Archives of Oral Biology 46 (2001) 973–981

The spatial and temporal expression of calretinin indeveloping rat molars (Rattus nor�egicus)

Dharmesh Mistry a, Mario Altini a,*, Hedley G. Coleman a, Hasiena Ali a,Eugenio Maiorano b

a Department of Anatomical Pathology, Di�ision of Oral Pathology, School of Oral Health Sciences, Faculty of Health Sciences,Uni�ersity of the Witwatersrand, 7 York Road, Parktown 2193, South Africa

b Department of Pathological Anatomy and Genetics, Uni�ersity of Bari, Policlinico, Piazza G. Cesare 11, 70124 Bari, Italy

Accepted 27 March 2001

Abstract

Calretinin is a 29-kDa calcium-binding protein abundantly expressed in central and peripheral neural tissues. Theaim here was to determine its expression during various stages of odontogenesis. Five categories of embryonic (E) andpostnatal (P) rats at various ages (E17, E18, E20, P0, and P7), both male and female, were used to represent thevarious stages of molar tooth development. The heads of the experimental animals were harvested at the appropriatetime and each was cut mid-sagittally and coronally to locate the tooth germs. Selected sections were stainedimmunohistochemically with polyclonal rabbit anticalretinin at a concentration of 1:25 after microwave irradiation.The results showed that calretinin is distributed widely in epithelium-derived tissues during odontogenesis in rat molartooth germs. It was expressed focally in the dental lamina, outer enamel epithelium, stellate reticulum and stratumintermedium at different stages. In contrast, it was expressed diffusely and intensely in the inner enamel epitheliumand presecretory ameloblasts, although it was discontinuous over the cusp tips. In the secretory ameloblasts, thestaining was less intense, being restricted to the cytoplasm, including Tomes’ processes. This distribution suggests thatcalretinin may play a part in enamel formation. © 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Calretinin; Calcium-binding proteins; CaBP; Odontogenesis; Tooth development; Rat

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1. Introduction

Advances in the unraveling of amino acid sequencesand three-dimensional structures have led to the de-scription of two families of calcium-binding proteins,the EF-hand homologue family and the annexin family.The EF-hand motif consists of two �-helices, ‘E’ and‘F’, joined by a Ca2+-binding loop. Functional EF-hands always seem to occur in pairs. The EF-hand

homologue family contains more than 200 differentcalcium-binding proteins; among them are calretinin,calmodulins, troponin C, myosin-regulatory light chain,parvalbumin, S-100 proteins and calbindins 9- and 28-kDa. The most striking feature of the EF-hand familyis their ability to modulate the activity of a number ofenzymes (Wieman, 1991).

Calretinin is a calcium-binding protein of 29 kDa,although other forms of lower molecular weight result-ing from alternative splicing have been described(Schwaller et al., 1995). The calretinin gene was isolatedinitially from a cDNA clone from the chick retina andshows 60% homology with the calbindin gene (Parmen-

* Corresponding author. Fax: +27-11-7172146.E-mail address: 083opds@cosmos.wits.ac.za (M. Altini).

0003-9969/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0003 -9969 (01 )00046 -2

D. Mistry et al. / Archi�es of Oral Biology 46 (2001) 973–981974

tier, 1989; Rogers et al., 1989; Parmentier and Lefort,1991). Calretinin is expressed abundantly in central andperipheral neural tissues, particularly in the retina andin neurones of the sensory pathways (Andressen et al.,1993; Schwaller et al., 1993). Calretinin expression out-side the central nervous system has been characterizedless extensively. Initially, it was described only in ep-ithelial cells of the guinea-pig ear and interstitial cells ofthe rat testis, but more recently it has been described inmesothelial cells and in such diverse tissues as the pilarinfundibulum, eccrine glands, convoluted tubules of thekidney, Leydig and Sertoli cells of the testis, epitheliumof the rete ovary, adrenal cortex, epithelial cells of thethymus and adipocytes (Doglioni et al., 1996; Dei Tosand Doglioni, 1998).

