervical loop are downregulated in Ring1a(-/-);Ring1b(cko/cko) incisors. In addition, expression of other genes of the PRC1 complex is also altered. We also identified an essential postnatal requirement for Ring1 proteins in molar root formation. These results show that the PRC1 complex regulates the transit amplifying cell compartment of the dental mesenchymal stem cell niche and cell differentiation in developing mouse incisors and is required for molar root formation.     

Expression patterns of ABCG2, Bmi-1, Oct-3/4, and Yap in the developing mouse incisor

Recent studies have demonstrated the existence of dental stem cells in the continuously growing tooth. However, much remains to be learned about the complex mechanism involving stem cells during tooth development. We determined the expression patterns of four stem cell markers ABCG2, Bmi-1, Oct-3/4, and Yap in the developing mouse incisors between embryonic day (E) 11 and postnatal day (PN) 20. ABCG2 was localized strongly in the perivascular region of the incisor mesenchyme from E11 to PN20, and in the odontoblasts from E18 to PN20. Bmi-1 was expressed in both the dental epithelium and mesenchyme from E11 to E14. The expression of Bmi-1 was noticeably reduced at E16, and was restricted to the apical bud from E16 to PN20. Oct-3/4 was localized in the nucleus of the cells in the superficial layer and stellate reticulum within the dental epithelium from E11 to E14 and in the apical bud from E16 to PN20. Meanwhile, once the ameloblasts and odontoblasts began to appear at E16, they expressed Oct-3/4 in the cytoplasm. Yap was expressed in most of the basal cells of the incisor dental epithelium from E11 to E14, but was expressed mainly in the transit-amplifying (TA) cells within the basal cell layer from E16 to PN20. The unique and overlapping expression patterns of ABCG2, Bmi-1, Oct-3/4, and Yap suggest the independent and interactive functions of the four stem cell markers in the developing mouse incisor.     

Interaction of junctional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin

Junctional adhesion molecule (JAM) is an integral membrane protein that has been reported to colocalize with the tight junction molecules occludin, ZO-1, and cingulin. However, evidence for the association of JAM with these molecules is missing. Transfection of Chinese hamster ovary cells with JAM (either alone or in combination with occludin) resulted in enhanced junctional localization of both endogenous ZO-1 and cotransfected occludin. Additionally, JAM was coprecipitated with ZO-1 in the detergent-insoluble fraction of Caco-2 epithelial cells. A putative PDZ-binding motif at the cytoplasmic carboxyl terminus of JAM was required for mediating the interaction of JAM with ZO-1, as assessed by in vitro binding and coprecipitation experiments. JAM was also coprecipitated with cingulin, another cytoplasmic component of tight junctions, and this association required the amino-terminal globular head of cingulin. Taken together, these data indicate that JAM is a component of the multiprotein complex of tight junctions, which may facilitate junction assembly.     

Expression and localization of Nell-1 during murine molar development

Nel-like molecule-1 (Nell-1) is a recently discovered secreted protein that plays an important role in osteoblast differentiation, bone formation, and bone regeneration. However, its expression and distribution during tooth development are largely unknown. The aim of this study was to investigate the expression patterns of Nell-1 during murine molar development by immunohistochemistry. Nell-1 protein was expressed during molar development in embryonic and postnatal Kunming mice, but its expression levels and patterns at various developmental stages differed. At embryonic day 13.5 (E13.5) and E14.5, Nell-1 was found in both the entire enamel organ and the underlying mesenchyme. At E16.5, it was detected in the inner and outer enamel epithelia, stratum intermedium, secondary enamel knot, and dental papilla. At E18.5, Nell-1 was expressed in the differentiating ameloblasts, differentiating odontoblasts, and stratum intermedium. Positive staining was also found in the outer enamel epithelium. At postnatal day 2.5 (P2.5), P5, and P7, Nell-1 appeared in the secretory and mature ameloblasts and odontoblasts (odontoblastic bodies and processes) as well as immature enamel. Hertwig’s epithelial root sheath also stained positively at P7. At P13.5, positive staining was restricted to the reduced dental epithelium and odontoblasts, whereas Nell-1 disappeared in the mature enamel. During tooth eruption, Nell-1 was observed only in the odontoblastic bodies, odontoblastic processes, and endothelial cells of blood vessels. The spatiotemporal expression patterns of Nell-1 during murine tooth development suggest that it might play an important role in ameloblast and odontoblast differentiation, secretion and mineralization of the extracellular enamel matrix, molar crown morphogenesis, as well as root formation.