Ichikawa et al. (1992, 1994, 1996) have studied theimmunoreactivity of calretinin and other calcium-bind-ing proteins, restricting their studies to neuralelements in teeth and oral tissues in the adult rat. Theydemonstrated that calretinin immunoreactivity is dis-tributed across the entire size range of the tri-geminal ganglion neurones. It was also observed innerve fibers surrounding neuronal cell bodies in auto-nomic ganglia, and nerve endings in the lip, tongue,incisal papilla, soft palate, pharynx and epiglottis. Inthe tooth pulp, myelinated and unmyelinated calretinin-immunoreactive fibers were seen, which projected asvaricose terminals to the subodontoblastic and odonto-blastic layers.

Calretinin has been identified as a definitivemarker for mesotheliomas, and has also been demon-strated in some carcinomas and adenocarcinomas oflung, breast, pancreas and ovary. There was only par-tial correlation between staining of normal tissues andtheir neoplastic counterparts. In some tumors, calre-tinin immunoreactivity appeared to be acquiredfollowing neoplastic transformation (Doglioni et al.,1996).

Calretinin expression has also been studied recently,in our department, in odontogenic tumors and cysts.We have demonstrated calretinin in both unicystic andconventional multicystic ameloblastomas, but not invarious other odontogenic cysts (Altini et al., 2000;Coleman et al., 2001).

As odontogenic tumors are known to recapitulatenormal odontogenesis, we have now decided to investi-gate whether calretinin is expressed in odontogenictissues during normal tooth development in the rat.This would allow comparison with the calretinin ex-pression observed in ameloblastomas, thus providinggreater understanding of the potential use of this im-munohistochemical marker in the diagnosis of odonto-genic lesions. In addition, insight might be obtainedinto the possible role of this calcium-binding protein innormal tooth development.

2. Materials and methods

2.1. Experimental animals

The study was approved by the animal ethics screen-ing committee of the University of the Witwatersrand,Johannesburg, South Africa. The experimental animalwas the Norwegian rat (Rattus nor�egicus) of theSprague–Dawley strain. Forty fetal rats (17, 18 and 20days of gestation) and 22 pups (0 and 7 days) wereused. The fetal rats were harvested from time-mated(vaginal plug=day 0) mature females representing thedifferent date categories mentioned above. Each preg-nant female and the pups were killed by overdose (2 ml)of sodium pentobarbitone (Eutha-naze®; CentaurLabs). Thereafter the fetal rats were dissected andtogether with the pups were fixed for 48 h in a solutionof 10% phosphate-buffered formalin.

2.2. Histology

Each head was cut in half mid-sagittally, and onehalf was further divided in three places coronally, ap-proximately in the location of the dentition, thus allow-ing for evaluation of both longitudinal and transversesections of developing molars. The specimens were notdemineralized, as a pilot study had shown that regard-less of the demineralization system used, an increase inbackground staining and a decrease in staining intensityoccurred. After routine histological processing andparaffin wax embedding, serial sections 4-�m thick werecut and stained with haematoxylin and eosin.

2.3. Immunohistochemistry

Selected sections were placed on 3-aminopropyltri-ethoxysilane-coated glass slides at room temperatureovernight and stained immunohistochemically withpolyclonal rabbit anticalretinin (Zymed LaboratoriesInc.) using the streptavidin–biotin complex im-munoperoxidase technique (StreptABComplex/HRPDuet, mouse/rabbit; DAKO).

Deparaffinized sections (xylene, 2×5 min) were mi-crowaved (800 W, medium power) in 0.01 M citratebuffer (pH 6.0) for 2×5 min. When cool (after 20min), they were quenched in 3% methanol–hydrogenperoxide for 30 min, washed in water and rinsed inphosphate-buffered saline (pH 7.6), incubated with nor-mal goat serum (1:5) for 20 min and then treated withthe primary antiserum (1:25) at room temperature for 1h. They were then incubated with biotinylated sec-ondary antibody for 30 min. After washing with phos-phate-buffered saline, the sections were further treatedwith StreptABC for 30 min at room temperature. Theywere then washed with phosphate-buffered saline.Bound peroxidase was visualized with 3,3�-diaminoben-

D. Mistry et al. / Archi�es of Oral Biology 46 (2001) 973–981 975

zidine hydrochloride (Sigma) for 5 min, resulting in abrown reaction product. Sections were counterstainedwith Meyer’s haematoxylin (5 min). All reagent dilu-tions were prepared with 3% bovine serum albuminfactor V (Boehringer Mannheim). Positive controls con-sisting of rat neurolfactory epithelium and brain wereused throughout the study. Negative controls wereachieved by substituting the primary specific antibodieswith non-immune serum.

2.4. E�aluation

The presence and distribution of immunohistochemi-cally stained cells and the intensity, pattern and local-ization of their staining were determined usingconventional light microscopy. The tissues evaluatedfor calretinin were oral epithelium, dental lamina, ec-tomesenchyme, outer enamel epithelium, inner enamelepithelium, stellate reticulum, stratum intermedium,dental papilla, odontoblasts, and ameloblasts. Whencalretinin immunostaining was detected, a descriptivegrading of weak, moderate or intense was used. Local-ization of the calretinin staining was also evaluated asbeing either single, focal, or diffuse, and whether it wasnuclear, cytoplasmic or both.

3. Results

The distribution patterns and staining intensities ofcalretinin in various odontogenic tissues at differentstages of development in rat molar teeth are summa-rized in Table 1. Where positive, calretinin was ex-pressed generally in both the cytoplasm and nucleus.

3.1. Oral epithelium

No calretinin immunostaining was seen in the oralepithelium for the developmental period studied (Fig.2b and c).

3.2. Dental lamina

Calretinin immunostaining of the dental lamina wasfirst noticed in a few specimens in the early cap stage(E17) of tooth development. Positive cells were alsoseen in approximately half of the specimens in the cap(E18) and late cap (E20) stages, and in the majority ofthe specimens in the early (P0) and late bell (P7) stages.Staining varied from weak to intense, and was alwaysfocally distributed in basal cells at the periphery of thelamina (Fig. 2c).

3.3. Ectomesenchyme

No calretinin immunostaining was seen in the ec-tomesenchyme for the developmental period studied(Fig. 1a).

3.4. Outer enamel epithelium

Immunopositive cells were first seen in only onespecimen in the early cap (E17) stage. In the cap (E18)and late cap (E20) stages, approximately half of thespecimens showed immunopositive cells, while in theearly (P0) and late bell (P7) stages the majority showedpositive cells. As tooth development progressed thestaining intensity increased, from weak to moderate inthe cap (E17 and E20) stages to moderate or intense in

Table 1Summary of results showing the distribution pattern and staining intensity of calretinin in various tissues at different stages ofdevelopment in rat molars

E20E18E17 P0 P7

– –Oral epithelium – ––(f), +++(f), +Dental lamina (f), ++(f), ++(f), +–Ectomesenchyme ––––

(f), + (f), +Outer enamel epithelium (f), ++ (f), ++ (f), +++Inner enamel epithelium/pre-secretory ameloblasts – – (f), + (f/d), ++ (d), +++

(s/f), ++Dental papilla (s/f), ++– (f), +–Stratum intermedium (f), +—

(f), +++(f), ++(f), +Stellate reticulum (f), +– –Odontoblasts

Ameloblasts (d), ++(d), +++

Distribution pattern: (s), single; (f), focal; (d), diffuse. Staining intensity: +, weak; ++, moderate; +++, intense.

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Fig. 1.

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the bell stages (P0 and P7). The staining pattern wasagain focal and was concentrated in the part of theouter enamel epithelium, where it originated from thedental lamina (Figs. 1e, 2b and c).

3.5. Inner enamel epithelium/presecretory ameloblasts

Calretinin immunostaining was present in the innerenamel epithelium in most of the specimens from thelate cap stage onwards (E20, P0 and P7) (Fig. 1c– f).Staining progressed from weak in the late cap stage(E20) to moderate and intense in the early bell stage(P0). All specimens in the late bell stage (P7) werestained intensely (Fig. 1e). The distribution pattern ofthe immunostained cells was focal in the early develop-mental stages but became diffuse as maturation pro-gressed (Fig. 1c and e). The majority of the specimensexamined showed intense staining of the inner enamelepithelium/presecretory ameloblasts in the pit andfissure regions, but over the cusp tips these tissuesremained negative (Fig. 2a).

3.6. Stellate reticulum

In the cap (E18) and late cap (E20) stages, approxi-mately two-thirds of the specimens stained positivelyfor calretinin in the stellate reticulum, while this num-ber increased to more than 90% for the early (P0) andlate bell (P7) stages. Staining again progressed fromweak in the late cap (E20) to moderate or intense in theearly (P0) and late (P7) bell stages. The distribution wasfocal and staining was never diffuse. In some cells thecytoplasmic processes also stained intensely (Figs. 1b, e,f and 2a).

3.7. Stratum intermedium

Stratum intermedium stained only weakly in a fewspecimens in the early bell stage (P0) and the stainingpattern was focal (Fig. 1e).

3.8. Dental papilla

Immunostaining was first observed in the dentalpapilla in the late cap stage (E20) when approximatelyhalf of the specimens showed calretinin-positive cells.As maturation progressed, only a few of the specimensremained immunoreactive. The positive cells were either

focal or isolated and stained weakly to moderately.Some were located superficially in the dental papillawhile others were deeper (Fig. 2b). The possibility thatthe superficial stained cells were preodontoblasts wasconsidered but it seemed more likely that they weredeveloping neural elements of the subodontoblasticnerve plexus (Fig. 1d). The stromal cells did not expresscalretinin at any stage of tooth development.

3.9. Odontoblasts

No staining of odontoblasts was seen at any stage(Fig. 2d).

3.10. Ameloblasts

Most of the specimens in the early bell (P0) and all ofthe specimens in the late bell (P7) stages showed posi-tive staining. It was at its most intense in presecretoryameloblasts before enamel production (Fig. 1e). Onceenamel formation was present the staining intensitydecreased, so that in the mature functioning layer ofameloblasts it was diffuse, weak to moderate, andlocated only in the cytoplasm, including the Tomesprocesses (Fig. 2c and d).

4. Discussion

We show that calretinin is distributed widely in epi-thelium-derived tissues during odontogenesis in theenamel organs of rat molar teeth. In the dental lamina,outer enamel epithelium and stellate reticulum, theexpression of calretinin was focal and generally weaklyto moderately stained throughout all the stages of toothdevelopment studied. In the inner enamel epithelium,the staining intensity and distribution increased withadvancing differentiation, reaching a maximum whenpresecretory ameloblasts were formed. In ameloblasts,staining was at its most intense before the deposition ofenamel matrix and thereafter decreased, so that in themature cells it was restricted to the cytoplasm and wasgraded only as weak to moderate. Calretinin was notexpressed in the ectomesenchyme and odontoblasts. Inthe dental papilla, staining was observed in the late capand bell stages, both superficially and deeper, inter-preted as representing neural elements including thesubodontoblastic plexus.

Fig. 1. Calretinin immunostaining: (a) A tooth germ (E17) showing no immunostaining of the ectomesenchyme and tooth bud(256× ). (b) Cap stage of tooth development (E18) showing immunostaining of stellate reticulum (256× ). (c) Molar tooth germ(E20) showing immunostaining of inner and outer enamel epithelia (256× ). (d) Molar tooth germ (E20) showing immunostainingof the inner and outer enamel epithelia and superficial cells of the dental papilla (160× ). (e) Molar tooth germ (P0) showingimmunostaining of the presecretory ameloblasts, outer enamel epithelium and stratum intermedium (arrow) (256× ). (f) Molar toothgerm (P0) showing immunostaining of inner enamel epithelium and stellate reticulum (400× ).

D. Mistry et al. / Archi�es of Oral Biology 46 (2001) 973–981978

Fig. 2. Calretinin immunostaining: (a) Molar tooth germ (P0) showing discontinuous immunostaining of inner enamel epitheliumover the cusp tips (160× ). (b) Molar tooth germ (P0) showing immunostaining of outer and inner enamel epithelia and dentalpapilla (256× ). (c) Molar tooth germ (P7) showing moderate immunostaining of the dental lamina, outer enamel epithelium andameloblasts (400× ). (d) Tooth germ (P7) showing moderate immunostaining of ameloblasts; the mature odontoblasts are negative(640× ).

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To the best of our knowledge there are no previouslypublished reports on calretinin expression in odontogenicepithelium and ectomesenchyme during tooth develop-ment. There are, however, several papers reporting theexpression of different calcium-binding proteins, such ascalbindin 28 kDa (Celio et al., 1984; Taylor et al., 1984;Magloire et al., 1988; Berdal et al., 1989a,b, 1991a,b,1993, 1996; Hotton et al., 1995; Bailleul-Forestier et al.,1996; Onishi et al., 1999), calbindin 9 kDa (Taylor et al.,1984; Berdal et al., 1989b, 1991a,b, 1993, 1996; Balmain,1991; Hotton et al., 1995), calmodulin (Goldberg et al.,1987; Sasaki and Garant, 1987), parvalbumin (Celio etal., 1984; Davideau et al., 1993), and annexins (Goldberget al., 1989, 1990, 1991), using various laboratory tech-niques including immunohistochemistry, immuno-elec-tron microscopy and immunoblotting. Most of thesestudies have concentrated on the localization of thecalcium-binding proteins within ameloblasts or odonto-blasts. Because calcium-binding proteins other thancalretinin were examined in these studies we cannot makea direct comparison between their results and our own.Nevertheless, their findings are of considerable interest.

Taylor (1984), Taylor et al. (1984), Elms and Taylor(1987), Berdal et al. (1989a, 1991a,b, 1993, 1996), andBailleul-Forestier et al. (1996) investigated the expressionof calbindin in developing rat teeth. They found thatcalbindin 28 kDa was expressed in both ameloblasts andodontoblasts, while calbindin 9 kDa was expressed onlyin the ameloblasts. The calbindin was relatively uniformin its distribution within the cytoplasm but was muchlower in concentration or absent from nuclei. Theexpression of calbindin appeared to be controlled devel-opmentally, as it varied with the cell stage and thefunctional steps of amelogenesis. During the maturationstage, calbindin expression was discontinuous, whichcould be associated with cyclical changes in matureameloblasts. Expression as early as the presecretory stagewas noted. Calbindin has not been observed in other cellsinvolved in early tooth formation, including pulpal cells,odontoblasts, stratum intermedium, stellate reticulum,and the outer enamel epithelium. Onishi et al. (1999)detected calbindin 28 kDa reactivity during root forma-tion in some cells of the epithelial rests of Malassez atthe bifurcation region, in certain cells between rootdentine and cementum at the apical region, in Hertwig’sroot sheath when it was fragmented, and in preodonto-blasts and odontoblasts.

Although there is a high degree of homology betweencalretinin and calbindin, with cross-reactivity havingbeen demonstrated (Resibois et al., 1989), the antiserumwe use does not cross-react with calbindin onimmunoblots.

Goldberg et al. (1987) and Sasaki and Garant (1987)studied the localization of calmodulin in rat incisorameloblasts and odontoblasts during early stages of

tooth development. They showed that it was present inthe presecretory and secretory ameloblasts, Tomes’ pro-cesses and odontoblasts.

Celio et al. (1984) and Davideau et al. (1993) confirmedthe presence of parvalbumin in ameloblasts in the secre-tory stage. It was also found in maturation-stageameloblasts, and in other cells, such as odontoblasts andstratum intermedium.

Goldberg et al. (1990) studied the expression ofannexins in ameloblasts and odontoblasts. They foundthat annexins I–VI were present in the cytosol insecretory vesicles in the ameloblasts. The forming enamelalso expressed these proteins.

The different subcellular, cellular and developmentallyrelated distributions of these calcium-binding proteinssuggest they have significantly different functions.Calmodulins may control Ca-ATPase in ameloblasts(Sasaki and Garant, 1987), annexin VI may be involvedwith actin during ameloblast and odontoblast secretion(Goldberg et al., 1991), while the calbindins mightmediate the regulation of amelogenesis by vitamin D(Berdal et al., 1989b).

A problem experienced in our study was variablestaining patterns, with some specimens staining diffuselyand intensely while in others the staining was focal andweak or none occurred at all. In fact, even in the samesection, variation occurred between the various toothgerms present. A possible explanation is that not allspecimens were stained at the same time but consecutivebatches were stained on different occasions, or that suchvariation is a result of cyclical changes in matureameloblasts. A further problem, as previously men-tioned, was that the specimens were not demineralized.Without demineralization, considerable tearing and lift-ing of the tissues was seen, especially in the seven-dayspecimens. We did not study older animals, in whichthere would be root formation and tooth eruption, andso a complete view of calretinin expressivity throughoutthe entire period of tooth development and eruption wasnot obtained.

Our findings show that the major sites of expressionof calretinin are the inner enamel epithelium, presecre-tory and secretory ameloblasts, suggesting that thisprotein may play a part in enamel formation. Over thecusp tips, where enamel is not normally present in maturerat molar teeth, calretinin expression remained negative,but if there is a relation between the absence of enameland lack of calretinin it has yet to be established. In allother tissues, staining was focal and less intense, andtherefore possibly of no biological significance.

A possible explanation for the dynamic spatial andtemporal distribution exhibited by calretinin is that thisprotein too, as suggested for calbindin by Elms andTaylor (1987), may be present only in cells that residedirectly in the path of calcium in transition on its wayto the enamel matrix, acting as a ‘calcium ferry’.

D. Mistry et al. / Archi�es of Oral Biology 46 (2001) 973–981980

The physiological function of calcium-bindingproteins is achieved via calcium-dependent interactionswith other proteins, thereby regulating their activity.Their major role is assumed to be buffering, transport ofcalcium ions, and regulation of various enzyme systems(Heizmann, 1992). Also, a role in electrical transmissionwould be consistent with the distribution of this proteinin the nervous system. Because there is no generalagreement on the biochemical role of calretinin in theother systems in which it has been studied more exten-sively, its role in odontogenesis, and especially in themineralization of enamel, also remains speculative.

Recent work in our department has shown thatcalretinin is expressed consistently in the epithelium ofboth unicystic and solid and multicystic ameloblastomas,but not in other odontogenic epithelia lining odontogenickeratocysts, dentigerous cysts and residual cysts (Altiniet al., 2000; Coleman et al., 2001). Based on these results,it was suggested that calretinin might be used as a specificimmunohistochemical marker for ameloblastomas andcould play an important part in the differential diagnosisof odontogenic tumors and cysts.

In the ameloblastomas, staining occurred mainly in thestellate reticulum-like tissues and not in the ameloblast-like cells; in the normal rat molars it occurred mainly inthe inner enamel epithelium, presecretory and secretoryameloblasts, but was also present in the stellate reticu-lum. It thus appears that there is no obvious correlationbetween the staining of normal odontogenic tissues andtheir neoplastic counterparts, but it is uncertain whetherresults obtained in rat tissues should be extrapolated tohuman odontogenic tumors.

Acknowledgements

We would like to thank Dr V. Gotzos of the Universityof Friborg for the opportunity of viewing photomicro-graphs of a similar study that was unpublished butapparently yielded similar results.

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